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Sample records for receptor tyrosine kinase-positive

  1. Characterization, biodistribution and small-animal SPECT of I-125-labeled c-Met binding peptide in mice bearing c-Met receptor tyrosine kinase-positive tumor xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Eun-Mi; Park, Eun-Hye; Cheong, Su-Jin; Lee, Chang-Moon; Kim, Dong Wook [Department of Nuclear Medicine, Chonbuk National University Medical School and Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Research Institute of Clinical Medicine, Chonbuk National University Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Cyclotron Research Center, Chonbuk National University Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Jeong, Hwan-Jeong [Department of Nuclear Medicine, Chonbuk National University Medical School and Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Research Institute of Clinical Medicine, Chonbuk National University Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Cyclotron Research Center, Chonbuk National University Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of)], E-mail: jayjeong@chonbuk.ac.kr; Lim, Seok Tae; Sohn, Myung-Hee [Department of Nuclear Medicine, Chonbuk National University Medical School and Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Research Institute of Clinical Medicine, Chonbuk National University Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Cyclotron Research Center, Chonbuk National University Hospital, Jeonju-si, Jeonbuk 561-712 (Korea, Republic of); Kim, Kisu; Chung, Junho [Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

    2009-05-15

    c-Met is a receptor tyrosine kinase involved in tumor cell growth, invasion, metastases and angiogenesis. Overexpression of c-Met is frequently observed in several tumor types. Here, we report the in vitro cell-binding properties and biodistribution and SPECT/CT imaging in glioma (U87MG) xenograft-bearing mice of {sup 125}I-labeled c-Met-binding peptides (cMBPs) including analogs conjugated to amino acid and aliphatic carbon linkers. In vitro assays showed that the peptide without any linker and those with GGG and 8-aminooctanoic acid linkers had low cellular internalization and that IC{sub 50} values of peptides were 1.5 {mu}M, 65 nM and 85.3 nM, respectively. Biodistribution studies showed the GGG-containing peptide had higher tumor uptake and a higher tumor-to-blood activity concentration ratio than other receptor-binding ligands. SPECT/CT studies with a dedicated small-animal imaging system were performed in U87MG-bearing athymic mice. Although U87MG tumor xenografts could be visualized by SPECT/micro-CT using the various {sup 125}I labeled cMBPs, image contrast and overall quality were unremarkable.

  2. 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

  3. 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.

  4. 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.

  5. Concomitant occurrence of EGFR (epidermal growth factor receptor) and KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) mutations in an ALK (anaplastic lymphoma kinase)-positive lung adenocarcinoma patient with acquired resistance to crizotinib

    DEFF Research Database (Denmark)

    Rossing, Henrik H; Grauslund, Morten; Urbanska, Edyta M

    2013-01-01

    , the events behind crizotinib-resistance currently remain largely uncharacterized. Thus, we report on an anaplastic lymphoma kinase-positive non-small cell lung carcinoma patient with concomitant occurrence of epidermal growth factor receptor and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog mutations...

  6. BRET biosensor analysis of receptor tyrosine kinase functionality

    Directory of Open Access Journals (Sweden)

    Sana eSiddiqui

    2013-04-01

    Full Text Available Bioluminescence resonance energy transfer (BRET is an improved version of earlier resonance energy transfer technologies used for the analysis of biomolecular protein interaction. BRET analysis can be applied to many transmembrane receptor classes, however the majority of the early published literature on BRET has focused on G protein-coupled receptor (GPCR research. In contrast, there is limited scientific literature using BRET to investigate receptor tyrosine kinase (RTK activity. This limited investigation is surprising as RTKs often employ dimerization as a key factor in their activation, as well as being important therapeutic targets in medicine, especially in the cases of cancer, diabetes, neurodegenerative and respiratory conditions. In this review, we consider an array of studies pertinent to RTKs and other non-GPCR receptor protein-protein signaling interactions; more specifically we discuss receptor-protein interactions involved in the transmission of signaling communication. We have provided an overview of functional BRET studies associated with the receptor tyrosine kinase (RTK super family involving: neurotrophic receptors (e.g. tropomyosin-related kinase (Trk and p75 neurotrophin receptor (p75NTR; insulinotropic receptors (e.g. insulin receptor (IR and insulin-like growth factor receptor (IGFR and growth factor receptors (e.g. ErbB receptors including the EGFR, the fibroblast growth factor receptor (FGFR, the vascular endothelial growth factor receptor (VEGFR and the c-kit and platelet-derived growth factor receptor (PDGFR. In addition, we review BRET-mediated studies of other tyrosine kinase-associated receptors including cytokine receptors, i.e. leptin receptor (OB-R and the growth hormone receptor (GHR. It is clear even from the relatively sparse experimental RTK BRET evidence that there is tremendous potential for this technological application for the functional investigation of RTK biology.

  7. A CSF-1 Receptor Phosphotyrosine 559 Signaling Pathway Regulates Receptor Ubiquitination and Tyrosine Phosphorylation*

    Science.gov (United States)

    Xiong, Ying; Song, Da; Cai, Yunfei; Yu, Wenfeng; Yeung, Yee-Guide; Stanley, E. Richard

    2011-01-01

    Receptor tyrosine kinase (RTK) activation involves ligand-induced receptor dimerization and transphosphorylation on tyrosine residues. Colony-stimulating factor-1 (CSF-1)-induced CSF-1 receptor (CSF-1R) tyrosine phosphorylation and ubiquitination were studied in mouse macrophages. Phosphorylation of CSF-1R Tyr-559, required for the binding of Src family kinases (SFKs), was both necessary and sufficient for these responses and for c-Cbl tyrosine phosphorylation and all three responses were inhibited by SFK inhibitors. In c-Cbl-deficient macrophages, CSF-1R ubiquitination and tyrosine phosphorylation were substantially inhibited. Reconstitution with wild-type, but not ubiquitin ligase-defective C381A c-Cbl rescued these responses, while expression of C381A c-Cbl in wild-type macrophages suppressed them. Analysis of site-directed mutations in the CSF-1R further suggests that activated c-Cbl-mediated CSF-1R ubiquitination is required for a conformational change in the major kinase domain that allows amplification of receptor tyrosine phosphorylation and full receptor activation. Thus the results indicate that CSF-1-mediated receptor dimerization leads to a Tyr-559/SFK/c-Cbl pathway resulting in receptor ubiquitination that permits full receptor tyrosine phosphorylation of this class III RTK in macrophages. PMID:21041311

  8. Retinitis pigmentosa: mutations in a receptor tyrosine kinase gene ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Biosciences; Volume 26; Issue 1. Clipboard: Retinitis pigmentosa: mutations in a receptor tyrosine kinase gene, MERTK. Arun Kumar. Volume 26 Issue 1 March 2001 pp 3-5. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/jbsc/026/01/0003-0005 ...

  9. DMPD: Receptor tyrosine kinases and the regulation of macrophage activation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14726496 Receptor tyrosine kinases and the regulation of macrophage activation. Cor...(.csml) Show Receptor tyrosine kinases and the regulation of macrophage activation. PubmedID 14726496 Title ...Receptor tyrosine kinases and the regulation of macrophage activation. Authors Co

  10. 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...

  11. 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......-binding activity, whereas the GH receptor mutant lacking all intracellular tyrosines was not. Synthetic tyrosine phosphorylated peptides corresponding to the GH receptor sequence around the three tyrosines inhibited Stat5 DNA-binding activity while their non-phosphorylated counterparts were ineffective. Tyrosine...... phosphorylated GST-GH receptor fusion proteins specifically bound to Stat5 in extracts from COS 7 cells transfected with Stat5 cDNA. This binding could be inhibited by tyrosine phosphorylated peptides derived from the GH receptor. This study thus demonstrated that specific GH receptor tyrosine residues...

  12. Cloning and expression of a widely expressed receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Sap, J; D'Eustachio, P; Givol, D

    1990-01-01

    and Bmp-2a loci. The corresponding mRNA (3.0 kilobases) is expressed in most murine tissues and most abundantly expressed in brain and kidney. Antibodies against a synthetic peptide of R-PTP-alpha identified a 130-kDa protein in cells transfected with the R-PTP-alpha cDNA.......We describe the identification of a widely expressed receptor-type (transmembrane) protein tyrosine phosphatase (PTPase; EC 3.1.3.48). Screening of a mouse brain cDNA library under low-stringency conditions with a probe encompassing the intracellular (phosphatase) domain of the CD45 lymphocyte...... antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

  13. G-protein-coupled receptors and tyrosine kinases: crossroads in cell signaling and regulation.

    Science.gov (United States)

    Gavi, Shai; Shumay, Elena; Wang, Hsien-yu; Malbon, Craig C

    2006-03-01

    G-protein-coupled receptors and protein tyrosine kinases represent two prominent pathways for cellular signaling. As our knowledge of cell signaling pathways mediated by the superfamily of G-protein-coupled receptors and the smaller family of receptor tyrosine kinases expands, so does our appreciation of how these two major signaling platforms share information and modulate each other, otherwise termed "cross-talk". Cross-talk between G-protein-coupled receptors and tyrosine kinases can occur at several levels, including the receptor-to-receptor level, and at crucial downstream points (e.g. phosphatidylinositol-3-kinase, Akt/protein kinase B and the mitogen-activated protein kinase cascade). Regulation of G-protein-coupled receptors by non-receptor tyrosine kinases, such as Src family members, also operates in signaling. A broader understanding of how G-protein-coupled receptors and tyrosine kinases cross-talk reveals new insights into signaling modalities in both health and disease.

  14. Receptor tyrosine kinases and schistosome reproduction: new targets for chemotherapy

    Directory of Open Access Journals (Sweden)

    Marion eMorel

    2014-07-01

    Full Text Available Schistosome parasites still represent a serious public health concern and a major economic problem in developing countries. Pathology of schistosomiasis is mainly due to massive egg production by these parasites and to inflammatory responses raised against the eggs which are trapped in host tissues. Tyrosine kinases (TKs are key molecules that control cell differentiation and proliferation and they already represent important targets in cancer therapy. During the recent years, it has been shown that receptor tyrosine kinases (RTK signaling was active in reproductive organs and that it could regulate sexual maturation of schistosomes and egg production. This opens interesting perspectives for the control of transmission and pathogenesis of schistosomiasis based on new therapies targeting schistosome RTKs. This review relates the numerous data showing the major roles of kinase signaling in schistosome reproduction. It describes the conserved and particular features of schistosome RTKs, their implication in gametogenesis and reproduction processes and summarizes recent works indicating that RTKs and their signaling partners are interesting chemotherapeutical targets in new programs of control.

  15. The Receptor Tyrosine Kinase AXL in Cancer Progression

    Directory of Open Access Journals (Sweden)

    Erinn B. Rankin

    2016-11-01

    Full Text Available The AXL receptor tyrosine kinase (AXL has emerged as a promising therapeutic target for cancer therapy. Recent studies have revealed a central role of AXL signaling in tumor proliferation, survival, stem cell phenotype, metastasis, and resistance to cancer therapy. Moreover, AXL is expressed within cellular components of the tumor microenvironment where AXL signaling contributes to the immunosuppressive and protumorigenic phenotypes. A variety of AXL inhibitors have been developed and are efficacious in preclinical studies. These agents offer new opportunities for therapeutic intervention in the prevention and treatment of advanced disease. Here we review the literature that has illuminated the cellular and molecular mechanisms by which AXL signaling promotes tumor progression and we will discuss the therapeutic potential of AXL inhibition for cancer therapy.

  16. Tyrosine Kinase Receptor Landscape in Lung Cancer: Therapeutical Implications

    Directory of Open Access Journals (Sweden)

    A. Quintanal-Villalonga

    2016-01-01

    Full Text Available Lung cancer is a heterogeneous disease responsible for the most cases of cancer-related deaths. The majority of patients are clinically diagnosed at advanced stages, with a poor survival rate. For this reason, the identification of oncodrivers and novel biomarkers is decisive for the future clinical management of this pathology. The rise of high throughput technologies popularly referred to as “omics” has accelerated the discovery of new biomarkers and drivers for this pathology. Within them, tyrosine kinase receptors (TKRs have proven to be of importance as diagnostic, prognostic, and predictive tools and, due to their molecular nature, as therapeutic targets. Along this review, the role of TKRs in the different lung cancer histologies, research on improvement of anti-TKR therapy, and the current approaches to manage anti-TKR resistance will be discussed.

  17. Receptor tyrosine kinase structure and function in health and disease

    Directory of Open Access Journals (Sweden)

    Oleg A. Karpov

    2015-09-01

    Full Text Available Receptor tyrosine kinases (RTKs are membrane proteins that control the flow of information through signal transduction pathways, impacting on different aspects of cell function. RTKs are characterized by a ligand-binding ectodomain, a single transmembrane α-helix, a cytosolic region comprising juxtamembrane and kinase domains followed by a flexible C-terminal tail. Somatic and germline RTK mutations can induce aberrant signal transduction to give rise to cardiovascular, developmental and oncogenic abnormalities. RTK overexpression occurs in certain cancers, correlating signal strength and disease incidence. Diverse RTK activation and signal transduction mechanisms are employed by cells during commitment to health or disease. Small molecule inhibitors are one means to target RTK function in disease initiation and progression. This review considers RTK structure, activation, and signal transduction and evaluates biological relevance to therapeutics and clinical outcomes.

  18. Tyrosine phosphorylation of the human guanylyl cyclase C receptor

    Indian Academy of Sciences (India)

    Unknown

    Tyrosine phosphorylation events are key components of several cellular signal transduction pathways. This study describes a novel method for identification of substrates for tyrosine kinases. Co-expression of the tyrosine kinase. EphB1 with the intracellular domain of guanylyl cyclase C (GCC) in Escherichia coli cells ...

  19. 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...

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

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    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.

  1. Evidence for association of the cloned liver growth hormone receptor with a tyrosine kinase

    DEFF Research Database (Denmark)

    Wang, X; Uhler, M D; Billestrup, N

    1992-01-01

    in a variety of cell types. The finding that the level of phosphorylation of GH receptor appears to vary with cell type is consistent with the cloned liver GH receptor being a substrate for an associated tyrosine kinase and with the amount of such a GH receptor-associated tyrosine kinase being cell type-specific.......The ability of the cloned liver growth hormone (GH) receptor, when expressed in mammalian cell lines, to copurify with tyrosine kinase activity and be tyrosyl phosphorylated was examined. 125I-human growth hormone-GH receptor complexes isolated from COS-7 cells transiently expressing high levels...... of the cloned liver GH receptor bound to anti-phosphotyrosine antibody, suggesting that the cloned GH receptor is tyrosyl phosphorylated in vivo. GH-GH receptor complexes purified from transfected COS-7 cells using anti-GH antibody incorporated 32P when incubated with [gamma-32P]ATP, indicating association...

  2. The tyrosine phosphatase STEP mediates AMPA receptor endocytosis after metabotropic glutamate receptor stimulation.

    Science.gov (United States)

    Zhang, Yang; Venkitaramani, Deepa V; Gladding, Clare M; Zhang, Yongfang; Kurup, Pradeep; Molnar, Elek; Collingridge, Graham L; Lombroso, Paul J

    2008-10-15

    Although it is well established that AMPA receptor (AMPAR) trafficking is a central event in several forms of synaptic plasticity, the mechanisms that regulate the surface expression of AMPARs are poorly understood. Previous work has shown that striatal-enriched protein tyrosine phosphatase (STEP) mediates NMDAR endocytosis. This protein tyrosine phosphatase is enriched in the synapses of the striatum, hippocampus, cerebral cortex, and other brain regions. In the present investigation, we have explored whether STEP also regulates AMPAR internalization. We found that (RS)-3,5-dihydroxyphenylglycine (DHPG) stimulation triggered a dose-dependent increase in STEP translation in hippocampal slices and synaptoneurosomes, a process that requires stimulation of mGluR5 (metabotropic glutamate receptor 5) and activation of mitogen-activated protein kinases and phosphoinositide-3 kinase pathways. DHPG-induced AMPAR internalization and tyrosine dephosphorylation of GluR2 (glutamate receptor 2) was blocked by a substrate-trapping TAT-STEP [C/S] protein in hippocampal slices and cultures. Moreover, DHPG-triggered AMPAR internalization was abolished in STEP knock-out mice and restored after replacement of wild-type STEP. These results suggest a role for STEP in the regulation of AMPAR trafficking.

  3. 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...

  4. 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...

  5. The LAR protein tyrosine phosphatase enables PDGF beta-receptor activation through attenuation of the c-Abl kinase activity.

    NARCIS (Netherlands)

    Zheng, W.; Lennartsson, J.; Hendriks, W.J.A.J.; Heldin, C.H.; Hellberg, C.

    2011-01-01

    The receptor tyrosine phosphatase (RPTP) LAR negatively regulates the activity of several receptor tyrosine kinases. To investigate if LAR affects the platelet-derived growth factor (PDGF) receptor signaling, mouse embryonic fibroblasts (MEFs) from mice where the LAR phosphatase domains were deleted

  6. The role of Ryk and Ror receptor tyrosine kinases in Wnt signal transduction

    NARCIS (Netherlands)

    Green, J.; Nusse, R.; van Amerongen, R.

    2014-01-01

    Receptor tyrosine kinases of the Ryk and Ror families were initially classified as orphan receptors because their ligands were unknown. They are now known to contain functional extracellular Wnt-binding domains and are implicated in Wnt-signal transduction in multiple species. Although their

  7. Abelson tyrosine kinase links PDGFbeta receptor activation to cytoskeletal regulation of NMDA receptors in CA1 hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Beazely Michael A

    2008-12-01

    Full Text Available Abstract Background We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl, control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood. Results Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation. Conclusion This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.

  8. Discoidin domain receptor (DDR) 1 and 2: collagen-activated tyrosine kinase receptors in the cornea.

    Science.gov (United States)

    Mohan, R R; Mohan, R R; Wilson, S E

    2001-01-01

    Discoidin domain receptor (DDR) 1 and 2 have recently been found to serve as receptors for several collagen types. These receptors have been found to modulate cell proliferation and metalloprotease expression in response to collagen stimulation. The purpose of this study was to examine expression of DDR1 and DDR2 in the cornea and to determine the effect of several collagen types on proliferation and response to pro-apoptotic cytokines by corneal fibroblasts. DDR1 and DDR2 mRNAs were detected by RT-PCR. Proteins were detected by immunocytochemistry and immunoprecipitation with Western blotting. Cell proliferation in response to acetic acid-solubilized collagen type I, II, IV, IX or X was determined by cell counting. The effect of these collagen types on Fas-stimulating antibody-induced cell death was determined by trypan blue assay. DDR1 and DDR2 mRNAs were detected in each major human cell type of the cornea. Both were also detected in ex vivo human corneal epithelium. DDR1 and DDR2 proteins were detected in all three major cell types in culture and in human corneal tissue. Collagen types I, II, IV, IX and X stimulated proliferation, but had no effect on Fas-mediated apoptosis, of corneal fibroblasts. DDR1 and DDR2 tyrosine kinase receptors are expressed in the cornea. Collagen-stimulated mitosis of corneal fibroblasts in culture is likely mediated by the DDR receptors. Collagen had no effect on Fas-mediated apoptosis of corneal fibroblasts. Copyright 2001 Academic Press.

  9. Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors

    DEFF Research Database (Denmark)

    Sorkin, A; Helin, K; Waters, C M

    1992-01-01

    The role of epidermal growth factor (EGF) receptor autophosphorylation sites in the regulation of receptor functions has been studied using cells transfected with mutant EGF receptors. Simultaneous point mutation of 4 tyrosines (Y1068, Y1086, Y1148, Y1173) to phenylalanine, as well as removal of ...

