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Sample records for metalloproteinase tyrosine phosphorylation

  1. Induction of matrix metalloproteinase-2 by tenascin-X deficiency is mediated through the c-Jun N-terminal kinase and protein tyrosine kinase phosphorylation pathway

    International Nuclear Information System (INIS)

    Matsumoto, Ken-ichi; Minamitani, Takeharu; Orba, Yasuko; Sato, Mami; Sawa, Hirofumi; Ariga, Hiroyoshi

    2004-01-01

    The results of our previous study showed that tumor invasion and metastasis are promoted in extracellular matrix (ECM) tenascin-X-deficient (TNX-/-) mice via increased expression of matrix metalloproteinases (MMPs). However, little is known about the relationship between TNX deficiency and activation of MMP genes. In this study, we investigated the molecular mechanism by which TNX deficiency activates the MMP-2 gene. We examined the intracellular signaling pathways that regulate gene expression of the proteinase in isolated fibroblasts. Results of gelatin zymography showed that MMP-2 was induced to a greater extent in TNX-/- fibroblasts embedded in type I collagen than in wild-type fibroblasts. RT-PCR analysis revealed that the increased level of MMP-2 expression was caused at the transcription level. Conversely, stable overexpression of TNX in a fibroblast cell line reduced MMP-2 expression and suppressed MMP-2 promoter activity. In addition, treatment of TNX-/- fibroblasts with SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and genistein, a tyrosine kinase inhibitor, suppressed the increased level of proMMP-2 and increased MMP-2 promoter activity in TNX-/- fibroblasts. Furthermore, increased activation of JNK and tyrosine phosphorylation of certain proteins were observed in TNX-/- fibroblasts. These findings suggest that induction of MMP-2 by TNX deficiency is mediated, at least in part, through the JNK and protein tyrosine kinase phosphorylation pathway

  2. Tyrosine phosphorylation in human lymphomas

    NARCIS (Netherlands)

    Haralambieva, E; Jones, M.; Roncador, GM; Cerroni, L; Lamant, L; Ott, G; Rosenwald, A; Sherman, C; Thorner, P; Kusec, R; Wood, KM; Campo, E; Falini, B; Ramsay, A; Marafioti, T; Stein, H; Kluin, PM; Pulford, K; Mason, DY

    2002-01-01

    In a previous study, we showed that the high level of protein tyrosine phosphorylation present in lymphomas containing an anaplastic lymphoma kinase (ALK) can be demonstrated in routinely processed paraffin tissue sections using immunolabelling techniques. In the present study we investigated

  3. Tyrosine phosphorylation in signal transduction

    International Nuclear Information System (INIS)

    Roberts, T.M.; Kaplan, D.; Morgan, W.; Keller, T.; Mamon, H.; Piwnica-Worms, H.; Druker, B.; Whitman, M.; Morrison, D.; Cohen, B.; Schaffhausen, B.; Cantley, L.; Rapp, U.

    1988-01-01

    Recent work has focused on the elucidation of the mechanisms by which membrane-bound tyrosine kinases transmit signals within the cell. To examine the role of tyrosine phosphorylation the authors have employed the following strategy. First, they have utilized antibodies to phosphotyrosine (anti-P.Tyr) to identify candidate substrates of various tyrosine kinases, such as pp60 c-src , the CSF- receptor, or the platelet-derived growth factor (PDGF) receptor. Second, they have attempted to characterize the biochemical properties of the putative substrates and to determine in what manner these properties are modified by phosphorylation on tyrosine residues. In this endeavor, they are recapitulating the classic biochemical analysis used to study the effect of kinases on metabolism. The final portion of our work consists of using modern molecular biological strategies to clone the genes or cDNAs for the substrates and overproduce the relevant proteins for studies in vitro in defined systems. This paper describes the first and second aspects of this strategy, the identification and characterization of novel substrate molecules

  4. Protein-Tyrosine Phosphorylation in Bacillus subtilis

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Bottini, N.

    2005-01-01

    of protein-tyrosine phosphorylation. We discuss the approaches currently used to chart this network: ranging from studies of substrate specifi city and the physiological role of tyrosine phosphorylation of individual enzymes to the global approaches at the level of systems biology....... on protein-tyrosine phosphorylation in this gram-positive model organism. With its two kinases, two kinase modulators, three phosphatases and at least four different tyrosine-phosphorylated substrates, B. subtilis is the bacterium with the highest number of presently known participants in the global network...

  5. Dityrosine formation is impaired by tyrosine phosphorylation.

    Science.gov (United States)

    Christian, S; Bernhard, G; Patrizia, R; Brigitte, M

    1992-10-15

    Using pure tyrosine and phosphotyrosine we have recently shown that phosphotyrosine is unable to form peroxidase catalyzed dimers (1989, FEBS Lett. 255, 395-397). In the present report, the effect of phosphotyrosine residues within a protein structure on dityrosine formation was studied using casein as a model protein. Dephosphorylation of casein resulted in a dose and time dependent increased synthesis of dityrosines following treatment with peroxidase/H2O2. The extent of crosslink formation was inversely related to the amount of phosphorylated tyrosine residues as quantitated by immunoblotting. Thus, phosphorylation of tyrosine residues could play a regulatory role in protein-crosslinking where dityrosine bonds are involved.

  6. SH3 domain tyrosine phosphorylation--sites, role and evolution.

    Directory of Open Access Journals (Sweden)

    Zuzana Tatárová

    Full Text Available BACKGROUND: SH3 domains are eukaryotic protein domains that participate in a plethora of cellular processes including signal transduction, proliferation, and cellular movement. Several studies indicate that tyrosine phosphorylation could play a significant role in the regulation of SH3 domains. RESULTS: To explore the incidence of the tyrosine phosphorylation within SH3 domains we queried the PhosphoSite Plus database of phosphorylation sites. Over 100 tyrosine phosphorylations occurring on 20 different SH3 domain positions were identified. The tyrosine corresponding to c-Src Tyr-90 was by far the most frequently identified SH3 domain phosphorylation site. A comparison of sequences around this tyrosine led to delineation of a preferred sequence motif ALYD(Y/F. This motif is present in about 15% of human SH3 domains and is structurally well conserved. We further observed that tyrosine phosphorylation is more abundant than serine or threonine phosphorylation within SH3 domains and other adaptor domains, such as SH2 or WW domains. Tyrosine phosphorylation could represent an important regulatory mechanism of adaptor domains. CONCLUSIONS: While tyrosine phosphorylation typically promotes signaling protein interactions via SH2 or PTB domains, its role in SH3 domains is the opposite - it blocks or prevents interactions. The regulatory function of tyrosine phosphorylation is most likely achieved by the phosphate moiety and its charge interfering with binding of polyproline helices of SH3 domain interacting partners.

  7. Tyrosine phosphorylation of Rab7 by Src kinase.

    Science.gov (United States)

    Lin, Xiaosi; Zhang, Jiaming; Chen, Lingqiu; Chen, Yongjun; Xu, Xiaohui; Hong, Wanjin; Wang, Tuanlao

    2017-07-01

    The small molecular weight GTPase Rab7 is a key regulator for late endosomal/lysosomal membrane trafficking, it was known that Rab7 is phosphorylated, but the corresponding kinase and the functional regulation of Rab7 phosphorylation remain unclear. We provide evidence here that Rab7 is a substrate of Src kinase, and is tyrosine-phosphorylated by Src, withY183 residue of Rab7 being the optimal phosphorylation site for Src. Further investigations demonstrated that the tyrosine phosphorylation of Rab7 depends on the guanine nucleotide binding activity of Rab7 and the activity of Src kinase. The tyrosine phosphorylation of Rab7 is physiologically induced by EGF, and impairs the interaction of Rab7 with RILP, consequently inhibiting EGFR degradation and sustaining Akt signaling. These results suggest that the tyrosine phosphorylation of Rab7 may be involved in coordinating membrane trafficking and cell signaling. Copyright © 2017. Published by Elsevier Inc.

  8. Cytochrome c Is Tyrosine 97 Phosphorylated by Neuroprotective Insulin Treatment

    Czech Academy of Sciences Publication Activity Database

    Sanderson, T. H.; Mahapatra, G.; Pecina, Petr; Ji, Q.; Yu, K.; Sinkler, Ch.; Varughese, A.; Kumar, R.; Bukowski, M. J.; Tousignant, R. N.; Salomon, A. R.; Lee, I.; Hüttemann, M.

    2013-01-01

    Roč. 8, č. 11 (2013), e78627 E-ISSN 1932-6203 Institutional support: RVO:67985823 Keywords : cytochrome c * tyrosine phosphorylation * brain ischemia * insulin Subject RIV: ED - Physiology Impact factor: 3.534, year: 2013

  9. Protein-Tyrosine Phosphorylation in Bacillus subtilis

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Bottini, N.

    2005-01-01

    phosphorylation, indicating that this post-translational modifi cation could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this fi eld was done in Bacillus subtilis , and we here present the current state of knowledge...

  10. Novel tyrosine phosphorylation sites in rat skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MS

    DEFF Research Database (Denmark)

    Zhang, Xiangmin; Højlund, Kurt; Luo, Moulun

    2012-01-01

    Tyrosine phosphorylation plays a fundamental role in many cellular processes including differentiation, growth and insulin signaling. In insulin resistant muscle, aberrant tyrosine phosphorylation of several proteins has been detected. However, due to the low abundance of tyrosine phosphorylation (...

  11. Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

    Directory of Open Access Journals (Sweden)

    Thomas H Sanderson

    Full Text Available Recent advancements in isolation techniques for cytochrome c (Cytc have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

  12. Proteomic analysis of tyrosine phosphorylation during human liver transplantation

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    Boutros Tarek

    2007-01-01

    Full Text Available Abstract Background Ischemia-reperfusion (I/R causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. Results Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH2/SH3 adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. Conclusion Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes.

  13. Protein tyrosine phosphorylation during meiotic divisions of starfish oocytes

    Energy Technology Data Exchange (ETDEWEB)

    Peaucellier, G.; Andersen, A.C.; Kinsey, W.H. (Univ. of Miami School of Medicine, FL (USA))

    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.

  14. Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators

    DEFF Research Database (Denmark)

    Kalantari, Aida; Derouiche, Abderahmane; Shi, Lei

    2015-01-01

    of residue, i.e. serine, threonine, tyrosine and cysteine, is also quite common. The phosphorylation of the ester type (phospho-serine/threonine/tyrosine) is more stable than the aspartate phosphorylation of TCSs. The kinases which catalyse these phosphorylation events (Hanks-type serine/threonine protein...

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

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Macek, B

    2006-01-01

    by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation...

  16. Peptide Microarray Analysis of the Cross-talk Between O-GlcNAcylation and Tyrosine Phosphorylation

    NARCIS (Netherlands)

    Shi, Jie; Tomašič, Tihomir; Sharif, Suhela; Brouwer, Arwin J; Anderluh, Marko; Ruijtenbeek, Rob; Pieters, Roland J

    2017-01-01

    O-GlcNAcylation of proteins regulates important cellular processes. A few reports noted that O-GlcNAcylation exhibits cross-talk with tyrosine phosphorylation. With an activity-based microarray analysis of 256 tyrosine kinase peptide substrates, we found that phosphorylation of 6 peptides by Jak2

  17. Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2

    DEFF Research Database (Denmark)

    Su, J; Batzer, A; Sap, J

    1994-01-01

    Receptor tyrosine phosphatases (R-PTPases) have generated interest because of their suspected involvement in cellular signal transduction. The adaptor protein Grb2 has been implicated in coupling receptor tyrosine kinases to Ras. We report that a ubiquitous R-PTPase, R-PTP-alpha, is tyrosine......-phosphorylated and associated in vivo with the Grb2 protein. This association can be reproduced in stably and transiently transfected cells, as well as in vitro using recombinant Grb2 protein. Association requires the presence of an intact SH2 domain in Grb2, as well as tyrosine phosphorylation of R-PTP-alpha. This observation...... links a receptor tyrosine phosphatase with a key component of a central cellular signalling pathway and provides a basis for addressing R-PTP-alpha function....

  18. Characterization of the hypertonically induced tyrosine phosphorylation of erythrocyte band 3.

    Science.gov (United States)

    Minetti, G; Seppi, C; Ciana, A; Balduini, C; Low, P S; Brovelli, A

    1998-01-01

    Human erythrocyte band 3 becomes rapidly phosphorylated on tyrosine residues after exposure of erythrocytes to hypertonic conditions. The driving force for this phosphorylation reaction seems to be a decrease in cell volume, because (1) changes in band 3 phosphotyrosine content accurately track repeated changes in erythrocyte volume through several cycles of swelling and shrinking; (2) the level of band 3 phosphorylation is independent of the osmolyte employed but strongly sensitive to the magnitude of cell shrinkage; and (3) exposure of erythrocytes to hypertonic buffers under conditions in which intracellular osmolarity increases but volume does not change (nystatin-treated cells) does not promote an increase in tyrosine phosphorylation. We hypothesize that shrinkage-induced tyrosine phosphorylation results either from an excluded-volume effect, stemming from an increase in intracellular crowding, or from changes in membrane curvature that accompany the decrease in cell volume. Although the net phosphorylation state of band 3 is shown to be due to a delicate balance between a constitutively active tyrosine phosphatase and constitutively active tyrosine kinase, the increase in phosphorylation during cell shrinkage was demonstrated to derive specifically from an activation of the latter. Further, a peculiar inhibition pattern of the volume-sensitive erythrocyte tyrosine kinase that matched that of p72syk, a tyrosine kinase already known to associate with band 3 in vivo, suggested the involvement of this kinase in the volume-dependent response. PMID:9761728

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

    Science.gov (United States)

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

    2009-01-01

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

  20. Reciprocal regulation of C-Maf tyrosine phosphorylation by Tec and Ptpn22.

    Science.gov (United States)

    Liu, Chih-Chun; Lai, Chen-Yen; Yen, Wei-Feng; Lin, Yu-Hsien; Chang, Hui-Hsin; Tai, Tzong-Shyuan; Lu, Yu-Jung; Tsao, Hsiao-Wei; Ho, I-Cheng; Miaw, Shi-Chuen

    2015-01-01

    C-Maf plays an important role in regulating cytokine production in TH cells. Its transactivation of IL-4 is optimized by phosphorylation at Tyr21, Tyr92, and Tyr131. However, the molecular mechanism regulating its tyrosine phosphorylation remains unknown. In this study, we demonstrate that Tec kinase family member Tec, but not Rlk or Itk, is a tyrosine kinase of c-Maf and that Tec enhances c-Maf-dependent IL-4 promoter activity. This effect of Tec is counteracted by Ptpn22, which physically interacts with and facilitates tyrosine dephosphorylation of c-Maf thereby attenuating its transcriptional activity. We further show that phosphorylation of Tyr21/92/131 of c-Maf is also critical for its recruitment to the IL-21 promoter and optimal production of this cytokine by TH17 cells. Thus, manipulating tyrosine phosphorylation of c-Maf through its kinases and phosphatases can have significant impact on TH cell-mediated immune responses.

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

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-09-01

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

  2. Calcium/phospholipid-dependent protein kinase (protein kinase C) phosphorylates and activates tyrosine hydroxylase.

    OpenAIRE

    Albert, K A; Helmer-Matyjek, E; Nairn, A C; Müller, T H; Haycock, J W; Greene, L A; Goldstein, M; Greengard, P

    1984-01-01

    Protein kinase C, purified to homogeneity, was found to phosphorylate and activate tyrosine hydroxylase that had been partially purified from pheochromocytoma PC 12 cells. These actions of protein kinase C required the presence of calcium and phospholipid. This phosphorylation of tyrosine hydroxylase reduced the Km for the cofactor 6-methyltetrahydropterine from 0.45 mM to 0.11 mM, increased the Ki for dopamine from 4.2 microM to 47.5 microM, and produced no change in the Km for tyrosine. Lit...

  3. Tyrosine Phosphorylation of Caspase-8 Abrogates Its Apoptotic Activity and Promotes Activation of c-Src

    Science.gov (United States)

    Tsang, Jennifer LY; Jia, Song Hui; Parodo, Jean; Plant, Pamela; Lodyga, Monika; Charbonney, Emmanuel; Szaszi, Katalin; Kapus, Andras; Marshall, John C.

    2016-01-01

    Src family tyrosine kinases (SFKs) phosphorylate caspase-8A at tyrosine (Y) 397 resulting in suppression of apoptosis. In addition, the phosphorylation of caspase-8A at other sites including Y465 has been implicated in the regulation of caspase-8 activity. However, the functional consequences of these modifications on caspase-8 processing/activity have not been elucidated. Moreover, various Src substrates are known to act as potent Src regulators, but no such role has been explored for caspase-8. We asked whether the newly identified caspase-8 phosphorylation sites might regulate caspase-8 activation and conversely, whether caspase-8 phosphorylation might affect Src activity. Here we show that Src phosphorylates caspase-8A at multiple tyrosine sites; of these, we have focused on Y397 within the linker region and Y465 within the p12 subunit of caspase-8A. We show that phosphomimetic mutation of caspase-8A at Y465 prevents its cleavage and the subsequent activation of caspase-3 and suppresses apoptosis. Furthermore, simultaneous phosphomimetic mutation of caspase-8A at Y397 and Y465 promotes the phosphorylation of c-Src at Y416 and increases c-Src activity. Finally, we demonstrate that caspase-8 activity prevents its own tyrosine phosphorylation by Src. Together these data reveal that dual phosphorylation converts caspase-8 from a pro-apoptotic to a pro-survival mediator. Specifically, tyrosine phosphorylation by Src renders caspase-8 uncleavable and thereby inactive, and at the same time converts it to a Src activator. This novel dynamic interplay between Src and caspase-8 likely acts as a potent signal-integrating switch directing the cell towards apoptosis or survival. PMID:27101103

  4. Boar sperm storage capacity of BTS and Androhep Plus: viability, motility, capacitation, and tyrosine phosphorylation.

    Science.gov (United States)

    Dubé, Charlotte; Beaulieu, Martin; Reyes-Moreno, Carlos; Guillemette, Christine; Bailey, Janice L

    2004-09-01

    Androhep Plus, a long-term extender (up to 7 days) and Beltsville Thawing Solution (BTS), a short-term extender (up to 3 days), are commonly used for liquid storage of porcine semen. To test the hypothesis that modifications in sperm viability, motility, chlortetracycline (CTC) fluorescence patterns, and protein tyrosine phosphorylation occur during semen storage in extenders, we compared these end points at different periods of storage in either Androhep Plus or BTS. Sperm from five boars were assessed daily over 12 days of storage (n = 5 ejaculates from different boars). Viability was not different (P BTS (P BTS. A tyrosine-phosphorylated protein of Mr 21,000 appeared after 10 days in sperm incubated in BTS, and was identified as a phospholipid hydroperoxide glutathione peroxidase. Therefore, modifications in viability, motility, CTC fluorescence patterns, and sperm protein tyrosine phosphorylation were apparent during sperm storage in extenders; these may affect the fertilizing capacity of the semen.

  5. Nicotinic stimulation of catecholamine synthesis and tyrosine hydroxylase phosphorylation in cervine adrenal medullary chromaffin cells.

    Science.gov (United States)

    Knowles, P J; Douglas, S A; Bunn, S J

    2011-03-01

    The synthesis and secretion of catecholamines by the adrenal medulla is of major importance in the stress response. Tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, has been extensively studied in adrenal medullary chromaffin cells from a number of species. Cervine chromaffin cells are of interest because the deer is known to be a relatively stress-prone reactive species. We report the first characterisation of tyrosine hydroxylase regulation in cervine chromaffin cells. Nicotinic receptor activation resulted in a time- and concentration-dependent increase in catecholamine synthesis, which was significantly reduced by the extracellular signal-regulated kinase (ERK)1/2 signalling pathway inhibitor PD98059 and the calcium/calmodulin protein kinase II inhibitor KN-93, but not by H89 or bisindolylmaleimide I, inhibitors of protein kinase A and C, respectively. Nicotinic stimulation also increased the phosphorylation of ERK1/2 and tyrosine hydroxylase. This latter response occurred on serine residues 19, 31 and 40 of the enzyme. The nicotinic-induced phosphorylation of ERK1/2 and serine 31 of tyrosine hydroxylase was suppressed by PD98059 but not bisindolylmaleimide I. These data indicate that nicotinic stimulation of tyrosine hydroxylase involves the phosphorylation of serine 31 via an ERK1/2-dependent, protein kinase C-independent pathway. Protein kinase C activation by phorbol 12-myristate 13-acetate also caused an ERK1/2-dependent increase in the serine 31 phosphorylation of tyrosine hydroxylase but, in contrast to the nicotinic response, was not accompanied by an increase in enzyme activity. Thus, ERK1/2-mediated serine 31 phosphorylation of tyrosine hydroxylase appears necessary but not sufficient for nicotinic activation of catecholamine synthesis in cervine chromaffin cells. These data present potentially important similarities and differences between the regulation of catecholamine synthesis in cervine and the more widely studied

  6. Protein tyrosine phosphorylation is involved in osmoregulation of ionic conductances

    NARCIS (Netherlands)

    B.C. Tilly (Bernard); N. van den Berghe (Nina); L.G. Tertoolen; M.J. Edixhoven (Marcel); H.R. de Jonge (Hugo)

    1993-01-01

    textabstractUsing the human Intestine 407 cell line as a model, we investigated a possible role for tyrosine kinase(s) in regulating the ion efflux pathways induced by hyposmotic stimulation (regulatory volume decrease, RVD). Pretreatment of 125I(-)-and 86Rb(+)-loaded

  7. Tyrosine phosphorylation of the human guanylyl cyclase C receptor

    Indian Academy of Sciences (India)

    Unknown

    5, in Luria Bertani broth ..... bacteria has been used by Larose et al (1993) to identify residues in the platelet derived growth factor ... GCC by EphB1 within bacterial cells suggests that GCC may be a substrate for the Eph family of tyrosine ...

  8. Insulin stimulates the tyrosine phosphorylation of a Mr = 160,000 glycoprotein in adipocyte plasma membranes

    International Nuclear Information System (INIS)

    Yu, K.T.; Khalaf, N.; Czech, M.P.

    1986-01-01

    In an attempt to identify putative substrates for the insulin receptor kinase, adipocyte plasma membranes were incubated with [γ- 32 P]ATP in the presence and absence of insulin. Insulin stimulates the tyrosine phosphorylation of its receptor β subunit but does not detectably alter the phosphorylation of other membrane proteins. In contrast, when plasma membranes from insulin-treated adipocytes are phosphorylated, the 32 P-labeling of a Mr=160,000 species (p160) and insulin receptor β subunit are markedly increased when compared to controls. p160 exhibits a rapid response (max. at 1 min) and high sensitivity (ED 50 = 2 x 10 -10 M) to insulin. The stimulatory effect of insulin on the phosphorylation of p160 is rapidly reversed following the addition of anti-insulin serum. Cold chase experiments indicate that insulin promotes the phosphorylation of p160 rather than inhibiting its dephosphorylation. p160 is a glycoprotein as evidenced by its adsorption to immobilized lectins and does not represent the insulin receptor precursor. The action of insulin on p160 tyrosine phosphorylation is mimicked by concanavalin A but not by EGF and other insulin-like agents such as hydrogen peroxide and vanadate. These results suggest that p160 tyrosine phosphorylation is an insulin receptor-mediated event and may participate in signalling by the insulin receptor

  9. Tyrosine 402 Phosphorylation of Pyk2 Is Involved in Ionomycin-Induced Neurotransmitter Release

    Science.gov (United States)

    Zhang, Zhao; Zhang, Yun; Mou, Zheng; Chu, Shifeng; Chen, Xiaoyu; He, Wenbin; Guo, Xiaofeng; Yuan, Yuhe; Takahashi, Masami; Chen, Naihong

    2014-01-01

    Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca2+ ionophore, concurrently induced asynchronous neurotransmitter release and phosphorylation of a non-receptor protein tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2), in clonal rat pheochromocytoma PC12 cells and cerebellar granule cells, whereas introduction of Pyk2 siRNA dramatically suppressed ionomycin-induced neurotransmitter release. Further study indicated that Tyr-402 (Y402) in Pyk2, instead of other tyrosine sites, underwent rapid phosphorylation after ionomycin induction in 1 min to 2 min. We demonstrated that the mutant of Pyk2 Y402 could abolish ionomycin-induced dopamine (DA) release by transfecting cells with recombinant Pyk2 and its mutants (Y402F, Y579F, Y580F, and Y881F). In addition, Src inhibition could prolong phosphorylation of Pyk2 Y402 and increase DA release. These findings suggested that Pyk2 was involved in ionomycin-induced neurotransmitter release through phosphorylation of Y402. PMID:24718602

  10. Irreversible AE1 tyrosine phosphorylation leads to membrane vesiculation in G6PD deficient red cells.

    Directory of Open Access Journals (Sweden)

    Antonella Pantaleo

    Full Text Available BACKGROUND: While G6PD deficiency is one of the major causes of acute hemolytic anemia, the membrane changes leading to red cell lysis have not been extensively studied. New findings concerning the mechanisms of G6PD deficient red cell destruction may facilitate our understanding of the large individual variations in susceptibility to pro-oxidant compounds and aid the prediction of the hemolytic activity of new drugs. METHODOLOGY/PRINCIPAL FINDINGS: Our results show that treatment of G6PD deficient red cells with diamide (0.25 mM or divicine (0.5 mM causes: (1 an increase in the oxidation and tyrosine phosphorylation of AE1; (2 progressive recruitment of phosphorylated AE1 in large membrane complexes which also contain hemichromes; (3 parallel red cell lysis and a massive release of vesicles containing hemichromes. We have observed that inhibition of AE1 phosphorylation by Syk kinase inhibitors prevented its clustering and the membrane vesiculation while increases in AE1 phosphorylation by tyrosine phosphatase inhibitors increased both red cell lysis and vesiculation rates. In control RBCs we observed only transient AE1 phosphorylation. CONCLUSIONS/SIGNIFICANCE: Collectively, our findings indicate that persistent tyrosine phosphorylation produces extensive membrane destabilization leading to the loss of vesicles which contain hemichromes. The proposed mechanism of hemolysis may be applied to other hemolytic diseases characterized by the accumulation of hemoglobin denaturation products.

  11. Growth hormone-dependent phosphorylation of tyrosine 333 and/or 338 of the growth hormone receptor

    DEFF Research Database (Denmark)

    VanderKuur, J A; Wang, X; Zhang, L

    1995-01-01

    Many signaling pathways initiated by ligands that activate receptor tyrosine kinases have been shown to involve the binding of SH2 domain-containing proteins to specific phosphorylated tyrosines in the receptor. Although the receptor for growth hormone (GH) does not contain intrinsic tyrosine...

  12. Src drives the Warburg effect and therapy resistance by inactivating pyruvate dehydrogenase through tyrosine-289 phosphorylation

    Science.gov (United States)

    Shenoy, Anitha K.; Lim, Sangbin; Zhang, Ying; Charles, Steve; Tarrash, Miriam; Fu, Xueqi; Kamarajugadda, Sushama; Trevino, Jose G.; Tan, Ming; Lu, Jianrong

    2016-01-01

    The Warburg effect, which reflects cancer cells' preference for aerobic glycolysis over glucose oxidation, contributes to tumor growth, progression and therapy resistance. The restraint on pyruvate flux into mitochondrial oxidative metabolism in cancer cells is in part attributed to the inhibition of pyruvate dehydrogenase (PDH) complex. Src is a prominent oncogenic non-receptor tyrosine kinase that promotes cancer cell proliferation, invasion, metastasis and resistance to conventional and targeted therapies. However, the potential role of Src in tumor metabolism remained unclear. Here we report that activation of Src attenuated PDH activity and generation of reactive oxygen species (ROS). Conversely, Src inhibitors activated PDH and increased cellular ROS levels. Src inactivated PDH through direct phosphorylation of tyrosine-289 of PDH E1α subunit (PDHA1). Indeed, Src was the main kinase responsible for PDHA1 tyrosine phosphorylation in cancer cells. Expression of a tyrosine-289 non-phosphorable PDHA1 mutant in Src-hyperactivated cancer cells restored PDH activity, increased mitochondrial respiration and oxidative stress, decreased experimental metastasis, and sensitized cancer cells to pro-oxidant treatment. The results suggest that Src contributes to the Warburg phenotype by inactivating PDH through tyrosine phosphorylation, and the metabolic effect of Src is essential for Src-driven malignancy and therapy resistance. Combination therapies consisting of both Src inhibitors and pro-oxidants may improve anticancer efficacy. PMID:26848621

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

    Science.gov (United States)

    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. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  14. Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

    Energy Technology Data Exchange (ETDEWEB)

    Coffey, Greg P.; Rajapaksa, Ranjani; Liu, Raymond; Sharpe, Orr; Kuo, Chiung-Chi; Wald Krauss, Sharon; Sagi, Yael; Davis, R. Eric; Staudt, Louis M.; Sharman, Jeff P.; Robinson, William H.; Levy, Shoshana

    2009-06-09

    CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of Blymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. Ezrin colocalized with CD81 and F-actin upon stimulation and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This may be a mechanism explaining the pleiotropic effects induced in response to stimulating cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

  15. Src Inhibits the Hippo Tumor Suppressor Pathway through Tyrosine Phosphorylation of Lats1.

    Science.gov (United States)

    Si, Yuan; Ji, Xinyan; Cao, Xiaolei; Dai, Xiaoming; Xu, Lingyi; Zhao, Hongxia; Guo, Xiaocan; Yan, Huan; Zhang, Haitao; Zhu, Chu; Zhou, Qi; Tang, Mei; Xia, Zongping; Li, Li; Cong, Yu-Sheng; Ye, Sheng; Liang, Tingbo; Feng, Xin-Hua; Zhao, Bin

    2017-09-15

    The Hippo pathway regulates cell proliferation, apoptosis, and stem cell self-renewal, and its inactivation in animal models causes organ enlargement followed by tumorigenesis. Hippo pathway deregulation occurs in many human cancers, but the underlying mechanisms are not fully understood. Here, we report tyrosine phosphorylation of the Hippo pathway tumor suppressor LATS1 as a mechanism underlying its regulation by cell adhesion. A tyrosine kinase library screen identified Src as the kinase to directly phosphorylate LATS1 on multiple residues, causing attenuated Mob kinase activator binding and structural alteration of the substrate-binding pocket in the kinase domain. Cell matrix adhesion activated the Hippo pathway effector transcription coactivator YAP partially through Src-mediated phosphorylation and inhibition of LATS1. Aberrant Src activation abolished the tumor suppressor activity of LATS1 and induced tumorigenesis in a YAP-dependent manner. Protein levels of Src in human breast cancer tissues correlated with accumulation of active YAP dephosphorylated on the LATS1 target site. These findings reveal tyrosine phosphorylation of LATS1 by Src as a novel mechanism of Hippo pathway regulation by cell adhesion and suggest Src activation as an underlying reason for YAP deregulation in tumorigenesis. Cancer Res; 77(18); 4868-80. ©2017 AACR . ©2017 American Association for Cancer Research.

  16. Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation

    DEFF Research Database (Denmark)

    Kirkegaard, Signe Skyum; Lambert, Ian Henry; Gammeltoft, Steen

    2010-01-01

    , it is demonstrated that mpV(pic) increased the volume-sensitive part of the K(+) efflux 1.3 times. To exclude K(+) efflux via a KCl cotransporter, cellular Cl(-) was substituted with NO(3)(-). Also under these conditions K(+) efflux was completely blocked by genistein. Thus tyrosine kinases seem to be involved...... together with Western blotting and antibodies against phosphotyrosines revealed a cell swelling-induced, time-dependent tyrosine phosphorylation of the channel. Even though we found an inhibiting effect of PP2 on RVD, neither Src nor the focal adhesion kinase (FAK) seem to be involved. Inhibitors...

  17. Chlamydia trachomatis serovar L2 induces protein tyrosine phosphorylation during uptake by HeLa cells

    DEFF Research Database (Denmark)

    Birkelund, Svend; Johnsen, H; Christiansen, Gunna

    1994-01-01

    . By use of a monoclonal antibody against phosphotyrosine, we showed that three classes of proteins are tyrosine phosphorylated: a triple band of 68, 66, and 64 kDa, a 97-kDa band, and a 140-kDa band. The phosphorylation could be detected by immunoblotting from 15 min after infection of HeLa cells. We...... inactive. Attachment of EBs to host cells is medicated by a heparan sulfate-like glycosaminoglycan. Following attachment, the EB is internalized within a membrane-bound vesicle, and during the first 8 h of infection the vesicles are transported to a perinuclear location where they aggregate and fuse...

  18. Protein phosphatase 2A is involved in the tyrosine hydroxylase phosphorylation regulated by α-synuclein.

    Science.gov (United States)

    Hua, Gao; Xiaolei, Lan; Weiwei, Yang; Hao, Wang; Yuangang, Zhu; Dongmei, Liu; Yazhuo, Zhang; Hui, Yang

    2015-03-01

    α-Synuclein (α-Syn) plays a crucial role in the pathophysiology of Parkinson's disease (PD), the degeneration of dopaminergic neurons. Previous studies have shown that α-Syn regulates dopamine synthesis by binding to and inhibiting tyrosine hydroxylase (TH). In neurons, protein phosphatases (PPs) play a prominent role in directing signaling toward survival or degeneration. This study was to re-evaluate whether α-Syn could regulate the tyrosine hydroxylase phosphorylation by protein phosphatase-2A (PP2A) in dopaminergic MN9D cells and cortex neurons. Our data demonstrated for the first time that α-Syn stimulates PP2A activity and reduces phosphorylation of TH through regulating the methylation of PP2A in dopaminergic MN9D cells and primary cortex neurons. Increased PP2A activity and reduced phosphorylation of PP2A at Y307 (inactive form of PP2A) were observed in α-Syn overexpression dopaminergic cells (Syn) and primary cortex neurons, and the TH phosphorylation relieved by enhancing PP2A methylation in Syn group could be abated by using PP inhibitors, okadaic acid (OKA). OKA could reduce the cell damage and cell apoptosis induced by α-Syn. Thus our findings may provide an insight into the complicated pathogenesis of PD as well as some clues to the development of novel therapeutic strategies targeting at PP2A.

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

    Science.gov (United States)

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

    2017-03-01

    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. 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. 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. 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 spread the infection.

  20. Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

    International Nuclear Information System (INIS)

    Zhong Li; Li Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.

    2008-01-01

    We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by ∼ 68% and ∼ 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy

  1. Insulin and Metabolic Stress Stimulate Multisite Serine/Threonine Phosphorylation of Insulin Receptor Substrate 1 and Inhibit Tyrosine Phosphorylation*

    Science.gov (United States)

    Hançer, Nancy J.; Qiu, Wei; Cherella, Christine; Li, Yedan; Copps, Kyle D.; White, Morris F.

    2014-01-01

    IRS1 and IRS2 are key substrates of the insulin receptor tyrosine kinase. Mass spectrometry reveals more than 50 phosphorylated IRS1 serine and threonine residues (Ser(P)/Thr(P) residues) in IRS1 from insulin-stimulated cells or human tissues. We investigated a subset of IRS1 Ser(P)/Thr(P) residues using a newly developed panel of 25 phospho-specific monoclonal antibodies (αpS/TmAbIrs1). CHO cells overexpressing the human insulin receptor and rat IRS1 were stimulated with insulin in the absence or presence of inhibitors of the PI3K → Akt → mechanistic target of rapamycin (mTOR) → S6 kinase or MEK pathways. Nearly all IRS1 Ser(P)/Thr(P) residues were stimulated by insulin and significantly suppressed by PI3K inhibition; fewer were suppressed by Akt or mTOR inhibition, and none were suppressed by MEK inhibition. Insulin-stimulated Irs1 tyrosine phosphorylation (Tyr(P)Irs1) was enhanced by inhibition of the PI3K → Akt → mTOR pathway and correlated with decreased Ser(P)-302Irs1, Ser(P)-307Irs1, Ser(P)-318Irs1, Ser(P)-325Irs1, and Ser(P)-346Irs1. Metabolic stress modeled by anisomycin, thapsigargin, or tunicamycin increased many of the same Ser(P)/Thr(P) residues as insulin, some of which (Ser(P)-302Irs1, Ser(P)-307Irs1, and four others) correlated significantly with impaired insulin-stimulated Tyr(P)Irs1. Thus, IRS1 Ser(P)/Thr(P) is an integrated response to insulin stimulation and metabolic stress, which associates with reduced Tyr(P)Irs1 in CHOIR/IRS1 cells. PMID:24652289

  2. Dopamine signaling negatively regulates striatal phosphorylation of Cdk5 at tyrosine 15 in mice.

    Directory of Open Access Journals (Sweden)

    Yukio eYamamura

    2013-02-01

    Full Text Available Striatal functions depend on the activity balance between the dopamine and glutamate neurotransmissions. Glutamate inputs activate cyclin-dependent kinase 5 (Cdk5, which inhibits postsynaptic dopamine signaling by phosphorylating DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, 32 kDa at Thr75 in the striatum. c-Abelson tyrosine kinase (c-Abl is known to phosphorylate Cdk5 at Tyr15 (Tyr15-Cdk5 and thereby facilitates the Cdk5 activity. We here report that Cdk5 with Tyr15 phosphorylation (Cdk5-pTyr15 is enriched in the mouse striatum, where dopaminergic stimulation inhibited phosphorylation of Tyr15-Cdk5 by acting through the D2 class dopamine receptors. Moreover, in the 1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine mouse model, dopamine deficiency caused increased phosphorylation of both Tyr15-Cdk5 and Thr75-DARPP-32 in the striatum, which could be attenuated by administration of L-3,4-dihydroxyphenylalanine and imatinib (STI-571, a selective c-Abl inhibitor. Our results suggest a functional link of Cdk5-pTyr15 with postsynaptic dopamine and glutamate signals through the c-Abl kinase activity in the striatum.

  3. Tyrosine 110 in the measles virus phosphoprotein is required to block STAT1 phosphorylation

    International Nuclear Information System (INIS)

    Devaux, Patricia; Messling, Veronika von; Songsungthong, Warangkhana; Springfeld, Christoph; Cattaneo, Roberto

    2007-01-01

    The measles virus (MV) P gene encodes three proteins: P, an essential polymerase cofactor, and C and V, which have multiple functions including immune evasion. We show here that the MV P protein also contributes to immune evasion, and that tyrosine 110 is required to block nuclear translocation of the signal transducer and activator of transcription factors (STAT) after interferon type I treatment. In particular, MV P inhibits STAT1 phosphorylation. This is shown not only by transient expression but also by reverse genetic analyses based on a new functional infectious cDNA derived from a MV vaccine vial (Moraten strain). Our study also identifies a conserved sequence around P protein tyrosine 110 as a candidate interaction site with a cellular protein

  4. Tyrosine phosphorylation of a 66KD soluble protein and augmentation of lectin induced mitogenesis by DMSO in human T lymphocytes

    International Nuclear Information System (INIS)

    Wedner, H.J.; Bass, G.

    1986-01-01

    The authors have demonstrated that induction of mitogenesis in human T lymphocytes is associated with the tyrosine phosphorylation of a 66KD soluble substrate-TPP 66. Since DMSO has been shown to be a non-specific stimulator of tyrosine protein kinases they have examined the effect of DMSO on both activation and tyrosine phosphorylation in human T cells. Human peripheral blood T lymphocytes were isolated by dextran sedimentation, Ficol/Paque centrifugation and nylon wool filtration. Phosphorylation was performed in cells incubated with [ 32 P] orthophosphate followed by DMSO for 30 min. TPP 66 was identified by 2-D PAGE, autoradiography, and HV electrophoresis of the hydrolyzed protein. Concentrations of DMSO from 1% to 50% induced the tyrosine phosphorylation of TPP 66 with maximal stimulation seen at 20%. DMSO alone did not activate the T cells (measured by [ 3 H] thymidine incorporation) when tested at high concentrations for 30 sec to 10 min. (longer incubations were markedly toxic) or low concentrations for 12 to 48 hrs. Low concentrations of DMSO 0.1%-0.5% did however, markedly augment [ 3 H] thymidine incorporation induced by PHA or Con A. These data suggest that tyrosine phosphorylation of TPP 66 alone may not constitute sufficient signal for the activation sequence to begin but the phosphorylation of this soluble substrate may be a critical factor in the propagation of the activation sequence

  5. Crk Tyrosine Phosphorylation Regulates PDGF-BB-inducible Src Activation and Breast Tumorigenicity and Metastasis.

    Science.gov (United States)

    Kumar, Sushil; Lu, Bin; Davra, Viralkumar; Hornbeck, Peter; Machida, Kazuya; Birge, Raymond B

    2018-01-01

    The activity of Src family kinases (Src being the prototypical member) is tightly regulated by differential phosphorylation on Tyr416 (positive) and Tyr527 (negative), a duet that reciprocally regulates kinase activity. The latter negative regulation of Src on Tyr527 is mediated by C-terminal Src kinase (CSK) that phosphorylates Tyr527 and maintains Src in a clamped negative regulated state by promoting an intramolecular association. Here it is demonstrated that the SH2- and SH3-domain containing adaptor protein CrkII, by virtue of its phosphorylation on Tyr239, regulates the Csk/Src signaling axis to control Src activation. Once phosphorylated, the motif (PIpYARVIQ) forms a consensus sequence for the SH2 domain of CSK to form a pTyr239-CSK complex. Functionally, when expressed in Crk -/- MEFs or in Crk +/+ HS683 cells, Crk Y239F delayed PDGF-BB-inducible Src Tyr416 phosphorylation. Moreover, expression of Crk Y239F in HS683 cells delayed Src kinase activation and suppressed the cell-invasive and -transforming phenotypes. Finally, through loss-of-function and epistasis experiments using CRISPR-Cas9-engineered 4T1 murine breast cancer cells, Crk Tyr239 is implicated in breast cancer tumor growth and metastasis in orthotopic immunocompetent 4T1 mice model of breast adenocarcinoma. These findings delineate a novel role for Crk Tyr239 phosphorylation in the regulation of Src kinases, as well as a potential molecular explanation for a long-standing question as to how Crk regulates the activation of Src kinases. Implications: These findings provide new perspectives on the versatility of Crk in cancer by demonstrating how Crk mechanistically drives, through a tyrosine phosphorylation-dependent manner, tumor growth, and metastasis. Mol Cancer Res; 16(1); 173-83. ©2017 AACR . ©2017 American Association for Cancer Research.

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

    Science.gov (United States)

    Naresh, Sai

    2016-02-01

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

  7. Extracellular phosphorylation of a receptor tyrosine kinase controls synaptic localization of NMDA receptors and regulates pathological pain

    Science.gov (United States)

    Sheffler-Collins, Sean I.; Xia, Nan L.; Henderson, Nathan; Tillu, Dipti V.; Hassler, Shayne; Spellman, Daniel S.; Zhang, Guoan; Neubert, Thomas A.; Price, Theodore J.

    2017-01-01

    Extracellular phosphorylation of proteins was suggested in the late 1800s when it was demonstrated that casein contains phosphate. More recently, extracellular kinases that phosphorylate extracellular serine, threonine, and tyrosine residues of numerous proteins have been identified. However, the functional significance of extracellular phosphorylation of specific residues in the nervous system is poorly understood. Here we show that synaptic accumulation of GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) and pathological pain are controlled by ephrin-B-induced extracellular phosphorylation of a single tyrosine (p*Y504) in a highly conserved region of the fibronectin type III (FN3) domain of the receptor tyrosine kinase EphB2. Ligand-dependent Y504 phosphorylation modulates the EphB-NMDAR interaction in cortical and spinal cord neurons. Furthermore, Y504 phosphorylation enhances NMDAR localization and injury-induced pain behavior. By mediating inducible extracellular interactions that are capable of modulating animal behavior, extracellular tyrosine phosphorylation of EphBs may represent a previously unknown class of mechanism mediating protein interaction and function. PMID:28719605

  8. Aging reveals a role for nigral tyrosine hydroxylase ser31 phosphorylation in locomotor activity generation.

    Directory of Open Access Journals (Sweden)

    Michael F Salvatore

    Full Text Available BACKGROUND: Tyrosine hydroxylase (TH regulates dopamine (DA bioavailability. Its product, L-DOPA, is an established treatment for Parkinson's disease (PD, suggesting that TH regulation influences locomotion. Site-specific phosphorylation of TH at ser31 and ser40 regulates activity. No direct evidence shows that ser40 phosphorylation is the dominating mechanism of regulating TH activity in vivo, and physiologically-relevant stimuli increase L-DOPA biosynthesis independent of ser40 phosphorylation. Significant loss of locomotor activity occurs in aging as in PD, despite less loss of striatal DA or TH in aging compared to the loss associated with symptomatic PD. However, in the substantia nigra (SN, there is equivalent loss of DA or TH in aging and at the onset of PD symptoms. Growth factors increase locomotor activity in both PD and aging models and increase DA bioavailability and ser31 TH phosphorylation in SN, suggesting that ser31 TH phosphorylation status in the SN, not striatum, regulates DA bioavailability necessary for locomotor activity. METHODOLOGY AND PRINCIPAL FINDINGS: We longitudinally characterized locomotor activity in young and older Brown-Norway Fischer 344 F(1 hybrid rats (18 months apart in age at two time periods, eight months apart. The aged group served as an intact and pharmacologically-naïve source of deficient locomotor activity. Following locomotor testing, we analyzed DA tissue content, TH protein, and TH phosphorylation in striatum, SN, nucleus accumbens, and VTA. Levels of TH protein combined with ser31 phosphorylation alone reflected inherent differences in DA levels among the four regions. Measures strictly pertaining to locomotor activity initiation significantly correlated to DA content only in the SN. Nigral TH protein and ser31 phosphorylation together significantly correlated to test subject's maximum movement number, horizontal activity, and duration. CONCLUSIONS/SIGNIFICANCE: Together, these results show ser

  9. Phosphorylation at serine 31 targets tyrosine hydroxylase to vesicles for transport along microtubules.

    Science.gov (United States)

    Jorge-Finnigan, Ana; Kleppe, Rune; Jung-Kc, Kunwar; Ying, Ming; Marie, Michael; Rios-Mondragon, Ivan; Salvatore, Michael F; Saraste, Jaakko; Martinez, Aurora

    2017-08-25

    Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine into l-DOPA, which is the rate-limiting step in the synthesis of catecholamines, such as dopamine, in dopaminergergic neurons. Low dopamine levels and death of the dopaminergic neurons are hallmarks of Parkinson's disease (PD), where α-synuclein is also a key player. TH is highly regulated, notably by phosphorylation of several Ser/Thr residues in the N-terminal tail. However, the functional role of TH phosphorylation at the Ser-31 site (THSer(P)-31) remains unclear. Here, we report that THSer(P)-31 co-distributes with the Golgi complex and synaptic-like vesicles in rat and human dopaminergic cells. We also found that the TH microsomal fraction content decreases after inhibition of cyclin-dependent kinase 5 (Cdk5) and ERK1/2. The cellular distribution of an overexpressed phospho-null mutant, TH1-S31A, was restricted to the soma of neuroblastoma cells, with decreased association with the microsomal fraction, whereas a phospho-mimic mutant, TH1-S31E, was distributed throughout the soma and neurites. TH1-S31E associated with vesicular monoamine transporter 2 (VMAT2) and α-synuclein in neuroblastoma cells, and endogenous THSer(P)-31 was detected in VMAT2- and α-synuclein-immunoprecipitated mouse brain samples. Microtubule disruption or co-transfection with α-synuclein A53T, a PD-associated mutation, caused TH1-S31E accumulation in the cell soma. Our results indicate that Ser-31 phosphorylation may regulate TH subcellular localization by enabling its transport along microtubules, notably toward the projection terminals. These findings disclose a new mechanism of TH regulation by phosphorylation and reveal its interaction with key players in PD, opening up new research avenues for better understanding dopamine synthesis in physiological and pathological states. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Casein kinase 2 dependent phosphorylation of neprilysin regulates receptor tyrosine kinase signaling to Akt.

    Directory of Open Access Journals (Sweden)

    Martin Siepmann

    2010-10-01

    Full Text Available Neprilysin (NEP is a type II membrane metalloproteinase that cleaves physiologically active peptides at the cell surface thus regulating the local concentration of these peptides available for receptor binding and signal transduction. In addition, the cytoplasmic N-terminal domain of NEP interacts with the phosphatase and tensin homologue deleted on chromosome 10 (PTEN thereby regulating intracellular signaling via Akt. Thus, NEP serves dual functions in extracellular and intracellular signal transduction. Here, we show that NEP undergoes phosphorylation at serine residue 6 within the N-terminal cytoplasmic domain. In vitro and cell culture experiments demonstrate that Ser 6 is efficiently phosphorylated by protein kinase CK2. The phosphorylation of the cytoplasmic domain of NEP inhibits its interaction with PTEN. Interestingly, expression of a pseudophosphorylated NEP variant (Ser6Asp abrogates the inhibitory effect of NEP on insulin/insulin-like growth factor-1 (IGF-1 stimulated activation of Akt. Thus, our data demonstrate a regulatory role of CK2 in the interaction of NEP with PTEN and insulin/IGF-1 signaling.

  11. Tyrosine phosphorylation of HSC70 and its interaction with RFC mediates methotrexate resistance in murine L1210 leukemia cells.

    Science.gov (United States)

    Liu, Tuoen; Singh, Ratan; Rios, Zechary; Bhushan, Alok; Li, Mengxiong; Sheridan, Peter P; Bearden, Shawn E; Lai, James C K; Agbenowu, Senyo; Cao, Shousong; Daniels, Christopher K

    2015-02-01

    We previously identified and characterized a 66-68 kDa membrane-associated, tyrosine phosphorylated protein in murine leukemia L1210 cells as HSC70 which is a methotrexate (MTX)-binding protein. In order to further characterize the functional role of HSC70 in regulating MTX resistance in L1210 cells, we first showed that HSC70 colocalizes and interacts with reduced folate carrier (RFC) in L1210 cells by confocal laser scanning microscopy and Duolink in situ proximity ligation assay. The tyrosine phosphorylation status of HSC70 found in the membrane fraction was different from the parental L1210/0 and cisplatin (CDDP)-MTX cross resistant L1210/DDP cells. In MTX-binding assays, HSC70 from L1210/DDP cells showed less affinity for MTX-agarose beads than that of L1210/0 cells. In addition, genistein (a tyrosine phosphorylation inhibitor) significantly enhanced the resistance of L1210/0 cells to MTX. Moreover, site-directed mutation studies indicated the importance of tyrosine phosphorylation of HSC70 in regulating its binding to MTX. These findings suggest that tyrosine phosphorylation of HSC70 regulates the transportation of MTX into the cells via the HSC70-RFC system and contributes to MTX resistance in L1210 cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  12. Activation of Bacillus subtilis Ugd by the BY-Kinase PtkA Proceeds via Phosphorylation of Its Residue Tyrosine 70

    DEFF Research Database (Denmark)

    Petranovic, Dina; Grangeasse, C.; Macek, B.

    2009-01-01

    The phosphorylation-dependent activation of bacterial UDP-glucose dehydrogenases by BY-kinases has been previously described in several bacterial model organisms, but the identity of phosphorylated tyrosine(s) and the exact activation mechanism remained unknown. A recent site-specific phosphoprot......The phosphorylation-dependent activation of bacterial UDP-glucose dehydrogenases by BY-kinases has been previously described in several bacterial model organisms, but the identity of phosphorylated tyrosine(s) and the exact activation mechanism remained unknown. A recent site...

  13. Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2

    DEFF Research Database (Denmark)

    Su, J; Batzer, A; Sap, J

    1994-01-01

    Receptor tyrosine phosphatases (R-PTPases) have generated interest because of their suspected involvement in cellular signal transduction. The adaptor protein Grb2 has been implicated in coupling receptor tyrosine kinases to Ras. We report that a ubiquitous R-PTPase, R-PTP-alpha, is tyrosine-phos...

  14. c-Abl mediated tyrosine phosphorylation of paxillin regulates LPS-induced endothelial dysfunction and lung injury

    Science.gov (United States)

    Usatyuk, Peter V.; Lele, Abhishek; Harijith, Anantha; Gregorio, Carol C.; Garcia, Joe G. N.; Salgia, Ravi; Natarajan, Viswanathan

    2015-01-01

    Paxillin is phosphorylated at multiple residues; however, the role of tyrosine phosphorylation of paxillin in endothelial barrier dysfunction and acute lung injury (ALI) remains unclear. We used siRNA and site-specific nonphosphorylable mutants of paxillin to abrogate the function of paxillin to determine its role in lung endothelial permeability and ALI. In vitro, lipopolysaccharide (LPS) challenge of human lung microvascular endothelial cells (HLMVECs) resulted in enhanced tyrosine phosphorylation of paxillin at Y31 and Y118 with no significant change in Y181 and significant barrier dysfunction. Knockdown of paxillin with siRNA attenuated LPS-induced endothelial barrier dysfunction and destabilization of VE-cadherin. LPS-induced paxillin phosphorylation at Y31 and Y118 was mediated by c-Abl tyrosine kinase, but not by Src and focal adhesion kinase. c-Abl siRNA significantly reduced LPS-induced endothelial barrier dysfunction. Transfection of HLMVECs with paxillin Y31F, Y118F, and Y31/118F double mutants mitigated LPS-induced barrier dysfunction and VE-cadherin destabilization. In vivo, the c-Abl inhibitor AG957 attenuated LPS-induced pulmonary permeability in mice. Together, these results suggest that c-Abl mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates LPS-mediated pulmonary vascular permeability and injury. PMID:25795725

  15. Analysis of tyrosine phosphorylation sites in signaling molecules by a phosphotyrosine-specific immonium ion scanning method

    DEFF Research Database (Denmark)

    Steen, Hanno; Pandey, Akhilesh; Andersen, Jens S

    2002-01-01

    Signal transduction pathways involve cascades of events, such as formation of second messengers and protein complexes that alter the activities of proteins. This can ultimately lead to changes in gene expression in response to the stimuli. Reversible phosphorylation of proteins is an important......-phosphorylated peptides are identified, they can be directly sequenced in the same experiment to localize the phosphorylation site. We provide protocols for isolation and preparation of samples for analysis, and detailed instructions for operation of a quadrupole time-of-flight (TOF) mass spectrometer for this method...... mechanism for activating or inhibiting enzymes and for the assembly of multiprotein complexes. Here, we describe a mass spectrometry-based phosphotyrosine-specific immonium ion scanning (PSI scanning) method for selective detection of tyrosine-phosphorylated peptides. Once the tyrosine...

  16. BAZ1B is dispensable for H2AX phosphorylation on Tyrosine 142 during spermatogenesis

    Directory of Open Access Journals (Sweden)

    Tyler J. Broering

    2015-07-01

    Full Text Available Meiosis is precisely regulated by the factors involved in DNA damage response in somatic cells. Among them, phosphorylation of H2AX on Serine 139 (γH2AX is an essential signal for the silencing of unsynapsed sex chromosomes during male meiosis. However, it remains unknown how adjacent H2AX phosphorylation on Tyrosine 142 (pTyr142 is regulated in meiosis. Here we investigate the meiotic functions of BAZ1B (WSTF, the only known Tyr142 kinase in somatic cells, using mice possessing a conditional deletion of BAZ1B. Although BAZ1B deletion causes ectopic γH2AX signals on synapsed autosomes during the early pachytene stage, BAZ1B is dispensable for fertility and critical events during spermatogenesis. BAZ1B deletion does not alter events on unsynapsed axes and pericentric heterochromatin formation. Furthermore, BAZ1B is dispensable for localization of the ATP-dependent chromatin remodeling protein SMARCA5 (SNF2h during spermatogenesis despite the complex formation between BAZ1B and SMARCA5, known as the WICH complex, in somatic cells. Notably, pTyr142 is regulated independently of BAZ1B and is dephosphorylated on the sex chromosomes during meiosis in contrast with the presence of adjacent γH2AX. Dephosphorylation of pTyr142 is regulated by MDC1, a binding partner of γH2AX. These results reveal the distinct regulation of two adjacent phosphorylation sites of H2AX during meiosis, and suggest that another kinase mediates Tyr142 phosphorylation.

  17. Redox modulation of tyrosine phosphorylation-dependent neutrophil adherence to endothelial cells

    International Nuclear Information System (INIS)

    Thibodeau, Paul A.; Gozin, Alexia; Gougerot-Pocidalo, Marie-Anne; Pasquier, Catherine

    2005-01-01

    Reactive oxygen species (ROS) are now well known to be involved in an increased interaction between neutrophils and endothelial cells. Previously, we have shown that the increased adhesion of neutrophils to ROS-stimulated endothelial cells involves an increase in tyrosine phosphorylation of the focal adhesion kinase, p125 FAK , and several cytoskeleton proteins. This review article focuses on the involvement of adhesion molecules in the increased adhesion of neutrophils to ROS-stimulated endothelial cells, on the oxygen species responsible for this adhesion, and on the intracellular signaling pathway leading to the modification of the cytoskeleton by ROS. The evidence from our laboratory and others describing these events is summarized. Finally, the future perspectives that need to be explored in order to inhibit or reduce the ROS-mediated adhesion of neutrophils to endothelial cells are addressed

  18. Tyrosine Phosphorylation of Rac1: A Role in Regulation of Cell Spreading

    Science.gov (United States)

    Chang, Fumin; Lemmon, Christopher; Lietha, Daniel; Eck, Michael; Romer, Lewis

    2011-01-01

    Rac1 influences a multiplicity of vital cellular- and tissue-level control functions, making it an important candidate for targeted therapeutics. The activity of the Rho family member Cdc42 has been shown to be modulated by tyrosine phosphorylation at position 64. We therefore investigated consequences of the point mutations Y64F and Y64D in Rac1. Both mutations altered cell spreading from baseline in the settings of wild type, constitutively active, or dominant negative Rac1 expression, and were accompanied by differences in Rac1 targeting to focal adhesions. Rac1-Y64F displayed increased GTP-binding, increased association with βPIX, and reduced binding with RhoGDI as compared with wild type Rac1. Rac1-Y64D had less binding to PAK than Rac1-WT or Rac1-64F. In vitro assays demonstrated that Y64 in Rac1 is a target for FAK and Src. Taken together, these data suggest a mechanism for the regulation of Rac1 activity by non-receptor tyrosine kinases, with consequences for membrane extension. PMID:22163037

  19. Tyrosine phosphorylation of Rac1: a role in regulation of cell spreading.

    Directory of Open Access Journals (Sweden)

    Fumin Chang

    Full Text Available Rac1 influences a multiplicity of vital cellular- and tissue-level control functions, making it an important candidate for targeted therapeutics. The activity of the Rho family member Cdc42 has been shown to be modulated by tyrosine phosphorylation at position 64. We therefore investigated consequences of the point mutations Y64F and Y64D in Rac1. Both mutations altered cell spreading from baseline in the settings of wild type, constitutively active, or dominant negative Rac1 expression, and were accompanied by differences in Rac1 targeting to focal adhesions. Rac1-Y64F displayed increased GTP-binding, increased association with βPIX, and reduced binding with RhoGDI as compared with wild type Rac1. Rac1-Y64D had less binding to PAK than Rac1-WT or Rac1-64F. In vitro assays demonstrated that Y64 in Rac1 is a target for FAK and Src. Taken together, these data suggest a mechanism for the regulation of Rac1 activity by non-receptor tyrosine kinases, with consequences for membrane extension.

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

    Science.gov (United States)

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

  1. Evidence for requirement of tyrosine phosphorylation in endothelial P2Y- and P2U- purinoceptor stimulation of prostacyclin release.

    Science.gov (United States)

    Bowden, A.; Patel, V.; Brown, C.; Boarder, M. R.

    1995-01-01

    1. The release of prostacyclin (PGI2) from vascular endothelial cells is stimulated by ATP acting at G protein-coupled P2-purinoceptors. Here we investigate the hypothesis that tyrosine protein phosphorylations are involved in this response. 2. The use of Western blots with anti-phosphotyrosine antibodies showed that 30 microM 2MeSATP (selective for P2Y-purinoceptors), 300 microM UTP (selective for P2U-purinoceptors) and 300 microM ATP (effective at both these purinoceptors), each stimulate the tyrosine phosphorylation of proteins in bovine cultured aortic endothelial cells. Each of these agonists also stimulates 6-keto PGF1 alpha accumulation in the medium (an index of PGI2 release) in these cells in the same period. 3. The tyrosine kinase inhibitor, genistein, inhibits the 6-keto PGF1 alpha response with the same concentration-dependency (1-100 microM) as the tyrosine phosphorylation response. 4. Tyrphostin, a structurally and functionally distinct tyrosine kinase inhibitor, is also a potent inhibitor (0.1-10 microM) of the 6-keto PGF1 alpha response. 5. Neither tyrphostin nor genistein inhibit the phospholipase C response to P2-purinoceptor stimulation. Furthermore, these inhibitors do not affect the 6-keto PGF1 alpha response to ionomycin. 6. These results show that the regulation of vascular endothelial cells by ATP acting at both P2Y- and P2U-purinoceptors involves the stimulation of tyrosine phosphorylation, and suggest that this is a necessary event for the purinoceptor-mediated stimulation of PGI2 production. Images Figure 1 Figure 5 PMID:8590971

  2. Tyrosine phosphorylation of 3BP2 is indispensable for the interaction with VAV3 in chicken DT40 cells

    International Nuclear Information System (INIS)

    Chihara, Kazuyasu; Kimura, Yukihiro; Honjoh, Chisato; Yamauchi, Shota; Takeuchi, Kenji; Sada, Kiyonao

    2014-01-01

    Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr 174 , Tyr 183 and Tyr 446 in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphorylation of Tyr 183 and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells. In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutational analysis showed that Tyr 174 , Tyr 183 and Tyr 426 of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr 426 is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr 426 was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr 426 following BCR stimulation. - Highlights: • 3BP2 is phosphorylated by Syk, but not Abl family kinases in BCR signaling. • Tyr183 and Tyr426 in chicken 3BP2 are the major phosphorylation sites by Syk. • The SH2 domain of 3BP2 is critical for tyrosine phosphorylation of 3BP2. • Phosphorylation of Tyr426 in 3BP2 is required for the inducible binding with Vav3. • 3BP2 is involved in the regulation of BCR-mediated Rac1 activation

  3. Tyrosine phosphorylation of 3BP2 is indispensable for the interaction with VAV3 in chicken DT40 cells

    Energy Technology Data Exchange (ETDEWEB)

    Chihara, Kazuyasu [Division of Genome Science and Microbiology, Department of Pathological Sciences, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Organization for Life Science Advancement Programs, University of Fukui, Fukui 910-1193 (Japan); Kimura, Yukihiro [Division of Genome Science and Microbiology, Department of Pathological Sciences, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Division of Otorhinolaryngology Head and Neck Surgery, Department of Sensory and Locomotor Medicine, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Honjoh, Chisato [Division of Genome Science and Microbiology, Department of Pathological Sciences, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Third Department of Internal Medicine, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Yamauchi, Shota; Takeuchi, Kenji [Division of Genome Science and Microbiology, Department of Pathological Sciences, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Organization for Life Science Advancement Programs, University of Fukui, Fukui 910-1193 (Japan); Sada, Kiyonao, E-mail: ksada@u-fukui.ac.jp [Division of Genome Science and Microbiology, Department of Pathological Sciences, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Organization for Life Science Advancement Programs, University of Fukui, Fukui 910-1193 (Japan)

    2014-03-10

    Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr{sup 174}, Tyr{sup 183} and Tyr{sup 446} in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphorylation of Tyr{sup 183} and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells. In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutational analysis showed that Tyr{sup 174}, Tyr{sup 183} and Tyr{sup 426} of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr{sup 426} is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr{sup 426} was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr{sup 426} following BCR stimulation. - Highlights: • 3BP2 is phosphorylated by Syk, but not Abl family kinases in BCR signaling. • Tyr183 and Tyr426 in chicken 3BP2 are the major phosphorylation sites by Syk. • The SH2 domain of 3BP2 is critical for tyrosine phosphorylation of 3BP2. • Phosphorylation of Tyr426 in 3BP2 is required for the inducible binding with Vav3. • 3BP2 is involved in the regulation of BCR-mediated Rac1 activation.

  4. Phosphatase of regenerating liver-3 directly interacts with integrin β1 and regulates its phosphorylation at tyrosine 783

    Directory of Open Access Journals (Sweden)

    Tian Wei

    2012-10-01

    Full Text Available Abstract Background Phosphatase of regenerating liver-3 (PRL-3 or PTP4A3 has been implicated in controlling cancer cell proliferation, motility, metastasis, and angiogenesis. Deregulated expression of PRL-3 is highly correlated with cancer progression and predicts poor survival. Although PRL-3 was categorized as a tyrosine phosphatase, its cellular substrates remain largely unknown. Results We demonstrated that PRL-3 interacts with integrin β1 in cancer cells. Recombinant PRL-3 associates with the intracellular domain of integrin β1 in vitro. Silencing of integrin α1 enhances PRL-3-integrin β1 interaction. Furthermore, PRL-3 diminishes tyrosine phosphorylation of integrin β1 in vitro and in vivo. With site-specific anti-phosphotyrosine antibodies against residues in the intracellular domain of integrin β1, tyrosine-783, but not tyrosine-795, is shown to be dephosphorylated by PRL-3 in a catalytic activity-dependant manner. Phosphorylation of Y783 is potentiated by ablation of PRL-3 or by treatment with a chemical inhibitor of PRL-3. Conversely, depletion of integrin α1 decreases the phosphorylation of this site. Conclusions Our results revealed a direct interaction between PRL-3 and integrin β1 and characterized Y783 of integrin β1 as a bona fide substrate of PRL-3, which is negatively regulated by integrin α1.

  5. Phosphorylated hepatocyte growth factor receptor/c-Met is associated with tumor growth and prognosis in patients with bladder cancer: correlation with matrix metalloproteinase-2 and -7 and E-cadherin.

