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Sample records for 2-containing protein tyrosine

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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

  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. Membrane palmitoylated protein 2 is a synaptic scaffold protein required for synaptic SK2-containing channel function

    PubMed Central

    Kim, Gukhan; Luján, Rafael; Schwenk, Jochen; Kelley, Melissa H; Aguado, Carolina; Watanabe, Masahiko; Fakler, Bernd; Maylie, James; Adelman, John P

    2016-01-01

    Mouse CA1 pyramidal neurons express apamin-sensitive SK2-containing channels in the post-synaptic membrane, positioned close to NMDA-type (N-methyl-D-aspartate) glutamate receptors. Activated by synaptically evoked NMDAR-dependent Ca2+ influx, the synaptic SK2-containing channels modulate excitatory post-synaptic responses and the induction of synaptic plasticity. In addition, their activity- and protein kinase A-dependent trafficking contributes to expression of long-term potentiation (LTP). We have identified a novel synaptic scaffold, MPP2 (membrane palmitoylated protein 2; p55), a member of the membrane-associated guanylate kinase (MAGUK) family that interacts with SK2-containing channels. MPP2 and SK2 co-immunopurified from mouse brain, and co-immunoprecipitated when they were co-expressed in HEK293 cells. MPP2 is highly expressed in the post-synaptic density of dendritic spines on CA1 pyramidal neurons. Knocking down MPP2 expression selectively abolished the SK2-containing channel contribution to synaptic responses and decreased LTP. Thus, MPP2 is a novel synaptic scaffold that is required for proper synaptic localization and function of SK2-containing channels. DOI: http://dx.doi.org/10.7554/eLife.12637.001 PMID:26880549

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. The association between the SH2-containing inositol polyphosphate 5-Phosphatase 2 (SHIP2) and the adaptor protein APS has an impact on biochemical properties of both partners.

    PubMed

    Onnockx, Sheela; De Schutter, Julie; Blockmans, Marianne; Xie, Jingwei; Jacobs, Christine; Vanderwinden, Jean-Marie; Erneux, Christophe; Pirson, Isabelle

    2008-01-01

    SHIP2 (SH2-containing inositol polyphosphate 5-phosphatase 2) is a phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase containing various motifs susceptible to mediate protein-protein interaction. In cell models, SHIP2 negatively regulates insulin signalling through its catalytic PtdIns(3,4,5)P(3) 5-phosphatase activity. We have previously reported that SHIP2 interacts with the c-Cbl associated protein (CAP) and c-Cbl, proteins implicated in the insulin cellular response regulating the small G protein TC10. The first steps of the TC10 pathway are the recruitment and tyrosine phosphorylation by the insulin receptor of the adaptor protein with Pleckstrin Homology and Src Homology 2 domains (APS). Herein, we show that SHIP2 can directly interact with APS in 3T3-L1 adipocytes and in transfected CHO-IR cells (Chinese hamster ovary cells stably transfected with the insulin receptor). Upon insulin stimulation, APS and SHIP2 are recruited to cell membranes as seen by immunofluorescence studies, which is consistent with their interaction. We also observed that SHIP2 negatively regulates APS insulin-induced tyrosine phosphorylation and consequently inhibits APS association with c-Cbl. APS, which specifically interacts with SHIP2, but not PTEN, in turn, increases the PtdIns(3,4,5)P(3) 5-phosphatase activity of SHIP2 in an inositol phosphatase assay. Co-transfection of SHIP2 and APS in CHO-IR cells further increases the inhibitory effect of SHIP2 on Akt insulin-induced phosphorylation. Therefore, the interaction between APS and SHIP2 provides to both proteins potential negative regulatory mechanisms to act on the insulin cascade.

  7. 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.

  8. 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

  9. SmShb, the SH2-Containing Adaptor Protein B of Schistosoma mansoni Regulates Venus Kinase Receptor Signaling Pathways.

    PubMed

    Morel, Marion; Vanderstraete, Mathieu; Cailliau, Katia; Hahnel, Steffen; Grevelding, Christoph G; Dissous, Colette

    2016-01-01

    Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) formed by an extracellular Venus Fly Trap (VFT) ligand binding domain associated via a transmembrane domain with an intracellular tyrosine kinase (TK) domain. Schistosoma mansoni VKRs, SmVKR1 and SmVKR2, are both implicated in reproductive activities of the parasite. In this work, we show that the SH2 domain-containing protein SmShb is a partner of the phosphorylated form of SmVKR1. Expression of these proteins in Xenopus oocytes allowed us to demonstrate that the SH2 domain of SmShb interacts with the phosphotyrosine residue (pY979) located in the juxtamembrane region of SmVKR1. This interaction leads to phosphorylation of SmShb on tyrosines and promotes SmVKR1 signaling towards the JNK pathway. SmShb transcripts are expressed in all parasite stages and they were found in ovary and testes of adult worms, suggesting a possible colocalization of SmShb and SmVKR1 proteins. Silencing of SmShb in adult S. mansoni resulted in an accumulation of mature sperm in testes, indicating a possible role of SmShb in gametogenesis.

  10. SmShb, the SH2-Containing Adaptor Protein B of Schistosoma mansoni Regulates Venus Kinase Receptor Signaling Pathways

    PubMed Central

    Morel, Marion; Vanderstraete, Mathieu; Cailliau, Katia; Hahnel, Steffen; Grevelding, Christoph G.; Dissous, Colette

    2016-01-01

    Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) formed by an extracellular Venus Fly Trap (VFT) ligand binding domain associated via a transmembrane domain with an intracellular tyrosine kinase (TK) domain. Schistosoma mansoni VKRs, SmVKR1 and SmVKR2, are both implicated in reproductive activities of the parasite. In this work, we show that the SH2 domain-containing protein SmShb is a partner of the phosphorylated form of SmVKR1. Expression of these proteins in Xenopus oocytes allowed us to demonstrate that the SH2 domain of SmShb interacts with the phosphotyrosine residue (pY979) located in the juxtamembrane region of SmVKR1. This interaction leads to phosphorylation of SmShb on tyrosines and promotes SmVKR1 signaling towards the JNK pathway. SmShb transcripts are expressed in all parasite stages and they were found in ovary and testes of adult worms, suggesting a possible colocalization of SmShb and SmVKR1 proteins. Silencing of SmShb in adult S. mansoni resulted in an accumulation of mature sperm in testes, indicating a possible role of SmShb in gametogenesis. PMID:27636711

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. [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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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 ...

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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...

  19. 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.

  20. 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

  1. 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.

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

    PubMed

    Madl, Christopher M; Heilshorn, Sarah C

    2017-02-02

    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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  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. 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

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. Electrogenerated chemiluminescence. 80. C-reactive protein determination at high amplification with [Ru(bpy)3]2+-containing microspheres.

    PubMed

    Miao, Wujian; Bard, Allen J

    2004-12-01

    Biotinylated anti-C-reactive protein (CRP) species were attached to the surface of streptavidin-coated magnetic beads (MB) and avidin-coated polystyrene microspheres/beads (PSB) entrapping a large number of electrogenerated chemiluminescence (ECL) labels ( approximately 10(9) Ru(bpy)(3)[B(C(6)F(5))(4)](2)/bead) to form anti-CRP<-->MB and Ru(II) subsetPSB/avidin<-->anti-CRP conjugates, respectively. Sandwich-type Ru(II) subsetPSB/avidin<-->anti-CRP CRP anti-CRP<-->MB aggregates were formed when Ru(II) subsetPSB/avidin<-->anti-CRP was mixed with anti-CRP<-->MB conjugates in the presence of analyte CRP. The newly formed aggregates were magnetically separated from the reaction media and dissolved in MeCN containing tri-n-propylamine as an ECL coreactant. ECL was carried out with a potential scan from 0 to 2.8 V vs Ag/Ag(+), and the ECL intensity was found to be proportional to the analyte CRP concentration over the range of 0.010-10 mug/mL. The CRP concentration of an unknown human plasma specimen was measured by the standard addition method based on this technique. Elimination of the nonspecific adsorption of the CRP system with several different blocking agents was also studied, and 2.0% bovine serum albumin was found to be best.

  3. 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.

  4. An isoform of microtubule-associated protein 2 (MAP2) containing four repeats of the tubulin-binding motif.

    PubMed

    Doll, T; Meichsner, M; Riederer, B M; Honegger, P; Matus, A

    1993-10-01

    Microtubule-associated protein 2 (MAP2) exists in both high- and low-molecular mass isoforms, each of which has a tubulin-binding domain consisting of 3 imperfect tandem repeats of 31 amino acids containing a more highly conserved 18 amino acid 'core' sequence. We describe here a novel form of low molecular mass MAP2 (MAP2c) that contains an additional 4th repeat of this tubulin-binding motif. Like the 3 previously known repeat sequences, this 4th copy is highly conserved between MAP2 and the two other known members of the same gene family, tau and MAP4. In each of these three genes the additional 4th repeat is inserted between the 1st and 2nd repeats of the 3-repeat form of the molecule. Experiments with brain cell cultures, in which the relative proportions of neurons and glia had been manipulated by drug treatment, showed that 4-repeat MAP2c is associated with glial cells whereas 3-repeat MAP2c is expressed in neurons. Whereas 3-repeat MAP2c is expressed early in development and then declines, the level of 4-repeat MAP2c increases later in development, corresponding to the relatively late differentiation of glial cells compared to neurons. When transfected into non-neuronal cells, the 4-repeat version of MAP2c behaved indistinguishably from the 3-repeat form in stabilising and rearranging cellular microtubules. The presence of an additional 4th repeat of the tubulin-binding motif in all three members of the MAP2 gene family suggests that this variant arose prior to their differentiation from an ancestral gene.

  5. 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

  6. 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.

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

    PubMed Central

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

    2013-01-01

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

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  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. Infection with Chlamydia trachomatis alters the tyrosine phosphorylation and/or localization of several host cell proteins including cortactin.

    PubMed Central

    Fawaz, F S; van Ooij, C; Homola, E; Mutka, S C; Engel, J N

    1997-01-01

    Infection of epithelial cells by two biovars of Chlamydia trachomatis results in the tyrosine phosphorylation of several host proteins. The most prominent change in host protein tyrosine phosphorylation involves a complex of proteins with molecular masses of 75 to 85 kDa (pp75/85) and 100 kDa (pp100). The C. trachomatis-induced tyrosine phosphorylation of pp75/85 and pp100 is observed in several cell lines, including epithelial cells, fibroblasts, and macrophages. Subcellular fractionation and detergent solubility properties of pp75/85 are consistent with its association with the cytoskeleton. Phosphoamino acid analysis demonstrates that the pp75/85 complex is phosphorylated on both tyrosine and serine residues. Immunofluorescence studies of chlamydia-infected cells by using fluorescein isothiocyanate-phalloidin and antibodies to phosphotyrosine and cortactin demonstrate that tyrosine-phosphorylated proteins, as well as cortactin, are localized to the chlamydial vacuole and that this process is facilitated by actin. PMID:9393830

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  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

    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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Role of Non-receptor Protein Tyrosine Kinases During Phospholipase C-γ1 Related Uterine Contractions in the Rat

    PubMed Central

    Phillippe, Mark; Sweet, Leigh M.; Bradley, Diana F.; Engle, Daniel

    2011-01-01

    Activated phospholipase Cγ1 (PLC-γ1), produced in response to tyrosine phosphorylation, appears to play an important role during uterine contractions. These studies sought to determine which non-receptor protein tyrosine kinases (PTKs) are involved in the tyrosine phosphorylation and activation of PLC-γ1 in uterine tissue from the rat. In vitro uterine contraction studies were performed utilizing isoform specific PTK inhibitors. Western blots were performed utilizing antibodies to phosphotyrosine-PLC-γ1, total PLC-γ1, c-Src kinase and Lck kinase. Spontaneous, stretch-stimulated, and bpV(phen) (a tyrosine phosphatase inhibitor) enhanced uterine contractions were significantly suppressed in response to Damnacanthal (a Lck kinase inhibitor) and PP1 (a c-Src kinase inhibitor); whereas, several other PTK isoform inhibitors had no significant effect. Damnacanthal and PP1 also significantly suppressed bpV(phen)-enhanced tyrosine phosphorylation of PLC-γ1 compared to other PTK isoform inhibitors. Western blots confirmed expression of the Lck and c-Src kinases in uterine tissue. In conclusion, the Lck and c-Src kinases appear to play an important role in regulating tyrosine phosphorylation of PLC-γ1 and contractile activity in the rat uterus. PMID:19208792

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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

  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. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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).

  16. 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.

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

    PubMed

    Katsube, Takanori; Tsuji, Hideo; Onoda, Makoto

    2007-06-01

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

  18. Protein tyrosine kinase signaling in the mouse oocyte cortex during sperm-egg interactions and anaphase resumption.

    PubMed

    McGinnis, Lynda K; Luo, Jinping; Kinsey, William H

    2013-04-01

    Fertilization triggers activation of a series of pre-programmed signal transduction pathways in the oocyte that establish a block to polyspermy, induce meiotic resumption, and initiate zygotic development. Fusion between sperm and oocyte results in rapid changes in oocyte intracellular free-calcium levels, which in turn activate multiple protein kinase cascades in the ooplasm. The present study examined the possibility that sperm-oocyte interaction involves localized activation of oocyte protein tyrosine kinases, which could provide an alternative signaling mechanism to that triggered by the fertilizing sperm. Confocal immunofluorescence analysis with antibodies to phosphotyrosine and phosphorylated protein tyrosine kinases allowed detection of minute signaling events localized to the site of sperm-oocyte interaction that were not amenable to biochemical analysis. The results provide evidence for localized accumulation of phosphotyrosine at the site of sperm contact, binding, or fusion, which suggests active protein tyrosine kinase signaling prior to and during sperm incorporation. The PYK2 kinase was found to be concentrated and activated at the site of sperm-oocyte interaction, and likely participates in this response. Widespread activation of PYK2 and FAK kinases was subsequently observed within the oocyte cortex, indicating that sperm incorporation is followed by more global signaling via these kinases during meiotic resumption. The results demonstrate an alternate signaling pathway triggered in mammalian oocytes by sperm contact, binding, or fusion with the oocyte.

  19. Channel-anchored Protein Kinase CK2 and Protein Phosphatase 1 Reciprocally Regulate KCNQ2-containing M-channels via Phosphorylation of Calmodulin*

    PubMed Central

    Kang, Seungwoo; Xu, Mingxuan; Cooper, Edward C.; Hoshi, Naoto

    2014-01-01

    M-type potassium channels, encoded by the KCNQ family genes (KCNQ2–5), require calmodulin as an essential co-factor. Calmodulin bound to the KCNQ2 subunit regulates channel trafficking and stabilizes channel activity. We demonstrate that phosphorylation of calmodulin by protein kinase CK2 (casein kinase 2) rapidly and reversibly modulated KCNQ2 current. CK2-mediated phosphorylation of calmodulin strengthened its binding to KCNQ2 channel, caused resistance to phosphatidylinositol 4,5-bisphosphate depletion, and increased KCNQ2 current amplitude. Accordingly, application of CK2-selective inhibitors suppressed KCNQ2 current. This suppression was prevented by co-expression of CK2 phosphomimetic calmodulin mutants or pretreatment with a protein phosphatase inhibitor, calyculin A. We also demonstrated that functional CK2 and protein phosphatase 1 (PP1) were selectively tethered to the KCNQ2 subunit. We identified a functional KVXF consensus site for PP1 binding in the N-terminal tail of KCNQ2 subunit: mutation of this site augmented current density. CK2 inhibitor treatment suppressed M-current in rat superior cervical ganglion neurons, an effect negated by overexpression of phosphomimetic calmodulin or pretreatment with calyculin A Furthermore, CK2 inhibition diminished the medium after hyperpolarization by suppressing the M-current. These findings suggest that CK2-mediated phosphorylation of calmodulin regulates the M-current, which is tonically regulated by CK2 and PP1 anchored to the KCNQ2 channel complex. PMID:24627475

  20. Effects of Vascular-Endothelial Protein Tyrosine Phosphatase Inhibition on Breast Cancer Vasculature and Metastatic Progression

    PubMed Central

    2013-01-01

    Background The solid tumor microvasculature is characterized by structural and functional abnormality and mediates several deleterious aspects of tumor behavior. Here we determine the role of vascular endothelial protein tyrosine phosphatase (VE-PTP), which deactivates endothelial cell (EC) Tie-2 receptor tyrosine kinase, thereby impairing maturation of tumor vessels. Methods AKB-9778 is a first-in-class VE-PTP inhibitor. We examined its effects on ECs in vitro and on embryonic angiogenesis in vivo using zebrafish assays. We studied the impact of AKB-9778 therapy on the tumor vasculature, tumor growth, and metastatic progression using orthotopic models of murine mammary carcinoma as well as spontaneous and experimental metastasis models. Finally, we used endothelial nitric oxide synthase (eNOS)–deficient mice to establish the role of eNOS in mediating the effects of VE-PTP inhibition. All statistical tests were two-sided. Results AKB-9778 induced ligand-independent Tie-2 activation in ECs and impaired embryonic zebrafish angiogenesis. AKB-9778 delayed the early phase of mammary tumor growth by maintaining vascular maturity (P < .01, t test); slowed growth of micrometastases (P < .01, χ2 test) by preventing extravasation of tumor cells (P < 0.01, Fisher exact test), resulting in a trend toward prolonged survival (27.0 vs 36.5 days; hazard ratio of death = 0.33, 95% confidence interval = 0.11 to 1.03; P = .05, Mantel–Cox test); and stabilized established primary tumor blood vessels, enhancing tumor perfusion (P = .03 for 4T1 tumor model and 0.05 for E0771 tumor model, by two-sided t tests) and, hence, radiation response (P < .01, analysis of variance; n = 7 mice per group). The effects of AKB-9778 on tumor vessels were mediated in part by endothelial nitric oxide synthase activation. Conclusions Our results demonstrate that pharmacological VE-PTP inhibition can normalize the structure and function of tumor vessels through Tie-2 activation, which delays tumor

  1. Human cervical cancer cells use Ca2+ signalling, protein tyrosine phosphorylation and MAP kinase in regulatory volume decrease

    PubMed Central

    Shen, Meng-Ru; Chou, Cheng-Yang; Browning, Joseph A; Wilkins, Robert J; Ellory, J Clive

    2001-01-01

    This study was aimed at identifying the signalling pathways involved in the activation of volume-regulatory mechanisms of human cervical cancer cells. Osmotic swelling of human cervical cancer cells induced a substantial increase in intracellular Ca2+ ([Ca2+]i) by the activation of Ca2+ entry across the cell membrane, as well as Ca2+ release from intracellular stores. This Ca2+ signalling was critical for the normal regulatory volume decrease (RVD) response. The activation of swelling-activated ion and taurine transport was significantly inhibited by tyrosine kinase inhibitors (genistein and tyrphostin AG 1478) and potentiated by the tyrosine phosphatase inhibitor Na3VO4. However, the Src family of tyrosine kinases was not involved in regulation of the swelling-activated Cl− channel. Cell swelling triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) and p38 kinase. The volume-responsive ERK1/ERK2 signalling pathway linked with the activation of K+ and Cl− channels, and taurine transport. However, the volume-regulatory mechanism was independent of the activation of p38 MAP kinase. The phosphorylated ERK1/ERK2 expression following a hypotonic shock was up-regulated by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and down-regulated by PKC inhibitor staurosporine. The response of ERK activation to hypotonicity also required Ca2+ entry and depended on tyrosine kinase and mitogen-activated/ERK-activating kinase (MEK) activity. Considering the results overall, osmotic swelling promotes the activation of tyrosine kinase and ERK1/ERK2 and raises intracellular Ca2+, all of which play a crucial role in the volume-regulatory mechanism of human cervical cancer cells. PMID:11731569

  2. Activation of Helicobacter pylori CagA by tyrosine phosphorylation is essential for dephosphorylation of host cell proteins in gastric epithelial cells.

    PubMed

    Püls, Jurgen; Fischer, Wolfgang; Haas, Rainer

    2002-02-01

    Helicobacter pylori type I strains harbour the cag pathogenicity island (cag-PAI), a 37 kb sequence,which encodes the components of a type IV secretion system. CagA, the first identified effector protein of the cag-PAI, is translocated into eukaryotic cells and tyrosine phosphorylated (CagAP-tyr) by a host cell tyrosine kinase. Translocation of CagA induces the dephosphorylation of a set of phosphorylated host cell proteins of unknown identity. CagA proteins of independent H. pylori strains vary in sequence and thus in the number and composition of putative tyrosine phosphorylation motifs (TPMs). The CagA protein of H. pylori strain J99 (CagAJ99) does not carry any of three putative tyrosine phosphorylation motifs (TPM-A, TPM-B or TPM-C) predicted by the MOTIF algorithm in CagA proteins. CagA,n is not tyrosine phosphorylated and is inactive in the dephosphorylation of host cell proteins. By site-specific mutagenesis,we introduced a TPM-C into CagA,. by replacing a single lysine with a tyrosine. This slight modification resulted in tyrosine phosphorylation of CagAJ99 and host cell protein dephosphorylation. In contrast, the removal of the indigenous TPM-C from CagAP12 did not abolish its tyrosine phosphorylation, suggesting that further phosphorylated sites are present in CagAP12. By generation of hybrid CagA proteins, a phosphorylation of the most N-terminal TPM-A could be excluded. Our data suggest that tyrosine phosphorylation at TPM-C is sufficient, but not exclusive,to activate translocated CagA. Activated CagAPtr might either convert into a phosphatase itself or activate a cellular phosphatase to dephosphorylate cellular phosphoproteins and modulate cellular signalling cascades of the host.

  3. The UL12 Protein of Herpes Simplex Virus 1 Is Regulated by Tyrosine Phosphorylation

    PubMed Central

    Fujii, Hikaru; Kato, Akihisa; Mugitani, Michio; Kashima, Yukie; Oyama, Masaaki; Kozuka-Hata, Hiroko; Arii, Jun

    2014-01-01

    ABSTRACT The herpes simplex virus 1 (HSV-1) UL12 protein (pUL12) is a nuclease that is critical for viral replication in vitro and neurovirulence in vivo. In this study, mass spectrometric analysis of pUL12 and phosphate-affinity SDS-polyacrylamide gel electrophoresis analysis identified tyrosine at pUL12 residue 371 (Tyr-371) as a pUL12 phosphorylation site: Tyr-371 is conserved in pUL12 homologs in herpesviruses in all Herpesviridae subfamilies. Replacement of Tyr-371 with phenylalanine (Y371F) in pUL12 (i) abolished its exonuclease activity in HSV-1-infected Vero, HEL, and A549 cells, (ii) reduced viral replication, cell-cell spread, and pUL12 expression in infected cells in a cell type-dependent manner, (iii) led to aberrant subcellular localization of pUL12 in infected cells in a cell type-dependent manner, and (iv) reduced HSV-1 neurovirulence in mice. The effects of the pUL12 Y371F mutation in cell cultures and mice were similar to those of a nuclease-dead double mutation in pUL12, although the Y371F mutation reduced viral replication severalfold more than the nuclease-dead double mutation in a cell type- and multiplicity-of-infection-dependent manner. Replacement of Tyr-371 with glutamic acid, which mimics constitutive phosphorylation, restored the wild-type phenotype in cell cultures and mice. These results suggested that phosphorylation of pUL12 Tyr-371 was essential for pUL12 to express its nuclease activity in HSV-1-infected cells and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and neurovirulence in mice mainly by upregulating pUL12 nuclease activity and, in part, by regulating the subcellular localization and expression of pUL12 in HSV-1-infected cells. IMPORTANCE Herpesviruses encode a considerable number of enzymes for their replication. Like cellular enzymes, the viral enzymes need to be properly regulated in infected cells. Although the functional aspects of herpesvirus enzymes have gradually been

  4. Protein tyrosine phosphatase 1B inhibitory activities of ursane- and lupane-type triterpenes from Sorbus pohuashanensis.

    PubMed

    Li, Dongxia; Li, Wei; Higai, Koji; Koike, Kazuo

    2014-04-01

    Protein tyrosine phosphatase 1B (PTP1B) is a non-transmembrane protein tyrosine phosphatase, and has received much attention as a molecular target for the treatment of insulin resistance diseases because of its critical roles in negatively regulating insulin- and leptin-signaling cascades. Six ursane-type triterpenes, 3β-acetoxy-urs-12-ene-28-oic acid (1), pomolic acid-3β-acetate (2), pomolic acid (3), ursolaldehyde (4), euscaphic acid (5) and 3β-acetoxy-urs-11-en-28,13-olide (6), and a lupane-type triterpene, betulinic acid (7), from the fruits of Sorbus pohuashanensis, exhibited significant PTP1B inhibitory activity, with IC50 values ranging from 3.5 to 54.8 μM. Kinetics analyses revealed that compounds 2, 3, and 7 are non-competitive PTP1B inhibitors, and compounds 1 and 6 are mixed-type PTP1B inhibitors.

  5. S-nitrosylation of endogenous protein tyrosine phosphatases in endothelial insulin signaling.

    PubMed

    Hsu, Ming-Fo; Pan, Kuan-Ting; Chang, Fan-Yu; Khoo, Kay-Hooi; Urlaub, Henning; Cheng, Ching-Feng; Chang, Geen-Dong; Haj, Fawaz G; Meng, Tzu-Ching

    2016-10-01

    Nitric oxide (NO) exerts its biological function through S-nitrosylation of cellular proteins. Due to the labile nature of this modification under physiological condition, identification of S-nitrosylated residue in enzymes involved in signaling regulation remains technically challenging. The present study investigated whether intrinsic NO produced in endothelium-derived MS-1 cells response to insulin stimulation might target endogenous protein tyrosine phosphatases (PTPs). For this, we have developed an approach using a synthetic reagent that introduces a phenylacetamidyl moiety on S-nitrosylated Cys, followed by detection with anti-phenylacetamidyl Cys (PAC) antibody. Coupling with sequential blocking of free thiols with multiple iodoacetyl-based Cys-reactive chemicals, we employed this PAC-switch method to show that endogenous SHP-2 and PTP1B were S-nitrosylated in MS-1 cells exposed to insulin. The mass spectrometry detected a phenylacetamidyl moiety specifically present on the active-site Cys463 of SHP-2. Focusing on the regulatory role of PTP1B, we showed S-nitrosylation to be the principal Cys reversible redox modification in endothelial insulin signaling. The PAC-switch method in an imaging format illustrated that a pool of S-nitrosylated PTP1B was colocalized with activated insulin receptor to the cell periphery, and that such event was endothelial NO synthase (eNOS)-dependent. Moreover, ectopic expression of the C215S mutant of PTP1B that mimics the active-site Cys215 S-nitrosylated form restored insulin responsiveness in eNOS-ablated cells, which was otherwise insensitive to insulin stimulation. This work not only introduces a new method that explores the role of physiological NO in regulating signal transduction, but also highlights a positive NO effect on promoting insulin responsiveness through S-nitrosylation of PTP1B's active-site Cys215.

  6. Differential protein stability of EGFR mutants determines responsiveness to tyrosine kinase inhibitors

    PubMed Central

    Ray, Paramita; Tan, Yee Sun; Somnay, Vishal; Mehta, Ranjit; Sitto, Merna; Ahsan, Aarif; Nyati, Shyam; Naughton, John P.; Bridges, Alexander; Zhao, Lili; Rehemtulla, Alnawaz; Lawrence, Theodore S.; Ray, Dipankar; Nyati, Mukesh K.

    2016-01-01

    Non-small cell lung cancer (NSCLC) patients carrying specific EGFR kinase activating mutations (L858R, delE746-A750) respond well to tyrosine kinase inhibitors (TKIs). However, drug resistance develops within a year. In about 50% of such patients, acquired drug resistance is attributed to the enrichment of a constitutively active point mutation within the EGFR kinase domain (T790M). To date, differential drug-binding and altered ATP affinities by EGFR mutants have been shown to be responsible for differential TKI response. As it has been reported that EGFR stability plays a role in the survival of EGFR driven cancers, we hypothesized that differential TKI-induced receptor degradation between the sensitive L858R and delE746-A750 and the resistant T790M may also play a role in drug responsiveness. To explore this, we have utilized an EGFR-null CHO overexpression system as well as NSCLC cell lines expressing various EGFR mutants and determined the effects of erlotinib treatment. We found that erlotinib inhibits EGFR phosphorylation in both TKI sensitive and resistant cells, but the protein half-lives of L858R and delE746-A750 were significantly shorter than L858R/T790M. Third generation EGFR kinase inhibitor (AZD9291) inhibits the growth of L858R/T790M-EGFR driven cells and also induces EGFR degradation. Erlotinib treatment induced polyubiquitination and proteasomal degradation, primarily in a c-CBL-independent manner, in TKI sensitive L858R and delE746-A750 mutants when compared to the L858R/T790M mutant, which correlated with drug sensitivity. These data suggest an additional mechanism of TKI resistance, and we postulate that agents that degrade L858R/T790M-EGFR protein may overcome TKI resistance. PMID:27612423

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

    SciTech Connect

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

    2010-11-26

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

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

    PubMed Central

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

    1987-01-01

    It is speculated that the transmission of an insulin signal across the plasma membrane of cells occurs through activation of the tyrosine-specific receptor kinase, autophosphorylation of the receptor, and subsequent phosphorylation of unidentified substrates in the cell. In an attempt to identify possible substrates, we labeled intact rat fat cells with [32P]orthophosphate and used an antiphosphotyrosine antibody to identify proteins that become phosphorylated on tyrosine residues in an insulin-stimulated way. In the membrane fraction of the fat cells, we found, in addition to the 95-kDa beta-subunit of the receptor, a 46-kDa phosphoprotein that is phosphorylated exclusively on tyrosine residues. This protein is not immunoprecipitated by antibodies against different regions of the insulin receptor and its HPLC tryptic peptide map is different from the tryptic peptide map of the insulin receptor, suggesting that it is not derived from the receptor beta-subunit. Insulin stimulates the tyrosine phosphorylation of the 46-kDa protein within 150 sec in the intact cell 3- to 4-fold in a dose-dependent way at insulin concentrations between 0.5 nM and 100 nM. The insulin effect starts after 30 sec, is maximal at 150 sec, and declines to almost basal values by 5 min. Furthermore, the antiphosphotyrosine antibody precipitated at least five proteins in the soluble fraction of the fat cell. Insulin (0.5 nM, 100 nM) stimulated within 2 min the 32P incorporation into a 116-kDa band, a 62-kDa band, and three bands between 45 kDa and 50 kDa 2- to 10-fold. We suggest that the 46-kDa membrane protein and possibly also the soluble proteins are endogenous substrates of the receptor tyrosine kinase in fat cells and that their phosphorylation is an early step in insulin signal transmission. Images PMID:3540953

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

    PubMed

    Singh, Neetu; Yadav, Smita; Rathaur, Sushma

    2015-11-01

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

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

    PubMed Central

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

    1988-01-01

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

  11. (S)-α-Chlorohydrin Inhibits Protein Tyrosine Phosphorylation through Blocking Cyclic AMP - Protein Kinase A Pathway in Spermatozoa

    PubMed Central

    Zheng, Weiwei; Yang, Bei; Pi, Jingbo; He, Gengsheng; Qu, Weidong

    2012-01-01

    α-Chlorohydrin is a common contaminant in food. Its (S)-isomer, (S)-α-chlorohydrin (SACH), is known for causing infertility in animals by inhibiting glycolysis of spermatozoa. The aim of present work was to examine the relationship between SACH and protein tyrosine phosphorylation (PTP), which plays a critical role in regulating mammalian sperm capacitation. In vitro exposure of SACH 50 µM to isolated rat epididymal sperm inhibited PTP. Sperm-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) activities, the intracellular adenosine 5′-triphosphate (ATP) levels, 3′-5′-cyclic adenosine monophosphate (cAMP) levels and phosphorylation of protein kinase A (PKA) substrates in rat sperm were diminished dramatically, indicating that both glycolysis and the cAMP/PKA signaling pathway were impaired by SACH. The inhibition of both PTP and phosphorylation of PKA substrates by SACH could be restored by addition of cAMP analog dibutyryl-cAMP (dbcAMP) and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Moreover, addition of glycerol protected glycolysis, ATP levels, phosphorylation of PKA substrates and PTP against the influence of SACH. These results suggested SACH inhibited PTP through blocking cAMP/PKA pathway in sperm, and PTP inhibition may play a role in infertility associated with SACH. PMID:22916194

  12. Juvenile hormone stimulated tyrosine kinase-mediated protein phosphorylation in the CNS of the silk worm, Bombyx mori.

    PubMed

    Arif, A; Shanavas, A; Murthy, Ch R K; Dutta-Gupta, Aparna

    2002-07-01

    In vitro studies with the larval CNS of the silkworm, Bombyx mori revealed the phosphorylation of a 48-kDa protein, which was not dependent on cyclic nucleotides. Studies also revealed modest phosphorylation of this protein by a calcium-dependent but calmodulin-independent mechanism. However, phosphorylation of this protein was greatly enhanced in the presence of juvenile hormone (JH) I by a calcium-independent mechanism. This stimulatory effect of JH was seen in both homogenates as well as in intact CNS of Bombyx. Immunoblotting studies revealed the cross-reaction of this 48-kDa protein with phosphotyrosine monoclonal antibody and the phosphorylation of this protein was inhibited by genistein. This study suggests that the 48-kDa protein is a substrate for tyrosine kinase. The phosphorylation of this protein was also observed in other larval tissues such as salivary gland, fat body, and epidermis of Bombyx.

  13. Photochemical tyrosine oxidation in the structurally well-defined α3Y protein: proton-coupled electron transfer and a long-lived tyrosine radical.

    PubMed

    Glover, Starla D; Jorge, Christine; Liang, Li; Valentine, Kathleen G; Hammarström, Leif; Tommos, Cecilia

    2014-10-08

    Tyrosine oxidation-reduction involves proton-coupled electron transfer (PCET) and a reactive radical state. These properties are effectively controlled in enzymes that use tyrosine as a high-potential, one-electron redox cofactor. The α3Y model protein contains Y32, which can be reversibly oxidized and reduced in voltammetry measurements. Structural and kinetic properties of α3Y are presented. A solution NMR structural analysis reveals that Y32 is the most deeply buried residue in α3Y. Time-resolved spectroscopy using a soluble flash-quench generated [Ru(2,2'-bipyridine)3](3+) oxidant provides high-quality Y32-O• absorption spectra. The rate constant of Y32 oxidation (kPCET) is pH dependent: 1.4 × 10(4) M(-1) s(-1) (pH 5.5), 1.8 × 10(5) M(-1) s(-1) (pH 8.5), 5.4 × 10(3) M(-1) s(-1) (pD 5.5), and 4.0 × 10(4) M(-1) s(-1) (pD 8.5). k(H)/k(D) of Y32 oxidation is 2.5 ± 0.5 and 4.5 ± 0.9 at pH(D) 5.5 and 8.5, respectively. These pH and isotope characteristics suggest a concerted or stepwise, proton-first Y32 oxidation mechanism. The photochemical yield of Y32-O• is 28-58% versus the concentration of [Ru(2,2'-bipyridine)3](3+). Y32-O• decays slowly, t1/2 in the range of 2-10 s, at both pH 5.5 and 8.5, via radical-radical dimerization as shown by second-order kinetics and fluorescence data. The high stability of Y32-O• is discussed relative to the structural properties of the Y32 site. Finally, the static α3Y NMR structure cannot explain (i) how the phenolic proton released upon oxidation is removed or (ii) how two Y32-O• come together to form dityrosine. These observations suggest that the dynamic properties of the protein ensemble may play an essential role in controlling the PCET and radical decay characteristics of α3Y.

  14. Identification of GIT1/Cat-1 as a substrate molecule of protein tyrosine phosphatase zeta /beta by the yeast substrate-trapping system.

    PubMed

    Kawachi, H; Fujikawa, A; Maeda, N; Noda, M

    2001-06-05

    We used a genetic method, the yeast substrate-trapping system, to identify substrates for protein tyrosine phosphatases zeta (PTPzeta/RPTPbeta). This method is based on the yeast two-hybrid system, with two essential modifications: conditional expression of protein tyrosine kinase v-src (active src) to tyrosine-phosphorylate the prey proteins and screening by using a substrate-trap mutant of PTPzeta (PTPzeta-D1902A) as bait. By using this system, several substrate candidates for PTPzeta were isolated. Among them, GIT1/Cat-1 (G protein-coupled receptor kinase-interactor 1/Cool-associated, tyrosine-phosphorylated 1) was examined further. GIT1/Cat-1 bound to PTPzeta-D1902A dependent on the substrate tyrosine phosphorylation. Tyrosine-phosphorylated GIT1/Cat-1 was dephosphorylated by PTPzeta in vitro. Immunoprecipitation experiments indicated that PTPzeta-D1902A and GIT1/Cat-1 form a stable complex also in mammalian cells. Immunohistochemical analyses revealed that PTPzeta and GIT1/Cat-1 were colocalized in the processes of pyramidal cells in the hippocampus and neocortex in rat brain. Subcellular colocalization was further verified in the growth cones of mossy fibers from pontine explants and in the ruffling membranes and processes of B103 neuroblastoma cells. Moreover, pleiotrophin, a ligand for PTPzeta, increased tyrosine phosphorylation of GIT1/Cat-1 in B103 cells. All these results indicate that GIT1/Cat-1 is a substrate molecule of PTPzeta.