  10. Essential domain of receptor tyrosine phosphatase beta (RPTPbeta) for interaction with Helicobacter pylori vacuolating cytotoxin

    DEFF Research Database (Denmark)

    Yahiro, Kinnosuke; Wada, Akihiro; Yamasaki, Eiki

    2004-01-01

    Helicobacter pylori produces a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Although VacA was able to interact with two receptor-like protein tyrosine phosphatases, RPTPbeta and RPTPalpha, RPTPbeta was found to be responsible for gastric damage caused...

  11. Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase

    DEFF Research Database (Denmark)

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

    1994-01-01

    Growth hormone (GH) has recently been shown to activate the GH receptor (GHR)-associated tyrosine kinase JAK2. In the present study, regions of the GHR required for JAK2 association with GHR were identified. GH-dependent JAK2 association with GHR was detected in Chinese hamster ovary (CHO) cells...

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

    Science.gov (United States)

    Perception of pathogen-associated molecular patterns (PAMPs) by surface-localised pattern-recognition receptors (PRRs) is a key component of plant innate immunity. Most known plant PRRs are receptor kinases and initiation of PAMP-triggered immunity (PTI) signalling requires phosphorylation of the PR...

  13. BIOLUMINISCENCE RESONANCE ENERGY TRANSFER (BRET) METHODS TO STUDY G PROTEIN-COUPLED RECEPTOR - RECEPTOR TYROSINE KINASE HETERORECEPTOR COMPLEXES

    OpenAIRE

    Borroto-Escuela, Dasiel O.; Flajolet, Marc; Agnati, Luigi F.; Greengard, Paul; Fuxe, Kjell

    2013-01-01

    A large body of evidence indicates that G protein-coupled receptors (GPCRs) and Receptor tyrosine kinases (RTKs) can form heteroreceptor complexes. In these complexes, the signalling from each interacting protomer is modulated to produce an integrated and therefore novel response upon agonist(s) activation. In the GPCR-RTK heteroreceptor complexes, GPCRs can activate RTK in the absence of added growth factor through the use of RTK signalling molecules. This integrative phenomenon is reciproca...

  14. Fatty acylated caveolin-2 is a substrate of insulin receptor tyrosine kinase for insulin receptor substrate-1-directed signaling activation.

    Science.gov (United States)

    Kwon, Hayeong; Lee, Jaewoong; Jeong, Kyuho; Jang, Donghwan; Pak, Yunbae

    2015-05-01

    Here, we demonstrate that insulin receptor (IR) tyrosine kinase catalyzes Tyr-19 and Tyr-27 phosphorylation of caveolin-2 (cav-2), leading to stimulation of signaling proteins downstream of IR, and that the catalysis is dependent on fatty acylation status of cav-2, promoting its interaction with IR. Cav-2 is myristoylated at Gly-2 and palmitoylated at Cys-109, Cys-122, and Cys-145. The fatty acylation deficient mutants are unable to localize in the plasma membrane and not phosphorylated by IR tyrosine kinase. IR interacts with the C-terminal domain of cav-2 containing the cysteines for palmitoylation. IR mutants, Y999F and K1057A, but not W1220S, fail interaction with cav-2. Insulin receptor substrate-1 (IRS-1) is recruited to interact with the IR-catalyzed phospho-tyrosine cav-2, which facilitates IRS-1 association with and activation by IR to initiate IRS-1-mediated downstream signaling. Cav-2 fatty acylation and tyrosine phosphorylation are necessary for the IRS-1-dependent PI3K-Akt and ERK activations responsible for glucose uptake and cell survival and proliferation. In conclusion, fatty acylated cav-2 is a new substrate of IR tyrosine kinase, and the fatty acylation and phosphorylation of cav-2 present novel mechanisms by which insulin signaling is activated. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Application of computational approaches to study signalling networks of nuclear and Tyrosine kinase receptors

    Directory of Open Access Journals (Sweden)

    Rebaï Ahmed

    2010-10-01

    Full Text Available Abstract Background Nuclear receptors (NRs and Receptor tyrosine kinases (RTKs are essential proteins in many cellular processes and sequence variations in their genes have been reported to be involved in many diseases including cancer. Although crosstalk between RTK and NR signalling and their contribution to the development of endocrine regulated cancers have been areas of intense investigation, the direct coupling of their signalling pathways remains elusive. In our understanding of the role and function of nuclear receptors on the cell membrane the interactions between nuclear receptors and tyrosine kinase receptors deserve further attention. Results We constructed a human signalling network containing nuclear receptors and tyrosine kinase receptors that identified a network topology involving eleven highly connected hubs. We further developed an integrated knowledge database, denominated NR-RTK database dedicated to human RTKs and NRs and their vertebrate orthologs and their interactions. These interactions were inferred using computational tools and those supported by literature evidence are indicated. NR-RTK database contains links to other relevant resources and includes data on receptor ligands. It aims to provide a comprehensive interaction map that identifies complex dynamics and potential crosstalk involved. Availability: NR-RTK database is accessible at http://www.bioinfo-cbs.org/NR-RTK/ Conclusions We infer that the NR-RTK interaction network is scale-free topology. We also uncovered the key receptors mediating the signal transduction between these two types of receptors. Furthermore, NR-RTK database is expected to be useful for researchers working on various aspects of the molecular basis of signal transduction by RTKs and NRs. Reviewers This article was reviewed by Professor Paul Harrison (nominated by Dr. Mark Gerstein, Dr. Arcady Mushegian and Dr. Anthony Almudevar.

  16. Tyrosine Kinase Ligand-Receptor Pair Prediction by Using Support Vector Machine

    Directory of Open Access Journals (Sweden)

    Masayuki Yarimizu

    2015-01-01

    Full Text Available Receptor tyrosine kinases are essential proteins involved in cellular differentiation and proliferation in vivo and are heavily involved in allergic diseases, diabetes, and onset/proliferation of cancerous cells. Identifying the interacting partner of this protein, a growth factor ligand, will provide a deeper understanding of cellular proliferation/differentiation and other cell processes. In this study, we developed a method for predicting tyrosine kinase ligand-receptor pairs from their amino acid sequences. We collected tyrosine kinase ligand-receptor pairs from the Database of Interacting Proteins (DIP and UniProtKB, filtered them by removing sequence redundancy, and used them as a dataset for machine learning and assessment of predictive performance. Our prediction method is based on support vector machines (SVMs, and we evaluated several input features suitable for tyrosine kinase for machine learning and compared and analyzed the results. Using sequence pattern information and domain information extracted from sequences as input features, we obtained 0.996 of the area under the receiver operating characteristic curve. This accuracy is higher than that obtained from general protein-protein interaction pair predictions.

  17. Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

    DEFF Research Database (Denmark)

    Jacob, K K; Sap, J; Stanley, F M

    1998-01-01

    A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin...... to increase prolactin gene expression but potentiates the effects of epidermal growth factor and cAMP on prolactin promoter activity. RPTPalpha was the only protein-tyrosine phosphatase tested that did this. Thus, the effect of RPTPalpha on prolactin-chloramphenicol acetyltransferase (CAT) promoter activity...... is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due...

  18. Changes in insulin receptor tyrosine kinase activity associated with metformin treatment of type 2 diabetes.

    Science.gov (United States)

    Santos, R F; Nomizo, R; Wajhenberg, B L; Reaven, G M; Azhar, S

    1995-10-01

    This study was performed to define the effect of metformin on glycaemic control and erythrocyte insulin receptor tyrosine kinase activity in patients with non-insulin-dependent (Type 2) diabetes mellitus. A case-control study of the effect of metformin treatment in hyperglycaemic patients with Type 2 diabetes was conducted in outpatients of the Diabetes Clinical Center. The study population consisted of 14 patients with Type 2 diabetes (5 males, 9 females) whose hyperglycaemia was uncontrolled by diet. Patients were treated with metformin 850 mg twice daily for 2 1/2 months. Fasting plasma glucose concentrations decreased from 8.9 to 6.4 mmol/L after 10 weeks of metformin treatment (p metformin treatment. There was no change in erythrocyte insulin receptor binding associated with metformin treatment, but both basal and insulin-stimulated insulin receptor tyrosine kinase activities of solubilized erythrocyte insulin receptors were significantly higher after 10 weeks of metformin treatment. It is concluded that the increase in insulin-stimulated tyrosine kinase activity contributed to the improvement in glucose insulin and lipoprotein metabolism associated with metformin treatment of Type 2 diabetes.

  19. SOCS proteins in regulation of receptor tyrosine kinase signaling

    DEFF Research Database (Denmark)

    Kazi, Julhash U.; Kabir, Nuzhat N.; Flores Morales, Amilcar

    2014-01-01

    proteins, SOCS1-7, and cytokine-inducible SH2-containing protein (CIS). A key feature of this family of proteins is the presence of an SH2 domain and a SOCS box. Recent studies suggest that SOCS proteins also play a role in RTK signaling. Activation of RTK results in transcriptional activation of SOCS......-encoding genes. These proteins associate with RTKs through their SH2 domains and subsequently recruit the E3 ubiquitin machinery through the SOCS box, and thereby limit receptor stability by inducing ubiquitination. In a similar fashion, SOCS proteins negatively regulate mitogenic signaling by RTKs. It is also...

  20. Eph receptor tyrosine kinase-mediated formation of a topographic map in the Drosophila visual system.

    Science.gov (United States)

    Dearborn, Richard; He, Qi; Kunes, Sam; Dai, Yong

    2002-02-15

    Roles for Eph receptor tyrosine kinase signaling in the formation of topographic patterns of axonal connectivity have been well established in vertebrate visual systems. Here we describe a role for a Drosophila Eph receptor tyrosine kinase (EPH) in the control of photoreceptor axon and cortical axon topography in the developing visual system. Although uniform across the developing eye, EPH is expressed in a concentration gradient appropriate for conveying positional information during cortical axon guidance in the second-order optic ganglion, the medulla. Disruption of this graded pattern of EPH activity by double-stranded RNA interference or by ectopic expression of wild-type or dominant-negative transgenes perturbed the establishment of medulla cortical axon topography. In addition, abnormal midline fasciculation of photoreceptor axons resulted from the eye-specific expression of the dominant-negative EPH transgene. These observations reveal a conserved role for Eph kinases as determinants of topographic map formation in vertebrates and invertebrates.

  1. Receptor Tyrosine Kinases as Targets for Treatment of Peripheral Nerve Sheath Tumors in NF 1 Patients

    Science.gov (United States)

    2010-03-01

    a pharmacologic IC50 of < 2 μM (Holtkamp et al. 2006). Also gefinitib reduced vitality of the MPNST cells (Fig. 1) Figure 1. Effect of imatinib and...recommendations in pretreatment solution (Abbott, Ludwigshafen, Ger- many) and then in protease or pepsin . The LSI EGFR SpectrumOrange/CEP 7...Hidalgo M, Siu LL, Nemunaitis J, et al. Phase I and pharmacologic study of OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in

  2. 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...

  3. Identification of a Novel Series of Potent TrkA Receptor Tyrosine Kinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Stéphane L. Raeppel

    2012-01-01

    Full Text Available A novel series of N-(3-(6-substituted-aminopyridin-3-yloxyphenyl-2-oxo-3-phenylimidazolidine-1-carboxamides targeting TrkA receptor tyrosine kinase was identified. SAR study of the series allowed us to design and synthesize compounds possessing inhibitory activity of TrkA kinase enzyme in the low nanomolar range with low residual activity against c-Met and with no significant activity against VEGFR2.

  4. Tyrosine Kinase Domain Gene Polymorphism of Epidermal Growth Factor Receptor in Gastric Cancer in Northern Iran

    Directory of Open Access Journals (Sweden)

    Jeivad F

    2012-01-01

    Full Text Available Background: Gastric cancer is one of the most common diseases of digestive system with a low 5-year survival rate and metastasis is the main cause of death. Multi-factors, such as changes in molecular pathways and deregulation of cells are involved in the disease development. Epidermal growth factor receptor pathway (EGFR which is associated with cell proliferation and survival can influence cancer development. EGFR function is governed by its genetic polymorphism; thus, we aimed to study the tyrosine kinase domain gene mutations of the receptor in patients with gastric cancer.Methods : In this experimental study, 123 subjects (83 patients with gastric cancer and 40 normal subjects were investigated in north of Iran for EGFR gene polymorphisms during 1 year. Genomic DNA was extracted by DNA extraction kit according to the manufacture's protocol. Polymerase chain reaction single-stranded conformation polymorphism (PCR-SSCP and silver staining were performed for investigating EGFR gene polymorphisms. Results : The participants included 72 men and 44 women. Gene polymorphism in exon 18 was present in 10% of the study population but SSCP pattern in exon 19 did not show different migrate bands neither in patients nor in normal subjects.Conclusion: It seems that screening for tyrosine kinas gene polymorphism of epidermal growth factor receptor in patients with gastric cancer and use of tyrosine kinas inhibitors could be useful in the prevention of disease progress and improvement of treatment process for a better quality of life in these patients.

  5. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying-Nan P.; LaMarche, Matthew J.; Chan, Ho Man; Fekkes, Peter; Garcia-Fortanet, Jorge; Acker, Michael G.; Antonakos, Brandon; Chen, Christine Hiu-Tung; Chen, Zhouliang; Cooke, Vesselina G.; Dobson, Jason R.; Deng, Zhan; Fei, Feng; Firestone, Brant; Fodor, Michelle; Fridrich, Cary; Gao, Hui; Grunenfelder, Denise; Hao, Huai-Xiang; Jacob, Jaison; Ho, Samuel; Hsiao, Kathy; Kang, Zhao B.; Karki, Rajesh; Kato, Mitsunori; Larrow, Jay; La Bonte, Laura R.; Lenoir, Francois; Liu, Gang; Liu, Shumei; Majumdar, Dyuti; Meyer, Matthew J.; Palermo, Mark; Perez, Lawrence; Pu, Minying; Price, Edmund; Quinn, Christopher; Shakya, Subarna; Shultz, Michael D.; Slisz, Joanna; Venkatesan, Kavitha; Wang, Ping; Warmuth, Markus; Williams, Sarah; Yang, Guizhi; Yuan, Jing; Zhang, Ji-Hu; Zhu, Ping; Ramsey, Timothy; Keen, Nicholas J.; Sellers, William R.; Stams, Travis; Fortin, Pascal D.

    2016-06-29

    The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase1. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma1, 2, 3, 4, 5. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway2, 3. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways6, 7. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy8, 9. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

  6. EGF-receptor tyrosine kinase inhibition induces keratinocyte growth arrest and terminal differentiation.

    Science.gov (United States)

    Peus, D; Hamacher, L; Pittelkow, M R

    1997-12-01

    Epidermal keratinocyte growth and differentiation are regulated by specific families of growth factors and receptors. Peptide growth factors of the epidermal growth factor family stimulate proliferation of clonal density human keratinocytes and suppress markers of terminal differentiation in confluent cultures of human keratinocytes. We present evidence that selected inhibitors of activation of the type I human epidermal growth factor receptor (EGFR or HER-1), namely, neutralizing monoclonal antibody to HER-1/EGFR and the specific tyrosine kinase inhibitor PD 153035, potently inhibit proliferation of human keratinocytes in autonomously replicating subconfluent cultures. Coupled to growth arrest is the suppression of HER-1 tyrosine autophosphorylation in inhibitor-treated human keratinocytes. Proliferation and tyrosine autophosphorylation are initially reversible following removal of the inhibitor and restimulation of cells with epidermal growth factor. Sustained inactivation of HER-1 in autonomously replicating cultures of human keratinocytes induces expression of keratin 1 and keratin 10 genes, early markers of terminal differentiation. Reversal of growth inhibition by epidermal growth factor suppresses keratin 1 and keratin 10 expression. These results demonstrate that human keratinocyte terminal differentiation as well as proliferation are mediated by HER-1. Co-expression of autocrine epidermal growth factor-related ligands as well as HER-1 by human keratinocyte may function as part of the signal transduction network in epidermis to regulate cell number, replication rate, and terminal differentiation.

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

    DEFF Research Database (Denmark)

    Petrone, Angiola; Battaglia, Fortunato; Wang, Cheng

    2003-01-01

    Despite clear indications of their importance in lower organisms, the contributions of protein tyrosine phosphatases (PTPs) to development or function of the mammalian nervous system have been poorly explored. In vitro studies have indicated that receptor protein tyrosine phosphatase alpha (RPTPa....... However, these synapses are unable to undergo long-term potentiation. Mice lacking RPTPalpha also underperform in the radial-arm water-maze test. These studies identify RPTPalpha as a key mediator of neuronal migration and synaptic plasticity....... neuronal migration. The migratory abnormality likely results from a radial glial dysfunction rather than from a neuron-autonomous defect. In spite of this aberrant development, basic synaptic transmission from the Schaffer collateral pathway to CA1 pyramidal neurons remains intact in Ptpra(-/-) mice...