    Science.gov (United States)

    Miyata, Yasuyoshi; Sagara, Yuji; Kanda, Shigeru; Hayashi, Tomayoshi; Kanetake, Hiroshi

    2009-04-01

    Hepatocyte growth factor receptor/c-Met is associated with malignant aggressiveness and survival in various cancers including bladder cancer. Although phosphorylation of hepatocyte growth factor receptor/c-Met is essential for its function, the pathologic significance of phosphorylated hepatocyte growth factor receptor/c-Met in bladder cancer remains elusive. We investigated the clinical significance of its expression, and its correlation with cancer cell progression-related molecules. The expression levels of 2 tyrosine residues of hepatocyte growth factor receptor/c-Met (pY1234/1235 and pY1349) were examined immunohistochemically in 133 specimens with nonmetastatic bladder cancer. We also investigated their correlation with matrix metalloproteinase-1, -2, -7, and -14; urokinase-type plasminogen activator; E-cadherin; CD44 standard, variant 3, and variant 6; and vascular endothelial growth factor. Expression of phosphorylated hepatocyte growth factor receptor/c-Met was detected in cancer cells, but was rare in normal urothelial cells. Although hepatocyte growth factor receptor/c-Met, pY1234/1235 hepatocyte growth factor receptor/c-Met, and pY1349 hepatocyte growth factor receptor/c-Met were associated with pT stage, multivariate analysis identified pY1349 hepatocyte growth factor receptor/c-met expression only as a significant factor for high pT stage. Expression of pY1349 hepatocyte growth factor receptor/c-Met was a marker of metastasis and (P = .001) and cause-specific survival (P = .003). Expressions of matrix metalloproteinase-2, matrix metalloproteinase-7, and E-cadherin correlated with pY1349 hepatocyte growth factor receptor/c-Met expression. Our results demonstrated that pY1349 hepatocyte growth factor receptor/c-Met plays an important role in tumor development, and its expression is a significant predictor of metastasis and survival of patients with bladder cancer. The results suggest that these activities are mediated, at least in part, by matrix

  6. Resistance to EGF receptor inhibitors in glioblastoma mediated by phosphorylation of the PTEN tumor suppressor at tyrosine 240.

    Science.gov (United States)

    Fenton, Tim R; Nathanson, David; Ponte de Albuquerque, Claudio; Kuga, Daisuke; Iwanami, Akio; Dang, Julie; Yang, Huijun; Tanaka, Kazuhiro; Oba-Shinjo, Sueli Mieko; Uno, Miyuki; Inda, Maria del Mar; Wykosky, Jill; Bachoo, Robert M; James, C David; DePinho, Ronald A; Vandenberg, Scott R; Zhou, Huilin; Marie, Suely K N; Mischel, Paul S; Cavenee, Webster K; Furnari, Frank B

    2012-08-28

    Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.

  7. The Pattern of Tyrosine Phosphorylation in Human Sperm in Response to Binding to Zona Pellucida or Hyaluronic Acid

    Science.gov (United States)

    Sati, Leyla; Cayli, Sevil; Delpiano, Elena; Sakkas, Denny

    2014-01-01

    In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-related manner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck, regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of sperm activation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP. PMID:24077441

  8. SH2 Superbinder Modified Monolithic Capillary Column for the Sensitive Analysis of Protein Tyrosine Phosphorylation.

    Science.gov (United States)

    Yao, Yating; Bian, Yangyang; Dong, Mingming; Wang, Yan; Lv, Jiawen; Chen, Lianfang; Wang, Hongwei; Mao, Jiawei; Dong, Jing; Ye, Mingliang

    2018-01-05

    In this study, we present a method to specifically capture phosphotyrosine (pTyr) peptides from minute amount of sample for the sensitive analysis of protein tyrosine phosphorylation. We immobilized SH2 superbinder on a monolithic capillary column to construct a microreactor to enrich pTyr peptides. It was found that the synthetic pTyr peptide could be specifically enriched by the microreactor from the peptide mixture prepared by spiking of the synthetic pTyr peptide into the tryptic digests of α-casein and β-casein with molar ratios of 1:1000:1000. The microreactor was further applied to enrich pTyr peptides from pervanadate-treated HeLa cell digests for phosphoproteomics analysis, which resulted in the identification of 796 unique pTyr sites. In contrast, the conventional SH2 superbinder-based method identified 41 pTyr sites for the same sample, only 5.2% of the number achieved by the microreactor. Finally, this microreactor was also applied to analyze the pTyr in Shc1 complex, an immunopurified protein complex, which resulted in the identification of 15 pTyr sites. Together, this technique is best fitted to analyze the pTyr in minute amount of sample and will have broad application in fields where only a limited amount of sample is available.

  9. Inhibition of deubiquitinating activity of USP14 decreases tyrosine hydroxylase phosphorylated at Ser19 in PC12D cells.

    Science.gov (United States)

    Nakashima, Akira; Ohnuma, Syuhei; Kodani, Yu; Kaneko, Yoko S; Nagasaki, Hiroshi; Nagatsu, Toshiharu; Ota, Akira

    2016-04-15

    Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its stability is a fundamental factor to maintain the level of the catecholamines in cells. However, the intracellular stability determined by the degradation pathway remains unknown. In this study, we investigated the mechanism by which phosphorylation of TH affected the proteasome pathway. The inhibition of proteasomes by MG-132 increased the percentage of TH molecules phosphorylated at their Ser19, Ser31 and/or Ser40 among the total TH proteins to about 70% in PC12D cells over a 24-hr period; although the percentage of phosphorylated TH molecules was about 20% under basal conditions. Moreover, the inhibition of proteasomes by epoxomicin with high specificity increased primarily the quantity of TH molecules phosphorylated at their Ser19. The phosphorylation of Ser19 potentiated Ser40 phosphorylation in cells by a process known as hierarchical phosphorylation. Therefore, the proteasome inhibition might result in an increase in the levels of all 3 phosphorylated TH forms, thus complicating interpretation of data. Conversely, activation of proteasome degradation by IU-1, which is an inhibitor for the deubiquitinating activity of USP14, decreased only the quantity of TH molecules phosphorylated at their Ser19, although it did not decrease that of TH phosphorylated at its Ser31 and Ser40 or that of TH molecules. These results suggest that the phosphorylation of Ser19 in the N-terminal portion of TH is critical as a trigger for the degradation of this enzyme by the ubiquitin-proteasome pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Rapid tyrosine phosphorylation of Lck following ligation of the tumor-associated cell surface molecule A6H

    DEFF Research Database (Denmark)

    Labuda, T; Gerwien, J; Ødum, Niels

    1999-01-01

    and the TCR-CD3 complex takes place and which signaling pathway might be involved. Here we show that ligation of the A6H antigen with mAb induces tyrosine phosphorylation of the Lck protein tyrosine kinase (PTK). Co-ligation of the A6H antigen with CD3 resulted in augmented Lck phosphorylation and mitogenesis....... In addition, A6H ligation induced an up-regulation of CD3-mediated phosphorylation of the 23 kDa high mol. wt form of TCR zeta and the zeta-associated protein, ZAP-70. Co-precipitation of Lck and ZAP-70 was only seen in T cells activated by combined A6H and anti-CD3 stimulation. In contrast, another Src...... family PTK, Fyn, was not affected by A6H ligation. In conclusion, we now demonstrate, for the first time, that A6H ligation triggers Lck phosphorylation, and that cross-talk between A6H and the TCR-CD3 complex involves Lck, ZAP-70 and the slow migrating isoform of TCR zeta. These results further suggests...

  11. Enterohemorrhagic Escherichia coli O157:H7 Produces Tir, Which Is Translocated to the Host Cell Membrane but Is Not Tyrosine Phosphorylated

    OpenAIRE

    DeVinney, Rebekah; Stein, Markus; Reinscheid, Dieter; Abe, Akio; Ruschkowski, Sharon; Finlay, B. Brett

    1999-01-01

    Intimate attachment to the host cell leading to the formation of attaching and effacing (A/E) lesions is an essential feature of enterohemorrhagic Escherichia coli (EHEC) O157:H7 pathogenesis. In a related pathogen, enteropathogenic E. coli (EPEC), this activity is dependent upon translocation of the intimin receptor, Tir, which becomes tyrosine phosphorylated within the host cell membrane. In contrast, the accumulation of tyrosine-phosphorylated proteins beneath adherent EHEC bacteria does n...

  12. Btk29A-mediated tyrosine phosphorylation of armadillo/β-catenin promotes ring canal growth in Drosophila oogenesis.

    Science.gov (United States)

    Hamada-Kawaguchi, Noriko; Nishida, Yasuyoshi; Yamamoto, Daisuke

    2015-01-01

    Drosophila Btk29A is the ortholog of mammalian Btk, a Tec family nonreceptor tyrosine kinase whose deficit causes X-linked agammaglobulinemia in humans. The Btk29AficP mutation induces multiple abnormalities in oogenesis, including the growth arrest of ring canals, large intercellular bridges that allow the flow of cytoplasm carrying maternal products essential for embryonic development from the nurse cells to the oocyte during oogenesis. In this study, inactivation of Parcas, a negative regulator of Btk29A, was found to promote Btk29A accumulation on ring canals with a concomitant increase in the ring canal diameter, counteracting the Btk29AficP mutation. This mutation markedly reduced the accumulation of phosphotyrosine on ring canals and in the regions of cell-cell contact, where adhesion-supporting proteins such as DE-cadherin and β-catenin ortholog Armadillo (Arm) are located. Our previous in vitro and in vivo analyses revealed that Btk29A directly phosphorylates Arm, leading to its release from DE-cadherin. In the present experiments, immunohistological analysis revealed that phosphorylation at tyrosine 150 (Y150) and Y667 of Arm was diminished in Btk29AficP mutant ring canals. Overexpression of an Arm mutant with unphosphorylatable Y150 inhibited ring canal growth. Thus Btk29A-induced Y150 phosphorylation is necessary for the normal growth of ring canals. We suggest that the dissociation of tyrosine-phosphorylated Arm from DE-cadherin allows dynamic actin to reorganize, leading to ring canal expansion and cell shape changes during the course of oogenesis.

  13. Btk29A-mediated tyrosine phosphorylation of armadillo/β-catenin promotes ring canal growth in Drosophila oogenesis.

    Directory of Open Access Journals (Sweden)

    Noriko Hamada-Kawaguchi

    Full Text Available Drosophila Btk29A is the ortholog of mammalian Btk, a Tec family nonreceptor tyrosine kinase whose deficit causes X-linked agammaglobulinemia in humans. The Btk29AficP mutation induces multiple abnormalities in oogenesis, including the growth arrest of ring canals, large intercellular bridges that allow the flow of cytoplasm carrying maternal products essential for embryonic development from the nurse cells to the oocyte during oogenesis. In this study, inactivation of Parcas, a negative regulator of Btk29A, was found to promote Btk29A accumulation on ring canals with a concomitant increase in the ring canal diameter, counteracting the Btk29AficP mutation. This mutation markedly reduced the accumulation of phosphotyrosine on ring canals and in the regions of cell-cell contact, where adhesion-supporting proteins such as DE-cadherin and β-catenin ortholog Armadillo (Arm are located. Our previous in vitro and in vivo analyses revealed that Btk29A directly phosphorylates Arm, leading to its release from DE-cadherin. In the present experiments, immunohistological analysis revealed that phosphorylation at tyrosine 150 (Y150 and Y667 of Arm was diminished in Btk29AficP mutant ring canals. Overexpression of an Arm mutant with unphosphorylatable Y150 inhibited ring canal growth. Thus Btk29A-induced Y150 phosphorylation is necessary for the normal growth of ring canals. We suggest that the dissociation of tyrosine-phosphorylated Arm from DE-cadherin allows dynamic actin to reorganize, leading to ring canal expansion and cell shape changes during the course of oogenesis.

  14. An Evolution-Guided Analysis Reveals a Multi-Signaling Regulation of Fas by Tyrosine Phosphorylation and its Implication in Human Cancers

    Science.gov (United States)

    Chakrabandhu, Krittalak; Huault, Sébastien; Durivault, Jérôme; Lang, Kévin; Ta Ngoc, Ly; Bole, Angelique; Doma, Eszter; Dérijard, Benoit; Gérard, Jean-Pierre; Pierres, Michel; Hueber, Anne-Odile

    2016-01-01

    Demonstrations of both pro-apoptotic and pro-survival abilities of Fas (TNFRSF6/CD95/APO-1) have led to a shift from the exclusive “Fas apoptosis” to “Fas multisignals” paradigm and the acceptance that Fas-related therapies face a major challenge, as it remains unclear what determines the mode of Fas signaling. Through protein evolution analysis, which reveals unconventional substitutions of Fas tyrosine during divergent evolution, evolution-guided tyrosine-phosphorylated Fas proxy, and site-specific phosphorylation detection, we show that the Fas signaling outcome is determined by the tyrosine phosphorylation status of its death domain. The phosphorylation dominantly turns off the Fas-mediated apoptotic signal, while turning on the pro-survival signal. We show that while phosphorylations at Y232 and Y291 share some common functions, their contributions to Fas signaling differ at several levels. The findings that Fas tyrosine phosphorylation is regulated by Src family kinases (SFKs) and the phosphatase SHP-1 and that Y291 phosphorylation primes clathrin-dependent Fas endocytosis, which contributes to Fas pro-survival signaling, reveals for the first time the mechanistic link between SFK/SHP-1-dependent Fas tyrosine phosphorylation, internalization route, and signaling choice. We also demonstrate that levels of phosphorylated Y232 and Y291 differ among human cancer types and differentially respond to anticancer therapy, suggesting context-dependent involvement of Fas phosphorylation in cancer. This report provides a new insight into the control of TNF receptor multisignaling by receptor phosphorylation and its implication in cancer biology, which brings us a step closer to overcoming the challenge in handling Fas signaling in treatments of cancer as well as other pathologies such as autoimmune and degenerative diseases. PMID:26942442

  15. KIT(D816V) Induces SRC-Mediated Tyrosine Phosphorylation of MITF and Altered Transcription Program in Melanoma

    DEFF Research Database (Denmark)

    Phung, Bengt; Kazi, Julhash U; Lundby, Alicia

    2017-01-01

    The oncogenic D816V mutation of the KIT receptor is well characterized in systemic mastocytosis and acute myeloid leukemia. Although KIT(D816V) has been found in melanoma, its function and involvement in this malignancy is not understood. Here we show that KIT(D816V) induces tyrosine phosphorylat.......Implications: This study demonstrates that an oncogenic tyrosine kinase mutant, KIT(D816V), can alter the transcriptional program of the transcription factor MITF in melanoma Mol Cancer Res; 15(9); 1265-74. ©2017 AACR....... complex formation, thus preventing MITF phosphorylation, the cells became hypersensitive to SRC inhibitors. We have therefore delineated a mechanism behind the oncogenic effects of KIT(D816V) in melanoma and provided a rationale for the heightened SRC inhibitor sensitivity in KIT(D816V) transformed cells...

  16. Manganese overload affects p38 MAPK phosphorylation and metalloproteinase activity during sea urchin embryonic development.

    Science.gov (United States)

    Pinsino, A; Roccheri, M C; Matranga, V

    2014-02-01

    In the marine environment, manganese represents a potential emerging contaminant, resulting from an increased production of manganese-containing compounds. In earlier reports we found that the exposure of Paracentrotus lividus sea urchin embryos to manganese produced phenotypes with no skeleton. In addition, manganese interfered with calcium uptake, perturbed extracellular signal-regulated kinase (ERK) signaling, affected the expression of skeletogenic genes, and caused an increase of the hsc70 and hsc60 protein levels. Here, we extended our studies focusing on the temporal activation of the p38 mitogen-activated protein kinase (p38 MAPK) and the proteolytic activity of metalloproteinases (MMPs). We found that manganese affects the stage-dependent dynamics of p38 MAPK activation and inhibits the total gelatin-auto-cleaving activity of MMPs, with the exclusion of the 90-85 kDa and 68-58 kDa MMPs, whose levels remain high all throughout development. Our findings correlate, for the first time to our knowledge, an altered activation pattern of the p38 MAPK with an aberrant MMP proteolytic activity in the sea urchin embryo. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    John G Koland

    2014-01-01

    Full Text Available 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

  18. The Effects of Insulin-Induced Hypoglycaemia on Tyrosine Hydroxylase Phosphorylation in Rat Brain and Adrenal Gland.

    Science.gov (United States)

    Senthilkumaran, Manjula; Johnson, Michaela E; Bobrovskaya, Larisa

    2016-07-01

    In this study we investigated the effects of insulin-induced hypoglycaemia on tyrosine hydroxylase (TH) protein and TH phosphorylation in the adrenal gland, C1 cell group, locus coeruleus (LC) and midbrain dopaminergic cell groups that are thought to play a role in response to hypoglycaemia and compared the effects of different concentrations of insulin in rats. Insulin (1 and 10 U/kg) treatment caused similar reductions in blood glucose concentration (from 7.5-9 to 2-3 mmol/L); however, plasma adrenaline concentration was increased 20-30 fold in response to 10 U/kg insulin and only 14 fold following 1 U/kg. Time course studies (at 10 U/kg insulin) revealed that in the adrenal gland, Ser31 phosphorylation was increased between 30 and 90 min (4-5 fold), implying that TH was activated to increase catecholamine synthesis in adrenal medulla to replenish the stores. In the brain, Ser19 phosphorylation was limited to certain dopaminergic groups in the midbrain, while Ser31 phosphorylation was increased in most catecholaminergic regions at 60 min (1.3-2 fold), suggesting that Ser31 phosphorylation may be an important mechanism to maintain catecholamine synthesis in the brain. Comparing the effects of 1 and 10 U/kg insulin revealed that Ser31 phosphorylation was increased to similar extent in the adrenal gland and C1 cell group in response to both doses whereas Ser31 and Ser19 phosphorylation were only increased in response to 1 U/kg insulin in LC and in response to 10 U/kg insulin in most midbrain regions. Thus, the adrenal gland and some catecholaminergic brain regions become activated in response to insulin administration and brain catecholamines may be important for initiation of physiological defences against insulin-induced hypoglycaemia.

  19. Thy-1/CD3 coengagement promotes TCR signaling and enhances particularly tyrosine phosphorylation of the raft molecule LAT.

    Science.gov (United States)

    Leyton, L; Quest, A F; Bron, C

    1999-08-01

    Clustering of the glycosyl-phosphatidylinositol (GPI)-anchored protein Thy-1 on the cell surface leads to T cell activation. However, despite the similarity to TCR-mediated events, cell signaling triggered by Thy-1 crosslinking, reportedly occurs in a manner independent of the TCR/CD3 complex. To investigate the relationship between responses resulting from Thy-1 or TCR engagement, a biochemically well defined system employing only affinity purified antibodies was used to crosslink these surface molecules and activation was assessed by monitoring tyrosine phosphorylation, intracellular calcium influx and IL-2 production. By these criteria, anti-CD3 mAbs moderately activated EL-4 thymoma or 2B4 hybridoma cell lines, while costimulation with anti-Thy-1-mAb strongly enhanced TCR signaling. Furthermore, a Thy-1 loss mutant cell line, did not respond to stimulation through CD3 despite expressing all essential signaling molecules. Together these results emphasized the existence of a poorly appreciated mutual interdependence between Thy-1 and CD3 for efficient cellular signaling. Thy-1/CD3-mediated activation enhanced mostly tyrosine phosphorylation of a 40 kDa protein which was identified as a transmembrane protein lacking N-linked oligosaccharides. These biochemical properties are identical to those described for a recently cloned adaptor protein called 'Linker for Activation of T cells' (LAT). Indeed, polyclonal Abs raised against a LAT-peptide (amino acids 103-131) specifically recognized the 40 kDa protein. LAT is present in microdomains of the plasma membrane enriched in sphingolipids, cholesterol, GPI-anchored proteins and a variety of signaling molecules. By contrast, the TCR/CD3 complex is excluded from these domains at least until stimulation takes place. Hence, we propose that Thy-1 promotes TCR/CD3 dependent signaling by facilitating LAT phosphorylation on tyrosine and the subsequent recruitment of downstream effector molecules.

  20. Lemur tyrosine kinase-2 signalling regulates kinesin-1 light chain-2 phosphorylation and binding of Smad2 cargo.

    LENUS (Irish Health Repository)

    Manser, C

    2012-05-31

    A recent genome-wide association study identified the gene encoding lemur tyrosine kinase-2 (LMTK2) as a susceptibility gene for prostate cancer. The identified genetic alteration is within intron 9, but the mechanisms by which LMTK2 may impact upon prostate cancer are not clear because the functions of LMTK2 are poorly understood. Here, we show that LMTK2 regulates a known pathway that controls phosphorylation of kinesin-1 light chain-2 (KLC2) by glycogen synthase kinase-3β (GSK3β). KLC2 phosphorylation by GSK3β induces the release of cargo from KLC2. LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3β on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2. Smad2 signals to the nucleus in response to transforming growth factor-β (TGFβ) receptor stimulation and transport of Smad2 by kinesin-1 is required for this signalling. We show that small interfering RNA loss of LMTK2 not only reduces binding of Smad2 to KLC2, but also inhibits TGFβ-induced Smad2 signalling. Thus, LMTK2 may regulate the activity of kinesin-1 motor function and Smad2 signalling.

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Spermatozoa with high mitochondrial membrane potential and low tyrosine phosphorylation preferentially bind to oviduct explants in the water buffalo (Bubalus bubalis).

    Science.gov (United States)

    Saraf, Kaustubh Kishor; Kumaresan, Arumugam; Chhillar, Shivani; Nayak, Samiksha; Lathika, Sreela; Datta, Tirtha Kumar; Gahlot, Subhash Chand; Karan, Prabha; Verma, Kiran; Mohanty, Tushar Kumar

    2017-05-01

    Although it is understood that spermatozoa are subjected to selection processes to form a functional sperm reservoir in the oviduct, the mechanism remains obscure. With the aim to understand the sperm selection process in the oviduct, in the present in vitro study, we analyzed mitochondrial membrane potential and tyrosine phosphorylation status in oviduct-explants bound and unbound spermatozoa. Frozen semen from Murrah buffalo bulls (n=10) used under progeny testing programme were utilized for the study. Oviduct explants were prepared by overnight culture of epithelial cells in TCM- 199 and washed spermatozoa were added to the oviduct explants and incubated for 4h. Mitochondrial membrane potential (MMP) and tyrosine phosphorylation status of bound and unbound spermatozoa were assessed at 1h and 4h of incubation. The proportion of spermatozoa with high MMP was significantly higher (Pspermatozoa (range 84.67-96.56%) compared to unbound (range 8.70-21.03%) spermatozoa. The proportion of tyrosine phosphorylated spermatozoa was significantly higher (Pspermatozoa displaying tyrosine phosphorylation at acrosomal area was significantly (Pspermatozoa with high MMP and low tyrosine phosphorylation were preferred for oviduct-explants binding in the buffalo. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Studies on the mechanism of rapid activation of protein tyrosine phosphorylation activities, particularly c-Src kinase, by TCDD in MCF10A.

    Science.gov (United States)

    Mazina, Olga; Park, Sujin; Sano, Hiromi; Wong, Patrick; Matsumura, Fumio

    2004-01-01

    While the process of the Ah receptor activation leading to cytochrome P450 induction has been well studied, the mechanism and the process through which the Ah receptor activates tyrosine kinases, within a few minutes of its ligand binding, is not known. Previously, it was reported by Tannheimer et al. (Carcinogenesis 1998; 19:1291-1297) that TCDD causes rapid induction of tyrosine phosphorylation activities in the MCF10A human mammary epithelial cell line. To study the mechanistic aspect of this phenomenon, particularly that occurs within a few minutes after administration, we first studied the effect of insulin on MCF10A under serum free conditions with added EGF. The addition of insulin induced a rapid (5 min) tyrosine phosphorylation on several 160-190 kDa proteins which was followed by significant dephosphorylation activities on these proteins by 15 min. TCDD increased the rate of tyrosine phosphorylation on those proteins but at 15 min, the level of phosphorylation was still high. When insulin and TCDD were added together, the ability of insulin to induce de-phosphorylation by 15 min disappeared. Such an action of TCDD was accompanied by an increase in the titer of the activated form of Src kinase (i.e. c-Src protein with 418 tyrosine phosphorylation), and a concomitant decrease in the level of 529 tyrosine phosphorylated form (an inactivated form). The TCDD-induced activation of c-Src could be blocked by pretreated MCF10A cells with antisense oligonucleotides against c-src or with a specific inhibitor of Src kinase, PP-2. These results support the conclusion that c-Src kinase is at least one of the earliest and the most upstream components of toxic signaling of the Ah-receptor activated by TCDD through the post-transcriptional process.

  4. The absence of phosphorylated tyrosine hydroxylase expression in the purkinje cells of the ataxic mutant pogo mouse.

    Science.gov (United States)

    Lee, N S; Kim, C T; Han, S Y; Kawk, J H; Sawada, K; Fukui, Y; Jeong, Y G

    2006-06-01

    The pogo mouse is a new ataxic autosomal recessive mutant that arose in Korean wild mice (KJR/Mskist). Its ataxic phenotype includes difficulty in maintaining a normal posture and the inability to walk in a straight line. Several studies have reported that tyrosine hydroxylase (TH) is persistently ectopically expressed in particular subsets of Purkinje cells in a parasagittal banding pattern in several ataxic mutant mice, e.g. tottering alleles and pogo mice. In this present study, we examined the expression of an enzymatically active form of TH and phosphorylated TH at Ser(40) (phospho-TH) by using immunohistochemistry and double immunofluorescence in the cerebellum of pogo mice. TH immunostaining appeared in some Purkinje cells in pogo, but in only a few of Purkinje cells of their heterozygous littermate controls. In all groups of mice, no phospho-TH immunoreactive Purkinje cells were observed in the cerebellum, although subsets of TH immunoreactive Purkinje cells were found in adjacent sections. This study suggests that TH expression in the Purkinje cells of pogo abnormally increases without activation of this enzyme by phosphorylation. This may mean that TH in the Purkinje cells of these mutants does not catalyse the conversion of tyrosine to l-DOPA, and is not related to catecholamine synthesis.

  5. Tyrosine Phosphorylation of the UDP-Glucose Dehydrogenase of Escherichia coli Is at the Crossroads of Colanic Acid Synthesis and Polymyxin Resistance

    DEFF Research Database (Denmark)

    Lacour, S.; Bechet, E.; Cozzone, A.J.

    2008-01-01

    to participate in the regulation of the amount of the exopolysaccharide colanic acid, whereas Etk-mediated Ugd phosphorylation appeared to participate in the resistance of E. coli to the antibiotic polymyxin. Conclusions/Significance: Ugd phosphorylation seems to be at the junction between two distinct...... shown to phosphorylate the UDP-glucose dehydrogenase Ugd in vitro. Not only is Ugd involved in the biosynthesis of extracellular polysaccharides, but also in the production of UDP-4-amino-4-deoxy-L-arabinose, a compound that renders E. coli resistant to cationic antimicrobial peptides. Methodology....../Principal Findings: Here, we studied the role of Ugd phosphorylation. We first confirmed in vivo the phosphorylation of Ugd by Wzc and we demonstrated that Ugd is also phosphorylated by Etk, the other BY-kinase identified in E. coli. Tyrosine 71 (Tyr71) was characterized as the Ugd site phosphorylated by both Wzc...

  6. Evaluation of L-selectin expression and assessment of protein tyrosine phosphorylation in bovine polymorphonuclear neutrophil leukocytes around parturition.

    Science.gov (United States)

    Monfardini, Erica; Paape, Max J; Wang, Yan; Capuco, Anthony V; Husheem, Michael; Wood, Larry; Burvenich, Christian

    2002-01-01

    Impaired polymorphonuclear neutrophil leukocyte (PMN) function around parturition has been associated with increased clinical mastitis in dairy cows. Rolling and attachment of PMN to the endothelium is the first step in the recruitment process and is accomplished by interaction between L-selectin on PMN and its ligand on endothelial cells. Furthermore, tyrosine phosphorylation is involved in the initiation of many PMN functions. The objective of this work was to determine changes in expression of L-selectin and tyrosine phosphorylation in the perinatal period. Eight clinically healthy Holstein cows were used as PMN donors at d-21, -14, -7,0 (calving), +1, +2, +7, +14, +28. Evaluation of L-selectin expression was carried out on activated and resting PMN. Anti-bovine L-selectin monoclonal antibody (MAB) and flow cytometric analysis were used to measure the percentage of PMN fluorescing and receptor expression (log mean fluorescent channel, LMFC). Activated and resting PMN showed similar trends in % PMN fluorescence and LM FC. The percentage of PMN fluorescing tended to decrease at parturition, followed by a significant increase at d +14 and +28 (P varied in intensity over time. The intensity of the 42-44 kDa band gradually increased from d -7, peaked at d +7 (P < 0.03), and steadily decreased to d +28 (P < 0.02). Antibody to activated mitogen protein kinase reacted with the 42-44 kDa band. Reduced PMN function during the periparturient period could be related to reduced L-selectin adhesion molecules on the cell surface, and to modulation in the phosphorylation of functionally important molecules.

  7. Cryopreservation of common carp (Cyprinus carpio L.) sperm induces protein phosphorylation in tyrosine and threonine residues

    Czech Academy of Sciences Publication Activity Database

    Li, P.; Hulák, M.; Li, Z.; H.; Šulc, Miroslav; Pšenička, M.; Rodina, M.; Gela, D.; Linhart, O.

    2013-01-01

    Roč. 80, č. 2 (2013), s. 84-89 ISSN 0093-691X Institutional support: RVO:61388971 Keywords : Cryopreservation * Sperm * Phosphorylation Subject RIV: CE - Biochemistry Impact factor: 1.845, year: 2013

  8. PTP-S2, a nuclear tyrosine phosphatase, is phosphorylated and ...

    Indian Academy of Sciences (India)

    PTP-S2 is a tyrosine specific protein phosphatase that binds to DNA and is localized to the nucleus in association with chromatin. It plays a role in the regulation of cell proliferation. Here we show that the subcellular distribution of this protein changes during cell division. While PTP-S2 was localized exclusively to the ...

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

    DEFF Research Database (Denmark)

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

    1997-01-01

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

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

    Science.gov (United States)

    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.

  11. Crystal Structure of Human Dual-Specificity Tyrosine-Regulated Kinase 3 Reveals New Structural Features and Insights into its Auto-phosphorylation.

    Science.gov (United States)

    Kim, Kuglae; Cha, Jeong Seok; Cho, Yong-Soon; Kim, Hoyoung; Chang, Nienping; Kim, Hye-Jung; Cho, Hyun-Soo

    2018-04-07

    Dual-specificity tyrosine-regulated kinases (DYRKs) auto-phosphorylate a critical tyrosine residue in their activation loop and phosphorylate their substrate on serine and threonine residues. The auto-phosphorylation occurs intramolecularly and is a one-off event. DYRK3 is selectively expressed at a high level in hematopoietic cells and attenuates erythroblast development, leading to anemia. In the present study, we determined the crystal structure of the mature form of human DYRK3 in complex with harmine, an ATP competitive inhibitor. The crystal structure revealed a phosphorylation site, residue S350, whose phosphorylation increases the stability of DYRK3 and enhances its kinase activity. In addition, our structural and biochemical assays suggest that the N-terminal auto-phosphorylation accessory domain stabilizes the DYRK3 protein, followed by auto-phosphorylation of the tyrosine of the activation loop, which is important for kinase activity. Finally, our docking analysis provides information for the design of novel and potent therapeutics to treat anemia. Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. FAK tyrosine phosphorylation is regulated by AMPK and controls metabolism in human skeletal muscle

    DEFF Research Database (Denmark)

    Lassiter, David G; Nylén, Carolina; Sjögren, Rasmus J O

    2018-01-01

    and the direct role of FAK on glucose and lipid metabolism. We hypothesised that insulin treatment and AMPK activation would have opposing effects on FAK phosphorylation and that gene silencing of FAK would alter metabolism. METHODS: Human muscle was treated with insulin or the AMPK-activating compound 5......-aminoimadazole-4-carboxamide ribonucleotide (AICAR) to determine FAK phosphorylation and glucose transport. Primary human skeletal muscle cells were used to study the effects of insulin or AICAR treatment on FAK signalling during serum starvation, as well as to determine the metabolic consequences of silencing...... in various non-muscle cell types and plays a regulatory role during skeletal muscle differentiation. The role of FAK in skeletal muscle in relation to insulin stimulation or AMPK activation is unknown. We examined the effects of insulin or AMPK activation on FAK phosphorylation in human skeletal muscle...

  13. Membrane depolarization-induced RhoA/Rho-associated kinase activation and sustained contraction of rat caudal arterial smooth muscle involves genistein-sensitive tyrosine phosphorylation

    Science.gov (United States)

    Mita, Mitsuo; Tanaka, Hitoshi; Yanagihara, Hayato; Nakagawa, Jun-ichi; Hishinuma, Shigeru; Sutherland, Cindy; Walsh, Michael P.; Shoji, Masaru

    2013-01-01

    Rho-associated kinase (ROK) activation plays an important role in K+-induced contraction of rat caudal arterial smooth muscle (Mita et al., Biochem J. 2002; 364: 431–40). The present study investigated a potential role for tyrosine kinase activity in K+-induced RhoA activation and contraction. The non-selective tyrosine kinase inhibitor genistein, but not the src family tyrosine kinase inhibitor PP2, inhibited K+-induced sustained contraction (IC50 = 11.3 ± 2.4 µM). Genistein (10 µM) inhibited the K+-induced increase in myosin light chain (LC20) phosphorylation without affecting the Ca2+ transient. The tyrosine phosphatase inhibitor vanadate induced contraction that was reversed by genistein (IC50 = 6.5 ± 2.3 µM) and the ROK inhibitor Y-27632 (IC50 = 0.27 ± 0.04 µM). Vanadate also increased LC20 phosphorylation in a genistein- and Y-27632-dependent manner. K+ stimulation induced translocation of RhoA to the membrane, which was inhibited by genistein. Phosphorylation of MYPT1 (myosin-targeting subunit of myosin light chain phosphatase) was significantly increased at Thr855 and Thr697 by K+ stimulation in a genistein- and Y-27632-sensitive manner. Finally, K+ stimulation induced genistein-sensitive tyrosine phosphorylation of proteins of ∼55, 70 and 113 kDa. We conclude that a genistein-sensitive tyrosine kinase, activated by the membrane depolarization-induced increase in [Ca2+]i, is involved in the RhoA/ROK activation and sustained contraction induced by K+. Ca2+ sensitization, myosin light chain phosphatase, RhoA, Rho-associated kinase, tyrosine kinase PMID:24133693

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

    International Nuclear Information System (INIS)

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

    2011-01-01

    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

  15. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitors: a survey of recent patent literature.

    Science.gov (United States)

    Nguyen, Thu Lan; Fruit, Corinne; Hérault, Yann; Meijer, Laurent; Besson, Thierry

    2017-11-01

    Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a eukaryotic serine-threonine protein kinase belonging to the CMGC group. DYRK1A hyperactivity appears to contribute to the development of a number of human malignancies and to cognitive deficits observed in Down syndrome and Alzheimer's disease. As a result, the DYRK1A kinase represents an attractive target for the synthesis and optimization of pharmacological inhibitors of potential therapeutic interest. Like most tyrosine kinase inhibitors developed up to the market, DYRK1A inhibitors are essentially acting by competing with ATP for binding at the catalytic site of the kinase. Areas covered: This paper reviews patent activity associated with the discovery of synthetic novel heterocyclic molecules inhibiting the catalytic activity of DYRK1A. Expert opinion: Despite the important role of DYRK1A in biological processes and the growing interest in the design of new therapeutic drugs, there are only few patented synthetic DYRK1A inhibitors and most of them were and are still developed by academic research groups, sometimes with industrial partners.

  16. Influence of the O-phosphorylation of serine, threonine and tyrosine in proteins on the amidic N-15 chemical shielding anisotropy tensors

    Czech Academy of Sciences Publication Activity Database

    Emmer, Jiří; Vavrinská, A.; Sychrovský, V.; Benda, L.; Kříž, Z.; Koča, J.; Boelens, R.; Sklenář, V.; Trantírek, L.

    2013-01-01

    Roč. 55, č. 1 (2013), s. 59-70 ISSN 0925-2738 Institutional support: RVO:60077344 Keywords : csa * Phosphorylation * Amidic nitrogen * Serine * Threonine * Tyrosine * Protein * nmr Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 3.305, year: 2013

  17. Influence of the O-phosphorylation of serine, threonine and tyrosine in proteins on the amidic 15N chemical shielding anisotropy tensors

    NARCIS (Netherlands)

    Emmer, J.; Vavrinska, A.; Sychrovský, V.; Benda, L.; Kriz, Z.; Koca, J.; Boelens, R.; Sklenár, V.; Trantirek, L.

    2012-01-01

    Density functional theory was employed to study the influence of O-phosphorylation of serine, threonine, and tyrosine on the amidic 15N chemical shielding anisotropy (CSA) tensor in the context of the complex chemical environments of protein structures. Our results indicate that the amidic 15N CSA

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

    Czech Academy of Sciences Publication Activity Database

    Niksirat, H.; Vancová, Marie; Andersson, L.; James, P.; Kouba, A.; Kozák, P.

    2016-01-01

    Roč. 172, SEP (2016), s. 123-130 ISSN 0378-4320 R&D Projects: GA TA ČR(CZ) TE01020118 Institutional support: RVO:60077344 Keywords : microtubular radial arm * spermatozoon capacitation * tyrosine-phosphorylation * ultrastructural localization Subject RIV: EA - Cell Biology Impact factor: 1.605, year: 2016

  19. Metabotropic glutamate receptor 5 activation enhances tyrosine phosphorylation of the N-methyl-D-aspartate (NMDA) receptor and NMDA-induced cell death in hippocampal cultured neurons.

    Science.gov (United States)

    Takagi, Norio; Besshoh, Shintaro; Marunouchi, Tetsuro; Takeo, Satoshi; Tanonaka, Kouichi

    2012-01-01

    The activation of group I metabotropic glutamate receptors (mGluRs), which are coupled with Gq-protein, initiates a variety physiological responses in different types of cells. While Gq-protein-coupled receptors can upregulate N-methyl-D-aspartate (NMDA) receptor function, group I mGluR-mediated regulations of NMDA receptor function are not fully understood. To determine biochemical roles of group I mGluRs in the regulation of the NMDA receptor, we have investigated changes in tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B induced by a selective mGluR5 agonist, (RS)-chloro-5-hydroxyphenylglycine (CHPG) in hippocampal neuronal cultures. Activation of mGluR5 by CHPG increased active-forms of Src. CHPG also enhanced tyrosine phosphorylation of NR2A and NR2B in hippocampal neuronal cultures. In addition, NMDA-induced cell death was enhanced by CHPG-induced mGluR5 stimulation at the concentration, which increased tyrosine phosphorylation of Src and NR2A/2B but did not induce cell death. This effect was inhibited by selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP). The results suggest that in hippocampal neurons, mGluR5 may regulate NMDA receptor activity, involving tyrosine phosphorylation of NR2A and NR2B and may be involved in NMDA receptor-mediated cell injury.

  20. Identification of tyrosine-phosphorylated proteins associated with metastasis and functional analysis of FER in human hepatocellular carcinoma cells

    Directory of Open Access Journals (Sweden)

    Wang Yan

    2009-10-01

    Full Text Available Abstract Background- Aberrant activity of tyrosine-phosphorylated proteins is commonly associated with HCC metastasis. Cell signaling events driven by these proteins are implicated in numerous processes that alter cancer cell behavior. Exploring the activities and signaling pathways of these proteins in HCC metastasis may help in identifying new candidate molecules for HCC-targeted therapy. Methods- Hep3B (a nonmetastatic HCC cell line and MHCC97H (a highly metastatic HCC cell line were used in this study, and the tyrosine-phosphorylated proteins expressed in these cell lines were profiled by a phosphoproteomics technique based on LC-MS/MS. Protein-protein interaction and functional clustering analyses were performed to determine the activities of the identified proteins and the signaling pathways closely related to HCC metastasis. Results- In both cell lines, a total of 247 phosphotyrosine (pTyr proteins containing 281 pTyr sites were identified without any stimulation. The involvement of almost 30% of these in liver or liver cancer has not been reported previously. Biological process clustering analysis indicated that pTyr proteins involved in cell motility, migration, protein autophosphorylation, cell-cell communication, and antiapoptosis functions were overexpressed during metastasis. Pathway clustering analysis revealed that signaling pathways such as those involved in EGFR signaling, cytokine- and chemokine-mediated signal transduction, and the PI3K and JAK-STAT cascades were significantly activated during HCC metastasis. Moreover, noncanonical regulation of the JNK cascade might also provide new targets for HCC metastasis. After comparing the pTyr proteins that were differentially expressed during HCC cell metastasis, we selected FER, a nonreceptor tyrosine kinase, and validated its role in terms of both expression and function. The data confirmed that FER might play a critical role in the invasion and metastasis of HCC. Conclusion- The

  1. Retinol activates tyrosine hydroxylase acutely by increasing the phosphorylation of serine40 and then serine31 in bovine adrenal chromaffin cells.

    Science.gov (United States)

    Gelain, Daniel P; Moreira, Jose C F; Bevilaqua, Lia R M; Dickson, Phillip W; Dunkley, Peter R

    2007-12-01

    Tyrosine hydroxylase is the rate-limiting enzyme in the biosynthesis of the catecholamines. It has been reported that retinol (vitamin A) modulates tyrosine hydroxylase activity by increasing its expression through the activation of the nuclear retinoid receptors. In this study, we observed that retinol also leads to an acute activation of tyrosine hydroxylase in bovine adrenal chromaffin cells and this was shown to occur via two distinct non-genomic mechanisms. In the first mechanism, retinol induced an influx in extracellular calcium, activation of protein kinase C and serine40 phosphorylation, leading to tyrosine hydroxylase activation within 15 min. This effect then declined over time. The retinol-induced rise in intracellular calcium then led to a second slower mechanism; this involved an increase in reactive oxygen species, activation of extracellular signal-regulated kinase 1/2 and serine31 phosphorylation and the maintenance of tyrosine hydroxylase activation for up to 2 h. No effects were observed with retinoic acid. These results show that retinol activates tyrosine hydroxylase via two sequential non-genomic mechanisms, which have not previously been characterized. These mechanisms are likely to operate in vivo to facilitate the stress response, especially when vitamin supplements are taken or when retinol is used as a therapeutic agent.

  2. Influence of the O-phosphorylation of serine, threonine and tyrosine in proteins on the amidic ¹⁵N chemical shielding anisotropy tensors.

    Science.gov (United States)

    Emmer, Jiří; Vavrinská, Andrea; Sychrovský, Vladimír; Benda, Ladislav; Kříž, Zdeněk; Koča, Jaroslav; Boelens, Rolf; Sklenář, Vladimír; Trantírek, Lukáš

    2013-01-01

    Density functional theory was employed to study the influence of O-phosphorylation of serine, threonine, and tyrosine on the amidic (15)N chemical shielding anisotropy (CSA) tensor in the context of the complex chemical environments of protein structures. Our results indicate that the amidic (15)N CSA tensor has sensitive responses to the introduction of the phosphate group and the phosphorylation-promoted rearrangement of solvent molecules and hydrogen bonding networks in the vicinity of the phosphorylated site. Yet, the calculated (15)N CSA tensors in phosphorylated model peptides were in range of values experimentally observed for non-phosphorylated proteins. The extent of the phosphorylation induced changes suggests that the amidic (15)N CSA tensor in phosphorylated proteins could be reasonably well approximated with averaged CSA tensor values experimentally determined for non-phosphorylated amino acids in practical NMR applications, where chemical surrounding of the phosphorylated site is not known a priori in majority of cases. Our calculations provide estimates of relative errors to be associated with the averaged CSA tensor values in interpretations of NMR data from phosphorylated proteins.

  3. Stimulatory effect of nobiletin, a citrus polymethoxy flavone, on catecholamine synthesis through Ser19 and Ser40 phosphorylation of tyrosine hydroxylase in cultured bovine adrenal medullary cells.

    Science.gov (United States)

    Zhang, Han; Yanagihara, Nobuyuki; Toyohira, Yumiko; Takahashi, Keita; Inagaki, Hirohide; Satoh, Noriaki; Li, Xiaoja; Goa, Xiumei; Tsutsui, Masato; Takahaishi, Kojiro

    2014-01-01

    We previously reported the dual effects of nobiletin, a compound of polymethoxy flavones found in citrus fruits, on catecholamine secretion in cultured bovine adrenal medullary cells. Here, we report the effects of nobiletin on catecholamine synthesis in the cells. Nobiletin increased the synthesis of (14)C-catecholamines from [(14)C]tyrosine in a time (20-30 min)- and concentration (1.0-100 μM)-dependent manner. Nobiletin (10-100 μM) also activated tyrosine hydroxylase activity. The stimulatory effect of nobiletin on (14)C-catecholamine synthesis was not observed when extracellular Ca(2+) was not present in the incubation medium. Protein kinase inhibitors including H-89, an inhibitor of cyclic AMP-dependent protein kinase, and KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinase II, suppressed the stimulatory effects of nobiletin on catecholamine synthesis as well as tyrosine hydroxylase activity. Nobiletin also induced the phosphorylation of tyrosine hydroxylase at Ser(19) and Ser(40). Nobiletin (1.0-100 μM) inhibited (14)C-catecholamine synthesis induced by acetylcholine. The present findings suggest that nobiletin, by itself, stimulates catecholamine synthesis through tyrosine hydroxylase phosphorylation at Ser(19) and Ser(40), whereas it inhibits catecholamine synthesis induced by acetylcholine in bovine adrenal medulla.

  4. Sphingosine 1-Phosphate Induces Platelet/Endothelial Cell Adhesion Molecule-1 Tyrosine Phosphorylation in Bovine Aortic Endothelial Cells through a PP2-Inhibitable Mechanism

    Directory of Open Access Journals (Sweden)

    Yu-Ting Huang

    2007-12-01

    Full Text Available Sphingosine-1-phosphate (S1P is a low-molecular-weight phospholipid derivative released by activated platelets. S1P transduces signals through a family of G protein-coupled receptors to modulate various physiological behaviors of endothelial cells. Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31 is a 130-kDa protein expressed on the surfaces of leukocytes, platelets, and endothelial cells. Upon PECAM-1 activation, its cytoplasmic tyrosine residues become phosphorylated and bind with SH2 domain-containing proteins, thus leading to the downstream functions mediated by PECAM-1. In the present study, we found that S1P induced PECAM-1 tyrosine phosphorylation and SHP-2 association in bovine aortic endothelial cells (BAECs by immunoprecipitation and western blotting. The pretreatment of BAECs with a series of chemical inhibitors to determine the signaling pathway showed that the PECAM-1 phosphorylation was inhibited by PP2, indicating the participation of Src family kinases. These results demonstrated that S1P induced PECAM-1 tyrosine phosphorylation in BAECs through mediation of Src family kinases, and this may regulate the physiological behaviors of endothelial cells.

  5. Effect of cholesterol loaded cyclodextrin supplementation on tyrosine phosphorylation and apoptosis like changes in frozen thawed Hariana bull spermatozoa.

    Science.gov (United States)

    Yadav, Hanuman P; Kumar, Anuj; Shah, Nadeem; Chauhan, Dharmendra S; Saxena, Atul; Yadav, Sarvajeet; Swain, Dilip Kumar

    2017-07-01

    The beneficial effects of cholesterol loaded cyclodextrin (CLC) addition were evaluated in cryopreserved bull semen. Forty ejaculates were collected from Hariana bulls (n = 4), pooled and divided into 4 aliquots. All the aliquots were initially diluted in to egg yolk tris citrate and supplemented with CLC @ 0.5 mg (Group-II), 1.0 mg (G-III) and 2.0 mg (G-IV) CLC/120 × 10 6 spermatozoa or without CLC (G-I) that served as control. Extended semen was cryopreserved at -196 °C for 24 h. Seminal attributes like motility, viability, cryocapacitation like changes, tyrosine phosphorylation, apoptosis like changes in terms of mitochondrial transmembrane potential and DNA integrity were evaluated after equilibration and thawing. Results showed a significant increase in the motility, viability and acrosome intact spermatozoa in Group II as compared to other three groups. Further, the proportion of spermatozoa showing capacitation and acrosome reaction was also decreased (P spermatozoa. Addition of CLC significantly increased (P spermatozoa showing high transmembrane mitochondrial potential, and also, CLC @ 0.5 mg/120 × 10 6 in semen extender significantly decreased (P spermatozoa showing fragmented DNA after thawing as compared to control. Results of the present study indicate beneficial effects of CLC supplementation on cryodamage of spermatozoa by reducing the cryocapacitation and apoptosis like changes. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Tyrosine phosphorylation and proteolytic cleavage of Notch are required for non-canonical Notch/Abl signaling inDrosophilaaxon guidance.

    Science.gov (United States)

    Kannan, Ramakrishnan; Cox, Eric; Wang, Lei; Kuzina, Irina; Gu, Qun; Giniger, Edward

    2018-01-17

    Notch signaling is required for the development and physiology of nearly every tissue in metazoans. Much of Notch signaling is mediated by transcriptional regulation of downstream target genes, but Notch controls axon patterning in Drosophila by local modulation of Abl tyrosine kinase signaling, via direct interactions with the Abl co-factors Disabled and Trio. Here, we show that Notch-Abl axonal signaling requires both of the proteolytic cleavage events that initiate canonical Notch signaling. We further show that some Notch protein is tyrosine phosphorylated in Drosophila , that this form of the protein is selectively associated with Disabled and Trio, and that relevant tyrosines are essential for Notch-dependent axon patterning but not for canonical Notch-dependent regulation of cell fate. Based on these data, we propose a model for the molecular mechanism by which Notch controls Abl signaling in Drosophila axons. © 2018. Published by The Company of Biologists Ltd.

  7. Influence of the O-phosphorylation of serine, threonine and tyrosine in proteins on the amidic N-15 chemical shielding anisotropy tensors

    Czech Academy of Sciences Publication Activity Database

    Emmer, J.; Vavrinská, A.; Sychrovský, Vladimír; Benda, Ladislav; Kříž, Z.; Koča, J.; Boelens, R.; Sklenář, V.; Trantírek, L.

    2013-01-01

    Roč. 55, č. 1 (2013), s. 59-70 ISSN 0925-2738 R&D Projects: GA ČR GAP205/10/0228 Grant - others:CEITEC(XE) CZ.1.05/1.1.00/02.0068 Institutional support: RVO:61388963 Keywords : CSA * phosphorylation * amidic nitrogen * serine * threonine * tyrosine * protein * NMR Subject RIV: CE - Biochemistry Impact factor: 3.305, year: 2013

  8. Enterohemorrhagic Escherichia coli O157:H7 produces Tir, which is translocated to the host cell membrane but is not tyrosine phosphorylated.

    Science.gov (United States)

    DeVinney, R; Stein, M; Reinscheid, D; Abe, A; Ruschkowski, S; Finlay, B B

    1999-05-01

    Intimate attachment to the host cell leading to the formation of attaching and effacing (A/E) lesions is an essential feature of enterohemorrhagic Escherichia coli (EHEC) O157:H7 pathogenesis. In a related pathogen, enteropathogenic E. coli (EPEC), this activity is dependent upon translocation of the intimin receptor, Tir, which becomes tyrosine phosphorylated within the host cell membrane. In contrast, the accumulation of tyrosine-phosphorylated proteins beneath adherent EHEC bacteria does not occur, leading to questions about whether EHEC uses a Tir-based mechanism for adherence and A/E lesion formation. In this report, we demonstrate that EHEC produces a functional Tir that is inserted into host cell membranes, where it serves as an intimin receptor. However, unlike in EPEC, in EHEC Tir is not tyrosine phosphorylated yet plays a key role in both bacterial adherence to epithelial cells and pedestal formation. EHEC, but not EPEC, was unable to synthesize Tir in Luria-Bertani medium but was able to secrete Tir into M9 medium, suggesting that Tir synthesis and secretion may be regulated differently in these two pathogens. EHEC Tir and EPEC Tir both bind intimin and focus cytoskeletal rearrangements, indicating that tyrosine phosphorylation is not needed for pedestal formation. EHEC and EPEC intimins are functionally interchangeable, but EHEC Tir shows a much greater affinity for EHEC intimin than for EPEC intimin. These findings highlight some of the differences and similarities between EHEC and EPEC virulence mechanisms, which can be exploited to further define the molecular basis of pedestal formation.

  9. Protein phosphorylation on tyrosine restores expression and glycosylation of cyclooxygenase-2 by 2-deoxy-D-glucose-caused endoplasmic reticulum stress in rabbit articular chondrocyte

    Directory of Open Access Journals (Sweden)

    Seon-Mi Yu

    2012-05-01

    Full Text Available 2-deoxy-D-glucose(2DG-caused endoplasmic reticulum (ERstress inhibits protein phosphorylation at tyrosine residues.However, the accurate regulatory mechanisms, which determinethe inflammatory response of chondrocytes to ER stress via proteintyrosine phosphorylation, have not been systematicallyevaluated. Thus, in this study, we examined whether proteinphosphorylation at tyrosine residues can modulate the expressionand glycosylation of COX-2, which is reduced by 2DG-inducedER stress. We observed that protein tyrosine phosphatase (PTP inhibitors,sodium orthovanadate (SOV, and phenylarsine oxide(PAO significantly decreased expression of ER stress inducibleproteins, glucose-regulated protein 94 (GRP94, and CCAAT/ enhancer-binding-protein- related gene (GADD153, which was inducedby 2DG. In addition, we demonstrated that SOV and PAOnoticeably restored the expression and glycosylation of COX-2 aftertreatment with 2DG. These results suggest that protein phosphorylationof tyrosine residues plays an important role in theregulation of expression and glycosylation during 2DG-inducedER stress in rabbit articular chondrocytes. [BMB reports 2012;45(5: 317-322

  10. Interleukins 2, 4, 7, and 15 stimulate tyrosine phosphorylation of insulin receptor substrates 1 and 2 in T cells. Potential role of JAK kinases.

    Science.gov (United States)

    Johnston, J A; Wang, L M; Hanson, E P; Sun, X J; White, M F; Oakes, S A; Pierce, J H; O'Shea, J J

    1995-12-01

    The signaling molecules insulin receptor substrate (IRS)-1 and the newly described IRS-2 (4PS) molecule are major insulin and interleukin 4 (IL-4)-dependent phosphoproteins. We report here that IL-2, IL-7, and IL-15, as well as IL-4, rapidly stimulate the tyrosine phosphorylation of IRS-1 and IRS-2 in human peripheral blood T cells, NK cells, and in lymphoid cell lines. In addition, we show that the Janus kinases, JAK1 and JAK3, associate with IRS-1 and IRS-2 in T cells. Coexpression studies demonstrate that these kinases can tyrosine-phosphorylate IRS-2, suggesting a possible mechanism by which cytokine receptors may induce the tyrosine phosphorylation of IRS-1 and IRS-2. We further demonstrate that the p85 subunit of phosphoinositol 3-kinase associates with IRS-1 in response to IL-2 and IL-4 in T cells. Therefore, these data indicate that IRS-1 and IRS-2 may have important roles in T lymphocyte activation not only in response to IL-4, but also in response to IL-2, IL-7, and IL-15.

  11. Convulxin induces platelet activation by a tyrosine-kinase-dependent pathway and stimulates tyrosine phosphorylation of platelet proteins, including PLC gamma 2, independently of integrin alpha IIb beta 3.

    Science.gov (United States)

    Francischetti, I M; Ghazaleh, F A; Reis, R A; Carlini, C R; Guimarães, J A

    1998-05-15

    1Convulxin (Cvx) is a well-characterized platelet aggregating glycoprotein isolated from Crotalus durissus terrificus and C. d. cascavella venoms. In the present report we show that Cvx induces tyrosine phosphorylation of human platelet proteins, including phospholipase C-gamma 2 (PLC gamma 2), and also stimulates [3H]arachidonic acid ([3H]AA) mobilization, pleckstrin phosphorylation, and an increase in the cytosolic Ca2+ concentration ([Ca2+]in) due to both Ca2+ entry and internal Ca2+ mobilization. Staurosporine, a potent protein kinase inhibitor, and genistein, a specific inhibitor of protein tyrosine kinases (PTK), were used to evaluate the role of protein tyrosine phosphorylation (PTP) in the signal transduction evoked by Cvx. Staurosporine and genistein inhibited in a dose-dependent manner platelet aggregation induced by Cvx. Both inhibitors significantly blocked to near basal levels breakdown of phosphatidylinositol 4,5-bisphosphate from [myo-2-3H]inositol-labeled platelets and the production of [3H]AA metabolites from [3H]AA-labeled platelets after challenge with Cvx. Cvx provokes an increase in [Ca2+]in in Fura-2-loaded platelets that was abolished by concentrations of staurosporine which also inhibited Cvx-induced platelet aggregation. In addition, Cvx stimulates a rapid increase in tyrosine phosphorylation of human platelets proteins with molecular masses of 40, 72/74, 78/80, 105, 120, and 145 kDa, followed by dephosphorylation. Furthermore, Cvx stimulates a rapid tyrosyl phosphorylation of a 145-kDa molecular mass protein that was identified as PLC gamma 2. PTP induced by Cvx was not inhibited when platelets were stimulated in the presence of indomethacin, apyrase, EDTA, or RGDS peptide. These results indicate that PTP is chronologically proximal to Cvx binding to platelets, and is independent of aggregation or fibrinogen binding to the integrin alpha IIb beta 3. On the other hand, the dephosphorylation step is inhibited by RGDS peptide or EDTA

  12. Inhibitors of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) exert a strong anti-herpesviral activity.

    Science.gov (United States)

    Hutterer, Corina; Milbradt, Jens; Hamilton, Stuart; Zaja, Mirko; Leban, Johann; Henry, Christophe; Vitt, Daniel; Steingruber, Mirjam; Sonntag, Eric; Zeitträger, Isabel; Bahsi, Hanife; Stamminger, Thomas; Rawlinson, William; Strobl, Stefan; Marschall, Manfred

    2017-07-01

    Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in immunocompromised individuals and neonates. The success of (val)ganciclovir therapy is hampered by low drug compatibility and induction of viral resistance. A novel strategy of antiviral treatment is based on the exploitation of cell-directed signaling, e. g. pathways with a known relevance for carcinogenesis and tumor drug development. Here we describe a principle for putative antiviral drugs based on targeting dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). DYRKs constitute an evolutionarily conserved family of protein kinases with key roles in the control of cell proliferation and differentiation. Members of the DYRK family are capable of phosphorylating a number of substrate proteins, including regulators of the cell cycle, e.g. DYRK1B can induce cell cycle arrest, a critical step for the regulation of HCMV replication. Here we provide first evidence for a critical role of DYRKs during viral replication and the high antiviral potential of DYRK inhibitors (SC84227, SC97202 and SC97208, Harmine and AZ-191). Using established replication assays for laboratory and clinically relevant strains of HCMV, concentration-dependent profiles of inhibition were obtained. Mean inhibitory concentrations (EC50) of 0.98 ± 0.08 μM/SC84227, 0.60 ± 0.02 μM/SC97202, 6.26 ± 1.64 μM/SC97208, 0.71 ± 0.019 μM/Harmine and 0.63 ± 0.23 μM/AZ-191 were determined with HCMV strain AD169-GFP for the infection of primary human fibroblasts. A first analysis of the mode of antiviral action suggested a block of viral replication at the early-late stage of HCMV gene expression. Moreover, rhesus macaque cytomegalovirus (RhCMV), varicella-zoster virus (VZV) and herpes simplex virus (HSV-1) showed a similarly high sensitivity to these compounds. Thus, we conclude that DYRK signaling represents a promising target pathway for the development of novel anti

  13. The cyclolignan PPP induces activation loop-specific inhibition of tyrosine phosphorylation of the insulin-like growth factor-1 receptor. Link to the phosphatidyl inositol-3 kinase/Akt apoptotic pathway.

    Science.gov (United States)

    Vasilcanu, Daiana; Girnita, Ada; Girnita, Leonard; Vasilcanu, Radu; Axelson, Magnus; Larsson, Olle

    2004-10-14

    The insulin-like growth factor-1 receptor (IGF-1R) is crucial for many functions in neoplastic cells, for example, antiapoptosis. Recently, we demonstrated that the cyclolignan PPP efficiently inhibited phosphorylation of IGF-1R without interfering with insulin receptor activity. PPP preferentially reduced phosphorylated Akt, as compared to phosphorylated Erk1/2, and caused apoptosis. Now, we aimed to investigate how PPP inhibits the IGF-1R tyrosine kinase (IGF-1RTK) and the PI3K/Akt apoptotic pathway. Using a baculovirus driven IGF-1RTK we found that PPP interfered with tyrosine phosphorylation in the activation loop of the kinase domain. Specifically, it blocked phosphorylation of tyrosine (Y) 1136, while sparing the two others (Y1131 and Y1135). To explore the impact of inhibition of Y1136 on Akt phosphorylation we transfected P6 cells (overexpressing IGF-1R) and malignant melanoma cells with different IGF-1R mutants, including Y1136F (tyrosine replaced by phenylalanine). Y1136F was found to strongly decrease IGF-1 stimulated phosphorylation of Akt. Conversely, Akt phosphorylation was weakly affected in the Y1131F transfectant. Taken together, our data suggest that the preferential inhibition of phosphorylated Akt, after PPP treatment, may be due to specific inhibition of Y1136. PPP was proven not to interfere directly with Akt or any of its downstream molecules in the apoptotic pathway.

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

    Science.gov (United States)

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

    2013-01-01

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

  15. Semi-automatized segmentation method using image-based flow cytometry to study sperm physiology: the case of capacitation-induced tyrosine phosphorylation.

    Science.gov (United States)

    Matamoros-Volante, Arturo; Moreno-Irusta, Ayelen; Torres-Rodriguez, Paulina; Giojalas, Laura; Gervasi, María G; Visconti, Pablo E; Treviño, Claudia L

    2018-02-01

    Is image-based flow cytometry a useful tool to study intracellular events in human sperm such as protein tyrosine phosphorylation or signaling processes? Image-based flow cytometry is a powerful tool to study intracellular events in a relevant number of sperm cells, which enables a robust statistical analysis providing spatial resolution in terms of the specific subcellular localization of the labeling. Sperm capacitation is required for fertilization. During this process, spermatozoa undergo numerous physiological changes, via activation of different signaling pathways, which are not completely understood. Classical approaches for studying sperm physiology include conventional microscopy, flow cytometry and Western blotting. These techniques present disadvantages for obtaining detailed subcellular information of signaling pathways in a relevant number of cells. This work describes a new semi-automatized analysis using image-based flow cytometry which enables the study, at the subcellular and population levels, of different sperm parameters associated with signaling. The increase in protein tyrosine phosphorylation during capacitation is presented as an example. Sperm cells were isolated from seminal plasma by the swim-up technique. We evaluated the intensity and distribution of protein tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h under different experimental conditions. We used an antibody against FER kinase and pharmacological inhibitors in an attempt to identify the kinases involved in protein tyrosine phosphorylation during human sperm capacitation. Semen samples from normospermic donors were obtained by masturbation after 2-3 days of sexual abstinence. We used the innovative technique image-based flow cytometry and image analysis tools to segment individual images of spermatozoa. We evaluated and quantified the regions of sperm where protein tyrosine phosphorylation takes place at the

  16. Matrix metalloproteinase-2 and -9 are induced differently by metal nanoparticles in human monocytes: The role of oxidative stress and protein tyrosine kinase activation

    International Nuclear Information System (INIS)

    Wan Rong; Mo Yiqun; Zhang Xing; Chien Sufan; Tollerud, David J.; Zhang Qunwei

    2008-01-01

    Recently, many studies have shown that nanoparticles can translocate from the lungs to the circulatory system. As a particulate foreign body, nanoparticles could induce host responses such as reactive oxygen species (ROS) generation, inflammatory cytokine and matrix metalloproteinase (MMP) release which play a major role in tissue destruction and remodeling. However, the direct effects of nanoparticles on leukocytes, especially monocytes, are still unclear. The objective of the present study was to compare the ability of Nano-Co and Nano-TiO 2 to cause alteration of transcription and activity of MMPs and to explore possible mechanisms. We hypothesized that non-toxic doses of some transition metal nanoparticles stimulate an imbalance of MMP/TIMP that cause MMP production that may contribute to their health effects. To test this hypothesis, U937 cells were treated with Nano-Co and Nano-TiO 2 and cytotoxic effects and ROS generation were measured. The alteration of MMP-2 and MMP-9 expression and activity of MMP-2 and MMP-9 after exposure to these metal nanoparticles were subsequently determined. To investigate the potential signaling pathways involved in the Nano-Co-induced MMP activation, the ROS scavengers or inhibitors, AP-1 inhibitor, and protein tyrosine kinase (PTK) inhibitors were also used to pre-treat U937 cells. Our results demonstrated that exposure of U937 cells to Nano-Co, but not to Nano-TiO 2 , at a dose that does not cause cytotoxicity, resulted in ROS generation and up-regulation of MMP-2 and MMP-9 mRNA expression .. Our results also showed dose- and time-related increases in pro-MMP-2 and pro-MMP-9 gelatinolytic activities in conditioned media after exposure of U937 cells to Nano-Co, but not to Nano-TiO 2 . Nano-Co-induced pro-MMP-2 and pro-MMP-9 activity increases were inhibited by pre-treatment with ROS scavengers or inhibitors. We also demonstrated dose- and time-related decreases in tissue inhibitors of metalloproteinases 2 (TIMP-2) in U937 cells

  17. Phosphorylation of tyrosine residues 31 and 118 on paxillin regulates cell migration through an association with CRK in NBT-II cells.

    Science.gov (United States)

    Petit, V; Boyer, B; Lentz, D; Turner, C E; Thiery, J P; Vallés, A M

    2000-03-06

    Identification of signaling molecules that regulate cell migration is important for understanding fundamental processes in development and the origin of various pathological conditions. The migration of Nara Bladder Tumor II (NBT-II) cells was used to determine which signaling molecules are specifically involved in the collagen-mediated locomotion. We show here that paxillin is tyrosine phosphorylated after induction of motility on collagen. Overexpression of paxillin mutants in which tyrosine 31 and/or tyrosine 118 were replaced by phenylalanine effectively impaired cell motility. Moreover, stimulation of motility by collagen preferentially enhanced the association of paxillin with the SH2 domain of the adaptor protein CrkII. Mutations in both tyrosine 31 and 118 diminished the phosphotyrosine content of paxillin and prevented the formation of the paxillin-Crk complex, suggesting that this association is necessary for collagen-mediated NBT-II cell migration. Other responses to collagen, such as cell adhesion and spreading, were not affected by these mutations. Overexpression of wild-type paxillin or Crk could bypass the migration-deficient phenotype. Both the SH2 and the SH3 domains of CrkII are shown to play a critical role in this collagen-mediated migration. These results demonstrate the important role of the paxillin-Crk complex in the collagen-induced cell motility.

  18. Ligand binding affinity at the insulin receptor isoform A (IR-A and subsequent IR-A tyrosine phosphorylation kinetics are important determinants of mitogenic biological outcomes.