  15. Protein tyrosine kinase 6 promotes ERBB2-induced mammary gland tumorigenesis in the mouse

    PubMed Central

    Peng, M; Ball-Kell, S M; Tyner, A L

    2015-01-01

    Protein tyrosine kinase 6 (PTK6) expression, activation, and amplification of the PTK6 gene have been reported in ERBB2/HER2-positive mammary gland cancers. To explore contributions of PTK6 to mammary gland tumorigenesis promoted by activated ERBB2, we crossed Ptk6−/− mice with the mouse mammary tumor virus-ERBB2 transgenic mouse line expressing activated ERBB2 and characterized tumor development and progression. ERBB2-induced tumorigenesis was significantly delayed and diminished in mice lacking PTK6. PTK6 expression was induced in the mammary glands of ERBB2 transgenic mice before tumor development and correlated with activation of signal transducer and activator of transcription 3 (STAT3) and increased proliferation. Disruption of PTK6 impaired STAT3 activation and proliferation. Phosphorylation of the PTK6 substrates focal adhesion kinase (FAK) and breast cancer anti-estrogen resistance 1 (BCAR1; p130CAS) was decreased in Ptk6−/− mammary gland tumors. Reduced numbers of metastases were detected in the lungs of Ptk6−/− mice expressing activated ERBB2, compared with wild-type ERBB2 transgenic mice. PTK6 activation was detected at the edges of ERBB2-positive tumors. These data support roles for PTK6 in both ERBB2-induced mammary gland tumor initiation and metastasis, and identify STAT3, FAK, and BCAR1 as physiologically relevant PTK6 substrates in breast cancer. Including PTK6 inhibitors as part of a treatment regimen could have distinct benefits in ERBB2/HER2-positive breast cancers. PMID:26247733

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

    PubMed Central

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

    2015-01-01

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

  17. The protein tyrosine phosphatase PRL-2 interacts with the magnesium transporter CNNM3 to promote oncogenesis.

    PubMed

    Hardy, S; Uetani, N; Wong, N; Kostantin, E; Labbé, D P; Bégin, L R; Mes-Masson, A; Miranda-Saavedra, D; Tremblay, M L

    2015-02-19

    The three PRL (phosphatases of regenerating liver) protein tyrosine phosphatases (PRL-1, -2 and -3) have been identified as key contributors to metastasis in several human cancers, yet the molecular basis of their pro-oncogenic property is unclear. Among the subfamily of PRL phosphatases, overexpression of PRL-2 in breast cancer cells has been shown to promote tumor growth by a mechanism that remains to be uncovered. Here we show that PRL-2 regulates intracellular magnesium levels by forming a functional heterodimer with the magnesium transporter CNNM3. We further reveal that CNNM3 is not a phosphorylated substrate of PRL-2, and that the interaction occurs through a loop unique to the CBS pair domains of CNNM3 that exists only in organisms having PRL orthologs. Supporting the role of PRL-2 in cellular magnesium transport is the observation that PRL-2 knockdown results in a substantial decrease of cellular magnesium influx. Furthermore, in PRL-2 knockout mice, serum magnesium levels were significantly elevated as compared with control animals, indicating a pivotal role for PRL-2 in regulating cellular magnesium homeostasis. Although the expression levels of CNNM3 remained unchanged after magnesium depletion of various cancer cell lines, the interaction between endogenous PRL-2 and CNNM3 was markedly increased. Importantly, xenograft tumor assays with CNNM3 and a mutant form that does not associate with PRL-2 confirm that CNNM3 is itself pro-oncogenic, and that the PRL-2/CNNM3 association is important for conferring transforming activities. This finding is further confirmed from data in human breast cancer tissues showing that CNNM3 levels correlate positively with both PRL-2 expression and the tumor proliferative index. In summary, we demonstrate that oncogenic PRL-2 controls tumor growth by modulating intracellular magnesium levels through binding with the CNNM3 magnesium transporter.

  18. Evolution of Bacterial Protein-Tyrosine Kinases and Their Relaxed Specificity Toward Substrates

    PubMed Central

    Shi, Lei; Kolar-Znika, Lorena; Boskovic, Ana; Jadeau, Fanny; Combet, Christophe; Grangeasse, Christophe; Franjevic, Damjan; Talla, Emmanuel; Mijakovic, Ivan

    2014-01-01

    It has often been speculated that bacterial protein-tyrosine kinases (BY-kinases) evolve rapidly and maintain relaxed substrate specificity to quickly adopt new substrates when evolutionary pressure in that direction arises. Here, we report a phylogenomic and biochemical analysis of BY-kinases, and their relationship to substrates aimed to validate this hypothesis. Our results suggest that BY-kinases are ubiquitously distributed in bacterial phyla and underwent a complex evolutionary history, affected considerably by gene duplications and horizontal gene transfer events. This is consistent with the fact that the BY-kinase sequences represent a high level of substitution saturation and have a higher evolutionary rate compared with other bacterial genes. On the basis of similarity networks, we could classify BY kinases into three main groups with 14 subgroups. Extensive sequence conservation was observed only around the three canonical Walker motifs, whereas unique signatures proposed the functional speciation and diversification within some subgroups. The relationship between BY-kinases and their substrates was analyzed using a ubiquitous substrate (Ugd) and some Firmicute-specific substrates (YvyG and YjoA) from Bacillus subtilis. No evidence of coevolution between kinases and substrates at the sequence level was found. Seven BY-kinases, including well-characterized and previously uncharacterized ones, were used for experimental studies. Most of the tested kinases were able to phosphorylate substrates from B. subtilis (Ugd, YvyG, and YjoA), despite originating from very distant bacteria. Our results are consistent with the hypothesis that BY-kinases have evolved relaxed substrate specificity and are probably maintained as rapidly evolving platforms for adopting new substrates. PMID:24728941

  19. Evolutionary mechanisms driving the evolution of a large polydnavirus gene family coding for protein tyrosine phosphatases

    PubMed Central

    2012-01-01

    Background Gene duplications have been proposed to be the main mechanism involved in genome evolution and in acquisition of new functions. Polydnaviruses (PDVs), symbiotic viruses associated with parasitoid wasps, are ideal model systems to study mechanisms of gene duplications given that PDV genomes consist of virulence genes organized into multigene families. In these systems the viral genome is integrated in a wasp chromosome as a provirus and virus particles containing circular double-stranded DNA are injected into the parasitoids’ hosts and are essential for parasitism success. The viral virulence factors, organized in gene families, are required collectively to induce host immune suppression and developmental arrest. The gene family which encodes protein tyrosine phosphatases (PTPs) has undergone spectacular expansion in several PDV genomes with up to 42 genes. Results Here, we present strong indications that PTP gene family expansion occurred via classical mechanisms: by duplication of large segments of the chromosomally integrated form of the virus sequences (segmental duplication), by tandem duplications within this form and by dispersed duplications. We also propose a novel duplication mechanism specific to PDVs that involves viral circle reintegration into the wasp genome. The PTP copies produced were shown to undergo conservative evolution along with episodes of adaptive evolution. In particular recently produced copies have undergone positive selection in sites most likely involved in defining substrate selectivity. Conclusion The results provide evidence about the dynamic nature of polydnavirus proviral genomes. Classical and PDV-specific duplication mechanisms have been involved in the production of new gene copies. Selection pressures associated with antagonistic interactions with parasitized hosts have shaped these genes used to manipulate lepidopteran physiology with evidence for positive selection involved in adaptation to host targets. PMID

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

    PubMed

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

    2015-08-01

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

  1. EGF receptor-ligand interaction generates extracellular hydrogen peroxide that inhibits EGFR-associated protein tyrosine phosphatases.

    PubMed

    DeYulia, Garrett J; Cárcamo, Juan M

    2005-08-19

    Hydrogen peroxide (H(2)O(2)) has been shown to be an important modulator of intracellular phosphatase activity involved in cell signaling pathways, including signaling by members of the receptor tyrosine kinase family of receptors such as the epidermal growth factor receptor (EGFR). Intracellular H(2)O(2) can be generated by mitochondria-dependent pathways, whereas we recently showed that H(2)O(2) could be generated extracellularly by receptor-ligand interaction. Here, we show that H(2)O(2) produced by EGF-EGFR interaction can modulate the activity of intracellular protein tyrosine phosphatases (PTPs). Using purified proteins, we found that EGFR-ligand interaction generates H(2)O(2) that is capable of inhibiting the activity of PTP1B in vitro. Furthermore, the addition of catalase rescued phosphatase inhibition consequent to EGF-EGFR interaction. Using cells that overexpress EGFR, we found that the addition of extracellular catalase prevented EGF-induced inhibition of EGFR-associated phosphatase activity. Our findings suggest that extracellular H(2)O(2) generated by EGFR-ligand interaction permeates the plasma membrane and inhibits EGFR-associated tyrosine phosphatase activity, thereby modulating downstream signal transduction pathways.

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

    PubMed

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

    2016-09-01

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

  3. Autocatalytic tyrosine-phosphorylation of protein kinase CK2 alpha and alpha' subunits: implication of Tyr182.

    PubMed Central

    Donella-Deana, A; Cesaro, L; Sarno, S; Brunati, A M; Ruzzene, M; Pinna, L A

    2001-01-01

    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 mechanism of modulation is still obscure. Here we show that CK2 alpha/alpha' subunits undergo intermolecular (trans) tyrosine-autophosphorylation, which is dependent on intrinsic catalytic activity and is suppressed by the individual mutation of Tyr182, a crucial residue of the activation loop, to phenylalanine. At variance with serine-autophosphorylation, tyrosine-autophosphorylation of CK2alpha is reversed by ADP and GDP and is counteracted by the beta-subunit and by a peptide reproducing the activation loop of CK2alpha/alpha' (amino acids 175-201). These results disclose new perspectives about the mode of regulation of CK2 catalytic subunits. PMID:11439109

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

    PubMed Central

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

    2014-01-01

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

  5. Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.

    PubMed

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

    2014-08-01

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

  6. 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.

  7. IGF-1R modulation of acute GH-induced STAT5 signaling: role of protein tyrosine phosphatase activity.

    PubMed

    Gan, Yujun; Zhang, Yue; Buckels, Ashiya; Paterson, Andrew J; Jiang, Jing; Clemens, Thomas L; Zhang, Zhong-Yin; Du, Keyong; Chang, Yingzi; Frank, Stuart J

    2013-11-01

    GH is a potent anabolic and metabolic factor that binds its cell surface receptor (GHR), activating the GHR-associated tyrosine kinase, Janus kinase 2, which phosphorylates and activates the latent transcription factor, signal transducer and activator of transcription 5 (STAT5). Some GH actions are mediated by the elaboration of IGF-1, which exerts effects by binding and activating the heterotetrameric tyrosine kinase growth factor receptor, IGF-1R. In addition to this GH-GHR-IGF-1-IGF-1R scheme, we have demonstrated in primary osteoblasts and in islet β-cells that then deletion or silencing of IGF-1R results in diminished GH-induced STAT5 phosphorylation, suggesting that the presence of IGF-1R may facilitate GH signaling. In this study, we explore potential roles for protein tyrosine phosphatase activity in modulating GH-induced signaling, comparing conditions in which IGF-1R is present or diminished. We confirm that in mouse primary osteoblasts harboring loxP sites flanking the IGF-1R gene, infection with an adenovirus that expresses the Cre recombinase results in IGF-1R deletion and diminished acute GH-induced STAT5 phosphorylation. Furthermore, we present a new model of IGF-1R silencing, in which expression of short hairpin RNA directed at IGF-1R greatly reduces IGF-1R abundance in LNCaP human prostate cancer cells. In both models, treatment with a chemical inhibitor of protein tyrosine phosphatase-1B (PTP-1B), but not one of src homology region 2 domain-containing phosphotase-1 (SHP-1) and SHP-2, reverses the loss of GH-induced STAT5 phosphorylation in cells lacking IGF-1R but has no effect in cells with intact IGF-1R. Furthermore, expression of either a dominant-negative PTP-1B or the PTP-1B-interacting inhibitory protein, constitutive photomorphogenesis 1, also rescues acute GH-induced STAT5 signaling in IGF-1R-deficient cells but has no effect in IGF-1R replete cells. By expressing a substrate-trapping mutant PTP-1B, we demonstrate that tyrosine

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

    PubMed Central

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

    1995-01-01

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

  9. Kinetic and mechanistic characterization of a mammalian protein-tyrosine phosphatase, PTP1.

    PubMed

    Zhang, Z Y

    1995-05-12

    The kinetic mechanism of the hydrolysis of phosphate monoesters catalyzed by a soluble form of rat protein-tyrosine phosphatase (PTPase), PTP1, was probed with a variety of steady-state and pre-steady-state kinetic techniques. Product inhibition and 18O exchange experiments are consistent with the enzymatic reaction proceeding through two chemical steps, i.e. formation and breakdown of a covalent phosphoenzyme intermediate. The variation of kcat/Km with pH indicates that three ionizable groups are involved in enzyme substrate binding and catalysis. The first group must be deprotonated and is attributed to the second ionization of the substrate. The other two groups with pK alpha values of 5.1 and 5.5 correspond to two enzyme active site residues. The kcat-pH profiles for both p-nitrophenyl phosphate and beta-naphthyl phosphate are bell-shaped and are superimposable, with the apparent pK alpha values derived from the acidic limb and the basic limb of the profile being 4.4 and 6.8, respectively. This suggests that the rate-limiting step corresponds to the decomposition of the phosphoenzyme intermediate at all pH values. Results from leaving group dependence of kcat at two different pH values support the above conclusion. Furthermore, burst kinetics have been demonstrated with PTP1 using p-nitrophenyl phosphate as a substrate. The rate constants for the formation and the breakdown of the intermediate are 241 and 12 s-1, respectively, at pH 6.0 and 3.5 degrees C. A normal D2O solvent isotope effect (kcatH/kcatD = 1.5) is associated with the breakdown of the phosphoenzyme intermediate, indicating a solvent-derived proton in the transition state. The leaving group dependence of kcat/Km suggests that there is a strong electrophilic interaction between the enzyme and the leaving group oxygen in the transition state of the phosphorylation event. These results are compared with those of the Yersinia PTPase and suggest that the mechanism for PTPase-catalyzed phosphate

  10. Diverse Levels of Sequence Selectivity and Catalytic Efficiency of Protein-Tyrosine Phosphatases

    PubMed Central

    Selner, Nicholas G.; Luechapanichkul, Rinrada; Chen, Xianwen; Neel, Benjamin G.; Zhang, Zhong-Yin; Knapp, Stefan; Bell, Charles E.; Pei, Dehua

    2014-01-01

    The sequence selectivity of 14 classical protein-tyrosine phosphatases (PTPs) (PTPRA, PTPRB, PTPRC, PTPRD, PTPRO, PTP1B, SHP-1, SHP-2, HePTP, PTP-PEST, TCPTP, PTPH1, PTPD1, and PTPD2) was systematically profiled by screening their catalytic domains against combinatorial peptide libraries. All of the PTPs exhibit similar preference for pY peptides rich in acidic amino acids and disfavor positively charged sequences, but differ vastly in their degrees of preference/disfavor. Some PTPs (PTP-PEST, SHP-1, and SHP-2) are highly selective for acidic over basic (or neutral) peptides (by >105-fold), whereas others (PTPRA and PTPRD) show no to little sequence selectivity. PTPs also have diverse intrinsic catalytic efficiencies (kcat/KM values against optimal substrates), which differ by >105-fold due to different kcat and/or KM values. Moreover, PTPs show little positional preference for the acidic residues relative to the pY residue. Mutation of Arg47 of PTP1B, which is located near the pY-1 and pY-2 residues of a bound substrate, decreased the enzymatic activity by 3–18-fold toward all pY substrates containing acidic residues anywhere within the pY-6 to pY+5 region. Similarly, mutation of Arg24, which is situated near the C-terminus of a bound substrate, adversely affected the kinetic activity of all acidic substrates. A co-crystal structure of PTP1B bound with a nephrin pY1193 peptide suggests that Arg24 engages in electrostatic interactions with acidic residues at the pY+1, pY+2, and likely other positions. These results suggest that long-range electrostatic interactions between positively charged residues near the PTP active site and acidic residues on pY substrates allow a PTP to bind acidic substrates with similar affinities and the varying levels of preference for acidic sequences by different PTPs are likely caused by the different electrostatic potentials near their active sites. The implications of the varying sequence selectivity and intrinsic catalytic

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-09-16

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

  13. Copy number alterations of chromosomal regions enclosing protein tyrosine phosphatase receptor-like genes in colorectal cancer.

    PubMed

    Laczmanska, Izabela; Karpinski, Pawel; Kozlowska, Joanna; Bebenek, Marek; Ramsey, David; Sedziak, Tomasz; Ziolkowski, Piotr; Sasiadek, Maria M

    2014-12-01

    Protein tyrosine phosphatases that act in different cellular pathways are described most commonly as tumor suppressors, but also as oncogenes. Their role has previously been described in colorectal cancer, as well as in gastric, breast, thyroid, prostate, ovarian, pancreatic, glioma, liver, leukemia and many other cancers. In a previous study, we have described protein tyrosine phosphatase receptor type T, M, Z1 and Q genes (PTPRT, PTPRM, PTPRZ1 and PTPRQ) hypermethylated in sporadic colorectal cancer. Thus, in this study, we examined the relation of unbalanced chromosomal alterations within regions covering these four protein tyrosine phosphatase genes with this cancer. One hundred and two cancer tissues were molecularly characterized, including analysis of the BRAF and K-ras mutations and methylator phenotype. The analysis of chromosomal aberrations was performed using Comparative Genomic Hybridization. We observed amplification of three regions containing genes coding for PTPs, such as PTPRZ1 (7q31.3, amplified in 23.5% of cases), PTPRQ (12q21.2, amplified in 5.9% of cases), PTPRT (20q12, amplified in 29.4% of cases), along with deletions in the region of PTPRM (18p11.2, deleted in 21.6% of cases). These data may suggest that in sporadic colorectal cancer PTPRZ1, PTPRT, PTPRQ probably act as oncogenes, while PTPRM acts as a tumor suppressor gene. Our study also revealed that gains on chromosome 20q12 and losses on chromosome 18p11.2 are connected with the absence of the BRAF mutation and the conventional adenocarcinoma pathway.

  14. The mechanism of the phosphoryl transfer catalyzed by Yersinia protein-tyrosine phosphatase: a computational and isotope effect study.

    PubMed

    Czyryca, P G; Hengge, A C

    2001-06-11

    In order to evaluate various mechanistic proposals that have been made regarding the mechanism of the first step of the reaction catalyzed by protein-tyrosine phosphatases, new experimental data have been obtained, and some existing data have been carefully reevaluated. New kinetic isotope effect data for the uncatalyzed hydrolysis of p-nitrophenyl phosphate allow a better evaluation of previously reported data from enzymatic reactions with this substrate. The interpretation, and misinterpretation, of pH rate studies is considered. The pathway of this reaction has been modeled computationally and is found to be generally consistent with experimental studies, except for the extent of proton transfer to the leaving group.

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

    PubMed

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

    2008-02-15

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

  16. High-resolution crystal structure of the PDZ1 domain of human protein tyrosine phosphatase PTP-Bas.

    PubMed

    Lee, Sang-Ok; Lee, Mi-Kyung; Ku, Bonsu; Bae, Kwang-Hee; Lee, Sang Chul; Lim, Heon M; Kim, Seung Jun; Chi, Seung-Wook

    2016-09-23

    Protein tyrosine phosphatase-Basophil (PTP-Bas) is a membrane-associated protein tyrosine phosphatase with five PDZ domains and is involved in apoptosis, tumorigenesis, and insulin signaling. The interaction between PTP-Bas and tandem-PH-domain-containing protein 1/2 (TAPP1/2) plays an essential role in the regulation of insulin signaling. Despite its high sequence homology with the other PDZ domains, only the PDZ1 domain of PTP-Bas showed distinct binding specificity for TAPP1/2. Although the interaction between PTP-Bas PDZ1 and TAPP1/2 is a therapeutic target for diabetes, the structural basis for the interaction has not been elucidated. In the present study, we determined the crystal structure of the PTP-Bas PDZ1 domain at 1.6 Å resolution. In addition, we calculated the structural models of complexes of PTP-Bas PDZ1 and the C-terminal peptides of TAPP1/2 (referred to as TAPP1p/2p). Structural comparison with the PTP-Bas PDZ2/RA-GEF2 peptide complex revealed a structural basis for distinct binding specificity of PTP-Bas PDZ1 for TAPP1p/2p peptides. Our high-resolution crystal structure of PTP-Bas PDZ1 will serve as a useful template for rational structure-based design of novel anti-diabetes therapeutics.

  17. Regulation of ERBB Receptor Tyrosine Kinase Activities in Breast Cancer by the KEK Proteins

    DTIC Science & Technology

    1999-11-01

    can also inhibit transformation in mouse mammary tumor cells with deregulated expression of receptors and ligands of the ErbB family. 14. SUBJECT...Reportable outcomes Conclusions References Appendices 7 4 5. INTRODUCTION: In 20-30% of breast tumors , ErbB2, a receptor tyrosine kinase (RTK) of the...inhibit transformation in mouse mammary tumor cells with deregulated expression of receptors and ligands of the ErbB family. In the second year of

  18. Search for Physiological Substrates of Protein Tyrosine Phosphatase Epsilon in Mammary Tumor Cell Lines

    DTIC Science & Technology

    2000-12-01

    PTPe. 7. Reportable Outcomes: Manuscripts: 1. Peretz, A*., Gil-Henn, H*., Sobko, A., Shinder, V., Attali , B and Elson, A. (2000) - Hypomyelination and...contributing first authors). (listed as in preparation in previous report). 2. Tiran, Z., Peretz, A., Gil-Henn, H., Attali , B. and Elson, A. - Tyrosine 124 of...myelination of peripheral nerves in early post- natal mice. 21 Ari Elson, Bernard Attali , Asher Peretz, Hava Gil-Henn, Alex Sobko, and Vera Shinder. (Oral

  19. TLR4 Signaling Is Coupled to SRC Family Kinase Activation, Tyrosine Phosphorylation of Zonula Adherens Proteins, and Opening of the Paracellular Pathway in Human Lung Microvascular Endothelia*

    PubMed Central

    Gong, Ping; Angelini, Daniel J.; Yang, Shiqi; Xia, Guanjun; Cross, Alan S.; Mann, Dean; Bannerman, Douglas D.; Vogel, Stefanie N.; Goldblum, Simeon E.

    2008-01-01

    Bacterial lipopolysaccharide (LPS) is a key mediator in the vascular leak syndromes associated with Gram-negative bacterial infections. LPS opens the paracellular pathway in pulmonary vascular endothelia through protein tyrosine phosphorylation. We now have identified the protein-tyrosine kinases (PTKs) and their substrates required for LPS-induced protein tyrosine phosphorylation and opening of the paracellular pathway in human lung microvascular endothelial cells (HMVEC-Ls). LPS disrupted barrier integrity in a dose- and time-dependent manner, and prior broad spectrum PTK inhibition was protective. LPS increased tyrosine phosphorylation of zonula adherens proteins, VE-cadherin, γ-catenin, and p120ctn. Two SRC family PTK (SFK)-selective inhibitors, PP2 and SU6656, blocked LPS-induced increments in tyrosine phosphorylation of VE-cadherin and p120ctn and paracellular permeability. In HMVEC-Ls, c-SRC, YES, FYN, and LYN were expressed at both mRNA and protein levels. Selective small interfering RNA-induced knockdown of c-SRC, FYN, or YES diminished LPS-induced SRC Tyr416 phosphorylation, tyrosine phosphorylation of VE-cadherin and p120ctn, and barrier disruption, whereas knockdown of LYN did not. For VE-cadherin phosphorylation, knockdown of either c-SRC or FYN provided total protection, whereas YES knockdown was only partially protective. For p120ctn phosphorylation, knockdown of FYN, c-SRC, or YES each provided comparable but partial protection. Toll-like receptor 4 (TLR4) was expressed both on the surface and intracellular compartment of HMVEC-Ls. Prior knockdown of TLR4 blocked both LPS-induced SFK activation and barrier disruption. These data indicate that LPS recognition by TLR4 activates the SFKs, c-SRC, FYN, and YES, which, in turn, contribute to tyrosine phosphorylation of zonula adherens proteins to open the endothelial paracellular pathway. PMID:18326860

  20. Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39.

    PubMed

    Mai, Sanyue; Qu, Xiuhua; Li, Ping; Ma, Qingjun; Liu, Xuan; Cao, Cheng

    2016-04-22

    RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39.

  1. Immunoreactivity of protein tyrosine phosphatase A (PtpA) in sera from sheep infected with Mycobacterium avium subspecies paratuberculosis.

    PubMed

    Gurung, Ratna B; Begg, Douglas J; Purdie, Auriol C; Bach, Horacio; Whittington, Richard J

    2014-07-15

    Evasion of host defense mechanisms and survival inside infected host macrophages are features of pathogenic mycobacteria including Mycobacterium avium subspecies paratuberculosis, the causative agent of Johne's disease in ruminants. Protein tyrosine phosphatase A (PtpA) has been identified as a secreted protein critical for survival of mycobacteria within infected macrophages. The host may mount an immune response to such secreted proteins. In this study, the humoral immune response to purified recombinant M. avium subsp. paratuberculosis PtpA was investigated using sera from a cohort of sheep infected with M. avium subsp. paratuberculosis and compared with uninfected healthy controls. A significantly higher level of reactivity to PtpA was observed in sera collected from M. avium subspecies paratuberculosis infected sheep when compared to those from uninfected healthy controls. PtpA could be a potential candidate antigen for detection of humoral immune responses in sheep infected with M. avium subspecies paratuberculosis.

  2. Heat Shock Protein 90 Has Roles in Intracellular Calcium Homeostasis, Protein Tyrosine Phosphorylation Regulation, and Progesterone-Responsive Sperm Function in Human Sperm

    PubMed Central

    Chen, Aijun; Jiang, Youfang; Xie, Haifeng; Shi, Qixian; Zhang, Songying; Ni, Ya

    2014-01-01

    Heat shock protein 90 plays critical roles in client protein maturation, signal transduction, protein folding and degradation, and morphological evolution; however, its function in human sperm is not fully understood. Therefore, our objective in this study was to elucidate the mechanism by which heat shock protein 90 exerts its effects on human sperm function. By performing indirect immunofluorescence staining, we found that heat shock protein 90 was localized primarily in the neck, midpiece, and tail regions of human sperm, and that its expression increased with increasing incubation time under capacitation conditions. Geldanamycin, a specific inhibitor of heat shock protein 90, was shown to inhibit this increase in heat shock protein 90 expression in western blotting analyses. Using a multifunctional microplate reader to examine Fluo-3 AM-loaded sperm, we observed for the first time that inhibition of heat shock protein 90 by using geldanamycin significantly decreased intracellular calcium concentrations during capacitation. Moreover, western blot analysis showed that geldanamycin enhanced tyrosine phosphorylation of several proteins, including heat shock protein 90, in a dose-dependent manner. The effects of geldanamycin on human sperm function in the absence or presence of progesterone was evaluated by performing chlortetracycline staining and by using a computer-assisted sperm analyzer. We found that geldanamycin alone did not affect sperm capacitation, hyperactivation, and motility, but did so in the presence of progesterone. Taken together, these data suggest that heat shock protein 90, which increases in expression in human sperm during capacitation, has roles in intracellular calcium homeostasis, protein tyrosine phosphorylation regulation, and progesterone-stimulated sperm function. In this study, we provide new insights into the roles of heat shock protein 90 in sperm function. PMID:25541943

  3. Receptor protein tyrosine phosphatase α is essential for hippocampal neuronal migration and long-term potentiation

    PubMed Central

    Petrone, Angiola; Battaglia, Fortunato; Wang, Cheng; Dusa, Adina; Su, Jing; Zagzag, David; Bianchi, Riccardo; Casaccia-Bonnefil, Patrizia; Arancio, Ottavio; Sap, Jan

    2003-01-01

    Despite clear indications of their importance in lower organisms, the contributions of protein tyrosine phosphatases (PTPs) to development or function of the mammalian nervous system have been poorly explored. In vitro studies have indicated that receptor protein tyrosine phosphatase α (RPTPα) regulates SRC family kinases, potassium channels and NMDA receptors. Here, we report that absence of RPTPα compromises correct positioning of pyramidal neurons during development of mouse hippocampus. Thus, RPTPα is a novel member of the functional class of genes that control radial neuronal migration. The migratory abnormality likely results from a radial glial dysfunction rather than from a neuron-autonomous defect. In spite of this aberrant development, basic synaptic transmission from the Schaffer collateral pathway to CA1 pyramidal neurons remains intact in Ptpra–/– mice. However, these synapses are unable to undergo long-term potentiation. Mice lacking RPTPα also underperform in the radial-arm water-maze test. These studies identify RPTPα as a key mediator of neuronal migration and synaptic plasticity. PMID:12912911

  4. Protein tyrosine phosphatase SHP-1 modulates osteoblast differentiation through direct association with and dephosphorylation of GSK3β.

    PubMed

    Tang, Xiao-Lu; Wang, Chang-Nan; Zhu, Xiao-Yan; Ni, Xin

    2017-01-05

    SHP-1, the Src homology-2 (SH2) domain-containing phosphatase 1, is a cytosolic protein-tyrosine phosphatase (PTP) predominantly expressed in hematopoietic-derived cells. Previous studies have focused on the involvement of SHP-1 in osteoclastogenesis. Using primary cultured mouse fetal calvaria-derived osteoblasts as a model, this study aims to investigate the effects of SHP-1 on differentiation and mineralization of osteoblasts and elucidate the signaling pathways responsible for these effects. We found that osteoblasts treated by osteogenic media showed significant increase in SHP-1 expression, which contributed to osteoblastic differentiation and mineralization. Using immunoprecipitation assay, we found that a direct association between SHP-1 and glycogen synthase kinase (GSK)-3β could be detected in differentiated osteoblasts and was significantly inhibited by SHP-1 inhibitor NSC87877. Inhibition of SHP-1 activated GSK3β, thereby leading to suppression of osteoblast differentiation and mineralization, which could be rescued by the inhibitor of GSK3β. In addition, we found that rosiglitazone (RSG) treatment led to significant decrease in SHP-1 expression. Overexpression of SHP-1 reversed RSG-induced GSK3β activation, thus rescuing the inhibitory effect of RSG on osteoblast differentiation and mineralization. These findings suggest that protein tyrosine phosphatase SHP-1 may act as a positive regulator of osteoblast differentiation through direct association with and dephosphorylation of GSK3β. Downregulation of SHP-1 may contribute to RSG-induced inhibition of mouse calvaria osteoblast differentiation by activating GSK3β-dependent pathway.

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

    SciTech Connect

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

    2011-07-01

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

  6. The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a Gram-negative bacterium

    PubMed Central

    Pruitt, Rory N.; Schwessinger, Benjamin; Joe, Anna; Thomas, Nicholas; Liu, Furong; Albert, Markus; Robinson, Michelle R.; Chan, Leanne Jade G.; Luu, Dee Dee; Chen, Huamin; Bahar, Ofir; Daudi, Arsalan; De Vleesschauwer, David; Caddell, Daniel; Zhang, Weiguo; Zhao, Xiuxiang; Li, Xiang; Heazlewood, Joshua L.; Ruan, Deling; Majumder, Dipali; Chern, Mawsheng; Kalbacher, Hubert; Midha, Samriti; Patil, Prabhu B.; Sonti, Ramesh V.; Petzold, Christopher J.; Liu, Chang C.; Brodbelt, Jennifer S.; Felix, Georg; Ronald, Pamela C.

    2015-01-01

    Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21–amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals. PMID:26601222

  7. Nucleotide binding by the epidermal growth factor receptor protein-tyrosine kinase. Trinitrophenyl-ATP as a spectroscopic probe.

    PubMed

    Cheng, K; Koland, J G

    1996-01-05

    The nucleotide binding properties of the epidermal growth factor (EGF) receptor protein-tyrosine kinase were investigated with the fluorescent nucleotide analog 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP). TNP-ATP was found to be an active substrate for the autophosphorylation reaction of the recombinant EGF receptor protein-tyrosine kinase domain (TKD). Whereas the Vmax for the TNP-ATP-dependent autophosphorylation reaction was approximately 200-fold lower than that of ATP, the Km for this reaction was similar to that observed with ATP. The nucleotide analog was also shown to be an inhibitor of the ATP-dependent autophosphorylation and substrate phosphorylation reactions of the TKD. Spectroscopic studies demonstrated both a high affinity binding of TNP-ATP to the recombinant TKD and a markedly enhanced fluorescence of the bound nucleotide analog. The fluorescence of enzyme-bound TNP-ATP was attenuated in the presence of ATP, which enabled determination of the dissociation constants for both ATP and the Mn2+ complex of ATP. A truncated form of the EGF receptor TKD lacking the C-terminal autophosphorylation domain exhibited an enhanced affinity for TNP-ATP, which indicated that the autophosphorylation domain occupied the peptide substrate binding site of the TKD and modulated the binding of the nucleotide substrates.

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

    SciTech Connect

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

    2009-10-02

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

  9. MASK, a large ankyrin repeat and KH domain-containing protein involved in Drosophila receptor tyrosine kinase signaling.

    PubMed

    Smith, Rachel K; Carroll, Pamela M; Allard, John D; Simon, Michael A

    2002-01-01

    The receptor tyrosine kinases Sevenless (SEV) and the Epidermal growth factor receptor (EGFR) are required for the proper development of the Drosophila eye. The protein tyrosine phosphatase Corkscrew (CSW) is a common component of many RTK signaling pathways, and is required for signaling downstream of SEV and EGFR. In order to identify additional components of these signaling pathways, mutations that enhanced the phenotype of a dominant negative form of Corkscrew were isolated. This genetic screen identified the novel signaling molecule MASK, a large protein that contains two blocks of ankyrin repeats as well as a KH domain. MASK genetically interacts with known components of these RTK signaling pathways. In the developing eye imaginal disc, loss of MASK function generates phenotypes similar to those generated by loss of other components of the SEV and EGFR pathways. These phenotypes include compromised photoreceptor differentiation, cell survival and proliferation. Although MASK is localized predominantly in the cellular cytoplasm, it is not absolutely required for MAPK activation or nuclear translocation. Based on our results, we propose that MASK is a novel mediator of RTK signaling, and may act either downstream of MAPK or transduce signaling through a parallel branch of the RTK pathway.

  10. Palmitoylation of either Cys-3 or Cys-5 is required for the biological activity of the Lck tyrosine protein kinase.

    PubMed Central

    Yurchak, L K; Sefton, B M

    1995-01-01

    Palmitoylation can regulate both the affinity for membranes and the biological activity of proteins. To study the importance of the palmitoylation of the Src-like tyrosine protein kinase p56lck in the function of the protein, Cys-3, Cys-5, or both were mutated to serine, and the mutant proteins were expressed stably in fibroblasts and T cells. Both Cys-3 and Cys-5 were apparent sites of palmitoylation in Lck expressed in fibroblasts, as only the simultaneous mutation of both Cys-3 and Cys-5 caused a large reduction in the incorporation of [3H]palmitic acid. The double mutant S3/5Lck was no longer membrane bound when examined by either immunofluorescence or cell fractionation. This indicated that palmitoylation was required for association of Lck with the plasma membrane. Since the S3/5Lck protein was myristoylated, myristoylation of Lck is not sufficient for membrane binding. When Cys-3, Cys-5, or both Cys-3 and Cys-5 were changed to serine in activated F505Lck, palmitoylation of either Cys-3 or Cys-5 was found to be necessary and sufficient for the transformation of fibroblasts and for the induction of spontaneous, antigen-independent interleukin-2 production in the T-helper cell line DO-11.10. Nonpalmitoylated F505Lck exhibited little activity in vivo, where it did not induce elevated levels of tyrosine phosphorylation, and in vitro, where it was unable to phosphorylate angiotensin in an in vitro kinase assay. These findings suggest that F505Lck must be anchored stably to membranes to become activated. Because palmitoylation is dynamic, it may be involved in regulating the cellular localization of p56(lck), and consequently its activity, by altering the proximity of p56(lck) to its activators and/or targets. PMID:8524258

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

    PubMed

    Canto, Isabel; Trejo, JoAnn

    2013-05-31

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

  12. Identification of potential inhibitors based on compound proposal contest: Tyrosine-protein kinase Yes as a target.

    PubMed

    Chiba, Shuntaro; Ikeda, Kazuyoshi; Ishida, Takashi; Gromiha, M Michael; Taguchi, Y-H; Iwadate, Mitsuo; Umeyama, Hideaki; Hsin, Kun-Yi; Kitano, Hiroaki; Yamamoto, Kazuki; Sugaya, Nobuyoshi; Kato, Koya; Okuno, Tatsuya; Chikenji, George; Mochizuki, Masahiro; Yasuo, Nobuaki; Yoshino, Ryunosuke; Yanagisawa, Keisuke; Ban, Tomohiro; Teramoto, Reiji; Ramakrishnan, Chandrasekaran; Thangakani, A Mary; Velmurugan, D; Prathipati, Philip; Ito, Junichi; Tsuchiya, Yuko; Mizuguchi, Kenji; Honma, Teruki; Hirokawa, Takatsugu; Akiyama, Yutaka; Sekijima, Masakazu

    2015-11-26

    A search of broader range of chemical space is important for drug discovery. Different methods of computer-aided drug discovery (CADD) are known to propose compounds in different chemical spaces as hit molecules for the same target protein. This study aimed at using multiple CADD methods through open innovation to achieve a level of hit molecule diversity that is not achievable with any particular single method. We held a compound proposal contest, in which multiple research groups participated and predicted inhibitors of tyrosine-protein kinase Yes. This showed whether collective knowledge based on individual approaches helped to obtain hit compounds from a broad range of chemical space and whether the contest-based approach was effective.