  8. Macrophage Proliferation Is Regulated through CSF-1 Receptor Tyrosines 544, 559, and 807*

    Science.gov (United States)

    Yu, Wenfeng; Chen, Jian; Xiong, Ying; Pixley, Fiona J.; Yeung, Yee-Guide; Stanley, E. Richard

    2012-01-01

    Colony-stimulating factor-1 (CSF-1)-stimulated CSF-1 receptor (CSF-1R) tyrosine phosphorylation initiates survival, proliferation, and differentiation signaling pathways in macrophages. Either activation loop Y807F or juxtamembrane domain (JMD) Y559F mutations severely compromise CSF-1-regulated proliferation and differentiation. YEF, a CSF-1R in which all eight tyrosines phosphorylated in the activated receptor were mutated to phenylalanine, lacks in vitro kinase activity and in vivo CSF-1-regulated tyrosine phosphorylation. The addition of Tyr-807 alone to the YEF backbone (Y807AB) led to CSF-1-independent but receptor kinase-dependent proliferation, without detectable activation loop Tyr-807 phosphorylation. The addition of Tyr-559 alone (Y559AB) supported a low level of CSF-1-independent proliferation that was slightly enhanced by CSF-1, indicating that Tyr-559 has a positive Tyr-807-independent effect. Consistent with the postulated autoinhibitory role of the JMD Tyr-559 and its relief by ligand-induced Tyr-559 phosphorylation, the addition of Tyr-559 to the Y807AB background suppressed proliferation in the absence of CSF-1, but restored most of the CSF-1-stimulated proliferation. Full restoration of kinase activation and proliferation required the additional add back of JMD Tyr-544. Inhibitor experiments indicate that the constitutive proliferation of Y807AB macrophages is mediated by the phosphatidylinositol 3-kinase (PI3K) and ERK1/2 pathways, whereas proliferation of WT and Y559,807AB macrophages is, in addition, contributed to by Src family kinase (SFK)-dependent pathways. Thus Tyr-807 confers sufficient kinase activity for strong CSF-1-independent proliferation, whereas Tyr-559 maintains the receptor in an inactive state. Tyr-559 phosphorylation releases this restraint and may also contribute to the CSF-1-regulated proliferative response by activating Src family kinase. PMID:22375015

  9. Crystal structure of the Sema-PSI extracellular domain of human RON receptor tyrosine kinase.

    Directory of Open Access Journals (Sweden)

    Kinlin L Chao

    Full Text Available Human RON (Recepteur d'Origine Nantais receptor tyrosine kinase is a cell surface receptor for Macrophage Stimulating Protein (MSP. RON mediates signal transduction pathways that regulate cell adhesion, invasion, motility and apoptosis processes. Elevated levels of RON and its alternatively spliced variants are implicated in the progression and metastasis of tumor cells. The binding of MSP α/β heterodimer to the extracellular region of RON receptor induces receptor dimerization and activation by autophosphorylation of the intracellular kinase domains. The ectodomain of RON, containing the ligand recognition and dimerization domains, is composed of a semaphorin (Sema, Plexins-Semaphorins-Integrins domain (PSI, and four Immunoglobulins-Plexins-Transcription factor (IPT domains. High affinity association between MSP and RON is mediated by the interaction between MSP β-chain and RON Sema, although RON activation requires intact RON and MSP proteins. Here, we report the structure of RON Sema-PSI domains at 1.85 Å resolution. RON Sema domain adopts a seven-bladed β-propeller fold, followed by disulfide bond rich, cysteine-knot PSI motif. Comparison with the homologous Met receptor tyrosine kinase reveals that RON Sema-PSI contains distinguishing secondary structural features. These define the receptors' exclusive selectivity towards their respective ligands, RON for MSP and Met for HGF. The RON Sema-PSI crystal packing generates a homodimer with interface formed by the Sema domain. Mapping of the dimer interface using the RON homology to Met, MSP homology to Hepatocyte Growth Factor (HGF, and the structure of the Met/HGF complex shows the dimer interface overlapping with the putative MSPβ binding site. The crystallographically determined RON Sema-PSI homodimer may represent the dimer assembly that occurs during ligand-independent receptor activation and/or the inhibition of the constitutive activity of RONΔ160 splice variant by the soluble RON

  10. The role of Ryk and Ror receptor tyrosine kinases in Wnt signal transduction.

    Science.gov (United States)

    Green, Jennifer; Nusse, Roel; van Amerongen, Renée

    2014-02-01

    Receptor tyrosine kinases of the Ryk and Ror families were initially classified as orphan receptors because their ligands were unknown. They are now known to contain functional extracellular Wnt-binding domains and are implicated in Wnt-signal transduction in multiple species. Although their signaling mechanisms still remain to be resolved in detail, both Ryk and Ror control important developmental processes in different tissues. However, whereas many other Wnt-signaling responses affect cell proliferation and differentiation, Ryk and Ror are mostly associated with controlling processes that rely on the polarized migration of cells. Here we discuss what is currently known about the involvement of this exciting class of receptors in development and disease.

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

    Science.gov (United States)

    Ganugapati, Jayasree; Baldwa, Aashish; Lalani, Sarfaraz

    2012-01-01

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

  12. Dimerization of Receptor Protein-Tyrosine Phosphatase alpha in living cells

    Directory of Open Access Journals (Sweden)

    Gadella Theodorus WJ

    2001-06-01

    Full Text Available Abstract Background Dimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs, are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined. Results In order to assess RPTP dimerization, we have assayed Fluorescence Resonance Energy Transfer (FRET between chimeric proteins of cyan- and yellow-emitting derivatives of green fluorescent protein, fused to RPTPα, using three different techniques: dual wavelength excitation, spectral imaging and fluorescence lifetime imaging. All three techniques suggested that FRET occurred between RPTPα -CFP and -YFP fusion proteins, and thus that RPTPα dimerized in living cells. RPTPα dimerization was constitutive, extensive and specific. RPTPα dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker. Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization. Conclusions We demonstrate here that RPTPα dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs.

  13. Coexpression of receptor tyrosine kinase AXL and EGFR in human primary lung adenocarcinomas.

    Science.gov (United States)

    Wu, Zhenzhou; Bai, Fan; Fan, Liyun; Pang, Wenshuai; Han, Ruiyu; Wang, Juan; Liu, Yueping; Yan, Xia; Duan, Huijun; Xing, Lingxiao

    2015-12-01

    AXL has been identified as a tyrosine kinase switch that causes resistance to inhibitors targeting epidermal growth factor receptor (EGFR) signaling in non-small cell lung cancer (NSCLC). However, the relationship between 2 receptor tyrosine kinases, AXL and EGFR, and the relevance of AXL expression with EGFR mutation status in treatment-naive human NSCLCs remain uncertain. In this study, we evaluated the coexpression pattern of AXL, EGFR, and pEGFR(1068) in 109 lung adenocarcinoma patients with or without an EGFR mutation. There were 68 (62.4%) patients with tumors harboring EGFR mutations such as 19 del and/or L858R; 2 patients were T790M positive. The expression of AXL, EGFR, and pEGFR(1068) was detected in 60 (55%), 68 (62.4%), and 57 (52.3%) of 109 patients, respectively. The positive rates of EGFR and pEGFR(1068) were associated with the L858R mutation alone or with the 19 del and L858R mutation status. Further analysis indicated that the percentage of AXL(+)/EGFR(+)/pEGFR(1068) coexpression in 68 EGFR-activating mutations patients was significantly higher than that in 39 EGFR wild-type patients (30.9% versus 10.3%, P=.015). Furthermore, in the subgroup of AXL(+) patients (35 mutation(+) and 23 wild-type patients), the coexpression rates of AXL(+)/pEGFR(1068+) and AXL(+)/EGFR(+)/pEGFR(1068+) in patients with EGFR mutations were significantly higher compared with those in wild-type patients (both P<.05). Our study emphasized that the AXL and EGFR receptor tyrosine kinases were coexpressed in a subgroup of treatment-naive lung adenocarcinomas with or without EGFR mutations. Anti-AXL therapeutics delivered up front in combination with an EGFR inhibitor might prevent or delay resistance in patients with AXL-positive, EGFR-mutant, or wild-type NSCLC. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase

    Energy Technology Data Exchange (ETDEWEB)

    O' Bryan, J.P.; Frye, R.A.; Cogswell, P.C.; Neubauer, A.; Kitch, B.; Prokop, C.; Earp, H.S.; Liu, E.T. (Univ. of North Carolina, Chapel Hill (United States)); Espinosa, R. III; Le Beau, M.M. (Univ. of Chicago, IL (United States))

    1991-10-01

    Using a sensitive transfection-tumorigenicity assay, the authors have isolated a novel transforming gene from the DNA of two patients with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of the appropriate recombinant protein that is recognized by antophosphotyrosine antibodies, confirming that the axl protein is a tyrosine kinase. The juxtaposition of fibronectin type II and immunoglobulinlike repeats in the extracellular domain, as well as distinct amino acid sequences in the kinase domain, indicate that the axl protein represents a novel subclass of receptor tyrosine kinases.

  15. Linifanib--a multi-targeted receptor tyrosine kinase inhibitor and a low molecular weight gelator.

    Science.gov (United States)

    Marlow, Maria; Al-Ameedee, Mohammed; Smith, Thomas; Wheeler, Simon; Stocks, Michael J

    2015-04-14

    In this study we demonstrate that linifanib, a multi-targeted receptor tyrosine kinase inhibitor, with a key urea containing pharmacophore, self-assembles into a hydrogel in the presence of low amounts of solvent. We demonstrate the role of the urea functional group and that of fluorine substitution on the adjacent aromatic ring in promoting self-assembly. We have also shown that linifanib has superior mechanical strength to two structurally related analogues and hence increased potential for localisation at an injection site for drug delivery applications.

  16. 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.......1 promoter. Any of these three tyrosines is able to independently mediate GH-induced transcription, indicating redundancy in this part of the GH receptor. Tyrosine phosphorylation was not required for GH stimulation of mitogen-activated protein (MAP) kinase activity or for GH-stimulated Ca2+ channel...

  17. Bioluminescence resonance energy transfer methods to study G protein-coupled receptor-receptor tyrosine kinase heteroreceptor complexes.

    Science.gov (United States)

    Borroto-Escuela, Dasiel O; Flajolet, Marc; Agnati, Luigi F; Greengard, Paul; Fuxe, Kjell

    2013-01-01

    A large body of evidence indicates that G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) can form heteroreceptor complexes. In these complexes, the signaling from each interacting protomer is modulated to produce an integrated and therefore novel response upon agonist(s) activation. In the GPCR-RTK heteroreceptor complexes, GPCRs can activate RTK in the absence of added growth factor through the use of RTK signaling molecules. This integrative phenomenon is reciprocal and can place also RTK signaling downstream of GPCR. Formation of either stable or transient complexes by these two important classes of membrane receptors is involved in regulating all aspects of receptor function, from ligand binding to signal transduction, trafficking, desensitization, and downregulation among others. Functional phenomena can be modulated with conformation-specific inhibitors that stabilize defined GPCR states to abrogate both GPCR agonist- and growth factor-stimulated cell responses or by means of small interfering heteroreceptor complex interface peptides. The bioluminescence resonance energy transfer (BRET) technology has emerged as a powerful method to study the structure of heteroreceptor complexes closely associated with the study of receptor-receptor interactions in such complexes. In this chapter, we provide an overview of different BRET(2) assays that can be used to study the structure of GPCR-RTK heteroreceptor complexes and their functions. Various experimental designs for optimization of these experiments are also described. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. 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.

  19. Radiation induction of the receptor tyrosine kinase gene Ptk-3 in normal rat astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Sakuma, S.; Hideyuki, S.; Akihiro, I. [Univ. of Texas, Houston, TX (United States)] [and others

    1995-07-01

    Radiation-induced gene expression was examined in rat astrocyte cultures using differential display of mRNA via reverse transcriptase-polymerase chain reaction. A 0.3-kb cDNA that was consistently observed in irradiated cultures but not in unirradiated cultures was cloned and sequenced. It was found to be identical to Ptk-3, a receptor tyrosine kinase gene identified recently. The protein encoded by Ptk-3 is a member of a novel class of receptor tyrosine kinases whose extracellular domain contains regions of homology with coagulation factors V and VIII and complement component C1. Northern blot analysis revealed that the expression of Ptk-3 was increased in rat astrocytes by 0.5 h after exposure to 10 Gy and remained at the same elevated level for at least 24 h. The maximum increase occurred after 5 Gy cloning studies indicated the presence of at least two Ptk-3 mRNA transcripts, which are probable the result of an alternative splicing mechanism. The short isoform lacks a 37 amino acid sequence in the glycine/proline-rich juxtamembrane region. The splicing pattern of the Ptk-3 gene was not altered by radiation. However, the ratios of the longer to the shorter mRNA transcripts differed between adult cortex, neonatal cortex and in vitro astrocyte cultures. 36 refs., 5 figs.

  20. 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.

  1. Molecular Mechanism of 17-Allylamino-17-demethoxygeldanamycin (17-AAG)-induced AXL Receptor Tyrosine Kinase Degradation*

    Science.gov (United States)

    Krishnamoorthy, Gnana Prakasam; Guida, Teresa; Alfano, Luigi; Avilla, Elvira; Santoro, Massimo; Carlomagno, Francesca; Melillo, Rosa Marina

    2013-01-01

    The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [35S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL. PMID:23629654

  2. Expression of the HER-1-4 family of receptor tyrosine kinases in neuroendocrine tumours.

    Science.gov (United States)

    Srirajaskanthan, Rajaventhan; Shah, Tahir; Watkins, Jennifer; Marelli, Laura; Khan, Korsa; Caplin, Martyn E

    2010-04-01

    The type I receptor tyrosine kinase family comprises four homologous members: Epidermal growth factor receptor (EGFR), HER-2, HER-3 and HER-4. Studies have shown that EGFR and HER-2 play a critical role in oncogenesis. In this study we sought to determine the pattern of expression and the prognostic significance of EGFR, HER-2, HER-3 and HER-4 in a variety of neuroendocrine tumours using immunohistochemistry. HER family receptor expression in 82 paraffin-embedded specimens of neuroendocrine tumours using immunohistochemistry was examined. The pattern and protein expression levels for each receptor were correlated with clinical and pathological parameters. EGFR expression was identified in 86.6% samples, HER-2 was not expressed in any samples, HER-3 was expressed in 8.5% samples and HER-4 was expressed 91.5%. EGFR and HER-4 were co-expressed in 79.3% of cases. HER-3 was correlated with better survival. EGFR was not associated with poor prognosis. This study has demonstrated EGFR, HER-2 and HER-4 expression is not associated with poorer survival. HER-3 expression is correlated with better prognosis. Overexpression of EGFR and HER-4 may offer potential new therapeutic targets.

  3. 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.

  4. Fyn tyrosine kinase increases Apolipoprotein E Receptor 2 levels and phosphorylation.

    Directory of Open Access Journals (Sweden)

    Teal C Burrell

    Full Text Available Apolipoprotein E Receptor 2 (ApoER2 and the tyrosine kinase Fyn are both members of the Reelin pathway, a signaling pathway essential for the laminar formation of the cortex during development and proper dendritic spine density and long-term potential (LTP in the adult brain. In the presence of extracellular Reelin, ApoER2 binds the intracellular protein Dab1, an adaptor protein that is phosphorylated by Fyn. However, direct interactions between ApoER2 and Fyn are not well defined. Here, we show that total levels of ApoER2 and surface levels of ApoER2 are increased by active Fyn. Via a separate mechanism, ApoER2 is also phosphorylated by Fyn, an event that peaks in the postnatal cortex at day 5 and can occur at multiple ApoER2 tyrosine residues. Dab1 is also involved in this phosphorylation, promoting the phosphorylation of ApoER2 by Fyn when it is itself phosphorylated. These results elucidate some of the intracellular mechanisms that give rise to a functional Reelin pathway.

  5. Targeting FMS-related tyrosine kinase receptor 3 with the human immunoglobulin G1 monoclonal antibody IMC-EB10.

    Science.gov (United States)

    Youssoufian, Hagop; Rowinsky, Eric K; Tonra, James; Li, Yiwen

    2010-02-15

    FMS-related tyrosine kinase receptor 3 (FLT3) is a class III receptor tyrosine kinase that holds considerable promise as a therapeutic target in hematologic malignancies. Current efforts directed toward the development of small-molecule tyrosine kinase inhibitors of FLT3 may be limited by off-target toxicities and the development of drug resistance. Target-specific antibodies could overcome these hurdles and provide additional mechanisms to enhance the antitumor efficacy of FLT3 inhibitors. IMC-EB10 is a novel antibody directed against FLT3. The binding of IMC-EB10 to FLT3 results in antiproliferative effects in vitro and in mouse models engrafted with human leukemia cells that harbor wild-type or constitutively activated FLT3. Future clinical trials will test these notions formally and will identify the most appropriate opportunities for this member of a new generation of antileukemic therapies. (c) 2010 American Cancer Society.

  6. The TAM family receptor tyrosine kinase TYRO3 is a negative regulator of type 2 immunity

    Science.gov (United States)

    Chan, Pamela Y.; Carrera Silva, Eugenio A.; De Kouchkovsky, Dimitri; Joannas, Leonel D.; Hao, Liming; Hu, Donglei; Huntsman, Scott; Eng, Celeste; Licona-Limón, Paula; Weinstein, Jason S.; Herbert, De’Broski R.; Craft, Joseph E.; Flavell, Richard A.; Repetto, Silvia; Correale, Jorge; Burchard, Esteban G.; Torgerson, Dara G.; Ghosh, Sourav; Rothlin, Carla V.

    2016-01-01

    Host responses against metazoan parasites or an array of environmental substances elicit type 2 immunity. Despite its protective function, type 2 immunity also drives allergic diseases. The mechanisms that regulate the magnitude of the type 2 response remain largely unknown. Here, we show that genetic ablation of a receptor tyrosine kinase encoded by Tyro3 in mice or the functional neutralization of its ortholog in human dendritic cells resulted in enhanced type 2 immunity. Furthermore, the TYRO3 agonist PROS1 was induced in T cells by the quintessential type 2 cytokine, interleukin-4. T cell–specific Pros1 knockouts phenocopied the loss of Tyro3. Thus, a PROS1-mediated feedback from adaptive immunity engages a rheostat, TYRO3, on innate immune cells to limit the intensity of type 2 responses. PMID:27034374

  7. The transmembrane domain of the p75 neurotrophin receptor stimulates phosphorylation of the TrkB tyrosine kinase receptor.

    Science.gov (United States)

    Saadipour, Khalil; MacLean, Michael; Pirkle, Sean; Ali, Solav; Lopez-Redondo, Maria-Luisa; Stokes, David L; Chao, Moses V

    2017-10-06

    The function of protein products generated from intramembraneous cleavage by the γ-secretase complex is not well defined. The γ-secretase complex is responsible for the cleavage of several transmembrane proteins, most notably the amyloid precursor protein that results in Aβ, a transmembrane (TM) peptide. Another protein that undergoes very similar γ-secretase cleavage is the p75 neurotrophin receptor. However, the fate of the cleaved p75 TM domain is unknown. p75 neurotrophin receptor is highly expressed during early neuronal development and regulates survival and process formation of neurons. Here, we report that the p75 TM can stimulate the phosphorylation of TrkB (tyrosine kinase receptor B). In vitro phosphorylation experiments indicated that a peptide representing p75 TM increases TrkB phosphorylation in a dose- and time-dependent manner. Moreover, mutagenesis analyses revealed that a valine residue at position 264 in the rat p75 neurotrophin receptor is necessary for the ability of p75 TM to induce TrkB phosphorylation. Because this residue is just before the γ-secretase cleavage site, we then investigated whether the p75(αγ) peptide, which is a product of both α- and γ-cleavage events, could also induce TrkB phosphorylation. Experiments using TM domains from other receptors, EGFR and FGFR1, failed to stimulate TrkB phosphorylation. Co-immunoprecipitation and biochemical fractionation data suggested that p75 TM stimulates TrkB phosphorylation at the cell membrane. Altogether, our results suggest that TrkB activation by p75(αγ) peptide may be enhanced in situations where the levels of the p75 receptor are increased, such as during brain injury, Alzheimer's disease, and epilepsy. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Novel method demonstrates differential ligand activation and phosphatase-mediated deactivation of insulin receptor tyrosine-specific phosphorylation.