    Directory of Open Access Journals (Sweden)

    Harinda eRajapaksha

    2015-07-01

    Full Text Available The insulin receptor (IR is a tyrosine kinase receptor that can mediate both metabolic and mitogenic biological actions. The IR isoform-A (IR-A arises from alternative splicing of exon 11 and has different ligand binding and signalling properties compared to the IR isoform-B. The IR-A not only binds insulin but also insulin-like growth factor-II (IGF-II with high affinity. IGF-II acting through the IR-A promotes cancer cell proliferation, survival and migration by activating some unique signalling molecules compared to those activated by insulin. This observation led us to investigate whether the different IR-A signalling outcomes in response to IGF-II and insulin could be attributed to phosphorylation of a different subset of IR-A tyrosine residues or to the phosphorylation kinetics. We correlated IR-A phosphorylation to activation of molecules involved in mitogenic and metabolic signalling (MAPK and Akt and receptor internalisation rates (related to mitogenic signalling. We also extended this study to incorporate two ligands that are known to promote predominantly mitogenic ([His4, Tyr15, Thr49, Ile51] IGF-I, qIGF-I or metabolic (S597 peptide biological actions, to see if common mechanisms can be used to define mitogenic or metabolic signalling through the IR-A. The 3-fold lower mitogenic action of IGF-II compared to insulin was associated with a decreased potency in activation of Y960, Y1146, Y1150, Y1151, Y1316 and Y1322, in MAPK phosphorylation and in IR-A internalization. With the poorly mitogenic S597 peptide it was a decreased rate of tyrosine phosphorylation rather than potency that was associated with a low mitogenic potential. We conclude that both decreased affinity of IR-A binding and the kinetics of IR-A phosphorylation can independently lead to a lower mitogenic activity. None of the studied parameters could account for the lower metabolic activity of qIGF-I.

  19. Postsynaptic density protein 95-regulated NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in levodopa-induced dyskinesia rat models

    Directory of Open Access Journals (Sweden)

    Ba M

    2014-12-01

    Full Text Available Maowen Ba,1,* Min Kong,2,* Guozhao Ma3 1Department of Neurology, Yuhuangding Hospital, Yantai City, Shandong, People’s Republic of China; 2Department of Neurology, Yantaishan Hospital, Yantai City, Shandong, People’s Republic of China; 3Department of Neurology, Shandong Provincial Qianfoshan Hospital, Shandong University, Shandong, People’s Republic of China *These authors contributed equally to this work Context: Abnormality in interactions between N-methyl-d-aspartate (NMDA receptor and its signaling molecules occurs in the lesioned striatum in Parkinson’s disease (PD and levodopa-induced dyskinesia (LID. It was reported that Fyn-mediated NR2B tyrosine phosphorylation, can enhance NMDA receptor function. Postsynaptic density protein 95 (PSD-95, one of the synapse-associated proteins, regulates interactions between receptor and downstream-signaling molecules. In light of the relationship between PSD-95, NR2B, and Fyn kinases, does PSD-95 contribute to the overactivity of NMDA receptor function induced by dopaminergic treatment? To further prove the possibility, the effects of regulating the PSD-95 expression on the augmented NR2B tyrosine phosphorylation and on the interactions of Fyn and NR2B in LID rat models were evaluated.Methods: In the present study, parkinsonian rat models were established by injecting 6-hydroxydopamine. Subsequently, valid PD rats were treated with levodopa (50 mg/kg/day with benserazide 12.5 mg/kg/day, twice daily intraperitoneally for 22 days to create LID rat models. Then, the effect of pretreatment with an intrastriatal injection of the PSD-95mRNA antisense oligonucleotides (PSD-95 ASO on the rotational response to levodopa challenge was assessed. The effects of pretreatment with an intrastriatal injection of PSD-95 ASO on the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in the LID rat models were detected by immunoblotting and immunoprecipitation. Results: Levodopa

  20. Plasma adiponectin concentration is associated with skeletal muscle insulin receptor tyrosine phosphorylation, and low plasma concentration precedes a decrease in whole-body insulin sensitivity in humans

    DEFF Research Database (Denmark)

    Stefan, Norbert; Vozarova, Barbora; Funahashi, Tohru

    2002-01-01

    concentration and skeletal muscle IR tyrosine phosphorylation and 2) the prospective effect of plasma adiponectin concentration at baseline on change in insulin sensitivity. Fasting plasma adiponectin concentration, body composition (hydrodensitometry or dual energy X-ray absorptiometry), insulin sensitivity.......02, respectively). Longitudinally, after adjustment for age, sex, and percent body fat, low plasma adiponectin concentration at baseline was associated with a decrease in insulin sensitivity (P = 0.04). In conclusion, our cross-sectional data suggest a role of physiological concentration of fasting plasma...

  1. Helicobacter pylori VacA, acting through receptor protein tyrosine phosphatase ?, is crucial for CagA phosphorylation in human duodenum carcinoma cell line AZ-521

    OpenAIRE

    Nakano, Masayuki; Yahiro, Kinnosuke; Yamasaki, Eiki; Kurazono, Hisao; Akada, Junko; Yamaoka, Yoshio; Niidome, Takuro; Hatakeyama, Masanori; Suzuki, Hidekazu; Yamamoto, Taro; Moss, Joel; Isomoto, Hajime; Hirayama, Toshiya

    2016-01-01

    ABSTRACT Helicobacter pylori, a major cause of gastroduodenal diseases, produces vacuolating cytotoxin (VacA) and cytotoxin-associated gene A (CagA), which seem to be involved in virulence. VacA exhibits pleiotropic actions in gastroduodenal disorders via its specific receptors. Recently, we found that VacA induced the phosphorylation of cellular Src kinase (Src) at Tyr418 in AZ-521 cells. Silencing of receptor protein tyrosine phosphatase (RPTP)?, a VacA receptor, reduced VacA-induced Src ph...

  2. Csk-Induced Phosphorylation of Src at Tyrosine 530 is Essential for H2O2-Mediated Suppression of ERK1/2 in Human Umbilical Vein Endothelial Cells

    Science.gov (United States)

    Jeon, Bo Kyung; Kwon, Kihwan; Kang, Jihee Lee; Choi, Youn-Hee

    2015-01-01

    Mitogen-activated protein kinases (MAPKs) are key signal transducers involved in various cellular events such as growth, proliferation, and differentiation. Previous studies have reported that H2O2 leads to phosphorylation of extracellular signal-regulated kinase (ERK), one of the MAPKs in endothelial cells. The current study shows that H2O2 suppressed ERK1/2 activation and phosphorylation at specific concentrations and times in human umbilical vein endothelial cells but not in immortalized mouse aortic endothelial cells or human astrocytoma cell line CRT-MG. Phosphorylation of other MAPK family members (i.e., p38 and JNK) was not suppressed by H2O2. The decrease in ERK1/2 phosphorylation induced by H2O2 was inversely correlated with the level of phosphorylation of Src tyrosine 530. Using siRNA, it was found that H2O2-induced suppression of ERK1/2 was dependent on Csk. Physiological laminar flow abrogated, but oscillatory flow did not affect, the H2O2-induced suppression of ERK1/2 phosphorylation. In conclusion, H2O2-induced Csk translocation to the plasma membrane leads to phosphorylation of Src at the tyrosine 530 residue resulting in a reduction of ERK1/2 phosphorylation. Physiological laminar flow abrogates this effect of H2O2 by inducing phosphorylation of Src tyrosine 419. These findings broaden our understanding of signal transduction mechanisms in the endothelial cells against oxidative stress. PMID:26234813

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

    DEFF Research Database (Denmark)

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

    1995-01-01

    We have studied the biochemical signal pathway leading to a rise in intracellular free calcium concentration ([Ca2+]i) following cross-linking of MHC class I (MHC-I) molecules on human T leukemic Jurkat cells. Evidence is presented that MHC-I signaling is dependent on tyrosine kinase activity......). Collectively, these results indicate that the MHC-I signaling pathway is linked to activation of tyrosine kinase(s) in Jurkat cells....

  4. Inhibitory effect of SPE-39 due to tyrosine phosphorylation and ubiquitination on the function of Vps33B in the EGF-stimulated cells.

    Science.gov (United States)

    Ishii, Ayumi; Kamimori, Kanae; Hiyoshi, Mineyoshi; Kido, Hiroshi; Ohta, Takeshi; Konishi, Hiroaki

    2012-07-30

    Although SPE-39 is a binding protein to Vps33B that is one of the subunit in the mammalian HOPS complex, the elements of SPE-39 function remain unknown. Here, we show that tyrosine phosphorylation of SPE-39 following EGF stimulation plays a role in the stability of SPE-39 itself. Ubiquitination of the C-terminal region of SPE-39 was also elevated in response to EGF stimulation, and this process was regulated by the phosphorylation of Tyr-11 in SPE-39. However, association of Vps33B with SPE-39 inhibited the elevation of ubiquitination of SPE-39 following EGF stimulation, which might be responsible for the stabilization of SPE-39. Furthermore, an opposing functional relationship between SPE-39 and Vps33B on the downregulation of the EGF receptor was observed in EGF-stimulated COS-7 cells. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  5. Basic fibroblast growth factor induces matrix metalloproteinase-13 via ERK MAP kinase-altered phosphorylation and sumoylation of Elk-1 in human adult articular chondrocytes

    Directory of Open Access Journals (Sweden)

    Hee-Jeong Im

    2009-10-01

    Full Text Available Hee-Jeong Im,1–4 Andrew D Sharrocks,5 Xia Lin,6 Dongyao Yan,1 Jaesung Kim,1 Andre J van Wijnen,7 Robert A Hipskind81Departments of Biochemistry, 2Internal Medicine, 3Section of Rheumatology, Orthopedic Surgery, 4Rush University Medical Center, and Department of Bioengineering; University of Illinois at Chicago, IL USA; 5Faculty of Life Sciences, University of Manchester, Oxford Rd, Manchester, UK; 6Michael D DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, USA; 7Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA; 8Institute De Genetique Moleculaire de Montpellier, FranceAbstract: Degradation of the extracellular matrix (ECM by matrix metalloproteinases (MMPs and release of basic fibroblast growth factor (bFGF are principal aspects of the pathology of osteoarthritis (OA. ECM disruption leads to bFGF release, which activates the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK pathway and its downstream target the Ets-like transcription factor Elk-1. Previously we demonstrated that the bFGF-ERK-Elk-1 signaling axis is responsible for the potent induction of MMP-13 in human primary articular chondrocytes. Here we report that, in addition to phosphorylation of Elk-1, dynamic posttranslational modification of Elk-1 by small ubiquitin-related modifier (SUMO serves as an important mechanism through which MMP-13 gene expression is regulated. We show that bFGF activates Elk-1 mainly through the ERK pathway and that increased phosphorylation of Elk-1 is accompanied by decreased conjugation of SUMO to Elk-1. Reporter gene assays reveal that phosphorylation renders Elk-1 competent for induction of MMP-13 gene transcription, while sumoylation has the opposite effect. Furthermore, we demonstrate that the SUMO-conjugase Ubc9 acts as a key mediator for Elk-1 sumoylation. Taken together, our results suggest that sumoylation antagonizes the phosphorylation

  6. High STAT1 mRNA levels but not its tyrosine phosphorylation are associated with macrophage infiltration and bad prognosis in breast cancer

    International Nuclear Information System (INIS)

    Tymoszuk, Piotr; Fiegl, Heidi; Doppler, Wolfgang; Charoentong, Pornpimol; Hackl, Hubert; Spilka, Rita; Müller-Holzner, Elisabeth; Trajanoski, Zlatko; Obrist, Peter; Revillion, Françoise; Peyrat, Jean-Philippe

    2014-01-01

    STAT1 has been attributed a function as tumor suppressor. However, in breast cancer data from microarray analysis indicated a predictive value of high mRNA expression levels of STAT1 and STAT1 target genes belonging to the interferon-related signature for a poor response to therapy. To clarify this issue we have determined STAT1 expression levels and activation by different methods, and investigated their association with tumor infiltration by immune cells. Additionally, we evaluated the interrelationship of these parameters and their significance for predicting disease outcome. Expression of STAT1, its target genes SOCS1, IRF1, CXCL9, CXCL10, CXCL11, IFIT1, IFITM1, MX1 and genes characteristic for immune cell infiltration (CD68, CD163, PD-L1, PD-L2, PD-1, CD45, IFN-γ, FOXP3) was determined by RT-PCR in two independent cohorts comprising 132 breast cancer patients. For a subset of patients, protein levels of total as well as serine and tyrosine-phosphorylated STAT1 were ascertained by immunohistochemistry or immunoblotting and protein levels of CXCL10 by ELISA. mRNA expression levels of STAT1 and STAT1 target genes, as well as protein levels of total and serine-phosphorylated STAT1 correlated with each other in neoplastic tissue. However, there was no association between tumor levels of STAT1 mRNA and tyrosine-phosphorylated STAT1 and between CXCL10 serum levels and CXCL10 expression in the tumor. Tumors with increased STAT1 mRNA amounts exhibited elevated expression of genes characteristic for tumor-associated macrophages and immunosuppressive T lymphocytes. Survival analysis revealed an association of high STAT1 mRNA levels and bad prognosis in both cohorts. A similar prognostically relevant correlation with unfavorable outcome was evident for CXCL10, MX1, CD68, CD163, IFN-γ, and PD-L2 expression in at least one collective. By contrast, activation of STAT1 as assessed by the level of STAT1-Y701 phosphorylation was linked to positive outcome. In multivariate Cox

  7. Growth hormone-dependent phosphorylation of tyrosine 333 and/or 338 of the growth hormone receptor

    DEFF Research Database (Denmark)

    VanderKuur, J A; Wang, X; Zhang, L

    1995-01-01

    and a reduction of GH-dependent phosphorylation of the full-length receptor. Consistent with Tyr333 and/or Tyr338 serving as substrates of JAK2, these substitutions resulted in a loss of tyrosyl phosphorylation of truncated receptor in an in vitro kinase assay using substantially purified GH.GHR.JAK2 complexes...

  8. A phosphorylation site in Bruton's tyrosine kinase selectively regulates B cell calcium signaling efficiency by altering phospholipase C-γ activation

    Science.gov (United States)

    Guo, Shuling; Ferl, Gregory Z.; Deora, Rajendar; Riedinger, Mireille; Yin, Sheng; Kerwin, James L.; Loo, Joseph A.; Witte, Owen N.

    2004-01-01

    Loss of function of Bruton's tyrosine kinase (Btk) causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency in mice (xid). By using MS analysis and phosphopeptide-specific antibodies, we identified a tyrosine phosphorylation site (Y617) near the carboxyl terminus of the Btk domain from Btk expressed in 293T as well as DT-40 cells. Y617 is conserved in all Tec family kinases except murine Tec. Replacement of Y617 with a negatively charged glutamic acid (E) suppressed Btk-mediated phospholipase Cγ2 activation and calcium response in DT-40 cells, whereas Akt activation was not affected. The Btk Y617E mutant could partially restore conventional B cell development and proliferation in Btk–/Tec– mice but failed to rescue CD5+ B-1 cell development and the TI-II immune response to 2,4,6,-trinitrophenyl-Ficoll. These data suggest that Y617 phosphorylation or a negative charge at this site may down-regulate the function of Btk by selectively suppressing the B cell calcium signaling pathway. PMID:15375214

  9. Induction of Tyrosine Phosphorylation of UV-Activated EGFR by the Beta-Human Papillomavirus Type 8 E6 Leads to Papillomatosis

    Directory of Open Access Journals (Sweden)

    Stefanie Taute

    2017-11-01

    Full Text Available Epidemiological evidence is accumulating that beta-human papillomaviruses (HPV synergize with UV-light in the development of precancerous actinic keratosis, and cutaneous squamous cell carcinomas (cSCC, one of the most common cancers in the Caucasian population. We previously demonstrated the tumorigenic activity of beta-HPV type 8 (HPV8 in the skin of transgenic mice and its cooperation with UV-light. Analysis of underlying mechanisms now showed that in keratinocytes expressing the HPV8E6 protein a transient increase of tyrosine phosphorylated epidermal growth factor receptor (EGFR in response to UV-irradiation occurred, while EGFR tyrosine phosphorylation, i.e., receptor tyrosine kinase (RTK-activity was hardly affected in empty vector control cells. FACS and immunofluorescences revealed that the EGFR was internalized into early endosomes in response to UV-exposure in both, HPV8E6 positive and in control cells, yet with a higher rate in the presence of HPV8E6. Moreover, only in HPV8E6 expressing keratinocytes the EGFR was further sorted into CD63+ intraluminal vesicles, indicative for trafficking to late endosomes. The latter requires the ubiquitination of the EGFR, and in correlation, we could show that only in HPV8E6 positive keratinocytes the EGFR was ubiquitinated upon UV-exposure. HPV8E6 and tyrosine phosphorylated EGFR directly interacted which was enhanced by UV-irradiation. The treatment of K14-HPV8E6 transgenic mice with Canertinib, an inhibitor of the RTK-activity of the EGFR, suppressed skin papilloma growth in response to UV-irradiation. This confirms the crucial role of the RTK-activity of the EGFR in HPV8E6 and UV-mediated papillomatosis in transgenic mice. Taken together, our results demonstrate that HPV8E6 alters the signaling of the UV-activated EGFR and this is a critical step in papilloma formation in response to UV-light in transgenic mice. Our results provide a molecular basis how a beta-HPV type may support early steps of

  10. Impaired degradation followed by enhanced recycling of epidermal growth factor receptor caused by hypo-phosphorylation of tyrosine 1045 in RBE cells

    International Nuclear Information System (INIS)

    Gui, Anping; Kobayashi, Akira; Motoyama, Hiroaki; Kitazawa, Masato; Takeoka, Michiko; Miyagawa, Shinichi

    2012-01-01

    Since cholangiocarcinoma has a poor prognosis, several epidermal growth factor receptor (EGFR)-targeted therapies with antibody or small molecule inhibitor treatment have been proposed. However, their effect remains limited. The present study sought to understand the molecular genetic characteristics of cholangiocarcinoma related to EGFR, with emphasis on its degradation and recycling. We evaluated EGFR expression and colocalization by immunoblotting and immunofluorescence, cell surface EGFR expression by fluorescence-activated cell sorting (FACS), and EGFR ubiquitination and protein binding by immunoprecipitation in the human cholangiocarcinoma RBE and immortalized cholangiocyte MMNK-1 cell lines. Monensin treatment and Rab11a depletion by siRNA were adopted for inhibition of EGFR recycling. Upon stimulation with EGF, ligand-induced EGFR degradation was impaired and the expression of phospho-tyrosine 1068 and phospho-p44/42 MAPK was sustained in RBE cells as compared with MMNK-1 cells. In RBE cells, the process of EGFR sorting for lysosomal degradation was blocked at the early endosome stage, and non-degradated EGFR was recycled to the cell surface. A disrupted association between EGFR and the E3 ubiquitin ligase c-Cbl, as well as hypo-phosphorylation of EGFR at tyrosine 1045 (Tyr1045), were also observed in RBE cells. In RBE cells, up-regulation of EGFR Tyr1045 phosphorylation is a potentially useful molecular alteration in EGFR-targeted therapy. The combination of molecular-targeted therapy determined by the characteristics of individual EGFR phosphorylation events and EGFR recycling inhibition show promise in future treatments of cholangiocarcinoma

  11. The radioprotector O-phospho-tyrosine stimulates DNA-repair via epidermal growth factor receptor- and DNA-dependent kinase phosphorylation

    International Nuclear Information System (INIS)

    Dittmann, Klaus; Mayer, Claus; Wanner, Gabriele; Kehlbach, Rainer; Rodemann, H. Peter

    2007-01-01

    Background and purpose: Purpose of the study was to elucidate the underlying molecular mechanism of the radioprotector O-phospho-tyrosine (P-Tyr). Methods: Molecular effects of P-Tyr at the level of EGFR responses were investigated in vitro with bronchial carcinoma cell line A549. Nuclear EGFR transport and DNA-PK activation were quantified after Western blotting. Residual DNA-damages were quantified by help of γH 2 AX focus assay. Results: As determined by dose-response curves, treatment of cells with P-Tyr for 16 h before irradiation results in radioprotection. Simultaneous treatment with EGFR blocking antibody Cetuximab abolished P-Tyr associated radioprotection. At the molecular level P-Tyr mediated a general phosphorylation of EGFR and a pronounced phosphorylation of nuclear EGFR at residue Thr No. 654, also observed after treatment with ionizing radiation. This phosphorylation was associated with nuclear EGFR accumulation. Moreover, P-Tyr-triggered EGFR nuclear accumulation was associated with phosphorylation of DNA-PK at Thr 2609. This activated form of DNA-PK was not DNA associated, but after radiation, DNA binding increased, particularly after P-Tyr pre-treatment. These molecular effects of P-Tyr resulted in a reduction of residual DNA-damage after irradiation. Conclusions: Radioprotection by P-Tyr is mediated through its stimulation of nuclear EGFR transport and concurrent, but DNA-damage independent, activation of DNA-PK. Thus, subsequent irradiation results in increased binding of DNA-PK to DNA, improved DNA-repair and increased cell survival

  12. A specific A/T polymorphism in Western tyrosine phosphorylation B-motifs regulates Helicobacter pylori CagA epithelial cell interactions.

    Directory of Open Access Journals (Sweden)

    Xue-Song Zhang

    2015-02-01

    Full Text Available Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk.

  13. A Specific A/T Polymorphism in Western Tyrosine Phosphorylation B-Motifs Regulates Helicobacter pylori CagA Epithelial Cell Interactions

    Science.gov (United States)

    Zhang, Xue-Song; Tegtmeyer, Nicole; Traube, Leah; Jindal, Shawn; Perez-Perez, Guillermo; Sticht, Heinrich; Backert, Steffen; Blaser, Martin J.

    2015-01-01

    Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk. PMID:25646814

  14. Heat shock protein 90 has roles in intracellular calcium homeostasis, protein tyrosine phosphorylation regulation, and progesterone-responsive sperm function in human sperm.

    Science.gov (United States)

    Li, Kun; Xue, Yamei; 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.

  15. Dopaminergic and cholinergic regulation of Fyn tyrosine kinase phosphorylation in the rat striatum in vivo.

    Science.gov (United States)

    Mao, Li-Min; Wang, John Q

    2015-12-01

    Src and Fyn are two Src family kinase (SFK) members that are expressed in mammalian brains and play important roles in the regulation of a variety of neuronal and synaptic substrates. Here we investigated the responsiveness of these SFKs to changing dopamine receptor signals in dopamine responsive regions of adult rat brains in vivo. Pharmacological activation of dopamine D1 receptors (D1Rs) by a systemic injection of the selective agonist SKF81297 increased phosphorylation of SFKs at a conserved and activation-associated autophosphorylation site (Y416) in the striatum, indicating activation of SFKs following SKF81297 injection. The dopamine D2 receptor (D2R) agonist quinpirole had no effect. Blockade of D1Rs with an antagonist SCH23390 did not alter striatal Y416 phosphorylation, while the D2R antagonist eticlopride elevated it. Between Src and Fyn, SKF81297 seemed to preferentially facilitate Fyn phosphorylation. Activation of muscarinic acetylcholine M4 receptors (M4Rs) with a positive allosteric modulator VU0152100 suppressed SFK Y416 responses to SKF81297. Additionally, SKF81297 induced a correlated increase in phosphorylation of N-methyl-D-aspartate (NMDA) receptor GluN2B subunits at a Fyn site (Y1472), which was attenuated by VU0152100. SKF81297 also enhanced synaptic recruitments of active Fyn and GluN1/GluN2B-containing NMDA receptors. These data demonstrate that D1Rs regulate Fyn and downstream NMDA receptors in striatal neurons in vivo. Acetylcholine through activating M4Rs inhibits Fyn and NMDA receptors in their sensitivity to D1R signaling. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Dopamine or biopterin deficiency potentiates phosphorylation at (40)Ser and ubiquitination of tyrosine hydroxylase to be degraded by the ubiquitin proteasome system.

    Science.gov (United States)

    Kawahata, Ichiro; Ohtaku, Shiori; Tomioka, Yoshihisa; Ichinose, Hiroshi; Yamakuni, Tohru

    2015-09-11

    The protein amount of tyrosine hydroxylase (TH), that is the rate-limiting enzyme for the biosynthesis of dopamine (DA), should be tightly regulated, whereas its degradation pathway is largely unknown. In this study, we analyzed how the TH protein is chemically modified and subsequently degraded under deficiencies of DA and tetrahydrobiopterin (BH4), a cofactor for TH, by using pharmacological agents in PC12D cells and cultured mesencephalic neurons. When inhibition of DA- or BH4-synthesizing enzymes greatly reduced the DA contents in PC12D cells, a marked and persistent increase in phosphorylated TH at (40)Ser (p40-TH) was concomitantly observed. This phosphorylation was mediated by D2 dopamine auto-receptor and cAMP-dependent protein kinase (PKA). Our immunoprecipitation experiments showed that the increase in the p40-TH level was accompanied with its poly-ubiquitination. Treatment of PC12D cells with cycloheximide showed that total-TH protein level was reduced by the DA- or BH4-depletion. Notably, this reduction in the total-TH protein level was sensitive not only to a 26S proteasomal inhibitor, MG-132, but also to a PKA inhibitor, H-89. These data demonstrated that DA deficiency should induce compensatory activation of TH via phosphorylation at (40)Ser through D2-autoreceptor and PKA-mediated pathways, which in turn give a rise to its degradation through an ubiquitin-proteasome pathway, resulting in a negative spiral of DA production when DA deficiency persists. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Diana M. Dunn

    2015-08-01

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

  18. Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells

    Science.gov (United States)

    Descostes, Nicolas; Heidemann, Martin; Spinelli, Lionel; Schüller, Roland; Maqbool, Muhammad Ahmad; Fenouil, Romain; Koch, Frederic; Innocenti, Charlène; Gut, Marta; Gut, Ivo; Eick, Dirk; Andrau, Jean-Christophe

    2014-01-01

    In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation. Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of promoter (5′ associated) Pol II in mammalian cells, in contrast to what was described in yeast. Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers. Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the truncated Pol IIB form, and results in a lethal phenotype. Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription, and Pol II stability. DOI: http://dx.doi.org/10.7554/eLife.02105.001 PMID:24842994

  19. ZNF198-FGFR1 Transforms Ba/F3 Cells to Growth Factor Independence and Results in High Level Tyrosine Phosphorylation of STATS 1 and 5

    Directory of Open Access Journals (Sweden)

    Damian Smedley

    1999-10-01

    Full Text Available The ZNF198-FGFR1 fusion gene arises as a result of the t(8;13(p11;g12 in the 8p11 myeloproliferative syndrome. To determine the transforming properties of this chimeric protein we transfected ZNF198-FGFR1 into the interleukin (IL-3 dependent cell line Ba/F3. Growth factor independent subclones were obtained in which ZNF198-FGFR1, STAT1, STAT5 were constitutively tyrosine phosphorylated, as determined by immunoprecipitation and Western blot analysis. To test the hypothesis that constitutive activation of ZNF198-FGFR1 tyrosine kinase activity is a result of selfassociation of the fusion protein, we in vitro transcribed and translated ZNF198-FGFR1 and a derivative construct, ZNF198-FGFR1ΔC-myc, in which the C-terminal FGFR1 epitope was replaced by a c-myc tag. As expected, an anti-FGFR1 antibody immunoprecipitated ZNF198-FGFR1 but not ZNF198-FGFR1ΔC-myc. However when both products were translated together, both were coimmunoprecipitated by anti-FGFR1 antisera. Similar results were obtained by using an anti-myc antibody and demonstrated a physical interaction between the two proteins. Analysis of COS-7 cells transfected with ZNF198-FGFR1 demonstrated that the fusion gene, in contrast to normal FGFR1, is located in the cytoplasm. We conclude that ZNF198-FGFRi is a cytoplasmic protein that self-associates and has constitutive transformation activity. These data suggest that ZNF198-FGFR1 plays a primary role in the pathogenesis of the t(8;13 myeloproliferative syndrome and is the first report to implicate STAT proteins in FGFR1-mediated signaling.

  20. Gab2 is phosphorylated on tyrosine upon interleukin-2/interleukin-15 stimulation in mycosis-fungoides-derived tumor T cells and associates inducibly with SHP-2 and Stat5a

    DEFF Research Database (Denmark)

    Brockdorff, J L; Gu, H; Mustelin, T

    2001-01-01

    of cells with the Src family kinase inhibitor, PP1, surprisingly increased the IL-2- and IL-15-induced tyrosine phosphorylation of Gab2, indicating that an Src family kinase member negatively regulates IL-2 receptor signaling in MF T cells. Thus, although Gab2 seems to function normally in MF T cells...... compared to normal T cells, Gab2 itself might be abnormally regulated by an Src family kinase....

  1. Interferon-alpha signalling in bovine adrenal chromaffin cells: involvement of signal-transducer and activator of transcription 1 and 2, extracellular signal-regulated protein kinases 1/2 and serine 31 phosphorylation of tyrosine hydroxylase.

    Science.gov (United States)

    Douglas, S A; Bunn, S J

    2009-03-01

    Adrenal medullary chromaffin cells are an integral part of the neuroendocrine system, playing an important role in the physiological adaptation to stress. In response to a wide variety of stimuli, including acetylcholine released from the splanchnic nerve, hormones such as angiotensin II or paracrine signals such as prostaglandins, chromaffin cells synthesise and secrete catecholamines and a number of biologically active peptides. This adrenal medullary output mediates a complex and diverse stress response. We report that chromaffin cells also respond both acutely and chronically to interferon (IFN)-alpha, thus providing a mechanism of interaction between the immune system and the stress response. Incubation of isolated bovine chromaffin cells maintained in culture, with IFN-alpha resulted in a rapid, transient activation of the extracellular signal-regulated protein kinase (ERK)1/2, which was maximal after 5 min. IFN-alpha mediated activation of ERK1/2 appeared to be responsible for the increased phosphorylation of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. This tyrosine hydroxylase phosphorylation was exclusively on serine 31, with no change in the phosphorylation of serine 19 or 40. This increase in the serine 31 phosphorylation of tyrosine hydroxylase was prevented by inhibition of protein kinase C or ERK1/2 activation. Incubation with IFN-alpha also resulted in a time- and concentration-dependent phosphorylation and nuclear translocation of signal transducer and activator of transcription proteins (STAT)1 and 2. This response was maximal after approximately 60 min. Prolonged treatment with IFN-alpha (12-48 h) resulted in increased expression of STAT1 and, to a lesser extent, STAT2. Thus, these findings demonstrate that adrenal medullary chromaffin cells are responsive to IFN-alpha and provide a possible cellular mechanism by which this immune-derived signal can potentially influence and integrate with the stress response.

  2. The novel synthetic ether lipid inositol-C2-PAF inhibits phosphorylation of the tyrosine kinases Src and FAK independent of integrin activation in transformed skin cells.

    Science.gov (United States)

    Semini, Geo; Hildmann, Annette; Reissig, Hans-Ulrich; Reutter, Werner; Danker, Kerstin

    2011-04-15

    New alkyl-phospholipids that are structurally derived from platelet-activating factor are promising candidates for anticancer treatment. The mechanism of action of derivatives of the platelet-activating factor is distinctly different from that of known DNA- or tubulin-targeting anticancer agents because they are incorporated into cell membranes, where they accumulate and interfere with a wide variety of key enzymes. We recently presented evidence of a novel group of alkyl-phospholipids, glycosidated phospholipids that efficiently inhibit cell proliferation. One member of this group, inositol-C2-PAF (Ino-C2-PAF), displays high efficacy and low cytotoxicity in HaCaT-cells, an immortalized non-tumorigenic skin keratinocyte cell line. Here, we show that Ino-C2-PAF also inhibits the motility of the skin-derived transformed cell lines HaCaT and squamous cell carcinoma (SCC)-25. This decrease in motility is accompanied by an altered F-actin cytoskeleton, increased clustering of integrins, and increased cell-matrix adhesion. Despite enhanced integrin clustering and matrix adhesion, we observed less phosphorylation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and Src, key regulators of cellular motility, at focal adhesion sites. Transient transfection of constitutively active variants of FAK and Src could at least in part bybass this inhibitory effect of Ino-C2-PAF. This fact indicates that Ino-C2-PAF interferes with the fine-tuned balance between adhesion and migration. Ino-C2-PAF at least partially uncouples integrin-mediated attachment from subsequent integrin-dependent signaling steps, which inhibits migration in transformed keratinocyte cell lines. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Tyrosine phosphorylation of plant tubulin

    Czech Academy of Sciences Publication Activity Database

    Blume, Y. B.; Yemets, A.; Sulimenko, Vadym; Sulimenko, Tetyana; Chan, J.; Lloyd, C.; Dráber, Pavel

    2008-01-01

    Roč. 229, č. 1 (2008), s. 143-150 ISSN 0032-0935 R&D Projects: GA MŠk LC545; GA ČR GA204/05/2375 Grant - others:EU(XE) INTAS 03-51-6459 Institutional research plan: CEZ:AV0Z50520514 Keywords : plant microtubules * tubulin * post-translation modifications Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.088, year: 2008

  4. Protein-tyrosine phosphatases in zebrafish gastrulation

    NARCIS (Netherlands)

    van Eekelen, M.J.L.

    2011-01-01

    Protein tyrosine phosphorylation plays a key role in relaying external stimuli and signals into the cell towards the appropriate responses. This process is mediated by protein-tyrosine kinases adding a phosphor group to a tyrosine residue and protein-tyrosine phosphatases removing a phosphor group

  5. Loss of Nuclear Localized and Tyrosine Phosphorylated Stat5 in Breast Cancer Predicts Poor Clinical Outcome and Increased Risk of Antiestrogen Therapy Failure

    Science.gov (United States)

    Peck, Amy R.; Witkiewicz, Agnieszka K.; Liu, Chengbao; Stringer, Ginger A.; Klimowicz, Alexander C.; Pequignot, Edward; Freydin, Boris; Tran, Thai H.; Yang, Ning; Rosenberg, Anne L.; Hooke, Jeffrey A.; Kovatich, Albert J.; Nevalainen, Marja T.; Shriver, Craig D.; Hyslop, Terry; Sauter, Guido; Rimm, David L.; Magliocco, Anthony M.; Rui, Hallgeir

    2011-01-01

    Purpose To investigate nuclear localized and tyrosine phosphorylated Stat5 (Nuc-pYStat5) as a marker of prognosis in node-negative breast cancer and as a predictor of response to antiestrogen therapy. Patients and Methods Levels of Nuc-pYStat5 were analyzed in five archival cohorts of breast cancer by traditional diaminobenzidine-chromogen immunostaining and pathologist scoring of whole tissue sections or by immunofluorescence and automated quantitative analysis (AQUA) of tissue microarrays. Results Nuc-pYStat5 was an independent prognostic marker as measured by cancer-specific survival (CSS) in patients with node-negative breast cancer who did not receive systemic adjuvant therapy, when adjusted for common pathology parameters in multivariate analyses both by standard chromogen detection with pathologist scoring of whole tissue sections (cohort I; n = 233) and quantitative immunofluorescence of a tissue microarray (cohort II; n = 291). Two distinct monoclonal antibodies gave concordant results. A progression array (cohort III; n = 180) revealed frequent loss of Nuc-pYStat5 in invasive carcinoma compared to normal breast epithelia or ductal carcinoma in situ, and general loss of Nuc-pYStat5 in lymph node metastases. In cohort IV (n = 221), loss of Nuc-pYStat5 was associated with increased risk of antiestrogen therapy failure as measured by univariate CSS and time to recurrence (TTR). More sensitive AQUA quantification of Nuc-pYStat5 in antiestrogen-treated patients (cohort V; n = 97) identified by multivariate analysis patients with low Nuc-pYStat5 at elevated risk for therapy failure (CSS hazard ratio [HR], 21.55; 95% CI, 5.61 to 82.77; P < .001; TTR HR, 7.30; 95% CI, 2.34 to 22.78; P = .001). Conclusion Nuc-pYStat5 is an independent prognostic marker in node-negative breast cancer. If confirmed in prospective studies, Nuc-pYStat5 may become a useful predictive marker of response to adjuvant hormone therapy. PMID:21576635

  6. Collagen Type I Selectively Activates Ectodomain Shedding of the Discoidin Domain Receptor 1: Involvement of Src Tyrosine Kinase

    Science.gov (United States)

    Slack, Barbara E.; Siniaia, Marina S.; Blusztajn, Jan K.

    2008-01-01

    The discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that is highly expressed in breast carcinoma cells. Upon binding to collagen, DDR1 undergoes autophosphorylation followed by limited proteolysis to generate a tyrosine phosphorylated C-terminal fragment (CTF). Although it was postulated that this fragment is formed as a result of shedding of the N-terminal ectodomain, collagen-dependent release of the DDR1 extracellular domain has not been demonstrated. We now report that, in conjunction with CTF formation, collagen type I stimulates concentration-dependent, saturable shedding of the DDR1 ectodomain from two carcinoma cell lines, and from transfected cells. In contrast, collagen did not promote cleavage of other transmembrane proteins including the amyloid precursor protein (APP), ErbB2, and E-cadherin. Collagen-dependent tyrosine phosphorylation and proteolysis of DDR1 in carcinoma cells were reduced by a pharmacologic Src inhibitor. Moreover, expression of a dominant negative Src mutant protein in human embryonic kidney cells inhibited collagen-dependent phosphorylation and shedding of co-transfected DDR1. The hydroxamate-based metalloproteinase inhibitor TAPI-1 (tumor necrosis factor-α protease inhibitor-1), and tissue inhibitor of metalloproteinase (TIMP)-3, also blocked collagen-evoked DDR1 shedding, but did not reduce levels of the phosphorylated CTF. Neither shedding nor CTF formation were affected by the γ-secretase inhibitor, L-685,458. The results demonstrate that collagen-evoked ectodomain cleavage of DDR1 is mediated in part by Src-dependent activation or recruitment of a matrix- or disintegrin metalloproteinase, and that CTF formation can occur independently of ectodomain shedding. Delayed shedding of the DDR1 ectodomain may represent a mechanism that limits DDR1-dependent cell adhesion and migration on collagen matrices. PMID:16440311

  7. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten

    2010-01-01

    phosphorylation. Protein-tyrosine phosphorylation in bacteria is particular with respect to very low occupancy of phosphorylation sites in vivo; this has represented a major challenge for detection techniques. Only the recent breakthroughs in gel-free high resolution mass spectrometry allowed the systematic...... detection of phosphorylated tyrosines by phosphoprotomics studies in bacteria. Other pioneering studies conducted in recent years, such as the first structures of BY-kinases and biochemical and phyiological studies of new BY-kinase substrates significantly furthered our understanding of these enzymes...

  8. Suppressor of Cytokine Signaling 6 (SOCS6) Negatively Regulates Flt3 Signal Transduction through Direct Binding to Phosphorylated Tyrosines 591 and 919 of Flt3

    DEFF Research Database (Denmark)

    Kazi, Julhash U; Sun, Jianmin; Phung, Bengt

    2012-01-01

    The receptor tyrosine kinase Flt3 is an important growth factor receptor in hematopoiesis, and gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia. SOCS6 (suppressor of cytokine signaling 6) is a member of the SOCS family of E3 ubiquitin ligases...

  9. The effect of various capacitation active compounds and capacitation time on the in vitro fertility and protein tyrosine phosphorylation profiles of bovine sperm

    Czech Academy of Sciences Publication Activity Database

    Pavlok, Antonín; Kubelka, Michal; Pěknicová, J.

    č. 9 (2001), s. 25-38 ISSN 0967-1994 R&D Projects: GA ČR GV524/96/K162; GA AV ČR KSK5052113 Keywords : capacitation * phosphorylation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.800, year: 2001

  10. Curcumin inhibits phorbol ester-induced up-regulation of cyclooxygenase-2 and matrix metalloproteinase-9 by blocking ERK1/2 phosphorylation and NF-kappaB transcriptional activity in MCF10A human breast epithelial cells.

    Science.gov (United States)

    Lee, Ki Won; Kim, Jung-Hwan; Lee, Hyong Joo; Surh, Young-Joon

    2005-01-01

    Elevated levels of cyclooxygenase-2 (COX-2) and matrix metalloproteinases (MMPs) are often observed in various types of cancerous and transformed cells, and hence recognized as potential molecular targets for the chemoprevention. In the present study, we investigated the possible inhibitory effects of curcumin on the expression of COX-2 and MMP-9 induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) in MCF10A human breast epithelial (MCF10A) cells and the underlying mechanisms. Curcumin inhibited the TPA-induced COX-2 expression at both transcriptional and post-transcriptional levels, and reduced the synthesis of prostaglandin E(2), one of the major products of COX-2. Likewise, curcumin attenuated invasiveness and motility of MCF10A cells stimulated with TPA through suppression of MMP expression. Curcumin blocked TPA-induced activation of extracellular signal-regulated protein kinase (ERK1/2) and nuclear factor kappaB (NF-kappaB) transcriptional activity. Overexpression of the dominant negative forms of ERK2 abrogated the TPA-induced NF-kappaB transcriptional activity. Treatment of MCF10A cells with U0126, which is a pharmacological inhibitor of ERK1/2, reduced TPA-induced up-regulation of COX-2 and MMP-9. Taken together, these findings suggest that curcumin inhibits the TPA-induced up-regulation of COX-2 and MMP-9 by suppressing ERK1/2 phosphorylation and NF-kappaB trans-activation in human breast epithelial cells, which may contribute to its chemopreventive potential. Antioxid. Redox Signal. 7, 1612-1620.

  11. A Loss-of-Function Screen for Phosphatases that Regulate Neurite Outgrowth Identifies PTPN12 as a Negative Regulator of TrkB Tyrosine Phosphorylation

    DEFF Research Database (Denmark)

    Ambjørn, Malene; Dubreuil, Véronique; Miozzo, Federico

    2013-01-01

    Alterations in function of the neurotrophin BDNF are associated with neurodegeneration, cognitive decline, and psychiatric disorders. BDNF promotes axonal outgrowth and branching, regulates dendritic tree morphology and is important for axonal regeneration after injury, responses that largely...... that phosphatases belong to multiple independently evolved families, which are rarely studied together. We undertook a loss-of-function RNA-interference-based screen of virtually all known (254) human phosphatases to understand their function in BDNF/TrkB-mediated neurite outgrowth in differentiated SH-SY5Y cells....... This approach identified phosphatases from diverse families, which either positively or negatively modulate BDNF-TrkB-mediated neurite outgrowth, and most of which have little or no previously established function related to NT signaling. "Classical" protein tyrosine phosphatases (PTPs) accounted for 13...

  12. Bruton’s Tyrosine Kinase Phosphorylates DDX41 and Activates Its Binding of dsDNA and STING to Initiate Type 1 Interferon Response

    Directory of Open Access Journals (Sweden)

    Koon-Guan Lee

    2015-02-01

    Full Text Available The innate immune system senses cytosolic dsDNA and bacterial cyclic dinucleotides and initiates signaling via the adaptor STING to induce type 1 interferon (IFN response. We demonstrate here that BTK-deficient cells have impaired IFN-β production and TBK1/IRF3 activation when stimulated with agonists or infected with pathogens that activate STING signaling. BTK interacts with STING and DDX41 helicase. The kinase and SH3/SH2 interaction domains of BTK bind, respectively, the DEAD-box domain of DDX41 and transmembrane region of STING. BTK phosphorylates DDX41, and its kinase activities are critical for STING-mediated IFN-β production. We show that Tyr364 and Tyr414 of DDX41 are critical for its recognition of AT-rich DNA and binding to STING, and tandem mass spectrometry identifies Tyr414 as the BTK phosphorylation site. Modeling studies further indicate that phospho-Tyr414 strengthens DDX41’s interaction with STING. Hence, BTK plays a critical role in the activation of DDX41 helicase and STING signaling.

  13. Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity

    DEFF Research Database (Denmark)

    Argetsinger, Lawrence S; Kouadio, Jean-Louis K; Steen, Hanno

    2004-01-01

    or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone......, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX......[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570...

  14. Determination of HER2 phosphorylation at tyrosine 1221/1222 improves prediction of poor survival for breast cancer patients with hormone receptor-positive tumors

    DEFF Research Database (Denmark)

    Frogne, Thomas; Laenkholm, Anne-Vibeke; Lyng, Maria B

    2009-01-01

    INTRODUCTION: High expression of total HER2 protein confers poor prognosis for breast cancer patients. HER2 is a member of the HER family consisting of four receptors, HER1 to HER4. HER receptor activity is regulated by a variety of mechanisms, and phosphorylation of the C-terminal part of the HER......, who had received adjuvant tamoxifen therapy. The observed protein expression levels were analyzed for co-expression, for correlation to clinicopathological parameters and for prognostic value in relation to disease-free survival and overall survival. Lastly, the difference between protein levels...... in primary tumors versus metastasis was evaluated. RESULTS: In the primary tumors, 8%, 18%, 14% and 15% of cases were scored positive for total HER2, pHER1, pHER2 and pHER3 expression, respectively. HER4 was expressed with strong intensity in 68% and at moderate intensity in 29% of cases. The activated forms...

  15. DYRK1A (Dual-Specificity Tyrosine-Phosphorylated and -Regulated Kinase 1A: A Gene with Dosage Effect During Development and Neurogenesis

    Directory of Open Access Journals (Sweden)

    M. Dierssen

    2006-01-01

    Full Text Available DYRKs (dual-specificity tyrosine-regulated kinases are an emerging family of evolutionarily conserved dual-specificity kinases that play key roles in cell proliferation, survival, and development. The research in the last years suggests a relevant conserved function during neuronal development, related to proliferation and/or differentiation for DYRK1A. It is expressed in neural progenitor cells and has been proposed to participate in the signaling mechanisms that regulate dendrite differentiation. In Drosophila, disruption of the homolog minibrain gene results in flies with reduced neuroblast proliferation, decreased numbers of central brain neurons, and learning/memory deficits. Knockout DYRK1A mice are embryonic lethal, and heterozygotes show decreased viability and region-specific reductions in brain size. In humans, DYRK1A has been proposed to be involved in the neurodevelopmental alterations associated with Down syndrome. The large number of protein interaction and putative substrates described for DYRK1A suggest multiple pathways and functions to be involved in its developmental function. This review focuses on the functional role that DYRK1A plays in brain development.

  16. Antioxidant-induced INrf2 (Keap1) tyrosine 85 phosphorylation controls the nuclear export and degradation of the INrf2-Cul3-Rbx1 complex to allow normal Nrf2 activation and repression.

    Science.gov (United States)

    Kaspar, James W; Niture, Suryakant K; Jaiswal, Anil K

    2012-02-15

    INrf2 (Keap1) serves as a negative regulator of the cytoprotective transcription factor Nrf2. At basal levels, INrf2 functions as a substrate adaptor to sequester Nrf2 into the Cul3-Rbx1 E3 ligase complex for ubiquitylation and proteasomal degradation. In response to antioxidants, Nrf2 is released from the INrf2-Cul3-Rbx1 complex and translocates into the nucleus, where it activates ARE-mediated cytoprotective gene expression. The present studies demonstrate that INrf2, Cul3 and Rbx1 export out of the nucleus and are degraded during the early or pre-induction response to antioxidants. Mutation of Tyr85 in INrf2 stymied the nuclear export of INrf2, suggesting that tyrosine phosphorylation controls the pre-induction nuclear export and degradation in response to antioxidants. The nuclear export of Cul3-Rbx1 were also blocked when INrf2Tyr85 was mutated, suggesting that INrf2-Cul3-Rbx1 undergo nuclear export as a complex. INrf2 siRNA also inhibited the nuclear export of Cul3-Rbx1, confirming that Cul3-Rbx1 requires INrf2 for nuclear export. Newly synthesized INrf2-Cul3-Rbx1 is imported back into the nucleus during the post-induction period to ubiquitylate and degrade Nrf2. Mutation of INrf2Tyr85 had no effect on activation of Nrf2 but led to nuclear accumulation of Nrf2 during the post-induction period owing to reduced export and degradation of Nrf2. Our results also showed that nuclear export and degradation followed by the new synthesis of INrf2-Cul3-Rbx1 controls the cellular abundance of the proteins during different phases of antioxidant responses. In conclusion, the early or pre-induction nuclear export of INrf2 in response to antioxidants is controlled by tyrosine phosphorylation, whereas the nuclear export of Cul3 and Rbx1 is controlled by INrf2, allowing normal activation or repression of Nrf2.

  17. Effect of acute acid-base disturbances on ErbB1/2 tyrosine phosphorylation in rabbit renal proximal tubules.

    Science.gov (United States)

    Skelton, Lara A; Boron, Walter F

    2013-12-15

    The renal proximal tubule (PT) is a major site for maintaining whole body pH homeostasis and is responsible for reabsorbing ∼80% of filtered HCO3(-), the major plasma buffer, into the blood. The PT adapts its rate of HCO3(-) reabsorption (JHCO3(-)) in response to acute acid-base disturbances. Our laboratory previously showed that single isolated perfused PTs adapt JHCO3(-) in response to isolated changes in basolateral (i.e., blood side) CO2 and HCO3(-) concentrations but, surprisingly, not to pH. The response to CO2 concentration can be blocked by the ErbB family tyrosine kinase inhibitor PD-168393. In the present study, we exposed enriched rabbit PT suspensions to five acute acid-base disturbances for 5 and 20 min using a panel of phosphotyrosine (pY)-specific antibodies to determine the influence of each disturbance on pan-pY, ErbB1-specific pY (four sites), and ErbB2-specific pY (two sites). We found that each acid-base treatment generated a distinct temporal pY pattern. For example, the summated responses of the individual ErbB1/2-pY sites to each disturbance showed that metabolic acidosis (normal CO2 concentration and reduced HCO3(-) concentration) produced a transient summated pY decrease (5 vs. 20 min), whereas metabolic alkalosis produced a transient increase. Respiratory acidosis (normal HCO3(-) concentration and elevated CO2 concentration) had little effect on summated pY at 5 min but produced an elevation at 20 min, whereas respiratory alkalosis produced a reduction at 20 min. Our data show that ErbB1 and ErbB2 in the PT respond to acute acid-base disturbances, consistent with the hypothesis that they are part of the signaling cascade.

  18. Hologram QSAR Models of a Series of 6-Arylquinazolin-4-Amine Inhibitors of a New Alzheimer’s Disease Target: Dual Specificity Tyrosine-Phosphorylation-Regulated Kinase-1A Enzyme

    Science.gov (United States)

    Leal, Felipe Dias; da Silva Lima, Camilo Henrique; de Alencastro, Ricardo Bicca; Castro, Helena Carla; Rodrigues, Carlos Rangel; Albuquerque, Magaly Girão

    2015-01-01

    Dual specificity tyrosine-phosphorylation-regulated kinase-1A (DYRK1A) is an enzyme directly involved in Alzheimer’s disease, since its increased expression leads to β-amyloidosis, Tau protein aggregation, and subsequent formation of neurofibrillary tangles. Hologram quantitative structure-activity relationship (HQSAR, 2D fragment-based) models were developed for a series of 6-arylquinazolin-4-amine inhibitors (36 training, 10 test) of DYRK1A. The best HQSAR model (q2 = 0.757; SEcv = 0.493; R2 = 0.937; SE = 0.251; R2pred = 0.659) presents high goodness-of-fit (R2 > 0.9), as well as high internal (q2 > 0.7) and external (R2pred > 0.5) predictive power. The fragments that increase and decrease the biological activity values were addressed using the colored atomic contribution maps provided by the method. The HQSAR contribution map of the best model is an important tool to understand the activity profiles of new derivatives and may provide information for further design of novel DYRK1A inhibitors. PMID:25756379

  19. Hologram QSAR Models of a Series of 6-Arylquinazolin-4-Amine Inhibitors of a New Alzheimer’s Disease Target: Dual Specificity Tyrosine-Phosphorylation-Regulated Kinase-1A Enzyme

    Directory of Open Access Journals (Sweden)

    Felipe Dias Leal

    2015-03-01

    Full Text Available Dual specificity tyrosine-phosphorylation-regulated kinase-1A (DYRK1A is an enzyme directly involved in Alzheimer’s disease, since its increased expression leads to β-amyloidosis, Tau protein aggregation, and subsequent formation of neurofibrillary tangles. Hologram quantitative structure-activity relationship (HQSAR, 2D fragment-based models were developed for a series of 6-arylquinazolin-4-amine inhibitors (36 training, 10 test of DYRK1A. The best HQSAR model (q2 = 0.757; SEcv = 0.493; R2 = 0.937; SE = 0.251; R2pred = 0.659 presents high goodness-of-fit (R2 > 0.9, as well as high internal (q2 > 0.7 and external (R2pred > 0.5 predictive power. The fragments that increase and decrease the biological activity values were addressed using the colored atomic contribution maps provided by the method. The HQSAR contribution map of the best model is an important tool to understand the activity profiles of new derivatives and may provide information for further design of novel DYRK1A inhibitors.

  20. Receptor Tyrosine Kinases in Drosophila Development

    Science.gov (United States)

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

    Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

  1. Dual-specificity tyrosine-phosphorylated and regulated kinase 1A (DYRK1A) interacts with the phytanoyl-CoA alpha-hydroxylase associated protein 1 (PAHX-AP1), a brain specific protein.

    Science.gov (United States)

    Bescond, Marilyne; Rahmani, Zohra

    2005-04-01

    Down syndrome (DS) is the most common genetic defect correlated with mental retardation and delayed development. The specific genes responsible for these phenotypic alterations have not yet been defined. Dyrk1A (dual-specificity tyrosine-phosphorylated and regulated kinase 1A), the human ortholog of the Drosophila minibrain gene (mnb), maps to the Down syndrome critical region of human chromosome 21 and is overexpressed in Down syndrome fetal brain. In Drosophila, minibrain is involved in postembryonic neurogenesis. In human, DYRK1A encodes a serine-threonine kinase but despite its potential involvement in the neurobiological alterations associated with Down syndrome, its physiological function has not yet been defined. To gain some insight into its biological function, we used the yeast two-hybrid approach to identify binding partners of DYRK1A. We found that the C-terminal region of DYRK1A interacts with a brain specific protein, phytanoyl-CoA alpha-hydroxylase-associated protein 1 (PAHX-AP1, also named PHYHIP) which was previously shown to interact with phytanoyl-CoA alpha-hydroxylase (PAHX, also named PHYH), a Refsum disease gene product. This interaction was confirmed by co-immunoprecipitation of PC12 cells co-transfected with DYRK1A and PAHX-AP1. Furthermore, immunofluorescence analysis of PC12 cells co-transfected with both plasmids showed a re-distribution of DYRK1A from the nucleus to the cytoplasm where it co-localized with PAHX-AP1. Finally, in PC12 cells co-transfected with both plasmids, DYRK1A was no longer able to interact with the nuclear transcription factor CREB, thereby confirming that the intracellular localization of DYRK1A was changed from the nucleus to the cytoplasm in the presence of PAHX-AP1. Therefore, these data indicate that by inducing a re-localization of DYRK1A into the cytoplasm, PAHX-AP1 may contribute to new cellular functions of DYRK1A and suggest that PAHX-AP1 may be involved in the development of neurological abnormalities

  2. Phosphoproteomics identifies driver tyrosine kinases in sarcoma cell lines and tumors.

    Science.gov (United States)

    Bai, Yun; Li, Jiannong; Fang, Bin; Edwards, Arthur; Zhang, Guolin; Bui, Marilyn; Eschrich, Steven; Altiok, Soner; Koomen, John; Haura, Eric B

    2012-05-15

    Driver tyrosine kinase mutations are rare in sarcomas, and patterns of tyrosine phosphorylation are poorly understood. To better understand the signaling pathways active in sarcoma, we examined global tyrosine phosphorylation in sarcoma cell lines and human tumor samples. Anti-phosphotyrosine antibodies were used to purify tyrosine phosphorylated peptides, which were then identified by liquid chromatography and tandem mass spectrometry. The findings were validated with RNA interference, rescue, and small-molecule tyrosine kinase inhibitors. We identified 1,936 unique tyrosine phosphorylated peptides, corresponding to 844 unique phosphotyrosine proteins. In sarcoma cells alone, peptides corresponding to 39 tyrosine kinases were found. Four of 10 cell lines showed dependence on tyrosine kinases for growth and/or survival, including platelet-derived growth factor receptor (PDGFR)α, MET, insulin receptor/insulin-like growth factor receptor signaling, and SRC family kinase signaling. Rhabdomyosarcoma samples showed overexpression of PDGFRα in 13% of examined cases, and sarcomas showed abundant tyrosine phosphorylation and expression of a number of tyrosine phosphorylated tyrosine kinases, including DDR2, EphB4, TYR2, AXL, SRC, LYN, and FAK. Together, our findings suggest that integrating global phosphoproteomics with functional analyses with kinase inhibitors can identify drivers of sarcoma growth and survival. ©2012 AACR.

  3. Phosphorylation site prediction in plants.

    Science.gov (United States)

    Yao, Qiuming; Schulze, Waltraud X; Xu, Dong

    2015-01-01

    Protein phosphorylation events on serine, threonine, and tyrosine residues are the most pervasive protein covalent bond modifications in plant signaling. Both low and high throughput studies reveal the importance of phosphorylation in plant molecular biology. Although becoming more and more common, the proteome-wide screening on phosphorylation by experiments remains time consuming and costly. Therefore, in silico prediction methods are proposed as a complementary analysis tool to enhance the phosphorylation site identification, develop biological hypothesis, or help experimental design. These methods build statistical models based on the experimental data, and they do not have some of the technical-specific bias, which may have advantage in proteome-wide analysis. More importantly computational methods are very fast and cheap to run, which makes large-scale phosphorylation identifications very practical for any types of biological study. Thus, the phosphorylation prediction tools become more and more popular. In this chapter, we will focus on plant specific phosphorylation site prediction tools, with essential illustration of technical details and application guidelines. We will use Musite, PhosPhAt and PlantPhos as the representative tools. We will present the results on the prediction of the Arabidopsis protein phosphorylation events to give users a general idea of the performance range of the three tools, together with their strengths and limitations. We believe these prediction tools will contribute more and more to the plant phosphorylation research community.

  4. Snake Venom Metalloproteinases

    Directory of Open Access Journals (Sweden)

    Gâz Florea Şerban Andrei

    2016-03-01

    Full Text Available As more data are generated from proteome and transcriptome analysis revealing that metalloproteinases represent most of the Viperid and Colubrid venom components authors decided to describe in a short review a classification and some of the multiple activities of snake venom metalloproteinases. SVMPs are classified in three major classes (P-I, P-II and P-III classes based on the presence of various domain structures and according to their domain organization. Furthermore, P-II and P-III classes were separated in subclasses based on distinctive post-translational modifications. SVMPs are synthesized in a latent form, being activated through a Cys-switch mechanism similar to matrix metalloproteinases. Most of the metalloproteinases of the snake venom are responsible for the hemorrhagic events but also have fibrinogenolytic activity, poses apoptotic activity, activate blood coagulation factor II and X, inhibit platelet aggregation, demonstrating that SVMPs have multiple functions in addition to well-known hemorrhagic function.

  5. Snake Venom Metalloproteinases

    OpenAIRE

    Gâz Florea Şerban Andrei; Gâz Florea Adriana; Kelemen Hajnal; Muntean Daniela-Lucia

    2016-01-01

    As more data are generated from proteome and transcriptome analysis revealing that metalloproteinases represent most of the Viperid and Colubrid venom components authors decided to describe in a short review a classification and some of the multiple activities of snake venom metalloproteinases. SVMPs are classified in three major classes (P-I, P-II and P-III classes) based on the presence of various domain structures and according to their domain organization. Furthermore, P-II and P-III clas...

  6. Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc

    NARCIS (Netherlands)

    Kranenburg, O.; Verlaan, I.; Hordijk, P. L.; Moolenaar, W. H.

    1997-01-01

    Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known

  7. Loss of Function Studies in Mice and Genetic Association Link Receptor Protein Tyrosine Phosphatase a to Schizophrenia

    DEFF Research Database (Denmark)

    Takahashi, Nagahide; Nielsen, Karin Sandager; Aleksic, Branko

    2011-01-01

    Solid evidence links schizophrenia (SZ) susceptibility to neurodevelopmental processes involving tyrosine phosphorylation-mediated signaling. Mouse studies implicate the Ptpra gene, encoding protein tyrosine phosphatase RPTPa, in the control of radial neuronal migration, cortical cytoarchitecture...

  8. A snake venom metalloproteinase, kistomin, cleaves platelet glycoprotein VI and impairs platelet functions.

    Science.gov (United States)

    Hsu, C C; Wu, W B; Huang, T F

    2008-09-01

    Injuries to the vessel wall and subsequent exposure of the matrix of the subendothelial layer resulted in thrombus formation. Platelet glycoprotein (GP) Ib and VI play a crucial role in matrix-induced activation and aggregation of platelets. In the present study, we reported that the GPIb-cleaving snake venom metalloproteinase (SVMP), kistomin, inhibited collagen-induced platelet aggregation. Moreover, kistomin inhibited platelet aggregation induced by convulxin (CVX, a GPVI agonist) and a GPVI-specific antibody in a concentration and time-dependent manner. Kistomin treatment decreased platelet GPVI but not integrin alpha2beta1 and alphaIIbbeta3, accompanied with the formation of GPVI cleavage fragments, as determined by flow cytometric and Western blot analyses. In addition, intact platelet GPVI and recombinant GPVI were digested by kistomin to release 25- and 35-kDa fragments, suggesting that kistomin cleaved GPVI near the mucin-like region. We designed four synthetic peptides ranging from Leu180 to Asn249 as the substrates for kistomin and found that kistomin cleaved these synthetic peptides at FSE205/A206TA and NKV218/F219TT, as analyzed by MALDI-TOF-MS. In addition, GPVI-specific antibody-induced tyrosine kinase phosphorylation in platelets was reduced after kistomin pretreatment, and platelet adhesion to collagen but not to fibrinogen was attenuated by kistomin. We provided here the first evidence that a P-I snake venom metalloproteinase, kistomin, inhibits the interaction between collagen and platelet GPVI through its proteolytic activity on GPVI, thus providing an alternative strategy for developing new anti-thrombotic agents.

  9. A P387L variant in protein tyrosine phosphatase-1B (PTP-1B) is associated with type 2 diabetes and impaired serine phosphorylation of PTP-1B in vitro

    DEFF Research Database (Denmark)

    Echwald, Søren M; Riis, Helle Bach; Vestergaard, Henrik

    2002-01-01

    In the present study, we tested the hypothesis that variability in the protein tyrosine phosphatase-1B (PTP-1B) gene is associated with type 2 diabetes. Using single-strand conformational polymorphism analysis, we examined cDNA of PTP-1B from 56 insulin-resistant patients with type 2 diabetes......, G381S and T420M. The G381S and 3'UTR+104insG insertion variants were not associated with type 2 diabetes. In an association study, the P387L variant was found in 14 of 527 type 2 diabetic subjects (allelic frequency 1.4%, 0.4-2.4 CI) and in 5 of 542 glucose-tolerant control subjects (allelic...... frequency 0.5%, CI 0.1-1.1), showing a significant association to type 2 diabetes (P = 0.036). In vitro, p34 cell division cycle (p34(cdc2)) kinase-directed incorporation of [gamma-(32)P]ATP was reduced in a mutant peptide compared with native peptide (387P: 100% vs. 387L: 28.4 +/- 5.8%; P = 0...

  10. Mechanism of protein tyrosine phosphatase 1B-mediated inhibition of leptin signalling

    DEFF Research Database (Denmark)

    Lund, I K; Hansen, J A; Andersen, H S

    2005-01-01

    Upon leptin binding, the leptin receptor is activated, leading to stimulation of the JAK/STAT signal transduction cascade. The transient character of the tyrosine phosphorylation of JAK2 and STAT3 suggests the involvement of protein tyrosine phosphatases (PTPs) as negative regulators of this sign...

  11. From immune response to cancer : a spot on the low molecular weight protein tyrosine phosphatase

    NARCIS (Netherlands)

    Souza, A. C. S.; Azoubel, S.; Queiroz, K. C. S.; Peppelenbosch, M. P.; Ferreira, C. V.

    Reversible tyrosine phosphorylation is a key posttranslational regulatory modification of proteins in all eukaryotic cells in normal and pathological processes. Recently a pivotal janus-faced biological role of the low molecular weight protein tyrosine phosphatase (LMWPTP) has become clear. On the

  12. Tissue inhibitor of metalloproteinase-3 (TIMP3) promotes endothelial apoptosis via a caspase-independent mechanism.

    Science.gov (United States)

    Qi, Jian Hua; Anand-Apte, Bela

    2015-04-01

    Tissue inhibitor of metalloproteinases-3 (TIMP3) is a tumor suppressor and a potent inhibitor of angiogenesis. TIMP3 exerts its anti-angiogenic effect via a direct interaction with vascular endothelial growth factor (VEGF) receptor-2 (KDR) and inhibition of proliferation, migration and tube formation of endothelial cells (ECs). TIMP3 has also been shown to induce apoptosis in some cancer cells and vascular smooth muscle cells via MMP inhibition and caspase-dependent mechanisms. In this study, we examined the molecular mechanisms of TIMP3-mediated apoptosis in endothelial cells. We have previously demonstrated that mice developed smaller tumors with decreased vascularity when injected with breast carcinoma cells overexpressing TIMP3, than with control breast carcinoma cells. TIMP3 overexpression resulted in increased apoptosis in human breast carcinoma (MDA-MB435) in vivo but not in vitro. However, TIMP3 could induce apoptosis in ECs in vitro. The apoptotic activity of TIMP3 in ECs appears to be independent of MMP inhibitory activity. Furthermore, the equivalent expression of functional TIMP3 promoted apoptosis and caspase activation in ECs expressing KDR (PAE/KDR), but not in ECs expressing PDGF beta-receptor (PAE/β-R). Surprisingly, the apoptotic activity of TIMP3 appears to be independent of caspases. TIMP3 inhibited matrix-induced focal adhesion kinase (FAK) tyrosine phosphorylation and association with paxillin and disrupted the incorporation of β3 integrin, FAK and paxillin into focal adhesion contacts on the matrix, which were not affected by caspase inhibitors. Thus, TIMP3 may induce apoptosis in ECs by triggering a caspase-independent cell death pathway and targeting a FAK-dependent survival pathway.

  13. The CD45 protein tyrosine phosphatase is required for the completion of the activation program leading to lymphokine production in the Jurkat human T cell line

    NARCIS (Netherlands)

    Peyron, J. F.; Verma, S.; de Waal Malefyt, R.; Sancho, J.; Terhorst, C.; Spits, H.