  13. Chronic Oxidative Stress Causes Amplification and Overexpression of ptprz1 Protein Tyrosine Phosphatase to Activate β-Catenin Pathway

    PubMed Central

    Liu, Yu-Ting; Shang, Donghao; Akatsuka, Shinya; Ohara, Hiroki; Dutta, Khokon Kumar; Mizushima, Katsura; Naito, Yuji; Yoshikawa, Toshikazu; Izumiya, Masashi; Abe, Kouichiro; Nakagama, Hitoshi; Noguchi, Noriko; Toyokuni, Shinya

    2007-01-01

    Ferric nitrilotriacetate induces oxidative renal tubular damage via Fenton-reaction, which subsequently leads to renal cell carcinoma (RCC) in rodents. Here, we used gene expression microarray and array-based comparative genomic hybridization analyses to find target oncogenes in this model. At the common chromosomal region of amplification (4q22) in rat RCCs, we found ptprz1, a tyrosine phosphatase (also known as protein tyrosine phosphatase ζ or receptor tyrosine phosphatase β) highly expressed in the RCCs. Analyses revealed genomic amplification up to eightfold. Despite scarcity in the control kidney, the amounts of PTPRZ1 were increased in the kidney after 3 weeks of oxidative stress, and mRNA levels were increased 16∼552-fold in the RCCs. Network analysis of the expression revealed the involvement of the β-catenin pathway in the RCCs. In the RCCs, dephosphorylated β-catenin was translocated to nuclei, resulting in the expression of its target genes cyclin D1, c-myc, c-jun, fra-1, and CD44. Furthermore, knockdown of ptprz1 with small interfering RNA (siRNA), in FRCC-001 and FRCC-562 cell lines established from the induced RCCs, decreased the amounts of nuclear β-catenin and suppressed cellular proliferation concomitant with a decrease in the expression of target genes. These results demonstrate that chronic oxidative stress can induce genomic amplification of ptprz1, activating β-catenin pathways without the involvement of Wnt signaling for carcinogenesis. Thus, iron-mediated persistent oxidative stress confers an environment for gene amplification. PMID:18055543

  14. Chronic oxidative stress causes amplification and overexpression of ptprz1 protein tyrosine phosphatase to activate beta-catenin pathway.

    PubMed

    Liu, Yu-Ting; Shang, Donghao; Akatsuka, Shinya; Ohara, Hiroki; Dutta, Khokon Kumar; Mizushima, Katsura; Naito, Yuji; Yoshikawa, Toshikazu; Izumiya, Masashi; Abe, Kouichiro; Nakagama, Hitoshi; Noguchi, Noriko; Toyokuni, Shinya

    2007-12-01

    Ferric nitrilotriacetate induces oxidative renal tubular damage via Fenton-reaction, which subsequently leads to renal cell carcinoma (RCC) in rodents. Here, we used gene expression microarray and array-based comparative genomic hybridization analyses to find target oncogenes in this model. At the common chromosomal region of amplification (4q22) in rat RCCs, we found ptprz1, a tyrosine phosphatase (also known as protein tyrosine phosphatase zeta or receptor tyrosine phosphatase beta) highly expressed in the RCCs. Analyses revealed genomic amplification up to eightfold. Despite scarcity in the control kidney, the amounts of PTPRZ1 were increased in the kidney after 3 weeks of oxidative stress, and mRNA levels were increased 16 approximately 552-fold in the RCCs. Network analysis of the expression revealed the involvement of the beta-catenin pathway in the RCCs. In the RCCs, dephosphorylated beta-catenin was translocated to nuclei, resulting in the expression of its target genes cyclin D1, c-myc, c-jun, fra-1, and CD44. Furthermore, knockdown of ptprz1 with small interfering RNA (siRNA), in FRCC-001 and FRCC-562 cell lines established from the induced RCCs, decreased the amounts of nuclear beta-catenin and suppressed cellular proliferation concomitant with a decrease in the expression of target genes. These results demonstrate that chronic oxidative stress can induce genomic amplification of ptprz1, activating beta-catenin pathways without the involvement of Wnt signaling for carcinogenesis. Thus, iron-mediated persistent oxidative stress confers an environment for gene amplification.

  15. Expression of protein tyrosine phosphatase alpha (RPTPalpha) in human breast cancer correlates with low tumor grade, and inhibits tumor cell growth in vitro and in vivo.

    PubMed

    Ardini, E; Agresti, R; Tagliabue, E; Greco, M; Aiello, P; Yang, L T; Ménard, S; Sap, J

    2000-10-12

    Tyrosine phosphorylation is controlled by a balance of tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Whereas the contribution of PTKs to breast tumorigenesis is the subject of intense scrutiny, the potential role of PTPs is poorly known. RPTPalpha is implicated in the activation of Src family kinases, and regulation of integrin signaling, cell adhesion, and growth factor responsiveness. To explore its potential contribution to human neoplasia, we surveyed RPTPalpha protein levels in primary human breast cancer. We found RPTPalpha levels to vary widely among tumors, with 29% of cases manifesting significant overexpression. High RPTPalpha protein levels correlated significantly with low tumor grade and positive estrogen receptor status. Expression of RPTPalpha in breast carcinoma cells led to growth inhibition, associated with increased accumulation in G0 and G1, and delayed tumor growth and metastasis. To our knowledge, this is the first example of a study correlating expression level of a specific bona fide PTP with neoplastic disease status in humans.

  16. Functional analysis of the T-cell-restricted protein tyrosine kinase Txk.

    PubMed Central

    Ellis, J H; Sutmuller, R P; Sims, M J; Cooksley, S

    1998-01-01

    T lymphocytes express a range of tyrosine kinases that are involved in signalling processes driving cell activation, proliferation and differentation. Two tyrosine kinases expressed only in T cells, the Itk/Emt and Txk gene products, are members of the Tec family of kinases. The role of Tec kinases in cellular function is poorly understood, although a Tec kinase specific to B cells, Btk, is essential for B-cell development. To explore the contribution of the T-cell-specific Tec kinases to lymphocyte function, we have expressed human Txk in the baculovirus system and conducted the first characterization of its activity. We find that Txk exhibits a substrate preference in vitro quite distinct from that of the major T-cell kinases Lck and ZAP70, suggesting that Tec-family kinases might act on a distinct range of substrates. We also investigated the interactions of Txk with the cytoplasmic domains of the key signalling molecules CD3zeta, CD28 and CTLA4 and find that none of these are phosphorylated by Txk, nor are they ligands for the SH2 or SH3 domains of Txk. We conclude that it is unlikely that Txk has a role in the early signal transduction events associated with these key pathways controlling T-cell activation. PMID:9761724

  17. A Tyrosine-Rich Cell Surface Protein in the Diatom Amphora coffeaeformis Identified through Transcriptome Analysis and Genetic Transformation

    PubMed Central

    Buhmann, Matthias T.; Poulsen, Nicole; Klemm, Jennifer; Kennedy, Matthew R.; Sherrill, C. David; Kröger, Nils

    2014-01-01

    Diatoms are single-celled eukaryotic microalgae that are ubiquitously found in almost all aquatic ecosystems, and are characterized by their intricately structured SiO2 (silica)-based cell walls. Diatoms with a benthic life style are capable of attaching to any natural or man-made submerged surface, thus contributing substantially to both microbial biofilm communities and economic losses through biofouling. Surface attachment of diatoms is mediated by a carbohydrate- and protein- based glue, yet no protein involved in diatom underwater adhesion has been identified so far. In the present work, we have generated a normalized transcriptome database from the model adhesion diatom Amphora coffeaeformis. Using an unconventional bioinformatics analysis we have identified five proteins that exhibit unique amino acid sequences resembling the amino acid composition of the tyrosine-rich adhesion proteins from mussel footpads. Establishing the first method for the molecular genetic transformation of A. coffeaeformis has enabled investigations into the function of one of these proteins, AC3362, through expression as YFP fusion protein. Biochemical analysis and imaging by fluorescence microscopy revealed that AC3362 is not involved in adhesion, but rather plays a role in biosynthesis and/or structural stability of the cell wall. The methods established in the present study have paved the way for further molecular studies on the mechanisms of underwater adhesion and biological silica formation in the diatom A. coffeaeformis. PMID:25372470

  18. A tyrosine-rich cell surface protein in the diatom Amphora coffeaeformis identified through transcriptome analysis and genetic transformation.

    PubMed

    Buhmann, Matthias T; Poulsen, Nicole; Klemm, Jennifer; Kennedy, Matthew R; Sherrill, C David; Kröger, Nils

    2014-01-01

    Diatoms are single-celled eukaryotic microalgae that are ubiquitously found in almost all aquatic ecosystems, and are characterized by their intricately structured SiO2 (silica)-based cell walls. Diatoms with a benthic life style are capable of attaching to any natural or man-made submerged surface, thus contributing substantially to both microbial biofilm communities and economic losses through biofouling. Surface attachment of diatoms is mediated by a carbohydrate- and protein- based glue, yet no protein involved in diatom underwater adhesion has been identified so far. In the present work, we have generated a normalized transcriptome database from the model adhesion diatom Amphora coffeaeformis. Using an unconventional bioinformatics analysis we have identified five proteins that exhibit unique amino acid sequences resembling the amino acid composition of the tyrosine-rich adhesion proteins from mussel footpads. Establishing the first method for the molecular genetic transformation of A. coffeaeformis has enabled investigations into the function of one of these proteins, AC3362, through expression as YFP fusion protein. Biochemical analysis and imaging by fluorescence microscopy revealed that AC3362 is not involved in adhesion, but rather plays a role in biosynthesis and/or structural stability of the cell wall. The methods established in the present study have paved the way for further molecular studies on the mechanisms of underwater adhesion and biological silica formation in the diatom A. coffeaeformis.

  19. Involvement of proline-rich tyrosine kinase 2 in platelet activation: tyrosine phosphorylation mostly dependent on alphaIIbbeta3 integrin and protein kinase C, translocation to the cytoskeleton and association with Shc through Grb2.

    PubMed Central

    Ohmori, T; Yatomi, Y; Asazuma, N; Satoh, K; Ozaki, Y

    2000-01-01

    Proline-rich tyrosine kinase 2 (Pyk2) (also known as RAFTK, CAKbeta or CADTK) has been identified as a member of the focal adhesion kinase (FAK) family of protein-tyrosine kinases and it has been suggested that the mode of Pyk2 activation is distinct from that of FAK. In the present study we investigated the mode of Pyk2 activation in human platelets. When platelets were stimulated with thrombin, Pyk2, as well as FAK, was markedly tyrosine-phosphorylated, in a manner mostly dependent on alphaIIbbeta3 integrin-mediated aggregation. The residual Pyk2 tyrosine phosphorylation observed in the absence of platelet aggregation was completely abolished by pretreatment with BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester]. The Pyk2 phosphorylation was inhibited by protein kinase C (PKC) inhibitors at concentrations that inhibited platelet aggregation. In contrast, direct activation of PKC with the active phorbol ester PMA induced the tyrosine phosphorylation of Pyk2 and FAK but only when platelets were fully aggregated with the exogenous addition of fibrinogen (the ligand for alphaIIbbeta3 integrin). Furthermore, PMA-induced Pyk2 (and FAK) tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. The activation of the von Willebrand factor (vWF)--glycoprotein Ib pathway with botrocetin together with vWF failed to induce Pyk2 (and FAK) tyrosine phosphorylation. Most Pyk2 and FAK was present in the cytosol and membrane skeleton fractions in unstimulated platelets. When platelets were stimulated with thrombin, both Pyk2 and FAK were translocated to the cytoskeleton in an aggregation-dependent manner. In immunoprecipitation studies, Pyk2, as well as FAK, seemed to associate with Shc through Grb2. With the use of glutathione S-transferase fusion proteins containing Shc-SH2, Grb2-SH2, and Grb2 N-terminal and C-terminal SH3 domains, it was implied that the proline-rich region of Pyk2 (and FAK) binds to the N

  20. Mussel-inspired new approach for polymerization and cross-linking of peptides and proteins containing tyrosines by Fremy's salt oxidation.

    PubMed

    Wilchek, Meir; Miron, Talia

    2015-03-18

    Our objective was to develop a method mimicking the natural process of coherence in marine mollusks, by direct chemical conversion of protein tyrosine residues to DOPA-o-quinones, which consequently generates polymerization and cross-linking. Fremy's salt, (ON(SO3K)2, was used to convert tyrosine residues in peptides and proteins to reactive o-quinones. The conversion of tyrosines to DOPA-o-quinones, and their ability to polymerize or cross-link, was tested on tyramine, peptides, and proteins. The peptides tested were as follows: biotin-PEG4-tyramine (PEG-BT), and two decapeptides (identical to the repeating units comprising the mussel's adhesive protein). The proteins tested were as follows: bovine pancreatic ribonuclease A (RNase), lysozyme, IgG, avidin, and streptavidin. The oxidized peptides and proteins were all shown to incorporate oxygen atoms and undergo polymerization and cross-linking, depending on the availability of nucleophiles, mostly lysine amino groups of proteins. All the peptides and the noninteracting proteins such as RNase and lysozyme underwent homopolymerization upon Fremy's salt oxidation. When Fremy's salt oxidaized PEG-BT was mixed with the above proteins, it did not react with any of these proteins because PEG-BT underwent fast self-polymerization. Conversely, streptavidin or avidin cross-linked with PEG-BT after preincubation, thus showing that biorecognition is a prerequisite for cross-linking. Polymerization and cross-linking also occurred, following Fremy's salt oxidation of interacting proteins such as avidin and strepavidin with biotinyilated lysozyme or biotinylated RNase. This indicates that only proteins in very close proximity readily cross-link and polymerize via tyrosine residues. Attempts to convert DOPA-quinone to DOPA by reduction with sodium dithionite (Na2S2O4), was successful as far as small peptides were used. Fremy's salt oxidation can serve as an easy and useful tool to polymerize and cross-link proteins, for

  1. Biochemical characteristics of cytosolic and particulate forms of protein tyrosine kinases from methyl nitrosourea (MNU)-induced rat mammary carcinoma

    SciTech Connect

    Srivastava, A.K.; Chiasson, J.C.; Chiasson, J.L.; Lacroix, A.; Windisch, L. )

    1991-03-11

    Protein tyrosine kinase (PTK) activities in MNU-induced rat mammary carcinoma has been investigated by using poly (glu: tyr; 4:1) as an exogenous substrate. The PTK activity of the mammary carcinoma was about equally distributed between the particulate and cytosolic fractions at 110 000 x g. Both particulate and cytosolic PTKs catalyzed the phosphorylation of several tyrosine containing synthetic substrates to various degrees, however, poly (glu: tyr; 4:1) was the best substrate. Both the forms utilized ATP as the phosphoryl group donor. Among various divalent cations tested, Co{sup 2+}, Mn{sup 2+} and Mg{sup 2+} were able to fulfill the divalent cation requirement. Poly-lysine exerted a stimulatory effect on the particulate, but not on the cytosolic form. On the other hand, though heparin and quercetin inhibited both the forms in a concentration dependent manner, the particulate form was more sensitive to inhibition. These data indicate that MNU-induced rat mammary carcinoma expresses both particulate and cytosolic forms of PTKs and that there are significant differences in the properties of the two forms. Differential differences in the properties of the two forms. Differential effects of some agents on mammary carcinoma PTKs suggest that these enzymes may be acutely regulated in vivo and could play important role in mammary carcinogenesis.

  2. Electroacupuncture Improves Insulin Resistance by Reducing Neuroprotein Y/Agouti-Related Protein Levels and Inhibiting Expression of Protein Tyrosine Phosphatase 1B in Diet-induced Obese Rats.

    PubMed

    Liu, Xia; He, Jun-Feng; Qu, Ya-Ting; Liu, Zhi-Jun; Pu, Qing-Yang; Guo, Sheng-Tong; Du, Jia; Jiang, Peng-Fei

    2016-04-01

    Electroacupuncture (EA) has been shown to exert beneficial effects on obesity, but the mechanism is unclear. This study investigated the effects of EA on diet-induced obese (DIO) rats. Fifty male Sprague-Dawley rats were randomly divided into low-fat diet (LFD, 10 rats) and high-fat diet (HFD, 40 rats) groups. After the DIO models had been established, successful model rats were randomly divided into HFD, EA, and orlistat (OLST) groups. The EA group received EA at Zusanli (ST36) and Quchi (LI11) for 20 minutes once per day for 28 days. The OLST group was treated with orlistat by gavage. The body weight, homeostasis model assessment-insulin resistance index, adipocyte diameters, and neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B levels were significantly lower in the EA group than in the HFD group. The rats of the OLST group showed watery stools and yellow hairs whereas those of the EA group had regular stools and sleek coats. The effect of EA on weight loss may be related to improved insulin resistance caused by changes in the adipocyte size and by reductions in the expressions of neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B. This study indicates that EA may be a better method of alternative therapy for treating obesity and other metabolic diseases.

  3. Retinal axon target selection in Drosophila is regulated by a receptor protein tyrosine phosphatase.

    PubMed

    Garrity, P A; Lee, C H; Salecker, I; Robertson, H C; Desai, C J; Zinn, K; Zipursky, S L

    1999-04-01

    Different Drosophila photoreceptors (R cells) connect to neurons in different optic lobe layers. R1-R6 axons project to the lamina; R7 and R8 axons project to separate layers of the medulla. We show a receptor tyrosine phosphatase, PTP69D, is required for lamina target specificity. In Ptp69D mutants, R1-R6 project through the lamina, terminating in the medulla. Genetic mosaics, transgene rescue, and immunolocalization indicate PTP69D functions in R1-R6 growth cones. PTP69D overexpression in R7 and R8 does not respecify their connections, suggesting PTP69D acts in combination with other factors to determine target specificity. Structure-function analysis indicates the extracellular fibronectin type III domains and intracellular phosphatase activity are required for targeting. We propose PTP69D promotes R1-R6 targeting in response to extracellular signals by dephosphorylating substrate(s) in R1-R6 growth cones.

  4. Protein tyrosine phosphatase-1B contributes to LPS-induced leptin resistance in male rats.

    PubMed

    Borges, Beatriz de Carvalho; Rorato, Rodrigo C; Uchoa, Ernane Torres; Marangon, Paula B; Elias, Carol F; Antunes-Rodrigues, Jose; Elias, Lucila L K

    2015-01-01

    Leptin resistance is induced by the feedback inhibitors tyrosine phosphatase-1B (PTP1B) and decreased Src homology 2 domain-containing tyrosine phosphatase-2 (SHP-2) signaling. To investigate the participation of PTP1B and SHP-2 in LPS-induced leptin resistance, we injected repeated (6-LPS) intraperitoneal LPS doses (100 μg/kg ip) for comparison with a single (1-LPS) treatment and evaluated the expression of SHP-2, PTP1B, p-ERK1/2, and p-STAT3 in the hypothalamus of male Wistar rats. The single LPS treatment increased the expression of p-STAT3 and PTP1B but not SHP-2. The repeated LPS treatment reduced SHP-2, increased PTP1B, and did not change p-STAT3. We observed that the PTP1B expression induced by the endotoxin was highly colocalized with leptin receptor cells in the hypothalamus of LepRb-IRES-Cre-tdTomato reporter mice. The single, but not the repeated, LPS treatment decreased the food intake and body weight. Leptin had no stimulatory effect on the hypophagia, body weight loss, or pSTAT3 expression in 6-LPS rats, indicating leptin unresponsiveness. Notably, the PTP1B inhibitor (3.0 nmol/rat in 5 μl icv) restored the LPS-induced hypophagia in 6-LPS rats and restored the ability of leptin to reduce food intake and body weight as well as to phosphorylate STAT3 in the arcuate, paraventricular, and ventromedial nuclei of the hypothalamus. The present data suggest that an increased PTP1B expression in the hypothalamus underlies the development of leptin resistance during repeated exposure to LPS. Our findings contribute to understanding the mechanisms involved in leptin resistance during low-grade inflammation as seen in obesity.

  5. Identification and characterization of a novel SH3-domain binding protein, Sab, which preferentially associates with Bruton's tyrosine kinase (BtK).

    PubMed

    Matsushita, M; Yamadori, T; Kato, S; Takemoto, Y; Inazawa, J; Baba, Y; Hashimoto, S; Sekine, S; Arai, S; Kunikata, T; Kurimoto, M; Kishimoto, T; Tsukada, S

    1998-04-17

    Protein interaction cloning method was used to identify a novel molecule, Sab, which binds to the SH3 domain of Bruton's tyrosine kinase (Btk), the deficient cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia and murine X-linked immunodeficiency. Immunoprecipitation using the anti-Sab antibody identified the protein product of the gene as a 70 kDa molecule. While Sab does not have a proline-rich sequence, it was shown to bind to Btk through the commonly conserved structure among SH3 domains. Remarkably, Sab exhibited a high preference for binding to Btk rather than to other cytoplasmic tyrosine kinases, which suggests a unique role of Sab in the Btk signal transduction pathway.

  6. Experimental and Theoretical Study of the Movement of the Wpd Flexible Loop of Human Protein Tyrosine Phosphatase PTP1B in Complex with Halide Ions

    NASA Astrophysics Data System (ADS)

    Katz, Aline; Saenz-Méndez, Patricia; Cousido-Siah, Alexandra; Podjarny, Alberto D.; Ventura, Oscar N.

    2012-11-01

    Protein tyrosine phosphorylation is a post-translational modification mechanism, crucial for the regulation of nearly all aspects of cell life. This dynamic, reversible process is regulated by the balanced opposing activity of protein tyrosine kinases and protein tyrosine phosphatases. In particular, the protein tyrosine phosphatase 1B (PTP1B) is implicated in the regulation of the insulin-receptor activity, leptin-stimulated signal transduction pathways and other clinically relevant metabolic routes, and it has been found overexpressed or overregulated in human breasts, colon and ovary cancers. The WPD loop of the enzyme presents an inherent flexibility, and it plays a fundamental role in the enzymatic catalysis, turning it into a potential target in the design of new efficient PTP1B inhibitors. In order to determine the interactions that control the spatial conformation adopted by the WPD loop, complexes between the enzyme and halide ions (Br- and I- in particular) were crystallized and their crystallographic structure determined, and the collective movements of the aforementioned complexes were studied through Molecular Dynamics (MD) simulations. Both studies yielded concordant results, indicating the existence of a relationship between the identity of the ion present in the complex and the strength of the interactions it establishes with the surrounding protein residues.

  7. Bioassay-guided isolation of an active compound with protein tyrosine phosphatase 1B inhibitory activity from Sargassum fusiforme by high-speed counter-current chromatography.

    PubMed

    Wang, Miao; Gu, Dongyu; Guo, Xinfeng; Li, Haoquan; Wang, Yi; Guo, Hong; Yang, Yi; Tian, Jing

    2016-11-01

    A rapid and efficient method using high-speed counter-current chromatography was established for the bioassay-guided separation of an active compound with protein tyrosine phosphatase 1B inhibitory activity from Sargassum fusiforme. Under the bioassay guidance, the ethyl acetate extract with the best IC50 value of 0.37 ± 0.07 μg/mL exhibited a potential protein tyrosine phosphatase 1B inhibitory activity, which was further separated by high-speed counter-current chromatography. The separation was performed with a two-phase solvent system composed of n-hexane/methanol/water (5:4:1, v/v). As a result, dibutyl phthalate (19.7 mg) with the purity of 95.3% was obtained from 200 mg of the ethyl acetate extract. Its IC50 was 14.05 ± 0.06 μM, which was further explained by molecular docking. The result of molecular docking showed that dibutyl phthalate enfolded in the catalytic site of protein tyrosine phosphatase 1B. The main force between dibutyl phthalate and protein tyrosine phosphatase 1B was the hydrogen bond interaction with Gln266. In addition, hydrogen bond, van der Waals force and hydrophobic interaction with the amino acids (Ala217, Ile219, and Gly220) were also responsible for the stable protein-ligand complex.

  8. Alpha 2-chimerin, an SH2-containing GTPase-activating protein for the ras-related protein p21rac derived by alternate splicing of the human n-chimerin gene, is selectively expressed in brain regions and testes.

    PubMed Central

    Hall, C; Sin, W C; Teo, M; Michael, G J; Smith, P; Dong, J M; Lim, H H; Manser, E; Spurr, N K; Jones, T A

    1993-01-01

    n-Chimerin (alpha 1-chimerin) is a brain GTPase-activating protein (GAP) for the ras-related p21rac. We now report the occurrence of another form of chimerin, termed alpha 2-chimerin. This is the product of an alternately spliced transcript of the human n-chimerin gene encoding an N-terminal SH2 (src homology 2) domain in addition to the phorbol ester receptor and GAP domains. alpha 1- and alpha 2-chimerin mRNAs were expressed differently. In the rat brain, only alpha 1-chimerin mRNA was expressed in cerebellar Purkinje cells, although both alpha 1- and alpha 2-chimerin mRNAs occurred in neurons in the cerebral cortex, hippocampus, and thalamus. Only alpha 2-chimerin RNA was expressed in rat testes, in early pachytene spermatocytes. A 45-kDa SH2-containing chimerin corresponding to the alpha 2 form was purified from rat brain. As with Escherichia coli 45-kDa recombinant alpha 2-chimerin, purified brain alpha 2-chimerin exhibited racGAP activity which was stimulated by phosphatidylserine. The recombinant SH2 domain bound several 32P-labelled phosphoproteins of PC12 cells, whose phosphorylation increased in response to trophic factors, including nerve growth factor. To examine the relationships of alpha 1- and alpha 2-chimerin transcripts, human genomic DNA clones were characterized. In alpha 2-chimerin mRNA, a 3' splice acceptor site within exon 1 of alpha 1-chimerin mRNA was used, replacing its 5' untranslated region and N-terminal coding sequence. The single human n-chimerin gene was mapped to chromosome 2q31-q32.1, colocalizing with the CRE-BP1 transcription factor gene (2q32). It contained several splice junctions conserved with the sequence-related protein kinase C and bcr genes. alpha 2-Chimerin is only the second SH2-containing GAP and the first example of an SH2 domain generated by alternate splicing. Images PMID:8336731

  9. Protein tyrosine phosphatase 1B (PTP1B)-inhibiting constituents from the leaves of Syzygium polyanthum.

    PubMed

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

    2012-08-01

    A methanol extract of the leaves of Syzygium polyanthum (Wight) Walp. afforded four new acylbenzene derivatives (1-4) together with seven known compounds (5-11). The structures of 1-11 were elucidated by extensive spectroscopic methods and comparison with the literature data. The new compounds 1-3 and a known compound, campest-4-en-3-one (10), exhibited a significant protein tyrosine phosphatase 1B inhibitory activity with IC₅₀ values of 13.1 ± 0.1, 5.77 ± 0.15, 4.01 ± 0.26, and 10.4 ± 0.5 µM, respectively. The inhibitory potency of the new compounds 2 and 3 was comparable to that of a positive control RK-682 (IC₅₀, 5.51 ± 0.04 µM).

  10. Triterpenoids from the leaves of Diospyros kaki (persimmon) and their inhibitory effects on protein tyrosine phosphatase 1B.

    PubMed

    Thuong, Phuong Thien; Lee, Chul Ho; Dao, Trong Tuan; Nguyen, Phi Hung; Kim, Wan Gi; Lee, Sang Jun; Oh, Won Keun

    2008-10-01

    Phytochemical study on a methanol-soluble extract of the leaves of persimmon (Diospyros kaki) resulted in the isolation of two new ursane-type triterpenoids, 3alpha,19alpha-dihydroxyurs-12,20(30)-dien-24,28-dioic acid (1) and 3alpha,19alpha-dihydroxyurs-12-en-24,28-dioic acid (2), together with 12 known ursane- and oleanane-type triterpenoids (3-14). Triterpenoids with a 3beta-hydroxy group were found to inhibit protein tyrosine phosphatase 1B (PTP1B) activity, with IC50 values ranging from 3.1+/-0.2 to 18.8+/-1.3 microM, whereas those with a 3alpha-hydroxy moiety were not active.

  11. Discovery of new potent human protein tyrosine phosphatase inhibitors via pharmacophore and QSAR analysis followed by in silico screening.

    PubMed

    Taha, Mutasem O; Bustanji, Yasser; Al-Bakri, Amal G; Yousef, Al-Motassem; Zalloum, Waleed A; Al-Masri, Ihab M; Atallah, Naji

    2007-03-01

    A pharmacophoric model was developed for human protein tyrosine phosphatase 1B (h-PTP 1B) inhibitors utilizing the HipHop-REFINE module of CATALYST software. Subsequently, genetic algorithm and multiple linear regression analysis were employed to select an optimal combination of physicochemical descriptors and pharmacophore hypothesis that yield consistent QSAR equation of good predictive potential (r = 0.87,F-statistic = 69.13,r(BS)2 = 0.76,r(LOO)2 = 0.68). The validity of the QSAR equation and the associated pharmacophoric hypothesis was experimentally established by the identification of five new h-PTP 1B inhibitors retrieved from the National Cancer Institute (NCI) database.

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

    PubMed

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

    2016-07-22

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

  13. Design, Synthesis and Biological Evaluation of Stilbene Derivatives as Novel Inhibitors of Protein Tyrosine Phosphatase 1B.

    PubMed

    He, Haibing; Ge, Yinghua; Dai, Hong; Cui, Song; Ye, Fei; Jin, Jia; Shi, Yujun

    2016-12-16

    By imitating the scaffold of lithocholic acid (LCA), a natural steroidal compound displaying Protein Tyrosine Phosphatase 1B (PTP1B) inhibitory activity, a series of stilbene derivatives containing phenyl-substituted isoxazoles were designed and synthesized. The structures of the title compounds were confirmed by ¹H-NMR, (13)C-NMR and HRMS. Activities of the title compounds were evaluated on PTP1B and the homologous enzyme TCPTP by using a colorimetric assay. Most of the target compounds had good activities against PTP1B. Among them, compound 29 (IC50 = 0.91 ± 0.33 μM), characterized by a 5-(2,3-dichlorophenyl) isoxazole moiety, exhibited an activity about 14-fold higher than the lead compound LCA and a 4.2-fold selectivity over TCPTP. Compound 29 was identified as a competitive inhibitor of PTP1B with a Ki value of 0.78 μM in enzyme kinetic studies.

  14. Beta2-containing nicotinic acetylcholine receptors mediate calcium/calmodulin-dependent protein kinase-II and synapsin I protein levels in the nucleus accumbens after nicotine withdrawal in mice.

    PubMed

    Jackson, Kia J; Imad Damaj, M

    2013-02-15

    Nicotinic acetylcholine receptors are calcium-permeable and the initial targets for nicotine. Studies suggest that calcium-dependent mechanisms mediate some behavioral responses to nicotine; however, the post-receptor calcium-dependent mechanisms associated with chronic nicotine and nicotine withdrawal remain unclear. The proteins calcium/calmodulin-dependent protein kinase II (CaMKII) and synapsin I are essential for neurotransmitter release and were shown to be involved in drug dependence. In the current study, using pharmacological techniques, we sought to (a) complement previously published behavioral findings from our lab indicating a role for calcium-dependent signaling in nicotine dependence and (b) expand on previously published acute biochemical and pharmacological findings indicating the relevance of calcium-dependent mechanisms in acute nicotine responses by evaluating the function of CaMKII and synapsin I after chronic nicotine and withdrawal in the nucleus accumbens, a brain region implicated in drug dependence. Male mice were chronically infused with nicotine for 14 days, and treated with the β2-selective antagonist dihydro-β-erythroidine (DHβE), or the α7 antagonist, methyllycaconitine citrate (MLA) 20min prior to dissection of the nucleus accumbens. Results show that phosphorylated and total CaMKII and synapsin I protein levels were significantly increased in the nucleus accumbens after chronic nicotine infusion, and reduced after treatment with DHβE, but not MLA. A spontaneous nicotine withdrawal assessment also revealed significant reductions in phosphorylated CaMKII and synapsin I levels 24h after cessation of nicotine treatment. Our findings suggest that post-receptor calcium-dependent mechanisms associated with nicotine withdrawal are mediated through β2-containing nicotinic receptors.

  15. Development of natural product-derived receptor tyrosine kinase inhibitors based on conservation of protein domain fold.

    PubMed

    Kissau, Lars; Stahl, Petra; Mazitschek, Ralph; Giannis, Athannasios; Waldmann, Herbert

    2003-07-03

    Receptor tyrosine kinases (RTKs) such as Tie-2, IGF1R, Her-2/Neu, EGFR, and VEGFR1-3 play crucial roles in the control of cell growth and differentiation. Inhibition of such RTKs has become a major focus of current anticancer drug development, and therefore the discovery of new classes of inhibitors for these signal-transducing proteins is of prime importance. We have recently proposed a novel concept for improving the hit-finding process by employing natural products as biologically validated starting points in structural space for compound library development. In this concept, natural products are regarded as evolutionary chosen ligands for protein domains which are structurally conserved yet genetically mobile. Here we report on the discovery of novel and highly selective VEGFR-2 and -3, Tie-2, and IGF1R inhibitors derived from the naturally occurring Her-2/Neu kinase inhibitor nakijiquinone C and developed on the basis of this concept. Based on the structure of the natural product, a small library (74 members) was synthesized and investigated for inhibition of kinases with highly similar ATP-binding domains. The library yielded inhibitors with IC(50)s in the low micromolar range with high frequency (7 out of 74). In particular, four inhibitors of Tie-2 were found, a kinase critically involved in the formation of new blood vessels from preexisting ones (angiogenesis) and believed to be a new promising target in antitumor therapy. These results support the "domain concept". To advance the development of improved inhibitors, extensive molecular modeling studies were undertaken, including the construction of new homology models for VEGFR-2 and Tie-2. These studies revealed residues in the kinase structure which are crucial to the development of tailor-made receptor tyrosine kinase inhibitors.

  16. Crosstalk between G protein-coupled receptors (GPCRs) and tyrosine kinase receptor (TXR) in the heart after morphine withdrawal.

    PubMed

    Almela, Pilar; García-Carmona, Juan-Antonio; Martínez-Laorden, Elena; Milanés, María-Victoria; Laorden, María-Luisa

    2013-01-01

    G protein-coupled receptors (GPCRs) comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth, and cell differentiation among others. Some of the effects of GPCRs are known to be mediated by the activation of mitogen-activated extracellular kinase (MAPK) pathways. Cross-talk among various signal pathways plays an important role in activation of intracellular and intranuclear signal transduction cascades. Naloxone-induced morphine withdrawal leads to an up-regulation of adenyl cyclase-mediated signaling, resulting in high expression of protein kinase (PK) A. In addition, there is also an increased expression of extracellular signal regulated kinase (ERK), one member of MAPK. For this reason, the crosstalk between these GPCRs and receptors with tyrosine kinase activity (TKR) can be considered a possible mechanism for adaptive changes that occurs after morphine withdrawal. Morphine withdrawal activates ERK1/2 and phosphorylated tyrosine hydroxylase (TH) at Ser31 in the right and left ventricle. When N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), a PKA inhibitor was infused, the ability of morphine withdrawal to activate ERK, which phosphorylates TH at Ser31, was reduced. The present finding demonstrated that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation of TH. Increasing understanding of the mechanisms that interconnect the two pathway regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies.

  17. Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate.

    PubMed

    Kumaresan, A; Siqueira, A P; Hossain, M S; Bergqvist, A S

    2011-12-01

    Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PTP and intracellular calcium ([Ca(2+)]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between P1 and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (P<0.05) in SRF-P1 compared to P1. There were no significant differences in percentages of spermatozoa with high [Ca(2+)]i between P1 and SRF-P1 in fresh as well as in frozen-thawed semen. A higher (P<0.001) proportion of spermatozoa displayed PTP during the course of cryopreservation indicating a definite effect of the cryopreservation process on sperm PTP. The proportion of spermatozoa with PTP did not differ significantly between portions of the boar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF.

  18. Crosstalk between G protein-coupled receptors (GPCRs) and tyrosine kinase receptor (TXR) in the heart after morphine withdrawal

    PubMed Central

    Almela, Pilar; García-Carmona, Juan-Antonio; Martínez-Laorden, Elena; Milanés, María-Victoria; Laorden, María-Luisa

    2013-01-01

    G protein-coupled receptors (GPCRs) comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth, and cell differentiation among others. Some of the effects of GPCRs are known to be mediated by the activation of mitogen-activated extracellular kinase (MAPK) pathways. Cross-talk among various signal pathways plays an important role in activation of intracellular and intranuclear signal transduction cascades. Naloxone-induced morphine withdrawal leads to an up-regulation of adenyl cyclase-mediated signaling, resulting in high expression of protein kinase (PK) A. In addition, there is also an increased expression of extracellular signal regulated kinase (ERK), one member of MAPK. For this reason, the crosstalk between these GPCRs and receptors with tyrosine kinase activity (TKR) can be considered a possible mechanism for adaptive changes that occurs after morphine withdrawal. Morphine withdrawal activates ERK1/2 and phosphorylated tyrosine hydroxylase (TH) at Ser31 in the right and left ventricle. When N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), a PKA inhibitor was infused, the ability of morphine withdrawal to activate ERK, which phosphorylates TH at Ser31, was reduced. The present finding demonstrated that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation of TH. Increasing understanding of the mechanisms that interconnect the two pathway regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies. PMID:24409147

  19. Essential role of two tyrosines and two tryptophans on the photoprotection activity of the Orange Carotenoid Protein.