    Science.gov (United States)

    Cieniewicz, Anne M; Cooper, Philip R; McGehee, Jennifer; Lingham, Russell B; Kihm, Anthony J

    2016-08-01

    Insulin receptor signaling is a complex cascade leading to a multitude of intracellular functional responses. Three natural ligands, insulin, IGF1 and IGF2, are each capable of binding with different affinities to the insulin receptor, and result in variable biological responses. However, it is likely these affinity differences alone cannot completely explain the myriad of diverse cellular outcomes. Ligand binding initiates activation of a signaling cascade resulting in phosphorylation of the IR itself and other intracellular proteins. The direct catalytic activity along with the temporally coordinated assembly of signaling proteins is critical for insulin receptor signaling. We hypothesized that determining differential phosphorylation among individual tyrosine sites activated by ligand binding or dephosphorylation by phosphatases could provide valuable insight into insulin receptor signaling. Here, we present a sensitive, novel immunoassay adapted from Meso Scale Discovery technology to quantitatively measure changes in site-specific phosphorylation levels on endogenous insulin receptors from HuH7 cells. We identified insulin receptor phosphorylation patterns generated upon differential ligand activation and phosphatase-mediated deactivation. The data demonstrate that insulin, IGF1 and IGF2 elicit different insulin receptor phosphorylation kinetics and potencies that translate to downstream signaling. Furthermore, we show that insulin receptor deactivation, regulated by tyrosine phosphatases, occurs distinctively across specific tyrosine residues. In summary, we present a novel, quantitative and high-throughput assay that has uncovered differential ligand activation and site-specific deactivation of the insulin receptor. These results may help elucidate some of the insulin signaling mechanisms, discriminate ligand activity and contribute to a better understanding of insulin receptor signaling. We propose this methodology as a powerful approach to characterize

  9. DMPD: Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflammatory activities. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available regulators of macrophage inflammatory activities. Wang MH, Zhou YQ, Chen YQ. Scand J Immunol. 2002 Dec;56(6)... potentialregulators of macrophage inflammatory activities. PubmedID 12472665 Tit...le Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflammatory activities

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

    Science.gov (United States)

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

    2011-01-01

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

  11. The receptor protein tyrosine phosphatase PTPRB negatively regulates FGF2-dependent branching morphogenesis.

    Science.gov (United States)

    Soady, Kelly J; Tornillo, Giusy; Kendrick, Howard; Meniel, Valerie; Olijnyk-Dallis, Daria; Morris, Joanna S; Stein, Torsten; Gusterson, Barry A; Isacke, Clare M; Smalley, Matthew J

    2017-10-15

    PTPRB is a transmembrane protein tyrosine phosphatase known to regulate blood vessel remodelling and angiogenesis. Here, we demonstrate that PTPRB negatively regulates branching morphogenesis in the mouse mammary epithelium. We show that Ptprb is highly expressed in adult mammary stem cells and also, although at lower levels, in oestrogen receptor-positive luminal cells. During mammary development, Ptprb expression is downregulated during puberty, a period of extensive ductal outgrowth and branching. In vivo shRNA knockdown of Ptprb in the cleared mammary fat pad transplant assay resulted in smaller epithelial outgrowths with an increased branching density and also increased branching in an in vitro organoid assay. Organoid branching was dependent on stimulation by FGF2, and Ptprb knockdown in mammary epithelial cells resulted in a higher level of fibroblast growth factor receptor (FGFR) activation and ERK1/2 phosphorylation, both at baseline and following FGF2 stimulation. Therefore, PTPRB regulates branching morphogenesis in the mammary epithelium by modulating the response of the FGFR signalling pathway to FGF stimulation. Considering the importance of branching morphogenesis in multiple taxa, our findings have general importance outside mammary developmental biology. © 2017. Published by The Company of Biologists Ltd.

  12. SYNAPTIC TRANSLATION OF STRIATAL-ENRICHED TYROSINE PHOSPHATASE (STEP) AFTER β1-ADRENERGIC RECEPTOR STIMULATION

    Science.gov (United States)

    Hu, Yaer; Zhang, Yang; Venkitaramani, Deepa V.; Lombroso, Paul J.

    2009-01-01

    The β-adrenergic system is implicated in long-term synaptic plasticity in the central nervous system, a process that requires protein synthesis. To identify proteins that are translated in response to β-adrenergic receptor stimulation and the pathways that regulate this process, we investigated the effects of isoproterenol on the translation of striatal-enriched protein tyrosine phosphatase (STEP) in both cortico-striatal slices and primary neuronal cultures. Isoproterenol stimulation induced a rapid dose-dependent increase in STEP expression. Anisomycin blocked the increase in STEP expression while actinomycin D had no effect, suggesting a translation-dependent mechanism. Isoproterenol-induced STEP translation required activation of β1 receptors. Application of the MEK inhibitor SL327 blocked both isoproterenol-induced activation of pERK and subsequent STEP translation. Inhibitors of PI3K (LY294002) or mTOR (rapamycin) also completely blocked STEP translation. These results suggest that co-activation of both the ERK and PI3K-Akt-mTOR pathways are required for STEP translation. As the substrates of STEP include ERK itself, these results suggest that STEP is translated upon β-adrenergic activation as part of a negative feedback mechanism. PMID:17623046

  13. Translation of striatal-enriched protein tyrosine phosphatase (STEP) after beta1-adrenergic receptor stimulation.

    Science.gov (United States)

    Hu, Yaer; Zhang, Yang; Venkitaramani, Deepa V; Lombroso, Paul J

    2007-10-01

    The beta-adrenergic system is implicated in long-term synaptic plasticity in the CNS, a process that requires protein synthesis. To identify proteins that are translated in response to beta-adrenergic receptor stimulation and the pathways that regulate this process, we investigated the effects of isoproterenol on the translation of striatal-enriched protein tyrosine phosphatase (STEP) in both cortico-striatal slices and primary neuronal cultures. Isoproterenol stimulation induced a rapid dose-dependent increase in STEP expression. Anisomycin blocked the increase in STEP expression while actinomycin D had no effect, suggesting a translation-dependent mechanism. Isoproterenol-induced STEP translation required activation of beta1-receptors. Application of the MAPK/ERK kinase (MEK) inhibitor SL327 blocked both isoproterenol-induced activation of pERK and subsequent STEP translation. Inhibitors of PI3K (LY294002) or mTOR (rapamycin) also completely blocked STEP translation. These results suggest that co-activation of both the ERK and PI3K-Akt-mTOR pathways are required for STEP translation. As one of the substrates of STEP includes ERK itself, these results suggest that STEP is translated upon beta-adrenergic activation as part of a negative feedback mechanism.

  14. Advances in mass spectrometry based strategies to study receptor tyrosine kinases

    Directory of Open Access Journals (Sweden)

    Simon Vyse

    2017-03-01

    Full Text Available Receptor tyrosine kinases (RTKs are key transmembrane environmental sensors that are capable of transmitting extracellular information into phenotypic responses, including cell proliferation, survival and metabolism. Advances in mass spectrometry (MS-based phosphoproteomics have been instrumental in providing the foundations of much of our current understanding of RTK signalling networks and activation dynamics. Furthermore, new insights relating to the deregulation of RTKs in disease, for instance receptor co-activation and kinome reprogramming, have largely been identified using phosphoproteomic-based strategies. This review outlines the current approaches employed in phosphoproteomic workflows, including phosphopeptide enrichment and MS data-acquisition methods. Here, recent advances in the application of MS-based phosphoproteomics to bridge critical gaps in our knowledge of RTK signalling are focused on. The current limitations of the technology are discussed and emerging areas such as computational modelling, high-throughput phosphoproteomic workflows and next-generation single-cell approaches to further our understanding in new areas of RTK biology are highlighted.

  15. The tyrosine kinase receptor ROR1 is constitutively phosphorylated in chronic lymphocytic leukemia (CLL cells.

    Directory of Open Access Journals (Sweden)

    Mohammad Hojjat-Farsangi

    Full Text Available Phosphorylation of receptor tyrosine kinases (RTKs has a key role in cellular functions contributing to the malignant phenotype of tumor cells. We and others have previously demonstrated that RTK ROR1 is overexpressed in chronic lymphocytic leukemia (CLL. Silencing siRNA downregulated ROR1 and induced apoptosis of CLL cells. In the present study we analysed ROR1 isoforms and the phosphorylation pattern in CLL cells (n=38 applying western blot and flow-cytometry using anti-ROR1 antibodies and an anti-phospho-ROR1 antibody against the TK domain. Two major ROR1 bands with the size of 105 and 130 kDa respectively were identified, presumably representing unglycosylated (immature and glycosylated (mature ROR1 respectively as well as a 260 kDa band which may represent dimerized ROR1. A ROR1 band of 64 kDa that may correspond to a C-terminal fragment was also noted, present only in the nucleus. The 105 kDa ROR1 isoform was more frequently expressed in non-progressive as compared to progressive CLL patients (p=0.03. The 64, 105, 130 and 260 kDa bands were constitutively phosphorylated both at tyrosine and serine residues. Phosphorylation intensity of the mature (130 kDa isoform was significantly higher in progressive than in non-progressive disease (p<0.001. Incubation of CLL cells with a mouse anti-ROR1 KNG or an anti-ROR1 CRD mAb respectively induced dephosphorylation of ROR1 before entering apoptosis. In conclusion CLL cells expressed different isoforms of ROR1 which were constitutively phosphorylated. The mature, phosphorylated ROR1 isoform was associated with a progressive disease stage. Targeting ROR1 by mAbs induced specific dephosphorylation and leukemic cell death. ROR1 might be an interesting therapeutic target.

  16. The Plasticity of Oncogene Addiction: Implications for Targeted Therapies Directed to Receptor Tyrosine Kinases

    Directory of Open Access Journals (Sweden)

    Vinochani Pillay

    2009-05-01

    Full Text Available A common mutation of the epidermal growth factor receptor (EGFR in glioblastoma multiforme (GBM is an extracellular truncation known as the de2-7 EGFR (or EGFRvIII. Hepatocyte growth factor (HGF is the ligand for the receptor tyrosine kinase (RTK c-Met, and this signaling axis is often active in GBM. The expression of the HGF/c-Met axis or de2-7 EGFR independently enhances GBMgrowth and invasiveness, particularly through the phosphatidylinositol-3 kinase/pAkt pathway. Using RTK arrays, we show that expression of de2-7 EGFR in U87MG GBM cells leads to the coactivation of several RTKs, including platelet-derived growth factor receptor β and c-Met. A neutralizing antibody to HGF (AMG102 did not inhibit de2-7 EGFR-mediated activation of c-Met, demonstrating that it is ligand-independent. Therapy for parental U87MG xenografts with AMG 102 resulted in significant inhibition of tumor growth, whereas U87MG.Δ2-7 xenografts were profoundly resistant. Treatment of U87MG.Δ2-7 xenografts with panitumumab, an anti-EGFR antibody, only partially inhibited tumor growth as xenografts rapidly reverted to the HGF/c-Met signaling pathway. Cotreatment with panitumumab and AMG 102 prevented this escape leading to significant tumor inhibition through an apoptotic mechanism, consistent with the induction of oncogenic shock. This observation provides a rationale for using panitumumab and AMG 102 in combination for the treatment of GBM patients. These results illustrate that GBM cells can rapidly change the RTK driving their oncogene addiction if the alternate RTK signals through the same downstream pathway. Consequently, inhibition of a dominant oncogene by targeted therapy can alter the hierarchy of RTKs resulting in rapid therapeutic resistance.

  17. Tyrosine kinase receptor inhibitor-targeted combined chemotherapy for metastatic bladder cancer

    Directory of Open Access Journals (Sweden)

    Chia-Lun Wu

    2012-04-01

    Full Text Available Overexpression of hypoxia-inducible factor-1 alpha is noted during the invasive and metastatic process of transitional cell carcinoma. It will upregulate vascular endothelial growth factor (VEGF and drive proliferation, invasiveness, metastasis, and antiapoptotic ability of cancer cells. We proposed that tyrosine kinase receptor inhibitor, sunitinib malate—(Sutent; Pfizer Inc., Taiwan, combined with chemotherapeutic drug may present synergistic cytotoxic enhancement to transitional cell carcinoma cells with subsequent inhibition of their cellular behaviors, including proliferation, invasiveness, and metastatic activity. The contents of VEGF-A in mouse bladder tumor cells (MBT-2 and culture medium were detected by quantification-polymerase chain reaction and Western blot individually. The inhibitory concentrations of various chemotherapeutic drugs, sunitinib, and their combination treatment in MBT-2 were determined by 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT assay. Microchamber transmembrane migration assay was applied in evaluation of the inhibitory effects of different dosages of sunitinib and combination treatment on tumor cells. The cell cycle and apoptosis were analyzed after combination therapy by flow cytometry. Variation in apoptotic pathway was elucidated by Western blot using specific antibodies with cleaved PARP and caspase-3. Metastatic animal model mimicked by tail vein injection of MBT-2 cells was used to evaluate the treatment efficiency in tumor weight and survival rate. The mRNA and protein level of VEGF-A in MBT-2 cells increased by 70% at 48 hours interval under hypoxia stress condition. In MTT assay, MBT-2 cells had shown the highest sensitivity to epirubicin. Sunitinib combined with epirubicin had shown a synergistic cytotoxic effect to MBT-2 cells. Sunitinib and its combination with epirubicin showed significant inhibition on MBT-2 cells migration in microchambers. G2/M phase arrest and

  18. Identification of the insulin receptor tyrosine residues undergoing insulin-stimulated phosphorylation in intact rat hepatoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Tornqvist, H.E.; Gunsalus, J.R.; Nemenoff, R.A.; Frackelton, A.R.; Pierce, M.W.; Avruch,J.

    1988-01-05

    Tyr(P)-containing proteins were purified from extracts of insulin-treated rat hepatoma cells (H4-II-E-C3) by antiphosphotyrosine immunoaffinity chromatography. Two major insulin-stimulated, Tyr(P) proteins were recovered: an M/sub r/ 95,000 protein (identified as the insulin receptor ..beta.. subunit by its immunoprecipitation by a patient-derived anti-insulin receptor serum and several anti-insulin receptor (peptide) anti-sera) and an M/sub r/ 180,000 protein. After purification and tryptic digestion of the M/sub r/ 95,000 protein, tryptic peptides containing Tyr (P) were purified by sequential antiphosphotyrosine immunoaffinity, reversed-phase, anion-exchange chromatography. Approximately 80% of all ..beta.. subunit (/sup 32/P)Tyr(P) resides on two tryptic peptides: 50-60% of (/sup 32/P)Tyr(P) is found on the tryptic peptide Asp-Ile-Try-Glu-Thr-Asp-Try-Try-Arg from the tyrosine kinase domain. A second tryptic peptide is located near the carboxyl terminus; this contains 20-30% of ..beta.. subunit (/sup 32/P)Tyr(P) and is identified primarily in a double phosphorylated form. In a summary, the insulin-stimulated tyrosine phosphorylation of the insulin receptor in intact rat hepatoma cells involves at least 6 of the 13 tyrosine residues located on the ..beta.. subunit intracellular extension. These tyrosines are clustered in several domains in a distribution virtually identical to that previously found for partially purified human insulin receptor autophosphorylated in vitro in the presence of insulin.

  19. Insulin receptor binding and tyrosine kinase activity in skeletal muscle from normal pregnant women and women with gestational diabetes

    DEFF Research Database (Denmark)

    Damm, P; Handberg, A; Kühl, C

    1993-01-01

    OBJECTIVE: To ascertain whether the decreased glucose tolerance and insulin resistance found in normal and gestational diabetic pregnancy might be associated with changes in insulin receptor function. METHODS: Eight nonpregnant healthy women (nonpregnant controls), eight healthy pregnant women...... (pregnant controls), and eight women with gestational diabetes were investigated. All were non-obese. Muscle biopsies were obtained from the vastus lateralis muscle, and insulin binding and tyrosine kinase activities in partially purified skeletal muscle insulin receptors were studied. The pregnant controls...... with gestational diabetes compared to nonpregnant controls (P pregnant women did not differ from the other two groups. Postpartum, no differences in insulin binding were found between the groups. Basal and maximal tyrosine kinase activities toward the exogenous substrate poly(Glu4Tyr1) were...

  20. Mammalian motoneuron axon targeting requires receptor protein tyrosine phosphatases sigma and delta.

    Science.gov (United States)

    Uetani, Noriko; Chagnon, Mélanie J; Kennedy, Timothy E; Iwakura, Yoichiro; Tremblay, Michel L

    2006-05-31

    The leukocyte common antigen-related (LAR) subfamily of receptor protein tyrosine phosphatases (RPTPs), LAR, RPTP-sigma, and RPTP-delta, regulate neuroendocrine development, axonal regeneration, and hippocampal long-term potentiation in mammals. In Drosophila, RPTPs are required for appropriate axon targeting during embryonic development. In contrast, deletion of any one of the three LAR-RPTP family members in mammals does not result in gross axon targeting defects. Both RPTP-sigma and RPTP-delta are highly expressed in the developing mammalian nervous system, suggesting they might be functionally redundant. To test this hypothesis, we generated RPTP-sigma and RPTP-delta (RPTP-sigma/delta) double-mutant mice. Although embryonic day 18.5 RPTP-sigma and RPTP-delta single-mutant embryos were viable, RPTP-sigma/delta double mutants were paralyzed, were never observed to draw a breath, and died shortly after cesarean section. RPTP-sigma/delta double mutants exhibit severe muscle dysgenesis and severe loss of motoneurons in the spinal cord. Detailed analysis of the projections of phrenic nerves in RPTP-sigma/delta double mutants indicated that these motoneuron axons emerge normally from the cervical spinal cord, but stall on reaching the diaphragm. Our results demonstrate that RPTP-sigma and RPTP-delta complement each other functionally during mammalian development, and reveal an essential contribution of RPTP-sigma and RPTP-delta to appropriate motoneuron axon targeting during mammalian axonogenesis.

  1. Reactive Neutrophil Responses Dependent on the Receptor Tyrosine Kinase c-MET Limit Cancer Immunotherapy.

    Science.gov (United States)

    Glodde, Nicole; Bald, Tobias; van den Boorn-Konijnenberg, Debby; Nakamura, Kyohei; O'Donnell, Jake S; Szczepanski, Sabrina; Brandes, Maria; Eickhoff, Sarah; Das, Indrajit; Shridhar, Naveen; Hinze, Daniel; Rogava, Meri; van der Sluis, Tetje C; Ruotsalainen, Janne J; Gaffal, Evelyn; Landsberg, Jennifer; Ludwig, Kerstin U; Wilhelm, Christoph; Riek-Burchardt, Monika; Müller, Andreas J; Gebhardt, Christoffer; Scolyer, Richard A; Long, Georgina V; Janzen, Viktor; Teng, Michele W L; Kastenmüller, Wolfgang; Mazzone, Massimiliano; Smyth, Mark J; Tüting, Thomas; Hölzel, Michael

    2017-10-17

    Inhibitors of the receptor tyrosine kinase c-MET are currently used in the clinic to target oncogenic signaling in tumor cells. We found that concomitant c-MET inhibition promoted adoptive T cell transfer and checkpoint immunotherapies in murine cancer models by increasing effector T cell infiltration in tumors. This therapeutic effect was independent of tumor cell-intrinsic c-MET dependence. Mechanistically, c-MET inhibition impaired the reactive mobilization and recruitment of neutrophils into tumors and draining lymph nodes in response to cytotoxic immunotherapies. In the absence of c-MET inhibition, neutrophils recruited to T cell-inflamed microenvironments rapidly acquired immunosuppressive properties, restraining T cell expansion and effector functions. In cancer patients, high serum levels of the c-MET ligand HGF correlated with increasing neutrophil counts and poor responses to checkpoint blockade therapies. Our findings reveal a role for the HGF/c-MET pathway in neutrophil recruitment and function and suggest that c-MET inhibitor co-treatment may improve responses to cancer immunotherapy in settings beyond c-MET-dependent tumors. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Expression of receptor tyrosine kinase RYK in developing rat central nervous system.

    Science.gov (United States)

    Kamitori, K; Machide, M; Osumi, N; Kohsaka, S

    1999-04-12

    Receptor tyrosine kinase RYK is a mammalian homologue of Drosophila Lio, which is involved in learning and memory and in axon guidance. We cloned a rat ryk gene and characterized its expression pattern in the central nervous system. Northern blot analysis of the whole brain revealed that the RYK mRNA was abundant during the period from 13 to 18 embryonic days (E13-18) and it decreased by E20. In the postnatal brain, the RYK signal was higher in postnatal one week (P1W) cerebrum and in P2W cerebellum than in later stages. In situ hybridization revealed that RYK was expressed throughout the central nervous system, mainly in the ventricular zone on E11 and E13. On E18 and E20, the remarkable level of RYK mRNA was detected in the ventricular zone as well as in the cortical plate of the forebrain. These two regions overlapped the immunoreactive areas of nestin and MAP2, a neural stem cell marker and a mature neural marker, respectively. Moreover, the double-labeling analysis showed that the same cells expressed both RYK and nestin in the ventricular zone. In the postnatal brain, RYK was predominantly expressed in neurons of various regions. These observations suggest that RYK plays a contributory role as a multifunctional molecule in the differentiation and maturation of neuronal cells in the central nervous system. Copyright 1999 Elsevier Science B.V.