    1991-01-01

    Stimulation of the T cell antigen receptor, TCR-CD3, induces tyrosine phosphorylation of specific cellular proteins through activation of a tyrosine kinase. The possible regulatory role of the CD45 protein tyrosine phosphatase in this process was explored by studying the functional properties of

  14. Tyrosine 769 of the keratinocyte growth factor receptor is required for receptor signaling but not endocytosis

    International Nuclear Information System (INIS)

    Ceridono, Mara; Belleudi, Francesca; Ceccarelli, Simona; Torrisi, Maria Rosaria

    2005-01-01

    Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells which belongs to the family of fibroblast growth factor receptors (FGFRs). Following ligand binding, KGFR is rapidly autophosphorylated on specific tyrosine residues in the intracellular domain, recruits substrate proteins, and is rapidly internalized by clathrin-mediated endocytosis. The role of different autophosphorylation sites in FGFRs, and in particular the role of the tyrosine 766 in FGFR1, first identified as PLCγ binding site, has been extensively studied. We analyzed here the possible role of the tyrosine 769 in KGFR, corresponding to tyrosine 766 in FGFR1, in the regulation of KGFR signal transduction and MAPK activation as well as in the control of the endocytic process of KGFR. A mutant KGFR in which tyrosine 769 was substituted by phenylalanine was generated and transfected in NIH3T3 and HeLa cells. Our results indicate that tyrosine 769 is required for the binding to KGFR and tyrosine phosphorylation of PLCγ as well as for the full activation of MAPKs and for cell proliferation through the regulation of FRS2 tyrosine phosphorylation, suggesting that this residue represents a key regulator of KGFR signal transduction. Our data also show that tyrosine 769 is not involved in the regulation of the endocytic process of KGFR

  15. Cellular Functions Regulated by Phosphorylation of EGFR on Tyr845

    Directory of Open Access Journals (Sweden)

    Ken-ichi Sato

    2013-05-01

    Full Text Available The Src gene product (Src and the epidermal growth factor receptor (EGFR are prototypes of oncogene products and function primarily as a cytoplasmic non-receptor tyrosine kinase and a transmembrane receptor tyrosine kinase, respectively. The identification of Src and EGFR, and the subsequent extensive investigations of these proteins have long provided cutting edge research in cancer and other molecular and cellular biological studies. In 1995, we reported that the human epidermoid carcinoma cells, A431, contain a small fraction of Src and EGFR in which these two kinase were in physical association with each other, and that Src phosphorylates EGFR on tyrosine 845 (Y845 in the Src-EGFR complex. Y845 of EGFR is located in the activation segment of the kinase domain, where many protein kinases contain kinase-activating autophosphorylation sites (e.g., cAMP-dependent protein kinase, Src family kinases, transmembrane receptor type tyrosine kinases or trans-phosphorylation sites (e.g., cyclin-dependent protein kinase, mitogen-activated protein kinase, Akt protein kinase. A number of studies have demonstrated that Y845 phosphorylation serves an important role in cancer as well as normal cells. Here we compile the experimental facts involving Src phosphorylation of EGFR on Y845, by which cell proliferation, cell cycle control, mitochondrial regulation of cell metabolism, gamete activation and other cellular functions are regulated. We also discuss the physiological relevance, as well as structural insights of the Y845 phosphorylation.

  16. Regulation of PCNA Function by Tyrosine Phosphorylation in Prostate Cancer

    Science.gov (United States)

    2012-10-01

    Cell Cycle 2004;3:15–8. 7. Maga G, Hubscher U. Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J Cell Sci 2003;116:3051–60. 8...SUMO. Cell Cycle 3: 15–8. 13. Maga G, Hubscher U (2003) Proliferating cell nuclear antigen (PCNA): a dancer with many partners. J Cell Sci 116: 3051

  17. Tyrosine phosphorylation of the human guanylyl cyclase C receptor

    Indian Academy of Sciences (India)

    Unknown

    per the manufacturers' protocol, with slight modifications. TKB1 cells transformed with the plasmid of interest were grown to an A600 of 0⋅5, in Luria Bertani broth containing antibiotics at 37°C. Cells were collected by centrifugation and resuspended in induction medium, containing 1 mM. IPTG, to induce the gene of interest ...

  18. Identification and characterization of hydra metalloproteinase 2 (HMP2): a meprin-like astacin metalloproteinase that functions in foot morphogenesis.

    Science.gov (United States)

    Yan, L; Fei, K; Zhang, J; Dexter, S; Sarras, M P

    2000-01-01

    Several members of the newly emerging astacin metalloproteinase family have been shown to function in a variety of biological events, including cell differentiation and morphogenesis during both embryonic development and adult tissue differentiation. We have characterized a new astacin proteinase, hydra metalloproteinase 2 (HMP2) from the Cnidarian, Hydra vulgaris. HMP2 is translated from a single mRNA of 1.7 kb that contains a 1488 bp open reading frame encoding a putative protein product of 496 amino acids. The overall structure of HMP2 most closely resembles that of meprins, a subgroup of astacin metalloproteinases. The presence of a transient signal peptide and a putative prosequence indicates that HMP2 is a secreted protein that requires post-translational processing. The mature HMP2 starts with an astacin proteinase domain that contains a zinc binding motif characteristic of the astacin family. Its COOH terminus is composed of two potential protein-protein interaction domains: an "MAM" domain (named after meprins, A-5 protein and receptor protein tyrosine phosphatase mu) that is only present in meprin-like astacin proteinases; and a unique C-terminal domain (TH domain) that is also present in another hydra metalloproteinase, HMP1, in Podocoryne metalloproteinase 1 (PMP1) of jellyfish and in toxins of sea anemone. The spatial expression pattern of HMP2 was determined by both mRNA whole-mount in situ hybridization and immunofluorescence studies. Both morphological techniques indicated that HMP2 is expressed only by the cells in the endodermal layer of the body column of hydra. While the highest level of HMP2 mRNA expression was observed at the junction between the body column and the foot process, immunofluorescence studies indicated that HMP2 protein was present as far apically as the base of the tentacles. In situ analysis also indicated expression of HMP2 during regeneration of the foot process. To test whether the higher levels of HMP2 mRNA expression at

  19. The role of low-molecular-weight protein tyrosine phosphatase (LMW-PTP ACP1) in oncogenesis.

    Science.gov (United States)

    Alho, Irina; Costa, Luís; Bicho, Manuel; Coelho, Constança

    2013-08-01

    Protein tyrosine phosphorylation is a crucial cellular event that is involved in the most important processes of cellular metabolism. Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) is a tyrosine phosphatase that presents two active distinct isoforms and is regulated through cysteine oxidation and tyrosine phosphorylation. This enzyme has been linked to tumorigenesis, but its role is considered controversial: it may be considered oncogenic or anti-oncogenic depending on its interaction with different substrates. Furthermore, recent studies have demonstrated that LMW-PTP is involved in epithelial cell migration, a characteristic of tumor cells. This fact strengthens the importance of this enzyme in the oncogenic process and opens new avenues for future research. The study of LMW-PTP and its pathways may enhance therapeutic strategies that target tyrosine phosphorylation and its substrates. In this review, we try to clarify the importance of this protein in carcinogenesis through the analysis of LMW-PTP interaction with different substrates.

  20. Exploring oxidative modifications of tyrosine

    DEFF Research Database (Denmark)

    Houée-Lévin, C; Bobrowski, K; Horakova, L

    2015-01-01

    residues are oxidised in vivo with impact on cellular homeostasis and redox signalling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different...... effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor...... processes are important in vivo and can contribute to cellular pathology....

  1. Phosphatidylinositol 3'-kinase and tyrosine-phosphatase activation positively modulate Convulxin-induced platelet activation. Comparison with collagen.

    Science.gov (United States)

    Lagrue, A H; Francischetti, I M; Guimarães, J A; Jandrot-Perrus, M

    1999-04-01

    In this report we have studied the role of phosphatidylinositol 3'-kinase (PI3-K) and tyrosine phosphatase activation on platelet activation by Convulxin (Cvx). Wortmannin, a specific PI3-K inhibitor, and phenylarsine oxide (PAO), a sulfhydryl reagent that inhibits tyrosine phosphatase (PTPase), block Cvx-induced platelet aggregation, granule secretion, inositol phosphate production, and increase in [Ca2+]i. However, PAO does not inhibit Cvx-induced tyrosine phosphorylation of platelet proteins, including Syk and PLCgamma2, but blocked collagen-induced platelet aggregation as well as tyrosine phosphorylation of PLCgamma2. In contrast, Cvx-induced PLCgamma2 tyrosyl phosphorylation was partially inhibited by wortmannin. We conclude that (i) although Cvx and collagen activate platelets by a similar mechanism, different regulatory processes are specific to each agonist; (ii) mechanisms other than tyrosine phosphorylation regulate PLCgamma2 activity; and (iii) besides protein tyrosine kinases, PI3-K (and PTPase) positively modulate platelet activation by both Cvx and collagen, and this enzyme is required for effective transmission of GPVI-Fc receptor gamma chain signal to result in full activation and tyrosine phosphorylation of PLCgamma2 in Cvx-stimulated platelets.

  2. Cloning of a novel phosphotyrosine binding domain containing molecule, Odin, involved in signaling by receptor tyrosine kinases

    DEFF Research Database (Denmark)

    Pandey, A.; Blagoev, B.; Kratchmarova, I.

    2002-01-01

    We have used a proteomic approach using mass spectrometry to identify signaling molecules involved in receptor tyrosine kinase signaling pathways. Using affinity purification by anti-phosphotyrosine antibodies to enrich for tyrosine phosphorylated proteins, we have identified a novel signaling mo...

  3. Phospho.ELM: A database of experimentally verified phosphorylation sites in eukaryotic proteins

    DEFF Research Database (Denmark)

    Diella, F.; Cameron, S.; Gemund, C.

    2004-01-01

    Background: Post-translational phosphorylation is one of the most common protein modifications. Phosphoserine, threonine and tyrosine residues play critical roles in the regulation of many cellular processes. The fast growing number of research reports on protein phosphorylation points to a gener...

  4. Tyrosine supplementation for phenylketonuria.

    Science.gov (United States)

    Webster, Diana; Wildgoose, Joanne

    2013-06-05

    Phenylketonuria is an inherited disease for which the main treatment is the dietary restriction of the amino acid phenylalanine. The diet has to be initiated in the neonatal period to prevent or reduce mental handicap. However, the diet is very restrictive and unpalatable and can be difficult to follow. A deficiency of the amino acid tyrosine has been suggested as a cause of some of the neuropsychological problems exhibited in phenylketonuria. Therefore, this review aims to assess the efficacy of tyrosine supplementation for phenylketonuria. To assess the effects of tyrosine supplementation alongside or instead of a phenylalanine-restricted diet for people with phenylketonuria, who commenced on diet at diagnosis and either continued on the diet or relaxed the diet later in life. To assess the evidence that tyrosine supplementation alongside, or instead of a phenylalanine-restricted diet improves intelligence, neuropsychological performance, growth and nutritional status, mortality rate and quality of life. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Trials Register which is comprised of references identified from comprehensive electronic database searches, handsearches of relevant journals and abstract books of conference proceedings. Additional studies were identified from handsearches of the Journal of Inherited Metabolic Disease (from inception in 1978 to 1998). The manufacturers of prescribable dietary products used in the treatment of phenylketonuria were also contacted for further references.Date of the most recent search of the Group's Inborn Errors of Metabolism Trials Register: 28 June 2012. All randomised or quasi-randomised trials investigating the use of tyrosine supplementation versus placebo in people with phenylketonuria in addition to, or instead of, a phenylalanine-restricted diet. People treated for maternal phenylketonuria were excluded. Two authors independently assessed the trial eligibility, methodological quality

  5. Constitutive phosphorylation of eps8 in tumor cell lines: relevance to malignant transformation

    DEFF Research Database (Denmark)

    Matoskova, B; Wong, W T; Salcini, A E

    1995-01-01

    eps8, a recently identified tyrosine kinase substrate, has been shown to augment epidermal growth factor (EGF) responsiveness, implicating it in EGF receptor (EGFR)-mediated mitogenic signaling. We investigated the status of eps8 phosphorylation in normal and transformed cells and the role of eps8...... in transformation. In NIH 3T3 cells overexpressing EGFR (NIH-EGFR), eps8 becomes rapidly phosphorylated upon EGF stimulation. At receptor-saturating doses of EGF, approximately 30% of the eps8 pool is tyrosine phosphorylated. Under physiological conditions of activation (i.e., at low receptor occupancy...... in NIH-EGFR cells. Overexpression of eps8 was able to transform NIH 3T3 cells under limiting conditions of activation of the EGFR pathway. Concomitant tyrosine phosphorylation of eps8 and shc, but not of rasGAP, phospholipase C-gamma, and eps15, was frequently detected in tumor cells. This suggested...

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

    Science.gov (United States)

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

    2016-03-11

    Cellular signaling through protein tyrosine phosphorylation is well established in mammalian cells. Although lacking the classic tyrosine kinases present in humans, plants have a tyrosine phospho-proteome that rivals human cells. Here we report a novel plant tyrosine phosphatase from Arabidopsis thaliana (AtRLPH2) that, surprisingly, has the sequence hallmarks of a phospho-serine/threonine phosphatase belonging to the PPP family. Rhizobiales/Rhodobacterales/Rhodospirillaceae-like phosphatases (RLPHs) are conserved in plants and several other eukaryotes, but not in animals. We demonstrate that AtRLPH2 is localized to the plant cell cytosol, is resistant to the classic serine/threonine phosphatase inhibitors okadaic acid and microcystin, but is inhibited by the tyrosine phosphatase inhibitor orthovanadate and is particularly sensitive to inhibition by the adenylates, ATP and ADP. AtRLPH2 displays remarkable selectivity toward tyrosine-phosphorylated peptides versus serine/threonine phospho-peptides and readily dephosphorylates a classic tyrosine phosphatase protein substrate, suggesting that in vivo it is a tyrosine phosphatase. To date, only one other tyrosine phosphatase is known in plants; thus AtRLPH2 represents one of the missing pieces in the plant tyrosine phosphatase repertoire and supports the concept of protein tyrosine phosphorylation as a key regulatory event in plants. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. The Transmodulation of HER2 and EGFR by Substance P in Breast Cancer Cells Requires c-Src and Metalloproteinase Activation.

    Directory of Open Access Journals (Sweden)

    Susana Garcia-Recio

    Full Text Available Substance P (SP is a pleiotropic cytokine/neuropeptide that enhances breast cancer (BC aggressiveness by transactivating tyrosine kinase receptors like EGFR and HER2. We previously showed that SP and its cognate receptor NK-1 (SP/NK1-R signaling modulates the basal phosphorylation of HER2 and EGFR in BC, increasing aggressiveness and drug resistance. In order to elucidate the mechanisms responsible for NK-1R-mediated HER2 and EGFR transactivation, we investigated the involvement of c-Src (a ligand-independent mediator and of metalloproteinases (ligand-dependent mediators in HER2/EGFR activation.Overexpression of NK-1R in MDA-MB-231 and its chemical inhibition in SK-BR-3, BT-474 and MDA-MB-468 BC cells significantly modulated c-Src activation, suggesting that this protein is a mediator of NK-1R signaling. In addition, the c-Src inhibitor 4-(4'-phenoxyanilino-6,7-dimethoxyquinazoline prevented SP-induced activation of HER2. On the other hand, SP-dependent phosphorylation of HER2 and EGFR decreased substantially in the presence of the MMP inhibitor 1-10, phenanthroline monohydrate, and the dual inhibition of both c-Src and MMP almost abolished the activation of HER2 and EGFR. Moreover, the use of these inhibitors demonstrated that this Src and MMP-dependent signaling is important to the cell viability and migration capacity of HER2+ and EGFR+ cell lines.Our results indicate that the transactivation of HER2 and EGFR by the pro-inflammatory cytokine/neuropeptide SP in BC cells is a c-Src and MMP-dependent process.

  8. O-GlcNAc modification: why so intimately associated with phosphorylation?

    Directory of Open Access Journals (Sweden)

    Ande Sudharsana R

    2011-01-01

    Full Text Available Abstract Post-translational modification of proteins at serine and threonine side chains by β-N-acetylglucosamine (O-GlcNAc mediated by the enzyme β-N-acetylglucosamine transferase has been emerging as a fundamental regulatory mechanism encompassing a wide range of proteins involved in cell division, metabolism, transcription and cell signaling. Furthermore, an extensive interplay between O-GlcNAc modification and serine/threonine phosphorylation in a variety of proteins has been reported to exist. However, our understanding of the regulatory mechanisms involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins is still elusive. Recent success in the mapping of O-GlcNAc modification sites in proteins as a result of technological advancement in mass spectrometry have revealed two important clues which may be inherently connected to the regulation of O-GlcNAc modification and its interplay with phosphorylation in proteins. First, almost all O-GlcNAc modified proteins are known phospho proteins. Second, the prevalence of tyrosine phosphorylation among O-GlcNAc modified proteins is exceptionally higher (~68% than its normal occurrence (~2% alone. We hypothesize that phosphorylation may be a requisite for O-GlcNAc modification and tyrosine phosphorylation plays a role in the interplay between O-GlcNAc modification and serine/threonine phosphorylation in proteins. In other words, the interplay between O-GlcNAc modification and phosphorylation is not limited to serine/threonine phosphorylation but also includes tyrosine phosphorylation. Our hypothesis provides an opportunity to understand the underlying mechanism involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins. Furthermore, implication of our hypothesis extends to tyrosine kinase signaling.

  9. Tyrosine Modifications in Aging

    OpenAIRE

    Feeney, Maria B.; Schöneich, Christian

    2012-01-01

    Significance: The understanding of physiological and pathological processes involving protein oxidation, particularly under conditions of aging and oxidative stress, can be aided by proteomic identification of proteins that accumulate oxidative post-translational modifications only if these detected modifications are connected to functional consequences. The modification of tyrosine (Tyr) residues can elicit significant changes in protein structure and function, which, in some cases, may cont...

  10. The oncogenic tyrosine kinase Lyn impairs the pro-apoptotic function of Bim.

    Science.gov (United States)

    Aira, Lazaro E; Villa, Elodie; Colosetti, Pascal; Gamas, Parvati; Signetti, Laurie; Obba, Sandrine; Proics, Emma; Gautier, Fabien; Bailly-Maitre, Béatrice; Jacquel, Arnaud; Robert, Guillaume; Luciano, Frédéric; Juin, Philippe P; Ricci, Jean-Ehrland; Auberger, Patrick; Marchetti, Sandrine

    2018-02-02

    Phosphorylation of Ser/Thr residues is a well-established modulating mechanism of the pro-apoptotic function of the BH3-only protein Bim. However, nothing is known about the putative tyrosine phosphorylation of this Bcl-2 family member and its potential impact on Bim function and subsequent Bax/Bak-mediated cytochrome c release and apoptosis. As we have previously shown that the tyrosine kinase Lyn could behave as an anti-apoptotic molecule, we investigated whether this Src family member could directly regulate the pro-apoptotic function of Bim. In the present study, we show that Bim is phosphorylated onto tyrosine residues 92 and 161 by Lyn, which results in an inhibition of its pro-apoptotic function. Mechanistically, we show that Lyn-dependent tyrosine phosphorylation of Bim increases its interaction with anti-apoptotic members such as Bcl-xL, therefore limiting mitochondrial outer membrane permeabilization and subsequent apoptosis. Collectively, our data uncover one molecular mechanism through which the oncogenic tyrosine kinase Lyn negatively regulates the mitochondrial apoptotic pathway, which may contribute to the transformation and/or the chemotherapeutic resistance of cancer cells.

  11. [Tyrosine hydroxylase of the blood leukocytes].

    Science.gov (United States)

    Mineeva, M F

    1987-07-01

    Tyrosine hydroxylase activity has been established in blood plasma leucocytes of rat, cat and man. Tyrosine precursors and some nuclear erythroid cells. GFU-GM did hydroxylase activity in leucocytes shows the Km for tyrosine inhibited by high concentrations of L6 tyrosine (substrate inhibition), alpha-methyl-para-tyrosine dopamine. The kinetic properties of leucocyte tyrosine hydroxylase are qualitatively similar to the properties of brain tyrosine hydroxylase.

  12. Roles of the C-terminal tyrosine residues of LAT in GPVI-induced platelet activation: insights into the mechanism of PLC gamma 2 activation.

    Science.gov (United States)

    Ragab, Ashraf; Séverin, Sonia; Gratacap, Marie-Pierre; Aguado, Enrique; Malissen, Marie; Jandrot-Perrus, Martine; Malissen, Bernard; Ragab-Thomas, Jeannie; Payrastre, Bernard

    2007-10-01

    Linker for activation of T cells (LAT) is an adaptor protein required for organization of the signaling machinery downstream of the platelet collagen receptor, the glycoprotein VI (GPVI). Here, we investigated the effect of LAT mutations on specific signaling pathways and on platelet functions in response to GPVI triggering by convulxin (Cvx). Using mice containing tyrosine to phenylalanine mutations of the adaptor, we show the crucial role played by the tyrosine residues at positions 175, 195, and 235 in the phosphorylation of LAT and in the whole pattern of protein tyrosine phosphorylation in response to Cvx. These 3 C-terminal tyrosine residues are important to recruit the tyrosine kinase Fyn, which may be involved in LAT phosphorylation. Efficient phosphoinositide 3-kinase (PI3K) activation requires the 3 C-terminal tyrosine residues of LAT but not its tyrosine 136. Interestingly, single mutation of the tyrosine 136 results in the loss of phospholipase C gamma2 (PLCgamma2) activation without affecting its PI3K-dependent membrane association, and is sufficient to impair platelet responses to Cvx. Thus, activation of PLCgamma2 via GPVI is dependent on 2 complementary events: its interaction with the tyrosine 136 of LAT and its membrane location, which itself requires events mediated by the 3 C-terminal tyrosines of LAT.

  13. Phosphotyrosine enrichment identifies focal adhesion kinase and other tyrosine kinases for targeting in canine hemangiosarcoma.

    Science.gov (United States)

    Marley, K; Maier, C S; Helfand, S C

    2012-09-01

    Canine hemangiosarcoma (HSA) is an endothelial cell malignancy driven, in part, by activating mutations in receptor and non-receptor tyrosine kinases. Proteomics, Western blots and a tyrosine kinase inhibitor were used to elucidate activating mechanisms in HSA cell lines. Phosphotyrosine peptides from focal adhesion kinase (FAK) STAT3, Lyn, Fyn and other signal transduction kinases were identified by mass spectrometry. FAK was constitutively activated at tyrosine 397, the autophosphorylation site, and this was reversible with high concentrations of a FAK inhibitor. FAK inhibitor-14 suppressed migration and phosphorylation of FAK tyrosine 397 and tyrosines 576/577 and was cytotoxic to HSA cells suggesting FAK signalling may be an important contributor to canine HSA survival. © 2012 Blackwell Publishing Ltd.

  14. Role of insulin receptor phosphorylation in the insulinomimetic effects of hydrogen peroxide

    International Nuclear Information System (INIS)

    Hayes, G.R.; Lockwood, D.H.

    1987-01-01

    The oxidant H 2 O 2 has many insulin-like effects in rat adipocytes. To determine whether these effects could be mediated by the tyrosine kinase activity of the insulin receptor, the ability of H 2 O 2 to stimulate receptor phosphorylation in intact adipocytes and partially purified insulin receptors has been examined. Phosphorylation of the β subunit of the insulin receptor was increased. Stimulation of receptor phosphorylation was rapid, reaching maximal levels within 5 min, and preceded activation of glucose transport. Phosphoamino acid analysis of insulin receptors from H 2 O 2 -treated adipocytes showed that 32 P incorporation into phosphotyrosine and phosphoserine residues of the β subunit was enhanced. Furthermore, partially purified receptors from H 2 O 2 -treated cells exhibit increased tyrosine kinase activity, as measured by phosphorylation of the peptide Glu 80 Tyr 20 . To define the factors involved in H 2 O 2 's effect, the authors have examined receptor phosphorylation in fat cell homogenates and purified plasma membranes. Although insulin stimulated receptor phosphorylation in both of these systems, H 2 O 2 was only effective in the cell homogenates. These data demonstrate that, under certain conditions, H 2 O 2 stimulates insulin receptor phosphorylation and tyrosine kinase activity, suggesting that the insulin-like effects of H 2 O 2 may be mediated by stimulation of insulin receptor phosphorylation. This does not appear to be a direct effect of H 2 O 2 on the insulin receptor and requires nonplasma membrane cellular constituents

  15. Enhanced cerebrovascular expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 via the MEK/ERK pathway during cerebral ischemia in the rat

    DEFF Research Database (Denmark)

    Maddahi, Aida; Chen, Qingwen; Edvinsson, Lars

    2009-01-01

    microscopy revealed enhanced expression of MMP-9, TIMP-1, and phosphorylated ERK1/2 in the smooth muscle cells of the ischemic MCA and associated intracerebral microvessels. The specific MEK1/2 inhibitor U0126, given intraperitoneal zero or 6 hours after the ischemic event, reduced the infarct volume...... of metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). RESULTS: Here, we found an infarct volume of 24.8 +/- 2% and a reduced neurological function after two hours of middle cerebral artery occlusion (MCAO), followed by 48 hours of recirculation in rat. Immunocytochemistry and confocal...... significantly (11.8 +/- 2% and 14.6 +/- 3%, respectively; P hours after MCAO did not alter the expression of MMP-9...

  16. Phospho-Tyrosine(s) vs. Phosphatidylinositol Binding in Shc Mediated Integrin Signaling

    OpenAIRE

    Lin, Xiaochen; Vinogradova, Olga

    2015-01-01

    The Shc adaptor protein, particularly its p52 isoform, has been identified as a primary signaling partner for the tyrosine(s)-phosphorylated cytoplasmic tails of activated ? 3 integrins. Inspired by our recent structure of the Shc PTB domain in complex with a bi-phosphorylated peptide derived from ? 3 cytoplasmic tail, we have initiated the investigation of Shc interaction with phospholipids of the membrane. We are particularly focused on PtdIns and their effects on Shc mediated integrin sign...

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

    DEFF Research Database (Denmark)

    Petrone, A; Sap, J

    2000-01-01

    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...

  18. Growth hormone, interferon-gamma, and leukemia inhibitory factor promoted tyrosyl phosphorylation of insulin receptor substrate-1

    DEFF Research Database (Denmark)

    Argetsinger, L S; Hsu, G W; Myers, M G

    1995-01-01

    The identification of JAK2 as a growth hormone (GH) receptor-associated, GH-activated tyrosine kinase has established tyrosyl phosphorylation as a signaling mechanism for GH. In the present study, GH is shown to stimulate tyrosyl phosphorylation of insulin receptor substrate 1 (IRS-1), the princi...... characterized insulin-like metabolic effects of GH observed in a variety of cell types....

  19. Identification of tyrosine residues in the intracellular domain of the growth hormone receptor required for transcriptional signaling and Stat5 activation

    DEFF Research Database (Denmark)

    Hansen, L. H.; Wang, X.; Kopchick, J J

    1996-01-01

    phosphorylation in intracellular signaling, we constructed GH receptors in which combinations of tyrosines were mutated to phenylalanines. We identified three tyrosine residues at positions 534, 566, and 627 that were required for activation of GH-stimulated transcription of the serine protease inhibitor (Spi) 2...

  20. Identification of tyrosine residues in the intracellular domain of the growth hormone receptor required for transcriptional signaling and Stat5 activation

    DEFF Research Database (Denmark)

    Hansen, L. H.; Wang, X.; Kopchick, J J

    1996-01-01

    The binding of growth hormone (GH) to its receptor results in its dimerization followed by activation of Jak2 kinase and tyrosine phosphorylation of the GH receptor itself, as well as Jak2 and the transcription factors Stat1, -3, and -5. In order to study the role of GH receptor tyrosine phosphor...

  1. Tau Phosphorylation by GSK3 in Different Conditions

    Directory of Open Access Journals (Sweden)

    Jesús Avila

    2012-01-01

    Full Text Available Almost a 20% of the residues of tau protein are phosphorylatable amino acids: serine, threonine, and tyrosine. In this paper we comment on the consequences for tau of being a phosphoprotein. We will focus on serine/threonine phosphorylation. It will be discussed that, depending on the modified residue in tau molecule, phosphorylation could be protective, in processes like hibernation, or toxic like in development of those diseases known as tauopathies, which are characterized by an hyperphosphorylation and aggregation of tau.

  2. Extracellular metalloproteinases in Phytomonas serpens.

    Science.gov (United States)

    Vermelho, Alane B; Almeida, Flávia V S; Bronzato, Leandro S; Branquinha, Marta H

    2003-03-01

    The detection of extracellular proteinases in Phytomonas serpens, a trypanosomatid isolated from tomato fruits, is demonstrated in this paper. Maximal production occurred at the end of the logarithmic phase of growth. These enzymes exhibited selective substrate utilization in SDS-PAGE, being more active with gelatin; hemoglobin and bovine serum albumin were not degraded. Three proteinases were detected in SDS-PAGE-gelatin, with apparent molecular masses between 94 and 70 kDa. The proteolytic activity was completely blocked by 1,10-phenanthroline and strongly inhibited by EDTA, whereas a partial inhibition was observed with trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) and soybean trypsin inhibitor; phenylmethylsulfonyl fluoride weakly inhibited the enzymes. This inhibition profile indicated that these extracellular proteinases belong to the metalloproteinase class.

  3. MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of phosphopeptides and regulation of activity.

    OpenAIRE

    Gardner, A M; Vaillancourt, R R; Lange-Carter, C A; Johnson, G L

    1994-01-01

    MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphory...

  4. Quantitative Tyrosine Phosphoproteomics of Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor-treated Lung Adenocarcinoma Cells Reveals Potential Novel Biomarkers of Therapeutic Response.

    Science.gov (United States)

    Zhang, Xu; Maity, Tapan; Kashyap, Manoj K; Bansal, Mukesh; Venugopalan, Abhilash; Singh, Sahib; Awasthi, Shivangi; Marimuthu, Arivusudar; Charles Jacob, Harrys Kishore; Belkina, Natalya; Pitts, Stephanie; Cultraro, Constance M; Gao, Shaojian; Kirkali, Guldal; Biswas, Romi; Chaerkady, Raghothama; Califano, Andrea; Pandey, Akhilesh; Guha, Udayan

    2017-05-01

    Mutations in the Epidermal growth factor receptor (EGFR) kinase domain, such as the L858R missense mutation and deletions spanning the conserved sequence 747 LREA 750 , are sensitive to tyrosine kinase inhibitors (TKIs). The gatekeeper site residue mutation, T790M accounts for around 60% of acquired resistance to EGFR TKIs. The first generation EGFR TKIs, erlotinib and gefitinib, and the second generation inhibitor, afatinib are FDA approved for initial treatment of EGFR mutated lung adenocarcinoma. The predominant biomarker of EGFR TKI responsiveness is the presence of EGFR TKI-sensitizing mutations. However, 30-40% of patients with EGFR mutations exhibit primary resistance to these TKIs, underscoring the unmet need of identifying additional biomarkers of treatment response. Here, we sought to characterize the dynamics of tyrosine phosphorylation upon EGFR TKI treatment of mutant EGFR-driven human lung adenocarcinoma cell lines with varying sensitivity to EGFR TKIs, erlotinib and afatinib. We employed stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry to identify and quantify tyrosine phosphorylated peptides. The proportion of tyrosine phosphorylated sites that had reduced phosphorylation upon erlotinib or afatinib treatment correlated with the degree of TKI-sensitivity. Afatinib, an irreversible EGFR TKI, more effectively inhibited tyrosine phosphorylation of a majority of the substrates. The phosphosites with phosphorylation SILAC ratios that correlated with the TKI-sensitivity of the cell lines include sites on kinases, such as EGFR-Y1197 and MAPK7-Y221, and adaptor proteins, such as SHC1-Y349/350, ERRFI1-Y394, GAB1-Y689, STAT5A-Y694, DLG3-Y705, and DAPP1-Y139, suggesting these are potential biomarkers of TKI sensitivity. DAPP1, is a novel target of mutant EGFR signaling and Y-139 is the major site of DAPP1 tyrosine phosphorylation. We also uncovered several off-target effects of these TKIs, such as MST1R-Y1238

  5. Tyrosine dephosphorylation regulates AMPAR internalisation in mGluR-LTD.

    Science.gov (United States)

    Gladding, Clare M; Collett, Valerie J; Jia, Zhengping; Bashir, Zafar I; Collingridge, Graham L; Molnár, Elek

    2009-02-01

    Long-term depression (LTD) can be induced at hippocampal CA1 synapses by activation of either NMDA receptors (NMDARs) or group I metabotropic glutamate receptors (mGluRs), using their selective agonists NMDA and (RS)-3,5-dihydroxyphenylglycine (DHPG), respectively. Recent studies revealed that DHPG-LTD is dependent on activation of postsynaptic protein tyrosine phosphatases (PTPs), which transiently dephosphorylate tyrosine residues in AMPA receptors (AMPARs). Here we show that while both endogenous GluR2 and GluR3 AMPAR subunits are tyrosine phosphorylated at basal activity, only GluR2 is dephosphorylated in DHPG-LTD. The tyrosine dephosphorylation of GluR2 does not occur in NMDA-LTD. Conversely, while NMDA-LTD is associated with the dephosphorylation of GluR1-serine-845, DHPG-LTD does not alter the phosphorylation of this site. The increased AMPAR endocytosis in DHPG-LTD is PTP-dependent and involves tyrosine dephosphorylation of cell surface AMPARs. Together, these results indicate that the subunit selective tyrosine dephosphorylation of surface GluR2 regulates AMPAR internalisation in DHPG-LTD but not in NMDA-LTD in the hippocampus.

  6. Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Vanesa Olivares-Illana

    2008-06-01

    Full Text Available Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.

  7. Mutation of the SHP-2 binding site in growth hormone (GH) receptor prolongs GH-promoted tyrosyl phosphorylation of GH receptor, JAK2, and STAT5B

    DEFF Research Database (Denmark)

    Stofega, M R; Herrington, J; Billestrup, Nils

    2000-01-01

    Binding of GH to GH receptor (GHR) rapidly and transiently activates multiple signal transduction pathways that contribute to the growth-promoting and metabolic effects of GH. While the events that initiate GH signal transduction, such as activation of the Janus tyrosine kinase JAK2, are beginning...... to be understood, the signaling events that terminate GH signaling, such as dephosphorylation of tyrosyl-phosphorylated signaling molecules, are poorly understood. In this report, we examine the role of the SH2 (Src homology-2) domain-containing protein tyrosine phosphatase SHP-2 in GH signaling. We demonstrate...... that the SH2 domains of SHP-2 bind directly to tyrosyl phosphorylated GHR from GH-treated cells. Tyrosine-to-phenylalanine mutation of tyrosine 595 of rat GHR greatly diminishes association of the SH2 domains of SHP-2 with GHR, and tyrosine-to-phenylalanine mutation of tyrosine 487 partially reduces...

  8. Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27.

    Science.gov (United States)

    Freshney, N W; Rawlinson, L; Guesdon, F; Jones, E; Cowley, S; Hsuan, J; Saklatvala, J

    1994-09-23

    An IL-1-stimulated protein kinase cascade resulting in phosphorylation of the small heat shock protein hsp27 has been identified in KB cells. It is distinct from the p42 MAP kinase cascade. An upstream activator kinase phosphorylated a 40 kDa kinase (p40) upon threonine and tyrosine residues, which in turn phosphorylated a 50 kDa kinase (p50) upon threonine (and some serine) residues. p50 phosphorylated hsp27 upon serine. p40 and p50 were purified to near homogeneity. All three components were inactivated by protein phosphatase 2A, and p40 was inactivated by protein tyrosine phosphatase 1B. The substrate specificity of p40 differed from that of p42 and p54 MAP kinases. The upstream activator was not a MAP kinase kinase. p50 resembled MAPKAPK-2 and may be identical.

  9. Substrate specificities of tyrosine-specific protein kinases toward cytoskeletal proteins in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Akiyama, T.; Kadowaki, T.; Nishida, E.; Kadooka, T.; Ogawara, H.; Fukami, Y.; Sakai, H.; Takaku, F.; Kasuga, M.

    1986-11-05

    The authors have previously reported that fodrin (..beta.. subunit), tubulin (..cap alpha.. subunit) and microtubule-associated proteins (MAPs; MAP2 and tau) are good substrates for the purified insulin receptor kinase. In this study, to investigate the substrate specificities of tyrosine kinases, they have examined the actions of the purified epidermal growth factor (EGF) receptor kinase and Rous sarcoma virus src kinase on purified microfilament- and microtubule-related proteins. Among microfilament-related proteins examined, the purified EGF receptor kinase phosphorylated the ..beta.. subunit, but not the ..cap alpha.. subunit, of fodrin on tyrosine residues with a K/sub m/ below the micromolar range. The fodrin phosphorylation by the EGF receptor kinase was markedly inhibited by F-actin. In contrast, the purified src kinase preferentially phosphorylated the ..cap alpha.. subunit of fodrin on tyrosine residues. Fodrin phosphorylation by the src kinase was not inhibited by F-actin. Among microtubule proteins examined, MAP-2 was the best substrate for the EGF receptor kinase. The peptide mapping of MAP2 phosphorylated by the EGF receptor kinase and by the insulin receptor kinase produced very similar patterns of phosphopeptides, while that of MAP2 phosphorylated by the src kinase gave a distinctly different pattern. When the phosphorylation of the tubulin subunits was examined, the EGF receptor kinase preferred ..beta.. subunit to ..cap alpha.. subunit, but the src kinase phosphorylated both ..cap alpha.. and ..beta.. subunits to a similar extent. These results, together with our previous results, indicate that the substrate specificities of the EGF receptor kinase and the insulin receptor kinase are very similar, but not identical, while that of the src kinase is distinctly different from that of these growth factor receptor kinases.

  10. Cellular progesterone receptor phosphorylation in response to ligands activating protein kinases

    Energy Technology Data Exchange (ETDEWEB)

    Rao, K.V.; Peralta, W.D.; Greene, G.L.; Fox, C.F.

    1987-08-14

    Progesterone receptors were immunoprecipitated with monoclonal antibodies KD68 from lysates of human breast carcinoma T47D cells labelled to steady state specific activity with /sup 32/Pi. The 120 kDa /sup 32/P-labelled progesterone receptor band was resolved by polyacrylamide gel electrophoresis and identified by autoradiography. Phosphoamino acid analysis revealed serine phosphorylation, but no threonine or tyrosine phosphorylation. Treatment of the /sup 32/Pi-labelled cells with EGF, TPA or dibutyryl cAMP had no significant quantitative effect on progesterone receptor phosphorylation, though the EGF receptor and the cAMP-dependent protein kinases have been reported to catalyze phosphorylation of purified avian progesterone receptor preparations in cell free systems. Progesterone receptor phosphorylation on serine residues was increased by 2-fold in cells treated with 10 nM progesterone; EGF had no effect on progesterone-mediated progesterone receptor phosphorylation.

  11. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko

    2011-08-01

    Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

  12. Tyrosine modifications in aging.

    Science.gov (United States)

    Feeney, Maria B; Schöneich, Christian

    2012-12-01

    The understanding of physiological and pathological processes involving protein oxidation, particularly under conditions of aging and oxidative stress, can be aided by proteomic identification of proteins that accumulate oxidative post-translational modifications only if these detected modifications are connected to functional consequences. The modification of tyrosine (Tyr) residues can elicit significant changes in protein structure and function, which, in some cases, may contribute to biological aging and age-related pathologies, such as atherosclerosis, neurodegeneration, and cataracts. Studies characterizing proteins in which Tyr has been modified to 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, 3,3'-dityrosine and other cross-links, or 3-chlorotyrosine are reviewed, with an emphasis on structural and functional consequences. Distinguishing between inconsequential modifications and functionally significant ones requires careful biochemical and biophysical analysis of target proteins, as well as innovative methods for isolating the effects of the multiple modifications that often occur under oxidizing conditions. The labor-intensive task of isolating and characterizing individual modified proteins must continue, especially given the expanding list of known modifications. Emerging approaches, such as genetic and metabolic incorporation of unnatural amino acids, hold promise for additional focused studies of this kind.

  13. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in gingival crevicular fluid during orthodontic tooth movement

    NARCIS (Netherlands)

    Bildt, Miriam; Bloemen, M; Kuijpers-Jagtman, A.M.; Von Den Hoff, Johannes W

    2009-01-01

    Orthodontic tooth movement requires extensive re-modelling of the periodontium. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during re-modelling, while their activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). The aim of this study was to investigate

  14. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in gingival crevicular fluid during orthodontic tooth movement.

    NARCIS (Netherlands)

    Bildt, M.M.; Bloemen, M.; Kuijpers-Jagtman, A.M.; Hoff, J.W. Von den

    2009-01-01

    Orthodontic tooth movement requires extensive re-modelling of the periodontium. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during re-modelling, while their activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). The aim of this study was to investigate

  15. Protein tyrosine kinases p53/56lyn and p72syk in MHC class I-mediated signal transduction in B lymphoma cells

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Bregenholt, S; Skov, S

    1998-01-01

    Crosslinking of major histocompatibility complex class I (MHC-I) molecules on the surface of human B lymphoma cells was shown to induce protein tyrosine phosphorylation and mobilization of intracellular free calcium. Immunoprecipitations indicated that the protein tyrosine kinases p53/56lyn and p72......syk are among the tyrosine-phosphorylated proteins. The kinetics of phosphorylation of these kinases after MHC-I crosslinking differ from the kinetics observed after crosslinking of the B cell antigen receptor (BCR). Additional experiments were performed with chicken lyn- and syk-negative DT40 B cells...... and the results indicate that these two kinases have different substrate specificity and regulate intracellular free calcium differently in response to MHC-I crosslinking. In addition MHC-I crosslinking of a sIgM-negative DT40 chicken B cell variant results in less activity of tyrosine kinases and less...

  16. Complexes of γ-tubulin with nonreceptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells

    International Nuclear Information System (INIS)

    Kukharskyy, Vitaliy; Sulimenko, Vadym; Macurek, Libor; Sulimenko, Tetyana; Draberova, Eduarda; Draber, Pavel

    2004-01-01

    Nonreceptor protein tyrosine kinases of the Src family have been shown to play an important role in signal transduction as well as in regulation of microtubule protein interactions. Here we show that γ-tubulin (γ-Tb) in P19 embryonal carcinoma cells undergoing neuronal differentiation is phosphorylated and forms complexes with protein tyrosine kinases of the Src family, Src and Fyn. Elevated expression of both kinases during differentiation corresponded with increased level of proteins phosphorylated on tyrosine. Immunoprecipitation experiments with antibodies against Src, Fyn, γ-tubulin, and with anti-phosphotyrosine antibody revealed that γ-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in γ-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated γ-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing γ-tubulin bound to SH2 domains and that these interactions were of SH2-phosphotyrosine type. The combined data suggest that Src family kinases might have an important role in the regulation of γ-tubulin interaction with tubulin dimers or other proteins during neurogenesis

  17. Protein phosphorylation in bcterial signaling and regulation

    KAUST Repository

    Mijakovic, Ivan

    2016-01-26

    In 2003, it was demonstrated for the first time that bacteria possess protein-tyrosine kinases (BY-kinases), capable of phosphorylating other cellular proteins and regulating their activity. It soon became apparent that these kinases phosphorylate a number of protein substrates, involved in different cellular processes. More recently, we found out that BY-kinases can be activated by several distinct protein interactants, and are capable of engaging in cross-phosphorylation with other kinases. Evolutionary studies based on genome comparison indicate that BY-kinases exist only in bacteria. They are non-essential (present in about 40% bacterial genomes), and their knockouts lead to pleiotropic phenotypes, since they phosphorylate many substrates. Surprisingly, BY-kinase genes accumulate mutations at an increased rate (non-synonymous substitution rate significantly higher than other bacterial genes). One direct consequence of this phenomenon is no detectable co-evolution between kinases and their substrates. Their promiscuity towards substrates thus seems to be “hard-wired”, but why would bacteria maintain such promiscuous regulatory devices? One explanation is the maintenance of BY-kinases as rapidly evolving regulators, which can readily adopt new substrates when environmental changes impose selective pressure for quick evolution of new regulatory modules. Their role is clearly not to act as master regulators, dedicated to triggering a single response, but they might rather be employed to contribute to fine-tuning and improving robustness of various cellular responses. This unique feature makes BY-kinases a potentially useful tool in synthetic biology. While other bacterial kinases are very specific and their signaling pathways insulated, BY-kinase can relatively easily be engineered to adopt new substrates and control new biosynthetic processes. Since they are absent in humans, and regulate some key functions in pathogenic bacteria, they are also very promising

  18. Cholinergic regulation of protein phosphorylation in bovine adrenal chromaffin cells

    International Nuclear Information System (INIS)

    Haycock, J.W.; Browning, M.D.; Greengard, P.

    1988-01-01

    Chromaffin cells were isolated from bovine adrenal medullae and maintained in primary culture. After prelabeling with 32 PO 4 , exposure of the chromaffin cells to acetylcholine increased the phosphorylation of a M/sub r/ ≅ 100,000 protein and a M/sub r/ ≅ 60,000 protein (tyrosine hydroxylase), visualized after separation of total cellular proteins in NaDodSO 4 /polyacrylamide gels. Immunoprecipitation with antibodies to three known phosphoproteins (100-kDa, 87-kDa, and protein III) revealed an acetylcholine-dependent phosphorylation of these proteins. These three proteins were also shown to be present in bovine adrenal chromaffin cells by immunolabeling techniques. 100-kDa is a M/sub r/ ≅ 100,000 protein selectively phosphorylated by calcium/calmodulin-dependent protein kinase III, 87-kDa is a M/sub r/ ≅ 87,000 protein selectively phosphorylated by protein kinase C, and protein III is a phosphoprotein doublet of M/sub r/ ≅ 74,000 (IIIa) and M/sub r/ ≅ 55,000 (IIIb) phosphorylated by cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase I. The data demonstrate that cholinergic activation of chromaffin cells increases the phosphorylation of several proteins and that several protein kinase systems may be involved in these effects

  19. Phosphoproteome analysis of E-coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation

    DEFF Research Database (Denmark)

    Macek, B.; Gnad, F.; Soufi, Boumediene

    2008-01-01

    Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is generally considered the major regulatory posttranslational modification in eukaryotic cells. Increasing evidence at the genome and proteome level shows that this modification is also present and functional in prokaryotes...

  20. A study on expression levels of matrix metalloproteinases and their ...

    African Journals Online (AJOL)

    Results: The prevailing MMP-9 and TIMP-1 expressions in the glandular epithelium and inflammatory cells were ... controlling ulcerative colitis. Keywords: Ulcerative colitis, Matrix metalloproteinases, Tissue inhibitors of metalloproteinases, Lamina propria, Inflammation ... cells, neutrophils, macrophages, neutrophils and.

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  2. Implications of tyrosine phosphoproteomics in cervical carcinogenesis

    Directory of Open Access Journals (Sweden)

    DeFord James

    2008-01-01

    Full Text Available Abstract Background Worldwide cervical cancer remains a leading cause of mortality from gynecologic malignancies. The link between cervical cancer and persistent infection with HPV has been established. At a molecular level little is known about the transition from the precancerous state to invasive cancer. To elucidate this process, cervical biopsies from human specimens were obtained from precancerous state to stage III disease. Methods Cervical biopsies were obtained from patients with a diagnosis of cervical cancer undergoing definitive surgery or staging operation. Biopsies were obtained from patients with precancerous lesions at the time of their excisional procedure. Control samples were obtained from patients undergoing hysterectomy for benign conditions such as fibroids. Samples were subjected to proteomic profiling using two dimensional gel electrophoresis with subsequent trypsin digestion followed by MALDI-TOF protein identification. Candidate proteins were then further studied using western blotting, immunoprecipitation and immunohistochemistry. Results Annexin A1 and DNA-PKcs were found to be differentially expressed. Phosphorylated annexin A1 was up regulated in diseased states in comparison to control and its level was strongly detected in the serum of cervical cancer patients compared to controls. DNA-PKcs was noted to be hyperphosphorylated and fragmented in cancer when compared to controls. By immunohistochemistry annexin A1 was noted in the vascular environment in cancer and certain precancerous samples. Conclusion This study suggests a probable role for protein tyrosine phosphorylation in cervical carcinogenesis. Annexin A1 and DNA-PK cs may have synergistic effects with HPV infection. Precancerous lesions that may progress to cervical cancer may be differentiated from lesions that will not base on similar immunohistochemical profile to invasive squamous cell carcinoma.

  3. Identification of novel Ack1-interacting proteins and Ack1 phosphorylated sites in mouse brain by mass spectrometry

    OpenAIRE

    del Mar Masdeu, Maria; Armendáriz, Beatriz G.; Torre, Anna La; Soriano, Eduardo; Burgaya, Ferran; Ureña, Jesús Mariano

    2017-01-01

    Ack1 (activated Cdc42-associated tyrosine kinase) is a non-receptor tyrosine kinase that is highly expressed in brain. This kinase contains several protein-protein interaction domains and its action is partially regulated by phosphorylation. As a first step to address the neuronal functions of Ack1, here we screened mouse brain samples to identify proteins that interact with this kinase. Using mass spectrometry analysis, we identified new putative partners for Ack1 including cytoskeletal prot...

  4. MHC class I ligation of human T cells activates the ZAP70 and p56lck tyrosine kinases, leads to an alternative phenotype of the TCR/CD3 zeta-chain, and induces apoptosis

    DEFF Research Database (Denmark)

    Skov, S; Bregenholt, S; Claesson, Mogens Helweg

    1997-01-01

    Cross-linking of MHC class I (MHC-I) molecules on human T cells induces signal-transduction events, including activation of tyrosine kinases, tyrosine phosphorylation of phospholipase C-gamma 1, and elevation of the intracellular free calcium concentration. In this study, we demonstrate that the ...

  5. Distinct phosphorylation requirements regulate cortactin activation by TirEPEC and its binding to N-WASP

    Directory of Open Access Journals (Sweden)

    Martinez-Quiles Narcisa

    2009-05-01

    Full Text Available Abstract Background Cortactin activates the actin-related 2/3 (Arp2/3 complex promoting actin polymerization to remodel cell architecture in multiple processes (e.g. cell migration, membrane trafficking, invadopodia formation etc.. Moreover, it was called the Achilles' heel of the actin cytoskeleton because many pathogens hijack signals that converge on this oncogenic scaffolding protein. Cortactin is able to modulate N-WASP activation in vitro in a phosphorylation-dependent fashion. Thus Erk-phosphorylated cortactin is efficient in activating N-WASP through its SH3 domain, while Src-phosphorylated cortactin is not. This could represent a switch on/off mechanism controlling the coordinated action of both nucleator promoting factors (NPFs. Pedestal formation by enteropathogenic Escherichia coli (EPEC requires N-WASP activation. N-WASP is recruited by the cell adapter Nck which binds a major tyrosine-phosphorylated site of a bacterial injected effector, Tir (translocated intimin receptor. Tir-Nck-N-WASP axis defines the current major pathway to actin polymerization on pedestals. In addition, it was recently reported that EPEC induces tyrosine phosphorylation of cortactin. Results Here we demonstrate that cortactin phosphorylation is absent on N-WASP deficient cells, but is recovered by re-expression of N-WASP. We used purified recombinant cortactin and Tir proteins to demonstrate a direct interaction of both that promoted Arp2/3 complex-mediated actin polymerization in vitro, independently of cortactin phosphorylation. Conclusion We propose that cortactin binds Tir through its N-terminal part in a tyrosine and serine phosphorylation independent manner while SH3 domain binding and activation of N-WASP is regulated by tyrosine and serine mediated phosphorylation of cortactin. Therefore cortactin could act on Tir-Nck-N-WASP pathway and control a possible cycling activity of N-WASP underlying pedestal formation.

  6. Using Phos-Tag in Western Blotting Analysis to Evaluate Protein Phosphorylation.

    Science.gov (United States)

    Horinouchi, Takahiro; Terada, Koji; Higashi, Tsunehito; Miwa, Soichi

    2016-01-01

    Protein phosphorylation has traditionally been detected by radioisotope phosphate labeling of proteins with radioactive ATP. Several nonradioactive assays with phosphorylation site-specific antibodies are now available for the analysis of phosphorylation status at target sites. However, due to their high specificity, these antibodies they cannot be used to detect unidentified phosphorylation sites. Recently, Phos-tag technology has been developed to overcome the disadvantages and limitations of phosphospecific antibodies. Phos-tag and its derivatives conjugated to biotin, acrylamide, or agarose, form alkoxide-bridged dinuclear metal complexes, which can capture phosphate monoester dianions bound to serine, threonine, and tyrosine residues, in an amino acid sequence-independent manner. Here, we describe our method, which is based on in vitro kinase assay and Western blotting analysis using biotinylated Phos-tag and horseradish peroxidase-conjugated streptavidin, to determine the sites of TRPC6 (transient receptor potential canonical 6) channel phosphorylated by protein kinase A.

  7. HER2 Phosphorylates and Destabilizes Pro-Apoptotic PUMA, Leading to Antagonized Apoptosis in Cancer Cells

    Science.gov (United States)

    Carpenter, Richard L.; Han, Woody; Paw, Ivy; Lo, Hui-Wen

    2013-01-01

    HER2 is overexpressed in 15–20% of breast cancers. HER2 overexpression is known to reduce apoptosis but the underlying mechanisms for this association remain unclear. To elucidate the mechanisms for HER2-mediated survival, we investigated the relationship between HER2 and p53 upregulated modulator of apoptosis (PUMA), a potent apoptosis inducer. Our results showed that HER2 interacts with PUMA, which was independent of HER2 activation. In addition, we observed that HER2 interacted with PUMA in both mitochondrial and non-mitochondrial compartments. We next examined whether HER2 phosphorylates PUMA. Notably, PUMA tyrosine phosphorylation has never been reported. Using an intracellular assay, we found PUMA to be phosphorylated in breast cancer cells with activated HER2. Via cell-free HER2 kinase assay, we observed that PUMA was directly phosphorylated by HER2. Activation of HER2 decreased PUMA protein half-life. To identify which of the three tyrosines within PUMA are targeted by HER2, we generated three PUMA non-phosphorylation mutants each with a single Tyr→Phe substitution. Results indicated that each PUMA single mutant had lost some, but not all phosphorylation by HER2 indicating that HER2 targets all three tyrosines. Consequently, we created an additional PUMA mutant with all three tyrosines mutated (TM-PUMA) that could not be phosphorylated by HER2. Importantly, TM-PUMA was found to have a longer half-life than PUMA. An inverse association was observed between HER2 and PUMA in 93 invasive breast carcinoma samples. We further found that TM-PUMA suppressed growth of breast cancer cells to a greater degree than PUMA. Also, TM-PUMA had a stronger propensity to induce apoptosis than PUMA. Together, our results demonstrate, for the first time, that PUMA can be tyrosine phosphorylated and that HER2-mediated phosphorylation destabilizes PUMA protein. The HER2-PUMA interplay represents a novel mechanism by which PUMA is regulated and a new molecular basis for HER2

  8. Identification of accelerated evolution in the metalloproteinase ...

    African Journals Online (AJOL)

    Computational protein sequence analysis is one of the most important tools used for understanding the evolution of closely related proteins sequences including snake venom metalloproteinase sequences (SVMPs) which give valuable information regarding genetic variations. The fundamental objective of the present study ...

  9. Effect of matrix metalloproteinase promoter polymorphisms on ...

    Indian Academy of Sciences (India)

    large-scale studies regarding gene–gene and gene–environment interactions are needed in the future. [Ye H., He Y., Wang J., Song T., Lan Z., Zhao Y. and Xi M. 2016 Effect of matrix metalloproteinase promoter polymorphisms on endometriosis and adenomyosis risk: evidence from a meta-analysis. J. Genet. 95, 611–619].

  10. Identification of accelerated evolution in the metalloproteinase ...

    African Journals Online (AJOL)

    U

    2016-02-24

    Feb 24, 2016 ... nearly all organisms from animal to plants. However, apart from its different expression sites in different plants and animals for performing distinct physiological roles, metalloproteinase also exists in the toxin/venom of several venomous creatures (snake, caterpillar, scorpion etc.) to cause agony, suffering ...

  11. Matrix metalloproteinase-12 (MMP-12) in osteoclasts

    DEFF Research Database (Denmark)

    Hou, Peng; Troen, Tine; Ovejero, Maria C

    2004-01-01

    Osteoclasts require matrix metalloproteinase (MMP) activity and cathepsin K to resorb bone, but the critical MMP has not been identified. Osteoclasts express MMP-9 and MMP-14, which do not appear limiting for resorption, and the expression of additional MMPs is not clear. MMP-12, also called...

  12. Molecular dynamics simulations of protein-tyrosine phosphatase 1B. I. Ligand-induced changes in the protein motions

    DEFF Research Database (Denmark)

    Peters, Günther H. J.; Frimurer, T.M.; Andersen, J.N.

    1999-01-01

    molecular dynamics simulations of PTP1B and PTP1B complexed with a high-affinity peptide DADEpYL, where pY stands for phosphorylated tyrosine. The peptide sequence is derived from the epidermal growth factor receptor (EGFR(988-993)). Simulations were performed in water for 1 ns, and the concerted motions...

  13. The myeloperoxidase-derived oxidant hypothiocyanous acid inhibits protein tyrosine phosphatases via oxidation of key cysteine residues

    DEFF Research Database (Denmark)

    Cook, Naomi L.; Moeke, Cassidy H.; Fantoni, Luca I.

    2016-01-01

    Phosphorylation of protein tyrosine residues is critical to cellular processes, and is regulated by kinases and phosphatases (PTPs). PTPs contain a redox-sensitive active site Cys residue, which is readily oxidized. Myeloperoxidase, released from activated leukocytes, catalyzes thiocyanate ion (S...

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

    Science.gov (United States)

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

    2002-08-15

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

  15. Matrix Metalloproteinase 9 Exerts Antiviral Activity against Respiratory Syncytial Virus.

    Directory of Open Access Journals (Sweden)

    Abdoulaye J Dabo

    Full Text Available Increased lung levels of matrix metalloproteinase 9 (MMP9 are frequently observed during respiratory syncytial virus (RSV infection and elevated MMP9 concentrations are associated with severe disease. However little is known of the functional role of MMP9 during lung infection with RSV. To determine whether MMP9 exerted direct antiviral potential, active MMP9 was incubated with RSV, which showed that MMP9 directly prevented RSV infectivity to airway epithelial cells. Using knockout mice the effect of the loss of Mmp9 expression was examined during RSV infection to demonstrate MMP9's role in viral clearance and disease progression. Seven days following RSV infection, Mmp9-/- mice displayed substantial weight loss, increased RSV-induced airway hyperresponsiveness (AHR and reduced clearance of RSV from the lungs compared to wild type mice. Although total bronchoalveolar lavage fluid (BALF cell counts were similar in both groups, neutrophil recruitment to the lungs during RSV infection was significantly reduced in Mmp9-/- mice. Reduced neutrophil recruitment coincided with diminished RANTES, IL-1β, SCF, G-CSF expression and p38 phosphorylation. Induction of p38 signaling was required for RANTES and G-CSF expression during RSV infection in airway epithelial cells. Therefore, MMP9 in RSV lung infection significantly enhances neutrophil recruitment, cytokine production and viral clearance while reducing AHR.

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

    Science.gov (United States)

    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.

  17. Replacement of insulin receptor tyrosine residues 1162 and 1163 does not alter the mitogenic effect of the hormone

    International Nuclear Information System (INIS)

    Debant, A.; Clauser, E.; Ponzio, G.; Filloux, C.; Auzan, C.; Contreres, J.O.; Rossi, B.

    1988-01-01

    Chinese hamster ovary transfectants that express insulin receptors in which tyrosine residues 1162 and 1163 were replaced by phenylalanine exhibit a total inhibition of the insulin-mediated tyrosine kinase activity toward exogenous substrates; this latter activity is associated with total inhibition of the hypersensitivity reported for insulin in promoting 2-deoxyglucose uptake. The authors now present evidence that the twin tyrosines also control the insulin-mediated stimulation of glycogen synthesis. Surprisingly, this type of Chinese hamster ovary transfectant is as hypersensitive to insulin for its mitogenic effect as are Chinese hamster ovary cells expressing many intact insulin receptors. Such data suggest that (i) the insulin mitogenic effect routes through a different pathway than insulin uses to activate the transport and metabolism of glucose and (ii) the mitogenic effect of insulin is not controlled by the twin tyrosines. At the molecular level, the solubilized mutated receptor has not insulin-dependent tyrosine kinase activity, whereas this receptor displays measurable insulin-stimulated phosphorylation of its β subunit in 32 P-labeled cells. The authors therefore propose that the autocatalytic phosphorylating activity of the receptor reports a cryptic tyrosine kinase activity that cannot be visualized by the use of classical exogenous substrates

  18. Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts

    DEFF Research Database (Denmark)

    Su, J; Muranjan, M; Sap, J

    1999-01-01

    BACKGROUND: Fyn and c-Src are two of the most widely expressed Src-family kinases. Both are strongly implicated in the control of cytoskeletal organization and in the generation of integrin-dependent signalling responses in fibroblasts. These proteins are representative of a large family...... of tyrosine kinases, the activity of which is tightly controlled by inhibitory phosphorylation of a carboxyterminal tyrosine residue (Tyr527 in chicken c-Src); this phosphorylation induces the kinases to form an inactive conformation. Whereas the identity of such inhibitory Tyr527 kinases has been well...... established, no corresponding phosphatases have been identified that, under physiological conditions, function as positive regulators of c-Src and Fyn in fibroblasts. RESULTS: Receptor protein tyrosine phosphatase alpha (RPTPalpha) was inactivated by homologous recombination. Fibroblasts derived from...

  19. Identification by Hydrogen/Deuterium Exchange of Structural Changes in Tyrosine Hydroxylase Associated with Regulation

    Science.gov (United States)

    Wang, Shanzhi; Sura, Giri R.; Dangott, Lawrence J.; Fitzpatrick, Paul F.

    2009-01-01

    The activity of tyrosine hydroxylase is regulated by reversible phosphorylation of serine residues in an N-terminal regulatory domain and catecholamine inhibition at the active site. Catecholamines such as dopamine bind very tightly to the resting enzyme; phosphorylation of Ser40 decreases the affinity for catecholamines by three orders of magnitude. The effects of dopamine binding and phosphorylation of Ser40 on the kinetics of deuterium incorporation into peptide bonds were examined by mass spectrometry. When dopamine is bound, three peptic peptides show significantly slower deuterium incorporation, 35-41 and 42-71 in the regulatory domain and 295-299 in the catalytic domain. In the phosphorylated enzyme, peptide 295-299 shows more rapid incorporation of deuterium, while 35-41 and 42-71 can not be detected. These results are consistent with tyrosine hydroxylase existing in two different conformations. In the closed conformation, the regulatory domain lies across the active site loop containing residues 295-298; this is stabilized when dopamine is bound in the active site. In the open conformation, the regulatory domain has moved out of the active site, allowing substrates access; this conformation is favored by phosphorylation of Ser40. PMID:19371093

  20. Protein-Tyrosine Kinase Signaling in the Biological Functions Associated with Sperm

    Directory of Open Access Journals (Sweden)

    Takashi W. Ijiri

    2012-01-01

    Full Text Available In sexual reproduction, two gamete cells (i.e., egg and sperm fuse (fertilization to create a newborn with a genetic identity distinct from those of the parents. In the course of these developmental processes, a variety of signal transduction events occur simultaneously in each of the two gametes, as well as in the fertilized egg/zygote/early embryo. In particular, a growing body of knowledge suggests that the tyrosine kinase Src and/or other protein-tyrosine kinases are important elements that facilitate successful implementation of the aforementioned processes in many animal species. In this paper, we summarize recent findings on the roles of protein-tyrosine phosphorylation in many sperm-related processes (from spermatogenesis to epididymal maturation, capacitation, acrosomal exocytosis, and fertilization.

  1. DMPD: Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15081522 Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signall...ruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? PubmedID 15081522 Title Bruton...'s tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? Authors

  2. Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways

    DEFF Research Database (Denmark)

    Pandey, A; Fernandez, M M; Steen, H

    2000-01-01

    to epidermal growth factor receptor-induced phosphorylation, this required the ITAM domain since mutants lacking this region did not undergo tyrosine phosphorylation. Finally, overexpression of wild type STAM2 led to an increase in IL-2-mediated induction of c-Myc promoter activation indicating...

  3. Protein-tyrosine Phosphatase SHP2 Contributes to GDNF Neurotrophic Activity through Direct Binding to Phospho-Tyr687 in the RET Receptor Tyrosine Kinase*

    Science.gov (United States)

    Perrinjaquet, Maurice; Vilar, Marçal; Ibáñez, Carlos F.

    2010-01-01

    The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr687 in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr687 and association with components of the Tyr1062 signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser696, a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr687 as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions. PMID:20682772

  4. Protein-tyrosine phosphatase SHP2 contributes to GDNF neurotrophic activity through direct binding to phospho-Tyr687 in the RET receptor tyrosine kinase.

    Science.gov (United States)

    Perrinjaquet, Maurice; Vilar, Marçal; Ibáñez, Carlos F

    2010-10-08

    The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr(687) in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr(687) and association with components of the Tyr(1062) signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser(696), a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr(687) as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.

  5. Lipedema – lack of evidence for the involvement of tyrosine kinases.

    Science.gov (United States)

    Schneble, N; Wetzker, R; Wollina, U

    2016-01-01

    Lipedema is a chronic disorder characterized by abnormal distribution of subcutaneous adipose tissue on the proximal extremities, pain and capillary fragility. Its etiology is unknown but in analogy to central obesity, chronic low-level inflammation in adipose tissue has been suggested. There seems to be an increased propagation of pre-adipocytes into mature adipocytes contributing to the massive enlargement of subcutaneous adipose tissue. We investigated whether tyrosine kinases might be involved. Proteins from adipose tissue harvested during microcannular tumescent liposuction in lipedema and in lipomas were subjected to 10% polyacrylamide-gel, transferred to a polyvinylidenfluorid membrane and immunoblotted with indicated P-Tyr-100 antibody followed by enhanced chemiluminescence reaction. A survey of all blots did not reveal tyrosine-phosphorylated proteins with a molecular weight >100 kD in lipedema tissue and controls. These investigations suggest absence of activated growth factor receptors. Some signals indicating unspecific tyrosine-phosphorylation of smaller proteins were detected in tissue of both lipedema patients and controls. The present data suggest that there is no enduring activation of tyrosine kinase pathways of adipogenesis in lipedema as in lipoma controls.