    PubMed

    Wilson, Adjélé; Punginelli, Claire; Couturier, Mohea; Perreau, François; Kirilovsky, Diana

    2011-03-01

    Photosynthetic organisms have developed photoprotective mechanisms to protect themselves from lethal high light intensities. One of these mechanisms involves the dissipation of excess absorbed light energy into heat. In cyanobacteria, light activation of a soluble carotenoid protein, the Orange Carotenoid Protein (OCP), binding a keto carotenoid, is the key inducer of this mechanism. Blue-green light absorption triggers structural changes within the carotenoid and the protein, leading to the conversion of a dark orange form into a red active form. Here we report the role in photoconversion and photoprotection of individual conserved tyrosines and tryptophans surrounding the rings of the carotenoid. Our results demonstrate that the interaction between the keto group of the carotenoid and Tyr201 and Trp288 is essential for OCP photoactivity. In addition, these amino acids are responsible for carotenoid affinity and specificity. We have already demonstrated that the aromatic character of Tyr44 and Trp110 interacting with the hydroxyl ring is critical. Here we show that the replacement of Tyr44 by Ser affects the stability of the red form avoiding its accumulation at any temperature, while Trp110Ser is affected in the energy necessary to the orange to red conversion and in the interaction with the antenna. Collectively our data support the idea that the red form is essential for photoprotection but not sufficient. Specific conformational changes occurring in the protein seem to be critical to the events leading to energy dissipation.

  20. Toll-like receptor 4 signaling is coupled to src family kinase activation, tyrosine phosphorylation of zonula adherens proteins, and opening of the paracellular pathway in human lung microvascular endothelia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial lipopolysaccharide (LPS) is a key mediator in the vascular leak syndromes associated with Gram-negative bacterial infections. LPS opens the paracellular pathway in pulmonary vascular endothelia through protein tyrosine phosphorylation. We now have identified the protein tyrosine kinase (PT...

  1. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling in commercial and wild-type turkey leukocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases (PTK) and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on days...

  4. The protein tyrosine phosphatase SHP-1 modulates the suppressive activity of regulatory T cells

    PubMed Central

    Iype, Tessy; Sankarshanan, Mohan; Mauldin, Ileana S.; Mullins, David W.; Lorenz, Ulrike

    2010-01-01

    The importance of regulatory T cells (Treg) for immune tolerance is well recognized, yet the signaling molecules influencing their suppressive activity are relatively poorly understood. Here, through in vivo studies and complementary ex vivo studies, we make several important observations. First, we identify the cytoplasmic tyrosine phosphatase SHP-1 as a novel ‘endogenous brake’ and modifier of the suppressive ability of Treg cells; consistent with this notion, loss of SHP-1 expression strongly augments the ability of Treg cells to suppress inflammation in a mouse model. Second, specific pharmacological inhibition of SHP-1 enzymatic activity via the cancer drug sodium stibogluconate (SSG) potently augmented Treg cell suppressor activity both in vivo and ex vivo. Finally, through a quantitative imaging approach, we directly demonstrate that Treg cells prevent the activation of conventional T cells, and that SHP-1-deficient Treg cells are more efficient suppressors. Collectively, our data reveal SHP-1 as a critical modifier of Treg cell function, and a potential therapeutic target for augmenting Treg cell-mediated suppression in certain disease states. PMID:20952680

  5. Fibulin 2, a Tyrosine O-Sulfated Protein, Is Up-regulated Following Retinal Detachment*

    PubMed Central

    Kanan, Yogita; Brobst, Daniel; Han, Zongchao; Naash, Muna I.; Al-Ubaidi, Muayyad R.

    2014-01-01

    Retinal detachment is the physical separation of the retina from the retinal pigment epithelium. It occurs during aging, trauma, or during a variety of retinal disorders such as age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, or as a complication following cataract surgery. This report investigates the role of fibulin 2, an extracellular component, in retinal detachment. A major mechanism for detachment resolution is enhancement of cellular adhesion between the retina and the retinal pigment epithelium and prevention of its cellular migration. This report shows that fibulin 2 is mainly present in the retinal pigment epithelium, Bruch membrane, choriocapillary, and to a lesser degree in the retina. In vitro studies revealed the presence of two isoforms for fibulin 2. The small isoform is located inside the cell, and the large isoform is present inside and outside the cells. Furthermore, fibulin 2 is post-translationally modified by tyrosine sulfation, and the sulfated isoform is present outside the cell, whereas the unsulfated pool is internally located. Interestingly, sulfated fibulin 2 significantly reduced the rate of cellular growth and migration. Finally, levels of fibulin 2 dramatically increased in the retinal pigment epithelium following retinal detachment, suggesting a direct role for fibulin 2 in the re-attachment of the retina to the retinal pigment epithelium. Understanding the role of fibulin 2 in enhancing retinal attachment is likely to help improve the current therapies or allow the development of new strategies for the treatment of this sight-threatening condition. PMID:24692557

  6. The Laminin–Nidogen Complex is a Ligand for a Specific Splice Isoform of the Transmembrane Protein Tyrosine Phosphatase LAR

    PubMed Central

    O'Grady, Pauline; Thai, Tran Cam; Saito, Haruo

    1998-01-01

    Leukocyte antigen–related protein (LAR) is a prototype for a family of transmembrane protein tyrosine phosphatases whose extracellular domain is composed of three Ig and several fibronectin type III (FnIII) domains. Complex alternative splicing of the LAR-FnIII domains 4–8 has been observed. The extracellular matrix laminin–nidogen complex was identified as a ligand for the LAR-FnIII domain 5 (Fn5) using a series of GST-LAR-FnIII domain fusion proteins and testing them in in vitro ligand-binding assays. LAR– laminin–nidogen binding was regulated by alternative splicing of a small exon within the LAR-Fn5 so that inclusion of this exon sequence resulted in disruption of the laminin–nidogen-binding activity. Long cellular processes were observed when HeLa cells were plated on laminin–nidogen, but not when plated on a fibronectin surface. Indirect immunofluorescent antibody staining revealed high expression of LAR in a punctate pattern, throughout the length of these cellular processes observed on laminin–nidogen. Antibody-induced cross-linking of LAR inhibited formation of these cellular processes, and inhibition was correlated with changes in cellular actin cytoskeletal structure. Thus, LAR–laminin–nidogen binding may play a role in regulating cell signaling induced by laminin–nidogen, resulting in cell morphological changes. PMID:9647658

  7. Expression and function of the protein tyrosine phosphatase receptor J (PTPRJ) in normal mammary epithelial cells and breast tumors.

    PubMed

    Smart, Chanel E; Askarian Amiri, Marjan E; Wronski, Ania; Dinger, Marcel E; Crawford, Joanna; Ovchinnikov, Dmitry A; Vargas, Ana Cristina; Reid, Lynne; Simpson, Peter T; Song, Sarah; Wiesner, Christiane; French, Juliet D; Dave, Richa K; da Silva, Leonard; Purdon, Amy; Andrew, Megan; Mattick, John S; Lakhani, Sunil R; Brown, Melissa A; Kellie, Stuart

    2012-01-01

    The protein tyrosine phosphatase receptor J, PTPRJ, is a tumor suppressor gene that has been implicated in a range of cancers, including breast cancer, yet little is known about its role in normal breast physiology or in mammary gland tumorigenesis. In this paper we show that PTPRJ mRNA is expressed in normal breast tissue and reduced in corresponding tumors. Meta-analysis revealed that the gene encoding PTPRJ is frequently lost in breast tumors and that low expression of the transcript associated with poorer overall survival at 20 years. Immunohistochemistry of PTPRJ protein in normal human breast tissue revealed a distinctive apical localisation in the luminal cells of alveoli and ducts. Qualitative analysis of a cohort of invasive ductal carcinomas revealed retention of normal apical PTPRJ localization where tubule formation was maintained but that tumors mostly exhibited diffuse cytoplasmic staining, indicating that dysregulation of localisation associated with loss of tissue architecture in tumorigenesis. The murine ortholog, Ptprj, exhibited a similar localisation in normal mammary gland, and was differentially regulated throughout lactational development, and in an in vitro model of mammary epithelial differentiation. Furthermore, ectopic expression of human PTPRJ in HC11 murine mammary epithelial cells inhibited dome formation. These data indicate that PTPRJ may regulate differentiation of normal mammary epithelia and that dysregulation of protein localisation may be associated with tumorigenesis.

  8. [Tyrosine kinase inhibitors].

    PubMed

    Robert, Jacques

    2011-11-01

    Membrane receptors with tyrosine kinase activity and cytoplasmic tyrosine kinases have emerged as important potential targets in oncology. Starting from basic structures such as anilino-quinazoline, numerous compounds have been synthesised, with the help of tyrosine kinase crystallography, which has allowed to optimise protein-ligand interactions. The catalytic domains of all kinases present similar three-dimensional structures, which explains that it may be difficult to identify molecules having a high specificity for a given tyrosine kinase. Some tyrosine kinase inhibitors are relatively specific for epidermal growth factor receptor (EGFR) such as géfitinib and erlotinib; other are mainly active against platelet-derived growth factor receptor (PDGFR) and the receptor KIT, such as imatinib or nilotinib, and other against vascular endothelial growth factor (VEGF) receptors involved in angiogenesis, such as sunitinib and sorafenib. The oral formulation of tyrosine kinase inhibitors is well accepted by the patients but may generate sometimes compliance problems requiring pharmacokinetic monitoring. This chemical family is in full expansion and several dozens of compounds have entered clinical trials.

  9. Tyrosine Binding Protein Sites Regulate the Intracellular Trafficking and Processing of Amyloid Precursor Protein through a Novel Lysosome-Directed Pathway

    PubMed Central

    Tam, Joshua H. K.; Cobb, M. Rebecca; Seah, Claudia; Pasternak, Stephen H.

    2016-01-01

    The amyloid hypothesis posits that the production of β-amyloid (Aβ) aggregates leads to neurodegeneration and cognitive decline associated with AD. Aβ is produced by sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretase. While nascent APP is well known to transit to the endosomal/ lysosomal system via the cell surface, we have recently shown that APP can also traffic to lysosomes intracellularly via its interaction with AP-3. Because AP-3 interacts with cargo protein via interaction with tyrosine motifs, we mutated the three tyrosines motif in the cytoplasmic tail of APP. Here, we show that the YTSI motif interacts with AP-3, and phosphorylation of the serine in this motif disrupts the interaction and decreases APP trafficking to lysosomes. Furthermore, we show that phosphorylation at this motif can decrease the production of neurotoxic Aβ 42. This demonstrates that reducing APP trafficking to lysosomes may be a strategy to reduce Aβ 42 in Alzheimer’s disease. PMID:27776132

  10. The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex.

    PubMed Central

    Barber, E K; Dasgupta, J D; Schlossman, S F; Trevillyan, J M; Rudd, C E

    1989-01-01

    Many mammalian receptors have been found to regulate cell growth by virtue of a protein-tyrosine kinase domain in their cytoplasmic tail. We recently described an association of the CD4 antigen with a T-cell-specific protein-tyrosine kinase (p56lck; formerly termed pp58lck; EC 2.7.1.112). This interaction represents a potential mechanism by which T-cell growth may be regulated and offers a model by which other members of the src family (products of c-src, c-yes, c-fgr, etc.) may interact with mammalian growth factor receptors. As in the case of the CD4 antigen, the CD8 antigen appears to serve as a receptor for nonpolymorphic regions of products of the major histocompatibility complex and has been implicated in the regulation of T-cell growth. In this study, we reveal that the human CD8 antigen is also associated with the T-cell-specific protein-tyrosine kinase (p56lck). The associated p56lck kinase was detected by use of both in vitro and in vivo labeling regimes using an antiserum to the C terminus of p56lck. Two-dimensional nonequilibrium pH-gradient gel electrophoresis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated the similarity of p56lck to the protein-tyrosine kinase associated with the CD4 antigen. The catalytic activity of p56lck was revealed by the autophosphorylation of the 55- to 60-kDa kinase and the occasional labeling of a 35-kDa protein. Last, we demonstrate directly that members of the CD3 complex, including the gamma, delta, and epsilon chains, as well as a putative zeta subunit, can be phosphorylated at tyrosine residues by the CD4/CD8.p56lck complex. Images PMID:2470098

  11. T-Cell Protein Tyrosine Phosphatase Attenuates STAT3 and Insulin Signaling in the Liver to Regulate Gluconeogenesis

    PubMed Central

    Fukushima, Atsushi; Loh, Kim; Galic, Sandra; Fam, Barbara; Shields, Ben; Wiede, Florian; Tremblay, Michel L.; Watt, Matthew J.; Andrikopoulos, Sofianos; Tiganis, Tony

    2010-01-01

    OBJECTIVE Insulin-induced phosphatidylinositol 3-kinase (PI3K)/Akt signaling and interleukin-6 (IL-6)-instigated JAK/STAT3-signaling pathways in the liver inhibit the expression of gluconeogenic genes to decrease hepatic glucose output. The insulin receptor (IR) and JAK1 tyrosine kinases and STAT3 can serve as direct substrates for the T-cell protein tyrosine phosphatase (TCPTP). Homozygous TCPTP-deficiency results in perinatal lethality prohibiting any informative assessment of TCPTP's role in glucose homeostasis. Here we have used Ptpn2+/− mice to investigate TCPTP's function in glucose homeostasis. RESEARCH DESIGN AND METHODS We analyzed insulin sensitivity and gluconeogenesis in chow versus high-fat–fed (HFF) Ptpn2+/− and Ptpn2+/+ mice and insulin and IL-6 signaling and gluconeogenic gene expression in Ptpn2+/− and Ptpn2+/+ hepatocytes. RESULTS HFF Ptpn2+/− mice exhibited lower fasted blood glucose and decreased hepatic glucose output as determined in hyperinsulinemic euglycemic clamps and by the decreased blood glucose levels in pyruvate tolerance tests. The reduced hepatic glucose output coincided with decreased expression of the gluconeogenic genes G6pc and Pck1 and enhanced hepatic STAT3 phosphorylation and PI3K/Akt signaling in the fasted state. Insulin-induced IR-β–subunit Y1162/Y1163 phosphorylation and PI3K/Akt signaling and IL-6–induced STAT3 phosphorylation were also enhanced in isolated Ptpn2+/− hepatocytes. The increased insulin and IL-6 signaling resulted in enhanced suppression of G6pc and Pck1 mRNA. CONCLUSIONS Liver TCPTP antagonises both insulin and STAT3 signaling pathways to regulate gluconeogenic gene expression and hepatic glucose output. PMID:20484139

  12. Reduced protein tyrosine phosphatase (PTPase) activity of CD45 on peripheral blood lymphocytes in patients with systemic lupus erythematosus (SLE).

    PubMed

    Takeuchi, T; Pang, M; Amano, K; Koide, J; Abe, T

    1997-07-01

    To disclose the mechanism of aberrant function of peripheral blood lymphocytes (PBL) in SLE, we focused on the catalytic function of CD45, and determined the CD45 PTPase activity in SLE patients. The sample population consisted of 32 SLE patients with different disease activity. PTPase activity of cell lysates immunoprecipitated by anti-CD45 MoAb was assayed against phosphotyrosine analogue PNPP, followed by measuring the release of para-nitro phenol at 410 nm. CD45 PTPase activity of PBL was significantly decreased in SLE patients, compared with that of normal controls and patients with systemic sclerosis (964 +/- 265, 1202 +/- 172, 1210 +/- 125, respectively; SLE versus normal, P<0.05). It was correlated with SLE Disease Activity Index (SLEDAI) score (r = 0.597, P = 0.0006), but not with the dose of prednisolone (r = 0.214, P = 0.2657), indicating that CD45 PTPase activity became reduced when the disease was active, but it was not affected by prednisolone. Moreover, it was not corrected by in vitro culture with or without stimulation. The expression of CD45 on PBL was comparable between normal and SLE, raising a possibility that it may be due to aberrant regulation of catalytic function of CD45 in SLE. Given the evidence that tyrosine phosphorylation of cellular proteins by tyrosine kinases and phosphatases is one of the key biochemical events in the signal transduction pathway, the decreased CD45 PTPase activity in SLE may account for the defective signal transduction via TCR/CD3, leading to dysregulated effector function of the lymphocytes.

  13. Identification of genomic regions that interact with a viable allele of the Drosophila protein tyrosine phosphatase corkscrew.

    PubMed Central

    Firth, L; Manchester, J; Lorenzen, J A; Baron, M; Perkins, L A

    2000-01-01

    Signaling by receptor tyrosine kinases (RTKs) is critical for a multitude of developmental decisions and processes. Among the molecules known to transduce the RTK-generated signal is the nonreceptor protein tyrosine phosphatase Corkscrew (Csw). Previously, Csw has been demonstrated to function throughout the Drosophila life cycle and, among the RTKs tested, Csw is essential in the Torso, Sevenless, EGF, and Breathless/FGF RTK pathways. While the biochemical function of Csw remains to be unambiguously elucidated, current evidence suggests that Csw plays more than one role during transduction of the RTK signal and, further, the molecular mechanism of Csw function differs depending upon the RTK in question. The isolation and characterization of a new, spontaneously arising, viable allele of csw, csw(lf), has allowed us to undertake a genetic approach to identify loci required for Csw function. The rough eye and wing vein gap phenotypes exhibited by adult flies homo- or hemizygous for csw(lf) has provided a sensitized background from which we have screened a collection of second and third chromosome deficiencies to identify 33 intervals that enhance and 21 intervals that suppress these phenotypes. We have identified intervals encoding known positive mediators of RTK signaling, e.g., drk, dos, Egfr, E(Egfr)B56, pnt, Ras1, rolled/MAPK, sina, spen, Src64B, Star, Su(Raf)3C, and vein, as well as known negative mediators of RTK signaling, e.g., aos, ed, net, Src42A, sty, and su(ve). Of particular interest are the 5 lethal enhancing intervals and 14 suppressing intervals for which no candidate genes have been identified. PMID:11014820

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

    DTIC Science & Technology

    2006-07-01

    baculoviral expression system to prepare a full length recombinant protein in Sf9 insect cells. We reasoned that expression of such a large protein...baculovirus expressing PTPN4 6 wild type and active site mutants as N terminally six histidine tagged proteins in Sf9 cells to facilitate rapid one step...from infected Sf9 cells from both wild type and active site mutant (band at ~116kDa present only in cells infected with PTPN4 recombinant baculovirus

  15. Changes in labeling of soluble and insoluble rat brain proteins using [3H]-tyrosine as precursor during a learning experiment.

    PubMed

    Popov, N; Schulzeck, S; Schmidt, S; Matthies, H

    1975-01-01

    The incorporation of intraventricularly injected L-[3H]-tyrosine into proteins from three rat brain regions was studied during the acquisition of a shock-motivated brightness discrimination. Separating the proteins by means of polyacrylamide gel electrophoresis revealed an increased labeling confined to only a few of the resolved slow-moving bands of soluble and, especially insoluble hippocampus proteins. The labeling of visual cortex proteins showed only minute changes, while the auditory cortex proteins exhibited no differences between trained and nontrained animals.

  16. Characterization of a protein tyrosine phosphatase as a host factor promoting baculovirus replication in silkworm, Bombyx mori.

    PubMed

    Wang, Fei; Xue, Renju; Li, Xianyang; Hu, Cuimei; Xia, Qingyou

    2016-04-01

    The relevance of protein tyrosine phosphatase (PTP) to host-pathogen interaction is highlighted in mammalian studies, whereas less is known in insects. Here we presented the categorization of the PTP complement of silkworm and characterized their homologous relationship with human and fruit fly PTPs. Among the 36 PTP genes, ptp-h, which was proposed to be the origin of baculovirus ptp belongs to atypical VH1-like dual-specific PTP subset and encodes a catalytic active protein. The maximum expression level of Bmptp-h was at 5th instar and in fat body. Bombyx mori nucleopolyhedrovirus (BmNPV) infection potently induced its expression in silkworm larvae and in BmE cells. Knock-down of Bmptp-h by RNA interference significantly inhibited viral replication, and over-expression enhanced viral replication as determined by viral DNA abundance and BmNPV-GFP positive cells. These results suggest that BmPTP-h might be one of the host factors that is beneficial to baculovirus infection by promoting viral replication.

  17. Downregulation of protein tyrosine phosphatase PTPL1 alters cell cycle and upregulates invasion-related genes in prostate cancer cells.

    PubMed

    Castilla, Carolina; Flores, M Luz; Conde, José M; Medina, Rafael; Torrubia, Francisco J; Japón, Miguel A; Sáez, Carmen

    2012-04-01

    PTPL1, a non-receptor type protein tyrosine phosphatase, has been involved in the regulation of apoptosis and invasiveness of various tumour cell types, but its role in prostate cancer remained to be investigated. We report here that downregulation of PTPL1 by small interfering RNA in PC3 cells decreases cell proliferation and concomitantly reduces the expression of cell cycle-related proteins such as cyclins E and B1, PCNA, PTTG1 and phospho-histone H3. PTPL1 downregulation also increases the invasion ability of PC3 cells through Matrigel coated membranes. cDNA array of PTPL1-silenced PC3 cells versus control cells showed an upregulation of invasion-related genes such as uPA, uPAR, tPA, PAI-1, integrin α6 and osteopontin. This increased expression was also confirmed in PTPL1-silenced DU145 prostate cancer cells by quantitative real time PCR and western blot. These findings suggest that PTPL1 is an important mediator of central cellular processes such as proliferation and invasion.

  18. CCAAT/enhancer-binding protein β: its role in breast cancer and associations with receptor tyrosine kinases

    PubMed Central

    Zahnow, Cynthia A.

    2011-01-01

    The CCAAT/enhancer-binding proteins (C/EBPs) are a family of leucine-zipper transcription factors that regulate gene expression to control cellular proliferation, differentiation, inflammation and metabolism. Encoded by an intronless gene, C/EBPβ is expressed as several distinct protein isoforms (LAP1, LAP2, LIP) whose expression is regulated by the differential use of several in-frame translation start sites. LAP1 and LAP2 are transcriptional activators and are associated with differentiation, whereas LIP is frequently elevated in proliferative tissue and acts as a dominant-negative inhibitor of transcription. However, emerging evidence suggests that LIP can serve as a transcriptional activator in some cellular contexts, and that LAP1 and LAP2 might also have unique actions. The LIP:LAP ratio is crucial for the maintenance of normal growth and development, and increases in this ratio lead to aggressive forms of breast cancer. This review discusses the regulation of C/EBPβ activity by post-translational modification, the individual actions of LAP1, LAP2 and LIP, and the functions and downstream targets that are unique to each isoform. The role of the C/EBPβ isoforms in breast cancer is discussed and emphasis is placed on their interactions with receptor tyrosine kinases. PMID:19351437

  19. Deficiency of the protein-tyrosine phosphatase DEP-1/PTPRJ promotes matrix metalloproteinase-9 expression in meningioma cells.

    PubMed

    Petermann, Astrid; Stampnik, Yvonn; Cui, Yan; Morrison, Helen; Pachow, Doreen; Kliese, Nadine; Mawrin, Christian; Böhmer, Frank-D

    2015-05-01

    Brain-invasive growth of a subset of meningiomas is associated with less favorable prognosis. The molecular mechanisms causing invasiveness are only partially understood, however, the expression of matrix metalloproteinases (MMPs) has been identified as a contributing factor. We have previously found that loss of density enhanced phosphatase-1 (DEP-1, also designated PTPRJ), a transmembrane protein-tyrosine phosphatase, promotes meningioma cell motility and invasive growth in an orthotopic xenotransplantation model. We have now analyzed potential alterations of the expression of genes involved in motility control, caused by DEP-1 loss in meningioma cell lines. DEP-1 depleted cells exhibited increased expression of mRNA encoding MMP-9, and the growth factors EGF and FGF-2. The increase of MMP-9 expression in DEP-1 depleted cells was also readily detectable at the protein level by zymography. MMP-9 upregulation was sensitive to chemical inhibitors of growth factor signal transduction. Conversely, MMP-9 mRNA levels could be stimulated with growth factors (e.g. EGF) and inflammatory cytokines (e.g. TNFα). Increase of MMP-9 expression by DEP-1 depletion, or growth factor/cytokine stimulation qualitatively correlated with increased invasiveness in vitro scored as transmigration through matrigel-coated membranes. The studies suggest induction of MMP-9 expression promoted by DEP-1 deficiency, or potentially by growth factors and inflammatory cytokines, as a mechanism contributing to meningioma brain invasiveness.

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

    PubMed Central

    Doss, VA; Kuberapandian, Dharaniyambigai

    2016-01-01

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

  1. The neural receptor protein tyrosine phosphatase DPTP69D is required during periods of axon outgrowth in Drosophila.

    PubMed Central

    Desai, Chand; Purdy, Joy

    2003-01-01

    We have isolated and characterized a series of 18 chemically induced alleles of Ptp69D ranging in strength from viable to worse than null, which represent unique tools for probing the structure, function, and signaling pathway of DPTP69D. Three alleles are strongly temperature sensitive and were used to define the developmental periods requiring DPTP69D function; adult health requires DPTP69D during the mid- to late-pupal stage, eclosion requires DPTP69D during the early to mid-larval stage, and larval survival requires DPTP69D during embryogenesis. Mutations predicted to abolish the phosphatase activity of the membrane proximal D1 domain severely reduce but do not abolish DPTP69D function. Six alleles appear null; only 20% of null homozygotes pupate and <5% eclose, only to fall into the food and drown. One allele, Ptp69D(7), confers axon and viability defects more severe than those of the null phenotype. Sequence analysis predicts that Ptp69D(7) encodes a mutant protein that may bind but not release substrate. Like mutations in the protein tyrosine phosphatase gene Dlar, strong Ptp69D alleles cause the ISNb nerve to bypass its muscle targets. Genetic analysis reveals that the bypass defect in Dlar and Ptp69D mutants is dependent upon DPTP99A function, consistent with the hypothesis that DPTP69D and DLAR both counteract DPTP99A, allowing ISNb axons to enter their target muscle field. PMID:12807778

  2. Large, detergent-resistant complexes containing murine antigens Thy-1 and Ly-6 and protein tyrosine kinase p56lck.

    PubMed

    Bohuslav, J; Cinek, T; Horejsí, V

    1993-04-01

    A number of human and mouse leukocyte surface (glyco)proteins anchored in a membrane via glycosylphosphatidylinositol (GPI) moiety have been previously shown to be noncovalently associated with protein tyrosine kinases (Science 1991. 254: 1016; J. Biol. Chem. 1992. 267: 12317). Here we show that two murine antigens of this group, Thy-1 and Ly-6, implicated in the activation of the T cells, are associated with each other, with the kinase p56lck and with several of potential kinase substrates in very large, detergent-resistant complexes, the size of which is between 50 and 200 nm, as determined by ultrafiltration and gel chromatography. Experiments on simultaneous solubilization of mixed human and mouse cells rule out that the observed complexes are artifacts induced by the detergent. Complexes of similar composition and properties were obtained when either detergents Brij-58, Nonidet-P40 or 3-[(3-cholamidopropyl)-dimethylammonio]- 1-propane-sulfonate (Chaps) were used for solubilization of the cells, while octylglucoside at least partially dissociated them. These "GPI-complexes" may be essential for the well-known signal-transducing capacity of Thy-1 and Ly-6.

  3. A defined medium supports changes consistent with capacitation in stallion sperm, as evidenced by increases in protein tyrosine phosphorylation and high rates of acrosomal exocytosis.

    PubMed

    McPartlin, L A; Littell, J; Mark, E; Nelson, J L; Travis, A J; Bedford-Guaus, S J

    2008-03-15

    Efficient in vitro capacitation of stallion sperm has not yet been achieved, as suggested by low sperm penetration rates reported in in vitro fertilization (IVF) studies. Our objectives were to evaluate defined incubation conditions that would support changes consistent with capacitation in stallion sperm. Protein tyrosine phosphorylation events and the ability of sperm to undergo acrosomal exocytosis under various incubation conditions were used as end points for capacitation. Sperm incubated 4-6h in modified Whitten's (MW) with the addition of 25 mM NaHCO3 and 7 mg/mL BSA (capacitating medium) yielded high rates of protein tyrosine phosphorylation. Either HCO3(-) or BSA was required to support these changes, with the combination of both providing the most intense results. When a membrane-permeable form of cAMP and a phosphodiesterase inhibitor (IBMX) were added to MW in the absence of HCO3(-) and BSA, the tyrosine phosphorylation results obtained in our capacitating conditions could not be replicated, suggesting either effects apart from cAMP were responsible for tyrosine phosphorylation, or that stallion sperm might respond differently to these reagents as compared to sperm from other mammals. Sperm incubation in capacitating conditions was also associated with high percentages (Pprotein tyrosine phosphorylation in stallion sperm incubated in defined conditions, coupled with significant percentages of acrosome reacted sperm. The continuation of these studies might help to elucidate the conditions and pathways supporting sperm capacitation in the horse.

  4. Changes in Carboxy Methylation and Tyrosine Phosphorylation of Protein Phosphatase PP2A Are Associated with Epididymal Sperm Maturation and Motility

    PubMed Central

    Dudiki, Tejasvi; Kadunganattil, Suraj; Ferrara, John K.; Kline, Douglas W.; Vijayaraghavan, Srinivasan

    2015-01-01

    Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function. PMID:26569399

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

    PubMed Central

    Yamada, Yasuyuki; Sato, Fumihiko

    2016-01-01

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

  6. Search for Physiological Substrates of Protein Tyrosine Phosphatase Epsilon in Mammary Tumor Cell Lines

    DTIC Science & Technology

    2001-12-01

    BamHI/SmaI sites of pGEX2TK; the resulting plasmids were expressed in the BL-21 strain of E . coli bacteria . GST-PTPe fusion proteins were purified...attempted to overcome these difficulties by optimizing bacterial growth conditions. Several strains of E . coli bacteria were used to try and find one

  7. Chronic restraint stress induces sperm acrosome reaction and changes in testicular tyrosine phosphorylated proteins in rats

    PubMed Central

    Arun, Supatcharee; Burawat, Jaturon; Sukhorum, Wannisa; Sampannang, Apichakan; Maneenin, Chanwit; Iamsaard, Sitthichai

    2016-01-01

    Background: Stress is a cause of male infertility. Although sex hormones and sperm quality have been shown to be low in stress, sperm physiology and testicular functional proteins, such as phosphotyrosine proteins, have not been documented. Objective: To investigate the acrosome status and alterations of testicular proteins involved in spermatogenesis and testosterone synthesis in chronic stress in rats. Materials and Methods: In this experimental study, male rats were divided into 2 groups (control and chronic stress (CS), n=7). CS rats were immobilized (4 hr/day) for 42 consecutive days. The blood glucose level (BGL), corticosterone, testosterone, acrosome status, and histopathology were examined. The expressions of testicular steroidogenic acute regulatory (StAR), cytochrome P450 side chain cleavage (CYP11A1), and phosphorylated proteins were analyzed. Results: Results showed that BGL (71.25±2.22 vs. 95.60±3.36 mg/dl), corticosterone level (24.33±4.23 vs. 36.9±2.01 ng/ml), acrosome reacted sperm (3.25±1.55 vs. 17.71±5.03%), and sperm head abnormality (3.29±0.71 vs. 6.21±1.18%) were significantly higher in CS group in comparison with control. In contrast, seminal vesicle (0.41±0.05 vs. 0.24±0.07 g/100g), testosterone level (3.37±0.79 vs. 0.61±0.29 ng/ml), and sperm concentration (115.33±7.70 vs. 79.13±3.65×106 cells/ml) of CS were significantly lower (p<0.05) than controls. Some atrophic seminiferous tubules and low sperm mass were apparent in CS rats. The expression of CYP11A1 except StAR protein was markedly decreased in CS rats. In contrast, a 55 kDa phosphorylated protein was higher in CS testes. Conclusion: CS decreased the expression of CYP11A, resulting in decreased testosterone, and increased acrosome-reacted sperm, assumed to be the result of an increase of 55 kDa phosphorylated protein. PMID:27525328

  8. Sprouty-related Ena/Vasodilator-stimulated Phosphoprotein Homology 1-Domain-containing Protein (SPRED1), a Tyrosine-Protein Phosphatase Non-receptor Type 11 (SHP2) Substrate in the Ras/Extracellular Signal-regulated Kinase (ERK) Pathway*

    PubMed Central

    Quintanar-Audelo, Martina; Yusoff, Permeen; Sinniah, Saravanan; Chandramouli, Sumana; Guy, Graeme R.

    2011-01-01

    SHP2 is a tyrosine phosphatase involved in the activation of the Ras/ERK signaling pathway downstream of a number of receptor tyrosine kinases. One of the proposed mechanisms involving SHP2 in this context is to dephosphorylate and inactivate inhibitors of the Ras/ERK pathway. Two protein families bearing a unique, common domain, Sprouty and SPRED proteins, are possible candidates because they have been reported to inhibit the Ras/ERK pathway upon FGF activation. We tested whether any of these proteins are likely substrates of SHP2. Our findings indicate that Sprouty2 binds to the C-terminal tail of SHP2, which is an unlikely substrate binding site, whereas SPRED proteins bind to the tyrosine phosphatase domain that is known to be the binding site for its substrates. Overexpressed SHP2 was able to dephosphorylate SPREDs but not Sprouty2. Finally, we found two tyrosine residues on SPRED1 that are required, when phosphorylated, to inhibit Ras/ERK activation and identified Tyr-420 as a specific dephosphorylation target of SHP2. The evidence obtained indicates that SPRED1 is a likely substrate of SHP2, whose tyrosine dephosphorylation is required to attenuate the inhibitory action of SPRED1 in the Ras/ERK pathway. PMID:21531714

  9. Fps/Fes and Fer non-receptor protein-tyrosine kinases regulate collagen- and ADP-induced platelet aggregation.

    PubMed

    Senis, Y A; Sangrar, W; Zirngibl, R A; Craig, A W B; Lee, D H; Greer, P A

    2003-05-01

    Fps/Fes and Fer proto-oncoproteins are structurally related non-receptor protein-tyrosine kinases implicated in signaling downstream from cytokines, growth factors and immune receptors. We show that Fps/Fes and Fer are expressed in human and mouse platelets, and are activated following stimulation with collagen and collagen-related peptide (CRP), suggesting a role in GPVI receptor signaling. Fer was also activated following stimulation with thrombin and a protease-activated receptor4 (PAR4)-activating peptide, suggesting a role in signaling downstream from the G protein-coupled PAR4. There were no detectable perturbations in CRP-induced activation of Syk, PLCgamma2, cortactin, Erk, Jnk, Akt or p38 in platelets from mice lacking Fps/Fes, Fer, or both kinases. Platelets lacking Fps/Fes, from a targeted fps/fes null strain of mice, showed increased rates and amplitudes of collagen-induced aggregation, relative to wild-type platelets. P-Selectin expression was also elevated on the surface of Fps/Fes-null platelets in response to CRP. Fer-deficient platelets, from mice targeted with a kinase-inactivating mutation, disaggregated more rapidly than wild-type platelets in response to ADP. This report provides the first evidence that Fps/Fes and Fer are expressed in platelets and become activated downstream from the GPVI collagen receptor, and that Fer is activated downstream from a G-protein coupled receptor. Furthermore, using targeted mouse models we show that deficiency in Fps/Fes or Fer resulted in disregulated platelet aggregation and disaggregation, demonstrating a role for these kinases in regulating platelet functions.

  10. The Mouse Cytomegalovirus Gene m42 Targets Surface Expression of the Protein Tyrosine Phosphatase CD45 in Infected Macrophages

    PubMed Central

    Thiel, Nadine; Keyser, Kirsten A.; Oduro, Jennifer D.; Wagner, Karen; Halenius, Anne; Lenac Roviš, Tihana; Brinkmann, Melanie M.; Jonjić, Stipan; Cicin-Sain, Luka

    2016-01-01

    The receptor-like protein tyrosine phosphatase CD45 is expressed on the surface of cells of hematopoietic origin and has a pivotal role for the function of these cells in the immune response. Here we report that following infection of macrophages with mouse cytomegalovirus (MCMV) the cell surface expression of CD45 is drastically diminished. Screening of a set of MCMV deletion mutants allowed us to identify the viral gene m42 of being responsible for CD45 down-modulation. Moreover, expression of m42 independent of viral infection upon retroviral transduction of the RAW264.7 macrophage cell line led to comparable regulation of CD45 expression. In immunocompetent mice infected with an m42 deletion mutant lower viral titers were observed in all tissues examined when compared to wildtype MCMV, indicating an important role of m42 for viral replication in vivo. The m42 gene product was identified as an 18 kDa protein expressed with early kinetics and is predicted to be a tail-anchored membrane protein. Tracking of surface-resident CD45 molecules revealed that m42 induces internalization and degradation of CD45. The observation that the amounts of the E3 ubiquitin ligases Itch and Nedd4 were diminished in cells expressing m42 and that disruption of a PY motif in the N-terminal part of m42 resulted in loss of function, suggest that m42 acts as an activator or adaptor for these Nedd4-like ubiquitin ligases, which mark CD45 for lysosomal degradation. In conclusion, the down-modulation of CD45 expression in MCMV-infected myeloid cells represents a novel pathway of virus-host interaction. PMID:27926943

  11. Effects of dietary energy intake and cold exposure on kinetics of plasma phenylalanine, tyrosine and protein synthesis in sheep.

    PubMed

    Sano, Hiroaki; Murakami, Shingo; Sasaki, Satori; Al-Mamun, Mohammad

    2010-02-01

    An isotope dilution method of [2H5]phenylalanine (Phe) and [2H2]tyrosine (Tyr) was used to determine the effects of metabolisable energy (ME) intake and cold exposure on plasma Phe and Tyr turnover rates in sheep. Whole body protein synthesis (WBPS) was calculated with the [2H5]Phe model. Eight adult sheep were assigned to two dietary treatments receiving the same amount of crude protein and either 515 or 828 kJ x kg BW(-0.75) x d(-1) of ME (Me-ME diet and Hi-ME diet, respectively) with a crossover design for two 28 d periods. The sheep were exposed from a thermoneutral environment (23 +/- 1 degrees C) to a cold environment (2 +/- 1 to 4 +/- 1 degrees C) for 6 d for each dietary treatment. The primed-continuous infusion method of isotope dilution was conducted in both environmental temperatures. Plasma Phe turnover rate (PheTR) tended to be greater and plasma Tyr turnover rate (TyrTR) was greater (p = 0.03) for the Hi-ME diet compared with the Me-ME diet. Plasma PheTR increased (p = 0.04) and plasma TyrTR tended to increase during cold exposure. Whole body protein synthesis tended to be greater for the Hi-ME diet compared with the Me-ME diet and increased (p = 0.03) during cold exposure compared to the thermoneutral environment, but no interaction was detected. It was concluded that in sheep, plasma PheTR and WBPS (as determined by the [2H5]Phe model) tended to be influenced by and plasma TyrTR was influenced by ME intake. Further, plasma PheTR and WBPS increased and plasma TyrTR tended to increase during cold exposure.