  3. Dialkoxyquinazolines: Screening Epidermal Growth Factor ReceptorTyrosine Kinase Inhibitors for Potential Tumor Imaging Probes

    Energy Technology Data Exchange (ETDEWEB)

    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.

    2005-09-01

    The epidermal growth factor receptor (EGFR), a long-standingdrug development target, is also a desirable target for imaging. Sixteendialkoxyquinazoline analogs, suitable for labeling with positron-emittingisotopes, have been synthesized and evaluated in a battery of in vitroassays to ascertain their chemical and biological properties. Thesecharacteristics provided the basis for the adoption of a selection schemato identify lead molecules for labeling and in vivo evaluation. A newEGFR tyrosine kinase radiometric binding assay revealed that all of thecompounds possessed suitable affinity (IC50 = 0.4 - 51 nM) for the EGFRtyrosine kinase. All of the analogs inhibited ligand-induced EGFRtyrosine phosphorylation (IC50 = 0.8 - 20 nM). The HPLC-estimatedoctanol/water partition coefficients ranged from 2.0-5.5. Four compounds,4-(2'-fluoroanilino)- and 4-(3'-fluoroanilino)-6,7-diethoxyquinazoline aswell as 4-(3'-chloroanilino)- and4-(3'-bromoanilino)-6,7-dimethoxyquinazoline, possess the bestcombination of characteristics that warrant radioisotope labeling andfurther evaluation in tumor-bearing mice.

  4. Congenital central hypoventilation syndrome: Mutation analysis of the receptor tyrosine kinase RET

    Energy Technology Data Exchange (ETDEWEB)

    Bolk, S.; Angrist, M.; Schwartz, S.; Chakravarti, A. [Case Western Reserve Univ., Cleveland, OH (United States)]|[University Hospitals, Cleveland, OH (United States)] [and others

    1996-06-28

    Congenital central hypoventilation syndrome (CCHS) usually occurs as an isolated phenotype. However, 16% of the index cases are also affected with Hirschsprung disease (HSCR). Complex segregation analysis suggests that CCHS is familial and has the same inheritance pattern with or without HSCR. We postulate that alteration of normal function of the receptor tyrosine kinase, RET, may contribute to CCHS based on RET`s expression pattern and the identification of RET mutations in HSCR patients. To further explore the nature of the inheritance of CCHS, we have undertaken two main routes of investigation: cytogenetic analysis and mutation detection. Cytogenetic analysis of metaphase chromosomes showed normal karyotypes in 13 of the 14 evaluated index cases; one index case carried a familial pericentric inversion on chromosome 2. Mutation analysis showed no sequence changes unique to index cases, as compared to control individuals, and as studied by single strand conformational polymorphism (SSCP) analysis of the coding region of RET. We conclude that point mutations in the RET coding region cannot account for a substantial fraction of CCHS in this patient population, and that other candidate genes involved in neural crest cell differentiation and development must be considered. 54 refs.

  5. Protein tyrosine phosphatase non-receptor type 22 modulates NOD2-induced cytokine release and autophagy.

    Directory of Open Access Journals (Sweden)

    Marianne R Spalinger

    Full Text Available BACKGROUND: Variations within the gene locus encoding protein tyrosine phosphatase non-receptor type 22 (PTPN22 are associated with the risk to develop inflammatory bowel disease (IBD. PTPN22 is involved in the regulation of T- and B-cell receptor signaling, but although it is highly expressed in innate immune cells, its function in other signaling pathways is less clear. Here, we study whether loss of PTPN22 controls muramyl-dipeptide (MDP-induced signaling and effects in immune cells. MATERIAL & METHODS: Stable knockdown of PTPN22 was induced in THP-1 cells by shRNA transduction prior to stimulation with the NOD2 ligand MDP. Cells were analyzed for signaling protein activation and mRNA expression by Western blot and quantitative PCR; cytokine secretion was assessed by ELISA, autophagosome induction by Western blot and immunofluorescence staining. Bone marrow derived dendritic cells (BMDC were obtained from PTPN22 knockout mice or wild-type animals. RESULTS: MDP-treatment induced PTPN22 expression and activity in human and mouse cells. Knockdown of PTPN22 enhanced MDP-induced activation of mitogen-activated protein kinase (MAPK-isoforms p38 and c-Jun N-terminal kinase as well as canonical NF-κB signaling molecules in THP-1 cells and BMDC derived from PTPN22 knockout mice. Loss of PTPN22 enhanced mRNA levels and secretion of interleukin (IL-6, IL-8 and TNF in THP-1 cells and PTPN22 knockout BMDC. Additionally, loss of PTPN22 resulted in increased, MDP-mediated autophagy in human and mouse cells. CONCLUSIONS: Our data demonstrate that PTPN22 controls NOD2 signaling, and loss of PTPN22 renders monocytes more reactive towards bacterial products, what might explain the association of PTPN22 variants with IBD pathogenesis.

  6. Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins

    Directory of Open Access Journals (Sweden)

    Krause Michael

    2010-07-01

    Full Text Available Abstract Background Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Methods Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Results Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1, early growth response 1 (Egr1, osteopontin (Opn, insulin-like growth factor binding protein 3 (Igfbp3, dual-specificity phosphatase 4 (Dusp4, and tumor-associated antigen L6 (Taal6. Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Conclusion Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute

  7. Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins.

    Science.gov (United States)

    Teutschbein, Janka; Haydn, Johannes M; Samans, Birgit; Krause, Michael; Eilers, Martin; Schartl, Manfred; Meierjohann, Svenja

    2010-07-21

    Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1), early growth response 1 (Egr1), osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), dual-specificity phosphatase 4 (Dusp4), and tumor-associated antigen L6 (Taal6). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development

  8. Association between receptor protein-tyrosine phosphatase RPTPalpha and the Grb2 adaptor. Dual Src homology (SH) 2/SH3 domain requirement and functional consequences

    DEFF Research Database (Denmark)

    Su, J; Yang, L T; Sap, J

    1996-01-01

    Receptor protein-tyrosine phosphatase RPTPalpha is found associated in vivo with the adaptor protein Grb2. Formation of this complex, which contains no detectable levels of Sos, is known to depend on a C-terminal phosphorylated tyrosine residue (Tyr798) in RPTPalpha and on the Src homology (SH) 2...... in vivo. These observations constitute a novel mode of Grb2 association and suggest a model in which association with a tyrosine-phosphorylated protein restricts the repertoire of SH3 binding proteins with which Grb2 can simultaneously interact. The function of the Tyr798 tyrosine phosphorylation/Grb2...

  9. Stretch-Induced Mitogen-Activated Protein Kinase Activation in Lung Fibroblasts Is Independent of Receptor Tyrosine Kinases

    OpenAIRE

    Boudreault, Francis; Tschumperlin, Daniel J.

    2009-01-01

    Lung growth and remodeling are modulated by mechanical stress, with fibroblasts thought to play a leading role. Little mechanistic information is available about how lung fibroblasts respond to mechanical stress. We exposed cultured lung fibroblasts to tonic stretch and measured changes in phosphorylation status of mitogen-activated protein kinases (MAPKs), selected receptor tyrosine kinases (RTKs), and phospholipase Cγ1 (PLCγ1) and activation of the small G-protein Ras. Human lung fibroblast...

  10. Stat3 activates the receptor tyrosine kinase like orphan receptor-1 gene in chronic lymphocytic leukemia cells.

    Directory of Open Access Journals (Sweden)

    Ping Li

    Full Text Available BACKGROUND: The receptor tyrosine kinase like orphan receptor (ROR-1 gene is overexpressed in chronic lymphocytic leukemia (CLL. Because Stat3 is constitutively activated in CLL and sequence analysis revealed that the ROR1 promoter harbors gamma-interferon activation sequence-like elements typically activated by Stat3, we hypothesized that Stat3 activates ROR1. METHODOLOGY/PRINCIPAL FINDINGS: Because IL-6 induced Stat3 phosphorylation and upregulated Ror1 protein levels in MM1 cells, we used these cells as a model. We transfected MM1 cells with truncated ROR1 promoter luciferase reporter constructs and found that IL-6 induced luciferase activity of ROR1-195 and upstream constructs. Co-transfection with Stat3 siRNA reduced the IL-6-induced luciferase activity, suggesting that IL-6 induced luciferase activity by activating Stat3. EMSA and the ChIP assay confirmed that Stat3 binds ROR1, and EMSA studies identified two Stat3 binding sites. In CLL cells, EMSA and ChIP studies determined that phosphorylated Stat3 bound to the ROR1 promoter at those two ROR1 promoter sites, and ChIP analysis showed that Stat3 co-immunoprecipitated DNA of STAT3, ROR1, and several Stat3-regulated genes. Finally, like STAT3-siRNA in MM1 cells, STAT3-shRNA downregulated STAT3, ROR1, and STAT3-regulated genes and Stat3 and Ror1 protein levels in CLL cells. CONCLUSION/SIGNIFICANCE: Our data suggest that constitutively activated Stat3 binds to the ROR1 promoter and activates ROR1 in CLL cells.

  11. 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.

  12. Identification of in vivo brain-derived neurotrophic factor-stimulated autophosphorylation sites on the TrkB receptor tyrosine kinase by site-directed mutagenesis.

    Science.gov (United States)

    Guiton, M; Gunn-Moore, F J; Stitt, T N; Yancopoulos, G D; Tavaré, J M

    1994-12-02

    Brain-derived neurotrophic factor (BDNF) interacts with the TrkB receptor tyrosine kinase, the tyrosine kinase domain of which has homology with the insulin receptor subfamily of protein kinases. This includes the conservation of three regulatory tyrosines (residues 670, 674, and 675) known to play a crucial role in signal transmission by the insulin receptor (tyrosines 1158, 1162, and 1163). Wild-type TrkB and TrkB mutants with Y670F, Y674F/Y675F, Y751F (the tyrosine reported to be important in phosphatidylinositol 3-kinase binding (Obermeier, A., Lammers, R., Wiesmuller, K. H., June, G., Schlessinger, J., and Ullrich, A. (1993) J. Biol. Chem. 268, 22963-22966)), and K540R (consensus ATP binding lysine) substitutions were transiently expressed in COS cells for analysis of phosphorylation sites by two-dimensional phosphopeptide mapping. TrkB phosphorylation sites were also studied in MG86 cells stably expressing wild-type TrkB. In addition, the mutants were expressed in Chinese hamster ovary cells for analysis of the ability of the receptor to mediate BDNF-stimulated transcription from a 12-O-tetradecanoylphorbol-13-acetate response element (TRE). BDNF stimulated the phosphorylation of wild-type TrkB on multiple tyrosine and serine residues. This phosphorylation occurred on tyrosines 670, 674, and 675 plus two other tyrosines and at least two serines that were not unequivocally identified. Wild-type TrkB mediated a pronounced stimulation of TRE-dependent transcription. A Y674F/Y675F, but not Y670F, substitution dramatically inhibited this response. Surprisingly, in COS cells, a Y751F substitution induced dramatically lower tyrosine and serine phosphorylation at all sites but mediated a normal BDNF-stimulated activation of a TRE. Our results demonstrate a critical role for the phosphorylation of tyrosines 674 and 675 in BDNF-dependent signaling by wild-type TrkB.

  13. Epidermal Growth Factor Receptor Tyrosine Kinase: A Potential Target in Treatment of Non-Small-Cell Lung Carcinoma.

    Science.gov (United States)

    Prabhu, Venugopal Vinod; Devaraj, Niranjali

    2017-01-01

    Lung cancer is responsible for 1.6 million deaths. Approximately 80%-85% of lung cancers are of the non-small-cell variety, which includes squamous cell carcinoma, adenocarcinoma, and large-cell carcinoma. Knowing the stage of cancer progression is a requisite for determining which management approach-surgery, chemotherapy, radiotherapy, and/or immunotherapy-is optimal. Targeted therapeutic approaches with antiangiogenic monoclonal antibodies or tyrosine kinase inhibitors are one option if tumors harbor oncogene mutations. Another, newer approach is directed against cancer-specific molecules and signaling pathways and thus has more limited nonspecific toxicities. This approach targets the epidermal growth factor receptor (EGFR, HER-1/ErbB1), a receptor tyrosine kinase of the ErbB family, which consists of four closely related receptors: HER-1/ErbB1, HER-2/neu/ErbB2, HER-3/ErbB3, and HER-4/ErbB4. Because EGFR is expressed at high levels on the surface of some cancer cells, it has been recognized as an effective anticancer target. EGFR-targeted therapies include monoclonal antibodies (mAbs) and small-molecule tyrosine kinase inhibitors. Tyrosine kinases are an especially important target because they play an important role in the modulation of growth factor signaling. This review highlights various classes of synthetically derived molecules that have been reported in the last few years as potential EGFR-TK inhibitors (TKIs) and their targeted therapies in NSCLC, along with effective strategies for overcoming EGFR-TKI resistance and efforts to develop a novel potent EGFR-TKI as an efficient target of NSCLC treatment in the foreseeable future.

  14. Receptor tyrosine kinase (c-Kit inhibitors: a potential therapeutic target in cancer cells

    Directory of Open Access Journals (Sweden)

    Abbaspour Babaei M

    2016-08-01

    Full Text Available Maryam Abbaspour Babaei,1 Behnam Kamalidehghan,2,3 Mohammad Saleem,4–6 Hasniza Zaman Huri,1,7 Fatemeh Ahmadipour1 1Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; 2Department of Medical Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB, Shahrak-e Pajoohesh, 3Medical Genetics Department, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran; 4Department of Urology, 5Department of Laboratory Medicine and Pathology, Masonic Cancer Center, University of Minnesota, 6Section of Molecular Therapeutics & Cancer Health Disparity, The Hormel Institute, Austin, MN, USA; 7Clinical Investigation Centre, University Malaya Medical Centre, Lembah Pantai, Kuala Lumpur, Malaysia Abstract: c-Kit, a receptor tyrosine kinase, is involved in intracellular signaling, and the mutated form of c-Kit plays a crucial role in occurrence of some cancers. The function of c-Kit has led to the concept that inhibiting c-Kit kinase activity can be a target for cancer therapy. The promising results of inhibition of c-Kit for treatment of cancers have been observed in some cancers such as gastrointestinal stromal tumor, acute myeloid leukemia, melanoma, and other tumors, and these results have encouraged attempts toward improvement of using c-Kit as a capable target for cancer therapy. This paper presents the findings of previous studies regarding c-Kit as a receptor tyrosine kinase and an oncogene, as well as its gene targets and signaling pathways in normal and cancer cells. The c-Kit gene location, protein structure, and the role of c-Kit in normal cell have been discussed. Comprehending the molecular mechanism underlying c-Kit-mediated tumorogenesis is consequently essential and may lead to the identification of future novel drug targets. The potential mechanisms by which c-Kit induces cellular transformation have been described. This study aims to elucidate the function of c

  15. Quantitative assessment of intragenic receptor tyrosine kinase deletions in primary glioblastomas: their prevalence and molecular correlates.

    Science.gov (United States)

    Kastenhuber, Edward R; Huse, Jason T; Berman, Samuel H; Pedraza, Alicia; Zhang, Jianan; Suehara, Yoshiyuki; Viale, Agnes; Cavatore, Magali; Heguy, Adriana; Szerlip, Nicholas; Ladanyi, Marc; Brennan, Cameron W

    2014-05-01

    Intragenic deletion is the most common form of activating mutation among receptor tyrosine kinases (RTK) in glioblastoma. However, these events are not detected by conventional DNA sequencing methods commonly utilized for tumor genotyping. To comprehensively assess the frequency, distribution, and expression levels of common RTK deletion mutants in glioblastoma, we analyzed RNA from a set of 192 glioblastoma samples from The Cancer Genome Atlas for the expression of EGFRvIII, EGFRvII, EGFRvV (carboxyl-terminal deletion), and PDGFRAΔ8,9. These mutations were detected in 24, 1.6, 4.7, and 1.6 % of cases, respectively. Overall, 29 % (55/189) of glioblastomas expressed at least one RTK intragenic deletion transcript in this panel. For EGFRvIII, samples were analyzed by both quantitative real-time PCR (QRT-PCR) and single mRNA molecule counting on the Nanostring nCounter platform. Nanostring proved to be highly sensitive, specific, and linear, with sensitivity comparable or exceeding that of RNA seq. We evaluated the prognostic significance and molecular correlates of RTK rearrangements. EGFRvIII was only detectable in tumors with focal amplification of the gene. Moreover, we found that EGFRvIII expression was not prognostic of poor outcome and that neither recurrent copy number alterations nor global changes in gene expression differentiate EGFRvIII-positive tumors from tumors with amplification of wild-type EGFR. The wide range of expression of mutant alleles and co-expression of multiple EGFR variants suggests that quantitative RNA-based clinical assays will be important for assessing the relative expression of intragenic deletions as therapeutic targets and/or candidate biomarkers. To this end, we demonstrate the performance of the Nanostring assay in RNA derived from routinely collected formalin-fixed paraffin-embedded tissue.

  16. Axl receptor tyrosine kinase is up-regulated in metformin resistant prostate cancer cells.

    Science.gov (United States)

    Bansal, Nitu; Mishra, Prasun J; Stein, Mark; DiPaola, Robert S; Bertino, Joseph R

    2015-06-20

    Recent epidemiological studies showed that metformin, a widely used anti-diabetic drug might prevent certain cancers. Metformin also has an anti-proliferative effect in preclinical studies of both hematologic malignancies as well as solid cancers and clinical studies testing metformin as an anti-cancer drug are in progress. However, all cancer types do not respond to metformin with the same effectiveness or acquire resistance. To understand the mechanism of acquired resistance and possibly its mechanism of action as an anti-proliferative agent, we developed metformin resistant LNCaP prostate cancer cells. Metformin resistant LNCaP cells had an increased proliferation rate, increased migration and invasion ability as compared to the parental cells, and expressed markers of epithelial-mesenchymal transition (EMT). A detailed gene expression microarray comparing the resistant cells to the wild type cells revealed that Edil2, Ereg, Axl, Anax2, CD44 and Anax3 were the top up-regulated genes and calbindin 2 and TPTE (transmembrane phosphatase with tensin homology) and IGF1R were down regulated. We focused on Axl, a receptor tyrosine kinase that has been shown to be up regulated in several drug resistance cancers. Here, we show that the metformin resistant cell line as well as castrate resistant cell lines that over express Axl were more resistant to metformin, as well as to taxotere compared to androgen sensitive LNCaP and CWR22 cells that do not overexpress Axl. Forced overexpression of Axl in LNCaP cells decreased metformin and taxotere sensitivity and knockdown of Axl in resistant cells increased sensitivity to these drugs. Inhibition of Axl activity by R428, a small molecule Axl kinase inhibitor, sensitized metformin resistant cells that overexpressed Axl to metformin. Inhibitors of Axl may enhance tumor responses to metformin and other chemotherapy in cancers that over express Axl.