  6. Pro-Tumorigenic Phosphorylation of p120 Catenin in Renal and Breast Cancer.

    Directory of Open Access Journals (Sweden)

    Antonis Kourtidis

    Full Text Available Altered protein expression and phosphorylation are common events during malignant transformation. These perturbations have been widely explored in the context of E-cadherin cell-cell adhesion complexes, which are central in the maintenance of the normal epithelial phenotype. A major component of these complexes is p120 catenin (p120, which binds and stabilizes E-cadherin to promote its adhesive and tumor suppressing function. However, p120 is also an essential mediator of pro-tumorigenic signals driven by oncogenes, such as Src, and can be phosphorylated at multiple sites. Although alterations in p120 expression have been extensively studied by immunohistochemistry (IHC in the context of tumor progression, little is known about the status and role of p120 phosphorylation in cancer. Here we show that tyrosine and threonine phosphorylation of p120 in two sites, Y228 and T916, is elevated in renal and breast tumor tissue samples. We also show that tyrosine phosphorylation of p120 at its N-terminus, including at the Y228 site is required for its pro-tumorigenic potential. In contrast, phosphorylation of p120 at T916 does not affect this p120 function. However, phosphorylation of p120 at T916 interferes with epitope recognition of the most commonly used p120 antibody, namely pp120. As a result, this antibody selectively underrepresents p120 levels in tumor tissues, where p120 is phosphorylated. Overall, our data support a role of p120 phosphorylation as a marker and mediator of tumor transformation. Importantly, they also argue that the level and localization of p120 in human cancer tissues immunostained with pp120 needs to be re-evaluated.

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

    Energy Technology Data Exchange (ETDEWEB)

    Mai, Sanyue [Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850 (China); Qu, Xiuhua [General Navy Hospital of PLA, 6 Fucheng Rd, Haidian District, Beijing 100037 (China); Li, Ping; Ma, Qingjun [Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850 (China); Liu, Xuan, E-mail: liux931932@163.com [Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850 (China); Cao, Cheng, E-mail: cao_c@sohu.com [Beijing Institute of Biotechnology, 27 Taiping Rd, Haidian District, Beijing 100850 (China)

    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. - Highlights: • c-Abl interacts with RBM39. • RBM39 is phosphorylated by c-Abl. • c-Abl regulates transcriptional coactivation activity of RBM39 on the ERα and PRβ.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    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. - Highlights: • c-Abl interacts with RBM39. • RBM39 is phosphorylated by c-Abl. • c-Abl regulates transcriptional coactivation activity of RBM39 on the ERα and PRβ.

  9. Thermolysin activates equine lamellar hoof matrix metalloproteinases.

    Science.gov (United States)

    Mungall, B A; Pollitt, C C

    2002-01-01

    Cultured equine lamellar hoof explants secrete the pro-enzymes matrix metalloproteinase-2 (MMP-2, 72 kDa) and MMP-2 (92 kDa). Untreated explants remained intact when tested on a calibrated force transducer, but when treated with an MMP activator, developed "in-vitro laminitis", separating at the dermal-epidermal junction. Explants treated with the bacterial protease thermolysin separated dose-dependently; this was accompanied by activation of both MMP-2 and -9. Thermolysin-mediated MP activation did not occur in a cell-free system and was not inhibited by the addition of the MMP inhibitor and batimastat. These findings suggest that thermolysin-mediated gelatinase activation is not dependent on membrane-bound matrix metalloproteinase (MT-MMP) activation, providing further evidence that bacteria can produce potent MMP activators that probably facilitate host invasion.

  10. Multifaceted role of matrix metalloproteinases (MMPs)

    OpenAIRE

    Singh, Divya; Srivastava, Sanjeev K.; Chaudhuri, Tapas K.; Upadhyay, Ghanshyam

    2015-01-01

    Matrix metalloproteinases (MMPs), a large family of calcium-dependent zinc-containing endopeptidases, are involved in the tissue remodeling and degradation of the extracellular matrix. MMPs are widely distributed in the brain and regulate various processes including microglial activation, inflammation, dopaminergic apoptosis, blood-brain barrier disruption, and modulation of ?-synuclein pathology. High expression of MMPs is well documented in various neurological disorders including Parkinson...

  11. Detection of Matrix Metalloproteinases by Zymography.

    Science.gov (United States)

    Tajhya, Rajeev B; Patel, Rutvik S; Beeton, Christine

    2017-01-01

    Matrix metalloproteinases (MMPs) represent more than 20 zinc-containing endopeptidases that cleave internal peptide bonds, leading to protein degradation. They play a critical role in many physiological cell functions, including tissue remodeling, embryogenesis, and angiogenesis. They are also involved in the pathogenesis of a vast array of diseases, including but not limited to systemic inflammation, various cancers, and cardiovascular, neurological, and autoimmune diseases. Here, we describe gel zymography to detect MMPs in cell and tissue samples and in cell culture supernatants.

  12. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Adi, Y. A., E-mail: yudi.adi@math.uad.ac.id [Department of Mathematic Faculty of MIPA Universitas Ahmad Dahlan (Indonesia); Department of Mathematic Faculty of MIPA Universitas Gadjah Mada (Indonesia); Kusumo, F. A.; Aryati, L. [Department of Mathematic Faculty of MIPA Universitas Gadjah Mada (Indonesia); Hardianti, M. S. [Department of Internal Medicine, Faculty of Medicine, Universitas Gadjah Mada (Indonesia)

    2016-04-06

    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

  13. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

    Science.gov (United States)

    Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.

    2016-04-01

    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

  14. Matrix metalloproteinases in fish biology and matrix turnover.

    Science.gov (United States)

    Pedersen, Mona E; Vuong, Tram T; Rønning, Sissel B; Kolset, Svein O

    2015-01-01

    Matrix metalloproteinases have important functions for tissue turnover in fish, with relevance both for the fish industry and molecular and cellular research on embryology, inflammation and tissue repair. These metalloproteinases have been studied in different fish types, subjected to both aquaculture and experimental conditions. This review highlights studies on these metalloproteinases in relation to both fish quality and health and further, the future importance of fish for basic research studies. Copyright © 2015. Published by Elsevier B.V.

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

    DEFF Research Database (Denmark)

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

    1997-01-01

    Binding of platelet-derived growth factor (PDGF) to its receptors leads to the activation of members of the Src family of protein tyrosine kinases. We show here that Fyn, a member of the Src family, is phosphorylated on Tyr28 in the unique N-terminal part of the molecule after interaction...

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

    Science.gov (United States)

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

    2017-04-01

    Nitric oxide (NO) and related molecules (reactive nitrogen species) regulate diverse physiological processes mainly through posttranslational modifications such as protein tyrosine nitration (PTN). PTN is a covalent and specific modification of tyrosine (Tyr) residues resulting in altered protein structure and function. In the last decade, great efforts have been made to reveal candidate proteins, target Tyr residues and functional consequences of nitration in plants. This review intends to evaluate the accumulated knowledge about the biochemical mechanism, the structural and functional consequences and the selectivity of plants' protein nitration and also about the decomposition or conversion of nitrated proteins. At the same time, this review emphasizes yet unanswered or uncertain questions such as the reversibility/irreversibility of tyrosine nitration, the involvement of proteasomes in the removal of nitrated proteins or the effect of nitration on Tyr phosphorylation. The different NO producing systems of algae and higher plants raise the possibility of diversely regulated protein nitration. Therefore studying PTN from an evolutionary point of view would enrich our present understanding with novel aspects. Plant proteomic research can be promoted by the application of computational prediction tools such as GPS-YNO 2 and iNitro-Tyr software. Using the reference Arabidopsis proteome, Authors performed in silico analysis of tyrosine nitration in order to characterize plant tyrosine nitroproteome. Nevertheless, based on the common results of the present prediction and previous experiments the most likely nitrated proteins were selected thus recommending candidates for detailed future research. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  17. PTEN is a protein tyrosine phosphatase for IRS1.

    Science.gov (United States)

    Shi, Yuji; Wang, Junru; Chandarlapaty, Sarat; Cross, Justin; Thompson, Craig; Rosen, Neal; Jiang, Xuejun

    2014-06-01

    The biological function of the PTEN tumor suppressor is mainly attributed to its lipid phosphatase activity. This study demonstrates that mammalian PTEN is a protein tyrosine phosphatase that selectively dephosphorylates insulin receptor substrate-1 (IRS1), a mediator of insulin and IGF signals. IGF signaling was defective in cells lacking NEDD4, a PTEN ubiquitin ligase, whereas AKT activation triggered by EGF or serum was unimpaired. Defective IGF signaling caused by NEDD4 deletion, including phosphorylation of IRS1 and AKT, was rescued by PTEN ablation. We demonstrate the nature of PTEN as an IRS1 phosphatase by direct biochemical analysis and cellular reconstitution, showing that NEDD4 supports insulin-mediated glucose metabolism and is required for the proliferation of IGF1 receptor-dependent but not EGF receptor-dependent tumor cells. Thus, PTEN is a protein phosphatase for IRS1, and its antagonism by NEDD4 promotes signaling by IGF and insulin.

  18. Trihydrophobin 1 Phosphorylation by c-Src Regulates MAPK/ERK Signaling and Cell Migration

    Science.gov (United States)

    Wu, Weibin; Sun, Zhichao; Wu, Jingwen; Peng, Xiaomin; Gan, Huacheng; Zhang, Chunyi; Ji, Lingling; Xie, Jianhui; Zhu, Haiyan; Ren, Shifang

    2012-01-01

    c-Src activates Ras-MAPK/ERK signaling pathway and regulates cell migration, while trihydrophobin 1 (TH1) inhibits MAPK/ERK activation and cell migration through interaction with A-Raf and PAK1 and inhibiting their kinase activities. Here we show that c-Src interacts with TH1 by GST-pull down assay, coimmunoprecipitation and confocal microscopy assay. The interaction leads to phosphorylation of TH1 at Tyr-6 in vivo and in vitro. Phosphorylation of TH1 decreases its association with A-Raf and PAK1. Further study reveals that Tyr-6 phosphorylation of TH1 reduces its inhibition on MAPK/ERK signaling, enhances c-Src mediated cell migration. Moreover, induced tyrosine phosphorylation of TH1 has been found by EGF and estrogen treatments. Taken together, our findings demonstrate a novel mechanism for the comprehensive regulation of Ras/Raf/MEK/ERK signaling and cell migration involving tyrosine phosphorylation of TH1 by c-Src. PMID:22238675

  19. In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase.

    Science.gov (United States)

    Cable, Jennifer; Prutzman, Kirk; Gunawardena, Harsha P; Schaller, Michael D; Chen, Xian; Campbell, Sharon L

    2012-03-20

    Focal adhesion kinase (FAK), a key regulator of cell adhesion and migration, is overexpressed in many types of cancer. The C-terminal focal adhesion targeting (FAT) domain of FAK is necessary for proper localization of FAK to focal adhesions and subsequent activation. Phosphorylation of Y926 in the FAT domain by the tyrosine kinase Src has been shown to promote metastasis and invasion in vivo by linking the FAT domain to the MAPK pathway via its interaction with growth factor receptor-bound protein 2. Several groups have reported that inherent conformational dynamics in the FAT domain likely regulate phosphorylation of Y926; however, what regulates these dynamics is unknown. In this paper, we demonstrate that there are two sites of in vitro Src-mediated phosphorylation in the FAT domain: Y926, which has been shown to affect FAK function in vivo, and Y1008, which has no known biological role. The phosphorylation of these two tyrosine residues is pH-dependent, but this does not reflect the pH dependence of Src kinase activity. Circular dichroism and nuclear magnetic resonance data indicate that the stability and conformational dynamics of the FAT domain are sensitive to changes in pH over a physiological pH range. In particular, regions of the FAT domain previously shown to regulate phosphorylation of Y926 as well as regions near Y1008 show pH-dependent dynamics on the microsecond to millisecond time scale.

  20. Saitohin, which is nested within the tau gene, interacts with tau and Abl and its human-specific allele influences Abl phosphorylation

    Science.gov (United States)

    Wang, Yan; Gao, Lei; Conrad, Christopher G.; Andreadis, Athena

    2011-01-01

    Saitohin (STH) is a gene unique to humans and their closest relatives whose function is not yet known. STH contains a single polymorphism (Q7R); the Q allele is human-specific and confers susceptibility to several neurodegenerative diseases. In previous work, we discovered that STH interacts with Peroxiredoxin 6 (Prdx6), a unique member of that family which is bifunctional and whose levels increase in Pick’s disease. In this study, we report that STH also interacts with tau and the non-receptor tyrosine kinase c-Abl (Abl). Furthermore, Abl phosphorylates STH on its single tyrosine residue and STH increases tyrosine phosphorylation by Abl. The effect of Saitohin on Abl-mediated phosphorylation appears to be allele-specific, providing evidence for a new cellular function for STH. PMID:21769920

  1. Cdk5 targets active Src for ubiquitin-dependent degradation by phosphorylating Src(S75)

    OpenAIRE

    Pan, Q.; Qiao, F.; Gao, C.; Norman, B.; Optican, L.; Zelenka, Peggy S.

    2011-01-01

    The non-receptor tyrosine kinase Src is a critical regulator of cytoskeletal contraction, cell adhesion, and migration. In normal cells, Src activity is stringently controlled by Csk-dependent phosphorylation of Src(Y530), and by Cullin-5-dependent ubiquitinylation, which affects active Src(pY419) exclusively, leading to its degradation by the proteosome. Previous work has shown that Src activity is also limited by Cdk5, a proline-directed kinase, which has been shown to phosphorylate Src(S75...

  2. Quantitative maps of protein phosphorylation sites across 14 different rat organs and tissues

    DEFF Research Database (Denmark)

    Lundby, Alicia; Secher, Anna; Lage, Kasper

    2012-01-01

    across 14 rat organs and tissues. We provide the data set as an easily accessible resource via a web-based database, the CPR PTM Resource. A major fraction of the presented phosphorylation sites are tissue-specific and modulate protein interaction networks that are essential for the function...... of individual organs. For skeletal muscle, we find that phosphotyrosines are over-represented, which is mainly due to proteins involved in glycogenolysis and muscle contraction, a finding we validate in human skeletal muscle biopsies. Tyrosine phosphorylation is involved in both skeletal and cardiac muscle...

  3. Phospho-Tyrosine(s) vs. Phosphatidylinositol Binding in Shc Mediated Integrin Signaling.

    Science.gov (United States)

    Lin, Xiaochen; Vinogradova, Olga

    2015-04-01

    The Shc adaptor protein, particularly its p52 isoform, has been identified as a primary signaling partner for the tyrosine(s)-phosphorylated cytoplasmic tails of activated β 3 integrins. Inspired by our recent structure of the Shc PTB domain in complex with a bi-phosphorylated peptide derived from β 3 cytoplasmic tail, we have initiated the investigation of Shc interaction with phospholipids of the membrane. We are particularly focused on PtdIns and their effects on Shc mediated integrin signaling in vitro . Here we present thermodynamic profiles and molecular details of the interactions between Shc, integrin, and PtdIns, all of which have been studied by ITC and solution NMR methods. A model of p52 Shc interaction with phosphorylated β 3 integrin cytoplasmic tail at the cytosolic face of the plasma membrane is proposed based on these data.

  4. Chemically modified tetracyclines stimulate matrix metalloproteinase-s production by periodontal ligament cells

    NARCIS (Netherlands)

    Bildt, M.M.; Snoek-van Beurden, A.M.P.; Groot, J. de; El, B. van; Kuijpers-Jagtman, A.M.; Hoff, J.W. van den

    2006-01-01

    Background and Objective: The purpose of this study was to investigate the effects of chemically modified tetracyclines (CMTs) on the production of gelatinases [matrix metalloproteinase (MMP)-2 and -9] by human periodontal ligament (PDL) cells, and on the activity of recombinant gelatinases.

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

    International Nuclear Information System (INIS)

    Hussain, Alamdar; Faryal, Rani; Nore, Beston F.; Mohamed, Abdalla J.; Smith, C.I. Edvard

    2009-01-01

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

  6. Src homology 2 domain-based high throughput assays for profiling downstream molecules in receptor tyrosine kinase pathways.

    Science.gov (United States)

    Yaoi, Takuro; Chamnongpol, Sangpen; Jiang, Xin; Li, Xianqiang

    2006-05-01

    Src homology 2 (SH2) domains are evolutionary conserved small protein modules that bind specifically to tyrosine-phosphorylated peptides. More than 100 SH2 domains have been identified in proteins encoded by the human genome. The binding specificity of these domains plays a critical role in signaling within the cell, mediating the relocalization and interaction of proteins in response to changes in tyrosine phosphorylation states. Here we developed an SH2 domain profiling method based on a multiplexed fluorescent microsphere assay in which various SH2 domains are used to probe the global state of tyrosine phosphorylation within a cell and to screen synthetic peptides that specifically bind to each SH2 domain. The multiplexed, fluorescent microsphere-based assay is a recently developed technology that can potentially detect a wide variety of interactions between biological molecules. We constructed 25-plex SH2 domain-GST fusion protein-conjugated fluorescent microsphere sets to investigate phosphorylation-mediated cell signaling through the specific binding of SH2 domains to activated target proteins. The response of HeLa, COS-1, A431, and 293 cells and four breast cancer cell lines to epidermal growth factor and insulin were quantitatively profiled using this novel microsphere-based, multiplexed, high throughput assay system.

  7. Src tyrosine kinase inhibition prevents pulmonary ischemia-reperfusion-induced acute lung injury.

    Science.gov (United States)

    Oyaizu, Takeshi; Fung, Shan-Yu; Shiozaki, Atsushi; Guan, Zehong; Zhang, Qiao; dos Santos, Claudia C; Han, Bing; Mura, Marco; Keshavjee, Shaf; Liu, Mingyao

    2012-05-01

    Pulmonary ischemia-reperfusion is a pathological process seen in several clinical conditions, including lung transplantation, cardiopulmonary bypass, resuscitation for circulatory arrest, atherosclerosis, and pulmonary embolism. A better understanding of its molecular mechanisms is very important. Rat left lung underwent in situ ischemia for 60 min, followed by 2 h of reperfusion. The gene expression profiles and Src protein tyrosine kinase (PTK) phosphorylation were studied over time, and PP2, an Src PTK inhibitor, was intravenously administered 10 min before lung ischemia to determine the role of Src PTK in lung injury. Reperfusion following ischemia significantly changed the expression of 169 genes, with Mmp8, Mmp9, S100a9, and S100a8 being the most upregulated genes. Ischemia alone only affected expression of 9 genes in the lung. However, Src PTK phosphorylation (activation) was increased in the ischemic lung, mainly on the alveolar wall. Src PTK inhibitor pretreatment decreased phosphorylation of Src PTKs, total protein tyrosine phosphorylation, and STAT3 phosphorylation. It increased phosphorylation of the p85α subunit of PI3 kinase, a signal pathway that can inhibit coagulation and inflammation. PP2 reduced leukocyte infiltration in the lung, apoptotic cell death, fibrin deposition, and severity of acute lung injury after reperfusion. Src inhibition also significantly reduced CXCL1 (GRO/KI) and CCL2 (MCP-1) chemokine levels in the serum. During pulmonary ischemia, Src PTK activation, rather than alteration in gene expression, may play a critical role in reperfusion-induced lung injury. Src PTK inhibition presents a new prophylactic treatment for pulmonary ischemia-reperfusion-induced acute lung injury.

  8. Recruitment of SHP-1 protein tyrosine phosphatase and signalling by a chimeric T-cell receptor-killer inhibitory receptor

    DEFF Research Database (Denmark)

    Christensen, M D; Geisler, C

    2000-01-01

    recognize MHC class I molecules. Following coligation of KIR with an activating receptor, the tyrosine in the ITIM is phosphorylated and the cytoplasmic protein tyrosine phosphatase SHP-1 is recruited to the ITIM via its SH2 domains. It is still not clear how SHP-1 affects T-cell receptor (TCR) signalling....... In this study, we constructed a chimeric TCR-KIR receptor. We demonstrated that SHP-1 is recruited to the chimeric TCR-KIR receptor following T-cell stimulation with either anti-TCR monoclonal antibody (MoAb) or superantigen. However, in spite of this we could not detect any effect of SHP-1 on TCR signalling...

  9. Nonreceptor Tyrosine Kinases in Prostate

    Directory of Open Access Journals (Sweden)

    Cancer Yu-Ming Chang

    2007-02-01

    Full Text Available BACKGROUND: Carcinoma of the prostate (CaP is the most commonly diagnosed cancer in men in the United States. Signal transduction molecules such as tyrosine kinases play important roles in CaP. Src, a nonreceptor tyrosine kinase (NRTK and the first proto-oncogene discovered is shown to participate in processes such as cell proliferation and migration in CaP. Underscoring NRTK's and, specifically, Src's importance in cancer is the recent approval by the US Food and Drug Administration of dasatinib, the first commercial Src inhibitor for clinical use in chronic myelogenous leukemia (CML. In this review we will focus on NRTKs and their roles in the biology of CaP. MATERIALS AND METHODS: Publicly available literature from PubMed regarding the topic of members of NRTKs in CaP was searched and reviewed. RESULTS: Src, FAK, JaK1/2, and ETK are involved in processes indispensable to the biology of CaP: cell growth, migration, invasion, angiogenesis, and apoptosis. CONCLUSIONS: Src emerges as a common signaling and regulatory molecule in multiple biological processes in CaP. Src's relative importance in particular stages of CaP, however, required further definition. Continued investigation of NRTKs will increase our understanding of their biological function and potential role as new therapeutic targets.

  10. Assessment of Matrix Metalloproteinases by Gelatin Zymography.

    Science.gov (United States)

    Cathcart, Jillian

    2016-01-01

    Matrix metalloproteinases are endopeptidases responsible for remodeling of the extracellular matrix and have been identified as critical contributors to breast cancer progression. Gelatin zymography is a valuable tool which allows the analysis of MMP expression. In this approach, enzymes are resolved electrophoretically on a sodium dodecyl sulfate-polyacrylamide gel copolymerized with the substrate for the MMP of interest. Post electrophoresis, the enzymes are refolded in order for proteolysis of the incorporated substrate to occur. This assay yields valuable information about MMP isoforms or changes in activation and can be used to analyze the role of MMPs in normal versus pathological conditions.

  11. A study on the expression levels of matrix metalloproteinases and ...

    African Journals Online (AJOL)

    Conclusion: MMP-2, MMP-7, and MMP-9 are potential targets for therapeutic control of UC. Keywords: Glandular epithelium, Inflammatory cells, Inhibitors, Matrix metalloproteinases (MMPs),. Tissue inhibitors of metalloproteinases, Ulcerative colitis. Tropical Journal of Pharmaceutical Research is indexed by Science ...

  12. A study on the expression levels of matrix metalloproteinases and ...

    African Journals Online (AJOL)

    Purpose: To investigate the expression matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in ulcerative colitis (UC) patients admitted to The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China. Methods: The expression of MMPs and TIMPs) was evaluated in 50 ...

  13. Cdk5 targets active Src for ubiquitin-dependent degradation by phosphorylating Src(S75)

    Science.gov (United States)

    Pan, Q.; Qiao, F.; Gao, C.; Norman, B.; Optican, L.

    2011-01-01

    The non-receptor tyrosine kinase Src is a critical regulator of cytoskeletal contraction, cell adhesion, and migration. In normal cells, Src activity is stringently controlled by Csk-dependent phosphorylation of Src(Y530), and by Cullin-5-dependent ubiquitinylation, which affects active Src(pY419) exclusively, leading to its degradation by the proteosome. Previous work has shown that Src activity is also limited by Cdk5, a proline-directed kinase, which has been shown to phosphorylate Src(S75). Here we show that this phosphorylation promotes the ubiquitin-dependent degradation of Src, thus restricting the availability of active Src. We demonstrate that Src(S75) phosphorylation occurs in vivo in epithelial cells, and like ubiquitinylation, is associated only with active Src. Preventing Cdk5-dependent phosphorylation of Src(S75), by site-specific mutation of S75 or by Cdk5 inhibition or suppression, increases Src(Y419) phosphorylation and kinase activity, resulting in Src-dependent cytoskeletal changes. In transfected cells, ubiquitinylation of Src(S75A) is about 35% that of wild-type Src-V5, and its half-life is approximately 2.5-fold greater. Cdk5 suppression leads to a comparable decrease in the ubiquitinylation of endogenous Src and a similar increase in Src stability. Together, these findings demonstrate that Cdk5-dependent phosphorylation of Src(S75) is a physiologically significant mechanism of regulating intracellular Src activity. PMID:21442427

  14. Plk1 phosphorylation of IRS2 prevents premature mitotic exit via AKT inactivation

    Science.gov (United States)

    Chen, Long; Li, Zhiguo; Ahmad, Nihal; Liu, Xiaoqi

    2016-01-01

    Insulin receptor substrate (IRS) proteins play important roles by acting as a platform in transducing signals from transmembrane receptors upon growth factor stimulation. Although tyrosine phosphorylation on IRS proteins plays critical roles in signal transduction, phosphorylation of IRS proteins on serine/threonine residues are believed to play various regulatory roles on IRS protein function. However, studies on serine/threonine phosphorylation of IRS proteins are very limited, especially for insulin receptor substrate 2 (IRS2), one member of the IRS protein family. In this study, we identify Polo-like kinase 1 (Plk1) as the responsible kinase for phosphorylation of IRS2 on two serine residues, Ser 556 and Ser 1098. Phosphorylation of IRS2 on these two serine residues by Plk1 prevents the activation of the PI3K pathway upon growth factor stimulation by inhibiting the binding between IRS2 and the PI3K pathway components and increasing IRS2 protein degradation. Of significance, we show that IRS2 phosphorylation is cell cycle regulated and that Plk1 phosphorylation of IRS2 prevents premature mitotic exit via AKT inactivation. PMID:25830382

  15. Identification and functional analysis of novel phosphorylation sites in the RNA surveillance protein Upf1.

    Science.gov (United States)

    Lasalde, Clarivel; Rivera, Andrea V; León, Alfredo J; González-Feliciano, José A; Estrella, Luis A; Rodríguez-Cruz, Eva N; Correa, María E; Cajigas, Iván J; Bracho, Dina P; Vega, Irving E; Wilkinson, Miles F; González, Carlos I

    2014-02-01

    One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity.

  16. MHC class I ligation of human T cells activates the ZAP70 and p56lck tyrosine kinases, leads to an alternative phenotype of the TCR/CD3 zeta-chain, and induces apoptosis

    DEFF Research Database (Denmark)

    Skov, S; Bregenholt, S; Claesson, Mogens Helweg

    1997-01-01

    Cross-linking of MHC class I (MHC-I) molecules on human T cells induces signal-transduction events, including activation of tyrosine kinases, tyrosine phosphorylation of phospholipase C-gamma 1, and elevation of the intracellular free calcium concentration. In this study, we demonstrate...... that the ZAP70 tyrosine kinase is tyrosine phosphorylated in Jurkat T cells and in purified peripheral T cells after MHC-I ligation. The tyrosine-phosphorylated ZAP70 kinase exhibits a particular phenotype with low affinities for proteins at 21, 40, 60, and 120 kDa, proteins normally co-precipitated with ZAP70...... after TCR/CD3 stimulation. The phosphorylation of ZAP70 after MHC-I ligation was dependent on TCR/CD3 surface expression. One of the natural substrates for ZAP70 is the zeta-chain dimer of the TCR/CD3 complex. MHC-I cross-linking induces a phosphorylated zeta-protein that migrates as a dimer at 42 k...

  17. Serum matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 levels in patients with tick-borne encephalitis

    Czech Academy of Sciences Publication Activity Database

    Palus, Martin; Žampachová, E.; Elsterová, Jana; Růžek, Daniel

    2014-01-01

    Roč. 68, č. 2 (2014), s. 165-169 ISSN 0163-4453 R&D Projects: GA ČR GAP502/11/2116 Institutional support: RVO:60077344 Keywords : tick-borne encephalitis * matrix metalloproteinase-9 * tissue inhibitor of metalloproteinase-1 * bloodebrain barrier Subject RIV: EC - Immunology Impact factor: 4.441, year: 2014

  18. Tumorigenic Potential of Extracellular Matrix Metalloproteinase Inducer

    Science.gov (United States)

    Zucker, Stanley; Hymowitz, Michelle; Rollo, Ellen E.; Mann, Richard; Conner, Cathleen E.; Cao, Jian; Foda, Hussein D.; Tompkins, David C.; Toole, Bryan P.

    2001-01-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein present on the cancer cell plasma membrane, enhances fibroblast synthesis of matrix metalloproteinases (MMPs). The demonstration that peritumoral fibroblasts synthesize most of the MMPs in human tumors rather than the cancer cells themselves has ignited interest in the role of EMMPRIN in tumor dissemination. In this report we have demonstrated a role for EMMPRIN in cancer progression. Human MDA-MB-436 breast cancer cells, which are tumorigenic but slow growing in vivo, were transfected with EMMPRIN cDNA and injected orthotopically into mammary tissue of female NCr nu/nu mice. Green fluorescent protein was used to visualize metastases. In three experiments, breast cancer cell clones transfected with EMMPRIN cDNA were considerably more tumorigenic and invasive than plasmid-transfected cancer cells. Increased gelatinase A and gelatinase B expression (demonstrated by in situ hybridization and gelatin substrate zymography) was demonstrated in EMMPRIN-enhanced tumors. In contrast to de novo breast cancers in humans, human tumors transplanted into mice elicited minimal stromal or inflammatory cell reactions. Based on these experimental studies and our previous demonstration that EMMPRIN is prominently displayed in human cancer tissue, we propose that EMMPRIN plays an important role in cancer progression by increasing synthesis of MMPs. PMID:11395366

  19. Metazoan-like signaling in a unicellular receptor tyrosine kinase

    Directory of Open Access Journals (Sweden)

    Schultheiss Kira P

    2013-02-01

    Full Text Available Abstract Background Receptor tyrosine kinases (RTKs are crucial components of signal transduction systems in multicellular animals. Surprisingly, numerous RTKs have been identified in the genomes of unicellular choanoflagellates and other protists. Here, we report the first biochemical study of a unicellular RTK, namely RTKB2 from Monosiga brevicollis. Results We cloned, expressed, and purified the RTKB2 kinase, and showed that it is enzymatically active. The activity of RTKB2 is controlled by autophosphorylation, as in metazoan RTKs. RTKB2 possesses six copies of a unique domain (designated RM2 in its C-terminal tail. An isolated RM2 domain (or a synthetic peptide derived from the RM2 sequence served as a substrate for RTKB2 kinase. When phosphorylated, the RM2 domain bound to the Src homology 2 domain of MbSrc1 from M. brevicollis. NMR structural studies of the RM2 domain indicated that it is disordered in solution. Conclusions Our results are consistent with a model in which RTKB2 activation stimulates receptor autophosphorylation within the RM2 domains. This leads to recruitment of Src-like kinases (and potentially other M. brevicollis proteins and further phosphorylation, which may serve to increase or dampen downstream signals. Thus, crucial features of signal transduction circuitry were established prior to the evolution of metazoans from their unicellular ancestors.

  20. Dietary Tyrosine Benefits Cognitive and Psychomotor Performance During Body Cooling

    National Research Council Canada - National Science Library

    O'Brien, Catherine; Mahoney, Caroline; Tharion, William J; Sils, Ingrid V; Castellani, John W

    2007-01-01

    Supplemental tyrosine is effective at limiting cold-induced decreases in working memory, presumably by augmenting brain catecholamine levels, since tyrosine is a precursor for catecholamine synthesis...

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

    Science.gov (United States)

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

    1996-07-19

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

  2. Multiple tyrosine residues in the intracellular domain of the common ß subunit of the interleukin 5 receptor are involved in activation of STAT5

    NARCIS (Netherlands)

    Dijk, Thamar B. van; Caldenhoven, Eric; Raaijmakers, J.A.M.; Lammers, J.W.J.; Koenderman, L.; Groot, Rolf P. de

    1997-01-01

    Abstract In contrast to the general model of cytokine-induced JAK/STAT signaling, tyrosine phosphorylation of the IL-5R ß chain seems to be dispensable for STAT activation in cells overexpressing exogenous STAT proteins. In this study we expressed IL-5 receptor mutants in 293 cells and studied

  3. Eps15R is a tyrosine kinase substrate with characteristics of a docking protein possibly involved in coated pits-mediated internalization

    DEFF Research Database (Denmark)

    Coda, L; Salcini, A E; Confalonieri, S

    1998-01-01

    , and 76 kDa were specifically recognized by anti-eps15R sera. The 125-kDa species is a bona fide product of the eps15R gene, whereas p108 and p76 are most likely products of alternative splicing events. Eps15R protein(s) are tyrosine-phosphorylated following epidermal growth factor receptor activation...

  4. Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung

    Directory of Open Access Journals (Sweden)

    Sabrina V. Martini

    2016-07-01

    Full Text Available Background/Aims: Adeno-associated virus (AAV vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. Methods: Eighteen C57BL/6 mice were randomly assigned into three groups: (1 a control group (CTRL animals underwent intratracheal (i.t. instillation of saline, (2 the wild-type AAV9 group (WT-AAV9, 1010 vg, and (3 the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg, which received (i.t. self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP. Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. Results: No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30% compared with their wild-type counterparts, without eliciting an inflammatory response. Conclusion: Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy.

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

    Science.gov (United States)

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

    1996-09-01

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

  6. Levels of matrix metalloproteinase-1 and tissue inhibitors of metalloproteinase-1 in gastric cancer

    Science.gov (United States)

    Kemik, Ozgur; Kemik, Ahu Sarbay; Sümer, Aziz; Dulger, Ahmet Cumhur; Adas, Mine; Begenik, Huseyin; Hasirci, Ismail; Yilmaz, Ozkan; Purisa, Sevim; Kisli, Erol; Tuzun, Sefa; Kotan, Cetin

    2011-01-01

    AIM: To evaluate the levels of preoperative serum matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in gastric cancer. METHODS: One hundred gastric cancer patients who underwent gastrectomy were enrolled in this study. The serum concentrations of MMP-1 and TIMP-1 in these patients and in fifty healthy controls were determined using an enzyme-linked immunosorbent assay. RESULTS: Higher serum MMP-1 and TIMP-1 levels were observed in patients than in controls (P < 0.001). Serum MMP-1 and TIMP-1 levels were positively associated with morphological appearance, tumor size, depth of wall invasion, lymph node metastasis, liver metastasis, perineural invasion, and pathological stage. They were not significantly associated with age, gender, tumor location, or histological type. CONCLUSION: Increased MMP-1 and TIMP-1 were associated with gastric cancer. Although these markers are not good markers for diagnosis, these markers show in advanced gastric cancer. PMID:21547130

  7. Circulating levels of matrix metalloproteinases and tissue inhibitors of metalloproteinases in patients with incisional hernia

    DEFF Research Database (Denmark)

    Henriksen, Nadia A; Sørensen, Lars T; Jorgensen, Lars N

    2013-01-01

    for MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, MMP-13, TIMP-1, TIMP-2, and TIMP-4 using a multiplex sandwich fluorescent immunoassay supplemented with gelatin zymography. The screening indicated differences in serum MMP-9 and TIMP-1 levels. Consequently, MMP-9 and TIMP-1 levels were......Incisional hernia formation is a common complication to laparotomy and possibly associated with alterations in connective tissue metabolism. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are closely involved in the metabolism of the extracellular matrix. Our...... aim was to study serum levels of multiple MMPs and TIMPs in patients with and without incisional hernia. Out of 305 patients who underwent laparotomy, 79 (25.9%) developed incisional hernia over a median follow-up period of 3.7 years. Pooled sera from a subset (n = 72) of these patients were screened...

  8. The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration

    DEFF Research Database (Denmark)

    Voena, Claudia; Conte, Chiara; Ambrogio, Chiara

    2007-01-01

    , leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine......), formed a complex with Shp2, Gab2, and growth factor receptor binding protein 2 (Grb2), where Grb2 bound to the phosphorylated Shp2 through its SH2 domain. Shp2 knock down by specific shRNA decreased the phosphorylation of extracellular signal-regulated kinase 1/2 and of the tyrosine residue Y416...... in the activation loop of Src, resulting in impaired ALCL cell proliferation and growth disadvantage. Finally, migration of ALCL cells was reduced by Shp2 shRNA. These findings show a direct involvement of Shp2 in NPM-ALK lymphomagenesis, highlighting its critical role in lymphoma cell proliferation and migration....

  9. Identification of Tyrosine Kinase Src Responsible for Antimicrobial Peptides Production in Bombyx mori.

    Science.gov (United States)

    Li, Xianyang; Hua, Xiaoting; Song, Liang; Xia, Qingyou; Wang, Fei

    2017-01-01

    Src is a non-receptor protein tyrosine kinase ubiquitously expressed in animals. It is involved in various cellular processes, including the innate immune response in mammals. However, less is known about the function of insect Src. Here we presented a homologue of Src in silkworm (Bombyx mori), named as BmSrc by phylogenetic analysis, homologous comparison and domain prediction. BmSrc contains the conserved phosphorylation residues and possesses tyrosine kinase activity. The expression pattern of BmSrc mRNA was specific in developmental stages and tissues. The highest expression of BmSrc was detected in moth stage, and the gonads showed the highest expression during larval stage. We then found over-expression of BmSrc in BmE cell resulted in an increase of p38 mitogen-activated protein kinase (p38 MAPK) and Akt phosphorylation but a decrease in extracellular signal-regulated kinase (ERK) phosphorylation. Finally, we demonstrated that BmSrc promoted the production of antimicrobial peptides (AMPs). These results implied that BmSrc is involved in immune response of silkworm possibly through activating p38 MAPK and Akt signaling pathway. Our study may provide reference for further investigation of the biological function of BmSrc in Bombyx mori. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

    Science.gov (United States)

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

    2011-01-07

    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. 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(KM)) were used to examine the possible signaling pathways in which TEC is involved. The cell proliferation rate was evaluated by (3)H-TdR incorporation. 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. TEC promotes hepatocyte proliferation and regeneration and is involved in HGF-induced Erk signaling pathway. Copyright © 2010 Elsevier Inc. All rights reserved.

  11. Recruitment of SHP-1 protein tyrosine phosphatase and signalling by a chimeric T-cell receptor-killer inhibitory receptor

    DEFF Research Database (Denmark)

    Christensen, M D; Geisler, C

    2000-01-01

    Receptors expressing the immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic tail play an important role in the negative regulation of natural killer and B-cell activation. A subpopulation of T cells expresses the ITIM containing killer cell inhibitory receptor (KIR), which...... recognize MHC class I molecules. Following coligation of KIR with an activating receptor, the tyrosine in the ITIM is phosphorylated and the cytoplasmic protein tyrosine phosphatase SHP-1 is recruited to the ITIM via its SH2 domains. It is still not clear how SHP-1 affects T-cell receptor (TCR) signalling....... In this study, we constructed a chimeric TCR-KIR receptor. We demonstrated that SHP-1 is recruited to the chimeric TCR-KIR receptor following T-cell stimulation with either anti-TCR monoclonal antibody (MoAb) or superantigen. However, in spite of this we could not detect any effect of SHP-1 on TCR signalling...

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

    Science.gov (United States)

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

    2016-01-01

    Immunosuppression is a major complication of alcoholism that contributes to increased rates of opportunistic infections and sepsis in alcoholics. The NLRP3 inflammasome, a multi-protein intracellular pattern recognition receptor complex that facilitates the cleavage and secretion of the pro-inflammatory cytokines IL-1β and IL-18, can be inhibited by ethanol and we sought to better understand the mechanism through which this occurs and whether chemically similar molecules exert comparable effects. We show that ethanol can specifically inhibit activation of the NLRP3 inflammasome, resulting in attenuated IL-1β and caspase-1 cleavage and secretion, as well as diminished ASC speck formation, without affecting potassium efflux, in a mouse macrophage cell line (J774), mouse bone marrow derived dendritic cells, mouse neutrophils, and human PBMCs. The inhibitory effects on the Nlrp3 inflammasome were independent of GABAA receptor activation or NMDA receptor inhibition, but was associated with decreased oxidant production. Ethanol treatment markedly decreased cellular tyrosine phosphorylation, while administration of the tyrosine phosphatase inhibitor sodium orthovanadate prior to ethanol restored tyrosine phosphorylation and IL-1β secretion subsequent to ATP stimulation. Furthermore, sodium orthovanadate-induced phosphorylation of ASC Y144, necessary and sufficient for Nlrp3 inflammasome activation, and secretion of phosphorylated ASC, were inhibited by ethanol. Finally, multiple alcohol-containing organic compounds exerted inhibitory effects on the Nlrp3 inflammasome, whereas 2-methylbutane (isopentane), the analogous alkane of the potent inhibitor isoamyl alcohol (isopentanol), did not. Our results demonstrate that ethanol antagonizes the NLRP3 inflammasome at an apical event in its activation through the stimulation of protein tyrosine phosphatases, an effect shared by other short-chain alcohols. PMID:27421477

  13. I-mediated signaling events by Lyn kinase C-terminal tyrosine phosphorylation

    Czech Academy of Sciences Publication Activity Database

    Tolar, Pavel; Dráberová, Lubica; Tolarová, Helena; Dráber, Petr

    2004-01-01

    Roč. 34, č. 4 (2004), s. 1136-1145 ISSN 0014-2980 R&D Projects: GA MŠk LN00A026; GA ČR GA204/00/0204; GA ČR GA310/00/0205; GA ČR GA204/03/0594; GA ČR GA301/03/0596; GA AV ČR IAA5052005; GA AV ČR IAA7052006; GA AV ČR IAA5052310 Institutional research plan: CEZ:AV0Z5052915 Keywords : mast cell * Fc receptor * signal transduction Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.005, year: 2004

  14. A Study on Expression and Tyrosine 705 phosphorylation of STAT3 ...

    African Journals Online (AJOL)

    446. 22. Takagi Y, Harada J, Chiarugi A, Moskowitz MA. STAT1 is activated in neurons after ischemia and contributes to ischemic brain injury. J Cereb Blood Flow Metab 2002;. 22:1311-1318. 23. Stephanou A, Brar BK, Knight RA, Latchman ...

  15. PTP-S2, a nuclear tyrosine phosphatase, is phosphorylated and ...

    Indian Academy of Sciences (India)

    2.1 Cell lines. Rat fibroblast cell line F-111 and D3, a stable clone of HeLa cells expressing rat PTP-S2, were maintained in DMEM containing 10% fetal bovine serum. D3 cell line has been described previously (Radha et al 1997). Cells in exponential cultures were used as interphase cells. Metaphase-arrested cells were ...

  16. Oviduct Binding and Elevated Environmental pH Induce Protein Tyrosine Phosphorylation in Stallion Spermatozoa

    NARCIS (Netherlands)

    Leemans, B.; Gadella, B.M.; Sostaric, E.; Nelis, H.; Stout, T.A.E.; Hoogewijs, M.; van Soom, A.

    2014-01-01

    Sperm-oviduct binding is an essential step in the capacitation process preparing the sperm for fertilization in several mammalian species. In many species, capacitation can be induced in vitro by exposing spermatozoa to bicarbonate, Ca2+, and albumin; however, these conditions are insufficient in

  17. Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation

    DEFF Research Database (Denmark)

    Satpathy, Shankha; Wagner, Sebastian A; Beli, Petra

    2015-01-01

    B-cell receptor (BCR) signaling is essential for the development and function of B cells; however, the spectrum of proteins involved in BCR signaling is not fully known. Here we used quantitative mass spectrometry-based proteomics to monitor the dynamics of BCR signaling complexes (signalosomes......) and to investigate the dynamics of downstream phosphorylation and ubiquitylation signaling. We identify most of the previously known components of BCR signaling, as well as many proteins that have not yet been implicated in this system. BCR activation leads to rapid tyrosine phosphorylation and ubiquitylation...... of the receptor-proximal signaling components, many of which are co-regulated by both the modifications. We illustrate the power of multilayered proteomic analyses for discovering novel BCR signaling components by demonstrating that BCR-induced phosphorylation of RAB7A at S72 prevents its association...

  18. Glycogen phosphorylation and Lafora disease.

    Science.gov (United States)

    Roach, Peter J

    2015-12-01

    Covalent phosphorylation of glycogen, first described 35 years ago, was put on firm ground through the work of the Whelan laboratory in the 1990s. But glycogen phosphorylation lay fallow until interest was rekindled in the mid 2000s by the finding that it could be removed by a glycogen-binding phosphatase, laforin, and that mutations in laforin cause a fatal teenage-onset epilepsy, called Lafora disease. Glycogen phosphorylation is due to phosphomonoesters at C2, C3 and C6 of glucose residues. Phosphate is rare, ranging from 1:500 to 1:5000 phosphates/glucose depending on the glycogen source. The mechanisms of glycogen phosphorylation remain under investigation but one hypothesis to explain C2 and perhaps C3 phosphate is that it results from a rare side reaction of the normal synthetic enzyme glycogen synthase. Lafora disease is likely caused by over-accumulation of abnormal glycogen in insoluble deposits termed Lafora bodies in neurons. The abnormality in the glycogen correlates with elevated phosphorylation (at C2, C3 and C6), reduced branching, insolubility and an enhanced tendency to aggregate and become insoluble. Hyperphosphorylation of glycogen is emerging as an important feature of this deadly childhood disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Kinetic analysis of the inhibition of matrix metalloproteinases: lessons from the study of tissue inhibitors of metalloproteinases.

    Science.gov (United States)

    Willenbrock, Frances; Thomas, Daniel A; Amour, Augustin

    2010-01-01

    Tissue inhibitors of metalloproteinases (TIMPs) are a group of highly potent inhibitors of matrix metalloproteinases (MMPs) and disintegrin metalloproteinases (ADAMs). The high affinity and "tight-binding" nature of the inhibition of MMPs or ADAMs by TIMPs presents challenges for the determination of both equilibrium and dissociation rate constants of these inhibitory events. Methodologies that enable some of these challenges to be overcome are described in this chapter and represent valuable lessons for the in vitro assessment of MMP or ADAM inhibitors within a drug discovery context.

  20. LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation.

    Science.gov (United States)

    Belkina, Natalya V; Liu, Yin; Hao, Jian-Jiang; Karasuyama, Hajime; Shaw, Stephen

    2009-03-24

    ERM (ezrin-radixin-moesin) proteins mediate linkage of actin cytoskeleton to plasma membrane in many cells. ERM activity is regulated in part by phosphorylation at a C-terminal threonine, but the identity of ERM kinases is unknown in lymphocytes and incompletely defined in other mammalian cells. Our studies show that lymphocyte-oriented kinase (LOK) is an ERM kinase in vitro and in vivo. Mass spectrometric analysis indicates LOK is abundant at the lymphocyte plasma membrane and immunofluorescence studies show LOK enrichment at the plasma membrane near ERM. In vitro peptide specificity analyses characterize LOK as a basophilic kinase whose optimal substrate sequence resembles the ERM site, including unusual preference for tyrosine at P-2. LOK's activity on moesin peptide and protein was comparable to reported ERM kinases ROCK and PKC but unlike them LOK displayed preferential specificity for moesin compared to traditional basophilic kinase substrates. Two genetic approaches demonstrate a role for LOK in ERM phosphorylation: cell transfection with LOK kinase domain augments ERM phosphorylation and lymphocytes from LOK knockout mice have >50% reduction in ERM phosphorylation. The findings on localization and specificity argue that LOK is a direct ERM kinase. The knockout mice have normal hematopoietic cell development but notably lymphocyte migration and polarization in response to chemokine are enhanced. These functional alterations fit the current understanding of the role of ERM phosphorylation in regulating cortical reorganization. Thus, these studies identify a new ERM kinase of importance in lymphocytes and confirm the role of ERM phosphorylation in regulating cell shape and motility.

  1. Insulin increase in MAP kinase phosphorylation is shifted to early time-points by overexpressing APS, while Akt phosphorylation is not influenced.

    Science.gov (United States)

    Onnockx, Sheela; Xie, Jingwei; Degraef, Chantal; Erneux, Christophe; Pirson, Isabelle

    2009-09-10

    Upon insulin stimulation, the adaptor protein APS is recruited to the insulin receptor and tyrosine phosphorylated. APS initiates the insulin-induced TC10 cascade which participates to GLUT4 translocation to the plasma membrane. Nevertheless, the molecular mechanism that governs APS and its SH2 and PH domains action on the insulin transduction cascade is not yet fully understood. Here, we show that APS co-immunoprecipitates with the class I PI 3-kinase regulatory subunit p85, through its SH2 domain but that APS does not modulate neither PtdIns(3,4,5)P3 levels nor Akt phosphorylation provoked by insulin. We have confirmed a previously described positive effect of APS overexpression on insulin-induced MAPK phosphorylation upregulation. Consequently, we analyzed the role of SH2 and PH domains of APS in the APS increased MAPK phosphorylation observed upon insulin stimulation and correlated this with the membrane localization of the protein. The effect observed on MAPK phosphorylation requires the intact PH binding domain of APS as well as its SH2 domain.

  2. Spinal cord NR1 serine phosphorylation and NR2B subunit suppression following peripheral inflammation

    Directory of Open Access Journals (Sweden)

    Del Valle-Pinero Arseima Y

    2005-09-01

    Full Text Available Abstract Background Spinal cord N-methyl-D-aspartate (NMDA receptors are intimately involved in the development and maintenance of central sensitization. However, the mechanisms mediating the altered function of the NMDA receptors are not well understood. In this study the role of phosphorylation of NR1 splice variants and NR2 subunits was examined following hind paw inflammation in rats. We further examined the level of expression of these proteins following the injury. Results Lumbar spinal cord NR1 subunits were found to be phosphorylated on serine residues within two hours of the induction of hind paw inflammation with carrageenan. The enhanced NR1 serine phosphorylation reversed within six hours. No phosphorylation on NR1 threonine or tyrosine residues was observed. Likewise, no NR2 subunit phosphorylation was observed on serine, threonine or tyrosine residues. An analysis of NR1 and NR2 protein expression demonstrated no change in the levels of NR1 splice variants or NR2A following the inflammation. However, spinal cord NR2B expression was depressed by the hind paw inflammation. The expression of NR2B remained depressed for more than one week following initiation of the inflammation. Conclusion These data suggest that NR1 serine phosphorylation leads to an initial increase in NMDA receptor activity in the spinal cord following peripheral injury. The suppression of NR2B expression suggests compensation for the enhanced nociceptive activity. These data indicate that spinal cord NMDA receptors are highly dynamic in the development, maintenance and recovery from central sensitization following an injury. Thus, chronic pain therapies targeted to NMDA receptors should be designed for the exact configuration of NMDA receptor subunits and post-translational modifications present during specific stages of the disease.

  3. Identification of protein tyrosine phosphatase 1B and casein as substrates for 124-v-Mos

    Directory of Open Access Journals (Sweden)

    Stabel Silvia

    2002-04-01

    Full Text Available Abstract Background The mos proto-oncogene encodes a cytoplasmic serine/threonine-specific protein kinase with crucial function during meiotic cell division in vertebrates. Based on oncogenic amino acid substitutions the viral derivative, 124-v-Mos, displays constitutive protein kinase activity and functions independent of unknown upstream effectors of mos protein kinase. We have utilized this property of 124-v-Mos and screened for novel mos substrates in immunocomplex kinase assays in vitro. Results We generated recombinant 124-v-Mos using the baculovirus expression system in Spodoptera frugiperda cells and demonstrated constitutive kinase activity by the ability of 124-v-Mos to auto-phosphorylate and to phosphorylate vimentin, a known substrate of c-Mos. Using this approach we analyzed a panel of acidic and basic substrates in immunocomplex protein kinase assays and identified novel in vitro substrates for 124-v-Mos, the protein tyrosine phosphatase 1B (PTP1B, alpha-casein and beta-casein. We controlled mos-specific phosphorylation of PTP1B and casein in comparative assays using a synthetic kinase-inactive 124-v-Mos mutant and further, tryptic digests of mos-phosphorylated beta-casein identified a phosphopeptide specifically targeted by wild-type 124-v-Mos. Two-dimensional phosphoamino acid analyses showed that 124-v-mos targets serine and threonine residues for phosphorylation in casein at a 1:1 ratio but auto-phosphorylation occurs predominantly on serine residues. Conclusion The mos substrates identified in this study represent a basis to approach the identification of the mos-consensus phosphorylation motif, important for the development of specific inhibitors of the Mos protein kinase.

  4. Complement receptor-3 negatively regulates the phagocytosis of degenerated myelin through tyrosine kinase Syk and cofilin

    Directory of Open Access Journals (Sweden)

    Hadas Smadar

    2012-07-01

    Full Text Available Abstract Background Intact myelin, which normally surrounds axons, breaks down in Wallerian degeneration following axonal injury and during neurodegenerative diseases such as multiple sclerosis. Clearance of degenerated myelin by phagocytosis is essential since myelin impedes repair and exacerbates damage. CR3 (complement receptor-3 is a principal phagocytic receptor in myelin phagocytosis. We studied how tyrosine kinase Syk (spleen tyrosine kinase and cofilin control phagocytosis of degenerated myelin by CR3 in microglia and macrophages. Syk is a non-receptor tyrosine kinase that CR3 recruits to convey cellular functions. Cofilin is an actin-depolymerizing protein that controls F-actin (filamentous actin remodeling (i.e., disassembly and reassembly by shifting between active unphosphorylated and inactive phosphorylated states. Results Syk was continuously activated during prolonged phagocytosis. Phagocytosis increased when Syk activity and expression were reduced, suggesting that normally Syk down regulates CR3-mediated myelin phagocytosis. Levels of inactive p-cofilin (phosphorylated cofilin decreased transiently during prolonged phagocytosis. In contrast, p-cofilin levels decreased continuously when Syk activity and expression were continuously reduced, suggesting that normally Syk advances the inactive state of cofilin. Observations also revealed inverse relationships between levels of phagocytosis and levels of inactive p-cofilin, suggesting that active unphosphorylated cofilin advances phagocytosis. Active cofilin could advance phagocytosis by promoting F-actin remodeling, which supports the production of membrane protrusions (e.g., filopodia, which, as we also revealed, are instrumental in myelin phagocytosis. Conclusions CR3 both activates and downregulates myelin phagocytosis at the same time. Activation was previously documented. We presently demonstrate that downregulation is mediated through Syk, which advances the inactive

  5. Propofol directly increases tau phosphorylation.

    Directory of Open Access Journals (Sweden)

    Robert A Whittington

    2011-01-01

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

  6. Protein tyrosine nitration in the cell cycle

    International Nuclear Information System (INIS)

    Jia, Min; Mateoiu, Claudia; Souchelnytskyi, Serhiy

    2011-01-01

    Highlights: → Enrichment of 3-nitrotyrosine containing proteins from cells synchronized in different phases of the cell cycle. → Identification of 76 tyrosine nitrated proteins that change expression during the cell cycle. → 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.

  7. Pirenzepine Inhibits Myopia in Guinea Pig Model by Regulating the Balance of MMP-2 and TIMP-2 Expression and Increased Tyrosine Hydroxylase Levels.

    Science.gov (United States)

    Qian, Lifeng; Zhao, Hong; Li, Xiaoxia; Yin, Juanjuan; Tang, Wenjian; Chen, Peng; Wang, Qian; Zhang, Jinsong

    2015-04-01

    In this study, we investigated the effects and mechanism of action of pirenzepine in a guinea pig model of myopia induced by exposure to monochromatic light. It was observed that pirenzepine inhibited the increase of diopter and extension of ocular axial length. Immunohistochemistry staining showed that the number of tyrosine hydroxylase (TH)-positive cells in pirenzepine group was significantly higher compared to the other treatment groups pointing to a highly positive correlation between TH expression levels and the diopter and axial length change. RT-PCR analysis further showed that pirenzepine treatment reduced the expression of matrix metalloproteinase (MMP-2) and enhanced the expression of tissue inhibitors of metalloproteinase (TIMP-2) compared to the other treatment and control groups. To conclude, we demonstrate that pirenzepine may improve the prognosis of monochromatic light-induced myopia in guinea pigs, possibly by both regulating the balance of MMP-2 and TIMP-2 in sclera and increasing the TH expression in retina.

  8. Reappraising metalloproteinases in rheumatoid arthritis and osteoarthritis: destruction or repair?

    Science.gov (United States)

    Murphy, Gillian; Nagase, Hideaki

    2008-03-01

    Metalloproteinases such as the matrix metalloproteinases (MMPs) and disintegrin-metalloproteinases with thrombospondin motifs (ADAMTSs) have been implicated in the pathological destruction of joint tissues in rheumatoid arthritis and osteoarthritis. These enzymes degrade extracellular matrix macromolecules and modulate factors governing cell behavior. They may also be involved in tissue repair, but become a part of the destructive disease process due to overexpression. Studies investigating the roles of metalloproteinases have thrown light on the failure of the early clinical trials of MMP inhibitors as therapeutic agents in arthritic diseases. It is now clear that a more accurate knowledge of the enzymes in the different cells and their precise roles in the disease process is required for these approaches to be successful. The next generations of metalloproteinase inhibitors should have added specificity, gained from an understanding not only of the catalytic domain structures but the role of extracatalytic motifs in substrate binding, or by the generation of engineered tissue inhibitors of metalloproteinases. Inhibition of the enzymes by modulating gene expression or preventing protein activation could also be considered. Work on the development of effective biomarkers is also essential before an effective evaluation of the new generations of specific inhibitors can be made.

  9. No effect of oral tyrosine on total tyrosine levels in breast milk: implications for dietary supplementation in early postpartum.

    Science.gov (United States)

    Dowlati, Yekta; Ravindran, Arun V; Maheux, Maxim; Steiner, Meir; Stewart, Donna E; Meyer, Jeffrey H

    2014-12-01

    Postpartum depression (PPD) is the most common complication of childbearing with a 13 % prevalence rate, and there is no widespread approach for prevention. There is an appealing theoretical rationale for oral tyrosine to help prevent PPD. However, the effect of oral tyrosine on its total and free concentrations in breast milk and plasma of breastfeeding mothers is not known. Twenty-four healthy breastfeeding women were randomly assigned to 0, 2, 5, or 10 g of oral tyrosine. Free and total tyrosine in breast milk and free tyrosine in plasma were measured. Free tyrosine was also measured in 12 different infant formulas. Total tyrosine in breast milk did not rise, but there was a slight tendency towards a reduction (up to −12 %; repeated measures ANOVA (RMANOVA): p = 0.074). Maternal plasma tyrosine rose (RMANOVA: p oral tyrosine on its concentration in breast milk supports further development of oral tyrosine as part of a prevention strategy for PPD.

  10. SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

    Energy Technology Data Exchange (ETDEWEB)

    JOHN C WALKER

    2011-11-01

    Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

  11. Enkephalins affect hippocampal membrane phosphorylation

    NARCIS (Netherlands)

    Bär, P.R; Schotman, P.; Gispen, W.H.

    1980-01-01

    Slices of rat brain hippocampus were incubated with methionine-enkephalin, leucine-enkephalin, [Des-Tyr1] methionine-enkephalin or etorphin. After incubation the endogenous phosphorylation of proteins was measured using crude mitochhondrial fractions prepared from the incubated slices. Methione- and

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

    DEFF Research Database (Denmark)

    Lotti, L V; Lanfrancone, L; Migliaccio, E

    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...... 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....

  13. Evaluation of the inhibition of other metalloproteinases by matrix metalloproteinase inhibitors.

    Science.gov (United States)

    Marcotte, P A; Elmore, I N; Guan, Z; Magoc, T J; Albert, D H; Morgand, D W; Curtin, M L; Garland, R B; Guo, Y; Heyman, H R; Holms, J H; Sheppard, G S; Steinman, D H; Wada, C K; Davidsen, S K

    1999-01-01

    Two series of compounds synthesized as specific matrix metalloproteinase (MMP) inhibitors have been evaluated for their inhibition of non-MMPs. In a series of substituted succinyl hydroxamic acids, some were found to be significant (IC50 inhibitors of leucine (microsomal) aminopeptidase, neprilysin (3.4.24.11), and thermolysin. Macrocyclic compounds in which the alpha carbon of the succinyl hydroxamate is linked to the side chain of the P2' amino acid were found to be good inhibitors of aminopeptidase, but not of neprilysin or thermolysin. Compounds of neither series were found to be significant inhibitors of angiotensin converting enzyme or carboxypeptidase A.

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

    Science.gov (United States)

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

    2014-02-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  16. The matrix metalloproteinase in larynx cancer

    Directory of Open Access Journals (Sweden)

    Weronika Lucas Grzelczyk

    2016-12-01

    Full Text Available One of the most common carcinoma occurring in the head and neck is laryngeal cancer. Despite the rapid scientific advances in medicine the prognosis for patients with such type of disease is not satisfying. In the last few years matrix metalloproteinases ‑ MMPs and their tissue inhibitors – TIMPs, mostly MMP‑2 and MMP‑9, arouses a great interest, especially in the process of carcinogenesis. It seems that their impact in the formation and development of laryngeal cancer is significant. MMPs a group of zinc‑ and calcium‑ dependent endopeptidases play crucial role extracellular matrix collagen degradation. That are enzymes, that degrade and the basement membrane by facilitating tumor growth, cell migration and tumor invasion. They are implicated in metastasis and angiogenesis potentiate within the tumor. Clear tendency was observed towards the higher MMPs and TIMPs expression in larynx cancer than in the stroma. Recent studies show correlations between increased MMP‑2 gene expression in the tumor tissue and clinical status, histopathological grading and metastases occurrence. The similar MMP2 over expression dependence were found on tumor recurrence and survival. Many authors pointed out, significant higher MMP‑2 expression as a potential marker of tumor invasiveness and worse prognosis in patients with larynx cancer. However, association of MMP 9 gene expression with laryngeal cancer clinicopathological features and survival of patients are ambiguous. Although, numerous researches show that this relationship does exists. Similar correlations could be found in TIMPs, but further studies are necessary because of small amount of literature.

  17. Correlation between matrix metalloproteinase-9 and endometriosis.

    Science.gov (United States)

    Liu, Haiping; Wang, Jianye; Wang, Haiyu; Tang, Ning; Li, Yunfei; Zhang, Yan; Hao, Tianyu

    2015-01-01

    Endometrial implantation is the major cause of endometriosis (EMS). Matrix metalloproteinase (MMPs) can degrade multiple extracellular matrix and has been postulated to be related with EMC occurrence. This study thus investigated serum and ascites levels of MMP-9 in EMS patients, in an attempt to discuss the correlation between MMP-9 and EMS. A total of 100 EMS patients, including eutopic endometrium and ectopic endometrium, were recruited in this study along with hysteromyoma patients as the control group. Peripheral blood and ascites samples were collected and tested for MMP-9 levels using gelatin zymogram and enzyme-linked immunosorbent assay (ELISA). In EMS patients, MMP-9 levels in serum and ascites were 6.24 ± 0.53 mM and 38.57 ± 4.93 mM, respectively. Both of them were significantly higher than those in control group (P<0.05). Eutopic endometrium group had higher MMP-9 levels compared to those in ectopic endometrium ones (P<0.05). With advancement of disease stage, EMS patients had progressively elevated MMP-9 levels (P<0.05). Patients at proliferative stage had higher MMP-9 secretion (P<0.05). In summary, site of endometrium, clinical stage and proliferative cycle were independent risk factors for EMS. The elevation of serum and ascites MMP-9 existed in EMS patients, of which those had ectopic endometrium, advanced stage and at proliferative stage had higher MMP-9 expression.

  18. Matrix Metalloproteinases in Non-Neoplastic Disorders

    Science.gov (United States)

    Tokito, Akinori; Jougasaki, Michihisa

    2016-01-01

    The matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases belonging to the metzincin superfamily. There are at least 23 members of MMPs ever reported in human, and they and their substrates are widely expressed in many tissues. Recent growing evidence has established that MMP not only can degrade a variety of components of extracellular matrix, but also can cleave and activate various non-matrix proteins, including cytokines, chemokines and growth factors, contributing to both physiological and pathological processes. In normal conditions, MMP expression and activity are tightly regulated via interactions between their activators and inhibitors. Imbalance among these factors, however, results in dysregulated MMP activity, which causes tissue destruction and functional alteration or local inflammation, leading to the development of diverse diseases, such as cardiovascular disease, arthritis, neurodegenerative disease, as well as cancer. This article focuses on the accumulated evidence supporting a wide range of roles of MMPs in various non-neoplastic diseases and provides an outlook on the therapeutic potential of inhibiting MMP action. PMID:27455234

  19. Matrix metalloproteinase activity assays: Importance of zymography.

    Science.gov (United States)

    Kupai, K; Szucs, G; Cseh, S; Hajdu, I; Csonka, C; Csont, T; Ferdinandy, P

    2010-01-01

    Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases capable of degrading extracellular matrix, including the basement membrane. MMPs are associated with various physiological processes such as morphogenesis, angiogenesis, and tissue repair. Moreover, due to the novel non-matrix related intra- and extracellular targets of MMPs, dysregulation of MMP activity has been implicated in a number of acute and chronic pathological processes, such as arthritis, acute myocardial infarction, chronic heart failure, chronic obstructive pulmonary disease, inflammation, and cancer metastasis. MMPs are considered as viable drug targets in the therapy of the above diseases. For the development of selective MMP inhibitor molecules, reliable methods are necessary for target validation and lead development. Here, we discuss the major methods used for MMP assays, focusing on substrate zymography. We highlight some problems frequently encountered during sample preparations, electrophoresis, and data analysis of zymograms. Zymography is a widely used technique to study extracellular matrix-degrading enzymes, such as MMPs, from tissue extracts, cell cultures, serum or urine. This simple and sensitive technique identifies MMPs by the degradation of their substrate and by their molecular weight and therefore helps to understand the widespread role of MMPs in different pathologies and cellular pathways. Copyright 2010 Elsevier Inc. All rights reserved.

  20. Proanthocyanidins from the American Cranberry (Vaccinium macrocarpon) inhibit matrix metalloproteinase-2 and matrix metalloproteinase-9 activity in human prostate cancer cells via alterations in multiple cellular signalling pathways.