  12. Identification of an activator protein-1-like sequence as the glucocorticoid response element in the rat tyrosine hydroxylase gene.

    PubMed

    Rani, C S Sheela; Elango, Narayanasamy; Wang, Shou-Shu; Kobayashi, Kazuto; Strong, Randy

    2009-03-01

    Glucocorticoids (GCs) generally stimulate gene transcription via consensus glucocorticoid response elements (GREs) located in the promoter region. To identify the GRE in the rat tyrosine hydroxylase (TH) gene promoter, we transiently transfected PC12 cells with a 9-kilobase (kb) TH promoter-luciferase (Luc) construct. Dexamethasone (Dex) stimulated Luc activity, which was abolished by mifepristone (RU486). Serial deletion mutations revealed a Dex-responsive 7-base pair (bp) sequence, TGACTAA, located at -5734 to -5728. Deletion of just these seven nucleotides from the 9-kb promoter completely abolished the Dex response and partially reduced the response to phorbol ester but not to forskolin. The Dex response was fully retained in a construct in which most of the 9-kb promoter was deleted, except for 100 bp around the -5.7-kb region, clearly identifying this 7-bp sequence as solely responsible for GC responsiveness. Conversely, deletion of the proximal cAMP-response element (-45/-38) or activator protein-1 (AP-1) (-207/-201) sites in the 9-kb promoter did not affect Dex and phorbol ester responses. A radiolabeled 25-bp promoter fragment bearing the 7-bp TH-GRE/AP-1 showed specific binding to PC12 nuclear proteins. Using antibodies against the glucocorticoid receptors and AP-1 family of proteins and primers for the TH-GRE/AP-1 region, we detected a specific DNA amplicon in a chromatin immunoprecipitation assay. This 7-bp TH-GRE/AP-1 sequence (TGACTAA) does not bear similarity to any known GRE but closely resembles the consensus AP-1 binding site, TGACTCA. Our studies describe for the first time a novel GRE/AP-1 site present in the TH gene promoter that is critical for glucocorticoid regulation of the TH gene.

  13. Distance Mapping in Proteins Using Fluorescence Spectroscopy: Tyrosine, like Tryptophan, Quenches Bimane Fluorescence in a Distance-Dependent Manner

    PubMed Central

    2015-01-01

    Tryptophan-induced quenching of fluorophores (TrIQ) uses intramolecular fluorescence quenching to assess distances in proteins too small (<15 Å) to be easily probed by traditional Forster resonance energy transfer methods. A powerful aspect of TrIQ is its ability to obtain an ultrafast snapshot of a protein conformation, by identifying “static quenching” (contact between the Trp and probe at the moment of light excitation). Here we report new advances in this site-directed fluorescence labeling (SDFL) approach, gleaned from recent studies of T4 lysozyme (T4L). First, we show that like TrIQ, tyrosine-induced quenching (TyrIQ) occurs for the fluorophore bimane in a distance-dependent fashion, although with some key differences. The Tyr “sphere of quenching” for bimane (≤10 Å) is smaller than for Trp (≤15 Å, Cα–Cα distance), and the size difference between the quenching residue (Tyr) and control (Phe) differs by only a hydroxyl group. Second, we show how TrIQ and TyrIQ can be used together to assess the magnitude and energetics of a protein movement. In these studies, we placed a bimane (probe) and Trp or Tyr (quencher) on opposite ends of a “hinge” in T4L and conducted TrIQ and TyrIQ measurements. Our results are consistent with an ∼5 Å change in Cα–Cα distances between these sites upon substrate binding, in agreement with the crystal structures. Subsequent Arrhenius analysis suggests the activation energy barrier (Ea) to this movement is relatively low (∼1.5–2.5 kcal/mol). Together, these results demonstrate that TyrIQ, used together with TrIQ, significantly expands the power of quenching-based distance mapping SDFL studies. PMID:25144569

  14. Redox regulation of protein tyrosine phosphatase 1B by manipulation of dietary selenium affects the triglyceride concentration in rat liver.

    PubMed

    Mueller, Andreas S; Klomann, Sandra D; Wolf, Nicole M; Schneider, Sandra; Schmidt, Rupert; Spielmann, Julia; Stangl, Gabriele; Eder, Klaus; Pallauf, Josef

    2008-12-01

    Protein tyrosine phosphatase 1B (PTP1B) is a key enzyme in the counter-regulation of insulin signaling and in the stimulation of fatty acid synthesis. Selenium (Se), via the activities of glutathione peroxidase (GPx) and thioredoxin reductase (TrxR), is involved in the removal of H(2)O(2) and organic peroxides, which are critical compounds in the modulation of PTP1B activity via glutathionylation. Our study with growing rats investigated how the manipulation of dietary Se concentration influences the regulation of PTP1B and lipogenic effects mediated by PTP1B. Weanling albino rats were divided into 3 groups of 10. The negative control group (NC) was fed a Se-deficient diet for 8 wk. Rats in groups Se75 and Se150 received diets supplemented with 75 or 150 microg Se/kg. Se supplementation of the rats strongly influenced expression and activity of the selenoenzymes cytosolic GPx, plasma GPx, phospholipidhydroperoxide GPx, and cytosolic TrxR, and liver PTP1B. Liver PTP1B activity was significantly higher in groups Se75 and Se150 than in the NC group and this was attributed to a lowered inhibition of the enzyme by glutathionylation. The increased liver PTP1B activity in groups Se75 and Se150 resulted in 1.1- and 1.4-fold higher liver triglyceride concentrations than in the NC rats. The upregulation of the sterol regulatory element binding protein-1c and of fatty acid synthase, 2 PTP1B targets, provided a possible explanation for the lipogenic effect of PTP1B due to the manipulation of dietary Se. We therefore conclude that redox-regulated proteins, such as PTP1B, represent important interfaces between dietary antioxidants such as Se and the regulation of metabolic processes.

  15. Activation of proacrosin accompanies upregulation of sp32 protein tyrosine phosphorylation in pig sperm.

    PubMed

    Sun, P L; Yang, L X; Cui, J-J; Tian, Y; Liu, Y; Jin, Y

    2013-12-11

    This study investigated the relationship between acrosin activation and pig sperm proacrosin binding protein (sp32) phosphorylation levels. Differently processed pig spermatozoa (fresh semen sperm, capacitation sperm, acrosome reaction sperm, capacitation-like sperm, and thawed sperm) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. The fresh semen and capacitation sperm groups both produced proacrosin protein bands of 55 kDa; however, the result of the fresh semen sperm group was clearer than that of the capacitation sperm group. The thawed sperm group showed a shallow strip at 55 kDa. The capacitation and acrosome reaction sperm groups produced obvious proacrosin protein bands at 35 kDa, and the strips of the capacitation sperm group were again clearer. A faint band was visible at 32 kDa in the acrosome reaction sperm group. The capacitation, thawed, and acrosome reaction sperm groups showed significant strips in sp32, and the bands of the acrosome reaction sperm group were shallower than those of the 2 other groups. The capacitation and thawed sperm groups produced significant strips at 40 kDa, and the capacitation sperm group produced an additional strip at 55 kDa. In conclusion, sp32 phosphorylation levels can promote proacrosin activation into the active acrosin.

  16. A Rapid Lateral Flow Immunoassay for the Detection of Tyrosine Phosphatase-Like Protein IA-2 Autoantibodies in Human Serum

    PubMed Central

    Kikkas, Ingrid; Mallone, Roberto; Larger, Etienne; Volland, Hervé; Morel, Nathalie

    2014-01-01

    Type 1 diabetes (T1D) results from the destruction of pancreatic insulin-producing beta cells and is strongly associated with the presence of islet autoantibodies. Autoantibodies to tyrosine phosphatase-like protein IA-2 (IA-2As) are considered to be highly predictive markers of T1D. We developed a novel lateral flow immunoassay (LFIA) based on a bridging format for the rapid detection of IA-2As in human serum samples. In this assay, one site of the IA-2As is bound to HA-tagged-IA-2, which is subsequently captured on the anti-HA-Tag antibody-coated test line on the strip. The other site of the IA-2As is bound to biotinylated IA-2, allowing the complex to be visualized using colloidal gold nanoparticle-conjugated streptavidin. For this study, 35 serum samples from T1D patients and 44 control sera from non-diabetic individuals were analyzed with our novel assay and the results were correlated with two IA-2A ELISAs. Among the 35 serum samples from T1D patients, the IA-2A LFIA, the in-house IA-2A ELISA and the commercial IA-2A ELISA identified as positive 21, 29 and 30 IA-2A-positive sera, respectively. The major advantages of the IA-2A LFIA are its rapidity and simplicity. PMID:25047039

  17. DSD-1-Proteoglycan/Phosphacan and receptor protein tyrosine phosphatase-beta isoforms during development and regeneration of neural tissues.

    PubMed

    Faissner, Andreas; Heck, Nicolas; Dobbertin, Alexandre; Garwood, Jeremy

    2006-01-01

    Interactions between neurons and glial cells play important roles in regulating key events of development and regeneration of the CNS. Thus, migrating neurons are partly guided by radial glia to their target, and glial scaffolds direct the growth and directional choice of advancing axons, e.g., at the midline. In the adult, reactive astrocytes and myelin components play a pivotal role in the inhibition of regeneration. The past years have shown that astrocytic functions are mediated on the molecular level by extracellular matrix components, which include various glycoproteins and proteoglycans. One important, developmentally regulated chondroitin sulfate proteoglycan is DSD-1-PG/phosphacan, a glial derived proteoglycan which represents a splice variant of the receptor protein tyrosine phosphatase (RPTP)-beta (also known as PTP-zeta). Current evidence suggests that this proteoglycan influences axon growth in development and regeneration, displaying inhibitory or stimulatory effects dependent on the mode of presentation, and the neuronal lineage. These effects seem to be mediated by neuronal receptors of the Ig-CAM superfamily.

  18. Protein tyrosine phosphatase 1B negatively regulates S100A9-mediated lung damage during respiratory syncytial virus exacerbations

    PubMed Central

    Foronjy, Robert F.; Ochieng, Pius O.; Salathe, Matthias A.; Dabo, Abdoulaye J.; Eden, Edward; Baumlin, Nathalie; Cummins, Neville; Barik, Sailen; Campos, Michael; Thorp, Edward B.; Geraghty, Patrick

    2015-01-01

    Protein tyrosine phosphatase 1B (PTP1B) has anti-inflammatory potential but PTP1B responses are desensitized in the lung by prolonged cigarette smoke exposure. Here we investigate whether PTP1B expression impacts lung disease severity during respiratory syncytial viral (RSV) exacerbations of chronic obstructive pulmonary disease (COPD). Ptp1b-/- mice infected with RSV exhibit exaggerated immune cell infiltration, damaged epithelial cell barriers, cytokine production and increased apoptosis. Elevated expression of S100A9, a damage-associated molecular pattern molecule, was observed in the lungs of Ptp1b-/- mice during RSV infection. Utilizing a neutralizing anti-S100A9 IgG antibody, it was determined that extracellular S100A9 signaling significantly impacts lung damage during RSV infection. Pre-exposure to cigarette smoke desensitized PTP1B activity, which coincided with enhanced S100A9 secretion and inflammation in wild type animals during RSV infection. S100A9 levels in human bronchoalveolar lavage fluid had an inverse relationship with lung function in healthy subjects, smokers and COPD subjects. Fully differentiated human bronchial epithelial cells isolated from COPD donors cultured at the air liquid interface secreted more S100A9 than cells from healthy donors or smokers following RSV infection. Together, these findings show that reduced PTP1B responses contribute to disease symptoms in part by enhancing S100A9 expression during viral-associated COPD exacerbations. PMID:26813343

  19. Protein tyrosine phosphatase 1B negatively regulates S100A9-mediated lung damage during respiratory syncytial virus exacerbations.

    PubMed

    Foronjy, R F; Ochieng, P O; Salathe, M A; Dabo, A J; Eden, E; Baumlin, N; Cummins, N; Barik, S; Campos, M; Thorp, E B; Geraghty, P

    2016-09-01

    Protein tyrosine phosphatase 1B (PTP1B) has anti-inflammatory potential but PTP1B responses are desensitized in the lung by prolonged cigarette smoke exposure. Here we investigate whether PTP1B expression affects lung disease severity during respiratory syncytial viral (RSV) exacerbations of chronic obstructive pulmonary disease (COPD). Ptp1b(-/-) mice infected with RSV exhibit exaggerated immune cell infiltration, damaged epithelial cell barriers, cytokine production, and increased apoptosis. Elevated expression of S100A9, a damage-associated molecular pattern molecule, was observed in the lungs of Ptp1b(-/-) mice during RSV infection. Utilizing a neutralizing anti-S100A9 IgG antibody, it was determined that extracellular S100A9 signaling significantly affects lung damage during RSV infection. Preexposure to cigarette smoke desensitized PTP1B activity that coincided with enhanced S100A9 secretion and inflammation in wild-type animals during RSV infection. S100A9 levels in human bronchoalveolar lavage fluid had an inverse relationship with lung function in healthy subjects, smokers, and COPD subjects. Fully differentiated human bronchial epithelial cells isolated from COPD donors cultured at the air liquid interface secreted more S100A9 than cells from healthy donors or smokers following RSV infection. Together, these findings show that reduced PTP1B responses contribute to disease symptoms in part by enhancing S100A9 expression during viral-associated COPD exacerbations.

  20. Salicylic Acid Based Small Molecule Inhibitor for the Oncogenic Src Homology-2 Domain Containing Protein Tyrosine Phosphatase-2 (SHP2)

    SciTech Connect

    Zhang, Xian; He, Yantao; Liu, Sijiu; Yu, Zhihong; Jiang, Zhong-Xing; Yang, Zhenyun; Dong, Yuanshu; Nabinger, Sarah C.; Wu, Li; Gunawan, Andrea M.; Wang, Lina; Chan, Rebecca J.; Zhang, Zhong-Yin

    2010-08-13

    The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with Noonan syndrome, various kinds of leukemias, and solid tumors. Thus, there is considerable interest in SHP2 as a potential target for anticancer and antileukemia therapy. We report a salicylic acid based combinatorial library approach aimed at binding both active site and unique nearby subpockets for enhanced affinity and selectivity. Screening of the library led to the identification of a SHP2 inhibitor II-B08 (compound 9) with highly efficacious cellular activity. Compound 9 blocks growth factor stimulated ERK1/2 activation and hematopoietic progenitor proliferation, providing supporting evidence that chemical inhibition of SHP2 may be therapeutically useful for anticancer and antileukemia treatment. X-ray crystallographic analysis of the structure of SHP2 in complex with 9 reveals molecular determinants that can be exploited for the acquisition of more potent and selective SHP2 inhibitors.

  1. CuAAC click chemistry accelerates the discovery of novel chemical scaffolds as promising protein tyrosine phosphatases inhibitors.

    PubMed

    He, X-P; Xie, J; Tang, Y; Li, J; Chen, G-R

    2012-01-01

    Protein tyrosine phosphatases (PTPs) are crucial regulators for numerous biological processes in nature. The dysfunction and overexpression of many PTP members have been demonstrated to cause fatal human diseases such as cancers, diabetes, obesity, neurodegenerative diseases and autoimmune disorders. In the past decade, considerable efforts have been devoted to the production of PTPs inhibitors by both academia and the pharmaceutical industry. However, there are only limited drug candidates in clinical trials and no commercial drugs have been approved, implying that further efficient discovery of novel chemical entities competent for inhibition of the specific PTP target in vivo remains yet a challenge. In light of the click-chemistry paradigm which advocates the utilization of concise and selective carbon-heteroatom ligation reactions for the modular construction of useful compound libraries, the Cu(I)-catalyzed azidealkyne 1,3-dipolar cycloaddition reaction (CuAAC) has fueled enormous energy into the modern drug discovery. Recently, this ingenious chemical ligation tool has also revealed efficacious and expeditious in establishing large combinatorial libraries for the acquisition of novel PTPs inhibitors with promising pharmacological profiles. We thus offer here a comprehensive review highlighting the development of PTPs inhibitors accelerated by the CuAAC click chemistry.

  2. Identification of protein tyrosine phosphatase SHP-2 as a new target of perfluoroalkyl acids in HepG2 cells.

    PubMed

    Yang, Yu; Lv, Qi-Yan; Guo, Liang-Hong; Wan, Bin; Ren, Xiao-Min; Shi, Ya-Li; Cai, Ya-Qi

    2016-08-29

    Perfluoroalkyl acids (PFAAs) are widespread environmental contaminants which have been detected in humans and linked to adverse health effects. Previous toxicological studies mostly focused on nuclear receptor-mediated pathways and did not support the observed toxic effects. In this study, we aimed to investigate the molecular mechanisms of PFAA toxicities by identifying their biological targets in cells. Using a novel electrochemical biosensor, 16 PFAAs were evaluated for inhibition of protein tyrosine phosphatase SHP-2 activity. Their potency increased with PFAA chain length, with perfluorooctadecanoic acid (PFODA) showing the strongest inhibition. Three selected PFAAs, 25 μM perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid, and PFODA, also inhibited SHP-2 activity in HepG2 cells and increased paxillin phosphorylation level. PFOA was detected in the immunoprecipitated SHP-2 from the cells exposed to 250 μM PFOA, providing unequivocal evidence for the direct binding of PFOA with SHP-2 in the cell. Molecular docking rationalized the formation of PFAA/SHP-2 complex and chain length-dependent inhibition potency. Our results have established SHP-2 as a new cellular target of PFAAs.

  3. 1,2-Naphthoquinone activates vanilloid receptor 1 through increased protein tyrosine phosphorylation, leading to contraction of guinea pig trachea

    SciTech Connect

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

    2006-01-15

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

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

    NASA Astrophysics Data System (ADS)

    Cui, Yongchao; Shi, Dayong; Hu, Zhiqiang

    2011-11-01

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

  5. A rapid lateral flow immunoassay for the detection of tyrosine phosphatase-like protein IA-2 autoantibodies in human serum.

    PubMed

    Kikkas, Ingrid; Mallone, Roberto; Larger, Etienne; Volland, Hervé; Morel, Nathalie

    2014-01-01

    Type 1 diabetes (T1D) results from the destruction of pancreatic insulin-producing beta cells and is strongly associated with the presence of islet autoantibodies. Autoantibodies to tyrosine phosphatase-like protein IA-2 (IA-2As) are considered to be highly predictive markers of T1D. We developed a novel lateral flow immunoassay (LFIA) based on a bridging format for the rapid detection of IA-2As in human serum samples. In this assay, one site of the IA-2As is bound to HA-tagged-IA-2, which is subsequently captured on the anti-HA-Tag antibody-coated test line on the strip. The other site of the IA-2As is bound to biotinylated IA-2, allowing the complex to be visualized using colloidal gold nanoparticle-conjugated streptavidin. For this study, 35 serum samples from T1D patients and 44 control sera from non-diabetic individuals were analyzed with our novel assay and the results were correlated with two IA-2A ELISAs. Among the 35 serum samples from T1D patients, the IA-2A LFIA, the in-house IA-2A ELISA and the commercial IA-2A ELISA identified as positive 21, 29 and 30 IA-2A-positive sera, respectively. The major advantages of the IA-2A LFIA are its rapidity and simplicity.

  6. Structure of the Trypanosoma cruzi protein tyrosine phosphatase TcPTP1, a potential therapeutic target for Chagas' disease

    SciTech Connect

    Lountos, George T.; Tropea, Joseph E.; Waugh, David S.

    2013-06-05

    Chagas’ disease, a neglected tropical affliction transmitted by the flagellated protozoan Trypanosoma cruzi, is prevalent in Latin America and affects nearly 18 million people worldwide, yet few approved drugs are available to treat the disease. Moreover, the currently available drugs exhibit severe toxicity or are poorly effective in the chronic phase of the disease. This limitation, along with the large population at risk, underscores the urgent need to discover new molecular targets and novel therapeutic agents. Recently, the T. cruzi protein tyrosine phosphatase TcPTP1 has been implicated in the cellular differentiation and infectivity of the parasite and is therefore a promising target for the design of novel anti-parasitic drugs. Here, we report the X-ray crystal structure of TcPTP1 refined to a resolution of 2.18 Å, which provides structural insights into the active site environment that can be used to initiate structure-based drug design efforts to develop specific TcPTP1 inhibitors. Potential strategies to develop such inhibitors are also discussed.

  7. Sustained High Protein-tyrosine Phosphatase 1B Activity in the Sperm of Obese Males Impairs the Sperm Acrosome Reaction*

    PubMed Central

    Shi, Lei; Zhang, Qipeng; Xu, Binqiang; Jiang, Xiaohong; Dai, Yutian; Zhang, Chen-Yu; Zen, Ke

    2014-01-01

    Evidence of a causal link between male obesity and subfertility or infertility has been demonstrated previously. However, the mechanism underlying this link is incompletely understood. Here, we report that sustained high protein-tyrosine phosphatase 1B (PTP1B) activity in sperm of obese donors plays an essential role in coupling male obesity and subfertility or infertility. First, PTP1B level and activity were significantly higher in sperm from ob/ob mice than in wild-type littermates. High PTP1B level and activity in sperm was also observed in obese patients compared with non-obese donors. The enhanced sperm PTP1B level and activity in ob/ob mice and obese patients correlated with a defect of the sperm acrosome reaction (AR). Second, treating sperm from male ob/ob mice or obese men with a specific PTP1B inhibitor largely restored the sperm AR. Finally, blockade of sperm AR by enhanced PTP1B activity in male ob/ob mice or obese men was due to prolonged dephosphorylation of N-ethylmaleimide-sensitive factor by PTP1B, leading to the inability to reassemble the trans-SNARE complexes, which is a critical step in sperm acrosomal exocytosis. In summary, our study demonstrates for the first time that a sustained high PTP1B level or activity in the sperm of obese donors causes a defect of sperm AR and that PTP1B is a novel potential therapeutic target for male infertility treatment. PMID:24519936

  8. Identification of Phospholipase C gamma1 as a Protein Tyrosine Phosphatase mu Substrate that Regulates Cell Migration

    PubMed Central

    Phillips-Mason, Polly J.; Kaur, Harpreet; Burden-Gulley, Susan M.; Craig, Sonya E.L.; Brady-Kalnay, Susann M.

    2010-01-01

    The receptor protein tyrosine phosphatase PTPmu has a cell-adhesion molecule-like extracellular segment and a catalytically active intracellular segment. This structure gives PTPmu the ability to transduce signals in response to cell-cell adhesion. Full-length PTPmu is down-regulated in glioma cells by proteolysis which is linked to increased migration of these cells in the brain. To gain insight into the substrates PTPmu may be dephosphorylating to suppress glioma cell migration, we used a substrate trapping method to identify PTPmu substrates in tumor cell lines. We identified both PKCdelta and PLCgamma1 as PTPmu substrates. As PLCgamma1 activation is linked to increased invasion of cancer cells, we set out to determine whether PTPmu may be upstream of PLCgamma1 in regulating glioma cell migration. We conducted brain slice assays using U87-MG human glioma cells in which PTPmu expression was reduced by shRNA to induce migration. Treatment of the same cells with PTPmu shRNA and a PLCgamma1 inhibitor prevented migration of the cells within the brain slice. These data suggest that PLCgamma1 is downstream of PTPmu and that dephosphorylation of PLCgamma1 is likely to be a major pathway through which PTPmu suppresses glioma cell migration. PMID:20506511

  9. A complex between contactin-1 and the protein tyrosine phosphatase PTPRZ controls the development of oligodendrocyte precursor cells

    SciTech Connect

    Lamprianou, Smaragda; Chatzopoulou, Elli; Thomas, Jean-Léon; Bouyain, Samuel; Harroch, Sheila

    2013-09-23

    The six members of the contactin (CNTN) family of neural cell adhesion molecules are involved in the formation and maintenance of the central nervous system (CNS) and have been linked to mental retardation and neuropsychiatric disorders such as autism. Five of the six CNTNs bind to the homologous receptor protein tyrosine phosphatases gamma (PTPRG) and zeta (PTPRZ), but the biological roles of these interactions remain unclear. We report here the cocrystal structure of the carbonic anhydrase-like domain of PTPRZ bound to tandem Ig repeats of CNTN1 and combine these structural data with binding assays to show that PTPRZ binds specifically to CNTN1 expressed at the surface of oligodendrocyte precursor cells. Furthermore, analyses of glial cell populations in wild-type and PTPRZ-deficient mice show that the binding of PTPRZ to CNTN1 expressed at the surface of oligodendrocyte precursor cells inhibits their proliferation and promotes their development into mature oligodendrocytes. Overall, these results implicate the PTPRZ/CNTN1 complex as a previously unknown modulator of oligodendrogenesis.

  10. Protein tyrosine phosphatase PTPN3 promotes drug resistance and stem cell-like characteristics in ovarian cancer

    PubMed Central

    Li, Shuqin; Cao, Jian; Zhang, Wei; Zhang, Fan; Ni, Guantai; Luo, Qian; Wang, Man; Tao, Xiang; Xia, Hongping

    2016-01-01

    The current standard treatment for ovarian cancer is aggressive surgery followed by platinum-based combination chemotherapy. Recurrence and chemotherapeutic drug resistance are the two main factors that account for the high mortality of most ovarian cancers. Liposomal doxorubicin is primarily used for the treatment of ovarian cancer when the disease has progressed after platinum-based chemotherapy. However, relatively little is known about the genomic changes that contribute to both cisplatin and doxorubicin resistance in high-grade serous ovarian cancer (HGSC) under the selective pressure of chemotherapy. Here, we found that protein tyrosine phosphatase PTPN3 gene expression was substantially increased in both cisplatin and doxorubicin-resistant ovarian cancer cells. Silencing of PTPN3 restored sensitivity to cisplatin and doxorubicin in resistant ovarian cancer cells. Down-regulation of PTPN3 also inhibited cell cycle progression, migration, stemness in vitro and the tumorigenicity of resistant ovarian cancer cells in vivo. Meanwhile, the expression of PTPN3 was found to be regulated by miR-199 in resistant ovarian cancer cells. These findings suggest that PTPN3 promotes tumorigenicity, stemness and drug resistance in ovarian cancer, and thus is a potential therapeutic target for the treatment of ovarian cancer. PMID:27833130

  11. Kinetics, substrate specificity, and stereospecificity of two new protein tyrosine phosphatase-like inositol polyphosphatases from Selenomonas lacticifex.

    PubMed

    Puhl, Aaron A; Greiner, Ralf; Selinger, L Brent

    2008-08-01

    Inositol polyphosphatases (IPPases) play an important role in the metabolism of inositol polyphosphates, a class of molecules involved in signal transduction. Here we characterize 2 new protein tyrosine phosphatase-like IPPases (PhyAsl and PhyBsl) cloned from Selenomonas lacticifex that can hydrolyze myo-inositol hexakisphosphate (InsP6) in vitro. To determine their preferred substrates and stereospecificity of InsP6 dephosphorylation, a combination of kinetic and high-performance ion pair chromatography studies were conducted. Despite only 33% amino acid sequence identity between them, both enzymes display strict specificity for IPP substrates and cleave InsP6 primarily at the D-3-phosphate position (>90%). Furthermore, both enzymes predominantly degrade InsP6 to Ins(2)P via identical and very specific routes of dephosphorylation (3,4,5,6,1). Despite these similarities, PhylAsl is shown to have a slight kinetic preference for the major inositol pentakisphosphate intermediate in its InsP6 hydrolysis pathway, whereas PhyBsl displays a unique and substantial preference for an inositol tetrakisphosphate intermediate.

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

    PubMed

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

    2012-05-01

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

  13. Dynamic active-site protection by the M. tuberculosis protein tyrosine phosphatase PtpB lid domain.

    PubMed

    Flynn, E Megan; Hanson, Jeffrey A; Alber, Tom; Yang, Haw

    2010-04-07

    The Mycobacterium tuberculosis protein tyrosine phosphatase PtpB shows resistance to the oxidative conditions that prevail within an infected host macrophage, but the mechanism of this molecular adaptation is unknown. Crystal structures of PtpB revealed previously that a closed, two-helix lid covers the active site. By measuring single-molecule Forster-type resonance energy transfer to probe the dynamics of two helices that constitute the lid, we obtained direct evidence for large, spontaneous opening transitions of PtpB with the closed form of both helices favored approximately 3:1. Despite similar populations of conformers, the two helices move asynchronously as demonstrated by different opening and closing rates under our experimental conditions. Assuming that lid closure excludes oxidant, the rates of opening and closing quantitatively accounted for the slow observed rate of oxidative inactivation. Increasing solvent viscosity using glycerol but not PEG8000 resulted in higher rates of oxidative inactivation due to an increase in the population of open conformers. These results establish that the rapid conformational gating of the PtpB lid constitutes a reversible physical blockade that transiently masks the active site and retards oxidative inactivation.

  14. Isopetrosynol, a New Protein Tyrosine Phosphatase 1B Inhibitor, from the Marine Sponge Halichondria cf. panicea Collected at Iriomote Island.

    PubMed

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

    2016-01-01

    A new polyacetylene compound, isopetrosynol (1), was isolated from the Okinawan marine sponge Halichondria cf. panicea together with petrosynol (2), adociacetylene D (3), (5R)-3,15,27-triacontatriene-1,29-diyn-5-ol (4), and petrosterol (5). The structure of 1 was assigned on the basis of spectroscopic data for 1 and 2. Compound 1 inhibited protein tyrosine phosphatase 1B (PTP1B) activity with an IC50 value of 8.2±0.3 µM, while compound 2, a diastereomer of 1, showed only 28.9±4.5% inhibition at 21.6 µM. The IC50 values of compounds 3 and 4 were 7.8±0.5 and 12.2±0.5 µM, respectively. Oleanolic acid, a positive control, inhibited PTP1B activity at 0.7±0.1 µM (IC50) in the same experiment. The inhibitory activity of 1 was stronger than that of its diastereomer (2). This is the first study to show the inhibitory effects of polyacetylene compounds on PTP1B.

  15. Hepatic oxidative stress promotes insulin-STAT-5 signaling and obesity by inactivating protein tyrosine phosphatase N2

    PubMed Central

    Gurzov, Esteban N.; Tran, Melanie; Fernandez-Rojo, Manuel A; Merry, Troy L; Zhang, Xinmei; Xu, Yang; Fukushima, Atsushi; Waters, Michael J; Watt, Matthew J; Andrikopoulos, Sofianos; Neel, Benjamin G; Tiganis, Tony

    2015-01-01

    Hepatic insulin resistance is a key contributor to the pathogenesis of obesity and type 2 diabetes (T2D). Paradoxically the development of insulin resistance in the liver is not universal, but pathway-selective, such that insulin fails to suppress gluconeogenesis but promotes lipogenesis, contributing to the hyperglycemia, steatosis and hypertriglyceridemia that underpin the deteriorating glucose control and microvascular complications in T2D. The molecular basis for the pathway-specific insulin resistance remains unknown. Here we report that oxidative stress accompanying obesity inactivates protein-tyrosine phosphatases (PTPs) in the liver, which activates select signaling pathways that exacerbate disease progression. In obese mice, hepatic PTPN2 (TCPTP) inactivation promoted lipogenesis and steatosis and insulin-STAT-5 signaling. The enhanced STAT-5 signaling increased hepatic IGF-1 production, which suppressed central growth hormone release and exacerbated the development of obesity and T2D. Our studies define a mechanism for the development of selective insulin resistance with wide-ranging implications for diseases characterised by oxidative stress. PMID:24954415

  16. New approach for local structure analysis of the tyrosine domain in proteins by using a site-specific and polarity-sensitive fluorescent probe.

    PubMed

    Chen, Suming; Li, Xiaohua; Ma, Huimin

    2009-05-04

    Designer label: A newly developed polarity-sensitive fluorescent probe (DBHA) was combined with a tyrosine-specific labelling method that uses transition metal catalysis, and was successfully used in local structural analysis of the Tyr108 domain in Cu/Zn superoxide dismutase (SOD; see scheme). The strategy presented here provides a new approach for studying the local polarity and conformation changes of this tyrosine domain in SOD under acid or heat denaturation conditions. The design and synthesis of a novel long-wavelength polarity-sensitive fluorescence probe, 6-[9-(diethylamino)-5-oxo-5 H-benzo[alpha]phenoxazin-2-yloxy]hex-2-enyl acetate, for the selective modification of tyrosine residues with the goal of providing local information on tyrosine domains in proteins, is reported. This probe comprises a polarity-sensitive Nile red fluorophore and an active pi-allyl group that can form pi-allylpalladium complexes and react selectively with tyrosine residues. The probe has the following features: 1) it has a long-wavelength emission of >550 nm, thanks to which interference from short-wavelength fluorescence from common biological matrixes can be avoided; 2) the maximum emission wavelength is sensitive only to polarity and not to pH or temperature; this allows the accurate determination of local polarity; and 3) it is a neutral, uncharged molecule, and does not disturb the overall charge of the labelled protein. With this probe the polarity and conformation changes of the Tyr108 domain in native and in acid- and heat-denatured bovine Cu/Zn superoxide dismutase were detected for the first time. It was found that the polarity of the Tyr108 domain hardly alters on acid denaturation between pH 4 and 9. However, heat denaturation caused the Tyr108 domain to be more hydrophobic, and was accompanied by an irreversible aggregation of the protein. In addition, the probe-binding experiments revealed that the surface of the protein becomes more hydrophobic after thermal

  17. Immunohistochemical and Western blot analysis of two protein tyrosine phosphatase receptors, R and Z1, in colorectal carcinoma, colon adenoma and normal colon tissues.

    PubMed

    Woźniak, Marta; Gamian, Elżbieta; Łaczmańska, Izabela; Sąsiadek, Maria M; Duś-Szachniewicz, Kamila; Ziółkowski, Piotr

    2014-05-01

    Two classes of proteins, namely tyrosine kinases (PTK) and phosphatases (PTP), play an important role in cell proliferation and differentiation, thus leading to an acceleration or inhibition of tumour growth. The role of the above proteins in colorectal carcinoma (CRC) growth is a well-known event. In this study we carried out immunohistochemical and Western blot analysis of colorectal carcinoma, adenoma and normal colon tissue in relation to two protein tyrosine phosphatase receptors, R and Z1. Twenty-five cases of CRC were analyzed and the results were compared with similar data obtained in non-malignant tissues. High expression of both PTP receptors was observed in all examined cases of CRC, adenoma and normal colon tissue in this study. These results are not in line with recently published data, showing that genetic coding for PTPRR and PTPRZ1 were hypermethylated in CRC's. We presume that the protein tyrosine phosphatase overexpression in colorectal carcinoma is not enough to protect from the progression of disease.

  18. Examination of the transition state of the low-molecular mass small tyrosine phosphatase 1. Comparisons with other protein phosphatases.