  17. Molecular analysis of alternative transcripts of equine AXL receptor tyrosine kinase gene

    Directory of Open Access Journals (Sweden)

    Jeong-Woong Park

    2017-10-01

    Full Text Available Objective Since athletic performance is a most importance trait in horses, most research focused on physiological and physical studies of horse athletic abilities. In contrast, the molecular analysis as well as the regulatory pathway studies remain insufficient for evaluation and prediction of horse athletic abilities. In our previous study, we identified AXL receptor tyrosine kinase (AXL gene which was expressed as alternative spliced isoforms in skeletal muscle during exercise. In the present study, we validated two AXL alternative splicing transcripts (named as AXLa for long form and AXLb for short form in equine skeletal muscle to gain insight(s into the role of each alternative transcript during exercise. Methods We validated two isoforms of AXL transcripts in horse tissues by reverse transcriptase polymerase chain reaction (RT-PCR, and then cloned the transcripts to confirm the alternative locus and its sequences. Additionally, we examined the expression patterns of AXLa and AXLb transcripts in horse tissues by quantitative RT-PCR (qRT-PCR. Results Both of AXLa and AXLb transcripts were expressed in horse skeletal muscle and the expression levels were significantly increased after exercise. The sequencing analysis showed that there was an alternative splicing event at exon 11 between AXLa and AXLb transcripts. 3-dimentional (3D prediction of the alternative protein structures revealed that the structural distance of the connective region between fibronectin type 3 (FN3 and immunoglobin (Ig domain was different between two alternative isoforms. Conclusion It is assumed that the expression patterns of AXLa and AXLb transcripts would be involved in regulation of exercise-induced stress in horse muscle possibly through an NF-κB signaling pathway. Further study is necessary to uncover biological function(s and significance of the alternative splicing isoforms in race horse skeletal muscle.

  18. Spontaneous Immunity Against the Receptor Tyrosine Kinase ROR1 in Patients with Chronic Lymphocytic Leukemia.

    Directory of Open Access Journals (Sweden)

    Mohammad Hojjat-Farsangi

    Full Text Available ROR1 is a receptor tyrosine kinase expressed in chronic lymphocytic leukemia (CLL and several other malignancies but absent in most adult normal tissues. ROR1 is considered an onco-fetal antigen. In the present study we analysed spontaneous humoral and cellular immunity against ROR1 in CLL patients.Antibodies against ROR1 were analysed in 23 patients and 20 healthy donors by ELISA and Western blot. Purified serum IgG from patients was tested for cytotoxicity against CLL cells using the MTT viability assay. A cellular immune response against ROR1 derived HLA-A2 restricted 9 aa and 16 aa long peptides were analysed using peptide loaded dendritic cells co-cultured with autologous T cells from CLL patients (n = 9 and healthy donors (n = 6. IFN-γ, IL-5 and IL-17A-secreting T cells were assessed by ELISPOT and a proliferative response using a H3-thymidine incorporation assay.The majority of CLL patients had antibodies against ROR1. Significantly higher titers of anti-ROR1 antibodies were noted in patients with non-progressive as compared to progressive disease. The extracellular membrane-close ROR1 KNG domain seemed to be an immunodominant epitope. Ten patients with high titers of anti-ROR1 binding antibodies were tested for cytotoxicity. Five of those had cytotoxic anti-ROR1 antibodies against CLL cells. ROR1-specific IFN-γ and IL-17A producing T cells could be detected in CLL patients, preferentially in non-progressive as compared to patients with progressive disease (p<0.05.ROR1 seemed to spontaneously induce a humoral as well as a T cell response in CLL patients. The data support the notion that ROR1 might be a specific neo-antigen and may serve as a target for immunotherapy.

  19. Regulation of STEP61 and tyrosine-phosphorylation of NMDA and AMPA receptors during homeostatic synaptic plasticity.

    Science.gov (United States)

    Jang, Sung-Soo; Royston, Sara E; Xu, Jian; Cavaretta, John P; Vest, Max O; Lee, Kwan Young; Lee, Seungbae; Jeong, Han Gil; Lombroso, Paul J; Chung, Hee Jung

    2015-09-22

    Sustained changes in network activity cause homeostatic synaptic plasticity in part by altering the postsynaptic accumulation of N-methyl-D-aspartate receptors (NMDAR) and α-amino-3-hydroxyle-5-methyl-4-isoxazolepropionic acid receptors (AMPAR), which are primary mediators of excitatory synaptic transmission. A key trafficking modulator of NMDAR and AMPAR is STriatal-Enriched protein tyrosine Phosphatase (STEP61) that opposes synaptic strengthening through dephosphorylation of NMDAR subunit GluN2B and AMPAR subunit GluA2. However, the role of STEP61 in homeostatic synaptic plasticity is unknown. We demonstrate here that prolonged activity blockade leads to synaptic scaling, and a concurrent decrease in STEP61 level and activity in rat dissociated hippocampal cultured neurons. Consistent with STEP61 reduction, prolonged activity blockade enhances the tyrosine phosphorylation of GluN2B and GluA2 whereas increasing STEP61 activity blocks this regulation and synaptic scaling. Conversely, prolonged activity enhancement increases STEP61 level and activity, and reduces the tyrosine phosphorylation and level of GluN2B as well as GluA2 expression in a STEP61-dependent manner. Given that STEP61-mediated dephosphorylation of GluN2B and GluA2 leads to their internalization, our results collectively suggest that activity-dependent regulation of STEP61 and its substrates GluN2B and GluA2 may contribute to homeostatic stabilization of excitatory synapses.

  20. NR2B-NMDA receptor mediated modulation of the tyrosine phosphatase STEP regulates glutamate induced neuronal cell death

    Science.gov (United States)

    Poddar, Ranjana; Deb, Ishani; Mukherjee, Saibal; Paul, Surojit

    2011-01-01

    The present study examines the role of a neuron-specific tyrosine phosphatase (STEP) in excitotoxic cell death. Our findings demonstrate that p38 MAPK, a stress-activated kinase that is known to play a role in the etiology of excitotoxic cell death is a substrate of STEP. Glutamate-mediated NMDA receptor stimulation leads to rapid but transient activation of p38 MAPK, which is primarily dependent on NR2A-NMDA receptor activation. Conversely, activation of NR2B-NMDA receptors leads to dephosphorylation and subsequent activation of STEP, which in turn leads to inactivation of p38 MAPK. Thus during transient NMDA receptor stimulation, increases in STEP activity appears to limit the duration of activation of p38 MAPK and improves neuronal survival. However, if NR2B-NMDA receptor stimulation is sustained, protective effects of STEP activation are lost, as these stimuli cause significant degradation of active STEP, leading to secondary activation of p38 MAP kinase. Consistent with this observation, a cell transducible TAT-STEP peptide that constitutively binds to p38 MAPK attenuated neuronal cell death caused by sustained NMDA receptor stimulation. The findings imply that the activation and levels of STEP are dependent on the duration and magnitude of NR2B-NMDA receptor stimulation and STEP serves as a modulator of NMDA receptor dependent neuronal injury, through its regulation of p38 MAPK. PMID:21029094

  1. A point mutation at tyrosine-809 in the human colony-stimulating factor 1 receptor impairs mitogenesis without abrogating tyrosine kinase activity, association with phosphatidylinositol 3-kinase, or induction of c-fos and junB genes

    Energy Technology Data Exchange (ETDEWEB)

    Roussel, M.F. (Univ. of Tennessee, Memphis (USA)); Shurtleff, S.A.; Downing, J.R. (Saint Jude Children' s Research Hospital, Memphis, TN (USA)); Sherr, C.J. (Univ. of Tennessee College of Medicine, Memphis (USA) Saint Jude Children' s Research Hospital, Memphis, TN (USA))

    1990-09-01

    Substitution of phenylalanine for tyrosine-809 in the human colony-stimulating factor 1 receptor (CSF-1R) inhibited its ability to transduce ligand-dependent mitogenic signals in mouse NIH 3T3 cells. When combined with an activating mutation at codon 301 that induces constitutive CSF-1R tyrosine kinase activity, the codon 809 mutation suppressed ligand-independent cell transformation. Comparative mapping tryptic phosphopeptides from mutant and wild-type CSF-1R indicated that tyrosine-809 is a site of ligand-dependent receptor phosphorylation in vivo. The mutant receptor was active as a tyrosine kinase in vitro and in vivo, underwent CSF-1-dependent association with a phosphatidylinositol 3-kinase, and induced expression of the protooncogenes c-fos and junB, underscoring its ability to trigger some of the known cellular responses to CSF-1. The mutant receptor is likely to be impaired in its ability to interact with critical cellular effectors whose activity is required for mitogenesis.

  2. The Molecular Crosstalk between the MET Receptor Tyrosine Kinase and the DNA Damage Response — Biological and Clinical Aspects

    Energy Technology Data Exchange (ETDEWEB)

    Medová, Michaela, E-mail: michaela.medova@dkf.unibe.ch; Aebersold, Daniel M.; Zimmer, Yitzhak, E-mail: michaela.medova@dkf.unibe.ch [Department of Radiation Oncology, Inselspital, Bern University Hospital, and University of Bern, 3010 Bern (Switzerland); Department of Clinical Research, University of Bern, DKF, MEM-E807, Murtenstrasse 35, 3010 Bern (Switzerland)

    2013-12-19

    Radiation therapy remains an imperative treatment modality for numerous malignancies. Enduring significant technical achievements both on the levels of treatment planning and radiation delivery have led to improvements in local control of tumor growth and reduction in healthy tissue toxicity. Nevertheless, resistance mechanisms, which presumably also involve activation of DNA damage response signaling pathways that eventually may account for loco-regional relapse and consequent tumor progression, still remain a critical problem. Accumulating data suggest that signaling via growth factor receptor tyrosine kinases, which are aberrantly expressed in many tumors, may interfere with the cytotoxic impact of ionizing radiation via the direct activation of the DNA damage response, leading eventually to so-called tumor radioresistance. The aim of this review is to overview the current known data that support a molecular crosstalk between the hepatocyte growth factor receptor tyrosine kinase MET and the DNA damage response. Apart of extending well established concepts over MET biology beyond its function as a growth factor receptor, these observations directly relate to the role of its aberrant activity in resistance to DNA damaging agents, such as ionizing radiation, which are routinely used in cancer therapy and advocate tumor sensitization towards DNA damaging agents in combination with MET targeting.

  3. Receptor tyrosine phosphatase beta is expressed in the form of proteoglycan and binds to the extracellular matrix protein tenascin

    DEFF Research Database (Denmark)

    Barnea, G; Grumet, M; Milev, P

    1994-01-01

    The extracellular domain of receptor type protein tyrosine phosphatase beta (RPTP beta) exhibits striking sequence similarity with a soluble, rat brain chondroitin sulfate proteoglycan (3F8 PG). Immunoprecipitation experiments of cells transfected with RPTP beta expression vector and metabolically...... labeled with [35S]sulfate and [35S]methionine indicate that the transmembrane form of RPTP beta is indeed a chondroitin sulfate proteoglycan. The 3F8 PG is therefore a variant form composed of the entire extracellular domain of RPTP beta probably generated by alternative RNA splicing. Previous...

  4. Internalization and down-regulation of the human epidermal growth factor receptor are regulated by the carboxyl-terminal tyrosines

    DEFF Research Database (Denmark)

    Helin, K; Beguinot, L

    1991-01-01

    of EGF. The specific rate of internalization of the triple point mutant was reduced. By contrast, intracellular processing of ligand previously internalized at 20 degrees C was similar between wild type and mutant receptors. Taken together the data indicate that the delay in degradation observed in cells...... expressing the triple point mutant EGF-R can be attributed mainly to a slower removal from the cell surface. Our results show that in the full-length EGF-R all three C-terminal tyrosines are necessary for rapid internalization, suggesting that autophosphorylation is required for efficient EGF...

  5. Intimate association of Thy-1 and the T-cell antigen receptor with the CD45 tyrosine phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Volarevic, S.; Burns, C.M.; Sussman, J.J.; Ashwell, J.D. (National Cancer Inst., Bethesda, MD (USA))

    1990-09-01

    Immunoprecipitation of Thy-1 from Triton X-100 detergent lysates of surface-iodinated and chemically cross-linked T cells precipitated at least first major and discrete bands. Four of these bands were identified as Thy-1, CD45 (a trasmembrane tyrosine phosphatase), a major histocompatibility complex-encoded class I molecule, and {beta}{sub 2}-microglobulin. Similar analyses revealed that CD45 was coprecipitated from lysates of cross-linker-treated cells by antibodies to the T-cell antigen receptor (TCR). The same pattern of coprecipitated bands was observed when digitonin was used to lyse untreated cells. Immunoprecipitation of Thy-1 or the TCR from lysates of cross-linked T cells precipitated CD45 tyrosine phosphatase activity. Calculations based upon the amounts of coprecipitated enzymatic activity or TCR {zeta} chain indicate that a substantial fraction of Thy-1 and TCR complexes can be cross-linked to CD45. These data support a model in which the dependence of Thy-1 signaling on TCR coexpression is due to their common interaction with a tyrosine phosphatase and provide a possible structural basis for the influence of CD45 on TCR-mediated signaling.

  6. Association of protein tyrosine phosphatase, non-receptor type 22 +1858C→T polymorphism and susceptibility to vitiligo: Systematic review and meta-analysis.

    Science.gov (United States)

    Agarwal, Silky; Changotra, Harish

    2017-01-01

    Protein tyrosine phosphatase, non-receptor type 22 gene, which translates to lymphoid tyrosine phosphatase, is considered to be a susceptibility gene marker associated with several autoimmune diseases. Several studies have demonstrated the association of protein tyrosine phosphatase, non-receptor type 22 +1858C→T polymorphism with vitiligo. However, these studies showed conflicting results. Meta-analysis of the same was conducted earlier that included fewer number of publications in their study. We performed a meta-analysis of a total of seven studies consisting of 2094 cases and 3613 controls to evaluate the possible association of protein tyrosine phosphatase, non-receptor type 22 +1858C>T polymorphism with vitiligo susceptibility. We conducted a literature search in PubMed, Google Scholar and Dogpile for all published paper on protein tyrosine phosphatase, non-receptor type 22 +1858C→T polymorphism and vitiligo risk till June 2016. Data analysis was performed by RevMan 5.3 and comprehensive meta-analysis v3.0 software. Meta-analysis showed an overall significant association of protein tyrosine phosphatase, non- receptor type 22 +1858C→T polymorphism with vitiligo in all models (allelic model [T vs. C]: odds ratio = 1.50, 95% confidence interval [1.32-1.71], Pvitiligo-type are some limitations of the present meta-analysis. Stratifying data by ethnicity showed an association of protein tyrosine phosphatase, non-receptor type 22 +1858C→T with vitiligo in European population (odds ratio = 1.53, 95% confidence interval [1.34-1.75], Pvitiligo.

  7. The R3 receptor-like protein tyrosine phosphatase subfamily inhibits insulin signalling by dephosphorylating the insulin receptor at specific sites.

    Science.gov (United States)

    Shintani, Takafumi; Higashi, Satoru; Takeuchi, Yasushi; Gaudio, Eugenio; Trapasso, Francesco; Fusco, Alfredo; Noda, Masaharu

    2015-09-01

    The autophosphorylation of specific tyrosine residues occurs in the cytoplasmic region of the insulin receptor (IR) upon insulin binding, and this in turn initiates signal transduction. The R3 subfamily (Ptprb, Ptprh, Ptprj and Ptpro) of receptor-like protein tyrosine phosphatases (RPTPs) is characterized by an extracellular region with 6-17 fibronectin type III-like repeats and a cytoplasmic region with a single phosphatase domain. We herein identified the IR as a substrate for R3 RPTPs by using the substrate-trapping mutants of R3 RPTPs. The co-expression of R3 RPTPs with the IR in HEK293T cells suppressed insulin-induced tyrosine phosphorylation of the IR. In vitro assays using synthetic phosphopeptides revealed that R3 RPTPs preferentially dephosphorylated a particular phosphorylation site of the IR: Y960 in the juxtamembrane region and Y1146 in the activation loop. Among four R3 members, only Ptprj was co-expressed with the IR in major insulin target tissues, such as the skeletal muscle, liver and adipose tissue. Importantly, the activation of IR and Akt by insulin was enhanced, and glucose and insulin tolerance was improved in Ptprj-deficient mice. These results demonstrated Ptprj as a physiological enzyme that attenuates insulin signalling in vivo, and indicate that an inhibitor of Ptprj may be an insulin-sensitizing agent. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  8. Structure and Molecular Dynamics Simulations of Protein Tyrosine Phosphatase Non-Receptor 12 Provide Insights into the Catalytic Mechanism of the Enzyme

    OpenAIRE

    Hui Dong; Francesco Zonta; Shanshan Wang; Ke Song; Xin He; Miaomiao He; Yan Nie; Sheng Li

    2017-01-01

    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 r...

  9. Crystal Structure of the Frizzled-Like Cysteine-Rich Domain of the Receptor Tyrosine Kinase MuSK

    Energy Technology Data Exchange (ETDEWEB)

    Stiegler, A.; Burden, S; Hubbard, S

    2009-01-01

    Muscle-specific kinase (MuSK) is an essential receptor tyrosine kinase for the establishment and maintenance of the neuromuscular junction (NMJ). Activation of MuSK by agrin, a neuronally derived heparan-sulfate proteoglycan, and LRP4 (low-density lipoprotein receptor-related protein-4), the agrin receptor, leads to clustering of acetylcholine receptors on the postsynaptic side of the NMJ. The ectodomain of MuSK comprises three immunoglobulin-like domains and a cysteine-rich domain (Fz-CRD) related to those in Frizzled proteins, the receptors for Wnts. Here, we report the crystal structure of the MuSK Fz-CRD at 2.1 {angstrom} resolution. The structure reveals a five-disulfide-bridged domain similar to CRDs of Frizzled proteins but with a divergent C-terminal region. An asymmetric dimer present in the crystal structure implicates surface hydrophobic residues that may function in homotypic or heterotypic interactions to mediate co-clustering of MuSK, rapsyn, and acetylcholine receptors at the NMJ.

  10. Strategies of targeting the extracellular domain of RON tyrosine kinase receptor for cancer therapy and drug delivery.

    Science.gov (United States)

    Zarei, Omid; Benvenuti, Silvia; Ustun-Alkan, Fulya; Hamzeh-Mivehroud, Maryam; Dastmalchi, Siavoush

    2016-12-01

    Cancer is one of the most important life-threatening diseases in the world. The current efforts to combat cancer are being focused on molecular-targeted therapies. The main purpose of such approaches is based on targeting cancer cell-specific molecules to minimize toxicity for the normal cells. RON (Recepteur d'Origine Nantais) tyrosine kinase receptor is one of the promising targets in cancer-targeted therapy and drug delivery. In this review, we will summarize the available agents against extracellular domain of RON with potential antitumor activities. The presented antibodies and antibody drug conjugates against RON in this review showed wide spectrum of in vitro and in vivo antitumor activities promising the hope for them entering the clinical trials. Due to critical role of extracellular domain of RON in receptor activation, the development of therapeutic agents against this region could lead to fruitful outcome in cancer therapy.

  11. Bruton's tyrosine kinase mediates the synergistic signalling between TLR9 and the B cell receptor by regulating calcium and calmodulin.