    Science.gov (United States)

    Déziel, Bob A; Patel, Kunal; Neto, Catherine; Gottschall-Pass, Katherine; Hurta, Robert A R

    2010-10-15

    Prostate cancer is one of the most common cancers in the Western world, and it is believed that an individual's diet affects his risk of developing cancer. There has been an interest in examining phytochemicals, the secondary metabolites of plants, in order to determine their potential anti-cancer activities in vitro and in vivo. In this study we document the effects of proanthocyanidins (PACs) from the American Cranberry (Vaccinium macrocarpon) on matrix metalloproteinase (MMP) activity in DU145 human prostate cancer cells. Cranberry PACs decreased cellular viability of DU145 cells at a concentration of 25 µg/ml by 30% after 6 h of treatment. Treatment of DU145 cells with PACs resulted in an inhibition of both MMPs 2 and 9 activity. PACs increased the expression of TIMP-2, a known inhibitor of MMP activity, and decreased the expression of EMMPRIN, an inducer of MMP expression. PACs decreased the expression of PI-3 kinase and AKT proteins, and increased the phosphorylation of both p38 and ERK1/2. Cranberry PACs also decreased the translocation of the NF-κB p65 protein to the nucleus. Cranberry PACs increased c-jun and decreased c-fos protein levels. These results suggest that cranberry PACs decreases MMP activity through the induction and/or inhibition of specific temporal MMP regulators, and by affecting either the phosphorylation status and/or expression of MAP kinase, PI-3 kinase, NF-κB and AP-1 pathway proteins. This study further demonstrates that cranberry PACs are a strong candidate for further research as novel anti-cancer agents. © 2010 Wiley-Liss, Inc.

  1. Serum concentrations of metalloproteinase 2, metalloproteinase 9 and granzyme B in contact eczema patients

    Science.gov (United States)

    Żbikowska-Gotz, Magdalena; Czajkowski, Rafał; Bartuzi, Zbigniew

    2013-01-01

    Introduction Contact eczema is a common skin condition with complex etiology, variable clinical presentation and lengthy therapy duration. The mechanism of contact eczema is complex, since it is affected by multiple inflammatory mediators. Aim To assess concentrations of metalloproteinase 2 (MMP-2), metalloproteinase 9 (MMP-9) and granzyme B (GzmB) in patients with contact eczema. Material and methods Seventy patients with contact eczema and 30 healthy persons as controls were included in the study. In all subjects, MMP-2, MMP-9 and GzmB were determined using ELISA immunoassay. In study group patients, concentrations were assayed in periods of disease exacerbation and remission. Obtained results were analyzed statistically. Results Mean MMP-2 and GzmB concentrations were found to be significantly higher in the study group than in the control group. Mean MMP-2, MMP-9 and GzmB levels were also statistically significantly higher during skin lesion relapse compared to contact eczema remission periods. Conclusions The presented paper demonstrates that MMP-2, MMP-9 and GzmB are good markers of contact eczema exacerbations. PMID:24278051

  2. Matrix Metalloproteinases and Tissue Inhibitor of Metalloproteinases in Inflammation and Fibrosis of Skeletal Muscles.

    Science.gov (United States)

    Alameddine, Hala S; Morgan, Jennifer E

    2016-11-29

    In skeletal muscles, levels and activity of Matrix MetalloProteinases (MMPs) and Tissue Inhibitors of MetalloProteinases (TIMPs) have been involved in myoblast migration, fusion and various physiological and pathological remodeling situations including neuromuscular diseases. This has opened perspectives for the use of MMPs' overexpression to improve the efficiency of cell therapy in muscular dystrophies and resolve fibrosis. Alternatively, inhibition of individual MMPs in animal models of muscular dystrophies has provided evidence of beneficial, dual or adverse effects on muscle morphology or function. We review here the role played by MMPs/TIMPs in skeletal muscle inflammation and fibrosis, two major hurdles that limit the success of cell and gene therapy. We report and analyze the consequences of genetic or pharmacological modulation of MMP levels on the inflammation of skeletal muscles and their repair in light of experimental findings. We further discuss how the interplay between MMPs/TIMPs levels, cytokines/chemokines, growth factors and permanent low-grade inflammation favor cellular and molecular modifications resulting in fibrosis.

  3. Expressions of matrix metalloproteinase-2 and extracellular matrix metalloproteinase inducer in retinoblastoma

    Directory of Open Access Journals (Sweden)

    Yu-Hong Cheng

    2015-07-01

    Full Text Available AIM: To investigate expressions of matrix metalloproteinase-2(MMP-2and extracellular matrix metalloproteinase inducer(EMMPRINin retinoblastoma(Rband the relationships between MMP-2, EMMPRIN and tumor development.METHODS:Immunohistochemical technique was used to detect expressions of MMP-2 and EMMPRIN in 39 cases of paraffin embedded Rb samples. Quantitative analysis of expressions of MMP-2 and EMMPRIN were assessed by measuring the mean gray scale of Rb tissue with LEICA IM50 Color Pathologic Analysis System. The differences of expressions of MMP-2 and EMMPRIN in each clinical and pathological stage were statistically analyzed, and the same step was also undertaken to study the relationship between Rb with MMP-2 positive expression and that with EMMPRIN positive expression.RESULTS: The positive expression rate of MMP-2 was 90%(Gray value: 109.64±14.52; 35/39, and that of EMMPRIN was 85%(Gray value: 108.01±13.60; 33/39. The expressions of MMP-2 and EMMPRIN were significantly higher in tumors of glaucomatous stage(Gray value: 108.21±11.47 and 107.56±14.32than those in intraocular stage(Gray value: 121.13±11.32 and 119.34±12.66; PPPPPPCONCLUSION: The positive expression levels of MMP-2 and EMMPRIN may correlate with tumor infiltration and metastasis.

  4. Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Fukumoto, Yasunori, E-mail: fukumoto@faculty.chiba-u.jp; Kuki, Kazumasa; Morii, Mariko; Miura, Takahito; Honda, Takuya; Ishibashi, Kenichi; Hasegawa, Hitomi; Kubota, Sho; Ide, Yudai; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto, E-mail: nyama@faculty.chiba-u.jp

    2014-09-26

    Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.

  5. Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

    International Nuclear Information System (INIS)

    Fukumoto, Yasunori; Kuki, Kazumasa; Morii, Mariko; Miura, Takahito; Honda, Takuya; Ishibashi, Kenichi; Hasegawa, Hitomi; Kubota, Sho; Ide, Yudai; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto

    2014-01-01

    Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint

  6. c-Src phosphorylation and activation of hexokinase promotes tumorigenesis and metastasis

    Science.gov (United States)

    Zhang, Jia; Wang, Suili; Jiang, Bin; Huang, Lihong; Ji, Zhiliang; Li, Xiaotong; Zhou, Huamin; Han, Aidong; Chen, Ai; Wu, Yanan; Ma, Huanhuan; Zhao, Wentao; Zhao, Qingwen; Xie, Changchuan; Sun, Xiaoyan; Zhou, Yanming; Huang, Huiying; Suleman, Muhammad; Lin, Furong; Zhou, Lin; Tian, Fang; Jin, Meijun; Cai, Yana; Zhang, Nan; Li, Qinxi

    2017-01-01

    It is well known that c-Src has important roles in tumorigenesis. However, it remains unclear whether c-Src contributes to metabolic reprogramming. Here we find that c-Src can interact with and phosphorylate hexokinases HK1 and HK2, the rate-limiting enzymes in glycolysis. Tyrosine phosphorylation dramatically increases their catalytic activity and thus enhances glycolysis. Mechanistically, c-Src phosphorylation of HK1 at Tyr732 robustly decreases its Km and increases its Vmax by disrupting its dimer formation. Mutation in c-Src phosphorylation site of either HK1 or HK2 remarkably abrogates the stimulating effects of c-Src on glycolysis, cell proliferation, migration, invasion, tumorigenesis and metastasis. Due to its lower Km for glucose, HK1 rather than HK2 is required for tumour cell survival when glucose is scarce. Importantly, HK1-Y732 phosphorylation level remarkably correlates with the incidence and metastasis of various clinical cancers and may serve as a marker to predict metastasis risk of primary cancers. PMID:28054552

  7. Cellular regulation by protein phosphorylation.

    Science.gov (United States)

    Fischer, Edmond H

    2013-01-11

    A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca(2+) and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert. Copyright © 2012. Published by Elsevier Inc.

  8. Molecular mechanisms of the synergy between cysteinyl-leukotrienes and receptor tyrosine kinase growth factors on human bronchial fibroblast proliferation

    Directory of Open Access Journals (Sweden)

    Hajime Yoshisue

    2006-12-01

    Full Text Available We have reported that cysteinyl-leukotrienes (cys-LTs synergise not only with epidermal growth factor (EGF but also with platelet-derived growth factor (PDGF and fibroblast growth factor (FGF to induce mitogenesis in human bronchial fibroblasts. We now describe the molecular mechanisms underlying this synergism. Mitogenesis was assessed by incorporation of [3H]thymidine into DNA and changes in protein phosphorylation by Western blotting. Surprisingly, no CysLT receptor antagonists (MK-571, montelukast, BAY u9773 prevented the synergistic mitogenesis. LTD4 did not cause phosphorylation of EGFR nor did it augment EGF-induced phosphorylation of EGFR, and the synergy between LTD4 and EGF was not blocked by the metalloproteinase inhibitor GM6001 or by an HB-EGF neutralising antibody. The EGFR-selective kinase inhibitor, AG1478, suppressed the synergy by LTD4 and EGF, but had no effect on the synergy with PDGF and FGF. While inhibitors of mitogen-activated protein kinase, phosphatidylinositol 3-kinase and protein kinase C (PKC prevented the synergy, these drugs also inhibited mitogenesis elicited by EGF alone. In contrast, pertussis toxin (PTX efficiently inhibited the potentiating effect of LTD4 on EGF-induced mitogenesis, as well as that provoked by PDGF or FGF, but had no effect on mitogenesis elicited by the growth factors alone. Whereas LTD4 alone did not augment phosphorylation of extracellular signal-regulated kinase (Erk-1/2 and Akt, it increased phosphorylation of PKC in a Gi-dependent manner. Addition of LTD4 prolonged the duration of EGF-induced phosphorylation of Erk-1/2 and Akt, both of which were sensitive to PTX. The effect of cys-LTs involves a PTX-sensitive and PKC-mediated intracellular pathway leading to sustained growth factor-dependent phosphorylation of Erk-1/2 and Akt.

  9. Mechanisms of Peroxynitrite Mediated Nitration of Tyrosine

    Science.gov (United States)

    Gunaydin, Hakan; Houk, K. N.

    2009-01-01

    The mechanisms of tyrosine nitration by peroxynitrous acid or nitrosoperoxycarbonate were investigated with the CBS-QB3 method. Either the protonation of peroxynitrite, or a reaction with carbon dioxide gives a reactive peroxide intermediate. Peroxynitrous acid mediated nitration of phenol occurs via the unimolecular decomposition to give nitrogen dioxide and hydroxyl radicals. Nitrosoperoxycarbonate also undergoes unimolecular decomposition to give carbonate and nitrogen dioxide radicals. The reactions of tyrosine with the hydroxyl or carbonate radicals give a phenoxy radical intermediate. The reaction of the nitrogen dioxide with this radical intermediate followed by tautomerization gives nitrated tyrosine in both cases. According to CBS-QB3 calculations, the rate-limiting step for the nitration of phenol is the decomposition of peroxynitrous acid or of nitrosoperoxycarbonate. PMID:19374346

  10. Structure and Molecular Dynamics Simulations of Protein Tyrosine Phosphatase Non-Receptor 12 Provide Insights into the Catalytic Mechanism of the Enzyme

    Directory of Open Access Journals (Sweden)

    Hui Dong

    2017-12-01

    Full Text Available Protein tyrosine phosphatase non-receptor 12 (PTPN12 is an important protein tyrosine phosphatase involved in regulating cell adhesion and migration as well as tumorigenesis. Here, we solved a crystal structure of the native PTPN12 catalytic domain with the catalytic cysteine (residue 231 in dual conformation (phosphorylated and unphosphorylated. Combined with molecular dynamics simulation data, we concluded that those two conformations represent different states of the protein which are realized during the dephosphorylation reaction. Together with docking and mutagenesis data, our results provide a molecular basis for understanding the catalytic mechanism of PTPN12 and its role in tumorigenesis.

  11. Validation of Anti-CSPα, SNAP25, Tyrosine Hydroxylase, Ubiquitin, Cleaved Caspase 3, and pSer PKC Motif Antibodies for Utilization in Western Blotting.

    Science.gov (United States)

    Shirafuji, Toshihiko; Ueyama, Takehiko; Tanaka, Shigeru; Hide, Izumi; Saito, Naoaki; Sakai, Norio

    2017-12-26

    There are many commercial antibodies with little information provided by their suppliers as to their reliability. Accordingly, commercial antibodies require proper validation before being used in scientific research. In this study, we validated several commercial antibodies, including anti-CSPα, SNAP25, tyrosine hydroxylase, ubiquitin, cleaved caspase 3, and pSer PKC motif. Anti-CSPα, SNAP25, and tyrosine hydroxylase antibodies could detect their endogenous target proteins with some degree of cross-reactivity. Furthermore, clear SNAP25 staining was observed with SNAP25 antibody. Antibodies directed against ubiquitin, cleaved caspase 3, and pSer PKC motif could detect poly-ubiquitination, apoptosis, and phosphorylation, respectively.

  12. Impact of flavonoids on matrix metalloproteinase secretion and invadopodia formation in highly invasive A431-III cancer cells.

    Directory of Open Access Journals (Sweden)

    Yo-Chuen Lin

    Full Text Available Metastasis is a major cause of mortality in cancer patients. Invadopodia are considered to be crucial structures that allow cancer cells to penetrate across the extracellular matrix (ECM by using matrix metalloproteinases (MMPs. Previously, we isolated a highly invasive A431-III subline from parental A431 cells by Boyden chamber assay. The A431-III cells possess higher invasive and migratory abilities, elevated levels of MMP-9 and an enhanced epithelial-mesenchymal transition (EMT phenotype. In this study, we discovered that A431-III cells had an increased potential to form invadopodia and an improved capacity to degrade ECM compared with the original A431 cells. We also observed enhanced phosphorylation levels of cortactin and Src in A431-III cells; these phosphorylated proteins have been reported to be the main regulators of invadopodia formation. Flavonoids, almost ubiquitously distributed in food plants and plant food products, have been documented to exhibit anti-tumor properties. Therefore, it was of much interest to explore the effects of flavonoid antioxidants on the metastatic activity of A431-III cells. Exposure of A431-III cells to two potent dietary flavonoids, namely luteolin (Lu and quercetin (Qu, caused inhibition of invadopodia formation and decrement in ECM degradation. We conclude that Lu and Qu attenuate the phosphorylation of cortactin and Src in A431-III cells. As a consequence, there ensues a disruption of invadopodia generation and the suppression of MMP secretion. These changes, in concert, bring about a reduction in metastasis.

  13. Molecular imaging of matrix metalloproteinase in atherosclerotic lesions : resolution with dietary modification and statin therapy

    NARCIS (Netherlands)

    Fujimoto, Shinichiro; Hartung, Dagmar; Ohshima, Satoru; Edwards, D. Scott; Zhou, Jun; Yalamanchili, Padmaja; Azure, Michael; Fujimoto, Ai; Isobe, Satoshi; Matsumoto, Yuji; Boersma, Hendrikus; Wong, Nathan; Yamazaki, Junichi; Narula, Navneet; Petrov, Artiom; Narula, Jagat

    2008-01-01

    OBJECTIVES: This study sought to evaluate the feasibility of noninvasive detection of matrix metalloproteinase (MMP) activity in experimental atherosclerosis using technetium-99m-labeled broad matrix metalloproteinase inhibitor (MPI) and to determine the effect of dietary modification and statin

  14. Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors.

    NARCIS (Netherlands)

    Snoek, P.A.; Hoff, J.W. Von den

    2005-01-01

    The balance between matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), is largely responsible for the remodeling of tissues. Deregulation of this balance is a characteristic of extensive tissue degradation in certain degenerative diseases. To

  15. Modulating uranium binding affinity in engineered Calmodulin EF-hand peptides: effect of phosphorylation

    International Nuclear Information System (INIS)

    Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Guilloreau, Luc; Berthomieu, Catherine; Delangle, Pascale; Adriano, Jean-Marc

    2012-01-01

    To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T 9 TKE 12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K d =25±6 nM to K d =5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the sub-nanomolar range (K d = 0.25±0.06 nM). FTIR analyses showed that the phospho-threonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν as (P-O) and ν s (P-O) IR modes of phospho-threonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν as (UO 2 ) 2+ vibration (from 923 cm -1 to 908 cm -1 ) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. (authors)

  16. Modulating uranium binding affinity in engineered calmodulin EF-hand peptides: effect of phosphorylation.

    Directory of Open Access Journals (Sweden)

    Romain Pardoux

    Full Text Available To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T(9TKE(12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K(d = 25±6 nM to K(d = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (K(d = 0.25±0.06 nM. FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν(as(P-O and ν(s(P-O IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν(as(UO(2(2+ vibration (from 923 cm(-1 to 908 cm(-1 was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH.

  17. Nuclear localization of Src-family tyrosine kinases is required for growth factor-induced euchromatinization

    International Nuclear Information System (INIS)

    Takahashi, Akinori; Obata, Yuuki; Fukumoto, Yasunori; Nakayama, Yuji; Kasahara, Kousuke; Kuga, Takahisa; Higashiyama, Yukihiro; Saito, Takashi; Yokoyama, Kazunari K.; Yamaguchi, Naoto

    2009-01-01

    Src-family kinases (SFKs), which participate in various signaling events, are found at not only the plasma membrane but also several subcellular compartments, including the nucleus. Nuclear structural changes are frequently observed during transcription, cell differentiation, senescence, tumorigenesis, and cell cycle. However, little is known about signal transduction in the alteration of chromatin texture. Here, we develop a pixel imaging method for quantitatively evaluating chromatin structural changes. Growth factor stimulation increases euchromatic hypocondensation and concomitant heterochromatic hypercondensation in G 1 phase, and the levels reach a plateau by 30 min, sustain for at least 5 h and return to the basal levels after 24 h. Serum-activated SFKs in the nucleus were more frequently detected in the euchromatin areas than the heterochromatin areas. Nuclear expression of kinase-active SFKs, but not unrelated Syk kinase, drastically increases both euchromatinization and heterochromatinization in a manner dependent on the levels of nuclear tyrosine phosphorylation. However, growth factor stimulation does not induce chromatin structural changes in SYF cells lacking SFKs, and reintroduction of one SFK member into SYF cells can, albeit insufficiently, induce chromatin structural changes. These results suggest that nuclear tyrosine phosphorylation by SFKs plays an important role in chromatin structural changes upon growth factor stimulation.

  18. Immunohistochemical analysis of receptor tyrosine kinase signal transduction activity in chordoma.

    Science.gov (United States)

    Fasig, J H; Dupont, W D; LaFleur, B J; Olson, S J; Cates, J M M

    2008-02-01

    Currently, there are no effective chemotherapeutic protocols for chordoma. Reports of receptor tyrosine kinase (RTK) expression in chordoma suggest that these tumours may respond to kinase inhibitor therapy. However, RTK signalling activity has not been extensively investigated in chordoma. A tissue microarray containing 21 cases of chordoma was analysed for expression of a number of proteins involved in signal transduction from RTKs by immunohistochemistry. Platelet-derived growth factor receptor-beta, epidermal growth factor receptor (EGFR), KIT and HER2 were detected in 100%, 67%, 33% and 0% of cases, respectively. Platelet-derived growth factor receptor-beta staining was of moderate-to-strong intensity in 20 of 21 cases. In contrast, KIT immunoreactivity was weak and focal in each of the seven positive cases. Total EGFR staining was variable; weak staining for phosphorylated EGFR was detected in nine cases. Phosphorylated isoforms of p44/42 mitogen-activated protein kinase, Akt and STAT3, indicative of tyrosine kinase activity, were detected in 86%, 76% and 67% of cases, respectively. Chordomas commonly express RTKs and activated signal transduction molecules. Although there were no statistically significant correlations between the expression of any of the markers studied and disease-free survival or tumour location, the results nonetheless indicate that chordomas may respond to RTK inhibitors or modulators of other downstream signalling molecules.

  19. Lemur Tyrosine Kinase-3 Suppresses Growth of Prostate Cancer Via the AKT and MAPK Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Pengcheng Sun

    2017-08-01

    Full Text Available Background/Aims: Lemur tyrosine kinase (LMTK-3 is a member of the receptor tyrosine kinase (RTK family. Abnormal expression of LMTK-3 exists in various types of cancers, especially in endocrine-resistant breast cancers; however, the precise level of expression and the biological function in prostate cancer are poorly understood. Methods: In the present study, we determined the expression of LMTK-3 in prostate cancer using immunohistochemistry and Western blotting. We infected PC3 and LNCaP cells with lentivirus-LMTK-3 and observed the biologic characteristics of the PC3 and LNCaP cells in vitro with TUNEL, and migration and invasion assays, respectively. We also established a transplant tumor model of human prostate cancer with infected cells in 15 BALB/c-nu/nu nude mice. Results: LMTK-3 was expressed in prostate epithelial cells. There was a significant decline in the level of LMTK-3 expression in prostate cancers compared to normal tissues. LMTK-3 inhibited PC3 and LNCaP cell growth, migration, and invasion, and induced cell apoptosis in vitro. We also observed that LMTK-3 induced PC3 cell apoptosis in vivo. Further study showed that LMTK-3 inhibited phosphorylation of AKT and ERK, and promoted phosphorylation and activation of p38 kinase and Jun kinase (JNK. Conclusion: Recombinant lentivirus with enhanced expression of LMTK-3 inhibited prostate cancer cell growth and induced apoptosis in vitro and in vivo. AKT and MAPK signaling pathways may contribute to the process.

  20. HER2 oncogenic function escapes EGFR tyrosine kinase inhibitors via activation of alternative HER receptors in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Anthony Kong

    2008-08-01

    Full Text Available The response rate to EGFR tyrosine kinase inhibitors (TKIs may be poor and unpredictable in cancer patients with EGFR expression itself being an inadequate response indicator. There is limited understanding of the mechanisms underlying this resistance. Furthermore, although TKIs suppress the growth of HER2-overexpressing breast tumor cells, they do not fully inhibit HER2 oncogenic function at physiological doses.Here we have provided a molecular mechanism of how HER2 oncogenic function escapes TKIs' inhibition via alternative HER receptor activation as a result of autocrine ligand release. Using both Förster Resonance Energy Transfer (FRET which monitors in situ HER receptor phosphorylation as well as classical biochemical analysis, we have shown that the specific tyrosine kinase inhibitors (TKIs of EGFR, AG1478 and Iressa (Gefitinib decreased EGFR and HER3 phosphorylation through the inhibition of EGFR/HER3 dimerization. Consequent to this, we demonstrate that cleavage of HER4 and dimerization of HER4/HER2 occur together with reactivation of HER3 via HER2/HER3, leading to persistent HER2 phosphorylation in the now resistant, surviving cells. These drug treatment-induced processes were found to be mediated by the release of ligands including heregulin and betacellulin that activate HER3 and HER4 via HER2. Whereas an anti-betacellulin antibody in combination with Iressa increased the anti-proliferative effect in resistant cells, ligands such as heregulin and betacellulin rendered sensitive SKBR3 cells resistant to Iressa.These results demonstrate the role of drug-induced autocrine events leading to the activation of alternative HER receptors in maintaining HER2 phosphorylation and in mediating resistance to EGFR tyrosine kinase inhibitors (TKIs in breast cancer cells, and hence specify treatment opportunities to overcome resistance in patients.

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

    Energy Technology Data Exchange (ETDEWEB)

    Rehman, Kanwal [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Chen, Zhe [Zhejiang Hospital of Traditional Chinese Medicine, Zhejiang Chinese Medical University, Hangzhou (China); Wang, Wen Wen; Wang, Yan Wei [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Sakamoto, Akira [Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260‐8675 (Japan); Zhang, Yan Fang [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Naranmandura, Hua, E-mail: narenman@zju.edu.cn [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Suzuki, Noriyuki [Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260‐8675 (Japan)

    2012-09-15

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

  2. Dialkoxyquinazolines: Screening Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors for Potential Tumor Imaging Probes

    International Nuclear Information System (INIS)

    VanBrocklin, Henry F.; Lim, John K.; Coffing, Stephanie L.; Hom, Darren L.; Negash, Kitaw; Ono, Michele Y.; Hanrahan, Stephen M.; Taylor, Scott E.; Vanderpoel, Jennifer L.; Slavik, Sarah M.; Morris, Andrew B.; Riese II, David J.

    2005-01-01

    The epidermal growth factor receptor (EGFR), a long-standing drug development target, is also a desirable target for imaging. Sixteen dialkoxyquinazoline analogs, suitable for labeling with positron-emitting isotopes, have been synthesized and evaluated in a battery of in vitro assays to ascertain their chemical and biological properties. These characteristics provided the basis for the adoption of a selection schema to identify lead molecules for labeling and in vivo evaluation. A newEGFR tyrosine kinase radiometric binding assay revealed that all of the compounds possessed suitable affinity (IC50 = 0.4 - 51 nM) for the EGFR tyrosine kinase. All of the analogs inhibited ligand-induced EGFR tyrosine phosphorylation (IC50 = 0.8 - 20 nM). The HPLC-estimated octanol/water partition coefficients ranged from 2.0-5.5. Four compounds,4-(2'-fluoroanilino)- and 4-(3'-fluoroanilino)-6,7-diethoxyquinazoline as well as 4-(3'-chloroanilino)- and4-(3'-bromoanilino)-6,7-dimethoxyquinazoline, possess the best combination of characteristics that warrant radioisotope labeling and further evaluation in tumor-bearing mice

  3. Structural stability of human protein tyrosine phosphatase ρ catalytic domain: effect of point mutations.

    Directory of Open Access Journals (Sweden)

    Alessandra Pasquo

    Full Text Available 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.

  4. Cyclooxygenase-2 expression correlates with membrane-type-1 matrix metalloproteinase expression in colorectal cancer tissue.

    Science.gov (United States)

    Guo, Hongfei; Tatsuguchi, Atsushi; Shinji, Seiichi; Fujimori, Shunji; Tanaka, Shu; Gudis, Katya; Sugisaki, Yuichi; Furukawa, Kiyonori; Tajiri, Takashi; Fukuda, Yuh; Kishida, Teruyuki; Sakamoto, Choitsu

    2006-08-01

    Elevated expression of cyclooxygenase-2 has been found in colorectal cancer. One of the mechanisms through which cyclooxygenase-2 affects tumorigenesis is through its overexpression, which leads to increased invasiveness of cancer cells. A crucial step in this pathway is thought to be the induction of membrane-type-1 matrix metalloproteinase, which activates matrix metalloproteinase-2. However, to date there have been few clinicopathologic studies concerning cyclooxygenase-2-mediated invasiveness in human colorectal cancer tissues. We performed immunohistochemical analysis of the respective antigens on colorectal cancer specimens obtained by surgical resections from 96 patients with colorectal cancer. Cyclooxygenase-2 and membrane-type-1 matrix metalloproteinase expression was positive exclusively in cancer cells in 88 cases (92 percent) and 23 cases (24 percent), respectively. All 23 cases expressing membrane-type-1 matrix metalloproteinase also expressed cyclooxygenase-2. Matrix metalloproteinase-2 expression was positive in cancer cells in 20 cases (21 percent) and stromal cells in 52 cases (54 percent). Expression of matrix metalloproteinase-2 in cancer cells correlated with lymphatic invasion and local recurrence. Statistically, a significant correlation was found between cyclooxygenase-2 and membrane-type-1 matrix metalloproteinase expression, and membrane-type-1 matrix metalloproteinase and matrix metalloproteinase-2 expression in cancer cells. There was no association between cyclooxygenase-2 expression and matrix metalloproteinase-2 expression. However, immunostaining of serial sections revealed that in the majority of cases examined, nearly 100 percent of cancer cells expressing matrix metalloproteinase-2 also coexpressed cyclooxygenase-2. This study indicates strong association between both cyclooxygenase-2 and membrane-type-1 matrix metalloproteinase expression, and membrane-type-1 matrix metalloproteinase and matrix metalloproteinase-2 in colorectal

  5. FAK phosphorylation plays a central role in thrombin-induced RPE cell migration.

    Science.gov (United States)

    Aguilar-Solis, E D; Lee-Rivera, I; Álvarez-Arce, A; López, E; López-Colomé, A M

    2017-08-01

    The migration of retinal pigment epithelial (RPE) cells is an important step in various pathologic conditions including subretinal neovascularization (SRN), proliferative vitreoretinopathy (PVR) and, importantly, as a consequence of retinal surgery. Therefore, the elucidation of the mechanisms underlying RPE trans-differentiation and migration is essential for devising effective treatments aimed to the prevention of these disorders. A common event in these pathologies is the alteration of the blood-retina barrier (BRB), which allows the interaction of RPE cells with thrombin, a pro-inflammatory protease contained in serum. Our previous work has demonstrated that thrombin induces RPE cell cytoskeletal remodeling and migration, hallmark processes in the development of PVR; however, the molecular mechanisms involved are still unclear. Cell migration requires the disassembly of focal adhesions induced by Focal Adhesion Kinase (FAK) phosphorylation, together with the formation of actin stress fibers. The aim of the present work was to identify thrombin-activated signaling pathways leading to FAK phosphorylation and to determine FAK participation in thrombin-induced RPE cell migration. Results demonstrate that the activation of PAR1 by thrombin induces FAK autophosphorylation at Y397 and the subsequent phosphorylation of Y576/577 within the activation loop. FAK phosphorylation was shown to be under the control of c/nPKC and PI3K/PKC-ζ, as well as by Rho/ROCK, since the inhibition of these pathways prevented thrombin-induced FAK phosphorylation and the consequent disassembly of focal adhesions, in parallel to FAK-dependent actin stress fiber formation and RPE cell migration. These findings demonstrate, for the first time, that thrombin stimulation of RPE cell transformation and migration are regulated by FAK tyrosine phosphorylation. Thus, targeting FAK phosphorylation may provide a strategical basis for PVR treatment. Copyright © 2017. Published by Elsevier Inc.

  6. Phenylketonuria : tyrosine supplementation in phenylalanine-restricted diets

    NARCIS (Netherlands)

    van Spronsen, FJ; van Rijn, M; Bekhof, J; Koch, R; Smit, PGA

    Treatment of phenylketonuria (PKU) consists of restriction of natural protein and provision of a protein substitute that lacks phenylalanine but is enriched in tyrosine. Large and unexplained differences exist, however, in the tyrosine enrichment of the protein substitutes. Furthermore, some

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

    Science.gov (United States)

    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.

  8. Matrix metalloproteinase-8 overexpression prevents proper tissue repair

    DEFF Research Database (Denmark)

    Danielsen, Patricia L; Holst, Anders V; Maltesen, Henrik R

    2011-01-01

    The collagenolytic matrix metalloproteinase-8 (MMP-8) is essential for normal tissue repair but is often overexpressed in wounds with disrupted healing. Our aim was to study the impact of a local excess of this neutrophil-derived proteinase on wound healing using recombinant adenovirus-driven tra......The collagenolytic matrix metalloproteinase-8 (MMP-8) is essential for normal tissue repair but is often overexpressed in wounds with disrupted healing. Our aim was to study the impact of a local excess of this neutrophil-derived proteinase on wound healing using recombinant adenovirus...

  9. Neural Functions of Matrix Metalloproteinases: Plasticity, Neurogenesis, and Disease

    Directory of Open Access Journals (Sweden)

    Hiromi Fujioka

    2012-01-01

    Full Text Available The brain changes in response to experience and altered environment. To do that, the nervous system often remodels the structures of neuronal circuits. This structural plasticity of the neuronal circuits appears to be controlled not only by intrinsic factors, but also by extrinsic mechanisms including modification of the extracellular matrix. Recent studies employing a range of animal models implicate that matrix metalloproteinases regulate multiple aspects of the neuronal development and remodeling in the brain. This paper aims to summarize recent advances of our knowledge on the neuronal functions of matrix metalloproteinases and discuss how they might relate in neuronal disease.

  10. Overexpression of membrane sialic acid-specific sialidase Neu3 inhibits matrix metalloproteinase-9 expression in vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Moon, Sung-Kwon; Cho, Seung-Hak; Kim, Kyung-Woon; Jeon, Jae Heung; Ko, Jeong-Heon; Kim, Bo Yeon; Kim, Cheorl-Ho

    2007-01-01

    The ganglioside-specific sialidase Neu3 has been suggested to participate in cell growth, migration, and differentiation. Recent reports suggest that sialidase may be involved in intimal thickening, an early stage in the development of atherosclerosis. However, the role of the Neu3 gene in vascular smooth muscle cells (VSMC) responses has not yet been elucidated. To determine whether a Neu3 is able to modulate VSMC growth, the effect of overexpression of the Neu3 gene on cell proliferation was examined. However, the results show that the overexpression of this gene has no effect on DNA synthesis and ERK phosphorylation in cultured VSMC in the presence of TNF-α. Because atherogenic effects need not be limited to proliferation, we decided to examine whether Neu3 exerted inhibitory effects on matrix metalloproteinase-9 (MMP-9) activity in TNF-α-induced VSMC. The expression of the Neu3 gene led to the inhibition of TNF-α-induced matrix metalloproteinase-9 (MMP-9) expression in VSMC as determined by zymography and immunoblot. Furthermore, Neu3 gene expression strongly decreased MMP-9 promoter activity in response to TNF-α. This inhibition was characterized by the down-regulation of MMP-9, which was transcriptionally regulated at NF-κB and activation protein-1 (AP-1) sites in the MMP-9 promoter. These findings suggest that the Neu3 gene represents a physiological modulator of VSMC responses that may contribute to plaque instability in atherosclerosis

  11. Chlorinated tyrosine derivatives in insect cuticle

    DEFF Research Database (Denmark)

    Andersen, Svend Olav

    2004-01-01

    , not-yet sclerotized cuticle of adult femur and tibia, the amounts increased rapidly during the first 24 h after ecdysis and more slowly during the next two weeks. Control analyses using stable isotope dilution mass spectrometry have confirmed that the chlorinated tyrosines are not artifacts formed...

  12. Enzyme kinetic characterization of protein tyrosine phosphatases

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Branner, S.; Møller, K. B.

    2003-01-01

    Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta...

  13. Morphological Features of Tyrosine Hydroxylase Immunoreactive ...

    African Journals Online (AJOL)

    The current immunohistochemical study used the antibody against tyrosine hydroxylase (TH) to observe the immunoreactive elements in the mouse pancreas. The results indicated the presence of immunoreactive nerve fibers and endocrine cells. The immunopositive nerve fibers appeared as thick and thin bundles; thick ...

  14. Galangin and kaempferol suppress phorbol-12-myristate-13-acetate-induced matrix metalloproteinase-9 expression in human fibrosarcoma HT-1080 cells.

    Science.gov (United States)

    Choi, Yu Jung; Lee, Young Hun; Lee, Seung-Taek

    2015-01-01

    Matrix metalloproteinase (MMP)-9 degrades type IV collagen in the basement membrane and plays crucial roles in several pathological implications, including tumorigenesis and inflammation. In this study, we analyzed the effect of flavonols on MMP-9 expression in phorbol-12-myristate-13-acetate (PMA)-induced human fibrosarcoma HT-1080 cells. Galangin and kaempferol efficiently decreased MMP-9 secretion, whereas fisetin only weakly decreased its secretion. Galangin and kaempferol did not affect cell viability at concentrations up to 30 μM. Luciferase reporter assays showed that galangin and kaempferol decrease transcription of MMP-9 mRNA. Moreover, galangin and kaempferol strongly reduce IκBα phosphorylation and significantly decrease JNK phosphorylation. These results indicate that galangin and kaempferol suppress PMA-induced MMP-9 expression by blocking activation of NF-κB and AP-1. Therefore, these flavonols could be used as chemopreventive agents to lower the risk of diseases involving MMP-9.

  15. Phenylketonuria : Tyrosine beyond the phenylalanine-restricted diet

    NARCIS (Netherlands)

    van Spronsen, FJ; Smit, PGA; Koch, R

    Controversies exist on the role of tyrosine in the pathogenesis of phenylketonuria (PKU) and, consequently, on the therapeutic role of tyrosine. This review examines data and theoretical considerations on the role of tyrosine in the pathogenesis and treatment of PKU. It is concluded that treatment

  16. Requirements for superoxide-dependent tyrosine hydroperoxide formation in peptides

    DEFF Research Database (Denmark)

    Winterbourn, Christine C; Parsons-Mair, Helena N; Gebicki, Silvia

    2004-01-01

    requirements for hydroperoxide formation using tyrosine analogues and di- and tri-peptides. Superoxide and phenoxyl radicals were generated using xanthine oxidase, peroxidase and the respective tyrosine derivative, or by gamma-radiation. Peroxides were measured using FeSO4/Xylenol Orange. Tyrosine and tyramine...

  17. Detection of functional matrix metalloproteinases by zymography.

    Science.gov (United States)

    Hu, Xueyou; Beeton, Christine

    2010-11-08

    Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel. Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary

  18. Mimic Phosphorylation of a βC1 Protein Encoded by TYLCCNB Impairs Its Functions as a Viral Suppressor of RNA Silencing and a Symptom Determinant.

    Science.gov (United States)

    Zhong, Xueting; Wang, Zhan Qi; Xiao, Ruyuan; Cao, Linge; Wang, Yaqin; Xie, Yan; Zhou, Xueping

    2017-08-15

    Phosphorylation of the βC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNB-βC1) by SNF1-related protein kinase 1 (SnRK1) plays a critical role in defense of host plants against geminivirus infection in Nicotiana benthamiana However, how phosphorylation of TYLCCNB-βC1 impacts its pathogenic functions during viral infection remains elusive. In this study, we identified two additional tyrosine residues in TYLCCNB-βC1 that are phosphorylated by SnRK1. The effects of TYLCCNB-βC1 phosphorylation on its functions as a viral suppressor of RNA silencing (VSR) and a symptom determinant were investigated via phosphorylation mimic mutants in N. benthamiana plants. Mutations that mimic phosphorylation of TYLCCNB-βC1 at tyrosine 5 and tyrosine 110 attenuated disease symptoms during viral infection. The phosphorylation mimics weakened the ability of TYLCCNB-βC1 to reverse transcriptional gene silencing and to suppress posttranscriptional gene silencing and abolished its interaction with N. benthamiana ASYMMETRIC LEAVES 1 in N. benthamiana leaves. The mimic phosphorylation of TYLCCNB-βC1 had no impact on its protein stability, subcellular localization, or self-association. Our data establish an inhibitory effect of phosphorylation of TYLCCNB-βC1 on its pathogenic functions as a VSR and a symptom determinant and provide a mechanistic explanation of how SnRK1 functions as a host defense factor. IMPORTANCE Tomato yellow leaf curl China virus (TYLCCNV), which causes a severe yellow leaf curl disease in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). TYLCCNB encodes a single pathogenicity protein, βC1 (TYLCCNB-βC1), which functions as both a viral suppressor of RNA silencing (VSR) and a symptom determinant. Here, we show that mimicking phosphorylation of TYLCCNB-βC1 weakens its ability to reverse transcriptional gene silencing, to suppress posttranscriptional gene silencing, and to interact with N

  19. Protein tyrosine kinases p53/56lyn and p72syk in MHC class I-mediated signal transduction in B lymphoma cells

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Bregenholt, S; Skov, S

    1998-01-01

    syk are among the tyrosine-phosphorylated proteins. The kinetics of phosphorylation of these kinases after MHC-I crosslinking differ from the kinetics observed after crosslinking of the B cell antigen receptor (BCR). Additional experiments were performed with chicken lyn- and syk-negative DT40 B cells...... mobilization of intracellular free calcium compared with MHC-I crosslinking of wild-type DT40 cells. Thus, expression of BCR at the cell surface is likely to be important for the signal cascade initiated by MHC-I crosslinking. Our data suggest that signal transduction initiated through ligation of the MHC...

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  1. Phosphorylated nano-diamond/ Polyimide Nanocomposites

    International Nuclear Information System (INIS)

    Beyler-Çiǧil, Asli; Çakmakçi, Emrah; Kahraman, Memet Vezir

    2014-01-01

    In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased

  2. Single Tyrosine Mutation in AAV8 Vector Capsid Enhances Gene Lung Delivery and Does Not Alter Lung Morphofunction in Mice

    Directory of Open Access Journals (Sweden)

    Sabrina V. Martini

    2014-08-01

    Full Text Available Background/Aims: Vectors derived from adeno-associated viruses (AAVs are important gene delivery tools for treating pulmonary diseases. Phosphorylation of surface-exposed tyrosine residues from AAV2 capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. We evaluated the pulmonary transduction efficiency of AAV8 vectors containing point mutations in surface-exposed capsid tyrosine residues. Methods: Male C57BL/6 mice (20-25 g, n=24 were randomly assigned into three groups: control group animals received intratracheal (i.t. instillation of saline (50 μl, wild-type AAV8 group, and capsid mutant Y733F AAV8 group, which received (i.t. AAV8 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP. Four weeks after instillation, lung mechanics and morphometry, vector transduction (immunohistochemistry and mRNA expression of eGFP, and inflammatory cytokines and growth factor expression were analyzed. Results: Tyrosine-mutant AAV8 vectors displayed significantly increased transduction efficiency in the lung compared with their wild-type counterparts. No significant differences were observed in lung mechanics and morphometry between experimental groups. There was no evidence of inflammatory response in any group. Conclusion: AAV8 vectors may be useful for new therapeutic strategies for the treatment of pulmonary diseases.

  3. Design of a selective insulin receptor tyrosine kinase inhibitor and its effect on glucose uptake and metabolism in intact cells

    Energy Technology Data Exchange (ETDEWEB)

    Saperstein, R.; Vicario, P.P.; Strout, H.V.; Brady, E.; Slater, E.E.; Greenlee, W.J.; Onedyka, D.L.; Patchett, A.A.; Hangauer, D.G. (Merck Sharp and Dohme Research Labs., Rahway, NJ (USA))

    1989-06-27

    An inhibitor of the insulin receptor tyrosine kinase (IRTK), (hydroxy-2-napthalenylmethyl)phosphonic acid, was designed and synthesized and was shown to be an inhibitor of the biological effects of insulin in vitro. With a wheat germ purified human placental insulin receptor preparation, this compound inhibited the insulin-stimulated autophosphorylation of the 95-kDa {beta}-subunit of the insulin receptor. The ability of the kinase to phosphorylate an exogenous peptide substrate, angiotensin II, was also inhibited. Half-maximal inhibition of basal and insulin-stimulated human placental IRTK activity was found at concentrations of 150 and 100 {mu}M, respectively, with 2 mM angiotensin II as the peptide substrate. The inhibitor was found to be specific for tyrosine kinases over serine kinases and noncompetitive with ATP. The inhibitor was converted into various (acyloxy)methyl prodrugs in order to achieve permeability through cell membranes. These prodrugs inhibited insulin-stimulated autophosphorylation of the insulin receptor 95-kDa {beta}-subunit in intact CHO cells transfected with human insulin receptor. Inhibition of insulin-stimulated glucose oxidation in isolated rat adipocytes and 2-deoxyglucose uptake into CHO cells was observed with these prodrugs. The data provide additional evidence for the involvement of the insulin receptor tyrosine kinase in the regulation of glucose uptake and metabolism. These results and additional data reported herein suggest that this class of prodrugs and inhibitors will be useful for modulating the activity of a variety of tyrosine kinases.

  4. Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

    LENUS (Irish Health Repository)

    Meehan, Maria

    2012-02-05

    Abstract Background Protein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. Results As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA\\'s primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. Conclusions PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

  5. Inhibition of protein phosphatase 2A induces serine/threonine phosphorylation, subcellular redistribution, and functional inhibition of STAT3

    DEFF Research Database (Denmark)

    Woetmann, A; Nielsen, M; Christensen, S T

    1999-01-01

    STAT3. We show that an inhibitor of protein phosphatases (PPs) PP1/PP2A, calyculin A, induces (i) phosphorylation of STAT3 on serine and threonine residues, (ii) inhibition of STAT3 tyrosine phosphorylation and DNA binding activity, and (iii) relocation of STAT3 from the nucleus to the cytoplasm......, whereas inhibitors of serine/threonine kinases, such as mitogen-activated protein kinase-1 extracellular-regulated kinase-kinase, mitogen-activated protein p38 kinase, and phosphatidylinositol 3-kinase, did not. In conclusion, we provide evidence that PP2A plays a crucial role in the regulation of STAT3....... Similar results were obtained with other PP2A inhibitors (okadaic acid, endothall thioanhydride) but not with inhibitors of PP1 (tautomycin) or PP2B (cyclosporine A). Pretreatment with the broad serine/threonine kinase inhibitor staurosporine partly blocked the calyculin A-induced STAT3 phosphorylation...

  6. ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor

    Energy Technology Data Exchange (ETDEWEB)

    Inagaki, Junko; Takahashi, Katsuyuki; Ogawa, Hiroko; Asano, Keiichi; Faruk Hatipoglu, Omer; Zeynel Cilek, Mehmet; Obika, Masanari; Ohtsuki, Takashi [Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama (Japan); Hofmann, Matthias [Department of Dermatology, Venereology and Allergology, Goethe University, Frankfurt (Germany); Kusachi, Shozo [Department of Medical Technology, Okayama University Graduate School of Health Sciences, Okayama (Japan); Ninomiya, Yoshifumi [Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama (Japan); Hirohata, Satoshi, E-mail: hirohas@cc.okayama-u.ac.jp [Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama (Japan); International Center, Okayama University, Okayama (Japan)

    2014-05-01

    Angiogenesis and lymphangiogenesis play roles in malignant tumor progression, dissemination, and metastasis. ADAMTS1, a member of the matrix metalloproteinase family, is known to inhibit angiogenesis. Recombinant ADAMTS1 was shown to strongly inhibit angiogenesis. We investigated whether ADAMTS1 inhibited lymphangiogenesis in the present study. We examined cell proliferation and cell migration in normal human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) transduced with or without adenoviral human ADAMTS1 gene therapy. We then examined the VEGFC/VEGFR3 signal transduction pathway in ADAMTS1-transduced HMVEC-dLy. Cell proliferation and tube formation in Matrigel were significantly lower with transduced ADAMTS1 than with control (non-transduced HMVEC-dLy). The phosphorylation of VEGFR3 was also attenuated by ADAMTS1 gene therapy in HMVEC-dLy. Immunoprecipitation assays revealed that ADAMTS1 formed a complex with VEGFC. Our results demonstrated that ADAMTS1 inhibited lymphangiogenesis in vitro. The data highlight the new function of ADAMTS1 in the regulation of lymphangiogenesis and the therapeutic potential of ADAMTS1 in cancer therapy. - Highlights: • ADAMTS1 significantly inhibited tube formation and cell proliferation in HMVEC-dLy. • Reduced lymph endothelial cell migration in ADAMTS1 transduced co-culture systems. • VEGFC-stimulated phosphorylation of VEGFR3 is attenuated by ADAMTS1. • Reduced phosphorylation of Akt and ERK1/2 in ADAMTS1 treated HMVEC-dLy. • ADAMTS1 binds directly to VEGFC.

  7. Plasma tissue inhibitor of metalloproteinases-1 as a biological marker?

    DEFF Research Database (Denmark)

    Lomholt, Anne F.; Frederiksen, Camilla B.; Christensen, Ib J.

    2007-01-01

    Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) may be a valuable biological marker in Colorectal Cancer (CRC). However, prospective validation of TIMP-1 as a biological marker should include a series of pre-analytical considerations. TIMP-1 is stored in platelets, which may degranulate during...

  8. Venous aneurysm complicating arteriovenous fistula access and matrix metalloproteinases

    Directory of Open Access Journals (Sweden)

    Serra Raffaele

    2015-01-01

    Full Text Available Introduction: An arteriovenous fistula (AVF for placed for hemodialysis may be burdened by one particular complication-the formation of a venous aneurysm. It has been shown that matrix metalloproteinases (MMPs and neutrophil gelatinase-associated lipocalin (NGAL could represent markers of disease in both venous and arterial vessels.

  9. Snake venom metalloproteinases and disintegrins: interactions with cells

    Directory of Open Access Journals (Sweden)

    Kamiguti A.S.

    1998-01-01

    Full Text Available Metalloproteinases and disintegrins are important components of most viperid and crotalid venoms. Large metalloproteinases referred to as MDC enzymes are composed of an N-terminal Metalloproteinase domain, a Disintegrin-like domain and a Cys-rich C-terminus. In contrast, disintegrins are small non-enzymatic RGD-containing cysteine-rich polypeptides. However, the disintegrin region of MDC enzymes bears a high degree of structural homology to that of the disintegrins, although it lacks the RGD motif. Despite these differences, both components share the property of being able to recognize integrin cell surface receptors and thereby to inhibit integrin-dependent cell reactions. Recently, several membrane-bound MDC enzymes, closely related to soluble venom MDC enzymes, have been described in mammalian cells. This group of membrane-anchored mammalian enzymes is also called the ADAM family of proteins due to the structure revealing A Disintegrin And Metalloproteinase domains. ADAMs are involved in the shedding of molecules from the cell surface, a property which is also shared by some venom MDC enzymes.

  10. Role of matrix metalloproteinases in recurrent corneal melting

    Czech Academy of Sciences Publication Activity Database

    Brejchová, K.; Lisková, P.; Čejková, Jitka; Jirsová, K.

    2010-01-01

    Roč. 90, č. 5 (2010), s. 583-590 ISSN 0014-4835 Institutional research plan: CEZ:AV0Z50390512 Keywords : corneal melting * extracellular matrix degradation * matrix metalloproteinases Subject RIV: FF - HEENT, Dentistry Impact factor: 2.817, year: 2010

  11. Involvement of matrix metalloproteinases in chronic Q fever

    NARCIS (Netherlands)

    Jansen, A.F.M.; Schoffelen, T.; Textoris, J.; Mege, J.L.; Bleeker-Rovers, C.P.; Roest, H.I.J.; Wever, P.C.; Joosten, L.A.B.; Netea, M.G.; Vosse, van de E.; Deuren, van M.

    2017-01-01

    Objectives: Chronic Q fever is a persistent infection with the intracellular Gram-negative bacterium Coxiella burnetii, which can lead to complications of infected aneurysms. Matrix metalloproteinases (MMPs) cleave extracellular matrix and are involved in infections as well as aneurysms. We aimed to

  12. Inhibition of matrix metalloproteinase-14 in osteosarcoma cells by clodronate

    NARCIS (Netherlands)

    Heikkilä, P.; Teronen, O.; Hirn, M.Y.; Sorsa, T.; Tervahartiala, T.; Salo, T.; Konttinen, Y.T.; Halttunen, T.; Moilanen, M.; Hanemaaijer, R.; Laitinen, M.

    2003-01-01

    Background. Bisphosphonates reduce the bone metastasis formation and angiogenesis but the exact molecular mechanisms involved are unclear. Progelatinase A (proMMP-2; 78 KDa) is activated up during the tumor spread and metastasis by a cell surface-associated matrix metalloproteinase (membrane-type

  13. Identification of BCAP-{sub L} as a negative regulator of the TLR signaling-induced production of IL-6 and IL-10 in macrophages by tyrosine phosphoproteomics

    Energy Technology Data Exchange (ETDEWEB)

    Matsumura, Takayuki [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Oyama, Masaaki; Kozuka-Hata, Hiroko [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Ishikawa, Kosuke; Inoue, Takafumi [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Muta, Tatsushi [Laboratory of Cell Recognition and Response, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578 (Japan); Semba, Kentaro, E-mail: ksemba@waseda.jp [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Inoue, Jun-ichiro, E-mail: jun-i@ims.u-tokyo.ac.jp [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan)

    2010-09-17

    Research highlights: {yields} Twenty five tyrosine-phosphorylated proteins in LPS-stimulated macrophages were determined. {yields} BCAP is a novel tyrosine-phosphorylated protein in LPS-stimulated macrophages. {yields} BCAP-{sub L} inhibits IL-6 and IL-10 production in LPS-stimulated macrophages. -- Abstract: Toll-like receptor (TLR) signaling in macrophages is essential for anti-pathogen responses such as cytokine production and antigen presentation. Although numerous reports suggest that protein tyrosine kinases (PTKs) are involved in cytokine induction in response to lipopolysaccharides (LPS; TLR4 ligand) in macrophages, the PTK-mediated signal transduction pathway has yet to be analyzed in detail. Here, we carried out a comprehensive and quantitative dynamic tyrosine phosphoproteomic analysis on the TLR4-mediated host defense system in RAW264.7 macrophages using stable isotope labeling by amino acids in cell culture (SILAC). We determined the temporal profiles of 25 proteins based on SILAC-encoded peptide(s). Of these, we focused on the tyrosine phosphorylation of B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) because the function of BCAP remains unknown in TLR signaling in macrophages. Furthermore, Bcap has two distinct transcripts, a full-length (Bcap-{sub L}) and an alternatively initiated or spliced (Bcap-{sub S}) mRNA, and little is known about the differential functions of the BCAP-{sub L} and BCAP-{sub S} proteins. Our study showed, for the first time, that RNAi-mediated selective depletion of BCAP-{sub L} enhanced IL-6 and IL-10 production but not TNF-{alpha} production in TLR ligand-stimulated macrophages. We propose that BCAP-{sub L} (but not BCAP-{sub S}) is a negative regulator of the TLR-mediated host defense system in macrophages.

  14. A cross-talk between TrkB and Ret tyrosine kinases receptors mediates neuroblastoma cells differentiation.

    Directory of Open Access Journals (Sweden)

    Carla Lucia Esposito

    Full Text Available Understanding the interplay between intracellular signals initiated by multiple receptor tyrosine kinases (RTKs to give the final cell phenotype is a major pharmacological challenge. Retinoic acid (RA-treatment of neuroblastoma (NB cells implicates activation of Ret and TrkB RTKs as critical step to induce cell differentiation. By studying the signaling interplay between TrkB and Ret as paradigmatic example, here we demonstrate the existence of a cross-talk mechanism between the two unrelated receptors that is needed to induce the cell differentiation. Indeed, we show that TrkB receptor promotes Ret phosphorylation by a mechanism that does not require GDNF. This reveals to be a key mechanism, since blocking either TrkB or Ret by small interfering RNA causes a failure in NB biochemical and morphological differentiation. Our results provide the first evidence that a functional transactivation between distinct tyrosine kinases receptors is required for an important physiological process.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    Naudin, Cécile; Chevalier, Clément; Roche, Serge

    2016-03-08

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

  17. Aspirin Inhibits IKK-β-mediated Prostate Cancer Cell Invasion by Targeting Matrix Metalloproteinase-9 and Urokinase-Type Plasminogen Activator.

    Science.gov (United States)

    Shi, Chongjun; Zhang, Nini; Feng, Yang; Cao, Jiewei; Chen, Xuyi; Liu, Bin

    2017-01-01

    Aspirin has been demonstrated to possess potent chemopreventive and anticancer effects on prostate cancer. However, the more detailed molecular mechanisms of aspirin to suppress prostate cancer cell invasion have not been clearly elucidated. Transwell assays were performed to evaluate the effects of aspirin on cell invasion. Matrix metalloproteinases (MMPs) and serine proteinases activities in cell media were examined by gelatin zymography and ELISA. In addition, inhibitor of κB (IκB) kinase-β (IKK-β) phosphorylation and IKK-β kinase activity were measured to assess the effects of aspirin on IKK-β activation. We found that aspirin suppressed the invasion and attachment in human prostate cancer cells. Aspirin treatment significantly resulted in reduction of matrix metalloproteinase-9 (MMP-9) and upregulation of tissue inhibitors of metalloproteinase-1 (TIMP-1) activity, which are the proteolytic enzymes contributing to the degradation of extracellular matrix and basement membrane in cell invasion and metastasis. Our data further showed that aspirin was able to inhibit both urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) expression in the cells. In addition, aspirin treatment caused a strong decrease in nuclear factor-kappa B (NF-κB) activation, inhibitor of κB (IκB)-α phosphorylation together with translocation of NF-κB p65 to nucleus and IκB kinase (IKK)- β activation. Moreover, the inhibitory effects of aspirin on cell invasion were reversed by IKK-β overexpression, while the IKK inhibitor sensitizes the anti-invasive effect of aspirin in prostate cancer cells. The present research concluded that aspirin suppressed prostate cancer cell invasion by reducing MMP-9 activity and uPA expression through decreasing of IKK-β-mediated NF-κB activation, indicating that the ability of aspirin to inhibit cell invasion might be useful in the chemoprevention of metastatic prostate cancer. © 2017 The Author(s)Published by

  18. Aspirin Inhibits IKK-β-mediated Prostate Cancer Cell Invasion by Targeting Matrix Metalloproteinase-9 and Urokinase-Type Plasminogen Activator

    Directory of Open Access Journals (Sweden)

    Chongjun Shi

    2017-03-01

    Full Text Available Background/Aims: Aspirin has been demonstrated to possess potent chemopreventive and anticancer effects on prostate cancer. However, the more detailed molecular mechanisms of aspirin to suppress prostate cancer cell invasion have not been clearly elucidated. Methods: Transwell assays were performed to evaluate the effects of aspirin on cell invasion. Matrix metalloproteinases (MMPs and serine proteinases activities in cell media were examined by gelatin zymography and ELISA. In addition, inhibitor of κB (IκB kinase-β (IKK-β phosphorylation and IKK-β kinase activity were measured to assess the effects of aspirin on IKK-β activation. Results: We found that aspirin suppressed the invasion and attachment in human prostate cancer cells. Aspirin treatment significantly resulted in reduction of matrix metalloproteinase-9 (MMP-9 and upregulation of tissue inhibitors of metalloproteinase-1 (TIMP-1 activity, which are the proteolytic enzymes contributing to the degradation of extracellular matrix and basement membrane in cell invasion and metastasis. Our data further showed that aspirin was able to inhibit both urokinase-type plasminogen activator (uPA and plasminogen activator inhibitor-1 (PAI-1 expression in the cells. In addition, aspirin treatment caused a strong decrease in nuclear factor-kappa B (NF-κB activation, inhibitor of κB (IκB-α phosphorylation together with translocation of NF-κB p65 to nucleus and IκB kinase (IKK- β activation. Moreover, the inhibitory effects of aspirin on cell invasion were reversed by IKK-β overexpression, while the IKK inhibitor sensitizes the anti-invasive effect of aspirin in prostate cancer cells. Conclusion: The present research concluded that aspirin suppressed prostate cancer cell invasion by reducing MMP-9 activity and uPA expression through decreasing of IKK-β-mediated NF-κB activation, indicating that the ability of aspirin to inhibit cell invasion might be useful in the chemoprevention of

  19. Inhibition of matrix metalloproteinase 14 (MMP-14)-mediated cancer cell migration.

    Science.gov (United States)

    Zarrabi, Kevin; Dufour, Antoine; Li, Jian; Kuscu, Cem; Pulkoski-Gross, Ashleigh; Zhi, Jizu; Hu, Youjun; Sampson, Nicole S; Zucker, Stanley; Cao, Jian

    2011-09-23

    Matrix metalloproteinases (MMPs) have been shown to be key players in both extracellular matrix remodeling and cell migration during cancer metastasis. MMP-14, a membrane-anchored MMP, in particular, is closely associated with these processes. The hemopexin (PEX) domain of MMP-14 has been proposed as the modulating region involved in the molecular cross-talk that initiates cell migration through homodimerization of MMP-14 as well as heterodimerization with the cell surface adhesion molecule CD44. In this study, minimal regions required for function within the PEX domain were investigated through a series of substitution mutations. Blades I and IV were found to be involved in cell migration. We found that blade IV is necessary for MMP-14 homodimerization and that blade I is required for CD44 MMP-14 heterodimerization. Cross-talk between MMP-14 and CD44 results in phosphorylation of EGF receptor and downstream activation of the MAPK and PI3K signaling pathways involved in cell migration. Based on these mutagenesis analyses, peptides mimicking the essential outermost strand motifs within the PEX domain of MMP-14 were designed. These synthetic peptides inhibit MMP-14-enhanced cell migration in a dose-dependent manner but have no effect on the function of other MMPs. Furthermore, these peptides interfere with cancer metastasis without affecting primary tumor growth. Thus, targeting the MMP-14 hemopexin domain represents a novel approach to inhibit MMP-14-mediated cancer dissemination.

  20. Inhibition of Matrix Metalloproteinase 14 (MMP-14)-mediated Cancer Cell Migration*

    Science.gov (United States)

    Zarrabi, Kevin; Dufour, Antoine; Li, Jian; Kuscu, Cem; Pulkoski-Gross, Ashleigh; Zhi, Jizu; Hu, Youjun; Sampson, Nicole S.; Zucker, Stanley; Cao, Jian

    2011-01-01

    Matrix metalloproteinases (MMPs) have been shown to be key players in both extracellular matrix remodeling and cell migration during cancer metastasis. MMP-14, a membrane-anchored MMP, in particular, is closely associated with these processes. The hemopexin (PEX) domain of MMP-14 has been proposed as the modulating region involved in the molecular cross-talk that initiates cell migration through homodimerization of MMP-14 as well as heterodimerization with the cell surface adhesion molecule CD44. In this study, minimal regions required for function within the PEX domain were investigated through a series of substitution mutations. Blades I and IV were found to be involved in cell migration. We found that blade IV is necessary for MMP-14 homodimerization and that blade I is required for CD44 MMP-14 heterodimerization. Cross-talk between MMP-14 and CD44 results in phosphorylation of EGF receptor and downstream activation of the MAPK and PI3K signaling pathways involved in cell migration. Based on these mutagenesis analyses, peptides mimicking the essential outermost strand motifs within the PEX domain of MMP-14 were designed. These synthetic peptides inhibit MMP-14-enhanced cell migration in a dose-dependent manner but have no effect on the function of other MMPs. Furthermore, these peptides interfere with cancer metastasis without affecting primary tumor growth. Thus, targeting the MMP-14 hemopexin domain represents a novel approach to inhibit MMP-14-mediated cancer dissemination. PMID:21795678

  1. Oxidative Stress-Associated Protein Tyrosine Kinases and Phosphatases in Fanconi Anemia

    Science.gov (United States)

    Pang, Qishen

    2014-01-01

    Abstract Significance: Fanconi anemia (FA) is a genetic disorder featuring chromosomal instability, developmental defects, progressive bone marrow failure, and predisposition to cancer. Besides the predominant role in DNA damage response and/or repair, many studies have linked FA proteins to oxidative stress. Oxidative stress, defined as imbalance in pro-oxidant and antioxidant homeostasis, has been considered to contribute to disease development, including FA. Recent Advances: A variety of signaling pathways may be influenced by oxidative stress, particularly the equilibrium between protein kinases and phosphatases, consequently leading to an aberrant phosphorylation state of cellular proteins. Dysfunction of kinases/phosphatases has been implicated in the pathophysiology of human diseases. In FA, evidence is emerging that links abnormal phosphorylation/de-phosphorylation of signaling molecules to clinical complications and malformations. Critical Issues: In this study, we review the recent findings on the oxidative stress-related kinases and phosphatases, particularly tyrosine phosphatases in FA. Future Directions: Understanding the role of oxidative stress-related kinases and phosphatases in FA may provide unique and generic possibilities for the future development of therapeutic strategies by targeting the dysregulated protein kinases and phosphatases in a clinical setting. Antioxid. Redox Signal. 20, 2290–2301. PMID:24206276

  2. Peripheral Lipopolysaccharide Challenge Induces Long-Term Changes in Tyrosine Hydroxylase Regulation in the Adrenal Medulla.

    Science.gov (United States)

    Ong, Lin Kooi; Page, Scott; Briggs, Gabrielle D; Guan, Liying; Dun, Matthew D; Verrills, Nicole M; Dunkley, Peter R; Dickson, Phillip W

    2017-08-01

    Immune activation can alter the activity of adrenal chromaffin cells. The effect of immune activation by lipopolysaccharide (LPS) on the regulation of tyrosine hydroxylase (TH) in the adrenal medulla in vivo was determined between 1 day and 6 months after LPS injection. The plasma levels of eleven cytokines were reduced 1 day after LPS injection, whereas the level for interleukin-10 was increased. The levels of all cytokines remained at control levels until 6 months when the levels of interleukin-6 and -4 were increased. One day after LPS injection, there was a decrease in TH-specific activity that may be due to decreased phosphorylation of serine 31 and 40. This decreased phosphorylation of serine 31 and 40 may be due to an increased activation of the protein phosphatase PP2A. One week after LPS injection, there was increased TH protein and increased phosphorylation of serine 40 that this was not accompanied by an increase in TH-specific activity. All TH parameters measured returned to basal levels between 1 month and 3 months. Six months after injection there was an increase in TH protein. This was associated with increased levels of the extracellular regulated kinase isoforms 1 and 2. This work shows that a single inflammatory event has the capacity to generate both short-term and long-term changes in TH regulation in the adrenal medulla of the adult animal. J. Cell. Biochem. 118: 2096-2107, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

    Directory of Open Access Journals (Sweden)

    Rodrigo A Silva

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

  4. Protein phosphorylations in poliovirus infected cells.

    Science.gov (United States)

    James, L A; Tershak, D R

    1981-01-01

    In vivo phosphorylation of proteins that are associated with polysomes of poliovirus-infected VERO (African green monkey kidney) and HeLa (Henrietta Lacks) cells differed from phosphorylations observed with uninfected cells that were fed fresh medium. With both types of cells infection stimulated phosphorylation of proteins with molecular weights of 40 000-41 000, 39 000, 34 000, 32 000, and 24 000. Similarities of phosphorylations in VERO and HeLa cells suggest that they are a specific consequence of infection and might serve a regulatory function during protein synthesis.

  5. Ror receptor tyrosine kinases: orphans no more.

    Science.gov (United States)

    Green, Jennifer L; Kuntz, Steven G; Sternberg, Paul W

    2008-11-01

    Receptor tyrosine kinase-like orphan receptor (Ror) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including skeletal and neuronal development, cell movement and cell polarity. Although Ror proteins were originally named because the associated ligand and signaling pathway were unknown, recent studies in multiple species have now established that Ror proteins are Wnt receptors. Depending on the cellular context, Ror proteins can either activate or repress transcription of Wnt target genes and can modulate Wnt signaling by sequestering Wnt ligands. New evidence implicates Ror proteins in planar cell polarity, an alternative Wnt pathway. Here, we review the progress made in understanding these mysterious proteins and, in particular, we focus on their function as Wnt receptors.

  6. Analysis of tyrosine-O-sulfation

    DEFF Research Database (Denmark)

    Bundgaard, J.R.; Sen, J.W.; Johnsen, A.H.