    PubMed

    Hengge, A C; Zhao, Y; Wu, L; Zhang, Z Y

    1997-06-24

    The reactions of p-nitrophenyl phosphate (pNPP) with the low-molecular mass tyrosine phosphatase Stp1 and with the mutants D128N, D128A, D128E, and S18A have been studied by measurement of heavy-atom isotope effects in the substrate. The isotope effects were measured at the nonbridging oxygen atoms [18(V/K)nonbridge], at the bridging oxygen atom (the site of bond cleavage) [18(V/K)bridge], and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The results with native Stp1 were 1.0160 +/- 0.0005 for 18(V/K)bridge, 1.0007 +/- 0.0001 for 15(V/K), and 1.0018 +/- 0.0003 for 18(V/K)nonbridge. The values for 18(V/K)nonbridge and 15(V/K) differ from those previously measured with other protein-tyrosine phosphatases and from those of the aqueous hydrolysis reaction of pNPP. The values indicate that in the transition state of the native Stp1 reaction the leaving group bears a partial negative charge, and there is nucleophilic interaction between the Cys nucleophile, and the phosphoryl group, causing some decrease in the nonbridge P-O bond order. The transition state remains highly dissociative with respect to the degree of bond cleavage to the leaving group. Mutation of the general acid from aspartic acid to glutamic acid slows catalysis but causes no change in the isotope effects and thus does not alter the degree of proton transfer to the leaving group in the transition state. Mutations of this residue to asparagine or alanine give values for 18(V/K)bridge of about 1.029, for 15(V/K) of about 1.003, and for 18(V/K)nonbridge of 1.0010 (D128A) to 1.0024 (D128N). These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion and indicate more nucleophilic participation than in the aqueous hydrolysis of the pNPP dianion, just as in the native enzyme. The isotope effects with the S18A mutant, in which a hydrogen bonding stabilization of the anionic Cys nucleophile has been removed, were within experimental error of

  19. Mechanistic studies of protein tyrosine phosphatases YopH and Cdc25A with m-nitrobenzyl phosphate.

    PubMed

    McCain, Daniel F; Grzyska, Piotr K; Wu, Li; Hengge, Alvan C; Zhang, Zhong-Yin

    2004-06-29

    Protein tyrosine phosphatases (PTPs) constitute a large family of signaling enzymes that include both tyrosine specific and dual-specificity phosphatases that hydrolyze pSer/Thr in addition to pTyr. Previous mechanistic studies of PTPs have relied on the highly activated substrate p-nitrophenyl phosphate (pNPP), an aryl phosphate with a leaving group pK(a) of 7. In the study presented here, we employ m-nitrobenzyl phosphate (mNBP), an alkyl phosphate with a leaving group pK(a) of 14.9, which mimics the physiological substrates of the PTPs. We have carried out pH dependence and kinetic isotope effect measurements to characterize the mechanism of two important members of the PTP superfamily: Yersinia PTP (YopH) and Cdc25A. Both YopH and Cdc25A exhibit bell-shaped pH-rate profiles for the hydrolysis of mNBP, consistent with general acid catalysis. The slightly inverse (18)(V/K)(nonbridge) isotope effects (0.9999 for YopH and 0.9983 for Cdc25A) indicate a loose transition state with little nucleophilic participation for both enzymes. The smaller (18)(V/K)(bridge) primary isotope effects (0.9995 for YopH and 1.0012 for Cdc25A) relative to the corresponding isotope effects for pNPP hydrolysis suggest that protonation of the leaving group oxygen at the transition state by the general acid is ahead of P-O bond fission with the alkyl substrate, while general acid catalysis of pNPP by YopH is more synchronous with P-O bond fission. The isotope effect data also confirm findings from previous studies that Cdc25A utilizes general acid catalysis for substrates with a leaving group pK(a) of >8, but not for pNPP. Interestingly, the difference in the kinetic isotope effects for the reactions of aryl phosphate pNPP and alkyl phosphate mNBP by the PTPs parallels what is observed in the uncatalyzed reactions of their monoanions. In these reactions, the leaving group is protonated in the transition state, as is the case in PTP-catalyzed reactions. Also, the phosphoryl group in the

  20. The activation of the neutrophil respiratory burst by anti-neutrophil cytoplasm autoantibody (ANCA) from patients with systemic vasculitis requires tyrosine kinases and protein kinase C activation

    PubMed Central

    Radford, D J; Lord, J M; Savage, C O S

    1999-01-01

    The ability of antineutrophil cytoplasm autoantibodies (ANCA) from patients with systemic vasculitis to stimulate protein kinase C (PKC) and tyrosine kinases was examined in human neutrophils. Using the superoxide dismutase-inhibitable reduction of ferricytochrome C, the kinetics of ANCA-induced superoxide (O2−) production were characterized and subsequently manipulated by specific inhibitors of PKC and tyrosine kinases. With this approach, ANCA IgG, but not normal IgG or ANCA F(ab′)2 fragments caused a time and dose dependent release of O2− from TNF-α primed neutrophils. The kinetics of ANCA-induced O2− production showed an initial 10–15 min lag phase compared to the N-formyl-l-methionyl-l-leucyl-l-phenylalanine response, suggesting differences in the signalling pathways recruited by these two stimuli. Inhibitor studies revealed that ANCA-activation involved members of both the Ca2+-dependent and -independent PKC isoforms and also tyrosine kinases. ANCA IgG resulted in the translocation of the βII isoform of PKC at a time corresponding to the end of the lag phase of O2− production, suggesting that PKC activity may be instrumental in processes regulating the activity of the NADPH oxidase in response to ANCA. Tyrosine phosphorylation of numerous proteins also peaked 10–15 min after stimulation with ANCA but not normal IgG. These data suggest that PKC and tyrosine kinases regulate O2− production from neutrophils stimulated with autoantibodies from patients with systemic vasculitis. PMID:10540175

  1. Dissecting the Roles of Tyrosines 490 and 785 of TrkA Protein in the Induction of Downstream Protein Phosphorylation Using Chimeric Receptors*

    PubMed Central

    Biarc, Jordane; Chalkley, Robert J.; Burlingame, A. L.; Bradshaw, Ralph A.

    2013-01-01

    Receptor tyrosine kinases generally act by forming phosphotyrosine-docking sites on their own endodomains that propagate signals through cascades of post-translational modifications driven by the binding of adaptor/effector proteins. The pathways that are stimulated in any given receptor tyrosine kinase are a function of the initial docking sites that are activated and the availability of downstream participants. In the case of the Trk receptors, which are activated by nerve growth factor, there are only two established phosphotyrosine-docking sites (Tyr-490 and Tyr-785 on TrkA) that are known to be directly involved in signal transduction. Taking advantage of this limited repertoire of docking sites and the availability of PC12 cell lines stably transfected with chimeric receptors composed of the extracellular domain of the PDGF receptor and the transmembrane and intracellular domains of TrkA, the downstream TrkA-induced phosphoproteome was assessed for the “native” receptor and mutants lacking Tyr-490 or both Tyr-490 and Tyr-785. Basal phosphorylation levels were compared with those formed after 20 min of stimulation with PDGF. Several thousand phosphopeptides were identified after TiO2 enrichment, and many were up- or down-regulated by receptor activation. The modified proteins in the native sample contained many of the well established participants in TrkA signaling. The results from the mutant receptors allowed grouping of these downstream targets by their dependence on the two characterized docking site(s). A clear subset that was not dependent on either Tyr-490 or Tyr-785 emerged, providing direct evidence that there are other sites on TrkA that are involved in downstream signaling. PMID:23589303

  2. Involvement of protein kinase C, phospholipase C, and protein tyrosine kinase pathways in oxygen radical generation by asbestos-stimulated alveolar macrophage.

    PubMed

    Lim, Y; Kim, S H; Kim, K A; Oh, M W; Lee, K H

    1997-09-01

    Although asbestos stimulates oxygen radical generation in alveolar macrophages, the exact mechanism is still not clear. The purpose of this study was to compare the ability of three asbestos fibers (amosite, chrysotile, and crocidolite) to generate oxygen radicals in macrophages and examine the mechanism of this action. All asbestos fibers were able to induce chemiluminescence but chrysotile induced maximal chemiluminescence at higher concentrations than amosite and crocidolite. Protein kinase C (PKC) inhibitors (sphingosine and staurosporine) suppressed the ability of asbestos to induce oxygen radical generation. Phospholipase C (PLC) inhibitors (U73122 and neomycin) and protein tyrosine kinase (PTK) inhibitors (erbstatin and genistein) decreased oxygen radical generation of asbestos-stimulated alveolar macrophages. Oxygen radical generation was not suppressed by an adenylate cyclase activator (forskolin), a protein kinase A inhibitor (H-8), and a protein serine-threonine phosphatase inhibitor (okadaic acid). PLC and PTK inhibitors suppressed the increment of phosphoinositide turnover by amosite. These results suggest that asbestos fibers induce the generation of oxygen radicals through PTK, PLC, and PKC pathways in a dose-response pattern.

  3. Resveratrol inhibits collagen-induced platelet stimulation through suppressing NADPH oxidase and oxidative inactivation of SH2 domain-containing protein tyrosine phosphatase-2.

    PubMed

    Jang, Ji Yong; Min, Ji Hyun; Wang, Su Bin; Chae, Yun Hee; Baek, Jin Young; Kim, Myunghee; Ryu, Jae-Sang; Chang, Tong-Shin

    2015-12-01

    Reactive oxygen species (ROS) produced upon collagen stimulation are implicated in propagating various platelet-activating pathways. Among ROS-producing enzymes, NADPH oxidase (NOX) is largely responsible for collagen receptor-dependent ROS production. Therefore, NOX has been proposed as a novel target for the development of antiplatelet agent. We here investigate whether resveratrol inhibits collagen-induced NOX activation and further examine the effects of resveratrol on ROS-dependent signaling pathways in collagen-stimulated platelets. Collagen-induced superoxide anion production in platelets was inhibited by resveratrol. Resveratrol suppressed collagen-induced phosphorylation of p47(phox), a major regulatory subunit of NOX. Correlated with the inhibitory effects on NOX, resveratrol protected SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) from ROS-mediated inactivation and subsequently attenuated the specific tyrosine phosphorylation of key components (spleen tyrosine kinase, Vav1, Bruton's tyrosine kinase, and phospholipase Cγ2) for collagen receptor signaling cascades. Resveratrol also inhibited downstream responses such as cytosolic calcium elevation, P-selectin surface exposure, and integrin-αIIbβ3 activation. Furthermore, resveratrol inhibited platelet aggregation and adhesion in response to collagen. The antiplatelet effects of resveratrol through the inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2 suggest that resveratrol is a potential compound for prevention and treatment of thrombovascular diseases.

  4. Oncogenic function and prognostic significance of protein tyrosine phosphatase PRL-1 in hepatocellular carcinoma

    PubMed Central

    Jin, Shaowen; Wang, Kaimei; Xu, Kang; Xu, Junyao; Sun, Jian; Chu, Zhonghua; Lin, Dechen; Koeffler, Phillip H.; Wang, Jie; Yin, Dong

    2014-01-01

    Our SNP-Chip data demonstrated 7/60 (12%) hepatocellular carcinoma (HCC) patients had PRL-1 copy number amplification. However, its biological functions and signaling pathways in HCC are deficient. Here, we investigated its oncogenic function and prognostic significance in HCC. PRL-1 protein levels were examined in 167 HCC samples by immunohistochemisty (IHC). The relationship of PRL-1 expression and clinicopathological features was assessed by correlation, Kaplan-Meier and Cox regression analyses. The oncogenic function of PRL-1 in HCC cells and its underlying mechanism were investigated by ectopic overexpression and knockdown model. PRL-1 levels in primary HCC and metastatic intravascular cancer thrombus were also determined by IHC. PRL-1 levels were frequently elevated in HCC tissues (81%), and elevated expression of PRL-1 was significantly associated with more aggressive phenotype and poorer prognosis in HCC patients (p<0.05). Ectopic overexpression of PRL-1 markedly enhanced HCC cells migration and invasion. Furthermore, the oncogenic functions of PRL-1 were mediated by PI3K/AKT/GSK3β signaling pathway through inhibiting E-cadherin expression. Finally, PRL-1 protein levels in metastatic cancer thrombus were higher than that in primary HCC tissues (p<0.05). These data highlight the oncogenic function of PRL-1 in HCC invasion and metastasis implicating PRL-1 as a potential prognostic marker as well as therapeutic target in HCC. PMID:25003523

  5. Oncogenic function and prognostic significance of protein tyrosine phosphatase PRL-1 in hepatocellular carcinoma.

    PubMed

    Jin, Shaowen; Wang, Kaimei; Xu, Kang; Xu, Junyao; Sun, Jian; Chu, Zhonghua; Lin, Dechen; Koeffler, Phillip H; Wang, Jie; Yin, Dong

    2014-06-15

    Our SNP-Chip data demonstrated 7/60 (12%) hepatocellular carcinoma (HCC) patients had PRL-1 copy number amplification. However, its biological functions and signaling pathways in HCC are deficient. Here, we investigated its oncogenic function and prognostic significance in HCC. PRL-1 protein levels were examined in 167 HCC samples by immunohistochemisty (IHC). The relationship of PRL-1 expression and clinicopathological features was assessed by correlation, Kaplan-Meier and Cox regression analyses. The oncogenic function of PRL-1 in HCC cells and its underlying mechanism were investigated by ectopic overexpression and knockdown model. PRL-1 levels in primary HCC and metastatic intravascular cancer thrombus were also determined by IHC. PRL-1 levels were frequently elevated in HCC tissues (81%), and elevated expression of PRL-1 was significantly associated with more aggressive phenotype and poorer prognosis in HCC patients (p<0.05). Ectopic overexpression of PRL-1 markedly enhanced HCC cells migration and invasion. Furthermore, the oncogenic functions of PRL-1 were mediated by PI3K/AKT/GSK3β signaling pathway through inhibiting E-cadherin expression. Finally, PRL-1 protein levels in metastatic cancer thrombus were higher than that in primary HCC tissues (p<0.05). These data highlight the oncogenic function of PRL-1 in HCC invasion and metastasis implicating PRL-1 as a potential prognostic marker as well as therapeutic target in HCC.

  6. Protein tyrosine phosphatase 1B is a key regulator of IFNAR1 endocytosis and a target for antiviral therapies

    PubMed Central

    Carbone, Christopher J.; Zheng, Hui; Bhattacharya, Sabyasachi; Lewis, John R.; Reiter, Alexander M.; Henthorn, Paula; Zhang, Zhong-Yin; Baker, Darren P.; Ukkiramapandian, Radha; Bence, Kendra K.; Fuchs, Serge Y.

    2012-01-01

    Type 1 interferons (IFN1) elicit antiviral defenses by activating the cognate receptor composed of IFN-α/β receptor chain 1 (IFNAR1) and IFNAR2. Down-regulation of this receptor occurs through IFN1-stimulated IFNAR1 ubiquitination, which exposes a Y466-based linear endocytic motif within IFNAR1 to recruitment of the adaptin protein-2 complex (AP2) and ensuing receptor endocytosis. Paradoxically, IFN1-induced Janus kinase-mediated phosphorylation of Y466 is expected to decrease its affinity for AP2 and to inhibit the endocytic rate. To explain how IFN1 promotes Y466 phosphorylation yet stimulates IFNAR1 internalization, we proposed that the activity of a protein tyrosine phosphatase (PTP) is required to enable both events by dephosphorylating Y466. An RNAi-based screen identified PTP1B as a specific regulator of IFNAR1 endocytosis stimulated by IFN1, but not by ligand-independent inducers of IFNAR1 ubiquitination. PTP1B is a promising target for treatment of obesity and diabetes; numerous research programs are aimed at identification and characterization of clinically relevant inhibitors of PTP1B. PTP1B is capable of binding and dephosphorylating IFNAR1. Genetic or pharmacologic modulation of PTP1B activity regulated IFN1 signaling in a manner dependent on the integrity of Y466 within IFNAR1 in human cells. These effects were less evident in mouse cells whose IFNAR1 lacks an analogous motif. PTP1B inhibitors robustly augmented the antiviral effects of IFN1 against vesicular stomatitis and hepatitis C viruses in human cells and proved beneficial in feline stomatitis patients. The clinical significance of these findings in the context of using PTP1B inhibitors to increase the therapeutic efficacy of IFN against viral infections is discussed. PMID:23129613

  7. Stereospecificity of myo-inositol hexakisphosphate hydrolysis by a protein tyrosine phosphatase-like inositol polyphosphatase from Megasphaera elsdenii.

    PubMed

    Puhl, Aaron A; Greiner, Ralf; Selinger, L Brent

    2009-02-01

    Inositol polyphosphatases (IPPases), particularly those that can hydrolyze myo-inositol hexakisphosphate (Ins P(6)), are of biotechnological interest for their ability to reduce the metabolically unavailable organic phosphate content of feedstuffs and to produce lower inositol polyphosphates (IPPs) for research and pharmaceutical applications. Here, the gene coding for a new protein tyrosine phosphatase (PTP)-like IPPase was cloned from Megasphaera elsdenii (phyAme), and the biochemical properties of the recombinant protein were determined. The deduced amino acid sequence of PhyAme is similar to known PTP-like IPPases (29-44% identity), and the recombinant enzyme displayed strict specificity for IPP substrates. Optimal IPPase activity was displayed at an ionic strength of 250 mM, a pH of 5.0, and a temperature of 60 degrees C. In order to elucidate its stereospecificity of Ins P(6) dephosphorylation, a combination of high-performance ion-pair chromatography and kinetic studies was conducted. PhyAme displayed a stereospecificity that is unique among enzymes belonging to this class in that it preferentially cleaved Ins P(6) at one of two phosphate positions, 1D-3 or 1D-4. PhyAme followed two distinct and specific routes of hydrolysis, predominantly degrading Ins P(6) to Ins(2)P via: (a) 1D-Ins(1,2,4,5,6)P(5), 1D-Ins(1,2,5,6)P(4), 1D-Ins(1,2,6)P(3), and 1D-Ins(1,2)P(2) (60%) and (b) 1D-Ins(1,2,3,5,6)P(5), 1D-Ins(1,2,3,6)P(4), Ins(1,2,3)P(3), and D/L-Ins(1,2)P(2)(35%).

  8. Protein tyrosine phosphatases PTP-1B, SHP-2, and PTEN facilitate Rb/E2F-associated apoptotic signaling.

    PubMed

    Morales, Liza D; Casillas Pavón, Edgar A; Shin, Jun Wan; Garcia, Alexander; Capetillo, Mario; Kim, Dae Joon; Lieman, Jonathan H

    2014-01-01

    To maintain tissue homeostasis, apoptosis is functionally linked to the cell cycle through the retinoblastoma (Rb)/E2F pathway. When the Rb tumor suppressor protein is functionally inactivated, E2F1 elicits an apoptotic response through both intrinsic (caspase-9 mediated) and extrinsic (caspase-8 mediated) apoptotic pathways in order to eliminate hyperproliferative cells. Rb/E2F-associated apoptosis has been demonstrated to be associated with the loss of constitutive transcriptional repression by Rb/E2F complexes and mediated by caspase-8. Protein tyrosine phosphatases (PTPs) PTP-1B and SHP-2 have been previously shown to be directly activated by loss of Rb/E2F repression during Rb/E2F-associated apoptosis. In this current study, we demonstrate that the PTEN tumor suppressor is also directly activated by loss of Rb/E2F repression. We also demonstrate that PTP-1B, SHP-2, and PTEN play a functional role in Rb/E2F-associated apoptosis. Knockdown of PTP1B, SHP2, or PTEN expression with small interfering RNA (siRNA) in apoptotic cells increases cell viability and rescues cells from the Rb/E2F-associated apoptotic response. Furthermore, rescue from apoptosis coincides with inhibition of caspase-8 and caspase-3 cleavage (activation). Our results indicate PTP-1B, SHP-2, and PTEN all play a functional role in Rb/E2F-associated apoptotic signal transduction and provide further evidence that PTP-1B, SHP-2, and PTEN can contribute to tumor suppression through an Rb/E2F-associated mechanism.

  9. Protein tyrosine phosphatase 1B targets PITX1/p120RasGAP thus showing therapeutic potential in colorectal carcinoma

    PubMed Central

    Teng, Hao-Wei; Hung, Man-Hsin; Chen, Li-Ju; Chang, Mao-Ju; Hsieh, Feng-Shu; Tsai, Ming-Hsien; Huang, Jui-Wen; Lin, Chih-Lung; Tseng, Hsiang-Wen; Kuo, Zong-Keng; Jiang, Jeng-Kai; Yang, Shung-Haur; Shiau, Chung-Wai; Chen, Kuen-Feng

    2016-01-01

    Protein tyrosine phosphatase 1B (PTP1B) is known to promote the pathogenesis of diabetes and obesity by negatively regulating insulin and leptin pathways, but its role associated with colon carcinogenesis is still under debate. In this study, we demonstrated the oncogenic role of PTP1B in promoting colon carcinogenesis and predicting worse clinical outcomes in CRC patients. By co-immunoprecipitation, we showed that PITX1 was a novel substrate of PTP1B. Through direct dephosphorylation at Y160, Y175 and Y179, PTP1B destabilized PITX1, which resulted in downregulation of the PITX1/p120RasGAP axis. Interestingly, we found that regorafenib, the approved target agent for advanced CRC patients, exerted a novel property against PTP1B. By inhibiting PTP1B activity, regorafenib treatment augmented the stability of PITX1 protein and upregulated the expression of p120RasGAP in CRC. Importantly, we found that this PTP1B-dependant PITX1/p120RasGAP axis determines the in vitro anti-CRC effects of regorafenib. The above-mentioned effects of regorafenib were confirmed by the HT-29 xenograft tumor model. In conclusion, we demonstrated a novel oncogenic mechanism of PTP1B on affecting PITX1/p120RasGAP in CRC. Regorafenib inhibited CRC survival through reserving PTP1B-dependant PITX1/p120RasGAP downregulation. PTP1B may be a potential biomarker predicting regorafenib effectiveness, and a potential solution for CRC. PMID:27752061

  10. Long-term effects of amphetamine neurotoxicity on tyrosine hydroxylase mRNA and protein in aged rats.

    PubMed

    Bowyer, J F; Frame, L T; Clausing, P; Nagamoto-Combs, K; Osterhout, C A; Sterling, C R; Tank, A W

    1998-08-01

    Four injections (intraperitoneal) of 3 mg/kg amphetamine (2 hr apart) produced pronounced hyperthermia and sustained decreases in dopamine levels and tyrosine hydroxylase (TH) protein levels in the striatum of 15-month-old male rats. A partial recovery of striatal dopamine levels was observed at 4 months after amphetamine. In contrast, TH mRNA and TH protein levels in the midbrain were unaffected at all time points tested up to 4 months after amphetamine treatment. The number of TH-immunopositive cells in the midbrain was also unchanged at 4 months after amphetamine, even though the number of TH-positive axons in the striatum remained dramatically decreased at this time point. Interestingly, TH-immunopositive cell bodies were observed 4 months after amphetamine in the lateral caudate/putamen, defined anteriorly by the genu of the corpus collosum and posteriorly by the junction of the anterior commissures; these striatal TH-positive cells were not observed in saline- or amphetamine-treated rats that did not become hyperthermic. In addition, low levels (orders of magnitude lower than that present in the midbrain) of TH mRNA were detected using reverse transcription-polymerase chain reaction in the striatum of these amphetamine-treated rats. Our results suggest that even though there is a partial recovery of striatal dopamine levels, which occurs within 4 months after amphetamine treatment, this recovery is not associated with increased TH gene expression in the midbrain. Furthermore, new TH-positive cells are generated in the striatum at this 4-month time point.

  11. Herpes Simplex Virus 1 UL37 Protein Tyrosine Residues Conserved among All Alphaherpesviruses Are Required for Interactions with Glycoprotein K, Cytoplasmic Virion Envelopment, and Infectious Virus Production

    PubMed Central

    Chouljenko, Dmitry V.; Jambunathan, Nithya; Chouljenko, Vladimir N.; Naderi, Misagh; Brylinski, Michal; Caskey, John R.

    2016-01-01

    ABSTRACT The herpes simplex virus 1 (HSV-1) UL37 protein functions in virion envelopment at trans-Golgi membranes, as well as in retrograde and anterograde transport of virion capsids. Recently, we reported that UL37 interacts with glycoprotein K (gK) and its interacting partner protein UL20 (N. Jambunathan, D. Chouljenko, P. Desai, A. S. Charles, R. Subramanian, V. N. Chouljenko, and K. G. Kousoulas, J Virol 88:5927–5935, 2014, http://dx.doi.org/10.1128/JVI.00278-14), facilitating cytoplasmic virion envelopment. Alignment of UL37 homologs encoded by alphaherpesviruses revealed the presence of highly conserved residues in the central portion of the UL37 protein. A cadre of nine UL37 site-specific mutations were produced and tested for their ability to inhibit virion envelopment and infectious virus production. Complementation analysis revealed that replacement of tyrosines 474 and 480 with alanine failed to complement the UL37-null virus, while all other mutated UL37 genes complemented the virus efficiently. The recombinant virus DC474-480 constructed with tyrosines 474, 476, 477, and 480 mutated to alanine residues produced a gK-null-like phenotype characterized by the production of very small plaques and accumulation of capsids in the cytoplasm of infected cells. Recombinant viruses having either tyrosine 476 or 477 replaced with alanine produced a wild-type phenotype. Immunoprecipitation assays revealed that replacement of all four tyrosines with alanines substantially reduced the ability of gK to interact with UL37. Alignment of HSV UL37 with the human cytomegalovirus and Epstein-Barr virus UL37 homologs revealed that Y480 was conserved only for alphaherpesviruses. Collectively, these results suggest that the UL37 conserved tyrosine 480 residue plays a crucial role in interactions with gK to facilitate cytoplasmic virion envelopment and infectious virus production. IMPORTANCE The HSV-1 UL37 protein is conserved among all herpesviruses, functions in both

  12. Analysis of the binding of the Src homology 2 domain of Csk to tyrosine-phosphorylated proteins in the suppression and mitotic activation of c-Src.

    PubMed Central

    Sabe, H; Hata, A; Okada, M; Nakagawa, H; Hanafusa, H

    1994-01-01

    Csk (C-terminal Src kinase), a protein-tyrosine kinase, bearing the Src homology 2 and 3 (SH2 and SH3) domains, has been implicated in phosphorylation of c-Src Tyr-527, resulting in suppression of c-Src kinase activity. We found that mutations in the SH2 or SH3 domain of Csk, though they did not affect its kinase activity, resulted in a loss of suppression of c-Src activity in fibroblasts. In normal fibroblasts, tyrosine-phosphorylated paxillin and focal adhesion kinase pp125FAK, which colocalize at focal adhesion plaques, were the major proteins to which the Csk SH2 domain bound. Loss of binding to these proteins by the Csk SH2 mutants correlated with loss of the activity to suppress c-Src. Consistent with this observation, the levels of tyrosine phosphorylation of paxillin and pp125FAK were greatly reduced during mitosis, whereas the kinase activity of c-Src was elevated. We suggest that the SH2 domain is required for Csk to suppress c-Src, perhaps in combination with the SH3 domain, by anchoring Csk to a particular subcellular location where c-Src may exist. Our data also indicate that a certain fraction of the Csk and Src family kinases function at the focal adhesion plaques. The activity of the c-Src kinase localized at the focal adhesion plaques appears to be regulated by cell adhesion to the extracellular matrix. Images PMID:7513429

  13. Effects of protein tyrosine kinase inhibitors on cytokine-induced adhesion molecule expression by human umbilical vein endothelial cells.

    PubMed Central

    May, M. J.; Wheeler-Jones, C. P.; Pearson, J. D.

    1996-01-01

    1. Endothelial cells can be stimulated by the pro-inflammatory cytokines interleukin (IL)-1 alpha and tumour necrosis factor (TNF) alpha to express the leukocyte adhesion molecules E-selectin, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 but the intracellular signalling mechanisms leading to this expression are incompletely understood. We have investigated the role of protein tyrosine kinases (PTK) in adhesion molecule expression by cytokine-activated human umbilical vein endothelial cells (HUVEC) using the PTK inhibitors genistein and herbimycin A, and the protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate. 2. Maximal E-selectin expression induced by incubation of HUVEC for 4 h with IL-1 alpha (100 u ml-1) and TNF alpha (100 u ml-1) was dose-dependently inhibited by genistein and herbimycin A. Although similar effects were seen on phorbol 12-myristate, 13-acetate (PMA)-induced expression, this was not due to inhibition of protein kinase C (PKC) activity as the selective inhibitors of PKC, bisindolylmaleimide (BIM), Ro31-7549 or Ro31-8220 did not affect IL-1 alpha- or TNF alpha-induced E-selectin expression at concentrations which maximally inhibited PMA-induced expression. 3. Genistein inhibited VCAM-1 expression induced by incubation of HUVEC for 24 h with TNF alpha or IL-1 alpha whereas it did not affect ICAM-1 expression induced by 24 h incubation with either of these cytokines. Herbimycin A inhibited both VCAM-1 and ICAM-1 expression induced by TNF alpha. 4. Basal expression of E-selectin, VCAM-1 and ICAM-1 was dose-dependently enhanced by sodium orthovanadate. In contrast, vanadate differentially affected TNF alpha-induced expression of these molecules with maximal E-selectin and ICAM-1 expression being slightly enhanced and VCAM-1 expression dose-dependently reduced. 5. We also studied the effects of PTK and PTP inhibitors on adhesion of the human pre-myeloid cell line U937 to TNF alpha-stimulated HUVEC

  14. Specificity in substrate binding by protein folding catalysts: tyrosine and tryptophan residues are the recognition motifs for the binding of peptides to the pancreas-specific protein disulfide isomerase PDIp.

    PubMed Central

    Ruddock, L. W.; Freedman, R. B.; Klappa, P.

    2000-01-01

    Using a cross-linking approach, we recently demonstrated that radiolabeled peptides or misfolded proteins specifically interact in vitro with two luminal proteins in crude extracts from pancreas microsomes. The proteins were the folding catalysts protein disulfide isomerase (PDI) and PDIp, a glycosylated, PDI-related protein, expressed exclusively in the pancreas. In this study, we explore the specificity of these proteins in binding peptides and related ligands and show that tyrosine and tryptophan residues in peptides are the recognition motifs for their binding by PDIp. This peptide-binding specificity may reflect the selectivity of PDIp in binding regions of unfolded polypeptide during catalysis of protein folding. PMID:10794419

  15. Tyrosine-phosphorylated Galectin-3 Protein Is Resistant to Prostate-specific Antigen (PSA) Cleavage*

    PubMed Central

    Balan, Vitaly; Nangia-Makker, Pratima; Kho, Dhong Hyo; Wang, Yi; Raz, Avraham

    2012-01-01

    Galectin-3 is a chimeric carbohydrate-binding protein, which interacts with cell surface carbohydrate-containing molecules and extracellular matrix glycoproteins and has been implicated in various biological processes such as cell growth, angiogenesis, motility, and metastasis. It is expressed in a wide range of tumor cells and is associated with tumor progression. The functions of galectin-3 are dependent on its localization and post-translational modifications such as cleavage and phosphorylation. Recently, we showed that galectin-3 Tyr-107 is phosphorylated by c-Abl; concomitantly, it was also shown that galectin-3 can be cleaved at this site by prostate-specific antigen (PSA), a chymotrypsin-like serine protease, after Tyr-107, resulting in loss of galectin-3 multivalency while preserving its carbohydrate binding activity. Galectin-3 is largely a monomer in solution but may form a homodimer by self-association through its carbohydrate recognition domain, whereas, in the presence of a ligand, galectin-3 polymerizes up to pentamers utilizing its N-terminal domain. Oligomerization is a unique feature of secreted galectin-3, which allows its function by forming ordered galectin-glycan structures, i.e. lattices, on the cell surface or through direct engagement of specific cell surface glycoconjugates by traditional ligand-receptor binding. We questioned whether Tyr-107 phosphorylation by c-Abl affects galectin-3 cleavage by PSA. The data suggest a role for galectin-3 in prostate cells associated with increased activity of c-Abl kinase and loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activity. In addition, the ratio of phosphorylated/dephosphorylated galectin-3 might be used as a complementary value to that of PSA for prognosis of prostate cancer and a novel therapeutic target for the treatment of prostate cancer. PMID:22232548

  16. Protein tyrosine phosphatase SHP2 regulates TGF-β1 production in airway epithelia and asthmatic airway remodeling in mice

    PubMed Central

    Qin, X.-J.; Zhang, G.-S.; Zhang, X.; Qiu, Z.-W.; Wang, P.-L.; Li, Y.-W.; Li, W.; Xie, Q.-M.; Ke, Y.-H.; Lee, J. J.; Shen, H.-H.

    2014-01-01

    Background Transforming growth factor (TGF)-β1 produced in airway epithelia has been suggested as a contributor to the airway remodeling observed in asthma patients. The protein tyrosine phosphatase SHP2 is a demonstrable modulator of TGF-β1 production and thus a potential regulator of airway remodeling. Objectives To define the signal event by which SHP2 regulates asthmatic responses in airway epithelial cells by using a mouse model of experimental OVA-induced airway remodeling. Methods The airways of Shp2flox/flox mice were infected with recombinant adenovirus vectors expressing a Cre recombinase–green fluorescence protein (GFP) fusion protein as part of allergen provocation studies using mice sensitized with ovalbumin (OVA) and repeatedly challenged with OVA. Several endpoint pathologies were assessed, including airway hyper-responsiveness (AHR), lung inflammatory score, peribronchial collagen deposition, and α-smooth muscle actin (SMA) hyperplasia. In vitro studies using airway epithelial cells (BEAS-2B) were used to investigate the role of SHP2 in the regulation of pulmonary remodeling events, including the expression of collagen, α-SMA, and TGF-β1. Results Chronic OVA challenges in wild-type mice resulted in airway remodeling and lung dysfunction (e.g., increased inflammatory scores, collagen deposition (fibrosis), smooth muscle hyperplasia, and a significant increase in AHR). These endpoint pathology metrics were each significantly attenuated by conditional shp2 gene knockdown in airway epithelia. In vitro studies using BEAS-2B cells also demonstrated that the level of TGF-β1 production by these cells correlated with the extent of shp2 gene expression. Conclusions SHP2 activities in airway epithelial cells appear to modulate TGF-β1 production and, in turn, regulate allergic airway remodeling following allergen provocation. Clinical Implications Our findings identify SHP2 as a previously underappreciated contributor to the airway remodeling and lung

  17. Midgut-enriched receptor protein tyrosine phosphatase PTP52F is required for Drosophila development during larva-pupa transition.

    PubMed

    Santhanam, Abirami; Liang, Suh-Yuen; Chen, Dong-Yuan; Chen, Guang-Chao; Meng, Tzu-Ching

    2013-01-01

    To date our understanding of Drosophila receptor protein tyrosine phosphatases (R-PTPs) in the regulation of signal transduction is limited. Of the seven R-PTPs identified in flies, six are involved in the axon guidance that occurs during embryogenesis. However, whether and how R-PTPs may control key steps of Drosophila development is not clear. In this study we investigated the potential role of Drosophila R-PTPs in developmental processes outside the neuronal system and beyond the embryogenesis stage. Through systematic data mining of available microarray databases, we found the mRNA level of PTP52F to be highly enriched in the midgut of flies at the larva-pupa transition. This finding was confirmed by gut tissue staining with a specific antibody. The unique spatiotemporal expression of PTP52F suggests that it is possibly involved in regulating metamorphosis during the transformation from larva to pupa. To test this hypothesis, we employed RNA interference to examine the defects of transgenic flies. We found that ablation of endogenous PTP52F led to high lethality characterized by the pharate adult phenotype, occurring due to post pupal eclosion failure. These results show that PTP52F plays an indispensable role during the larva-pupa transition. We also found that PTP52F could be reclassified as a member of the subtype R3 PTPs instead of as an unclassified R-PTP without a human ortholog, as suggested previously. Together, these findings suggest that Drosophila R-PTPs may control metamorphosis and other biological processes beyond our current knowledge.

  18. Competitive protein tyrosine phosphatase 1B (PTP1B) inhibitors, prenylated caged xanthones from Garcinia hanburyi and their inhibitory mechanism.

    PubMed

    Tan, Xue Fei; Uddin, Zia; Park, Chanin; Song, Yeong Hun; Son, Minky; Lee, Keun Woo; Park, Ki Hun

    2017-04-15

    Protein tyrosine phosphatase 1B (PTP1B) plays important role in diabetes, obesity and cancer. The methanol extract of the gum resin of Garcinia hanburyi (G. hanburyi) showed potent PTP1B inhibition at 10µg/ml. The active compounds were identified as prenylated caged xanthones (1-9) which inhibited PTP1B in dose-dependent manner. Carboxybutenyl group within caged motif (A ring) was found to play a critical role in enzyme inhibition such as 1-6 (IC50s=0.47-4.69µM), whereas compounds having hydroxymethylbutenyl 7 (IC50=70.25µM) and methylbutenyl 8 (IC50>200µM) showed less activity. The most potent inhibitor, gambogic acid 1 (IC50=0.47µM) showed 30-fold more potency than ursolic acid (IC50=15.5µM), a positive control. In kinetic study, all isolated xanthones behaved as competitive inhibitors which were fully demonstrated with Km, Vmax and Kik/Kiv ratio. It was also proved that inhibitor 1 operated under the enzyme isomerization model having k5=0.0751µM(-)(1)S(-)(1), k6=0.0249µM(-)(1)S(-)(1) and Ki(app)=0.499µM. To develop a pharmacophore model, we explored the binding sites of compound 1 and 7 in PTP1B. These modeling results were in agreement with our findings, which revealed that the inhibitory activities are tightly related to caged motif and prenyl group in A ring.

  19. Ultrasensitive electrochemical detection of protein tyrosine kinase-7 by gold nanoparticles and methylene blue assisted signal amplification.

    PubMed

    Miao, Xiangmin; Li, Zongbing; Zhu, Aihua; Feng, Zhaozhong; Tian, Jun; Peng, Xue

    2016-09-15

    We present here an ultrasensitive and simple strategy for protein tyrosine kinase-7 (PTK7) detection based on the recognition-induced structure change of sgc8 aptamer, and the signal change of methylene blue (MB) that interacted with sandwiched DNA complex. To construct such a sensor, an homogeneous nano-surface was formed firstly on the glass carbon electrode (GCE) by using negatively charged Nafion (Nf) as the inner layer and positively charged gold nanoparticles ((+)AuNPs) as the outer layer, followed by the immobilization of sgc8 aptamer based on Au-S bond. In the presence of helper probe (HP), sandwiched DNA complex was formed between the sgc8 aptamer and the DNA modified gold nanoparticle probe (DNA-AuNPs). Then, a strong current signal was produced due to the capture of abundant MB molecules by both the sandwiched DNA complex and the multiple DNAs that modified on AuNPs surface. However, the specific binding of sgc8 aptamer with PNK7 would trigger a structure transition of it, and directly prevented the following formation of sandwiched structure and the capture of MB. Thus, PTK7 detection could be realized based on monitoring the signal reduction of MB upon incubation of sgc8 aptamer with PTK7. Under optimal conditions, a low detection limit of 372 fM was obtained for PNK7 detection. Due to the employment of sgc8 aptamer, the proposed biosensor exhibited high selectivity to PNK7. Moreover, satisfactory results were obtained when the proposed method was applied for PNK7 detection in cellular debris.

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    2012-11-09

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

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

    PubMed Central

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

    2014-01-01

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

  3. Regulation of Signaling at Regions of Cell-Cell Contact by Endoplasmic Reticulum-Bound Protein-Tyrosine Phosphatase 1B

    PubMed Central

    Haj, Fawaz G.; Sabet, Ola; Kinkhabwala, Ali; Wimmer-Kleikamp, Sabine; Roukos, Vassilis; Han, Hong-Mei; Grabenbauer, Markus; Bierbaum, Martin; Antony, Claude; Neel, Benjamin G.; Bastiaens, Philippe I.