    Directory of Open Access Journals (Sweden)

    Elaine F Kenny

    Full Text Available B cells signal through both the B cell receptor (BCR which binds antigens and Toll-like receptors (TLRs including TLR9 which recognises CpG DNA. Activation of TLR9 synergises with BCR signalling when the BCR and TLR9 co-localise within an auto-phagosome-like compartment. Here we report that Bruton's tyrosine kinase (BTK is required for synergistic IL6 production and up-regulation of surface expression of MHC-class-II, CD69 and CD86 in primary murine and human B cells. We show that BTK is essential for co-localisation of the BCR and TLR9 within a potential auto-phagosome-like compartment in the Namalwa human B cell line. Downstream of BTK we find that calcium acting via calmodulin is required for this process. These data provide new insights into the role of BTK, an important target for autoimmune diseases, in B cell activation.

  12. Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1 into Diverse Memory T-Cell Populations.

    Directory of Open Access Journals (Sweden)

    Drew C Deniger

    Full Text Available T cells modified with chimeric antigen receptors (CARs targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1 is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28 or CD137 (designated ROR1RCD137 and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC, which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.

  13. Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations.

    Science.gov (United States)

    Deniger, Drew C; Yu, Jianqiang; Huls, M Helen; Figliola, Matthew J; Mi, Tiejuan; Maiti, Sourindra N; Widhopf, George F; Hurton, Lenka V; Thokala, Radhika; Singh, Harjeet; Olivares, Simon; Champlin, Richard E; Wierda, William G; Kipps, Thomas J; Cooper, Laurence J N

    2015-01-01

    T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.

  14. ARQ 197, a novel and selective inhibitor of the human c-Met receptor tyrosine kinase with antitumor activity.

    Science.gov (United States)

    Munshi, Neru; Jeay, Sébastien; Li, Youzhi; Chen, Chang-Rung; France, Dennis S; Ashwell, Mark A; Hill, Jason; Moussa, Magdi M; Leggett, David S; Li, Chiang J

    2010-06-01

    The met proto-oncogene is functionally linked with tumorigenesis and metastatic progression. Validation of the receptor tyrosine kinase c-Met as a selective anticancer target has awaited the emergence of selective c-Met inhibitors. Herein, we report ARQ 197 as the first non-ATP-competitive small molecule that selectively targets the c-Met receptor tyrosine kinase. Exposure to ARQ 197 resulted in the inhibition of proliferation of c-Met-expressing cancer cell lines as well as the induction of caspase-dependent apoptosis in cell lines with constitutive c-Met activity. These cellular responses to ARQ 197 were phenocopied by RNAi-mediated c-Met depletion and further demonstrated by the growth inhibition of human tumors following oral administration of ARQ 197 in multiple mouse xenograft efficacy studies. Cumulatively, these data suggest that ARQ 197, currently in phase II clinical trials, is a promising agent for targeting cancers in which c-Met-driven signaling is important for their survival and proliferation.

  15. Isolation and functional characterization of peptide agonists of PTPRJ, a tyrosine phosphatase receptor endowed with tumor suppressor activity.

    Science.gov (United States)

    Paduano, Francesco; Ortuso, Francesco; Campiglia, Pietro; Raso, Cinzia; Iaccino, Enrico; Gaspari, Marco; Gaudio, Eugenio; Mangone, Graziella; Carotenuto, Alfonso; Bilotta, Anna; Narciso, Domenico; Palmieri, Camillo; Agosti, Valter; Artese, Anna; Gomez-Monterrey, Isabel; Sala, Marina; Cuda, Giovanni; Iuliano, Rodolfo; Perrotti, Nicola; Scala, Giuseppe; Viglietto, Giuseppe; Alcaro, Stefano; Croce, Carlo M; Novellino, Ettore; Fusco, Alfredo; Trapasso, Francesco

    2012-10-19

    PTPRJ is a receptor-type protein tyrosine phosphatase whose expression is strongly reduced in the majority of investigated cancer cell lines and tumor specimens. PTPRJ negatively interferes with mitogenic signals originating from several oncogenic receptor tyrosine kinases, including HGFR, PDGFR, RET, and VEGFR-2. Here we report the isolation and characterization of peptides from a random peptide phage display library that bind and activate PTPRJ. These agonist peptides, which are able to both circularize and form dimers in acqueous solution, were assayed for their biochemical and biological activity on both human cancer cells and primary endothelial cells (HeLa and HUVEC, respectively). Our results demonstrate that binding of PTPRJ-interacting peptides to cell cultures dramatically reduces the extent of both MAPK phosphorylation and total phosphotyrosine levels; conversely, they induce a significant increase of the cell cycle inhibitor p27(Kip1). Moreover, PTPRJ agonist peptides both reduce proliferation and trigger apoptosis of treated cells. Our data indicate that peptide agonists of PTPRJ positively modulate the PTPRJ activity and may lead to novel targeted anticancer therapies.

  16. Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site.

    Science.gov (United States)

    Braunger, J; Schleithoff, L; Schulz, A S; Kessler, H; Lammers, R; Ullrich, A; Bartram, C R; Janssen, J W

    1997-06-05

    Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

  17. A tyrosine kinase inhibitor-based high-affinity PET radiopharmaceutical targets vascular endothelial growth factor receptor.

    Science.gov (United States)

    Li, Feng; Jiang, Sheng; Zu, Youli; Lee, Daniel Y; Li, Zheng

    2014-09-01

    Tyrosine kinase receptors including vascular endothelial growth factor receptor (VEGFR) have gained significant attention as pharmacologic targets. However, clinical evaluation of small-molecule drugs or biologics that target these pathways has so far yielded mixed results in a variety of solid tumors. The reasons for response variability remain unknown, including the temporal and spatial patterns of receptor tyrosine kinase expression. Methods to detect and quantify the presence of such cellular receptors would greatly facilitate drug development and therapy response assessment. We aimed to generate specific imaging agents as potential companion diagnostics that could also be used for targeted radionuclide therapy. Here, we report on the synthesis and initial preclinical performance of (64)Cu-labeled probes that were based on the kinase inhibitor already in clinical use, vandetanib (ZD6474), as a VEGFR-selective theranostic radiopharmaceutical. A monomeric (ZD-G1) and a dimeric (ZD-G2) derivative of ZD6474 were synthesized and conjugated with DOTA for chelation with (64)Cu to produce the probes (64)Cu-DOTA-ZD-G1 and (64)Cu-DOTA-ZD-G2. The binding affinity and specificity to VEGFR were measured using U-87 MG cells known to overexpress VEGFR. Small-animal PET and biodistribution studies were performed with (64)Cu-labeled probes (3-4 MBq) intravenously administered in U-87 MG tumor-bearing mice with or without coinjection of unlabeled ZD-G2 for up to 24 h after injection. Receptor-binding assays yielded a mean equilibrium dissociation constant of 44.7 and 0.45 nM for monomeric and dimeric forms, respectively, indicating a synergistic effect in VEGFR affinity by multivalency. Small-animal PET/CT imaging showed rapid tumor accumulation of (64)Cu-DOTA-ZD-G2, with excellent tumor-to-normal tissue contrast by 24 h. Coinjection of the (64)Cu-DOTA-ZD-G2 with 50 nmol (60 μg) of nonradioactive ZD-G2 effectively blocked tumor uptake. A (64)Cu-labeled probe derived from an

  18. Tyrosine-based signal mediates LRP6 receptor endocytosis and desensitization of Wnt/β-catenin pathway signaling.

    Science.gov (United States)

    Liu, Chia-Chen; Kanekiyo, Takahisa; Roth, Barbara; Bu, Guojun

    2014-10-03

    Wnt/β-catenin signaling orchestrates a number of critical events including cell growth, differentiation, and cell survival during development. Misregulation of this pathway leads to various human diseases, specifically cancers. Endocytosis and phosphorylation of the LDL receptor-related protein 6 (LRP6), an essential co-receptor for Wnt/β-catenin signaling, play a vital role in mediating Wnt/β-catenin signal transduction. However, its regulatory mechanism is not fully understood. In this study, we define the mechanisms by which LRP6 endocytic trafficking regulates Wnt/β-catenin signaling activation. We show that LRP6 mutant with defective tyrosine-based signal in its cytoplasmic tail has an increased cell surface distribution and decreased endocytosis rate. These changes in LRP6 endocytosis coincide with an increased distribution to caveolae, increased phosphorylation, and enhanced Wnt/β-catenin signaling. We further demonstrate that treatment of Wnt3a ligands or blocking the clathrin-mediated endocytosis of LRP6 leads to a redistribution of wild-type receptor to lipid rafts. The LRP6 tyrosine mutant also exhibited an increase in signaling activation in response to Wnt3a stimulation when compared with wild-type LRP6, and this activation is suppressed when caveolae-mediated endocytosis is blocked. Our results reveal molecular mechanisms by which LRP6 endocytosis routes regulate its phosphorylation and the strength of Wnt/β-catenin signaling, and have implications on how this pathway can be modulated in human diseases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Thrombin-mediated Proteoglycan Synthesis Utilizes Both Protein-tyrosine Kinase and Serine/Threonine Kinase Receptor Transactivation in Vascular Smooth Muscle Cells*

    Science.gov (United States)

    Burch, Micah L.; Getachew, Robel; Osman, Narin; Febbraio, Mark A.; Little, Peter J.

    2013-01-01

    G protein-coupled receptor signaling is mediated by three main mechanisms of action; these are the classical pathway, β-arrestin scaffold signaling, and the transactivation of protein-tyrosine kinase receptors such as those for EGF and PDGF. Recently, it has been demonstrated that G protein-coupled receptors can also mediate signals via transactivation of serine/threonine kinase receptors, most notably the transforming growth factor-β receptor family. Atherosclerosis is characterized by the development of lipid-laden plaques in blood vessel walls. Initiation of plaque development occurs via low density lipoprotein retention in the neointima of vessels due to binding with modified proteoglycans secreted by vascular smooth muscle cells. Here we show that transactivation of protein-tyrosine kinase receptors is mediated by matrix metalloproteinase triple membrane bypass signaling. In contrast, serine/threonine kinase receptor transactivation is mediated by a cytoskeletal rearrangement-Rho kinase-integrin system, and both protein-tyrosine kinase and serine/threonine kinase receptor transactivation concomitantly account for the total proteoglycan synthesis stimulated by thrombin in vascular smooth muscle. This work provides evidence of thrombin-mediated proteoglycan synthesis and paves the way for a potential therapeutic target for plaque development and atherosclerosis. PMID:23335513

  20. 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.

  1. A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Park, So Jung; Park, Young Jun; Shin, Ji Hyun; Kim, Eun Sung [Graduate School of East-West Medical Science, Kyung Hee University, Gyeoggi-Do 446-701 (Korea, Republic of); Hwang, Jung Jin; Jin, Dong-Hoon; Kim, Jin Cheon [Institute for Innovative Cancer Research, Asan Medical Center, Seoul 138-736 (Korea, Republic of); Cho, Dong-Hyung, E-mail: dhcho@khu.ac.kr [Graduate School of East-West Medical Science, Kyung Hee University, Gyeoggi-Do 446-701 (Korea, Republic of)

    2011-05-13

    Highlights: {yields} We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. {yields} Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. {yields} Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. {yields} Our studies suggest that Tyrphostin A9 induces mitochondria fragmentation and apoptotic cell death via Drp1 dependently. -- Abstract: Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactive chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death.

  2. Ligand-mediated negative regulation of a chimeric transmembrane receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Desai, D M; Sap, J; Schlessinger, J

    1993-01-01

    inactivate the EGFR-CD45 chimera in a manner that is dependent on dimerization of the chimeric protein. Inactivation of EGFR-CD45 chimera function results in the loss of TCR signaling, indicating that CD45 function is continuously required for TCR-mediated proximal signaling events. These results suggest......CD45, a transmembrane protein tyrosine phosphatase (PTPase), is required for TCR signaling. Multiple CD45 isoforms, differing in the extracellular domain, are expressed in a tissue- and activation-specific manner, suggesting an important function for this domain. We report that a chimeric protein...

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

    Science.gov (United States)

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

    2009-01-01

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

  4. A new family of receptor tyrosine kinases with a venus flytrap binding domain in insects and other invertebrates activated by aminoacids.

    Directory of Open Access Journals (Sweden)

    Arnaud Ahier

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

  5. {delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    Energy Technology Data Exchange (ETDEWEB)

    Heiss, Anika; Ammer, Hermann [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany); Eisinger, Daniela A., E-mail: eisinger@pharmtox.vetmed.uni-muenchen.de [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany)

    2009-07-15

    {delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  6. The role of oestrogen receptor {alpha} in human thyroid cancer: contributions from coregulatory proteins and the tyrosine kinase receptor HER2.

    LENUS (Irish Health Repository)

    Kavanagh, Dara O

    2012-02-01

    Epidemiological, clinical, and molecular studies suggest a role for oestrogen in thyroid cancer. How oestrogen mediates its effects and the consequence of it on clinical outcome has not been fully elucidated. The participation of coregulatory proteins in modulating oestrogen receptor (ER) function and input of crosstalk with the tyrosine kinase receptor HER2 was investigated. Oestrogen induced cell proliferation in the follicular thyroid cancer (FTC)-133 cells, but not in the anaplastic 8305C cell line. Knockdown of the coactivator steroid receptor coactivator (SRC)-1 inhibited FTC-133 basal, but not oestrogen induced, cell proliferation. Oestrogen also increased protein expression of SRC-1 and the ER target gene cyclin D1 in the FTC-133 cell line. ERalpha, ERbeta, the coregulatory proteins SRC-1 and nuclear corepressor (NCoR), and the tyrosine kinase receptor HER2 were localised by immunohistochemistry and immnofluorescence in paraffin-embedded tissue from thyroid tumour patients (n=111). ERalpha was colocalised with both SRC-1 and NCoR to the nuclei of the tumour epithelial cells. Expression of ERalpha and NCoR was found predominantly in non-anaplastic tumours and was significantly associated with well-differentiated tumours and reduced incidence of disease recurrence. In non-anaplastic tumours, HER2 was significantly associated with SRC-1, and these proteins were associated with poorly differentiated tumours, capsular invasion and disease recurrence. Totally, 87% of anaplastic tumours were positive for SRC-1. Kaplan-Meier estimates of disease-free survival indicated that in thyroid cancer, SRC-1 strongly correlates with reduced disease-free survival (P<0.001), whereas NCoR predicted increased survival (P<0.001). These data suggest opposing roles for the coregulators SRC-1 and NCoR in thyroid tumour progression.

  7. Investigation of the vitamin D receptor gene (VDR) and its interaction with protein tyrosine phosphatase, non-receptor type 2 gene (PTPN2) on risk of islet autoimmunity and type 1 diabetes : The Diabetes Autoimmunity Study in the Young (DAISY)

    NARCIS (Netherlands)

    Frederiksen, B.; Liu, E.; Romanos, J.; Steck, A. K.; Yin, X.; Kroehl, M.; Fingerlin, T. E.; Erlich, H.; Eisenbarth, G. S.; Rewers, M.; Norris, J. M.

    The present study investigated the association between variants in the vitamin D receptor gene (VDR) and protein tyrosine phosphatase, non-receptor type 2 gene (PTPN2), as well as an interaction between VDR and PTPN2 and the risk of islet autoimmunity (IA) and progression to type 1 diabetes (T1D).

  8. Araguspongine C Induces Autophagic Death in Breast Cancer Cells through Suppression of c-Met and HER2 Receptor Tyrosine Kinase Signaling

    Directory of Open Access Journals (Sweden)

    Mohamed R. Akl

    2015-01-01

    Full Text Available Receptor tyrosine kinases are key regulators of cellular growth and proliferation. Dysregulations of receptor tyrosine kinases in cancer cells may promote tumorigenesis by multiple mechanisms including enhanced cell survival and inhibition of cell death. Araguspongines represent a group of macrocyclic oxaquinolizidine alkaloids isolated from the marine sponge Xestospongia species. This study evaluated the anticancer activity of the known oxaquinolizidine alkaloids araguspongines A, C, K and L, and xestospongin B against breast cancer cells. Araguspongine C inhibited the proliferation of multiple breast cancer cell lines in vitro in a dose-dependent manner. Interestingly, araguspongine C-induced autophagic cell death in HER2-overexpressing BT-474 breast cancer cells was characterized by vacuole formation and upregulation of autophagy markers including LC3A/B, Atg3, Atg7, and Atg16L. Araguspongine C-induced autophagy was associated with suppression of c-Met and HER2 receptor tyrosine kinase activation. Further in-silico docking studies and cell-free Z-LYTE assays indicated the potential of direct interaction between araguspongine C and the receptor tyrosine kinases c-Met and HER2 at their kinase domains. Remarkably, araguspongine C treatment resulted in the suppression of PI3K/Akt/mTOR signaling cascade in breast cancer cells undergoing autophagy. Induction of autophagic death in BT-474 cells was also associated with decreased levels of inositol 1,4,5-trisphosphate receptor upon treatment with effective concentration of araguspongine C. In conclusion, results of this study are the first to reveal the potential of araguspongine C as an inhibitor to receptor tyrosine kinases resulting in the induction of autophagic cell death in breast cancer cells.

  9. Primary cilia and coordination of receptor tyrosine kinase (RTK) and transforming growth factor β (TGF-β) signaling

    DEFF Research Database (Denmark)

    Christensen, Søren Tvorup; Morthorst, Stine Kjær; Mogensen, Johanne Bay

    2017-01-01

    Since the beginning of the millennium, research in primary cilia has revolutionized our way of understanding how cells integrate and organize diverse signaling pathways during vertebrate development and in tissue homeostasis. Primary cilia are unique sensory organelles that detect changes...... in their extracellular environment and integrate and transmit signaling information to the cell to regulate various cellular, developmental, and physiological processes. Many different signaling pathways have now been shown to rely on primary cilia to function properly, and mutations that lead to ciliary dysfunction...... are at the root of a pleiotropic group of diseases and syndromic disorders called ciliopathies. In this review, we present an overview of primary cilia-mediated regulation of receptor tyrosine kinase (RTK) and transforming growth factor β (TGF-β) signaling. Further, we discuss how defects in the coordination...

  10. Frontal affinity chromatography with MS detection of EphB2 tyrosine kinase receptor. 1. Comparison with conventional ELISA.

    Science.gov (United States)

    Slon-Usakiewicz, Jacek J; Ng, William; Foster, J Estelle; Dai, Jin-Rui; Deretey, Eugen; Toledo-Sherman, Leticia; Redden, Peter R; Pasternak, Andrew; Reid, Neil

    2004-10-07

    FAC-MS offers a convenient method for measuring the relative binding strengths of ligands in a mixture and enables a rapid ranking and identification of ligands in the mixture as potential hits against immobilized targets. Using immobilized EphB2 receptor tyrosine kinase as the target and known kinase inhibitors, the results of FAC-MS screening (% shift) have been shown to correlate with the binding constant, K(d), and with IC(50) results from the more traditional ELISA assay. Therefore, since FAC-MS can accommodate a wide variety of target proteins, its applications could play a broad role in drug discovery not only at the hit discovery stage but also during the subsequent more rigorous screening at the hit-to-lead and lead optimization stages.

  11. The receptor tyrosine kinase gene linotte is required for neuronal pathway selection in the Drosophila mushroom bodies.