    2008-01-01

    Tyrosine O-sulfation was first described about 50 years ago as a post-translational modification of fibrinogen. In the following 30 years it was considered to be a rare modification affecting only a few proteins and peptides. However, in the beginning of the 1980s tyrosine (Tyr) sulfation was shown...... to be a common modification and since then an increasing number of proteins have been identified as sulfated. The target proteins belong to the classes of secretory, plasma membrane, and lysosomal proteins, which reflects the intracellular localization of the enzymes catalyzing Tyr sulfation, the tyrosylprotein...... sulfotransferases (TPSTs).Traditionally, Tyr sulfation has been analyzed by incorporation of radiolabeled sulfate into target cells followed by purification of the target protein. Subsequently, the protein is degraded enzymatically or by alkaline hydrolysis followed by thin-layer electrophoresis to demonstrate...

  7. Analysis of tyrosine-O-sulfation

    DEFF Research Database (Denmark)

    Bundgaard, J.R.; Sen, J.W.; Johnsen, A.H.

    2008-01-01

    to be a common modification and since then an increasing number of proteins have been identified as sulfated. The target proteins belong to the classes of secretory, plasma membrane, and lysosomal proteins, which reflects the intracellular localization of the enzymes catalyzing Tyr sulfation, the tyrosylprotein......Tyrosine O-sulfation was first described about 50 years ago as a post-translational modification of fibrinogen. In the following 30 years it was considered to be a rare modification affecting only a few proteins and peptides. However, in the beginning of the 1980s tyrosine (Tyr) sulfation was shown...... sulfotransferases (TPSTs).Traditionally, Tyr sulfation has been analyzed by incorporation of radiolabeled sulfate into target cells followed by purification of the target protein. Subsequently, the protein is degraded enzymatically or by alkaline hydrolysis followed by thin-layer electrophoresis to demonstrate...

  8. Protein kinase C-dependent dephosphorylation of tyrosine hydroxylase requires the B56δ heterotrimeric form of protein phosphatase 2A.

    Directory of Open Access Journals (Sweden)

    Jung-Hyuck Ahn

    Full Text Available Tyrosine hydroxylase, which plays a critical role in regulation of dopamine synthesis, is known to be controlled by phosphorylation at several critical sites. One of these sites, Ser40, is phosphorylated by a number of protein kinases, including protein kinase A. The major protein phosphatase that dephosphorylates Ser40 is protein phosphatase-2A (PP2A. A recent study has also linked protein kinase C to the dephosphorylation of Ser40 [1], but the mechanism is unclear. PP2A isoforms are comprised of catalytic, scaffold, and regulatory subunits, the regulatory B subunits being able to influence cellular localization and substrate selection. In the current study, we find that protein kinase C is able to phosphorylate a key regulatory site in the B56δ subunit leading to activation of PP2A. In turn, activation of the B56δ-containing heterotrimeric form of PP2A is responsible for enhanced dephosphorylation of Ser40 of tyrosine hydroylase in response to stimulation of PKC. In support of this mechanism, down-regulation of B56δ expression in N27 cells using RNAi was found to increase dopamine synthesis. Together these studies reveal molecular details of how protein kinase C is linked to reduced tyrosine hydroxylase activity via control of PP2A, and also add to the complexity of protein kinase/protein phosphatase interactions.

  9. Tyrosine nitration affects thymidylate synthase properties.

    Science.gov (United States)

    Dąbrowska-Maś, Elżbieta; Frączyk, Tomasz; Ruman, Tomasz; Radziszewska, Karolina; Wilk, Piotr; Cieśla, Joanna; Zieliński, Zbigniew; Jurkiewicz, Agata; Gołos, Barbara; Wińska, Patrycja; Wałajtys-Rode, Elżbieta; Leś, Andrzej; Nizioł, Joanna; Jarmuła, Adam; Stefanowicz, Piotr; Szewczuk, Zbigniew; Rode, Wojciech

    2012-01-14

    Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.

  10. Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930.

    Science.gov (United States)

    Petti, Filippo; Thelemann, April; Kahler, Jen; McCormack, Siobhan; Castaldo, Linda; Hunt, Tony; Nuwaysir, Lydia; Zeiske, Lynn; Haack, Herbert; Sullivan, Laura; Garton, Andrew; Haley, John D

    2005-08-01

    OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3' kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non-receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed. Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell

  11. Integration of Receptor Tyrosine Kinases Determines Sensitivity to PI3Kα-selective Inhibitors in Breast Cancer.

    Science.gov (United States)

    Xu, Yi-Chao; Wang, Xiang; Chen, Yi; Chen, Si-Meng; Yang, Xin-Ying; Sun, Yi-Ming; Geng, Mei-Yu; Ding, Jian; Meng, Ling-Hua

    2017-01-01

    PI3Kα-selective inhibitor BYL719 is currently in phase II/III clinical trial for the treatment of breast cancer, but highly variable response has been observed among patients. We sought to discover predictive biomarker for the efficacy of BYL719 by dissecting the proliferative signaling pathway mediated by PI3K in breast cancer. BYL719 concurrently inhibited the phosphorylation of AKT and ERK in PIK3CA -mutated human breast cancer cells. PI3K-regulated ERK phosphorylation was independent of canonical PDK1/AKT/mTOR pathway, while it was associated with RAF/MEK. Hyper-activation of EGFR or RAS abrogated inhibition of ERK phosphorylation by BYL719. Furthermore, hyper-activation of receptor tyrosine kinases (RTKs) including EGFR, c-MET, FGFR and HER3 but not IGF-1R restored ERK phosphorylation and cell viability suppressed by BYL719, suggesting the discriminative functions of RTKs in cell signaling and proliferation. By profiling 22 breast cancer cell lines, we found that BYL719 was more potent in cell lines where phosphorylation of both AKT and ERK was attenuated than those where only AKT phosphorylation was inhibited. The potency of BYL719 was further found to be significantly correlated with the expression profile of RTKs in breast cancer cells. Specifically, overexpression of EGFR, c-MET and/or FGFR1 forecasted resistance, while overexpression of IGF-1R and/or HER2 predicted sensitivity to BYL719 in breast cancer cells. Similar correlation between BYL719 efficacy and expression profile of RTKs was found in patient-derived xenograft models of breast cancer. Thus, inhibition of ERK phosphorylation by PI3Kα inhibitor BYL719 contributes to its antitumor efficacy and is determined by the converged signaling from RTKs. The expression profile of RTKs in breast cancer tissue could be potentially developed as a predictive biomarker for the efficacy of PI3Kα inhibitors.

  12. TIMP-1 increases expression and phosphorylation of proteins associated with drug resistance in breast cancer cells

    DEFF Research Database (Denmark)

    Hekmat, Omid; Munk, Stephanie; Fogh, Louise

    2013-01-01

    spectrometry to analyze global proteome and phosphoproteome differences of MCF-7 breast cancer cells expressing high or low levels of TIMP-1. In TIMP-1 high expressing cells, 312 proteins and 452 phosphorylation sites were up-regulated. Among these were the cancer drug targets topoisomerase 1, 2A and 2B, which......Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a protein with a potential biological role in drug resistance. To elucidate the unknown molecular mechanisms underlying the association between high TIMP-1 levels and increased chemotherapy resistance, we employed SILAC-based quantitative mass...... may explain the resistance phenotype to topoisomerase inhibitors that was observed in cells with high TIMP-1 levels. Pathway analysis showed an enrichment of proteins from functional categories such as apoptosis, cell cycle, DNA repair, transcription factors, drug targets and proteins associated...

  13. Matrix metalloproteinase-7 and matrix metalloproteinase-25 in oral tongue squamous cell carcinoma.

    Science.gov (United States)

    Mäkinen, Laura K; Häyry, Valtteri; Hagström, Jaana; Sorsa, Timo; Passador-Santos, Fabricio; Keski-Säntti, Harri; Haukka, Jari; Mäkitie, Antti A; Haglund, Caj; Atula, Timo

    2014-12-01

    Predicting the clinical course of early-stage oral tongue squamous cell carcinoma (SCC) is challenging. As matrix metalloproteinases (MMPs) are enzymes associated with invasion, metastasis, and poor survival in many cancers, we examined MMP-7 and MMP-25 in oral tongue SCC. We used tissue microarray (TMA) technique and immunohistochemistry to study the expression of MMP-7 and MMP-25 in 73 patients with stage I to II oral tongue SCC and compared their immunoexpressions with clinical data. Immunohistochemistry revealed MMP-7 and MMP-25 expression in 90% (n = 63 of 70) and 90% (n = 64 of 71) of the tumors, respectively. MMP-7 protein expression was associated with presence of occult cervical metastases (odds ratio [OR], 3.67; p = .013), increased invasion depth (OR, 4.60; p = .005), and higher tumor grade (OR, 3.30; p = .007). MMP-7 expression was predictive for poor outcome (p = .021). Immunostaining of MMP-25 did not correlate with any clinical parameters. We conclude that MMP-7, but not MMP-25, expression may have prognostic significance in early-stage oral tongue SCC. © 2014 Wiley Periodicals, Inc.

  14. SIMAC - A phosphoproteomic strategy for the rapid separation of mono-phosphorylated from multiply phosphorylated peptides

    DEFF Research Database (Denmark)

    Thingholm, Tine E; Jensen, Ole N; Robinson, Phillip J

    2008-01-01

    spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy - SIMAC - for sequential separation of mono-phosphorylated peptides and multiply phosphorylated peptides from...... and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a three-fold increase in recovery of multiply phosphorylated peptides....

  15. Bruton's tyrosine kinase is essential for hydrogen peroxide-induced calcium signaling.

    Science.gov (United States)

    Qin, S; Chock, P B

    2001-07-10

    Using Btk-deficient DT40 cells and the transfectants expressing wild-type Btk or Btk mutants in either kinase (Arg(525) to Gln), Src homology 2 (SH2, Arg(307) to Ala), or pleckstrin homology (PH, Arg(28) to Cys) domains, we investigated the roles and structure-function relationships of Btk in hydrogen peroxide-induced calcium mobilization. Our genetic evidence showed that Btk deficiency resulted in a significant reduction in hydrogen peroxide-induced calcium response. This impaired calcium signaling is correlated with the complete elimination of IP3 production and the significantly reduced tyrosine phosphorylation of PLCgamma2 in Btk-deficient DT40 cells. All of these defects were fully restored by the expression of wild-type Btk in Btk-deficient DT40 cells. The data from the point mutation study revealed that a defect at any one of the three functional domains would prevent a full recovery of Btk-mediated hydrogen peroxide-induced intracellular calcium mobilization. However, mutation at either the SH2 or PH domain did not affect the hydrogen peroxide-induced activation of Btk. Mutation at the SH2 domain abrogates both IP3 generation and calcium release, while the mutant with the nonfunctional PH domain can partially activate PLCgamma2 and catalyze IP3 production but fails to produce significant calcium mobilization. Thus, these observations suggest that Btk-dependent tyrosine phosphorylation of PLCgamma2 is required but not sufficient for hydrogen peroxide-induced calcium mobilization. Furthermore, hydrogen peroxide stimulates a Syk-, but not Btk-, dependent tyrosine phosphorylation of B cell linker protein BLNK. The overall results, together with those reported earlier [Qin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 7118], are consistent with the notion that functional SH2 and PH domains are required for Btk to form a complex with PLCgamma2 through BLNK in order to position the Btk, PLCgamma2, and phosphatidylinositol 4,5-bisphosphate in close proximity for

  16. Platelet-activating factor stimulation of tyrosine kinase and its relationship to phospholipase C in rabbit platelets: Studies with genistein and monoclonal antibody to phosphotyrosine

    International Nuclear Information System (INIS)

    Dhar, A.; Paul, A.K.; Shukla, S.D.

    1990-01-01

    Platelet-activating factor (PAF) is a proinflammatory lipid that has platelet-stimulating property. PAF receptor-coupled activation of phosphoinositide-specific phospholipase C (PLC) and phosphorylation of several proteins has already been established in our laboratory. To investigate further the molecular mechanism and relationship between activation of PLC and protein phosphorylation, we have used Genistein (a putative inhibitor of tyrosine-specific protein kinases), phosphotyrosine antibody, and phosphoamino acid analysis to probe the involvement of tyrosine kinase in this process. Washed rabbit platelets were loaded with myo-[2-3H]inositol and challenged with PAF (100 nM) after pretreatment with Genistein. PLC-mediated production of radioactive inositol monophosphate, inositol diphosphate, and inositol triphosphate was monitored. PAF alone caused stimulation of PLC activity [( 3H]inositol triphosphate production), whereas pretreatment with Genistein (0.5 mM) diminished PAF-stimulated PLC activity to basal level. Genistein also blocked PAF-stimulated platelet aggregation at this dose. In contrast to Genistein, staurosporine which inhibits protein kinase C, potentiated PAF-stimulated [3H]inositol triphosphate production. Genistein substantially inhibited the combined effects of staurosporine and PAF on inositol triphosphate production. Genistein also reduced PAF-induced phosphorylation of Mr 20,000 and 50,000 proteins. Phorbol 12-myristate 13-acetate-induced Mr 40,000 protein phosphorylation was also affected by Genistein. The above results suggested that Genistein inhibited tyrosine kinase at an early stage of signal transduction by inhibiting PLC. This, in turn, decreased the activation of protein kinase C and, therefore, caused a reduction in Mr 40,000 protein phosphorylation

  17. Phosphorylation of FOXP3 by LCK downregulates MMP9 expression and represses cell invasion.

    Directory of Open Access Journals (Sweden)

    Kumiko Nakahira

    Full Text Available Forkhead Box P3 (FOXP3 is a member of the forkhead/winged helix family of the transcription factors and plays an important role not only as a master gene in T-regulatory cells, but also as a tumor suppressor. In this study, we identified lymphocyte-specific protein tyrosine kinase (LCK, which correlates with cancer malignancy, as a binding partner of FOXP3. FOXP3 downregulated LCK-induced MMP9, SKP2, and VEGF-A expression. We observed that LCK phosphorylated Tyr-342 of FOXP3 by immunoprecipitation and in vitro kinase assay, and the replacement of Tyr-342 with phenylalanine (Y342F abolished the ability to suppress MMP9 expression. Although FOXP3 decreased the invasive ability induced by LCK in MCF-7 cells, Y342F mutation in FOXP3 diminished this suppressive effect. Thus we demonstrate for the first time that LCK upregulates FOXP3 by tyrosine phosphorylation, resulting in decreased MMP9, SKP2, and VEGF-A expression, and suppressed cellular invasion. We consider that further clarification of transcriptional mechanism of FOXP3 may facilitate the development of novel therapeutic approaches to suppress cancer malignancy.

  18. Molecular dynamics simulations of protein-tyrosine phosphatase 1B: II. Substrate-enzyme interactions and dynamics

    DEFF Research Database (Denmark)

    Peters, Günther H.j.; Frimurer, T. M.; Andersen, J. N.

    2000-01-01

    Molecular dynamics simulations of protein tyrosine phosphatase 1B (PTP1B) complexed with the phosphorylated peptide substrate DADEpYL and the free substrate have been conducted to investigate 1) the physical forces involved in substrate-protein interactions, 2) the importance of enzyme...... for catalysis. Analysis of the individual enzyme-substrate interaction energies revealed that mainly electrostatic forces contribute to binding. Indeed, calculation of the electrostatic field of the enzyme reveals that only the field surrounding the binding pocket is positive, while the remaining protein...

  19. Nonreceptor protein tyrosine and lipid phosphatases in type I Fcepsilon receptor-mediated activation of mast and basophils

    Czech Academy of Sciences Publication Activity Database

    Heneberg, Petr; Dráber, Petr

    2002-01-01

    Roč. 128, č. 4 (2002), s. 253-263 ISSN 1018-2438 R&D Projects: GA MŠk LN00A026; GA ČR GA204/00/0204; GA ČR GA310/00/0205; GA AV ČR IAA5052005; GA AV ČR IAA7052006 Institutional research plan: CEZ:AV0Z5052915 Keywords : phosphatases * tyrosin kinases * lipid phosphorylation Subject RIV: EC - Immunology Impact factor: 1.828, year: 2002

  20. Matrix metalloproteinases in the brain and blood–brain barrier: Versatile breakers and makers

    Science.gov (United States)

    Rempe, Ralf G; Hartz, Anika MS

    2016-01-01

    Matrix metalloproteinases are versatile endopeptidases with many different functions in the body in health and disease. In the brain, matrix metalloproteinases are critical for tissue formation, neuronal network remodeling, and blood–brain barrier integrity. Many reviews have been published on matrix metalloproteinases before, most of which focus on the two best studied matrix metalloproteinases, the gelatinases MMP-2 and MMP-9, and their role in one or two diseases. In this review, we provide a broad overview of the role various matrix metalloproteinases play in brain disorders. We summarize and review current knowledge and understanding of matrix metalloproteinases in the brain and at the blood–brain barrier in neuroinflammation, multiple sclerosis, cerebral aneurysms, stroke, epilepsy, Alzheimer’s disease, Parkinson’s disease, and brain cancer. We discuss the detrimental effects matrix metalloproteinases can have in these conditions, contributing to blood–brain barrier leakage, neuroinflammation, neurotoxicity, demyelination, tumor angiogenesis, and cancer metastasis. We also discuss the beneficial role matrix metalloproteinases can play in neuroprotection and anti-inflammation. Finally, we address matrix metalloproteinases as potential therapeutic targets. Together, in this comprehensive review, we summarize current understanding and knowledge of matrix metalloproteinases in the brain and at the blood–brain barrier in brain disorders. PMID:27323783

  1. Mapping protein phosphorylation in zebrafish development

    NARCIS (Netherlands)

    Lemeer, S.M.

    2008-01-01

    Mapping protein phosphorylation in zebrafish development Reversible protein phosphorylation plays a key role in signaling processes that are vital for a cell and organism. It provides a rapid switch for protein activity as it often changes the conformation and function of a protein in the cell.

  2. Bacillus subtilis single-stranded DNA-binding protein SsbA is phosphorylated at threonine 38 by the serine/threonine kinase YabT

    DEFF Research Database (Denmark)

    Derouiche, Abderahmane; Petranovic, Dina; Macek, Boris

    2016-01-01

    Background and purpose: Single-stranded DNA-binding proteins participate in all stages of DNA metabolism that involve single-stranded DNA, from replication, recombination, repair of DNA damage, to natural competence in species such as Bacillus subtilis. B. subtilis single-stranded DNA-binding pro......Background and purpose: Single-stranded DNA-binding proteins participate in all stages of DNA metabolism that involve single-stranded DNA, from replication, recombination, repair of DNA damage, to natural competence in species such as Bacillus subtilis. B. subtilis single-stranded DNA...... assays.Results: In addition to the known tyrosine phosphorylation of SsbA on tyrosine 82, we identified a new phosphorylation site: threonine 38. The in vitro assays demonstrated that SsbA is preferentially phosphorylated by the B. subtilis Hanks-type kinase YabT, and phosphorylation of threonine 38...... leads to enhanced cooperative binding to DNA.Conclusions: Our findings contribute to the emerging picture that bacterial proteins, exemplified here by SsbA, undergo phosphorylation at multiple residues. This results in a complex regulation of cellular functions, and suggests that the complexity...

  3. Phosphorylation of a 72-kDa protein in PDGF-stimulated cells which forms complex with c-Crk, c-Fyn and Eps15

    DEFF Research Database (Denmark)

    Hansen, Klaus; Rönnstrand, L; Claesson-Welsh, L

    1997-01-01

    Ligand-induced activation of the beta-receptor for platelet-derived growth factor (PDGF) induces tyrosine phosphorylation of a number of downstream signaling proteins. In the present study, we used two-dimensional gel electrophoresis to characterize the spectrum of proteins phosphorylated...... in response to PDGF stimulation in porcine aortic endothelial cells expressing PDGF beta-receptors. Several previously known substrates for the PDGF beta-receptor were identified as well as a novel substrate of 72 kDa. The 72-kDa component could be co-immunoprecipitated in complex with the adaptor protein c...

  4. rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain.

    Science.gov (United States)

    Mark, M R; Scadden, D T; Wang, Z; Gu, Q; Goddard, A; Godowski, P J

    1994-04-08

    We have isolated cDNA clones that encode the human and murine forms of a novel receptor-type tyrosine kinase termed Rse. Sequence analysis indicates that human Rse contains 890 amino acids, with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains. Murine Rse contains 880 amino acids and shares 90% amino acid identity with its human counterpart. Rse is structurally similar to the receptor-type tyrosine kinase Axl/Ufo, and the two proteins have 35 and 63% sequence identity in their extracellular and intracellular domains, respectively. To study the synthesis and activation of this putative receptor-type tyrosine kinase, we constructed a version of Rse (termed gD-Rse, where gD represents glycoprotein D) that contains an NH2-terminal epitope tag. NIH3T3 cells were engineered to express gD-Rse, which could be detected at the cell surface by fluorescence-activated cell sorting. Moreover, gD-Rse was rapidly phosphorylated on tyrosine residues upon incubation of the cells with an antibody directed against the epitope tag, suggesting that rse encodes an active tyrosine kinase. In the human tissues we examined, the highest level of expression of rse mRNA was observed in the brain; rse mRNA was also detected in the premegakaryocytopoietic cell lines CMK11-5 and Dami. The gene for rse was localized to human chromosome 15.

  5. Effect of supplementation of taurine or trehalose in extender on immunolocalization of tyrosine phosphoproteins in buffalo and cattle (Karan Fries) cryopreserved spermatozoa.

    Science.gov (United States)

    Kumar, R; Singh, V K; Chhillar, S; Atreja, S K

    2013-06-01

    The present study assessed the effects of incorporation of Taurine or Trehalose in extender on immunolocalization of tyrosine phosphoproteins, Cryocapacitation and other sperm quality parameters (motility, viability and membrane integrity) in post-thawed sperm from Buffalo (Murrah) and Cattle (Karan Fries). Six ejaculates from six individual bulls from both species were chosen at random and split into four aliquots: one aliquot without dilution (fresh sample), another diluted in egg yolk tris-citrate (EYTC) extender and the rest of aliquots with EYTC dilution supplemented with taurine (50 mm) or trehalose (100 mm), respectively, and cryopreserved. Following cryopreservation, semen were thawed and assessed for standard semen quality parameters. Extent of capacitation in cryopreserved spermatozoa was measured by inducing in vitro acrosome reaction followed by dual staining. Immunolocalization of tyrosine phosphoproteins was carried out by immunocytochemistry using primary antibody clone pT-154 (anti-phosphotyrosine antibody) and FITC-conjugated secondary antibody. Immunofluorescent signals were analysed for level of protein tyrosine phosphorylation in spermatozoa. Post-thaw semen evaluation showed supplementation of taurine or trehalose to EYTC extender significantly (p taurine or trehalose to freezing extender changed the localization of tyrosine phosphoproteins in cryopreserved spermatozoa similar to fresh in both the species. The results obtained clearly indicated that supplementation of taurine or trehalose to EYTC prior to cryopreservation improves Buffalo and Cattle sperm quality in terms of cryocapacitation and immunolocalization of tyrosine phosphoproteins during freezing-thawing process. © 2012 Blackwell Verlag GmbH.

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

    Directory of Open Access Journals (Sweden)

    Ayrapetov Marina K

    2005-11-01

    Full Text Available Abstract 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.

  7. Global tyrosine kinome profiling of human thyroid tumors identifies Src as a promising target for invasive cancers

    International Nuclear Information System (INIS)

    Cho, Nancy L.; Lin, Chi-Iou; Du, Jinyan; Whang, Edward E.; Ito, Hiromichi; Moore, Francis D.; Ruan, Daniel T.

    2012-01-01

    Highlights: ► Kinome profiling is a novel technique for identifying activated kinases in human cancers. ► Src activity is increased in invasive thyroid cancers. ► Inhibition of Src activity decreased proliferation and invasion in vitro. ► Further investigation of Src targeted therapies in thyroid cancer is warranted. -- Abstract: Background: Novel therapies are needed for the treatment of invasive thyroid cancers. Aberrant activation of tyrosine kinases plays an important role in thyroid oncogenesis. Because current targeted therapies are biased toward a small subset of tyrosine kinases, we conducted a study to reveal novel therapeutic targets for thyroid cancer using a bead-based, high-throughput system. Methods: Thyroid tumors and matched normal tissues were harvested from twenty-six patients in the operating room. Protein lysates were analyzed using the Luminex immunosandwich, a bead-based kinase phosphorylation assay. Data was analyzed using GenePattern 3.0 software and clustered according to histology, demographic factors, and tumor status regarding capsular invasion, size, lymphovascular invasion, and extrathyroidal extension. Survival and invasion assays were performed to determine the effect of Src inhibition in papillary thyroid cancer (PTC) cells. Results: Tyrosine kinome profiling demonstrated upregulation of nine tyrosine kinases in tumors relative to matched normal thyroid tissue: EGFR, PTK6, BTK, HCK, ABL1, TNK1, GRB2, ERK, and SRC. Supervised clustering of well-differentiated tumors by histology, gender, age, or size did not reveal significant differences in tyrosine kinase activity. However, supervised clustering by the presence of invasive disease showed increased Src activity in invasive tumors relative to non-invasive tumors (60% v. 0%, p < 0.05). In vitro, we found that Src inhibition in PTC cells decreased cell invasion and proliferation. Conclusion: Global kinome analysis enables the discovery of novel targets for thyroid cancer

  8. Matrix metalloproteinases during and outside of migraine attacks without aura

    DEFF Research Database (Denmark)

    Ashina, M.; Tvedskov, J.F.; Thiesen, Kerstin Lipka

    2010-01-01

    Ashina M, Tvedskov JF, Lipka K, Bilello J, Penkowa M & Olesen J. Matrix metalloproteinases during and outside of migraine attacks without aura. Cephalalgia 2009. London. ISSN 0333-1024To test the hypothesis that permeability of the blood-brain barrier (BBB) is altered during migraine attack due...... to enhanced activation of matrix metalloproteinases (MMPs), we investigated MMP-3, MMP-9 and tissue inhibitor of metalloproteases (TIMP)-1 in the external jugular vein during and outside of migraine attacks in 21 patients with migraine without aura. In addition, we measured plasma levels of several other...... of MMP-3 in the external jugular (P = 0.002) and cubital (P = 0.008) vein during attacks compared with outside of attacks. We found no correlation of ictal or interictal MMP-3, MMP-9 and TIMP-1 to migraine duration and frequency analysed in 21 patients (P > 0.05). There was no difference between ictal...

  9. The radioenzymatic assay of matrix metalloproteinase-1 activity

    International Nuclear Information System (INIS)

    Wu Shiliang; Xu Lan; Zhou Yinghui; Wang Weiping; Shi Ning

    1998-12-01

    The radioenzymatic assay method for tissue collagenase, a metalloproteinase, activity in matrix was established. The matrix collagenase is the most vital catabolic enzyme of collagen in tissue. It mainly acts on type- I, II, III matrix collagen and is also called matrix metalloproteinase-1 (MMPs-1). The assay method for the matrix collagenase was as follows: After type-I collagen was prepared from calf skin and identified with HPLC, it was marked with 3 H-acetic anhydride as the substrate. Then a series of assays were performed in animal experiments and human cases, which showed that the matrix collagenase (MMPs-1) activity assay is feasible and gives reliable results in clinical biochemistry study

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

    Science.gov (United States)

    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

  11. Free Fatty Acids Inhibit Protein Tyrosine Phosphatase 1B and Activate Akt

    Directory of Open Access Journals (Sweden)

    Eisuke Shibata

    2013-09-01

    Full Text Available Background/Aims: Accumulating evidence has suggested that free fatty acids (FFAs interact with protein kinases and protein phosphatases. The present study examined the effect of FFAs on protein phosphatases and Akt. Methods: Activities of protein phosphatase 1 (PP1, protein phosphatase 2A (PP2A, and protein tyrosine phosphatase 1B (PTP1B were assayed under the cell-free conditions. Phosphorylation of Akt was monitored in MSTO-211H human malignant pleural mesothelioma cells without and with knocking-down phosphatidylinositol 3 kinase (PI3K or 3-phosphoinositide-dependent protein kinase-1 (PDK1. Results: In the cell-free assay, unsaturated FFAs (uFFAs such as oleic, linoleic and linolenic acid and saturated FFAs (sFFAs such as stearic, palmitic, myristic, and behenic acid markedly reduced PTP1B activity, with the potential for uFFAs greater than that for sFFAs. All the investigated sFFAs inhibited PP2A activity, but otherwise no inhibition was obtained with uFFAs. Both uFFAs and sFFAs had no effect on PP1 activity. Oleic acid phosphorylated Akt both on Thr308 and Ser473, while stearic acid phosphorylated Akt on Thr308 alone. The effects of oleic and stearic acid on Akt phosphorylation were abrogated by the PI3K inhibitor wortmannin or the PDK1 inhibitor BX912 and also by knocking-down PI3K or PDK1. Conclusion: The results of the present study indicate that uFFAs and sFFAs could activate Akt through a pathway along a PI3K/PDK1/Akt axis in association with PTP1B inhibition.

  12. αE-catenin inhibits a Src–YAP1 oncogenic module that couples tyrosine kinases and the effector of Hippo signaling pathway

    Science.gov (United States)

    Li, Peng; Silvis, Mark R.; Honaker, Yuchi; Lien, Wen-Hui; Arron, Sarah T.; Vasioukhin, Valeri

    2016-01-01

    Cell–cell adhesion protein αE-catenin inhibits skin squamous cell carcinoma (SCC) development; however, the mechanisms responsible for this function are not completely understood. We report here that αE-catenin inhibits β4 integrin-mediated activation of SRC tyrosine kinase. SRC is the first discovered oncogene, but the protein substrate critical for SRC-mediated transformation has not been identified. We found that YAP1, the pivotal effector of the Hippo signaling pathway, is a direct SRC phosphorylation target, and YAP1 phosphorylation at three sites in its transcription activation domain is necessary for SRC–YAP1-mediated transformation. We uncovered a marked increase in this YAP1 phosphorylation in human and mouse SCC tumors with low/negative expression of αE-catenin. We demonstrate that the tumor suppressor function of αE-catenin involves negative regulation of the β4 integrin–SRC signaling pathway and that SRC-mediated phosphorylation and activation of YAP1 are an alternative to the canonical Hippo signaling pathway that directly connect oncogenic tyrosine kinase signaling with YAP1. PMID:27013234

  13. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts.

    Science.gov (United States)

    Hasaneen, Nadia A; Cao, Jian; Pulkoski-Gross, Ashleigh; Zucker, Stanley; Foda, Hussein D

    2016-02-17

    Idiopathic pulmonary fibrosis (IPF) is a chronic progressively fatal disease. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) is a glycosylated transmembrane protein that induces the expression of some matrix metalloproteinase (MMP) in neighboring stromal cells through direct epithelial-stromal interactions. EMMPRIN is highly expressed in type II alveolar epithelial cells at the edges of the fibrotic areas in IPF lung sections. However, the exact role of EMMPRIN in IPF is unknown. To determine if EMMPRIN contributes to lung fibroblast proliferation, resistance to apoptosis, and differentiation to myofibroblasts, normal Human lung fibroblasts (NHLF) transiently transfected with either EMMPRIN/GFP or GFP were treated with TGF- β1 from 0 to 10 ng/ml for 48 h and examined for cell proliferation (thymidine incorporation), apoptosis (FACS analysis and Cell Death Detection ELISA assay), cell migration (Modified Boyden chamber) and differentiation to myofibroblasts using Western blot for α-smooth actin of cell lysates. The effect of EMMPRIN inhibition on NHLF proliferation, apoptosis, migration and differentiation to myofibroblasts after TGF- β1 treatment was examined using EMMPRIN blocking antibody. We examined the mechanism by which EMMPRIN induces its effects on fibroblasts by studying the β-catenin/canonical Wnt signaling pathway using Wnt luciferase reporter assays and Western blot for total and phosphorylated β-catenin. Human lung fibroblasts overexpressing EMMPRIN had a significant increase in cell proliferation and migration compared to control fibroblasts. Furthermore, EMMPRIN promoted lung fibroblasts resistance to apoptosis. Lung fibroblasts overexpressing EMMPRIN showed a significantly increased expression of α- smooth muscle actin, a marker of differentiation to myofibroblasts compared to control cells. TGF-β1 increased the expression of EMMPRIN in lung fibroblasts in a dose-dependent manner. Attenuation of EMMPRIN expression with the use of an

  14. Beneficial Regulation of Matrix Metalloproteinases for Skin Health

    Directory of Open Access Journals (Sweden)

    Neena Philips

    2011-01-01

    Full Text Available Matrix metalloproteinases (MMPs are essential to the remodeling of the extracellular matrix. While their upregulation facilitates aging and cancer, they are essential to epidermal differentiation and the prevention of wound scars. The pharmaceutical industry is active in identifying products that inhibit MMPs to prevent or treat aging and cancer and products that stimulate MMPs to prevent epidermal hyperproliferative diseases and wound scars.

  15. Discovery of novel plant peptides as strong inhibitors of metalloproteinases.

    Science.gov (United States)

    Carrilho, Dina M; Duarte, Isabel C; Francisco, Rita; Ricardo, Cândido P P; Duque-Magalhães, Maria C

    2009-01-01

    Five novel metalloproteinase protein inhibitors (MPIs) with molecular mass between 5.6 and 8.9 kDa and acid/neutral pI were detected in lupin seeds and exhibited strong inhibitory activities against thermolysin and/or gelatinase B. These novel peptides constitute not only the first MPIs described in plants but also the first plant peptides with inhibitory activity against a matrixin.

  16. Periodontal Disease, Matrix Metalloproteinases and Chemically Modified Tetracyclines

    OpenAIRE

    Steinsvoll, Svein

    2011-01-01

    Matrix metalloproteinases (MMPs) are crucial in the degradation of the main components in the extracellular matrix and thereby play important roles in cell migration, wound healing and tissue remodelling. MMPs have pathogenic roles in arthritis, periodontitis, hepatitis, glomerulonephritis, atherosclerosis and cancer cell invasion. MMPs are activators of pro-inflammatory mediators that occur in latent forms, such as interleukin (IL)-1β, membrane-bound tumour necrosis factor (TNF) and dif...

  17. Matrix Metalloproteinases as Therapeutic Targets for Idiopathic Pulmonary Fibrosis

    OpenAIRE

    Craig, Vanessa J.; Zhang, Li; Hagood, James S.; Owen, Caroline A.

    2015-01-01

    Idiopathic pulmonary fibrosis (IPF) is a restrictive lung disease that is associated with high morbidity and mortality. Current medical therapies are not fully effective at limiting mortality in patients with IPF, and new therapies are urgently needed. Matrix metalloproteinases (MMPs) are proteinases that, together, can degrade all components of the extracellular matrix and numerous nonmatrix proteins. MMPs and their inhibitors, tissue inhibitors of MMPs (TIMPs), have been implicated in the p...

  18. Matrix metalloproteinases in stem cell regulation and cancer

    OpenAIRE

    Kessenbrock, K; Wang, CY; Wang, CY; Werb, Z

    2014-01-01

    © 2015. Since Gross and Lapiere firstly discovered matrix metalloproteinases (MMPs) as important collagenolytic enzymes during amphibian tadpole morphogenesis in 1962, this intriguing family of extracellular proteinases has been implicated in various processes of developmental biology. However, the pathogenic roles of MMPs in human diseases such as cancer have also garnered widespread attention. The most straightforward explanation for their role in cancer is that MMPs, through extracellular ...

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

    Energy Technology Data Exchange (ETDEWEB)

    Li, Bin, E-mail: binli@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Eyer, Peter, E-mail: peter.eyer@lrz.uni-muenchen.de [Walther-Straub-Institut Für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, 80336 München (Germany); Eddleston, Michael, E-mail: M.Eddleston@ed.ac.uk [Clinical Pharmacology Unit, University of Edinburgh, Edinburgh (United Kingdom); Jiang, Wei, E-mail: wjiang@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Schopfer, Lawrence M., E-mail: lmschopf@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Lockridge, Oksana, E-mail: olockrid@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States)

    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.

  20. Tissue Inhibitor of Metalloproteinase-3 Promotes Schwann Cell Myelination.

    Science.gov (United States)

    Kim, Jihyun; Elias, Anthony; Lee, Taeweon; Maurel, Patrice; Kim, Haesun A

    2017-01-01

    Tissue inhibitor of metalloproteinase-3 (TIMP-3) inhibits the activities of various metalloproteinases including matrix metalloproteinases and ADAM family proteins. In the peripheral nervous system, ADAM17, also known as TNF-α converting enzyme (TACE), cleaves the extracellular domain of Nrg1 type III, an axonal growth factor that is essential for Schwann cell myelination. The processing by ADAM17 attenuates Nrg1 signaling and inhibits Schwann cell myelination. TIMP-3 targets ADAM17, suggesting a possibility that TIMP-3 may elicit a promyelinating function in Schwann cells by relieving ADAM17-induced myelination block. To investigate this, we used a myelinating coculture system to determine the effect of TIMP-3 on Schwann cell myelination. Treatment with TIMP-3 enhanced myelin formation in cocultures, evident by an increase in the number of myelin segments and upregulated expression of Krox20 and myelin protein. The effect of TIMP-3 was accompanied by the inhibition of ADAM17 activity and an increase in Nrg1 type III signaling in cocultures. Accordingly, the N-terminus fragment of TIMP-3, which exhibits a selective inhibitory function toward ADAM17, elicited a similar myelination-promoting effect and increased Nrg1 type III activity. TIMP-3 also enhanced laminin production in cocultures, which is likely to aid Schwann cell myelination.

  1. The insect neuropeptide PTTH activates receptor tyrosine kinase torso to initiate metamorphosis.

    Science.gov (United States)

    Rewitz, Kim F; Yamanaka, Naoki; Gilbert, Lawrence I; O'Connor, Michael B

    2009-12-04

    Holometabolous insects undergo complete metamorphosis to become sexually mature adults. Metamorphosis is initiated by brain-derived prothoracicotropic hormone (PTTH), which stimulates the production of the molting hormone ecdysone via an incompletely defined signaling pathway. Here we demonstrate that Torso, a receptor tyrosine kinase that regulates embryonic terminal cell fate in Drosophila, is the PTTH receptor. Trunk, the embryonic Torso ligand, is related to PTTH, and ectopic expression of PTTH in the embryo partially rescues trunk mutants. In larvae, torso is expressed specifically in the prothoracic gland (PG), and its loss phenocopies the removal of PTTH. The activation of Torso by PTTH stimulates extracellular signal-regulated kinase (ERK) phosphorylation, and the loss of ERK in the PG phenocopies the loss of PTTH and Torso. We conclude that PTTH initiates metamorphosis by activation of the Torso/ERK pathway.

  2. Euglena mitochondria and chloroplasts form tyrosine-O-sulfate

    Energy Technology Data Exchange (ETDEWEB)

    Saidha, T.; Hanfstingl, U.; Schiff, J.A. (Brandeis Univ., Waltham, MA (USA))

    1989-04-01

    Mitochondria from light-grown wild-type Euglena gracilis var. bacillaris Cori or dark-grown mutant W{sub 10}BSmL incubated with {sup 35}SO{sub 4}{sup 2{minus}} and ATP, or with {sup 14}C-tyrosine, non-radioactive sulfate and ATP accumulate a labeled compound in the medium. Since this compound shows exact coelectrophoresis with tyrosine-O-sulfate (TOS) at pH 2.0, 5.8 or 8.0., yields sulfate and tyrosine on acid hydrolysis, and treatment with aryl sulfatase from Aerobacter aerogenes yields sulfate and tyrosine but no tyrosine methyl ester, it is identified as TOS. No TOS is found outside purified developing chloroplasts incubated with {sup 35}SO{sub 4}{sup 2{minus}} and ATP, but both chloroplasts and mitochondria form to {sup 35}S externally when incubated with adenosine 3{prime} phosphate 5{prime}phospho({sup 35}S) sulfate (PAP{sup 35}S). Since no tyrosine need be added, tyrosine is provided from endogenous sources. Although TOS is found in the free pool of Euglena cells it cannot be detected in proteins of cells or mucus ruling our sulfation of tyrosine of protein or incorporation of TOS into proteins. The system forming TOS is membrane-bound and may be involved in tyrosine transport.

  3. Tyrosine metabolic enzymes from insects and mammals: a comparative perspective.

    Science.gov (United States)

    Vavricka, Christopher John; Han, Qian; Mehere, Prajwalini; Ding, Haizhen; Christensen, Bruce M; Li, Jianyong

    2014-02-01

    Differences in the metabolism of tyrosine between insects and mammals present an interesting example of molecular evolution. Both insects and mammals possess fine-tuned systems of enzymes to meet their specific demands for tyrosine metabolites; however, more homologous enzymes involved in tyrosine metabolism have emerged in many insect species. Without knowledge of modern genomics, one might suppose that mammals, which are generally more complex than insects and require tyrosine as a precursor for important catecholamine neurotransmitters and for melanin, should possess more enzymes to control tyrosine metabolism. Therefore, the question of why insects actually possess more tyrosine metabolic enzymes is quite interesting. It has long been known that insects rely heavily on tyrosine metabolism for cuticle hardening and for innate immune responses, and these evolutionary constraints are likely the key answers to this question. In terms of melanogenesis, mammals also possess a high level of regulation; yet mammalian systems possess more mechanisms for detoxification whereas insects accelerate pathways like melanogenesis and therefore must bear increased oxidative pressure. Our research group has had the opportunity to characterize the structure and function of many key proteins involved in tyrosine metabolism from both insects and mammals. In this mini review we will give a brief overview of our research on tyrosine metabolic enzymes in the scope of an evolutionary perspective of mammals in comparison to insects. © 2013 Institute of Zoology, Chinese Academy of Sciences.

  4. Dietary Tyrosine Benefits Cognitive and Psychomotor Performance During Body Cooling

    National Research Council Canada - National Science Library

    O'Brien, Catherine; Mahoney, Caroline; Tharion, William J; Sils, Ingrid V; Castellani, John W

    2007-01-01

    ... examined. This study evaluated the effect of tyrosine supplementation on cognitive, psychomotor, and physical performance following a cold water immersion protocol that lowered body core temperature...

  5. Overexpression of interleukin-1β and interferon-γ in type I thoracic aortic dissections and ascending thoracic aortic aneurysms: possible correlation with matrix metalloproteinase-9 expression and apoptosis of aortic media cells.

    Science.gov (United States)

    Zhang, Lei; Liao, Ming-fang; Tian, Lei; Zou, Si-li; Lu, Qing-sheng; Bao, Jun-min; Pei, Yi-fei; Jing, Zai-ping

    2011-07-01

    To examine the expression of interleukin-1β and interferon-γ and their possible roles in aortic dissections and aneurysms. Aortic specimens were obtained from patients with type I thoracic aortic dissection, ascending thoracic aortic aneurysms, and control organ donors. The expression of interleukin-1β, interferon-γ, matrix metalloproteinase-9, and signal transduction factors phospho-p38 and phosphorylated c-jun N-terminal kinase (phospho-JNK) were detected by real time reverse transcription-polymerase chain reaction (real time RT-PCR), Western blot, and immunohistochemistry, respectively. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining was performed to detect apoptosis of media cells. The correlation of these factors and apoptosis was also studied. Apoptosis in the media of thoracic aortic dissection and in ascending thoracic aortic aneurysms was dramatically higher than in the control group. The expression of interleukin-1β gradually increased from the control group, thoracic aortic dissection to ascending thoracic aortic aneurysms (p matrix metalloproteinase-9 was significantly increased in the media of thoracic aortic dissection and ascending thoracic aortic aneurysms compared with the control group (p correlations between interleukin-1β versus matrix metalloproteinase-9, interleukin-1β versus phospho-p38 in thoracic aortic dissection (p matrix metalloproteinase-9, interferon-γ versus phospho-JNK, interferon-γ versus apoptosis, and interleukin-1β versus apoptosis in ascending thoracic aortic aneurysms (p = 0.02, 0.02, p matrix metalloproteinase-9 and the apoptosis of media cells in humans. Copyright © 2010 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.

  6. Glomerular Protein Levels of Matrix Metalloproteinase-1 and Tissue Inhibitor of Metalloproteinase-1 Are Lower in Diabetic Subjects

    Science.gov (United States)

    Cornish, Toby C.; Bagnasco, Serena M.; Macgregor, Anne M.; Lu, Jie; Selvin, Elizabeth; Halushka, Marc K.

    2009-01-01

    Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix turnover throughout the body, including in renal glomeruli. We investigated protein levels of multiple MMPs (MMP-1, MMP-2, MMP-3, and MMP-9) and TIMP-1 in glomeruli and investigated whether disease phenotypes were associated with levels of these proteins. Renal cortex was collected from 100 adult autopsy subjects arrayed across 17 tissue microarrays. Immunohistochemical staining intensity for each MMP and TIMP-1 was determined using quantitative color deconvolution techniques. We observed significantly decreased glomerular MMP-1 and TIMP-1 staining in subjects with diabetes, hypertension, and an estimated glomerular filtration rate contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 57:995–1001, 2009) PMID:19506087

  7. Correlation between expression of extracellular matrix metalloproteinase inducer and matrix metalloproteinase-2 and cervical lymph node metastasis of nasopharyngeal carcinoma.

    Science.gov (United States)

    Huang, Tian; Chen, Mao-Huai; Wu, Ming-Yao; Wu, Xian-Ying

    2013-03-01

    We evaluated the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinase-2 (MMP-2) in nasopharyngeal carcinoma (NPC) and studied their relationship with cervical lymph node metastasis. Immunohistochemical staining was used to detect the expression of EMMPRIN and MMP-2 in specimens from patients with chronic nasopharyngitis (CN), nonmetastastic NPC (NM-NPC), and lymph node-metastatic NPC (LNM-NPC). The rates of positive EMMPRIN expression in CN, NM-NPC, and LNM-NPC were 13.3%, 30.0%, and 66.7%, respectively. Significant differences were found between the rates in CN and LNM-NPC (p correlated (rs = 0.466; p <0.01). Nasopharyngeal carcinoma cells may attain enhanced metastastic capability through the expression of MMP-2 induced by EMMPRIN.

  8. The presence of promatrix metalloproteinase-3 and its relation with different categories of coal workers' pneumoconiosis

    Directory of Open Access Journals (Sweden)

    Remzi Altin

    2004-01-01

    Full Text Available Extracellular matrix formation (ECM and remodeling are critical events related to the pathogenesis of pulmonary fibrosis. Matrix metalloproteinases play an essential role in degrading and remodeling the ECM. In this study, we tried to show the presence and correlation of promatrix metalloproteinase-3 (proMMP-3 (the inactive form of metalloproteinase-3 levels in coal workers' pneumoconiosis (CWP with different categories.

  9. Large daily fluctuations in plasma tyrosine in treated patients with phenylketonuria

    NARCIS (Netherlands)

    vanSpronsen, FJ; vanDijk, T; Smit, GPA; vanRijn, M; Reijngoud, DJ; Berger, Ruud; Heymans, HSA

    1996-01-01

    In patients with phenylketonuria (PKU), extra tyrosine supplementation is advocated in addition to tyrosine-enriched amino acid mixtures. PKU patients have low fasting plasma tyrosine concentrations, but little is known about tyrosine fluctuations during the day. Plasma tyrosine concentrations were

  10. Proto-Oncogenic Src Phosphorylates EB1 to Regulate the Microtubule-Focal Adhesion Crosstalk and Stimulate Cell Migration.

    Science.gov (United States)

    Zhang, Yijun; Luo, Youguang; Lyu, Rui; Chen, Jie; Liu, Ruming; Li, Dengwen; Liu, Min; Zhou, Jun

    2016-01-01

    Cell migration, a complex process critical for tumor progression and metastasis, requires a dynamic crosstalk between microtubules (MTs) and focal adhesions (FAs). However, the molecular mechanisms underlying this event remain elusive. Herein we identify the proto-oncogenic protein Src as an important player in the regulation of the MT-FA crosstalk. Src interacts with and phosphorylates end-binding protein 1 (EB1), a member of MT plus end-tracking proteins (+TIPs), both in cells and in vitro . Systematic mutagenesis reveals that tyrosine-247 (Y247) is the primary residue of EB1 phosphorylated by Src. Interestingly, both constitutively activated Src and Y247-phosphorylated EB1 localize to the centrosome and FAs. Src-mediated EB1 phosphorylation diminishes its interactions with other +TIPs, including adenomatous polyposis coli (APC) and mitotic centromere associated kinesin (MCAK). In addition, EB1 phosphorylation at Y247 enhances the rate of MT catastrophe and significantly stimulates cell migration. These findings thus demonstrate that the Src-EB1 axis plays a crucial role in regulating the crosstalk between MTs and FAs to promote cell migration.

  11. β-Integrin de-phosphorylation by the Density-Enhanced Phosphatase DEP-1 attenuates EGFR signaling in C. elegans.

    Directory of Open Access Journals (Sweden)

    Michael Walser

    2017-01-01

    Full Text Available Density-Enhanced Phosphatase-1 (DEP-1 de-phosphorylates various growth factor receptors and adhesion proteins to regulate cell proliferation, adhesion and migration. Moreover, dep-1/scc1 mutations have been detected in various types of human cancers, indicating a broad tumor suppressor activity. During C. elegans development, DEP-1 mediates binary cell fate decisions by negatively regulating EGFR signaling. Using a substrate-trapping DEP-1 mutant in a proteomics approach, we have identified the C. elegans β-integrin subunit PAT-3 as a specific DEP-1 substrate. DEP-1 selectively de-phosphorylates tyrosine 792 in the membrane-proximal NPXY motif to promote integrin activation via talin recruitment. The non-phosphorylatable β-integrin mutant pat-3(Y792F partially suppresses the hyperactive EGFR signaling phenotype caused by loss of dep-1 function. Thus, DEP-1 attenuates EGFR signaling in part by de-phosphorylating Y792 in the β-integrin cytoplasmic tail, besides the direct de-phosphorylation of the EGFR. Furthermore, in vivo FRAP analysis indicates that the αβ-integrin/talin complex attenuates EGFR signaling by restricting receptor mobility on the basolateral plasma membrane. We propose that DEP-1 regulates EGFR signaling via two parallel mechanisms, by direct receptor de-phosphorylation and by restricting receptor mobility through αβ-integrin activation.

  12. S1PR1 Tyr143 phosphorylation downregulates endothelial cell surface S1PR1 expression and responsiveness

    Science.gov (United States)

    Chavez, Alejandra; Schmidt, Tracy Thennes; Yazbeck, Pascal; Rajput, Charu; Desai, Bhushan; Sukriti, Sukriti; Giantsos-Adams, Kristina; Knezevic, Nebojsa; Malik, Asrar B.; Mehta, Dolly

    2015-01-01

    ABSTRACT Activation of sphingosine-1-phosphate receptor 1 (S1PR1) plays a key role in repairing endothelial barrier function. We addressed the role of phosphorylation of the three intracellular tyrosine residues of S1PR1 in endothelial cells in regulating the receptor responsiveness and endothelial barrier function regulated by sphingosine 1-phosphate (S1P)-mediated activation of S1PR1. We demonstrated that phosphorylation of only Y143 site was required for S1PR1 internalization in response to S1P. Maximal S1PR1 internalization was seen in 20 min but S1PR1 returned to the cell surface within 1 h accompanied by Y143-dephosphorylation. Cell surface S1PR1 loss paralleled defective endothelial barrier enhancement induced by S1P. Expression of phospho-defective (Y143F) or phospho-mimicking (Y143D) mutants, respectively, failed to internalize or showed unusually high receptor internalization, consistent with the requirement of Y143 in regulating cell surface S1PR1 expression. Phosphorylation of the five S1PR1 C-terminal serine residues did not affect the role of Y143 phosphorylation in signaling S1PR1 internalization. Thus, rapid reduction of endothelial cell surface expression of S1PR1 subsequent to Y143 phosphorylation is a crucial mechanism of modulating S1PR1 signaling, and hence the endothelial barrier repair function of S1P. PMID:25588843

  13. PTP1B Inhibition Causes Rac1 Activation by Enhancing Receptor Tyrosine Kinase Signaling

    Directory of Open Access Journals (Sweden)

    Ayako Tsuchiya

    2014-04-01

    Full Text Available Background/Aims: The present study investigated the signaling pathway underlying Rac1 activation induced by the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl-cyclopropyl]-octanoic acid (DCP-LA. Methods: Activity of protein tyrosine phosphatase 1B (PTP1B was assayed under cell-free conditions. Western blot was carried out to quantify phosphorylation of insulin receptor substrate-1 (IRS-1 and Akt in PC-12 cells. Rac1 activity was monitored in the föerster resonance energy transfer (FRET analysis using living and fixed PC-12 cells. Results: DCP-LA markedly suppressed PTP1B activity in a concentration (100 pM-100 µM-dependent manner. In the DCP-LA binding assay, fluorescein-conjugated DCP-LA produced a single fluorescent signal band at 60 kDa, corresponding to the molecule of PTP1B, and the signal was attenuated or abolished by co-treatment or pretreatment with non-conjugated DCP-LA. DCP-LA significantly enhanced nerve growth factor (NGF-stimulated phosphorylation of IRS-1 at Tyr1222 and Akt1/2 at Thr308/309 and Ser473/474 in PC-12 cells. In the FRET analysis, DCP-LA significantly enhanced NGF-stimulated Rac1 activation, which is abrogated by the phosphatidylinositol 3 kinase (PI3K inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase-1 (PDK1 inhibitor BX912, or the Akt inhibitor MK2206. Conclusion: The results of the present study show that DCP-LA-induced PTP1B inhibition, possibly through its direct binding, causes Rac1 activation by enhancing a pathway along a receptor tyrosine kinase (RTK/IRS-1/PI3K/Akt/Rac1 axis.

  14. Crystal-induced neutrophil activation: X. Proinflammatory role of the tyrosine kinase Tec.

    Science.gov (United States)

    Popa-Nita, Oana; Marois, Louis; Paré, Guillaume; Naccache, Paul H

    2008-06-01

    Monosodium urate monohydrate (MSU) crystals are among the most potent proinflammatory stimuli, and an innate immune inflammatory response to the crystal surface is involved in the pathogenesis of gouty arthritis. Release of the crystals into the joint cavity promotes an acute inflammation characterized by massive infiltration of neutrophils, which leads to tissue damage. The aim of the present study was to assess the involvement of the tyrosine kinase Tec in MSU crystal-initiated transduction events in human neutrophils. Immunoprecipitation and immunoblotting techniques were used for the cellular signaling studies. Chemotaxis and enzyme-linked immunosorbent assay techniques were used for the functional studies. Silencing of Tec expression using specific small interfering RNA was also performed. MSU crystals induced the phosphorylation and activation of Tec in a Src-dependent manner. This activation was necessary for the MSU crystal-induced secretion of interleukin-1beta (IL-1beta) and IL-8 and for the generation of chemotactic activity in supernatants of MSU crystal-stimulated neutrophils. In addition, colchicine, an effective drug for the treatment of gout, inhibited the MSU crystal-induced tyrosine phosphorylation of Tec, thus modulating its kinase activity. Our findings show that Tec is the principal kinase of the Tec family that plays a major role in the responses of human neutrophils to MSU crystals, which are likely to be involved in the initiation and perpetuation of gout. Our results suggest that the specific inhibition of Tec during the acute phase of MSU crystal-induced inflammation may be considered for the treatment of gouty arthritis.

  15. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome

    DEFF Research Database (Denmark)

    Mann, Matthias; Ong, Shao En; Grønborg, Mads

    2002-01-01

    In signal transduction in eukaryotes, protein phosphorylation is a key event. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling...

  16. Membrane phosphorylation and nerve cell function

    International Nuclear Information System (INIS)

    Baer, P.R.

    1982-01-01

    This thesis deals with the phosphorylation of membrane components. In part I a series of experiments is described using the hippocampal slice as a model system. In part II a different model system - cultured hybrid cells - is used to study protein and lipid phosphorylation, influenced by incubation with neuropeptides. In part III in vivo and in vitro studies are combined to study protein phosphorylation after neuroanatomical lesions. In a section of part II (Page 81-90) labelling experiments of the membrane inositol-phospholipids are described. 32 P-ATP was used to label phospholipids in intact hybrid cells, and short incubations were found to be the most favourable. (C.F.)

  17. Protein tyrosine phosphatase receptor type z negatively regulates oligodendrocyte differentiation and myelination.

    Directory of Open Access Journals (Sweden)

    Kazuya Kuboyama

    Full Text Available BACKGROUND: Fyn tyrosine kinase-mediated down-regulation of Rho activity through activation of p190RhoGAP is crucial for oligodendrocyte differentiation and myelination. Therefore, the loss of function of its counterpart protein tyrosine phosphatase (PTP may enhance myelination during development and remyelination in demyelinating diseases. To test this hypothesis, we investigated whether Ptprz, a receptor-like PTP (RPTP expressed abuntantly in oligodendrocyte lineage cells, is involved in this process, because we recently revealed that p190RhoGAP is a physiological substrate for Ptprz. METHODOLOGY/PRINCIPAL FINDINGS: We found an early onset of the expression of myelin basic protein (MBP, a major protein of the myelin sheath, and early initiation of myelination in vivo during development of the Ptprz-deficient mouse, as compared with the wild-type. In addition, oligodendrocytes appeared earlier in primary cultures from Ptprz-deficient mice than wild-type mice. Furthermore, adult Ptprz-deficient mice were less susceptible to experimental autoimmune encephalomyelitis (EAE induced by active immunization with myelin/oligodendrocyte glycoprotein (MOG peptide than were wild-type mice. After EAE was induced, the tyrosine phosphorylation of p190RhoGAP increased significantly, and the EAE-induced loss of MBP was markedly suppressed in the white matter of the spinal cord in Ptprz-deficient mice. Here, the number of T-cells and macrophages/microglia infiltrating into the spinal cord did not differ between the two genotypes after MOG immunization. All these findings strongly support the validity of our hypothesis. CONCLUSIONS/SIGNIFICANCE: Ptprz plays a negative role in oligodendrocyte differentiation in early central nervous system (CNS development and remyelination in demyelinating CNS diseases, through the dephosphorylation of substrates such as p190RhoGAP.

  18. Cytoplasmic retention of protein tyrosine kinase 6 promotes growth of prostate tumor cells.

    Science.gov (United States)

    Brauer, Patrick M; Zheng, Yu; Wang, Lin; Tyner, Angela L

    2010-10-15

    Protein tyrosine kinase 6 (PTK6) is an intracellular tyrosine kinase that is nuclear in epithelial cells of the normal prostate, but cytoplasmic in prostate tumors and in the PC3 prostate tumor cell line. The impact of altered PTK6 intracellular localization in prostate tumor cells has not been extensively explored. Knockdown of endogenous cytoplasmic PTK6 resulted in decreased PC3 cell proliferation and colony formation, suggesting that cytoplasmic PTK6 stimulates oncogenic pathways. In contrast, reintroduction of PTK6 into nuclei of PC3 cells had a negative effect on growth. Enhanced tyrosine phosphorylation of the PTK6 substrate Sam68 was detected in cells expressing nuclear-targeted PTK6. We found that mechanisms regulating nuclear localization of PTK6 are intact in PC3 cells. Transiently overexpressed PTK6 readily enters the nucleus. Ectopic expression of ALT-PTK6, a catalytically inactive splice variant of PTK6, did not affect localization of endogenous PTK6 in PC3 cells. Using leptomycin B, we confirmed that cytoplasmic localization of endogenous PTK6 is not due to Crm-1/exportin-1 mediated nuclear export. In addition, overexpression of the PTK6 nuclear substrate Sam68 is not sufficient to bring PTK6 into the nucleus. While exogenous PTK6 was readily detected in the nucleus when transiently expressed at high levels, low-level expression of inducible wild type PTK6 in stable cell lines resulted in its cytoplasmic retention. Our results suggest that retention of PTK6 in the cytoplasm of prostate cancer cells disrupts its ability to regulate nuclear substrates and leads to aberrant growth. In prostate cancer, restoring PTK6 nuclear localization may have therapeutic advantages.

  19. Matrix metalloproteinase 2 and tissue inhibitor of matrix metalloproteinases 2 in the diagnosis of colorectal adenoma and cancer patients.

    Directory of Open Access Journals (Sweden)

    Barbara Mroczko

    2011-04-01

    Full Text Available The aim of the study was to assess the importance of the measurement of matrix metalloproteinase 2 (MMP-2 and tissue inhibitor of matrix metalloproteinases 2 (TIMP-2 in patients with colorectal cancer (CRC in relation to clinicopathological features of tumor and patients' survival. Additionally, we determined serum MMP-2 and TIMP-2 in colorectal adenoma (CA patients and healthy controls and compared them with tumor markers, CEA and CA 19-9. The serum levels of MMP-2 and TIMP-2 in 91 CRC patients, 28 CA subjects and 91 healthy controls were determined by ELISA method, but concentrations of CEA and CA 19-9 using MEIA method. Nonparametric statistical analyses were used. Serum levels of MMP-2 and TIMP-2 were significantly lower in CRC patients than in healthy subjects and decreased with tumor stage. Additionally, MMP-2 concentrations were significantly lower in patients with CRC than in CA group. Diagnostic sensitivity of TIMP-2 (59% was the highest among biomarkers tested and increased in combined use with CEA (79%. Moreover, the area under ROC curve (AUC of TIMP-2 was larger than AUC of MMP-2 in differentiation between CRC and healthy subjects, but lower than AUC of matrix metalloproteinase 2 in differentiation between colorectal cancer and adenoma. Our findings suggest clinical usefulness of TIMP-2 as a biomarker in the diagnosis of CRC, especially in combination with CEA. However, further investigation is necessary.

  20. Matrix metalloproteinase 2 and tissue inhibitor of matrix metalloproteinases 2 in the diagnosis of colorectal adenoma and cancer patients

    Directory of Open Access Journals (Sweden)

    Magdalena Groblewska

    2010-04-01

    Full Text Available The aim of the study was to assess the importance of the measurement of matrix metalloproteinase 2 (MMP-2and tissue inhibitor of matrix metalloproteinases 2 (TIMP-2 in patients with colorectal cancer (CRC in relation to clinicopathologicalfeatures of tumor and patients' survival. Additionally, we determined serum MMP-2 and TIMP-2 in colorectaladenoma (CA patients and healthy controls and compared them with tumor markers, CEA and CA 19-9. The serum levelsof MMP-2 and TIMP-2 in 91 CRC patients, 28 CA subjects and 91 healthy controls were determined by ELISA method, butconcentrations of CEA and CA 19-9 using MEIA method. Nonparametric statistical analyses were used. Serum levels ofMMP-2 and TIMP-2 were significantly lower in CRC patients than in healthy subjects and decreased with tumor stage.Additionally, MMP-2 concentrations were significantly lower in patients with CRC than in CA group. Diagnostic sensitivityof TIMP-2 (59% was the highest among biomarkers tested and increased in combined use with CEA (79%. Moreover,the area under ROC curve (AUC of TIMP-2 was larger than AUC of MMP-2 in differentiation between CRC and healthysubjects, but lower than AUC of matrix metalloproteinase 2 in differentiation between colorectal cancer and adenoma. Ourfindings suggest clinical usefulness of TIMP-2 as a biomarker in the diagnosis of CRC, especially in combination with CEA.However, further investigation is necessary.

  1. Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

    International Nuclear Information System (INIS)

    Okabe, Seiichi; Tauchi, Tetsuzo; Tanaka, Yuko; Ohyashiki, Kazuma

    2013-01-01

    Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-01-01

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

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

    Science.gov (United States)

    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.

  4. Hypoxia modulates the activity of a series of clinically approved tyrosine kinase inhibitors.

    Science.gov (United States)

    Ahmadi, M; Ahmadihosseini, Z; Allison, S J; Begum, S; Rockley, K; Sadiq, M; Chintamaneni, S; Lokwani, R; Hughes, N; Phillips, R M

    2014-01-01

    Hypoxia in tumours is known to cause resistance to conventional chemotherapeutic drugs. In contrast, little is known about the effects of hypoxia on targeted anti-cancer drugs. This study evaluated the effect of hypoxia on a series of clinically approved tyrosine kinase inhibitors (TKIs). The effect of hypoxia (0.1% oxygen) on the activity of conventional cytotoxic drugs (5-fluorouracil, doxorubicin and vinblastine), the hypoxia-activated prodrug tirapazamine and 9 TKIs was determined in a panel of cell lines. Where hypoxia had a marked effect on chemosensitivity, Western blot analysis was conducted to determine the effect of hypoxia on target expression and the effect of TKIs on cell signalling response under aerobic and hypoxic conditions. Three patterns of chemosensitivity were observed: resistance under hypoxia, equitoxic activity against hypoxic and aerobic cells, and preferential cytotoxicity to hypoxic cells. Significant hypoxia selectivity (independent of HIF1) was observed in the case of dasatinib and this correlated with the ability of dasatinib to inhibit phosphorylation of Src at tyrosine 530. Sorafenib was significantly less effective under hypoxic conditions but resistance did not correlate with hypoxia-induced changes in Raf/MEK/ERK signalling. Hypoxia influences the activity of TKIs but in contrast to conventional cytotoxic drugs, preferential activity against hypoxic cells can occur. The search for hypoxia-targeted therapies has been long and fruitless and this study suggests that some clinically approved TKIs could preferentially target the hypoxic fraction of some tumour types. © 2013 The British Pharmacological Society.

  5. Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Okabe, Seiichi, E-mail: okabe@tokyo-med.ac.jp; Tauchi, Tetsuzo; Tanaka, Yuko; Ohyashiki, Kazuma

    2013-06-07

    Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance.