    2012-01-01

    Protein-tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed PTP that is anchored to the endoplasmic reticulum (ER). PTP1B dephosphorylates activated receptor tyrosine kinases after endocytosis, as they transit past the ER. However, PTP1B also can access some plasma membrane (PM)-bound substrates at points of cell-cell contact. To explore how PTP1B interacts with such substrates, we utilized quantitative cellular imaging approaches and mathematical modeling of protein mobility. We find that the ER network comes in close proximity to the PM at apparently specialized regions of cell-cell contact, enabling PTP1B to engage substrate(s) at these sites. Studies using PTP1B mutants show that the ER anchor plays an important role in restricting its interactions with PM substrates mainly to regions of cell-cell contact. In addition, treatment with PTP1B inhibitor leads to increased tyrosine phosphorylation of EphA2, a PTP1B substrate, specifically at regions of cell-cell contact. Collectively, our results identify PM-proximal sub-regions of the ER as important sites of cellular signaling regulation by PTP1B. PMID:22655028

  4. The protein tyrosine phosphatase PTP1B is a negative regulator of CD40 and BAFF-R signaling and controls B cell autoimmunity

    PubMed Central

    Hobeika, Elias; Biesen, Robert; Kollert, Florian; Taddeo, Adriano; Voll, Reinhard E.; Hiepe, Falk

    2014-01-01

    Tyrosine phosphorylation of signaling molecules that mediate B cell activation in response to various stimuli is tightly regulated by protein tyrosine phosphatases (PTPs). PTP1B is a ubiquitously expressed tyrosine phosphatase with well-characterized functions in metabolic signaling pathways. We show here that PTP1B negatively regulates CD40, B cell activating factor receptor (BAFF-R), and TLR4 signaling in B cells. Specifically, PTP1B counteracts p38 mitogen-activated protein kinase (MAPK) activation by directly dephosphorylating Tyr182 of this kinase. Mice with a B cell–specific PTP1B deficiency show increased T cell–dependent immune responses and elevated total serum IgG. Furthermore, aged animals develop systemic autoimmunity with elevated serum anti-dsDNA, spontaneous germinal centers in the spleen, and deposition of IgG immune complexes and C3 in the kidney. In a clinical setting, we observed that B cells of rheumatoid arthritis patients have significantly reduced PTP1B expression. Our data suggest that PTP1B plays an important role in the control of B cell activation and the maintenance of immunological tolerance. PMID:24590766

  5. Tyrosine phosphorylation of two cytosolic proteins of 50 kDa and 35 kDa in rat liver by insulin-receptor kinase in vitro.

    PubMed Central

    Kwok, Y C; Yip, C C

    1987-01-01

    Insulin-receptor tyrosine kinase can phosphorylate a variety of artificial substrates in vitro. Its physiological substrate(s), however, remains unknown. In the present study, we show that immobilized insulin receptors phosphorylate tyrosine residues of two cytosolic proteins of 50 kDa and 35 kDa in rat liver. Phosphorylation of these two proteins required Mn2+- or Mg2+-ATP as the phosphate donor. Phosphorylation was time- and temperature-dependent. Furthermore, the rate of phosphorylation of the two proteins was related to the autophosphorylated state of the insulin receptor. The pI of the phosphorylated 50 kDa and 35 kDa proteins was 5.4 and 5.6 respectively. These proteins were present in low abundance. They were not related to each other, nor to the insulin receptor, as demonstrated by in-gel proteolytic digestion and by immunoprecipitation using antibodies produced against them. They were specific substrates for the insulin receptor kinase, since they were not phosphorylated by epidermal-growth-factor-receptor kinase. These observations suggest that the 50 kDa and 35 kDa cytosolic proteins may be endogenous substrates for the insulin-receptor kinase. Images Fig. 1. Fig. 2. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:2829823

  6. The protein tyrosine phosphatase PTPN22 controls forkhead box protein 3 T regulatory cell induction but is dispensable for T helper type 1 cell polarization

    PubMed Central

    Fousteri, G; Jofra, T; Debernardis, I; Stanford, S M; Laurenzi, A; Bottini, N; Battaglia, M

    2014-01-01

    Protein tyrosine phosphatases (PTPs) regulate T cell receptor (TCR) signalling and thus have a role in T cell differentiation. Here we tested whether the autoimmune predisposing gene PTPN22 encoding for a PTP that inhibits TCR signalling affects the generation of forkhead box protein 3 (FoxP3)+ T regulatory (Treg) cells and T helper type 1 (Th1) cells. Murine CD4+ T cells isolated from Ptpn22 knock-out (Ptpn22KO) mice cultured in Treg cell polarizing conditions showed increased sensitivity to TCR activation compared to wild-type (WT) cells, and subsequently reduced FoxP3 expression at optimal-to-high levels of activation. However, at lower levels of TCR activation, Ptpn22KO CD4+ T cells showed enhanced expression of FoxP3. Similar experiments in humans revealed that at optimal levels of TCR activation PTPN22 knock-down by specific oligonucleotides compromises the differentiation of naive CD4+ T cells into Treg cells. Notably, in vivo Treg cell conversion experiments in mice showed delayed kinetic but overall increased frequency and number of Treg cells in the absence of Ptpn22. In contrast, the in vitro and in vivo generation of Th1 cells was comparable between WT and Ptpn22KO mice, thus suggesting PTPN22 as a FoxP3-specific regulating factor. Together, these results propose PTPN22 as a key factor in setting the proper threshold for FoxP3+ Treg cell differentiation. PMID:24905474

  7. 18F-labeled Single-Stranded DNA Aptamer for PET Imaging of Protein Tyrosine Kinase-7 Expression

    PubMed Central

    Wang, Lu; Wang, Zhe; Yang, Xiangyu; Dewhurst, Andrew; Ma, Ying; Zhu, Guizhi; Niu, Gang; Kiesewetter, Dale O.; Vasdev, Neil; Liang, Steven H.; Chen, Xiaoyuan

    2016-01-01

    Protein tyrosine kinase-7 (PTK7), a member of receptor tyrosine kinase superfamily initially identified as colon carcinoma kinase-4 (CCK-4), is highly expressed in various human malignancies. Its expression was found to correlate with aggressive biological behaviors such as increased cell proliferation, invasiveness and migration. Despite the importance and unmet need of imaging PTK7 in vivo, there is currently no clinically-relevant method to visualize tumoral PTK7 expression noninvasively such as PET or SPECT. This study aims to develop a specific, selective and high affinity PET radioligand based on single-stranded DNA (ssDNA) aptamer to address this challenge. Methods Sgc8, a 41-oligonucleotide that targets to PTK7, was labeled with F-18 using a two-step radiochemical synthesis, which featured a direct one-step radiofluorination on the distinctive spirocyclic hypervalent iodine(III) precursor to give 18F-fluorobenzyl azide followed by copper mediated “click” conjugation with Sgc8-alkyne. 18F-Sgc8 was evaluated in vitro and in vivo in two cell lines, HCT116 and U87MG, which express high and low amounts of PTK7, respectively. Results Sgc8 was labeled efficiently with F-18 in an isolated radiochemical yield of 62 ± 2%, non-decay-corrected (ndc) based on 18F-fluorobenzyl azide. 18F-Tr-Sgc8 was found to possess high affinity binding to both cell lines, with IC50 values for HCT116 as 2.7 ± 0.6 nM and U87MG as 16.9 ± 2.1 nM. In vivo PET imaging clearly visualized PTK7 expression in HCT116 xenografted mice with tumor uptake of 0.76 ± 0.09 %ID/g at 30 min post-injection (p.i.) for the subcutaneous tumor model and greater than 1.5 %ID/g for the liver metastasis model. U87MG xenograft tumors had much lower tracer accumulation (0.13 ± 0.06 %ID/g at 30 min p.i.), which was consistent with the lower expression of PTK7 in this tumor model. The labeled aptamer was rapidly cleared from the blood through the kidneys and bladder to give high tumor-to-blood and tumor

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

    PubMed

    Huang, Danzhi; Caflisch, Amedeo

    2015-01-01

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

  9. Visualisation of brain tumors and quantitation of the protein synthesis rate with L-1-[C-11]-tyrosine

    SciTech Connect

    Pruim, J.; Willemsen, A.T.M.; Waarde, A. van

    1994-05-01

    We have developed the tracer L-1-[C-11]-tyrosine (TYR) for the quantitation of the protein synthesis rate (PSR) in tumours. Here the first results with TYR in a group of patients with (suspected) primary or recurrent brain tumours are reported. Twenty-six patients were studied: 12 male, 14 female, age 45{plus_minus}16 (mean{plus_minus}S.D.) years. At the time of the study the diagnosis of a primary tumour or recurrent tumour was considered on the basis of clinical symptoms and CT/MRI. Patients received 237{plus_minus}111 MBq TYR i.v. Seventeen patients were studied in a dynamic mode (frame sequence: 10 x 0.5, 3 x 5, 3 x 10 minutes). During the studies, arterial blood samples were taken for measurement of the input function, and the assessment of metabolites ([C-11]CO{sub 2}, [C-11]proteins). ROIs were placed over the tumour and using a modified Patlak-analysis the PSR was calculated. In the other 9 patients a static emission scan was made, 20-40 min after injection. All images were corrected for attenuation via a transmission scan. Histology or cytology of the tumour was obtained shortly after the TYR-PET in 20 patients. The calculated PSR of the tumours was 1.0{plus_minus}0.6 nmol/ml/min. This is in range with our animal experiments. The PSR in brain tissue of the contralateral hemisphere was 0.7{plus_minus}0.4 nmol/ml/min. Sixteen of the turnouts were correctly identified with TYR-PET. Also, 2 benign lesions were correctly identified. TYR-PET gave 1 false-positive (infarction) and 1 false-negative (astrocytoma of intermediate malignancy) result. In a few patients with extensive peri-tumoural edema on MRI/CT, additional tumour locations were found with TYR-PET, proven to be malignant on biopsy. In conclusion: TYR is applicable for the visualisation of brain tumours. The possibility of calculating a PSR allows its use in the evaluation of therapy.

  10. IL-1 receptor accessory protein-like 1 associated with mental retardation and autism mediates synapse formation by trans-synaptic interaction with protein tyrosine phosphatase δ.

    PubMed

    Yoshida, Tomoyuki; Yasumura, Misato; Uemura, Takeshi; Lee, Sung-Jin; Ra, Moonjin; Taguchi, Ryo; Iwakura, Yoichiro; Mishina, Masayoshi

    2011-09-21

    Mental retardation (MR) and autism are highly heterogeneous neurodevelopmental disorders. IL-1-receptor accessory protein-like 1 (IL1RAPL1) is responsible for nonsyndromic MR and is associated with autism. Thus, the elucidation of the functional role of IL1RAPL1 will contribute to our understanding of the pathogenesis of these mental disorders. Here, we showed that knockdown of endogenous IL1RAPL1 in cultured cortical neurons suppressed the accumulation of punctate staining signals for active zone protein Bassoon and decreased the number of dendritic protrusions. Consistently, the expression of IL1RAPL1 in cultured neurons stimulated the accumulation of Bassoon and spinogenesis. The extracellular domain (ECD) of IL1RAPL1 was required and sufficient for the presynaptic differentiation-inducing activity, while both the ECD and cytoplasmic domain were essential for the spinogenic activity. Notably, the synaptogenic activity of IL1RAPL1 was specific for excitatory synapses. Furthermore, we identified presynaptic protein tyrosine phosphatase (PTP) δ as a major IL1RAPL1-ECD interacting protein by affinity chromatography. IL1RAPL1 interacted selectively with certain forms of PTPδ splice variants carrying mini-exon peptides in Ig-like domains. The synaptogenic activity of IL1RAPL1 was abolished in primary neurons from PTPδ knock-out mice. IL1RAPL1 showed robust synaptogenic activity in vivo when transfected into the cortical neurons of wild-type mice but not in PTPδ knock-out mice. These results suggest that IL1RAPL1 mediates synapse formation through trans-synaptic interaction with PTPδ. Our findings raise an intriguing possibility that the impairment of synapse formation may underlie certain forms of MR and autism as a common pathogenic pathway shared by these mental disorders.

  11. Fulvestrant-induced cell death and proteasomal degradation of estrogen receptor α protein in MCF-7 cells require the CSK c-Src tyrosine kinase.

    PubMed

    Yeh, Wei-Lan; Shioda, Keiko; Coser, Kathryn R; Rivizzigno, Danielle; McSweeney, Kristen R; Shioda, Toshi

    2013-01-01

    Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.

  12. Tyrosine kinases in inflammatory dermatologic disease

    PubMed Central

    Paniagua, Ricardo T.; Fiorentino, David; Chung, Lorinda; Robinson, William H.

    2010-01-01

    Tyrosine kinases are enzymes that catalyze the phosphorylation of tyrosine residues on protein substrates. They are key components of signaling pathways that drive an array of cellular responses including proliferation, differentiation, migration, and survival. Specific tyrosine kinases have recently been identified as critical to the pathogenesis of several autoimmune and inflammatory diseases. Small-molecule inhibitors of tyrosine kinases are emerging as a novel class of therapy that may provide benefit in certain patient subsets. In this review, we highlight tyrosine kinase signaling implicated in inflammatory dermatologic diseases, evaluate strategies aimed at inhibiting these aberrant signaling pathways, and discuss prospects for future drug development. PMID:20584561

  13. Melatonin attenuates methamphetamine-induced reduction of tyrosine hydroxylase, synaptophysin and growth-associated protein-43 levels in the neonatal rat brain.

    PubMed

    Kaewsuk, Sukit; Sae-ung, Kwankanit; Phansuwan-Pujito, Pansiri; Govitrapong, Piyarat

    2009-11-01

    Methamphetamine (METH) is a most commonly abused drug which damages nerve terminals by causing formation of reactive oxygen species (ROS), apoptosis, and finally neuronal damage. Fetal exposure to neurotoxic METH causes significant behavioral effects. The developing fetus is substantially deficient in most antioxidative enzymes, and may therefore be at high risk from both endogenous and drug-enhanced oxidative stress. Little is known about the effects of METH on vesicular proteins such as synaptophysin and growth-associated protein 43 (GAP-43) in the immature brain. The present study attempted to investigate the effects of METH-induced neurotoxicity in the dopaminergic system of the neonatal rat brain. Neonatal rats were subcutaneously exposed to 5-10mg/kg METH daily from postnatal day 4-10 for 7 consecutive days. The results showed that tyrosine hydroxylase enzyme levels were significantly decreased in the dorsal striatum, prefrontal cortex, nucleus accumbens and substantia nigra, synaptophysin levels decreased in the striatum and prefrontal cortex and growth-associated protein-43 (GAP-43) levels significantly decreased in the nucleus accumbens of neonatal rats. Pretreatment with 2mg/kg melatonin 30 min prior to METH administration prevented METH-induced reduction in tyrosine hydroxylase, synaptophysin and growth-associated protein-43 protein levels in different brain regions. These results suggest that melatonin provides a protective effect against METH-induced nerve terminal degeneration in the immature rat brain probably via its antioxidant properties.

  14. cDNA isolated from a human T-cell library encodes a member of the protein-tyrosine-phosphatase family

    SciTech Connect

    Cool, D.E.; Tonks, N.K.; Charbonneau, H.; Walsh, K.A.; Fischer, E.H.; Krebs, E.G. )

    1989-07-01

    A human peripheral T-cell cDNA library was screened with two labeled synthetic oligonucleotides encoding regions of a human placenta protein-tyrosine-phosphatase. One positive clone was isolated and the nucleotide sequence was determined. It contained 1,305 base pairs of open reading frame followed by a TAA stop codon and 978 base pairs of 3{prime} untranslated end, although a poly(A){sup +} tail was not found. An initiator methionine residue was predicted at position 61, which would result in a protein of 415 amino acid residues. This was supported by the synthesis of a M{sub r} 48,000 protein in an in vitro reticulocyte lysate translation system using RNA transcribed from the cloned cDNA and T7 RNA polymerase. The deduced amino acid sequence was compared to other known proteins revealing 65% identity to the low M{sub r} PTPase 1B isolated from placenta. In view of the high degree of similarity, the T-cell cDNA likely encodes a newly discovered protein-tyrosine-phosphatase, thus expanding this family of genes.

  15. Anti-Group B Streptococcus Glycan-Conjugate Vaccines Using Pilus Protein GBS80 As Carrier and Antigen: Comparing Lysine and Tyrosine-directed Conjugation.

    PubMed

    Nilo, Alberto; Morelli, Laura; Passalacqua, Irene; Brogioni, Barbara; Allan, Martin; Carboni, Filippo; Pezzicoli, Alfredo; Zerbini, Francesca; Maione, Domenico; Fabbrini, Monica; Romano, Maria Rosaria; Hu, Qi-Ying; Margarit, Immaculada; Berti, Francesco; Adamo, Roberto

    2015-07-17

    Gram-positive Streptococcus agalactiae or group B Streptococcus (GBS) is a leading cause of invasive infections in pregnant women, newborns, and elderly people. Vaccination of pregnant women represents the best strategy for prevention of neonatal disease, and GBS polysaccharide-based conjugate vaccines are currently under clinical testing. The potential of GBS pilus proteins selected by genome-based reverse vaccinology as protective antigens for anti-streptococcal vaccines has also been demonstrated. Dressing pilus proteins with surface glycan antigens could be an attractive approach to extend vaccine coverage. We have recently developed an efficient method for tyrosine-directed ligation of large glycans to proteins via copper-free azide-alkyne [3 + 2] cycloaddition. This method enables targeting of predetermined sites of the protein, ensuring that protein epitopes are preserved prior to glycan coupling and a higher consistency in glycoconjugate batches. Herein, we compared conjugates of the GBS type II polysaccharide (PSII) and the GBS80 pilus protein obtained by classic lysine random conjugation and by the recently developed tyrosine-directed ligation. PSII conjugated to CRM197, a carrier protein used for vaccines in the market, was used as a control. We found that the constructs made from PSII and GBS80 were able to elicit murine antibodies recognizing individually the glycan and protein epitopes on the bacterial surface. The generated antibodies were efficacious in mediating opsonophagocytic killing of strains expressing exclusively PSII or GBS80 proteins. The two glycoconjugates were also effective in protecting newborn mice against GBS infection following vaccination of the dams. Altogether, these results demonstrated that polysaccharide-conjugated GBS80 pilus protein functions as a carrier comparably to CRM197, while maintaining its properties of protective protein antigen. Glycoconjugation and reverse vaccinology can, therefore, be combined to design

  16. Tyrosine supplementation in chronic experimental uremia.

    PubMed

    Abitbol, C L; Mandel, S; Mrozinska, K; Wapnir, R A

    1983-08-01

    The occurrence of low tyrosine tissue levels in uremic subjects, possibly due to impaired phenylalanine hydroxylation, suggests that tyrosine may be an essential amino acid in uremia. Additional dietary tyrosine may thus re-dress the deficiency. This study examined growth and tyrosine/phenylalanine metabolism in uremic rats during tyrosine supplementation. Rats made uremic (U) by 7/8 nephrectomy were compared to pair-fed (CP) and ad libitum-fed (CA), sham-operated controls. Two sets of each group of rats were studied after 21 days on the respective diets: I = Purina Lab Chow; II = same + 3.5% tyrosine. Plasma tyrosine was below normal in U and CP-fed diet I. With diet II, the tyrosine:phenylalanine ratio in U was lower than both CA and CP. In rats fed diet II, the tyrosine:phenylalanine ratio became indistinguishable among the three groups. Growth parameters in U and CP were similar, regardless of the diet. Body weight gain, tibial length, muscle mass, and tissue protein did not improve in uremic animals supplemented with tyrosine. The specific activity of liver phenylalanine hydroxylase in U was not different from CA or CP. However, loss of cortical renal mass appeared to be the major determinant of decreased kidney phenylalanine hydroxylation in experimental uremia. This alteration is likely to be the greatest contributory factor to the alteration of plasma levels of tyrosine and phenylalanine. The data presented do not support a proposed essentiality of tyrosine in uremia.

  17. Regulation of the Src Kinase-associated Phosphoprotein 55 Homologue by the Protein Tyrosine Phosphatase PTP-PEST in the Control of Cell Motility*

    PubMed Central

    Ayoub, Emily; Hall, Anita; Scott, Adam M.; Chagnon, Mélanie J.; Miquel, Géraldine; Hallé, Maxime; Noda, Masaharu; Bikfalvi, Andreas; Tremblay, Michel L.

    2013-01-01

    PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis. PMID:23897807

  18. Regulation of the Src kinase-associated phosphoprotein 55 homologue by the protein tyrosine phosphatase PTP-PEST in the control of cell motility.

    PubMed

    Ayoub, Emily; Hall, Anita; Scott, Adam M; Chagnon, Mélanie J; Miquel, Géraldine; Hallé, Maxime; Noda, Masaharu; Bikfalvi, Andreas; Tremblay, Michel L

    2013-09-06

    PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis.

  19. Distinct Pathways Regulate Syk Protein Activation Downstream of Immune Tyrosine Activation Motif (ITAM) and hemITAM Receptors in Platelets*

    PubMed Central

    Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol; Eble, Johannes A.; Ellmeier, Wilfried; Kahn, Mark; Kunapuli, Satya P.

    2015-01-01

    Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an immune tyrosine activation motif (ITAM) and hemITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In this study, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3K, which demonstrates that PI3K regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus, our data show, for the first time, that PI3K and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of the CLEC-2 receptor. PMID:25767114

  20. An epigenetic modifier induces production of (10'S)-verruculide B, an inhibitor of protein tyrosine phosphatases by Phoma sp. nov. LG0217, a fungal endophyte of Parkinsonia microphylla.

    PubMed

    Gubiani, Juliana R; Wijeratne, E M Kithsiri; Shi, Taoda; Araujo, Angela R; Arnold, A Elizabeth; Chapman, Eli; Gunatilaka, A A Leslie

    2017-03-15

    Incorporation of the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), to a culture broth of the endophytic fungus Phoma sp. nov. LG0217 isolated from Parkinsonia microphylla changed its metabolite profile and resulted in the production of (10'S)-verruculide B (1), vermistatin (2) and dihydrovermistatin (3). When cultured in the absence of the epigenetic modifier, it produced a new metabolite, (S,Z)-5-(3',4'-dihydroxybutyldiene)-3-propylfuran-2(5H)-one (4) together with nafuredin (5). The structure of 4 was elucidated by spectroscopic analyses and its absolute configuration was determined by application of the modified Mosher's ester method. The absolute structure of (10'S)-verruculide B was determined as 5-[(10'S,2'E,6'E)-10',11'-dihydroxy-3',7',11'-trimethyldodeca-2',6'-dien-1'-yl]-(3R)-6,8-dihydroxy-3-methylisochroman-1-one (1) with the help of CD and NOE data. Compound 1 inhibited the activity of protein tyrosine phosphatases (PTPs) 1B (PTP1B), Src homology 2-containing PTP 1 (SHP1) and T-cell PTP (TCPTP) with IC50 values of 13.7±3.4, 8.8±0.6, and 16.6±3.8μM, respectively. Significance of these activities and observed modest selectivity of 1 for SHP1 over PTP1B and TCPTP is discussed.

  1. Diversity of the glycine/tyrosine-rich keratin-associated protein 6 gene (KAP6) family in sheep.

    PubMed

    Gong, Hua; Zhou, Huitong; Hickford, Jon G H

    2011-01-01

    Keratin-associated proteins (KAPs) are structural components of wool and variation in them may affect wool characteristics. In this study, we used PCR-SSCP to analyse the ovine KAP6 family which encodes glycine and tyrosine-rich KAPs. Five unique PCR-SSCP patterns were detected in the 250 sheep investigated. Between two and five patterns were observed in individual sheep and none with only one pattern was detected. This suggests the amplicons were heterogeneous and derived from more than one locus. To analyse these heterogeneous PCR amplicons, a sequencing approach using SSCP to separate individual amplified sequences, was developed. Using this approach, five DNA sequences (A-E) representing five unique PCR-SSCP patterns were obtained. D was identical to a published ovine KAP6-1 sequence (GenBank accession no. M95719), whereas the others were novel, but the closest homology was with KAP6 sequences from human, sheep, goats and cattle. The five ovine KAP6 sequences could be assigned into three distinct groups. B and D were identical to each other, with the exception of a 57-bp deletion/insertion and a single nucleotide polymorphism (SNP) in the 3'-UTR region. These appear to be allelic variants of ovine KAP6-1. A and C could form another group, as they were similar to each other (with only one synonymous SNP), but different to the other sequences. This group appears to be related to a sheep KAP6 amino acid sequence, and represent allelic variation at another KAP6 locus (designated KAP6-2). The remaining sequence E did not show high sequence homology with either the KAP6-1 or KAP6-2 sequences, but exhibited homology with a bovine KAP6-3 sequence, with the exception of a deletion/insertion of 30 nucleotides. This suggests that E represents ovine KAP6-3. This sequence was detected in only 11% of the sheep investigated, suggesting either a KAP6-3 null allele, or failure to amplify allleles. These results suggest that ovine KAP6 is a complex gene family, that is not only

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

    PubMed

    Saifudin, Azis; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2013-04-01

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

  3. Coarse-grained molecular simulation of epidermal growth factor receptor protein tyrosine kinase multi-site self-phosphorylation.

    PubMed

    Koland, John G

    2014-01-01

    Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR), the intrinsic protein tyrosine kinase (PTK) activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites) in either of the two C-terminal (CT) domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in molecules such as EGFR

  4. Coarse-Grained Molecular Simulation of Epidermal Growth Factor Receptor Protein Tyrosine Kinase Multi-Site Self-Phosphorylation

    PubMed Central

    Koland, John G.

    2014-01-01

    Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR), the intrinsic protein tyrosine kinase (PTK) activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites) in either of the two C-terminal (CT) domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in molecules such as EGFR

  5. New potential eukaryotic substrates of the mycobacterial protein tyrosine phosphatase PtpA: hints of a bacterial modulation of macrophage bioenergetics state.

    PubMed

    Margenat, Mariana; Labandera, Anne-Marie; Gil, Magdalena; Carrion, Federico; Purificação, Marcela; Razzera, Guilherme; Portela, María Magdalena; Obal, Gonzalo; Terenzi, Hernán; Pritsch, Otto; Durán, Rosario; Ferreira, Ana María; Villarino, Andrea

    2015-03-06

    The bacterial protein tyrosine phosphatase PtpA is a key virulence factor released by Mycobacterium tuberculosis in the cytosol of infected macrophages. So far only two unrelated macrophage components (VPS33B, GSK3α) have been identified as PtpA substrates. As tyrosine phosphatases are capable of using multiple substrates, we developed an improved methodology to pull down novel PtpA substrates from an enriched P-Y macrophage extract using the mutant PtpA D126A. This methodology reduced non-specific protein interactions allowing the identification of four novel putative PtpA substrates by MALDI-TOF-MS and nano LC-MS: three mitochondrial proteins - the trifunctional enzyme (TFP), the ATP synthase, and the sulfide quinone oxidoreductase - and the cytosolic 6-phosphofructokinase. All these proteins play a relevant role in cell energy metabolism. Using surface plasmon resonance, PtpA was found to bind immunopurified human TFP through its catalytic site since TFP-PtpA association was inhibited by a specific phosphatase inhibitor. Moreover, PtpA wt was capable of dephosphorylating immunopurified human TFP in vitro supporting that TFP may be a bona fide PtpA susbtrate. Overall, these results suggest a novel scenario where PtpA-mediated dephosphorylation may affect pathways involved in cell energy metabolism, particularly the beta oxidation of fatty acids through modulation of TFP activity and/or cell distribution.

  6. Role of protein tyrosine phosphatase non-receptor type 7 in the regulation of TNF-α production in RAW 264.7 macrophages.

    PubMed

    Seo, Huiyun; Lee, In-Seon; Park, Jae Eun; Park, Sung Goo; Lee, Do Hee; Park, Byoung Chul; Cho, Sayeon

    2013-01-01

    Protein tyrosine phosphatases play key roles in a diverse range of cellular processes such as differentiation, cell proliferation, apoptosis, immunological signaling, and cytoskeletal function. Protein tyrosine phosphatase non-receptor type 7 (PTPN7), a member of the phosphatase family, specifically inactivates mitogen-activated protein kinases (MAPKs). Here, we report that PTPN7 acts as a regulator of pro-inflammatory TNF-α production in RAW 264.7 cells that are stimulated with lipopolysaccharide (LPS) that acts as an endotoxin and elicits strong immune responses in animals. Stimulation of RAW 264.7 cells with LPS leads to a transient decrease in the levels of PTPN7 mRNA and protein. The overexpression of PTPN7 inhibits LPS-stimulated production of TNF-α. In addition, small interfering RNA (siRNA) analysis showed that knock-down of PTPN7 in RAW 264.7 cells increased TNF-α production. PTPN7 has a negative regulatory function to extracellular signal regulated kinase 1/2 (ERK1/2) and p38 that increase LPS-induced TNF-α production in macrophages. Thus, our data presents PTPN7 as a negative regulator of TNF-α expression and the inflammatory response in macrophages.

  7. Protein tyrosine phosphatases regulate arachidonic acid release, StAR induction and steroidogenesis acting on a hormone-dependent arachidonic acid-preferring acyl-CoA synthetase.

    PubMed

    Cano, Florencia; Poderoso, Cecilia; Cornejo Maciel, Fabiana; Castilla, Rocío; Maloberti, Paula; Castillo, Fernanda; Neuman, Isabel; Paz, Cristina; Podestá, Ernesto J

    2006-06-01

    The activation of the rate-limiting step in steroid biosynthesis, that is the transport of cholesterol into the mitochondria, is dependent on PKA-mediated events triggered by hormones like ACTH and LH. Two of such events are the protein tyrosine dephosphorylation mediated by protein tyrosine phosphatases (PTPs) and the release of arachidonic acid (AA) mediated by two enzymes, ACS4 (acyl-CoA synthetase 4) and Acot2 (mitochondrial thioesterase). ACTH and LH regulate the activity of PTPs and Acot2 and promote the induction of ACS4. Here we analyzed the involvement of PTPs on the expression of ACS4. We found that two PTP inhibitors, acting through different mechanisms, are both able to abrogate the hormonal effect on ACS4 induction. PTP inhibitors also reduce the effect of cAMP on steroidogenesis and on the level of StAR protein, which facilitates the access of cholesterol into the mitochondria. Moreover, our results indicate that exogenous AA is able to overcome the inhibition produced by PTP inhibitors on StAR protein level and steroidogenesis. Then, here we describe a link between PTP activity and AA release, since ACS4 induction is under the control of PTP activity, being a key event for AA release, StAR induction and steroidogenesis.

  8. A mechanism of immunoreceptor tyrosine-based activation motif (ITAM)-like sequences in the capsid protein VP2 in viral growth and pathogenesis of Coxsackievirus B3.

    PubMed

    Kim, Dae-Sun; Park, Jung-Hyun; Kim, Joo-Young; Kim, Dokeun; Nam, Jae-Hwan

    2012-04-01

    Coxsackievirus B3 (CVB3) is an RNA virus that mainly causes myocarditis. We have reported previously that immunoreceptor tyrosine-based activation motif (ITAM)-like sequences are contained in the capsid protein VP2 of CVB3. The substitution of two tyrosines for phenylalanines in the ITAM-like region causes attenuation of CVB3, possibly via defective viral assembly. In this study, we found that Syk, a downstream molecule of ITAM, interacts with the wild-type (WT) CVB3 VP0 protein, but not with the mutant CVB3 VP0 (called YYFF), and that an inhibitor of Syk reduced the growth of CVB3. The WT CVB3 activated nuclear factor kappa B (NF-κB), a protein activated by ITAM, and eventually induced the production of interleukin-6 (IL-6)-one of the proinflammatory cytokines induced by NF-κB-in macrophages. However, the YYFF form did not. In addition, viral VP2 protein may be dependent on the phosphorylation of an ITAM-like region that affected the activation of NF-κB. Taken together, these results suggest that the ITAM-like sequences in CVB3 VP2 can not only affect viral structure but also act as signals in pathogenesis.

  9. Effect of the tyrosine kinase inhibitor lapatinib on CUB-domain containing protein (CDCP1)-mediated breast cancer cell survival and migration

    SciTech Connect

    Seidel, Jeanette; Kunc, Klaudia; Possinger, Kurt; Jehn, Christian; Lueftner, Diana

    2011-10-14

    Highlights: {yields} CDCP1 downregulation reduces anchorage free survival of breast cancer cells. {yields} Anoikis of CDCP1-positive breast cancer cells is increased after CDCP1 downregulation. {yields} CDCP1 knockdown decreases migration and extensively reduces invasiveness in vitro. {yields} Proliferation rate does not correlate with CDCP1 expression. {yields} Lapatinib does not influence tyrosine kinases of CDCP1 signal transduction. -- Abstract: The surface receptor CUB domain-containing protein 1 (CDCP1) is highly expressed in several adenocarcinomas and speculated to participate in anchorage-independent cell survival and cell motility. Tyrosine kinase phosphorylation seems to be crucial for intracellular signaling of CDCP1. Lapatinib, a tyrosine kinase inhibitor (TKI), is approved for treatment of HER-2/neu overexpressing metastatic breast cancer and functions by preventing autophosphorylation following HER-2/neu receptor activation. This study aimed to investigate the effect of CDCP1 expression on anchorage-independent growth and cell motility of breast cancer cells. Moreover, studies were performed to examine if lapatinib provided any beneficial effect on HER-2/neu{sup (+)/-}/CDCP1{sup +} breast cancer cell lines. In our studies, we affirmed that CDCP1 prevents cells from undergoing apoptosis when cultured in the absence of cell-substratum anchorage and that migratory and invasive properties of these cells were decreased when CDCP1 was down-regulated. However, only HER-2/neu{sup +}, but not HER-2/neu{sup (+)/-} cells showed decreased proliferation and invasion and an enhanced level of apoptosis towards loss of anchorage when treated with lapatinib. Therefore, we conclude that CDCP1 might be involved in regulating adhesion and motility of breast cancer cells but that lapatinib has no effect on tyrosine kinases regulating CDCP1. Nonetheless, other TKIs might offer therapeutic approaches for CDCP1-targeted breast cancer therapy and should be studied

  10. Xanthium strumarium as an Inhibitor of α-Glucosidase, Protein Tyrosine Phosphatase 1β, Protein Glycation and ABTS⁺ for Diabetic and Its Complication.

    PubMed

    Hwang, Seung Hwan; Wang, Zhiqiang; Yoon, Ha Na; Lim, Soon Sung

    2016-09-16

    Phytochemical investigation of the natural products from Xanthium strumarium led to the isolation of fourteen compounds including seven caffeoylquinic acid (CQA) derivatives. The individual compounds were screened for inhibition of α-glucosidase, protein tyrosine phosphatase 1β (PTP1β), advanced glycation end products (AGEs), and ABTS⁺ radical scavenging activity using in vitro assays. Among the isolated compounds, methyl-3,5-di-caffeoyquinic acid exhibited significant inhibitory activity against α-glucosidase (18.42 μM), PTP1β (1.88 μM), AGEs (82.79 μM), and ABTS⁺ (6.03 μM). This effect was marked compared to that of the positive controls (acarbose 584.79 μM, sumarin 5.51 μM, aminoguanidine 1410.00 μM, and trolox 29.72 μM respectively). In addition, 3,5-di-O-CQA (88.14 μM) and protocatechuic acid (32.93 μM) had a considerable inhibitory effect against α-glucosidase and ABTS⁺. Based on these findings, methyl-3,5-di-caffeoyquinic acid was assumed to be potentially responsible for the anti-diabetic actions of X. strumarium.

  11. Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class.

    PubMed Central

    Buchdunger, E; Zimmermann, J; Mett, H; Meyer, T; Müller, M; Regenass, U; Lydon, N B

    1995-01-01

    The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation. Images Fig. 1 Fig. 2 Fig. 3 PMID:7708684

  12. Isolation of betulinic acid, its methyl ester and guaiane sesquiterpenoids with protein tyrosine phosphatase 1B inhibitory activity from the roots of Saussurea lappa C.B.Clarke.

    PubMed

    Choi, Ji Young; Na, Minkyun; Hyun Hwang, In; Ho Lee, Seung; Young Bae, Eun; Yeon Kim, Bo; Seog Ahn, Jong

    2009-01-08

    Activity-guided fractionation of a MeOH extract of the roots of Saussurea lappa C.B.Clarke (Compositae), using an in vitro protein tyrosine phosphatase 1B (PTP1B) inhibition assay, led to the isolation of four active constituents: betulinic acid (1), betulinic acid methyl ester (2), mokko lactone (3) and dehydrocostuslactone (4), along with nine inactive compounds. Our findings indicate that betulinic acid (1) and its methyl ester 2, as well as the two guaiane sesquiterpenoids 3 and 4 are potential lead moieties for the development of new PTP1B inhibitors.

  13. A selective Seoul-Fluor-based bioprobe, SfBP, for vaccinia H1-related phosphatase--a dual-specific protein tyrosine phosphatase.

    PubMed

    Jeong, Myeong Seon; Kim, Eunha; Kang, Hyo Jin; Choi, Eun Joung; Cho, Alvin R; Chung, Sang J; Park, Seung Bum

    2012-07-04

    We report a Seoul-Fluor-based bioprobe, SfBP, for selective monitoring of protein tyrosine phosphatases (PTPs). A rational design based on the structures at the active site of dual-specific PTPs can enable SfBP to selectively monitor the activity of these PTPs with a 93-fold change in brightness. Moreover, screening results of SfBP against 30 classical PTPs and 35 dual-specific PTPs show that it is selective toward vaccinia H1-related (VHR) phosphatase, a dual-specific PTP (DUSP-3).

  14. Crystal structure of the kinase domain of human protein tyrosine kinase 6 (PTK6) at 2.33 Å resolution.

    PubMed

    Thakur, Manish Kumar; Kumar, Amit; Birudukota, Swarnakumari; Swaminathan, Srinivasan; Tyagi, Rajiv; Gosu, Ramachandraiah

    2016-09-16

    Human Protein tyrosine kinase 6 (PTK6) (EC:2.7.10.2), also known as the breast tumor kinase (BRK), is an intracellular non-receptor Src-related tyrosine kinase expressed in a majority of human breast tumors and breast cancer cell lines, but its expression is low or completely absent in normal mammary glands. In the recent past, several studies have suggested that PTK6 is a potential therapeutic target in cancer. To understand its structural and functional properties, the PTK6 kinase domain (PTK6-KD) gene was cloned, overexpressed in a baculo-insect cell system, purified and crystallized at room temperature. X-ray diffraction data to 2.33 Å resolution was collected on a single PTK6-KD crystal, which belonged to the triclinic space group P1. The Matthews coefficient calculation suggested the presence of four protein molecules per asymmetric unit, with a solvent content of ∼50%.The structure has been solved by molecular replacement and crystal structure data submitted to the protein data bank under the accession number 5D7V. This is the first report of apo PTK6-KD structure crystallized in DFG-in and αC-helix-out conformation.

  15. Host-targeting protein 1 (SpHtp1) from the oomycete Saprolegnia parasitica translocates specifically into fish cells in a tyrosine-O-sulphate-dependent manner.

    PubMed

    Wawra, Stephan; Bain, Judith; Durward, Elaine; de Bruijn, Irene; Minor, Kirsty L; Matena, Anja; Löbach, Lars; Whisson, Stephen C; Bayer, Peter; Porter, Andrew J; Birch, Paul R J; Secombes, Chris J; van West, Pieter

    2012-02-07

    The eukaryotic oomycetes, or water molds, contain several species that are devastating pathogens of plants and animals. During infection, oomycetes translocate effector proteins into host cells, where they interfere with host-defense responses. For several oomycete effectors (i.e., the RxLR-effectors) it has been shown that their N-terminal polypeptides are important for the delivery into the host. Here we demonstrate that the putative RxLR-like effector, host-targeting protein 1 (SpHtp1), from the fish pathogen Saprolegnia parasitica translocates specifically inside host cells. We further demonstrate that cell-surface binding and uptake of this effector protein is mediated by an interaction with tyrosine-O-sulfate-modified cell-surface molecules and not via phospholipids, as has been reported for RxLR-effectors from plant pathogenic oomycetes. These results reveal an effector translocation route based on tyrosine-O-sulfate binding, which could be highly relevant for a wide range of host-microbe interactions.

  16. Host-targeting protein 1 (SpHtp1) from the oomycete Saprolegnia parasitica translocates specifically into fish cells in a tyrosine-O-sulphate–dependent manner

    PubMed Central

    Wawra, Stephan; Bain, Judith; Durward, Elaine; de Bruijn, Irene; Minor, Kirsty L.; Matena, Anja; Löbach, Lars; Whisson, Stephen C.; Bayer, Peter; Porter, Andrew J.; Birch, Paul R. J.; Secombes, Chris J.; van West, Pieter

    2012-01-01

    The eukaryotic oomycetes, or water molds, contain several species that are devastating pathogens of plants and animals. During infection, oomycetes translocate effector proteins into host cells, where they interfere with host-defense responses. For several oomycete effectors (i.e., the RxLR-effectors) it has been shown that their N-terminal polypeptides are important for the delivery into the host. Here we demonstrate that the putative RxLR-like effector, host-targeting protein 1 (SpHtp1), from the fish pathogen Saprolegnia parasitica translocates specifically inside host cells. We further demonstrate that cell-surface binding and uptake of this effector protein is mediated by an interaction with tyrosine-O-sulfate–modified cell-surface molecules and not via phospholipids, as has been reported for RxLR-effectors from plant pathogenic oomycetes. These results reveal an effector translocation route based on tyrosine-O-sulfate binding, which could be highly relevant for a wide range of host–microbe interactions. PMID:22308362

  17. 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.

  18. Protein kinase C and tyrosine kinase pathways regulate lipopolysaccharide-induced nitric oxide synthase activity in RAW 264.7 murine macrophages.

    PubMed Central

    Paul, A; Pendreigh, R H; Plevin, R

    1995-01-01

    1. In RAW 264.7 macrophages, lipopolysaccharide (LPS) and gamma-interferon (IFN gamma) alone or in combination stimulated the induction of nitric oxide synthase (iNOS) activity and increased the expression of the 130 kDa isoform of NOS. 2. LPS-induced NOS activity was reduced by incubation with CD14 neutralising antibodies and abolished in macrophages deprived of serum. 3. LPS stimulated a small increase in protein kinase C (PKC) activity in RAW 264.7 macrophages which was dependent on the presence of serum. However, IFN gamma did not potentiate LPS-stimulated PKC activity. 4. The protein kinase C inhibitor, Ro-318220, abolished both LPS- and IFN gamma-stimulated protein kinase C activity and the induction of NOS activity. 5. LPS- and IFN gamma-induced NOS activity was reduced by the tyrosine kinase inhibitor genestein. Genestein also reduced LPS-stimulated protein kinase C activity but did not affect the response to the protein kinase C activator, tetradecanoylphorbol acetate (TPA). 6. Nicotinamide, an inhibitor of poly-ADP ribosylation, abolished LPS- and IFN gamma-induced NOS activity. 7. Brefeldin A, an inhibitor of a factor which stimulates nucleotide exchange activity on the 21 kDa ADP-ribosylation factor, ARF, reduced LPS- and IFN gamma-induced NOS activity by approximately 80%. 8. These results suggest the involvement of protein kinase C, tyrosine kinase and poly-ADP ribosylation pathways in the regulation of the induction of nitric oxide synthase in RAW 264.7 macrophages by LPS and IFN gamma. Images Figure 2 PMID:7533621

  19. Activation of protein tyrosine kinases and matrix metalloproteinases causes blood-brain barrier injury: Novel mechanism for neurodegeneration associated with alcohol abuse.

    PubMed

    Haorah, James; Schall, Kathy; Ramirez, Servio H; Persidsky, Yuri

    2008-01-01

    Blood-brain barrier (BBB) formed by brain microvascular endothelial cells (BMVEC) regulates the passage of molecules and leukocytes in and out of the brain. Activation of matrix metalloproteinases (MMPs) and alteration of basement membrane (BM) associated with BBB injury was documented in stroke patients. While chronic alcoholism is a risk factor for developing stroke, underlying mechanisms are not well understood. We hypothesized that ethanol (EtOH)-induced protein tyrosine kinase (PTK) signaling resulted a loss of BBB integrity via MMPs activation and degradation of BM component, collagen IV. Treatment of BMVEC with EtOH or acetaldehyde (AA) for 2-48 h increased MMP-1, -2 and -9 activities or decreased the levels of tissue inhibitors of MMPs (TIMP-1, -2) in a PTK-dependent manner without affecting protein tyrosine phosphatase activity. Enhanced PTK activity after EtOH exposure correlated with increased phosphorylated proteins of selective receptor and nonreceptor PTKs. Up-regulation of MMPs activities and protein contents paralleled a decrease in collagen IV content, and inhibitors of EtOH metabolism, MMP-2 and -9, or PTK reversed all these effects. Using human BMVEC assembled into BBB models, we found that EtOH/AA diminished barrier tightness, augmented permeability, and monocyte migration across the BBB via activation of PTKs and MMPs. These findings suggest that alcohol associated BBB injury could be mediated by MMPs via BM protein degradation and could serve as a comorbidity factor for neurological disorders like stroke or neuroinflammation. Furthermore, our preliminary experiments indicated that human astrocytes secreted high levels of MMP-1 and -9 following exposure to EtOH, suggesting the role of BM protein degradation and BBB compromise as a result of glial activation by ethanol. These results provide better understanding of multifaceted effects of alcohol on the brain and could help develop new therapeutic interventions.

  20. A critical role for Syk protein tyrosine kinase in Fc receptor-mediated antigen presentation and induction of dendritic cell maturation.

    PubMed

    Sedlik, Christine; Orbach, Daniel; Veron, Philippe; Schweighoffer, Edina; Colucci, Francesco; Gamberale, Romina; Ioan-Facsinay, Andrea; Verbeek, Sjef; Ricciardi-Castagnoli, Paola; Bonnerot, Christian; Tybulewicz, Victor L J; Di Santo, James; Amigorena, Sebastian

    2003-01-15

    Dendritic cells (DCs) are the only APCs capable of initiating adaptive immune responses. The initiation of immune responses requires that DCs 1) internalize and present Ags; and 2) undergo a differentiation process, called "maturation", which transforms DCs into efficient APCs. DC maturation may be initiated by the engagement of different surface receptors, including certain cytokine receptors (such as TNFR), Toll-like receptors, CD40, and FcRs. The early activation events that link receptor engagement and DC maturation are not well characterized. We found that FcR engagement by immune complexes induced the phosphorylation of Syk, a protein tyrosine kinase acting immediately downstream of FcRs. Syk was dispensable for DC differentiation in vitro and in vivo, but was strictly required for immune complexes internalization and subsequent Ag presentation to T lymphocytes. Importantly, Syk was also required for the induction of DC maturation and IL-12 production after FcR engagement, but not after engagement of other surface receptors, such as TNFR or Toll-like receptors. Therefore, protein tyrosine phosphorylation by Syk represents a novel pathway for the induction of DC maturation.

  1. Protein tyrosine phosphatases from amphioxus, hagfish, and ray: divergence of tissue-specific isoform genes in the early evolution of vertebrates.

    PubMed

    Ono-Koyanagi, K; Suga, H; Katoh, K; Miyata, T

    2000-03-01

    Since separation from fungi and plants, multicellular animals evolved a variety of gene families involved in cell-cell communication from a limited number of ancestral precursors by gene duplications in two separate periods of animal evolution. In the very early evolution of animals before the separation of parazoans and eumetazoans, animals underwent extensive gene duplications by which different subtypes (subfamilies) with distinct functions diverged. The multiplicity of members (isoforms) in the same subtype increased by further gene duplications (isoform duplications) in the first half of chordate evolution before the fish-tetrapod split; different isoforms are virtually identical in structure and function but differ in tissue distribution. From cloning and phylogenetic analyses of four subfamilies of the protein tyrosine kinase (PTK) family, we recently showed extensive isoform duplications in a limited period around or just before the cyclostome-gnathostome split. To obtain a reliable estimate for the divergence time of vertebrate isoforms, we have conducted isolation of cDNAs encoding the protein tyrosine phosphatases (PTPs) from Branchiostoma belcheri, an amphioxus, Eptatretus burgeri, a hagfish, and Potamotrygon motoro, a ray. We obtained 33 different cDNAs in total, most of which belong to known PTP subfamilies. The phylogenetic analyses of five subfamilies based on the maximum likelihood method revealed frequent isoform duplications in a period around or just before the gnathostome-cyclostome split. An evolutionary implication was discussed in relation to the Cambrian explosion.

  2. Phosphorylation of actin-binding protein (ABP-280; filamin) by tyrosine kinase p56lck modulates actin filament cross-linking.

    PubMed

    Pal Sharma, C; Goldmann, Wolfgang H

    2004-01-01

    Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, we decided to investigate the possibility of whether it serves as substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins. The interaction of p56lck with membrane glycoproteins is important for cell development and functional activation. Here, we show that purified p56lck interacts and catalyzes in vitro kinase reactions. Tyrosine phosphorylation by p56lck is restricted to a single peptide of labeled ABP-280 shown by protease digest. The addition of phorbol ester to cells results in the inhibition of phosphorylation of ABP-280 by p56lck. These results show a decrease in phosphorylation suggesting conformationally induced regulation. Dynamic light scattering confirmed increased actin filament cross-linking due to phosphorylation of ABP-280 by p56lck.

  3. Synthesis and characterization of N-coumaroyltyramine as a potent phytochemical which arrests human transformed cells via inhibiting protein tyrosine kinases.

    PubMed

    Park, Jae B; Schoene, Norberta

    2002-04-12

    Numerous phytochemicals are believed to have beneficial effects on human health. N-Coumaroyltyramine accumulates in plants in response to wounding and pathogen attack. Due to the scarcity of N-coumaroyltyramine, its biological activities have not been studied in human cells. In this study, N-coumaroyltyramine was chemically synthesized and then purified by an HPLC with a UV-visible absorbance detector. Retention times of major peaks were 14.3 and 20.7 min, and the peak at 20.7 min was confirmed by LC-MS as N-coumaroyltyramine with a mass/charge (m/z) unit of 284.1. The synthesis procedure was relatively easy and had an acceptable yield (approximately 55%). The compound exhibited a new activity, suppression of growth of human tumor cells such as U937 and Jurkat cells. In addition, the suppressed growth of the cells was strongly associated with an increased percentage of cells in the S phase of the cell cycle progression. Furthermore, N-coumaroyltyramine was able to inhibit the protein tyrosine kinases including epidermal growth factor receptor (EGFR). This is the first report of the growth suppressing activity of N-coumaroyltyramine and its arrest of cells at the S phase of the cell cycle, possibly by inhibition of protein tyrosine kinases.

  4. Protein tyrosine phosphatase Meg2 dephosphorylates signal transducer and activator of transcription 3 and suppresses tumor growth in breast cancer

    PubMed Central

    2012-01-01

    Introduction Signal transducer and activator of transcription 3 (STAT3) is over-activated or phosphorylated in breast cancers. The hyper-phosphorylation of STAT3 was attributed to either up-regulated phosphorylation by several tyrosine-kinases or down-regulated activity of phosphatases. Although several factors have been identified to phosphorylate STAT3, it remains unclear how STAT3 is dephosphorylated by PTPMeg2. The aim of this study was to determine the role of PTPMeg2 as a phosphatase in regulation of the activity of STAT3 in breast cancers. Methods Immunoprecipitation assays were used to study the interaction of STAT3 with PTPMeg2. A series of biochemistry experiments were performed to evaluate the role of PTPMeg2 in the dephosphorylation of STAT3. Two breast cancer cell lines MCF7 (PTPMeg2 was depleted as it was endogenously high) and MDA-MB-231 (PTPMeg2 was overexpressed as it was endogenously low) were used to compare the level of phosphorylated STAT3 and the tumor growth ability in vitro and in vivo. Samples from breast carcinoma (n = 73) were subjected to a pair-wise Pearson correlation analysis for the correlation of levels of PTPMeg2 and phosphorylated STAT3. Results PTPMeg2 directly interacts with STAT3 and mediates its dephosphorylation in the cytoplasm. Over-expression of PTPMeg2 decreased tyrosine phosphorylation of STAT3 while depletion of PTPMeg2 increased its phosphorylation. The decreased tyrosine phosphorylation of STAT3 is coupled with suppression of STAT3 transcriptional activity and reduced tumor growth in vitro and in vivo. Levels of PTPMeg2 and phosphorylated STAT3 were inversely correlated in breast cancer tissues (P = 0.004). Conclusions PTPMeg2 is an important phosphatase for the dephosphorylation of STAT3 and plays a critical role in breast cancer development. PMID:22394684

  5. Protein Flexibility in Docking-Based Virtual Screening: Discovery of Novel Lymphoid-Specific Tyrosine Phosphatase Inhibitors Using Multiple Crystal Structures.

    PubMed

    Hou, Xuben; Li, Kangshuai; Yu, Xiao; Sun, Jin-peng; Fang, Hao

    2015-09-28

    Incorporating protein flexibility is a major challenge for docking-based virtual screening. With an increasing number of available crystal structures, ensemble docking with multiple protein structures is an efficient approach to deal with protein flexibility. Herein, we report the successful application of a docking-based virtual screen using multiple crystal structures to discover novel inhibitors of lymphoid-specific tyrosine phosphatase (LYP), a potential drug target for autoimmune diseases. The appropriate use of multiple protein structures allowed a better enrichment than a single structure in the recovery of known inhibitors. Subsequently, an optimal ensemble of LYP structures was selected and used in docking-based virtual screening. Eight novel LYP inhibitors (IC50 ranging from 7.95 to 56.6 μM) were identified among 23 hit compounds. Further studies demonstrated that the most active compound B15 possessed some selectivity over other protein phosphatases and could effectively up-regulate TCR (T cell receptor)-mediated signaling in Jurkat T cells. These novel hits not only provided good starting points for the development of therapeutic agents useful in autoimmune diseases but also demonstrated the advantages of choosing an appropriate ensemble of protein structures in docking-based virtual screening over using a single protein conformation.

  6. Discovering the first tyrosine kinase.

    PubMed

    Hunter, Tony

    2015-06-30

    In the middle of the 20th century, animal tumor viruses were heralded as possible models for understanding human cancer. By the mid-1970s, the molecular basis by which tumor viruses transform cells into a malignant state was beginning to emerge as the first viral genomic sequences were reported and the proteins encoded by their transforming genes were identified and characterized. This was a time of great excitement and rapid progress. In 1978, prompted by the discovery from Ray Erikson's group that the Rous sarcoma virus (RSV) v-Src-transforming protein had an associated protein kinase activity specific for threonine, my group at the Salk Institute set out to determine whether the polyomavirus middle T-transforming protein had a similar kinase activity. Here, I describe the experiments that led to the identification of a kinase activity associated with middle T antigen and our serendipitous discovery that this activity was specific for tyrosine in vitro, and how this in turn led to the fortuitous observation that the v-Src-associated kinase activity was also specific for tyrosine. Our finding that v-Src increased the level of phosphotyrosine in cellular proteins in RSV-transformed cells confirmed that v-Src is a tyrosine kinase and transforms cells by phosphorylating proteins on tyrosine. My colleague Bart Sefton and I reported these findings in the March issue of PNAS in 1980. Remarkably, all of the experiments in this paper were accomplished in less than one month.

  7. Discovering the first tyrosine kinase

    PubMed Central

    Hunter, Tony

    2015-01-01

    In the middle of the 20th century, animal tumor viruses were heralded as possible models for understanding human cancer. By the mid-1970s, the molecular basis by which tumor viruses transform cells into a malignant state was beginning to emerge as the first viral genomic sequences were reported and the proteins encoded by their transforming genes were identified and characterized. This was a time of great excitement and rapid progress. In 1978, prompted by the discovery from Ray Erikson’s group that the Rous sarcoma virus (RSV) v-Src–transforming protein had an associated protein kinase activity specific for threonine, my group at the Salk Institute set out to determine whether the polyomavirus middle T-transforming protein had a similar kinase activity. Here, I describe the experiments that led to the identification of a kinase activity associated with middle T antigen and our serendipitous discovery that this activity was specific for tyrosine in vitro, and how this in turn led to the fortuitous observation that the v-Src–associated kinase activity was also specific for tyrosine. Our finding that v-Src increased the level of phosphotyrosine in cellular proteins in RSV-transformed cells confirmed that v-Src is a tyrosine kinase and transforms cells by phosphorylating proteins on tyrosine. My colleague Bart Sefton and I reported these findings in the March issue of PNAS in 1980. Remarkably, all of the experiments in this paper were accomplished in less than one month. PMID:26130799

  8. Isolation and characterization of a gene from Trypanosoma cruzi encoding a 46-kilodalton protein with homology to human and rat tyrosine aminotransferase.

    PubMed

    Bontempi, E J; Búa, J; Aslund, L; Porcel, B; Segura, E L; Henriksson, J; Orn, A; Pettersson, U; Ruiz, A M

    1993-06-01

    The complete sequence of a gene encoding a 46-kDa protein of Trypanosoma cruzi is presented. The first ATG complies with the consensus sequence for initiation of translation. A single band of 2 kb was highlighted by hybridizing a probe from the 46-kDa protein gene to a Northern filter containing total T. cruzi RNA. The gene is present in 50-80 copies per cell and most of them are contained in 2 tandem arrays on large T. cruzi chromosomes (> 2000 kb). A strong homology with rat and human tyrosine aminotransferase was detected. Homology with a Trypanosoma brucei retrotransposon was found in the nonsense strand of the intergenic region.

  9. The Protein Tyrosine Phosphatase, Shp2, Positively Contributes to FLT3-ITD-Induced Hematopoietic Progenitor Hyperproliferation and Malignant Disease In Vivo

    PubMed Central

    Nabinger, Sarah C.; Li, XingJun; Ramdas, Baskar; He, Yantao; Zhang, Xian; Zeng, Lifan; Richine, Briana; Bowling, Joshua D.; Fukuda, Seiji; Goenka, Shreevrat; Liu, Ziyue; Feng, Gen-Sheng; Yu, Menggang; Sandusky, George E.; Boswell, H. Scott; Zhang, Zhong-Yin; Kapur, Reuben; Chan, Rebecca J.

    2014-01-01

    Internal tandem duplications in the fms-like tyrosine kinase receptor (FLT3-ITDs) confer a poor prognosis in acute myeloid leukemia. We hypothesized that increased recruitment of the protein tyrosine phosphatase, Shp2, to FLT3-ITDs contributes to FLT3 ligand (FL)-independent hyperproliferation and STAT5 activation. Co-immunoprecipitation demonstrated constitutive association of Shp2 with the FLT3-ITD, N51-FLT3, as well as with STAT5. Knock-down of Shp2 in Baf3/N51-FLT3 cells significantly reduced proliferation while having little effect on WT-FLT3-expressing cells. Consistently, mutation of N51-FLT3 tyrosine 599 to phenylalanine or genetic disruption of Shp2 in N51-FLT3-expressing bone marrow low density mononuclear cells reduced proliferation and STAT5 activation. In transplants, genetic disruption of Shp2 in vivo yielded increased latency to and reduced severity of FLT3-ITD-induced malignancy. Mechanistically, Shp2 co-localizes with nuclear phospho-STAT5, is present at functional interferon-γ activation sites (GAS) within the BCL2L1 promoter, and positively activates the human BCL2L1 promoter, suggesting that Shp2 works with STAT5 to promote pro-leukemogenic gene expression. Further, using a small molecule Shp2 inhibitor, the proliferation of N51-FLT3-expressing bone marrow progenitors and primary AML samples was reduced in a dose-dependent manner. These findings demonstrate that Shp2 positively contributes to FLT3-ITD-induced leukemia and suggest that Shp2 inhibition may provide a novel therapeutic approach to acute myeloid leukemia. PMID:23103841

  10. The immunomodulatory adapter proteins DAP12 and Fc receptor γ-chain (FcRγ) regulate development of functional osteoclasts through the Syk tyrosine kinase

    PubMed Central

    Mócsai, Attila; Humphrey, Mary Beth; Van Ziffle, Jessica A. G.; Hu, Yongmei; Burghardt, Andrew; Spusta, Steven C.; Majumdar, Sharmila; Lanier, Lewis L.; Lowell, Clifford A.; Nakamura, Mary C.

    2004-01-01

    Osteoclasts, the only bone-resorbing cells, are central to the pathogenesis of osteoporosis, yet their development and regulation are incompletely understood. Multiple receptors of the immune system use a common signaling paradigm whereby phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) within receptor-associated adapter proteins recruit the Syk tyrosine kinase. Here we demonstrate that a similar mechanism is required for development of functional osteoclasts. Mice lacking two ITAM-bearing adapters, DAP12 and the Fc receptor γ-chain (FcRγ), are severely osteopetrotic. DAP12-/-FcRγ-/- bone marrow cells fail to differentiate into multinucleated osteoclasts or resorb bone in vitro and show impaired phosphorylation of the Syk tyrosine kinase. syk-/- progenitors are similarly defective in osteoclast development and bone resorption. Intact SH2-domains of Syk, introduced by retroviral transduction, are required for functional reconstitution of syk-/- osteoclasts, whereas intact ITAM-domains on DAP12 are required for reconstitution of DAP12-/- FcRγ-/- cells. These data indicate that recruitment of Syk to phosphorylated ITAMs is critical for osteoclastogenesis. Although DAP12 appears to be primarily responsible for osteoclast differentiation in cultures directly stimulated with macrophage-colony stimulating factor and receptor activator of NF-κB ligand cytokines, DAP12 and FcRγ have overlapping roles in supporting osteoclast development in osteoblast–osteoclast cocultures, which mirrors their overlapping functions in vivo. These results provide new insight into the biology of osteoclasts and suggest novel therapeutic targets in diseases of bony remodeling. PMID:15073337

  11. Activation of protein phosphatase 2A in FLT3+ acute myeloid leukemia cells enhances the cytotoxicity of FLT3 tyrosine kinase inhibitors

    PubMed Central

    Lee, Erwin M.; Harrison, Celeste; Kahl, Richard; Flanagan, Hayley; Panicker, Nikita; Mashkani, Baratali; Don, Anthony S.; Morris, Jonathan; Toop, Hamish; Lock, Richard B.; Powell, Jason A.; Thomas, Daniel; Guthridge, Mark A.; Moore, Andrew; Ashman, Leonie K.; Skelding, Kathryn A.; Enjeti, Anoop; Verrills, Nicole M.

    2016-01-01

    Constitutive activation of the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3), via co-expression of its ligand or by genetic mutation, is common in acute myeloid leukemia (AML). In this study we show that FLT3 activation inhibits the activity of the tumor suppressor, protein phosphatase 2A (PP2A). Using BaF3 cells transduced with wildtype or mutant FLT3, we show that FLT3-induced PP2A inhibition sensitizes cells to the pharmacological PP2A activators, FTY720 and AAL(S). FTY720 and AAL(S) induced cell death and inhibited colony formation of FLT3 activated cells. Furthermore, PP2A activators reduced the phosphorylation of ERK and AKT, downstream targets shared by both FLT3 and PP2A, in FLT3/ITD+ BaF3 and MV4-11 cell lines. PP2A activity was lower in primary human bone marrow derived AML blasts compared to normal bone marrow, with blasts from FLT3-ITD patients displaying lower PP2A activity than WT-FLT3 blasts. Reduced PP2A activity was associated with hyperphosphorylation of the PP2A catalytic subunit, and reduced expression of PP2A structural and regulatory subunits. AML patient blasts were also sensitive to cell death induced by FTY720 and AAL(S), but these compounds had minimal effect on normal CD34+ bone marrow derived monocytes. Finally, PP2A activating compounds displayed synergistic effects when used in combination with tyrosine kinase inhibitors in FLT3-ITD+ cells. A combination of Sorafenib and FTY720 was also synergistic in the presence of a protective stromal microenvironment. Thus combining a PP2A activating compound and a FLT3 inhibitor may be a novel therapeutic approach for treating AML. PMID:27329844

  12. An immunoreceptor tyrosine activation motif in the mouse mammary tumor virus envelope protein plays a role in virus-induced mammary tumors.

    PubMed

    Ross, Susan R; Schmidt, John W; Katz, Elad; Cappelli, Laura; Hultine, Stacy; Gimotty, Phyllis; Monroe, John G

    2006-09-01

    Mouse mammary tumor virus (MMTV) induces breast cancer with almost 100% efficiency in susceptible strains through insertional activation of protooncogenes, such as members of the wnt and fibroblast growth factor (fgf) families. We previously showed that expression of the MMTV envelope protein (Env) in normal immortalized mammary epithelial cells grown in three-dimensional cultures caused their morphological transformation, and that this phenotype depended on an immunoreceptor tyrosine-based activation motif (ITAM) present in Env and signaling through the Syk tyrosine kinase (E. Katz, M. H. Lareef, J. C. Rassa, S. M. Grande, L. B. King, J. Russo, S. R. Ross, and J. G. Monroe, J. Exp. Med. 201:431-439, 2005). Here, we examined the role of the Env protein in virus-induced mammary tumorigenesis in vivo. Similar to the effect seen in vitro, Env expression in the mammary glands of transgenic mice bearing either full-length wild-type provirus or only Env transgenes showed increased lobuloalveolar budding. Introduction of the ITAM mutation into the env of an infectious, replication-competent MMTV or into MMTV/murine leukemia virus pseudotypes had no effect on incorporation of Env into virus particles or on in vitro infectivity. Moreover, replication-competent MMTV bearing the ITAM mutation in Env infected lymphoid and mammary tissue at the same level as wild-type MMTV and was transmitted through milk. However, mammary tumor induction was greatly attenuated, and the pattern of oncogene activation was altered. Taken together, these studies indicate that the MMTV Env protein participates in mammary epithelial cell transformation in vivo and that this requires a functional ITAM in the envelope protein.

  13. PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena.

    PubMed

    Hughes, Shannon K; Oudin, Madeleine J; Tadros, Jenny; Neil, Jason; Del Rosario, Amanda; Joughin, Brian A; Ritsma, Laila; Wyckoff, Jeff; Vasile, Eliza; Eddy, Robert; Philippar, Ulrike; Lussiez, Alisha; Condeelis, John S; van Rheenen, Jacco; White, Forest; Lauffenburger, Douglas A; Gertler, Frank B

    2015-11-01

    During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5' inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena(INV) is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor-induced signaling. Disruption of this attenuation by Mena(INV) sensitizes tumor cells to low-growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes.

  14. Tyrosine Kinase Inhibitors in Lung Cancer

    PubMed Central

    Thomas, Anish; Rajan, Arun; Giaccone, Giuseppe

    2012-01-01

    SYNOPSIS ‘Driver mutations’ are essential for carcinogenesis as well as tumor progression as they confer a selective growth advantage to cancer cells. Identification of driver mutations in growth related protein kinases, especially tyrosine kinases have led to clinical development of an array of tyrosine kinase inhibitors in various malignancies, including lung cancer. Inhibition of epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinases have proven to be of meaningful clinical benefit, while inhibition of several other tyrosine kinases have been of limited clinical benefit, thus far. An improved understanding of tyrosine kinase biology has also led to faster drug development, identification of resistance mechanisms and ways to overcome resistance. In this review, we discuss the clinical data supporting the use and practical aspects of management of patients on epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinase inhibitors. PMID:22520981

  15. An essential tyrosine residue of Aspergillus polygalacturonase.

    PubMed

    Stratilová, E; Dzúrová, M; Markovic, O; Jörnvall, H

    1996-03-11

    Based on strict conservation of a tyrosine residue in 24 polygalacturonases, tyrosine modification was assessed in two different forms of the Aspergillus enzyme. The second subform was unknown in structure but submitted to sequence analysis and was found also to have the conserved tyrosine residue. Results of chemical modifications are consistent in showing inactivation of the proteins with all tyrosine-reactive agents tested, acetic anhydride, N-acetyl imidazole, and tetranitromethane. Furthermore, after acetylation, regeneration of enzyme activity was possible with hydroxylamine. Spectrophotometric pH titration showed that one accessible tyrosine residue is ionized at pH 9.3-9.5, whereas the remaining, masked residues are all ionized at pH 10.5. It is concluded that one tyrosine residue is catalytically important, in agreement with the inactivation and reactivation data, that this residue is accessible, and that it is likely to correspond to the strictly conserved residue observed in all forms.

  16. Role of the protein tyrosine phosphatase SHP-1 (Src homology phosphatase-1) in the regulation of interleukin-3-induced survival, proliferation and signalling.

    PubMed Central

    Paling, Nicholas R D; Welham, Melanie J

    2002-01-01

    The tyrosine phosphatase SHP-1 (Src homology phosphatase-1) has been widely implicated as a negative regulator of signalling in immune cells. We have investigated in detail the role of SHP-1 in interleukin-3 (IL-3) signal transduction by inducibly expressing wild-type (WT), C453S (substrate-trapping) and R459M (catalytically inactive) forms of SHP-1 in the IL-3-dependent cell line BaF/3. Expression of WT SHP-1 had little impact on IL-3-induced proliferation, but enhanced apoptosis following IL-3 withdrawal. Expression of R459M SHP-1 increased the proliferative response of BaF/3 cells to IL-3 and increased cell survival at low doses of IL-3 and following IL-3 withdrawal. Investigation into the biochemical consequences resulting from expression of these SHP-1 variants demonstrated that the beta chain of the IL-3 receptor (Aic2A) was hypo-phosphorylated in cells expressing WT SHP-1 and hyper-phosphorylated in those expressing R459M SHP-1. Further, ectopic expression of the trapping mutant, C453S SHP-1, protected Aic2A from dephosphorylation, suggesting that Aic2A is a SHP-1 substrate in BaF/3 cells. Examination of overall levels of tyrosine phosphorylation demonstrated that they were not perturbed in these transfectants. Activation-specific phosphorylation of STAT (signal transducer and activator of transcription) 5a/b, protein kinase B and ERK (extracellular-signal-regulated kinase)-1 and -2 was also unaffected by expression of WT or R459M SHP-1. However, overall levels of IL-3-induced tyrosine phosphorylation of STAT5 were reduced upon expression of WT SHP-1 and increased when R459M SHP-1 was expressed, consistent with STAT5 being a potential SHP-1 substrate. These results demonstrate that SHP-1 acts to negatively regulate IL-3-driven survival and proliferation, potentially via regulation of tyrosine phosphorylation of Aic2A and STAT5. PMID:12220225

  17. Polyclonal antibody to soman-tyrosine

    PubMed Central

    Li, Bin; Duysen, Ellen G.; Froment, Marie-Thérèse; Masson, Patrick; Nachon, Florian; Jiang, Wei; Schopfer, Lawrence M.; Thiele, Geoffrey M.; Klassen, Lynell W.; Cashman, John; Williams, Gareth R.; Lockridge, Oksana

    2013-01-01

    Soman forms a stable, covalent bond with tyrosine 411 of human albumin, with tyrosines 257 and 593 in human transferrin, and with tyrosine in many other proteins. The pinacolyl group of soman is retained, suggesting that pinacolyl methylphosphonate bound to tyrosine could generate specific antibodies. Tyrosine in the pentapeptide RYGRK was covalently modified with soman simply by adding soman to the peptide. The phosphonylated-peptide was linked to keyhole limpet hemocyanin, and the conjugate was injected into rabbits. The polyclonal antiserum recognized soman-labeled human albumin, soman-mouse albumin, and soman human transferrin, but not non-phosphonylated control proteins. The soman-labeled tyrosines in these proteins are surrounded by different amino acid sequences, suggesting that the polyclonal recognizes soman-tyrosine independent of the amino acid sequence. Antiserum obtained after 4 antigen injections over a period of 18 weeks was tested in a competition ELISA where it had an IC50 of 10−11 M. The limit of detection on Western blots was 0.01 μg (15 picomoles) of soman-labeled albumin. In conclusion, a high-affinity, polyclonal antibody that specifically recognizes soman adducts on tyrosine in a variety of proteins has been produced. Such an antibody could be useful for identifying secondary targets of soman toxicity. PMID:23469927

  18. A (1)H NMR metabolic profiling to the assessment of protein tyrosine phosphatase 1B role in liver regeneration after partial hepatectomy.

    PubMed

    Samino, Sara; Revuelta-Cervantes, Jesús; Vinaixa, Maria; Rodríguez, Miguel Ángel; Valverde, Angela M; Correig, Xavier

    2013-04-01

    Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the tyrosine kinase growth factor signaling pathway, which is involved in major physiological mechanisms such as liver regeneration. We investigate early hepatic metabolic events produced by partial hepatectomy (PHx) for PTP1B deficient (PTP1B KO) and wild type (WT) mice using proton nuclear magnetic resonance spectroscopy. Metabolic response of the two genotypes produced 24 h upon PHx is compared using magic angle spinning high-resolution nuclear magnetic resonance ((1)H-HR-MAS-NMR) on intact liver tissues. In addition, genotype-associated metabolic profile changes were monitored during the first 48 h after PHx using high-resolution nuclear magnetic resonance ((1)H-HR-NMR) on liver extracts. A marked increase of lipid-related signals in regenerating livers was observed after 24 h PHx in either intact tissues or liver extracts studies. In spite of this common initial metabolic response, results obtained 48 h after PHx on liver extracts indicate a genotype-differential metabolic pattern. This metabolic pattern resulted in line with well known regenerative features such as more sustained cell proliferation, a better management of lipids as energy fuel and lessened liver injury for PTP1B KO mice as compared to WT. Taken together, these findings suggest the metabolic basis to the pivotal role of PTP1B in liver regeneration.

  19. New compounds from acid hydrolyzed products of the fruits of Momordica charantia L. and their inhibitory activity against protein tyrosine phosphatas 1B.

    PubMed

    Zeng, Ke; He, Yan-Ni; Yang, Di; Cao, Jia-Qing; Xia, Xi-Chun; Zhang, Shi-Jun; Bi, Xiu-Li; Zhao, Yu-Qing

    2014-06-23

    Four new cucurbitane-type triterpene sapogenins, compounds 1-4, together with other eight known compounds were isolated from the acid-hydrolyzed fruits extract of Momordica charantia L. Their chemical structures were established by NMR, mass spectrometry and X-ray crystallography. Compounds 1-7 and 9-12 were evaluated for their inhibitory activities toward protein tyrosine phosphatase 1B (PTP1B), a tyrosine phosphatase that has been implicated as a key target for therapy against type II diabetes. Compounds 1, 2, 4, 7 and 9 were shown inhibitory activities of 77%, 62%, 62% 60% and 68% against PTP1B, respectively. All of these tested compounds were exhibited higher PTP1B inhibition activities than that of the Na3VO4, a known PTP1B inhibitor used as positive control in present study. Structure activity relationship (SAR) analysis indicated that the inhibition activity of PTP1B was associated with the presence and number of -OH groups.

  20. Dynamic quantification of intracellular calcium and protein tyrosine phosphorylation in cryopreserved boar spermatozoa during short-time incubation with oviductal fluid.

    PubMed

    Kumaresan, A; González, R; Johannisson, A; Berqvist, A-S

    2014-11-01

    Freshly ejaculated boar spermatozoa require several hours of exposure to capacitating conditions to undergo capacitation. We hypothesized that cryopreserved boar spermatozoa might elicit a capacitation response after a relatively shorte