  6. Human RON receptor tyrosine kinase induces complete epithelial-to-mesenchymal transition but causes cellular senescence

    International Nuclear Information System (INIS)

    Cote, Marceline; Miller, A. Dusty; Liu, Shan-Lu

    2007-01-01

    The RON receptor tyrosine kinase is a member of the MET proto-oncogene family and is important for cell proliferation, differentiation, and cancer development. Here, we created a series of Madin-Darby canine kidney (MDCK) epithelial cell clones that express different levels of RON, and have investigated their biological properties. While low levels of RON correlated with little morphological change in MDCK cells, high levels of RON expression constitutively led to morphological scattering or complete and stabilized epithelial-to-mesenchymal transition (EMT). Unexpectedly, MDCK clones expressing higher levels of RON exhibited retarded proliferation and senescence, despite increased motility and invasiveness. RON was constitutively tyrosine-phosphorylated in MDCK cells expressing high levels of RON and undergoing EMT, and the MAPK signaling pathway was activated. This study reveals for the first time that RON alone is sufficient to induce complete and stabilized EMT in MDCK cells, and overexpression of RON does not cause cell transformation but rather induces cell cycle arrest and senescence, leading to impaired cell proliferation

  7. Flavonol-enriched fraction from Vaccinium macrocarpon fruit inhibits matrix metalloproteinase-2, matrix metalloproteinase-9 and urokinase-type plasminogen activator expression in human prostate cancer cells in vitro

    Directory of Open Access Journals (Sweden)

    James MacPhee

    2014-11-01

    Full Text Available Background: Prostate cancer, amongst other cancer types has a genetic and environmental component, which can contribute to prostate cancer development and progression. Vaccinum macrocarpon (American cranberry is a botanical that contains several phytochemicals which have been suggested to play a role in preventing cardiovascular disease, cancer, and urinary tract infections as well as in the maintenance of oral health. Context and purpose of this study: This investigation evaluated the effects of a flavonolenriched fraction (FL from the American cranberry (Vaccinium macrocarpon containing quercetin and myricetin glycosides on matrix metalloproteinase (MMP and urokinase-type plasminogen activator (uPA activities and their associated regulatory proteins in DU145 human prostate cancer cells in vitro. Results: A flavonol-enriched fraction (FL was prepared from Vaccinium macrocarpon berries and the effect of this fraction on prostate cancer cell behaviour was assessed using biochemical and molecular approaches including cytotoxicity assays and Western blot analysis to determine protein expression. Cranberry FL decreased cellular viability of DU145 cells at a concentration of 25 ug/ml by 20% after 6 hours of treatment. Further investigations determined that associated with this cytotoxicity, cranberry FL decreases matrix metalloproteinase (MMP ( specifically MMP-2 and MMP-9 activity and urokinase plasminogen activator (uPA activity through effects on specific temporal MMP regulators and uPA regulators and by affecting either the phosphorylation status and/or expression of specific MAP kinase, PI-3 kinase, NF-kB and AP-1 pathway associated proteins. Conclusion: This study demonstrates, for the first time, the ability of Vaccinium macrocarpon flavonols to modulate cellular pathways associated with migration, invasion, and proliferation, suggesting that cranberry (Vaccinium macrocarpon is a viable candidate for further research as a natural product that

  8. ROR-Family Receptor Tyrosine Kinases.

    Science.gov (United States)

    Stricker, Sigmar; Rauschenberger, Verena; Schambony, Alexandra

    2017-01-01

    ROR-family receptor tyrosine kinases form a small subfamily of receptor tyrosine kinases (RTKs), characterized by a conserved, unique domain architecture. ROR RTKs are evolutionary conserved throughout the animal kingdom and act as alternative receptors and coreceptors of WNT ligands. The intracellular signaling cascades activated downstream of ROR receptors are diverse, including but not limited to ROR-Frizzled-mediated activation of planar cell polarity signaling, RTK-like signaling, and antagonistic regulation of WNT/β-Catenin signaling. In line with their diverse repertoire of signaling functions, ROR receptors are involved in the regulation of multiple processes in embryonic development such as development of the axial and paraxial mesoderm, the nervous system and the neural crest, the axial and appendicular skeleton, and the kidney. In humans, mutations in the ROR2 gene cause two distinct developmental syndromes, recessive Robinow syndrome (RRS; MIM 268310) and dominant brachydactyly type B1 (BDB1; MIM 113000). In Robinow syndrome patients and animal models, the development of multiple organs is affected, whereas BDB1 results only in shortening of the distal phalanges of fingers and toes, reflecting the diversity of functions and signaling activities of ROR-family RTKs. In this chapter, we give an overview on ROR receptor structure and function. We discuss their signaling functions and role in vertebrate embryonic development with a focus on those developmental processes that are affected by mutations in the ROR2 gene in human patients. © 2017 Elsevier Inc. All rights reserved.

  9. Sympathetic Hyperactivity, Increased Tyrosine Hydroxylase and Exaggerated Corpus Cavernosum Relaxations Associated with Oxidative Stress Plays a Major Role in the Penis Dysfunction in Townes Sickle Cell Mouse.

    Directory of Open Access Journals (Sweden)

    Fábio H Silva

    Full Text Available Sickle cell disease patients display priapism that may progress to erectile dysfunction. However, little is known about the pathophysiological alterations of corpus cavernosum in sickle cell disease.Thus, this study aimed to evaluate the functional and molecular alterations of sympathetic machinery and nitric oxide-cyclic guanosine monophosphate signaling pathway in Townes transgenic sickle cell disease mice.Concentration-response curves to contractile (phenylephrine and relaxant agents (acetylcholine and sodium nitroprusside were obtained in corpus cavernosum strips from sickle and C57BL/6 (control mice. Neurogenic contractions and nitrergic relaxations were obtained using electrical-field stimulation. Measurements of endothelial nitric oxide synthase (eNOS, neuronal nitric oxide synthase (nNOS, phosphodiesterase-5 (PDE5 and α1A-, α1B- and α1D-adrenoceptor mRNA expressions and reactive-oxygen species were performed. Tyrosine hydroxylase phosphorylated at Ser-31 and total tyrosine hydroxylase protein expressions in cavernosal tissues were also measured.The neurogenic contractions were higher in the sickle cell disease group, in association with elevated tyrosine hydroxylase phosphorylated at Ser-31 and total tyrosine hydroxylase protein expression, as well as increased tyrosine hydroxylase mRNA expression. Likewise, phenylephrine-induced contractions were greater in the sickle mice, whereas α1A-, α1B- and α1D-adrenoceptor mRNA expression remained unchanged. Cavernosal relaxations to acetylcholine, sodium nitroprusside and EFS were higher in sickle mice, accompanied by decreased eNOS and nNOS, along with lower PDE5 mRNA expression. An increase of about 40% in reactive-oxygen species generation in corpus cavernosum from sickle mice was also detected.Our study shows that decreased nitric oxide bioavailability in erectile tissue due to increased oxidative stress leads to both sympathetic hyperactivity and dysregulation of nitric oxide

  10. ERK5 activity is required for nerve growth factor-induced neurite outgrowth and stabilization of tyrosine hydroxylase in PC12 cells.

    Science.gov (United States)

    Obara, Yutaro; Yamauchi, Arata; Takehara, Shin; Nemoto, Wataru; Takahashi, Maho; Stork, Philip J S; Nakahata, Norimichi

    2009-08-28

    Extracellular signal-regulated kinases (ERKs) play important physiological roles in proliferation, differentiation, and gene expression. ERK5 is approximately twice the size of ERK1/2, and its amino-terminal half contains the kinase domain that shares homology with ERK1/2 and TEY activation motif, whereas the carboxyl-terminal half is unique. In this study, we examined a physiological role of ERK5 in rat pheochromocytoma cells (PC12), comparing it with ERK1/2. Nerve growth factor (NGF) induced phosphorylation of both ERK5 and ERK1/2, whereas the cAMP analog dibutyryl cAMP (Bt(2)cAMP) caused only ERK1/2 phosphorylation. U0126, at 30 mum, that blocks ERK1/2 signaling selectively attenuated neurite outgrowth induced by NGF and Bt(2)cAMP, but BIX02188 and BIX02189, at 30 mum, that block ERK5 signaling and an ERK5 dominant-negative mutant suppressed only NGF-induced neurite outgrowth. Next, we examined the expression of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis. Both NGF and Bt(2)cAMP increased tyrosine hydroxylase gene promoter activity in an ERK1/2-dependent manner but was ERK5-independent. However, when both ERK5 and ERK1/2 signalings were inhibited, tyrosine hydroxylase protein up-regulation by NGF and Bt(2)cAMP was abolished, because of the loss of stabilization of tyrosine hydroxylase protein by ERK5. Taking these results together, ERK5 is involved in neurite outgrowth and stabilization of tyrosine hydroxylase in PC12 cells, and ERK5, along with ERK1/2, plays essential roles in the neural differentiation process.

  11. S137 phosphorylation of profilin 1 is an important signaling event in breast cancer progression.

    Directory of Open Access Journals (Sweden)

    Wasia Rizwani

    Full Text Available BACKGROUND: Profilins are actin-modulating proteins regulating many intracellular functions based on their multiple and diverse ligand interactions. They have been implicated to play a role in many pathological conditions such as allergies, cardiovascular diseases, muscular atrophy, diabetes, dementia and cancer. Post-translational modifications of profilin 1 can alter its properties and subsequently its function in a cell. In the present study, we identify the importance of phosphorylation of profilin 1 at serine 137 (S137 residue in breast cancer progression. METHODS/PRINCIPAL FINDINGS: We found elevated profilin 1 (PFN in human breast cancer tissues when compared to adjacent normal tissues. Overexpression of wild-type profilin 1 (PFN-WT in breast cancer MCF7 cells made them more migratory, invasive and adherent independent in comparison to empty vector transfected cells. Mutation in serine phosphorylation site (S137 of profilin 1 (PFN-S137A significantly abrogated these properties. Mutation affecting actin-binding ability (PFN-R74E of profilin 1 enhanced its tumorigenic function whereas mutation affecting its poly-L-proline binding function (PFN-H133S alleviated these mechanisms in breast cancer cells. PFN-WT was found to activate matrix metalloproteinases by zymography, MMP2 and MMP9 in presence of PDBu (phorbol 12, 13 dibutyrate, PI3K agonist to enhance migration and invasion in MCF7 cells while PFN-S137A did not. Phosphorylation increased migration and invasion in other mutants of profilin 1. Nuclear profilin levels also increased in the presence of PDBu. CONCLUSIONS: Previous studies show that profilin could be executing a dual role in cancer by either suppressing or promoting tumorigenesis in a context dependent manner. In this study we demonstrate for the first time that phosphorylation of profilin 1 at serine 137 enhances oncogenic properties in breast cancer cells. Inhibitors targeting profilin 1 phosphorylation directly or indirectly

  12. Mapping of p140Cap phosphorylation sites

    DEFF Research Database (Denmark)

    Repetto, Daniele; Aramu, Simona; Boeri Erba, Elisabetta

    2013-01-01

    Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation). p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes...

  13. Src SUMOylation Inhibits Tumor Growth Via Decreasing FAK Y925 Phosphorylation.

    Science.gov (United States)

    Wang, Jing; Deng, Rong; Cui, Nan; Zhang, Hailong; Liu, Tianqi; Dou, Jinzhuo; Zhao, Xian; Chen, Ran; Wang, Yanli; Yu, Jianxiu; Huang, Jian

    2017-12-01

    Src, a non-receptor tyrosine kinase protein, plays a critical role in cell proliferation and tumorigenesis. SUMOylation, a reversible ubiquitination-like post-translational modification, is vital for tumor progression. Here, we report that the Src protein can be SUMOylated at lysine 318 both in vitro and in vivo. Hypoxia can induce a decrease of Src SUMOylation along with an increase of Y419 phosphorylation, a phosphorylation event required for Src activation. On the other hand, treatment with hydrogen peroxide can enhance Src SUMOylation. Significantly, ectopic expression of SUMO-defective mutation, Src K318R, promotes tumor growth more potently than that of wild-type Src, as determined by migration assay, soft agar assay, and tumor xenograft experiments. Consistently, Src SUMOylation leads to a decrease of Y925 phosphorylation of focal adhesion kinase (FAK), an established regulatory event of cell migration. Our results suggest that SUMOylation of Src at lysine 318 negatively modulate its oncogenic function by, at least partially, inhibiting Src-FAK complex activity. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Phosphorylation of the dimeric cytoplasmic domain of the phytosulfokine receptor, PSKR1

    KAUST Repository

    Muleya, V.

    2016-08-04

    Phytosulfokines (PSKs) are plant peptide hormones that co-regulate plant growth, differentiation and defense responses. PSKs signal through a plasma membrane localized leucine-rich repeat receptor-like kinase (phytosulfokine receptor 1, PSKR1) that also contains a functional cytosolic guanylate cyclase with its cyclase catalytic center embedded within the kinase domain. To functionally characterize this novel type of overlapping dual catalytic function, we investigated the phosphorylation of PSKR1 in vitro Tandem mass spectrometry of the cytoplasmic domain of PSKR1 (PSKR1cd) revealed at least 11 phosphorylation sites (8 serines, 2 threonines and 1 tyrosine) within the PSKR1cd. Phosphomimetic mutations of three serine residues (Ser686, Ser696 and Ser698) in tandem at the juxta-membrane position resulted in enhanced kinase activity in the on-mutant that was suppressed in the off-mutant, but both mutations reduced guanylate cyclase activity. Both the on and off phosphomimetic mutations of the phosphotyrosine (Tyr888) residue in the activation loop suppressed kinase activity, while neither mutation affected guanylate cyclase activity. Size exclusion and analytical ultracentrifugation analysis of the PSKR1cd suggest that it is reversibly dimeric in solution, which was further confirmed by biflourescence complementation. Taken together, these data suggest that in this novel type of receptor domain architecture, specific phosphorylation and dimerization are possibly essential mechanisms for ligand-mediated catalysis and signaling.

  15. HGF-independent regulation of MET and GAB1 by nonreceptor tyrosine kinase FER potentiates metastasis in ovarian cancer.

    Science.gov (United States)

    Fan, Gaofeng; Zhang, Siwei; Gao, Yan; Greer, Peter A; Tonks, Nicholas K

    2016-07-01

    Ovarian cancer cells disseminate readily within the peritoneal cavity, which promotes metastasis, and are often resistant to chemotherapy. Ovarian cancer patients tend to present with advanced disease, which also limits treatment options; consequently, new therapies are required. The oncoprotein tyrosine kinase MET, which is the receptor for hepatocyte growth factor (HGF), has been implicated in ovarian tumorigenesis and has been the subject of extensive drug development efforts. Here, we report a novel ligand- and autophosphorylation-independent activation of MET through the nonreceptor tyrosine kinase feline sarcoma-related (FER). We demonstrated that the levels of FER were elevated in ovarian cancer cell lines relative to those in immortalized normal surface epithelial cells and that suppression of FER attenuated the motility and invasive properties of these cancer cells. Furthermore, loss of FER impaired the metastasis of ovarian cancer cells in vivo. Mechanistically, we demonstrated that FER phosphorylated a signaling site in MET: Tyr1349. This enhanced activation of RAC1/PAK1 and promoted a kinase-independent scaffolding function that led to recruitment and phosphorylation of GAB1 and the specific activation of the SHP2-ERK signaling pathway. Overall, this analysis provides new insights into signaling events that underlie metastasis in ovarian cancer cells, consistent with a prometastatic role of FER and highlighting its potential as a novel therapeutic target for metastatic ovarian cancer. © 2016 Fan et al.; Published by Cold Spring Harbor Laboratory Press.

  16. Regulation of Matrix Metalloproteinase-2 Activity by COX-2-PGE2-pAKT Axis Promotes Angiogenesis in Endometriosis

    Science.gov (United States)

    Ray, Amlan K.; DasMahapatra, Pramathes; Swarnakar, Snehasikta

    2016-01-01

    Endometriosis is characterized by the ectopic development of the endometrium which relies on angiogenesis. Although studies have identified the involvement of different matrix metalloproteinases (MMPs) in endometriosis, no study has yet investigated the role of MMP-2 in endometriosis-associated angiogenesis. The present study aims to understand the regulation of MMP-2 activity in endothelial cells and on angiogenesis during progression of ovarian endometriosis. Histological and biochemical data showed increased expressions of vascular endothelial growth factor (VEGF), VEGF receptor-2, cycloxygenase (COX)-2, von Willebrand factor along with angiogenesis during endometriosis progression. Women with endometriosis showed decreased MMP-2 activity in eutopic endometrium as compared to women without endometriosis. However, ectopic ovarian endometrioma showed significantly elevated MMP-2 activity with disease severity. In addition, increased MT1MMP and decreased tissue inhibitors of metalloproteinases (TIMP)-2 expressions were found in the late stages of endometriosis indicating more MMP-2 activation with disease progression. In vitro study using human endothelial cells showed that prostaglandin E2 (PGE2) significantly increased MMP-2 activity as well as tube formation. Inhibition of COX-2 and/or phosphorylated AKT suppressed MMP-2 activity and endothelial tube formation suggesting involvement of PGE2 in regulation of MMP-2 activity during angiogenesis. Moreover, specific inhibition of MMP-2 by chemical inhibitor significantly reduced cellular migration, invasion and tube formation. In ovo assay showed decreased angiogenic branching upon MMP-2 inhibition. Furthermore, a significant reduction of lesion numbers was observed upon inhibition of MMP-2 and COX-2 in mouse model of endometriosis. In conclusion, our study establishes the involvement of MMP-2 activity via COX-2-PGE2-pAKT axis in promoting angiogenesis during endometriosis progression. PMID:27695098

  17. Deuterium Labelling of L-Tyrosine with Raney Alloys in Alkaline Deuterium Oxide Solutions

    OpenAIRE

    Tsuzuki, Hirohisa; Mukumoto, Mamoru; Udagawa, Jun; Mataka, Shuntaro; Tashiro, Masashi

    1997-01-01

    The synthesis of deuteriated L-tyrosines with Raney alloys in alkaline deuterium oxide solutions, involving reductive debromination of brominated L-tyrosines and hydrogen-deuterium (H-D) exchange of L-tyrosines, without causing racemization, is presented.

  18. Determination of o-tyrosine in irradiated chicken

    International Nuclear Information System (INIS)

    Zoller, O.; Schoeni, D.; Zimmerli, B.

    1991-01-01

    The author explains his method to determine O-Tyrosine in irradiated chickens with a high-performance liquid chromatography. The method is simple and fast, but a proper chromatographic separation is difficult. The detection limit with a high sensitive detector is about 0.05-0.1 mg O-Tyrosine/kg meat (9 refs)

  19. Tyrosine improves working memory in a multitasking environment.

    Science.gov (United States)

    Thomas, J R; Lockwood, P A; Singh, A; Deuster, P A

    1999-11-01

    Previous studies indicate that tyrosine may prove useful in promoting improved performance in situations in which performance is compromised by stress. To extend the generality of previous tyrosine findings, the present study examined the effects of tyrosine ingestion on performance during both a Multiple Task and a Simple Task battery. The multiple task battery was designed to measure working memory, arithmetic skills, and visual and auditory monitoring simultaneously, whereas the simple task battery measured only working memory and visual monitoring. Ten men and 10 women subjects underwent these batteries 1 h after ingesting 150 mg/kg of l-tyrosine or placebo. Administration of tyrosine significantly enhanced accuracy and decreased frequency of list retrieval on the working memory task during the multiple task battery compared with placebo. However, tyrosine induced no significant changes in performance on the arithmetic, visual, or auditory tasks during the Multiple Task, or modified any performance measures during the Simple Task battery. Blood levels of ACTH and cortisol were not, but heart rate and blood pressure were significantly increased during the performance tasks. The present results indicate that tyrosine may sustain working memory when competing requirements to perform other tasks simultaneously degrade performance, and that supplemental tyrosine may be appropriate for maintaining performance when mild to severe decrements are anticipated.

  20. Endotoxin mediated-iNOS induction causes insulin resistance via ONOO⁻ induced tyrosine nitration of IRS-1 in skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Geneviève Pilon

    2010-12-01

    Full Text Available It is believed that the endotoxin lipopolysaccharide (LPS is implicated in the metabolic perturbations associated with both sepsis and obesity (metabolic endotoxemia. Here we examined the role of inducible nitric oxide synthase (iNOS in skeletal muscle insulin resistance using LPS challenge in rats and mice as in vivo models of endotoxemia.Pharmacological (aminoguanidine and genetic strategies (iNOS⁻/⁻ mice were used to counter iNOS induction in vivo. In vitro studies using peroxynitrite (ONOO⁻ or inhibitors of the iNOS pathway, 1400 W and EGCG were conducted in L6 myocytes to determine the mechanism by which iNOS mediates LPS-dependent insulin resistance. In vivo, both pharmacological and genetic invalidation of iNOS prevented LPS-induced muscle insulin resistance. Inhibition of iNOS also prevented insulin resistance in myocytes exposed to cytokine/LPS while exposure of myocytes to ONOO⁻ fully reproduced the inhibitory effect of cytokine/LPS on both insulin-stimulated glucose uptake and PI3K activity. Importantly, LPS treatment in vivo and iNOS induction and ONOO⁻ treatment in vitro promoted tyrosine nitration of IRS-1 and reduced insulin-dependent tyrosine phosphorylation.Our work demonstrates that iNOS-mediated tyrosine nitration of IRS-1 is a key mechanism of skeletal muscle insulin resistance in endotoxemia, and presents nitrosative modification of insulin signaling proteins as a novel therapeutic target for combating muscle insulin resistance in inflammatory settings.

  1. Measurement of optical purity of p-BPA-Tyrosine dipeptide

    Energy Technology Data Exchange (ETDEWEB)

    Yoshino, K.; Sato, N.; Kitta, K.; Saitake, Y. [Shinshu Univ., Faculty of Science, Matsumoto, Nagano (Japan); Hiratsuka, J. [Kawasaki Medical School, Dept. of Radiation Oncology, Kurashiki, Okayama (Japan); Ichihashi, M. [Kobe Univ. (Japan). School of Medicine

    2000-10-01

    Melanin biosynthesis is very active in melanoma cells, and tyrosine is one of the substrates of the melanin biosynthesis. Tyrosine is oxidized to dopa by tyrosinase at the beginning of melanin biosynthesis process. Therefore, p-boronophenylalanine (BPA)-tyrosine dipeptide is expected to be a substrate of melanin biosynthesis process, and the peptide will be incorporated in melanoma cells, and then tumor boron concentration lasts in their cells for long time. Since p-BPA tyrosine are amino acids, they have D, L isomers. Therefore, we have tried to synthesize four isomers (L-L, L-D, D-L, D-D) of p-BPA-Tyrosine dipeptide, and have measured their optical purity with HPLC. (author)

  2. The Sulfinator: predicting tyrosine sulfation sites in protein sequences.

    Science.gov (United States)

    Monigatti, Flavio; Gasteiger, Elisabeth; Bairoch, Amos; Jung, Eva

    2002-05-01

    Protein tyrosine sulfation is an important post-translational modification of proteins that go through the secretory pathway. No clear-cut acceptor motif can be defined that allows the prediction of tyrosine sulfation sites in polypeptide chains. The Sulfinator is a software tool that can be used to predict tyrosine sulfation sites in protein sequences with an overall accuracy of 98%. Four different Hidden Markov Models were constructed, each of them specialized to recognize sulfated tyrosine residues depending on their location within the sequence: near the N-terminus, near the C-terminus, in the center of a window with a size of at least 25 amino acids, as well as in windows containing several tyrosine residues. The Sulfinator is accessible at (http://www.expasy.org/tools/sulfinator/). Sulfinator documentation is accessible at (http://www.expasy.org/tools/sulfinator/sulfinator-doc.html).

  3. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    International Nuclear Information System (INIS)

    Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

    2015-01-01

    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression

  4. Ramipril inhibits AGE-RAGE-induced matrix metalloproteinase-2 activation in experimental diabetic nephropathy.

    Science.gov (United States)

    Fukami, Kei; Yamagishi, Sho-Ichi; Coughlan, Melinda T; Harcourt, Brooke E; Kantharidis, Phillip; Thallas-Bonke, Vicki; Okuda, Seiya; Cooper, Mark E; Forbes, Josephine M

    2014-01-01

    Advanced glycation end products (AGE)-receptor for AGE (RAGE) axis and renin-angiotensin system (RAS) play a role in diabetic nephropathy (DN). Matrix metalloproteinase-2 (MMP-2) activation also contributes to DN. However, the pathological interaction among AGE-RAGE, RAS and MMP-2 in DN remains unknown. We examined here the involvement of AGE and RAS in MMP-2 activation in streptozotocin (STZ)-induced diabetic rats and in AGE-exposed rat renal proximal tubular cells (RPTCs). Experimental diabetes was induced in 6-week-old male Sprague-Dawley (SD) rats by intravenous injection of STZ. Diabetic rats received ramipril (3 mg/kg body weight/day) or vehicle for 32 weeks. AGE-modified rat serum albumin (AGE-RSA) or RSA was intraperitoneally administrated to 6-week-old male SD rats for 16 weeks. RPTCs were stimulated with 100 μg/ml AGE-modified bovine serum albumin (AGE-BSA) or BSA in the presence or absence of 10(-7) M ramiprilat, an inhibitor of angiotensin-converting enzyme or 100 nM BAY11-7082, an IκB-α phosphorylation inhibitor. AGE and RAGE expression levels and MMP-2 activity in the tubules of diabetic rats was significantly increased in association with increased albuminuria, all of which were blocked by ramipril. AGE infusion induced tubular MMP-2 activation and RAGE gene expression in SD rats. Ramiprilat or BAY11-7082 inhibited the AGE-induced MMP-2 activation or reactive oxygen species generation in RPTCs. Angiotensin II increased MMP-2 gene expression in RPTCs, which was blocked by BAY11-7082. Our present study suggests the involvement of AGE-RAGE-induced, RAS-mediated MMP-2 activation in experimental DN. Blockade of AGE-RAGE axis by ramipril may protect against DN partly via suppression of MMP-2.

  5. Matrix metalloproteinase-14 mediates formation of bile ducts and hepatic maturation of fetal hepatic progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Otani, Satoshi [Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo (Japan); Kakinuma, Sei, E-mail: skakinuma.gast@tmd.ac.jp [Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo (Japan); Department for Liver Disease Control, Tokyo Medical and Dental University, Tokyo (Japan); Kamiya, Akihide [Institute of Innovative Science and Technology, Tokai University, Isehara (Japan); Goto, Fumio; Kaneko, Shun; Miyoshi, Masato; Tsunoda, Tomoyuki; Asano, Yu; Kawai-Kitahata, Fukiko; Nitta, Sayuri; Nakata, Toru; Okamoto, Ryuichi; Itsui, Yasuhiro; Nakagawa, Mina; Azuma, Seishin [Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo (Japan); Asahina, Yasuhiro [Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo (Japan); Department for Liver Disease Control, Tokyo Medical and Dental University, Tokyo (Japan); Yamaguchi, Tomoyuki [Division of Stem Cell Therapy, Institute of Medical Science, The University of Tokyo, Tokyo (Japan); Koshikawa, Naohiko [Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Tokyo (Japan); Seiki, Motoharu [Medical School, Kanazawa University, Kanazawa (Japan); Nakauchi, Hiromitsu [Division of Stem Cell Therapy, Institute of Medical Science, The University of Tokyo, Tokyo (Japan); and others

    2016-01-22

    Fetal hepatic stem/progenitor cells, called hepatoblasts, play central roles in liver development; however, the molecular mechanisms regulating the phenotype of these cells have not been completely elucidated. Matrix metalloproteinase (MMP)-14 is a type I transmembrane proteinase regulating pericellular proteolysis of the extracellular matrix and is essential for the activation of several MMPs and cytokines. However, the physiological functions of MMP-14 in liver development are unknown. Here we describe a functional role for MMP-14 in hepatic and biliary differentiation of mouse hepatoblasts. MMP-14 was upregulated in cells around the portal vein in perinatal stage liver. Formation of bile duct-like structures in MMP-14–deficient livers was significantly delayed compared with wild-type livers in vivo. In vitro biliary differentiation assays showed that formation of cholangiocytic cysts derived from MMP-14–deficient hepatoblasts was completely impaired, and that overexpression of MMP-14 in hepatoblasts promoted the formation of bile duct-like cysts. In contrast, the expression of molecules associated with metabolic functions in hepatocytes, including hepatic nuclear factor 4α and tryptophan 2,3-dioxygenase, were significantly increased in MMP-14–deficient livers. Expression of the epidermal growth factor receptor and phosphorylation of mitogen-activated protein kinases were significantly upregulated in MMP-14–deficient livers. We demonstrate that MMP-14–mediated signaling in fetal hepatic progenitor cells promotes biliary luminal formation around the portal vein and negatively controls the maturation of hepatocytes. - Highlights: • Loss of MMP-14 delayed formation of bile duct-like structures in perinatal liver. • Overexpression of MMP-14 in hepatobalsts promoted the biliary formation in vitro. • Loss of MMP-14 promoted hepatocyte maturation of hepatoblasts in vivo. • MMP-14–mediated signaling regulates terminal differentiation of

  6. Mixed lineage kinase 3 is required for matrix metalloproteinase expression and invasion in ovarian cancer cells

    International Nuclear Information System (INIS)

    Zhan, Yu; Abi Saab, Widian F.; Modi, Nidhi; Stewart, Amanda M.; Liu, Jinsong; Chadee, Deborah N.

    2012-01-01

    Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates MAPK signaling pathways and regulates cellular responses such as proliferation, migration and apoptosis. Here we report high levels of total and phospho-MLK3 in ovarian cancer cell lines in comparison to immortalized nontumorigenic ovarian epithelial cell lines. Using small interfering RNA (siRNA)-mediated gene silencing, we determined that MLK3 is required for the invasion of SKOV3 and HEY1B ovarian cancer cells. Furthermore, mlk3 silencing substantially reduced matrix metalloproteinase (MMP)-1, -2, -9 and -12 gene expression and MMP-2 and -9 activities in SKOV3 and HEY1B ovarian cancer cells. MMP-1, -2, -9 and-12 expression, and MLK3-induced activation of MMP-2 and MMP-9 requires both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities. In addition, inhibition of activator protein-1 (AP-1) reduced MMP-1, MMP-9 and MMP-12 gene expression. Collectively, these findings establish MLK3 as an important regulator of MMP expression and invasion in ovarian cancer cells. -- Highlights: ► Ovarian cancer cell lines have high levels of total and phosphorylated MLK3. ► MLK3 is required for MMP expression and activity in ovarian cancer cells. ► MLK3 is required for invasion of SKOV3 and HEY1B ovarian cancer cells. ► MLK3-dependent regulation of MMP-2 and MMP-9 activities requires ERK and JNK.

  7. Small molecule inhibition of Axl receptor tyrosine kinase potently suppresses multiple malignant properties of glioma cells

    Science.gov (United States)

    Vouri, Mikaella; An, Qian; Birt, Matthew; Pilkington, Geoffrey J.; Hafizi, Sassan

    2015-01-01

    Glioblastoma multiforme (GBM) often features a combination of tumour suppressor gene inactivation and multiple oncogene overactivation. The Axl receptor tyrosine kinase is found overexpressed in GBM and thought to contribute to invasiveness, chemoresistance and poor survival. Here, we have evaluated the effect of BGB324, a clinical candidate Axl-specific small molecule inhibitor, on the invasive behaviour of human GBM cells in vitro, as an indicator of its potential in GBM therapy and also to elucidate the role of Axl in GBM pathogenesis. Two cultured adult GBM cell lines, SNB-19 and UP007, were treated with Gas6 and/or BGB324, and analysed in assays for survival, 3D colony growth, motility, migration and invasion. Western blot was used to detect protein expression and signal protein phosphorylation. In both cell lines, BGB324 inhibited specifically phosphorylation of Axl as well as Akt kinase further downstream. BGB324 also inhibited survival and proliferation of both cell lines in a concentration-dependent manner, as well as completely suppressing migration and invasion. Furthermore, our results indicate co-operative activation between the Axl and Tyro3 receptors, as well as ligand-independent Axl signalling, to take place in GBM cells. In conclusion, small molecule inhibitor-led targeting of Axl may be a promising therapy for GBM progression. PMID:25980499

  8. Protein Tyrosine Nitration: Selectivity, physicochemical and biological consequences, denitration and proteomics methods for the identification of tyrosine-nitrated proteins

    NARCIS (Netherlands)

    Abello, N.; Kerstjens, H.A.M.; Postma, D.S; Bischoff, Rainer

    2009-01-01

    Protein tyrosine nitration (PTN) is a post-translational modification occurring under the action of a nitrating agent. Tyrosine is modified in the 3-position of the phenolic ring through the addition of a nitro group (NO2). In the present article, we review the main nitration reactions and elucidate

  9. Protein Tyrosine Nitration : Selectivity, Physicochemical and Biological Consequences, Denitration, and Proteomics Methods for the Identification of Tyrosine-Nitrated Proteins

    NARCIS (Netherlands)

    Abello, Nicolas; Kerstjens, Huib A. M.; Postma, Dirkje S.; Bischoff, Rainer

    Protein tyrosine nitration (PTN) is a post-translational modification occurring under the action of a nitrating agent. Tyrosine is modified in the 3-position of the phenolic ring through the addition of a nitro group (NO(2)). In the present article, we review the main nitration reactions and

  10. Prevention of pulmonary vascular and myocardial remodeling by the combined tyrosine and serine-/threonine kinase inhibitor, sorafenib, in pulmonary hypertension and right heart failure

    Directory of Open Access Journals (Sweden)

    M. Klein

    2008-06-01

    Full Text Available Inhibition of tyrosine kinases can reverse pulmonary hypertension but little is known about the role of serine-/threonine kinases in vascular and myocardial remodeling. We investigated the effects of sorafenib, an inhibitor of the tyrosine kinases VEGFR, PDGFR and c-kit as well as the serine-/threonine kinase Raf-1, in pulmonary hypertension and right ventricular (RV pressure overload. In monocrotaline treated rats, sorafenib (10 mg·kg–1·d–1 p.o. reduced pulmonary arterial pressure, pulmonary artery muscularization and RV hypertrophy, and improved systemic hemodynamics (table 1. Sorafenib prevented phosphorylation of Raf-1 and suppressed activation of downstream signaling pathways (Erk 1/2. After pulmonary banding, sorafenib, but not the PDGFR/c-KIT/ABL-inhibitor imatinib reduced RV mass and RV filling pressure significantly. Congruent with these results, sorafenib only prevented ERK phosphorylation and vasopressin induced hypertrophy of the cardiomyocyte cell line H9c2 dose dependently (IC50 = 300 nM. Combined inhibition of tyrosine and serine-/threonine kinases by sorafenib prevents vascular and cardiac remodeling in pulmonary hypertension, which is partly mediated via inhibition of the Raf kinase pathway.

  11. Matrix metalloproteinases in gastric inflammation and cancer : clinical relevance and prognostic impact

    NARCIS (Netherlands)

    Kubben, Francois Jozef Gerard Marie

    2007-01-01

    The studies in this thesis describe the clinical impact of several matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in H. pylori-induced gastritis and gastric cancer. In patients with H. pylori-induced gastritis, significantly increased mucosal MMP-9 levels were

  12. Regulation of brown fat adipogenesis by protein tyrosine phosphatase 1B.

    Directory of Open Access Journals (Sweden)

    Kosuke Matsuo

    2011-01-01

    Full Text Available Protein-tyrosine phosphatase 1B (PTP1B is a physiological regulator of insulin signaling and energy balance, but its role in brown fat adipogenesis requires additional investigation.To precisely determine the role of PTP1B in adipogenesis, we established preadipocyte cell lines from wild type and PTP1B knockout (KO mice. In addition, we reconstituted KO cells with wild type, substrate-trapping (D/A and sumoylation-resistant (K/R PTP1B mutants, then characterized differentiation and signaling in these cells. KO, D/A- and WT-reconstituted cells fully differentiated into mature adipocytes with KO and D/A cells exhibiting a trend for enhanced differentiation. In contrast, K/R cells exhibited marked attenuation in differentiation and lipid accumulation compared with WT cells. Expression of adipogenic markers PPARγ, C/EBPα, C/EBPδ, and PGC1α mirrored the differentiation pattern. In addition, the differentiation deficit in K/R cells could be reversed completely by the PPARγ activator troglitazone. PTP1B deficiency enhanced insulin receptor (IR and insulin receptor substrate 1 (IRS1 tyrosyl phosphorylation, while K/R cells exhibited attenuated insulin-induced IR and IRS1 phosphorylation and glucose uptake compared with WT cells. In addition, substrate-trapping studies revealed that IRS1 is a substrate for PTP1B in brown adipocytes. Moreover, KO, D/A and K/R cells exhibited elevated AMPK and ACC phosphorylation compared with WT cells.These data indicate that PTP1B is a modulator of brown fat adipogenesis and suggest that adipocyte differentiation requires regulated expression of PTP1B.

  13. Protein-tyrosine kinase activity profiling in knock down zebrafish embryos.

    Directory of Open Access Journals (Sweden)

    Simone Lemeer

    Full Text Available BACKGROUND: Protein-tyrosine kinases (PTKs regulate virtually all biological processes. PTKs phosphorylate substrates in a sequence-specific manner and relatively short peptide sequences determine selectivity. Here, we developed new technology to determine PTK activity profiles using peptide arrays. The zebrafish is an excellent model system to investigate signaling in the whole organism, given its wealth of genetic tools, including morpholino-mediated knock down technology. We used zebrafish embryo lysates to determine PTK activity profiles, thus providing the unique opportunity to directly compare the effect of protein knock downs on PTK activity profiles on the one hand and phenotypic changes on the other. METHODOLOGY: We used multiplex arrays of 144 distinct peptides, spotted on a porous substrate, allowing the sample to be pumped up and down, optimizing reaction kinetics. Kinase reactions were performed using complex zebrafish embryo lysates or purified kinases. Peptide phosphorylation was detected by fluorescent anti-phosphotyrosine antibody binding and the porous chips allowed semi-continuous recording of the signal. We used morpholinos to knock down protein expression in the zebrafish embryos and subsequently, we determined the effects on the PTK activity profiles. RESULTS AND CONCLUSION: Reproducible PTK activity profiles were derived from one-day-old zebrafiish embryos. Morpholino-mediated knock downs of the Src family kinases, Fyn and Yes, induced characteristic phenotypes and distinct changes in the PTK activity profiles. Interestingly, the peptide substrates that were less phosphorylated upon Fyn and Yes knock down were preferential substrates of purified Fyn and Yes. Previously, we demonstrated that Wnt11 knock down phenocopied Fyn/Yes knock down. Interestingly, Wnt11 knock down induced similar changes in the PTK activity profile as Fyn/Yes knock down. The control Nacre/Mitfa knock down did not affect the PTK activity profile

  14. The full oncogenic activity of Ret/ptc2 depends on tyrosine 539, a docking site for phospholipase Cgamma.

    Science.gov (United States)

    Borrello, M G; Alberti, L; Arighi, E; Bongarzone, I; Battistini, C; Bardelli, A; Pasini, B; Piutti, C; Rizzetti, M G; Mondellini, P; Radice, M T; Pierotti, M A

    1996-01-01

    RET/PTC oncogenes, generated by chromosomal rearrangements in papillary thyroid carcinomas, are constitutively activated versions of proto-RET, a gene coding for a receptor-type tyrosine kinase (TK) whose ligand is still unknown. RET/PTCs encode fusion proteins in which proto-RET TK and C-terminal domains are fused to different donor genes. The respective Ret/ptc oncoproteins display constitutive TK activity and tyrosine phosphorylation. We found that Ret/ptcs associate with and phosphorylate the SH2-containing transducer phospholipase Cgamma (PLCgamma). Two putative PLCgamma docking sites, Tyr-505 and Tyr-539, have been identified on Ret/ptc2 by competition experiments using phosphorylated peptides modelled on Ret sequence. Transfection experiments and biochemical analysis using Tyr-->Phe mutants of Ret/ptc2 allowed us to rule out Tyr-505 and to identify Tyr-539 as a functional PLCgamma docking site in vivo. Moreover, kinetic measurements showed that Tyr-539 is able to mediate high-affinity interaction with PLCgamma. Mutation of Tyr-539 resulted in a drastically reduced oncogenic activity of Ret/ptc2 on NIH 3T3 cells (75 to 90% reduction) both in vitro and in vivo, which correlates with impaired ability of Ret/ptc2 to activate PLCgamma. In conclusion, this paper demonstrates that Tyr-539 of Ret/ptc2 (Tyr-761 on the proto-RET product) is an essential docking site for the full transforming potential of the oncogene. In addition, the present data identify PLCgamma as a downstream effector of Ret/ptcs and suggest that this transducing molecule could play a crucial role in neoplastic signalling triggered by Ret/ptc oncoproteins. PMID:8628282

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

    Directory of Open Access Journals (Sweden)

    Silviya R Stateva

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

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

    Directory of Open Access Journals (Sweden)

    Pilar eAlmela

    2013-12-01

    Full Text Available 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 signalling, 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. BRD4 Phosphorylation Regulates HPV E2-Mediated Viral Transcription, Origin Replication, and Cellular MMP-9 Expression

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    Shwu-Yuan Wu

    2016-08-01

    Full Text Available Post-translational modification can modulate protein conformation and alter binding partner recruitment within gene regulatory regions. Here, we report that bromodomain-containing protein 4 (BRD4, a transcription co-factor and chromatin regulator, uses a phosphorylation-induced switch mechanism to recruit E2 protein encoded by cancer-associated human papillomavirus (HPV to viral early gene and cellular matrix metalloproteinase-9 (MMP-9 promoters. Enhanced MMP-9 expression, induced upon keratinocyte differentiation, occurs via BRD4-dependent recruitment of active AP-1 and NF-κB to their target sequences. This is triggered by replacement of AP-1 family members JunB and JunD by c-Jun and by re-localization of NF-κB from the cytoplasm to the nucleus. In addition, BRD4 phosphorylation is critical for E2- and origin-dependent HPV DNA replication. A class of phospho-BRD4-targeting compounds, distinct from the BET bromodomain inhibitors, effectively blocks BRD4 phosphorylation-specific functions in transcription and factor recruitment.

  18. Inhibitory effect of berberine on the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2

    International Nuclear Information System (INIS)

    Peng, P.-L.; Hsieh, Y.-S.; Wang, C.-J.; Hsu, J.-L.; Chou, F.-P.

    2006-01-01

    Berberine, a compound isolated from medicinal herbs, has been reported with many pharmacological effects related to anti-cancer and anti-inflammation capabilities. In this study, we observed that berberine exerted a dose- and time-dependent inhibitory effect on the motility and invasion ability of a highly metastatic A549 cells under non-cytotoxic concentrations. In cancer cell migration and invasion process, matrix-degrading proteinases are required. A549 cell treated with berberine at various concentrations showed reduced ECM proteinases including matrix metalloproteinase-2 (MMP2) and urokinase-plasminogen activator (u-PA) by gelatin and casein zymography analysis. The inhibitory effect is likely to be at the transcriptional level, since the reduction in the transcripts levels was corresponding to the proteins. Moreover, berberine also exerted its action via regulating tissue inhibitor of metalloproteinase-2 (TIMP-2) and urokinase-plasminogen activator inhibitor (PAI). The upstream mediators of the effect involved c-jun, c-fos and NF-κB, as evidenced by reduced phosphorylation of the proteins. These findings suggest that berberine possesses an anti-metastatic effect in non-small lung cancer cell and may, therefore, be helpful in clinical treatment

  19. Caffeic Acid Phenethyl Ester Inhibits Oral Cancer Cell Metastasis by Regulating Matrix Metalloproteinase-2 and the Mitogen-Activated Protein Kinase Pathway

    Directory of Open Access Journals (Sweden)

    Chih-Yu Peng

    2012-01-01

    Full Text Available Caffeic acid phenethyl ester (CAPE, an active component extracted from honeybee hives, exhibits anti-inflammatory and anticancer activities. However, the molecular mechanism by which CAPE affects oral cancer cell metastasis has yet to be elucidated. In this study, we investigated the potential mechanisms underlying the effects of CAPE on the invasive ability of SCC-9 oral cancer cells. Results showed that CAPE attenuated SCC-9 cell migration and invasion at noncytotoxic concentrations (0 μM to 40 μM. Western blot and gelatin zymography analysis findings further indicated that CAPE downregulated matrix metalloproteinase-2 (MMP-2 protein expression and inhibited its enzymatic activity. CAPE exerted its inhibitory effects on MMP-2 expression and activity by upregulating tissue inhibitor of metalloproteinase-2 (TIMP-2 and potently decreased migration by reducing focal adhesion kinase (FAK phosphorylation and the activation of its downstream signaling molecules p38/MAPK and JNK. These data indicate that CAPE could potentially be used as a chemoagent to prevent oral cancer metastasis.

  20. Osthole Suppresses the Migratory Ability of Human Glioblastoma Multiforme Cells via Inhibition of Focal Adhesion Kinase-Mediated Matrix Metalloproteinase-13 Expression

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    Cheng-Fang Tsai

    2014-03-01

    Full Text Available Glioblastoma multiforme (GBM is the most common type of primary and malignant tumor occurring in the adult central nervous system. GBM often invades surrounding regions of the brain during its early stages, making successful treatment difficult. Osthole, an active constituent isolated from the dried C. monnieri fruit, has been shown to suppress tumor migration and invasion. However, the effects of osthole in human GBM are largely unknown. Focal adhesion kinase (FAK is important for the metastasis of cancer cells. Results from this study show that osthole can not only induce cell death but also inhibit phosphorylation of FAK in human GBM cells. Results from this study show that incubating GBM cells with osthole reduces matrix metalloproteinase (MMP-13 expression and cell motility, as assessed by cell transwell and wound healing assays. This study also provides evidence supporting the potential of osthole in reducing FAK activation, MMP-13 expression, and cell motility in human GBM cells.

  1. Regulation of gap junctions by protein phosphorylation

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    J.C. Sáez

    1998-05-01

    Full Text Available Gap junctions are constituted by intercellular channels and provide a pathway for transfer of ions and small molecules between adjacent cells of most tissues. The degree of intercellular coupling mediated by gap junctions depends on the number of gap junction channels and their activity may be a function of the state of phosphorylation of connexins, the structural subunit of gap junction channels. Protein phosphorylation has been proposed to control intercellular gap junctional communication at several steps from gene expression to protein degradation, including translational and post-translational modification of connexins (i.e., phosphorylation of the assembled channel acting as a gating mechanism and assembly into and removal from the plasma membrane. Several connexins contain sites for phosphorylation for more than one protein kinase. These consensus sites vary between connexins and have been preferentially identified in the C-terminus. Changes in intercellular communication mediated by protein phosphorylation are believed to control various physiological tissue and cell functions as well as to be altered under pathological conditions.

  2. Phosphorylation and Internalization of Lysophosphatidic Acid Receptors LPA1, LPA2, and LPA3.

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    Rocío Alcántara-Hernández

    Full Text Available The lysophosphatidic acid receptors LPA1, LPA2, and LPA3 were individually expressed in C9 cells and their signaling and regulation were studied. Agonist-activation increases intracellular calcium concentration in a concentration-dependent fashion. Phorbol myristate acetate markedly inhibited LPA1- and LPA3-mediated effect, whereas that mediated by LPA2 was only partially diminished; the actions of the phorbol ester were inhibited by bisindolylmaleimide I and by overnight incubation with the protein kinase C activator, which leads to down regulation of this protein kinase. Homologous desensitization was also observed for the three LPA receptors studied, with that of LPA2 receptors being consistently of lesser magnitude; neither inhibition nor down-regulation of protein kinase C exerted any effect on homologous desensitization. Activation of LPA1-3 receptors induced ERK 1/2 phosphorylation; this effect was markedly attenuated by inhibition of epidermal growth factor receptor tyrosine kinase activity, suggesting growth factor receptor transactivation in this effect. Lysophosphatidic acid and phorbol myristate acetate were able to induce LPA1-3 phosphorylation, in time- and concentration-dependent fashions. It was also clearly observed that agonists and protein kinase C activation induced internalization of these receptors. Phosphorylation of the LPA2 subtype required larger concentrations of these agents and its internalization was less intense than that of the other subtypes.Our data show that these three LPA receptors are phosphoproteins whose phosphorylation state is modulated by agonist-stimulation and protein kinase C-activation and that differences in regulation and cellular localization exist, among the subtypes.

  3. Acknowledged Signatures of Matrix Metalloproteinases in Takayasu’s Arteritis

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    Gang Wu

    2014-01-01

    Full Text Available Takayasu’s arteritis (TA was reported as an eye disease in the year 1905 and later was confirmed as a vasculitis. Since then, the etiology of the disease remains unknown; however, characteristic clinical features suggest multiple causative factors. Recent progress in vascular biology and other disciplines enlightens the pathophysiology of TA and demonstrated induction of various nonspecific inflammatory symptoms and destruction of the arterial wall, which leads to aneurysms and rupture of the affected arteries. Matrix metalloproteinases (MMPs as an enzyme family have well-established roles in several vascular pathologies including intima formation, atherosclerosiss and aneurysms. MMPs have been proposed to be one of the molecules with a potential of having dual role in the course of TA, first as an active participant in pathophysiology and secondly as a diagnostic biomarker for TA disease. The desire to improve our understanding of the importance of MMPs and their endogenous inhibitors (TIMPs in TA disease and for the development of therapeutic agents has inspired basic and clinical scientists for over a decade. In the present paper, we summarized the scientific rationale which highlights the signatures of matrix metalloproteinases and their endogenous inhibitors in pathophysiology as well as their being a potential candidate as biomarker for Takayasu’s arteritis.

  4. New intracellular activities of matrix metalloproteinases shine in the moonlight.

    Science.gov (United States)

    Jobin, Parker G; Butler, Georgina S; Overall, Christopher M

    2017-11-01

    Adaption of a single protein to perform multiple independent functions facilitates functional plasticity of the proteome allowing a limited number of protein-coding genes to perform a multitude of cellular processes. Multifunctionality is achievable by post-translational modifications and by modulating subcellular localization. Matrix metalloproteinases (MMPs), classically viewed as degraders of the extracellular matrix (ECM) responsible for matrix protein turnover, are more recently recognized as regulators of a range of extracellular bioactive molecules including chemokines, cytokines, and their binders. However, growing evidence has convincingly identified select MMPs in intracellular compartments with unexpected physiological and pathological roles. Intracellular MMPs have both proteolytic and non-proteolytic functions, including signal transduction and transcription factor activity thereby challenging their traditional designation as extracellular proteases. This review highlights current knowledge of subcellular location and activity of these "moonlighting" MMPs. Intracellular roles herald a new era of MMP research, rejuvenating interest in targeting these proteases in therapeutic strategies. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Role of matrix metalloproteinases in the pathogenesis of childhood gastroenteritis.

    Science.gov (United States)

    Kawamura, Yoshiki; Gotoh, Kensei; Takeuchi, Nao; Miura, Hiroki; Nishimura, Naoko; Ozaki, Takao; Yoshikawa, Tetsushi

    2016-08-01

    Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been implicated in the pathogenesis of gastrointestinal diseases, such as rotavirus gastroenteritis (GE). Kinetics of these biomarkers were examined in paired serum samples collected from bacterial enteritis patients with Campylobacter (n = 2) and Salmonella (n = 4) and viral GE patients with rotavirus (n = 27), norovirus (n = 25), and adenovirus (n = 11). At the time of hospital admission, all viral GE patients demonstrated increased MMP-9 and decreased MMP-2 and TIMP-2 serum levels. In contrast to viral GE patients, serum MMP-9 levels were not elevated at the time of hospital admission but elevated at the time of discharge; serum MMP-2 and TIMP-2 levels were decreased both at the time of admission and discharge in bacterial enteritis patients. Interestingly, the kinetics of serum MMP-2, MMP-9, and TIMP-2 levels were similar among the viral GE patients but distinct from bacterial enteritis patients. Thus, the involvement of MMPs and TIMPs in the pathophysiology of gastrointestinal symptoms likely varies depending on the etiological agent. Further studies are required to verify whether the extent of the bacterial enteritis or age of the patients influences these serum biomarkers. J. Med. Virol. 88:1341-1346, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  6. A PLC-γ1 Feedback Pathway Regulates Lck Substrate Phosphorylation at the T-Cell Receptor and SLP-76 Complex.

    Science.gov (United States)

    Belmont, Judson; Gu, Tao; Mudd, Ashley; Salomon, Arthur R

    2017-08-04

    Phospholipase C gamma 1 (PLC-γ1) occupies a critically important position in the T-cell signaling pathway. While its functions as a regulator of both Ca 2+ signaling and PKC-family kinases are well characterized, PLC-γ1's role in the regulation of early T-cell receptor signaling events is incompletely understood. Activation of the T-cell receptor leads to the formation of a signalosome complex between SLP-76, LAT, PLC-γ1, Itk, and Vav1. Recent studies have revealed the existence of both positive and negative feedback pathways from SLP-76 to the apical kinase in the pathway, Lck. To determine if PLC-γ1 contributes to the regulation of these feedback networks, we performed a quantitative phosphoproteomic analysis of PLC-γ1-deficient T cells. These data revealed a previously unappreciated role for PLC-γ1 in the positive regulation of Zap-70 and T-cell receptor tyrosine phosphorylation. Conversely, PLC-γ1 negatively regulated the phosphorylation of SLP-76-associated proteins, including previously established Lck substrate phosphorylation sites within this complex. While the positive and negative regulatory phosphorylation sites on Lck were largely unchanged, Tyr 192 phosphorylation was elevated in Jgamma1. The data supports a model wherein Lck's targeting, but not its kinase activity, is altered by PLC-γ1, possibly through Lck Tyr 192 phosphorylation and increased association of the kinase with protein scaffolds SLP-76 and TSAd.

  7. Ligand-induced tyrosine phosphorylation of cysteinyl leukotriene receptor 1 triggers internalization and signaling in intestinal epithelial cells

    DEFF Research Database (Denmark)

    Parhamifar, Ladan; Sime, Wondossen; Yudina, Yuliana

    2010-01-01

    Leukotriene D(4) (LTD(4)) belongs to the bioactive lipid group known as eicosanoids and has implications in pathological processes such as inflammation and cancer. Leukotriene D(4) exerts its effects mainly through two different G-protein-coupled receptors, CysLT(1) and CysLT(2). The high affinit...

  8. Hyperglycemia-Induced Vasculopathy in the Murine Vitelline Vasculature : Correlation with PECAM-1/CD31 Tyrosine Phosphorylation State

    OpenAIRE

    Pinter, Emese; Mahooti, Sepi; Wang, Yi; Imhof, Beat A.; Madri, Joseph A.

    1999-01-01

    Maternal diabetes mellitus is associated with an increased incidence of congenital abnormalities as well as embryonic and perinatal lethality. In particular, a wide range of cardiovascular abnormalities have been noted in children of diabetic mothers and in the offspring of diabetic animals. The vascular system is the first organ system to develop in the embryo and is critical for normal organogenesis. The organization of mesodermal cells into endothelial and hematopoietic cells and into a co...

  9. What precedes the initial tyrosine phosphorylation of the high affinity IgE receptor in antigen-activated mast cell?

    Czech Academy of Sciences Publication Activity Database

    Bugajev, Viktor; Bambousková, Monika; Dráberová, Lubica; Dráber, Petr

    2010-01-01

    Roč. 584, č. 24 (2010), s. 4949-4955 ISSN 0014-5793 R&D Projects: GA MŠk 1M0506; GA MŠk LC545; GA ČR(CZ) GD204/05/H023; GA ČR GA301/09/1826; GA ČR GAP302/10/1759 Institutional research plan: CEZ:AV0Z50520514 Keywords : mast cell * cell signaling * plasma membrane Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.601, year: 2010

  10. Regulation of Ca2+ signaling in mast cells by tyrosine-phosphorylated and unphosphorylated non-T cell activation linker

    Czech Academy of Sciences Publication Activity Database

    Dráberová, Lubica; Shaik, Gouse Mohiddin; Lusková, Petra; Heneberg, Petr; Tůmová, Magda; Lebduška, Pavel; Korb, Jan; Dráber, Petr

    2007-01-01

    Roč. 179, č. 8 (2007), s. 5169-5180 ISSN 0022-1767 R&D Projects: GA MŠk 1M0506; GA MŠk LC545; GA ČR GD204/05/H023; GA ČR GA301/06/0361; GA AV ČR IAA5052310; GA AV ČR 1QS500520551 Institutional research plan: CEZ:AV0Z50520514 Keywords : mast cells * plasma membrane * adaptor protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.068, year: 2007

  11. Dual-specificity tyrosine phosphorylation-regulated kinase 2 regulates osteoclast fusion in a cell heterotypic manner.

    Science.gov (United States)

    Guterman-Ram, Gali; Pesic, Milena; Orenbuch, Ayelet; Czeiger, Tal; Aflalo, Anastasia; Levaot, Noam

    2018-01-01

    Monocyte fusion into osteoclasts, bone resorbing cells, plays a key role in bone remodeling and homeostasis; therefore, aberrant cell fusion may be involved in a variety of debilitating bone diseases. Research in the last decade has led to the discovery of genes that regulate osteoclast fusion, but the basic molecular and cellular regulatory mechanisms underlying the fusion process are not completely understood. Here, we reveal a role for Dyrk2 in osteoclast fusion. We demonstrate that Dyrk2 down regulation promotes osteoclast fusion, whereas its overexpression inhibits fusion. Moreover, Dyrk2 also promotes the fusion of foreign-body giant cells, indicating that Dyrk2 plays a more general role in cell fusion. In an earlier study, we showed that fusion is a cell heterotypic process initiated by fusion-founder cells that fuse to fusion-follower cells, the latter of which are unable to initiate fusion. Here, we show that Dyrk2 limits the expansion of multinucleated founder cells through the suppression of the fusion competency of follower cells. © 2017 Wiley Periodicals, Inc.

  12. Human platelets activation by convulxin is accompanied by tyrosyl-phosphorylation of PLCgamma2 and occurs independently of integrin alphaIIbbeta3.

    Science.gov (United States)

    Francischetti, I M; Carlini, C R; Guimàraes, J A

    1998-01-01

    In the present report we show that convulxin (Cvx), a C-type lectin from Crotalus durissus terrificus venom, induces platelet agregation and phospholipase C (PLC) activation by a protein tyrosine kinase (PTK)-dependent pathway. In addition, Cvx stimulates a rapid increase in tyrosine phosphorylation of human platelet proteins with molecular masses of 40, 72/74, 78/80 and 120 kDa, followed by dephosphorylation of some proteins. However, platelet aggregation was accompanied by the phosphorylation of a 105-kDa molecular mass protein. Furthermore, Cvx stimulates a rapid-tyrosyl phosphorylation of a 145-kDa protein that was identified as PLC gamma 2. Protein tyrose phosphatase (PTP) induced by Cvx was not blocked when platelets were stimulated in the presence of indomethacin, apyrase, EDTA or RGDS peptide, but inhibited by staurosporine and genistein. These results indicate that PTP is chronologically proximal to Cvx binding to platelets, and it is independent of platelet aggregation or fibrinogen binding to integrin alphaIIbbeta3. On the other hand, the phosphorylation step, and the phosphorylation of the 105-kDa protein, were both inhibited by RGDS and EDTA, which suggests that the integrin alphaIIbbeta3 beta is involved in these steps. Our results, taken together, show that Cvx induces platelet IIb 3 aggregation in a similar manner as collagen and collagen-related peptides that also trigger platelet aggregation by a PTK-dependent pathway, and stimulate tyrosyl-phosphorylation of PLC gamma 2. However, Cvx is unique among platelet receptor agonists, because under test-tube stirring conditions it induces a PTP profile independently of integrin alphaIIbbeta3.

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

    Directory of Open Access Journals (Sweden)

    Raabe Timothy D

    2003-04-01

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

  14. Differential Requirements for Src-Family Kinases in SYK or ZAP70-Mediated SLP-76 Phosphorylation in Lymphocytes.

    Science.gov (United States)

    Fasbender, Frank; Claus, Maren; Wingert, Sabine; Sandusky, Mina; Watzl, Carsten

    2017-01-01

    In a synthetic biology approach using Schneider (S2) cells, we show that SLP-76 is directly phosphorylated at tyrosines Y113 and Y128 by SYK in the presence of ITAM-containing adapters such as CD3ζ, DAP12, or FcεRγ. This phosphorylation was dependent on at least one functional ITAM and a functional SH2 domain within SYK. Inhibition of Src-kinases by inhibitors PP1 and PP2 did not reduce SLP-76 phosphorylation in S2 cells, suggesting an ITAM and SYK dependent, but Src-kinase independent signaling pathway. This direct ITAM/SYK/SLP-76 signaling pathway therefore differs from previously described ITAM signaling. However, the SYK-family kinase ZAP70 required the additional co-expression of the Src-family kinases Fyn or Lck to efficiently phosphorylate SLP-76 in S2 cells. This difference in Src-family kinase dependency of SYK versus ZAP70-mediated ITAM-based signaling was further demonstrated in human lymphocytes. ITAM signaling in ZAP70-expressing T cells was dependent on the activity of Src-family kinases. In contrast, Src-family kinases were partially dispensable for ITAM signaling in SYK-expressing B cells or in natural killer cells, which express SYK and ZAP70. This demonstrates that SYK can signal using a Src-kinase independent ITAM-based signaling pathway, which may be involved in calibrating the threshold for lymphocyte activation.

  15. Identification of new phosphorylation sites of AMPA receptors in the rat hippocampus--A resource for neuroscience research.

    Science.gov (United States)

    Ghafari, Maryam; Keihan Falsafi, Soheil; Höger, Harald; Bennett, Keiryn L; Lubec, Gert

    2015-10-01

    AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs) are glutamate-gated ion channels that mediate the majority of fast excitatory synaptic transmissions in the mammalian brain. A series of phosphorylation sites have been predicted or identified and knowledge on phosphorylations is mandatory for understanding receptor biology and functions. Immunoprecipitation from extracted hippocampal rat proteins was carried out using an antibody against the AMPAR GluA1 subunit, followed by identification of GluA1 and binding partners by MS. Bands from SDS-PAGE were picked, peptides were generated by trypsin and chymotrypsin digestion and identified by MS/MS (LTQ Orbitrap Velos). Using Mascot as a search engine, phosphorylation sites S506, S645, S720, S849, S863, S895, T858, Y228, Y419, and T734 were found on GluA1; S357, S513, S656, S727, T243, T420, T741, Y 143, Y301,Y426 on GluA2; S301, S516, S657, S732, T222, and T746 were observed on GluA3; and S514, S653 was phosphorylated on GluA4. A series of additional protein modifications were observed and in particular, tyrosine and tryptophan nitrations on GluA1 were detected that may raise questions on additional regulation mechanisms for AMPARs in addition to phosphorylations. The findings are relevant for interpretation of previous work and design of future studies using AMPAR serving as a resource for neuroscience research and indeed, phosphorylations and PTMs per se would have to be respected when neuropathological and neurological disorders are being studied. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. The plasma and peritoneal fluid concentrations of matrix metalloproteinase-9 are elevated in patients with endometriosis.

    Science.gov (United States)

    Liu, Haiping; Wang, Jianye; Wang, Haiyu; Tang, Ning; Li, Yunfei; Zhang, Yan; Hao, Tianyu

    2016-09-01

    Enzyme matrix metalloproteinase-9 is a member of the matrix metalloproteinase family, which is critical to normal tissue remodelling during embryogenesis and wound healing. In patients with endometriosis, increased expression and activity of matrix metalloproteinase-9 have been observed in ectopic endometrium, but the plasma and peritoneal fluid concentrations of matrix metalloproteinase-9 in patients with endometriosis and their relation to disease severity have not been clear. The aim of the study was to investigate the concentrations of matrix metalloproteinase-9 in plasma and peritoneal fluid of patients with endometriosis. A prospective case-control study was conducted in Jinan Military General Hospital between January 2010 and December 2013. Fifty patients with proven endometriosis and 26 endometriosis-free controls were enrolled in this study. Patients with endometriosis were evaluated and divided into moderate/severe endometriosis group (stage I-II, n = 26) and minimal/mild endometriosis group (stage III-IV, n = 24) according to the revised criteria of the American Society for Reproductive Medicine. Blood samples and peritoneal fluid were obtained from both patients and controls. Matrix metalloproteinase-9 was measured using enzyme-linked immunosorbent assay in plasma and peritoneal fluid. The concentration of matrix metalloproteinase-9 between different groups was compared and its correlation to disease severity was analysed. Plasma and peritoneal fluid concentrations of matrix metalloproteinase-9 in patients with endometriosis were higher than that in controls. In addition, those patients with moderate/severe endometriosis had significantly higher plasma and peritoneal fluid concentrations of matrix metalloproteinase-9 compared to those with minimal/mild endometriosis. Matrix metalloproteinase-9 concentrations in plasma and peritoneal fluid were both positively correlated with severity of endometriosis and plasma matrix metalloproteinase-9

  17. Matrix metalloproteinase 2 (MMP-2) levels are increased in active acromegaly patients.

    Science.gov (United States)

    Karci, Alper Cagri; Canturk, Zeynep; Tarkun, Ilhan; Cetinarslan, Berrin

    2017-07-01

    During follow-up of acromegaly patients, there is a discordance rate of 30% between the measurements of growth hormone and insulin-like growth factor-1 levels. Further tests are required to determine disease activity in patients with discordant results. This study was planned to investigate an association of serum levels of matrix metalloproteinase-2, matrix metalloproteinase-9, and cathepsin B with disease activity in acromegaly patients. In this study, 64 acromegaly patients followed in our clinic were divided into two groups according to the 2010 consensus criteria for cure of acromegaly as patients with active disease (n = 24) and patients with controlled disease (n = 40). Serum matrix metalloproteinase-2, matrix metalloproteinase-9, and cathepsin B levels were measured by the enzyme-linked immunosorbent assay method. The mean serum matrix metalloproteinase-2 level was significantly higher in the active acromegaly patients than in the controlled acromegaly patients (150.1 ± 54.5 ng/mL vs. 100.2 ± 44.6 ng/mL; p matrix metalloproteinase-9 and cathepsin B levels (p = 0.205 and p = 0.598, respectively). Serum matrix metalloproteinase-2 levels of 118.3 ng/mL and higher had a sensitivity of 75% and a specificity of 77.5% in determining active disease. The risk of active acromegaly was 3.3 fold higher in the patients with a matrix metalloproteinase-2 level of >118.3 ng/mL than in the patients with a matrix metalloproteinase-2 level of matrix metalloproteinase-2 level is increased in the active acromegaly patients and a threshold value in determining active disease was defined for serum matrix metalloproteinase-2 level. This study is the first to compare acromegaly patients having active or controlled disease in terms of matrix metalloproteinase-2 and matrix metalloproteinase-9 levels. The results need to be confirmed by a study that will be conducted in a larger patient group also including a healthy control group to demonstrate the

  18. Modulation of fatty acid synthase degradation by concerted action of p38 MAP kinase, E3 ligase COP1, and SH2-tyrosine phosphatase Shp2.

    Science.gov (United States)

    Yu, Jianxiu; Deng, Rong; Zhu, Helen H; Zhang, Sharon S; Zhu, Changhong; Montminy, Marc; Davis, Roger; Feng, Gen-Sheng

    2013-02-08

    The Src-homology 2 (SH2) domain-containing tyrosine phosphatase Shp2 has been known to regulate various signaling pathways triggered by receptor and cytoplasmic tyrosine kinases. Here we describe a novel function of Shp2 in control of lipid metabolism by mediating degradation of fatty acid synthase (FASN). p38-phosphorylated COP1 accumulates in the cytoplasm and subsequently binds FASN through Shp2 here as an adapter