    Science.gov (United States)

    Moreau-Fauvarque, C; Taillebourg, E; Boissoneau, E; Mesnard, J; Dura, J M

    1998-11-01

    The linotte (lio) mutant was first isolated as a memory mutant. The lio gene encodes a putative receptor tyrosine kinase (RTK), homologous to the human protein RYK. This gene has been independently identified in a screen for embryonic nervous system axonal guidance defects and called derailed (drl). Here, we report that linotte mutants present structural brain defects in the adult central complex (CX) and mushroom bodies (MB). linotte and derailed are allelic for this phenotype, which can be rescued by a drl+ transgene. The Lio RTK is expressed preferentially in the adult CX and MB. Our results suggest that, analogous to its role within the embryonic nervous system, the Lio RTK is involved in neuronal pathway selection during adult brain development. Copyright 1998 Elsevier Science Ireland Ltd. All Rights Reserved

  12. An in silico high-throughput screen identifies potential selective inhibitors for the non-receptor tyrosine kinase Pyk2

    Directory of Open Access Journals (Sweden)

    Meirson T

    2017-05-01

    Full Text Available Tomer Meirson, Abraham O Samson, Hava Gil-Henn Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Abstract: The non-receptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2 is a critical mediator of signaling from cell surface growth factor and adhesion receptors to cell migration, proliferation, and survival. Emerging evidence indicates that signaling by Pyk2 regulates hematopoietic cell response, bone density, neuronal degeneration, angiogenesis, and cancer. These physiological and pathological roles of Pyk2 warrant it as a valuable therapeutic target for invasive cancers, osteoporosis, Alzheimer’s disease, and inflammatory cellular response. Despite its potential as a therapeutic target, no potent and selective inhibitor of Pyk2 is available at present. As a first step toward discovering specific potential inhibitors of Pyk2, we used an in silico high-throughput screening approach. A virtual library of six million lead-like compounds was docked against four different high-resolution Pyk2 kinase domain crystal structures and further selected for predicted potency and ligand efficiency. Ligand selectivity for Pyk2 over focal adhesion kinase (FAK was evaluated by comparative docking of ligands and measurement of binding free energy so as to obtain 40 potential candidates. Finally, the structural flexibility of a subset of the docking complexes was evaluated by molecular dynamics simulation, followed by intermolecular interaction analysis. These compounds may be considered as promising leads for further development of highly selective Pyk2 inhibitors. Keywords: virtual screen, efficiency metrics, MM-GBSA, molecular dynamics

  13. 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

  14. 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

    2014-05-02

    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/TmAb(Irs1)). 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)-302(Irs1), Ser(P)-307(Irs1), Ser(P)-318(Irs1), Ser(P)-325(Irs1), and Ser(P)-346(Irs1). 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)-302(Irs1), Ser(P)-307(Irs1), 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 CHO(IR)/IRS1 cells.

  15. Effects of formaldehyde exposure on anxiety-like and depression-like behavior, cognition, central levels of glucocorticoid receptor and tyrosine hydroxylase in mice.

    Science.gov (United States)

    Li, Yani; Song, Zhuoyi; Ding, Yujuan; Xin, Ye; Wu, Tong; Su, Tao; He, Rongqiao; Tai, Fadao; Lian, Zhenmin

    2016-02-01

    Formaldehyde exposure is toxic to the brains of mammals, but the mechanism remains unclear. We investigated the effects of inhaled formaldehyde on anxiety, depression, cognitive capacity and central levels of glucocorticoid receptor and tyrosine hydroxylase in mice. After exposure to 0, 1 or 2 ppm gaseous formaldehyde for one week, we measured anxiety-like behavior using open field and elevated plus-maze tests, depression-like behavior using a forced swimming test, learning and memory using novel object recognition tests, levels of glucocorticoid receptors in the hippocampus and tyrosine hydroxylase in the Arc, MPOA, ZI and VTA using immuhistochemistry. We found that inhalation of 1 ppm formaldehyde reduced levels of anxiety-like behavior. Inhalation of 2 ppm formaldehyde reduced body weight, but increased levels of depression-like behavior, impaired novel object recognition, and lowered the numbers of glucocorticoid receptor immonureactive neurons in the hippocampus and tyrosine hydroxylase immonureactive neurons in the ventral tegmental area and the zona incerta, medial preoptic area. Different concentrations of gaseous formaldehyde result in different effects on anxiety, depression-like behavior and cognition ability which may be associated with alterations in hippocampal glucocorticoid receptors and brain tyrosine hydroxylase levels. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Endoscopy in patients with diarrhea during treatment with vascular endothelial growth factor receptor tyrosine kinase inhibitors: Is the cause in the mucosa?

    NARCIS (Netherlands)

    Boers-Sonderen, M.J.; Mulder, S.F.; Nagtegaal, I.D.; Derikx, L.A.A.P.; Wanten, G.J.A.; Mulders, P.F.A.; Graaf, W.T.A. van der; Hoentjen, F.; Herpen, C.M.L. van

    2016-01-01

    Background Diarrhea is a frequently occurring adverse event during treatment with vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR TKIs) and is mostly accompanied by abdominal cramps, flatulence and pyrosis. These complaints impair quality of life and lead to dose

  17. Interactions between a receptor tyrosine phosphatase and a cell surface ligand regulate axon guidance and glial-neuronal communication.

    Science.gov (United States)

    Lee, Hyung-Kook Peter; Cording, Amy; Vielmetter, Jost; Zinn, Kai

    2013-06-05

    We developed a screening method for orphan receptor ligands, in which cell-surface proteins are expressed in Drosophila embryos from GAL4-dependent insertion lines and ligand candidates identified by the presence of ectopic staining with receptor fusion proteins. Stranded at second (Sas) binds to the receptor tyrosine phosphatase Ptp10D in embryos and in vitro. Sas and Ptp10D can interact in trans when expressed in cultured cells. Interactions between Sas and Ptp10D on longitudinal axons are required to prevent them from abnormally crossing the midline. Sas is expressed on both neurons and glia, whereas Ptp10D is restricted to CNS axons. We conducted epistasis experiments by overexpressing Sas in glia and examining how the resulting phenotypes are changed by removal of Ptp10D from neurons. We find that neuronal Ptp10D restrains signaling by overexpressed glial Sas, which would otherwise produce strong glial and axonal phenotypes. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Breakdown of immune homeostasis in the testis of mice lacking Tyro3, Axl and Mer receptor tyrosine kinases.

    Science.gov (United States)

    Zhang, Yue; Li, Nan; Chen, Qiaoyuan; Yan, Keqin; Liu, Zhenghui; Zhang, Xiaoyan; Liu, Peng; Chen, Yongmei; Han, Daishu

    2013-07-01

    Tyro3, Axl and Mer (TAM) receptor tyrosine kinases triple knockout (TAM(-/-)) mice are male infertile due to impaired spermatogenesis. However, the mechanism by which TAM receptors regulate spermatogenesis remains unclear. In this study, we demonstrate that the testicular immune homeostasis was impaired in TAM(-/-) mice. As development after the onset of sexual maturity, germ cells were progressively degenerated. Macrophages and lymphocytes infiltrated into the testis as TAM(-/-) mice aged. Moreover, the integrity of blood-testis barrier was impaired, and the autoantibodies against germ cell antigens were produced. Major inflammatory cytokines, including tumor necrosis factor-α, interleukin-6 and monocyte chemotactic protein 1 were upregulated in the testis of TAM(-/-) mice, and predominantly located in Sertoli cells (SCs). In vitro assays showed that TAM(-/-) SCs secrete significantly high levels of inflammatory cytokines compared with wild-type SCs after coculture with apoptotic germ cells. These results suggest that TAM receptors are important in the maintenance of the immune homeostasis in the testis.

  19. Abeta-mediated NMDA receptor endocytosis in Alzheimer's disease involves ubiquitination of the tyrosine phosphatase STEP61.

    Science.gov (United States)

    Kurup, Pradeep; Zhang, Yongfang; Xu, Jian; Venkitaramani, Deepa V; Haroutunian, Vahram; Greengard, Paul; Nairn, Angus C; Lombroso, Paul J

    2010-04-28

    Amyloid beta (Abeta) is involved in the etiology of Alzheimer's disease (AD) and may contribute to cognitive deficits by increasing internalization of ionotropic glutamate receptors. Striatal-enriched protein tyrosine phosphatase 61 (STEP(61)), which is targeted in part to the postsynaptic terminal, has been implicated in this process. Here we show that STEP(61) levels are progressively increased in the cortex of Tg2576 mice over the first year, as well as in prefrontal cortex of human AD brains. The increased STEP(61) was associated with greater STEP activity, dephosphorylation of phospho-tyr(1472) of the NR2B subunit, and decreased NR1 and NR2B subunits on neuronal membranes. Treatment with Abeta-enriched medium also increased STEP(61) levels and decreased NR1/NR2B abundance in mouse cortical cultures as determined by biotinylation experiments. In STEP knock-out cultures, Abeta treatment failed to induce NMDA receptor internalization. The mechanism for the increase in STEP(61) levels appears to involve the ubiquitin proteasome system. Blocking the proteasome resulted in elevated levels of STEP(61). Moreover, STEP(61)-ubiquitin conjugates were increased in wild-type cortical slices upon Abeta treatment as well as in 12 month Tg2576 cortex. These findings reveal a novel mechanism by which Abeta-mediated accumulation of STEP(61) results in increased internalization of NR1/NR2B receptor that may contribute to the cognitive deficits in AD.

  20. Brain-derived neurotrophic factor and tyrosine kinase B receptor signalling in post-mortem brain of teenage suicide victims.

    Science.gov (United States)

    Pandey, Ghanshyam N; Ren, Xinguo; Rizavi, Hooriyah S; Conley, Robert R; Roberts, Rosalinda C; Dwivedi, Yogesh

    2008-12-01

    Teenage suicide is a major public health concern, but its neurobiology is not very well understood. Stress and major mental disorders are major risk factors for suicidal behaviour, and it has been shown that brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase B (TrkB) are not only regulated by stress but are also altered in these illnesses. We therefore examined if BDNF/TrkB signalling is altered in the post-mortem brain of teenage suicide victims. Protein and mRNA expression of BDNF and of TrkB receptors were determined in the prefrontal cortex (PFC), Brodmann's Area 9 (BA 9), and hippocampus obtained from 29 teenage suicide victims and 25 matched normal control subjects. Protein expression was determined using the Western blot technique; mRNA levels by a quantitative RT-PCR technique. The protein expression of BDNF was significantly decreased in the PFC of teenage suicide victims compared with normal control subjects, whereas no change was observed in the hippocampus. Protein expression of TrkB full-length receptors was significantly decreased in both PFC and hippocampus of teenage suicide victims without any significant changes in the truncated form of TrkB receptors. mRNA expression of both BDNF and TrkB was significantly decreased in the PFC and hippocampus of teenage suicide victims compared with normal control subjects. These studies indicate a down-regulation of both BDNF and its receptor TrkB in the PFC and hippocampus of teenage suicide victims, which suggests that stress and altered BDNF may represent a major vulnerability factor in teenage suicidal behaviour.

  1. Tyrosine agonists reverse the molecular defects associated with dominant-negative mutations in human peroxisome proliferator-activated receptor gamma.

    Science.gov (United States)

    Agostini, Maura; Gurnell, Mark; Savage, David B; Wood, Emily M; Smith, Aaron G; Rajanayagam, Odelia; Garnes, Keith T; Levinson, Sidney H; Xu, H Eric; Schwabe, John W R; Willson, Timothy M; O'Rahilly, Stephen; Chatterjee, V Krishna

    2004-04-01

    Loss-of-function mutations in the ligand-binding domain of human peroxisome proliferator-activated receptor gamma (PPARgamma) are associated with a novel syndrome characterized by partial lipodystrophy and severe insulin resistance. Here we have further characterized the properties of natural dominant-negative PPARgamma mutants (P467L, V290M) and evaluated the efficacy of putative natural ligands and synthetic thiazolidinedione (TZD) or tyrosine-based (TA) receptor agonists in rescuing mutant receptor function. A range of natural ligands failed to activate the PPARgamma mutants and their transcriptional responses to TZDs (e.g. pioglitazone, rosiglitazone) were markedly attenuated, whereas TAs (e.g. farglitazar) corrected defects in ligand binding and coactivator recruitment by the PPARgamma mutants, restoring transcriptional function comparable with wild-type receptor. Transcriptional silencing via recruitment of corepressor contributes to dominant-negative inhibition of wild type by the P467L and V290M mutants and the introduction of an artificial mutation (L318A) disrupting corepressor interaction abrogated their dominant-negative activity. More complete ligand-dependent corepressor release and reversal of dominant-negative inhibition was achieved with TA than TZD agonists. Modeling suggests a structural basis for these observations: both mutations destabilize helix 12 to favor receptor-corepressor interaction; conversely, farglitazar makes more extensive contacts than rosiglitazone within the ligand-binding pocket, to stabilize helix 12, facilitating corepressor release and transcriptional activation. Farglitazar was a more potent inducer of PPARgamma target gene (aP2) expression in peripheral blood mononuclear cells with the P467L mutation. Having shown that rosiglitazone is of variable and limited efficacy in these subjects, we suggest that TAs may represent a more rational therapeutic approach.

  2. Structural basis for the binding specificity of human Recepteur d'Origine Nantais (RON) receptor tyrosine kinase to macrophage-stimulating protein.

    Science.gov (United States)

    Chao, Kinlin L; Gorlatova, Natalia V; Eisenstein, Edward; Herzberg, Osnat

    2014-10-24

    Recepteur d'origine nantais (RON) receptor tyrosine kinase and its ligand, serum macrophage-stimulating protein (MSP), play important roles in inflammation, cell growth, migration, and epithelial to mesenchymal transition during tumor development. The binding of mature MSPαβ (disulfide-linked α- and β-chains) to RON ectodomain modulates receptor dimerization, followed by autophosphorylation of tyrosines in the cytoplasmic receptor kinase domains. Receptor recognition is mediated by binding of MSP β-chain (MSPβ) to the RON Sema. Here we report the structure of RON Sema-PSI-IPT1 (SPI1) domains in complex with MSPβ at 3.0 Å resolution. The MSPβ serine protease-like β-barrel uses the degenerate serine protease active site to recognize blades 2, 3, and 4 of the β-propeller fold of RON Sema. Despite the sequence homology between RON and MET receptor tyrosine kinase and between MSP and hepatocyte growth factor, it is well established that there is no cross-reactivity between the two receptor-ligand systems. Comparison of the structure of RON SPI1 in complex with MSPβ and that of MET receptor tyrosine kinase Sema-PSI in complex with hepatocyte growth factor β-chain reveals the receptor-ligand selectivity determinants. Analytical ultracentrifugation studies of the SPI1-MSPβ interaction confirm the formation of a 1:1 complex. SPI1 and MSPαβ also associate primarily as a 1:1 complex with a binding affinity similar to that of SPI1-MSPβ. In addition, the SPI1-MSPαβ ultracentrifuge studies reveal a low abundance 2:2 complex with ∼ 10-fold lower binding affinity compared with the 1:1 species. These results support the hypothesis that the α-chain of MSPαβ mediates RON dimerization. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Structural Basis for the Binding Specificity of Human Recepteur d'Origine Nantais (RON) Receptor Tyrosine Kinase to Macrophage-stimulating Protein*

    Science.gov (United States)

    Chao, Kinlin L.; Gorlatova, Natalia V.; Eisenstein, Edward; Herzberg, Osnat

    2014-01-01

    Recepteur d'origine nantais (RON) receptor tyrosine kinase and its ligand, serum macrophage-stimulating protein (MSP), play important roles in inflammation, cell growth, migration, and epithelial to mesenchymal transition during tumor development. The binding of mature MSPαβ (disulfide-linked α- and β-chains) to RON ectodomain modulates receptor dimerization, followed by autophosphorylation of tyrosines in the cytoplasmic receptor kinase domains. Receptor recognition is mediated by binding of MSP β-chain (MSPβ) to the RON Sema. Here we report the structure of RON Sema-PSI-IPT1 (SPI1) domains in complex with MSPβ at 3.0 Å resolution. The MSPβ serine protease-like β-barrel uses the degenerate serine protease active site to recognize blades 2, 3, and 4 of the β-propeller fold of RON Sema. Despite the sequence homology between RON and MET receptor tyrosine kinase and between MSP and hepatocyte growth factor, it is well established that there is no cross-reactivity between the two receptor-ligand systems. Comparison of the structure of RON SPI1 in complex with MSPβ and that of MET receptor tyrosine kinase Sema-PSI in complex with hepatocyte growth factor β-chain reveals the receptor-ligand selectivity determinants. Analytical ultracentrifugation studies of the SPI1-MSPβ interaction confirm the formation of a 1:1 complex. SPI1 and MSPαβ also associate primarily as a 1:1 complex with a binding affinity similar to that of SPI1-MSPβ. In addition, the SPI1-MSPαβ ultracentrifuge studies reveal a low abundance 2:2 complex with ∼10-fold lower binding affinity compared with the 1:1 species. These results support the hypothesis that the α-chain of MSPαβ mediates RON dimerization. PMID:25193665

  4. Monoclonal antibodies to the insulin receptor stimulate the intrinsic tyrosine kinase activity by cross-linking receptor molecules.

    Science.gov (United States)

    O'Brien, R M; Soos, M A; Siddle, K

    1987-12-20

    The effect of monoclonal anti-insulin receptor antibodies on the intrinsic kinase activity of solubilized receptor was investigated. Antibodies for six distinct epitopes stimulated receptor autophosphorylation and kinase activity towards exogenous substrates. This effect of antibodies was seen only within a narrow concentration range and monovalent antibody fragments were ineffective. Evidence was obtained by sucrose density-gradient centrifugation for the formation of antibody-receptor complexes which involved both inter- and intra-molecular cross-linking, although stimulation of autophosphorylation appeared to be preferentially associated with the latter. There was partial additivity between the effects of insulin and antibodies in stimulating autophosphorylation, although the sites of phosphorylation appeared identical on two-dimensional peptide maps. Antibodies for two further epitopes failed to activate receptor kinase, but inhibited its stimulation by insulin. The effects of antibodies on kinase activity paralleled their metabolic effects on adipocytes, except for one antibody which was potently insulin-like in its metabolic effects, but which antagonized insulin stimulation of kinase activity. It is concluded that antibodies activate the receptor by cross-linking subunits rather than by reacting at specific epitopes. The ability of some antibodies to activate receptor may depend on receptor environment as well as the disposition of epitopes.

  5. Structure and Molecular Dynamics Simulations of Protein Tyrosine Phosphatase Non-Receptor 12 Provide Insights into the Catalytic Mechanism of the Enzyme.

    Science.gov (United States)

    Dong, Hui; Zonta, Francesco; Wang, Shanshan; Song, Ke; He, Xin; He, Miaomiao; Nie, Yan; Li, Sheng

    2017-12-26

    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.

  6. 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.

  7. 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

  8. Ligand-induced tyrosine phosphorylation of cysteinyl leukotriene receptor 1 triggers internalization and signaling in intestinal epithelial cells.

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    Ladan Parhamifar

    Full Text Available BACKGROUND: 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 affinity LTD(4 receptor CysLT(1R exhibits tumor-promoting properties by triggering cell proliferation, survival, and migration in intestinal epithelial cells. In addition, increased expression and nuclear localization of CysLT(1R correlates with a poorer prognosis for patients with colon cancer. METHODOLOGY/PRINCIPAL FINDINGS: Using a proximity ligation assay and immunoprecipitation, this study showed that endogenous CysLT(1R formed heterodimers with its counter-receptor CysLT(2R under basal conditions and that LTD(4 triggers reduced dimerization of CysLTRs in intestinal epithelial cells. This effect was dependent upon a parallel LTD(4-induced increase in CysLT(1R tyrosine phosphorylation. Leukotriene D(4 also led to elevated internalization of CysLT(1Rs from the plasma membrane and a simultaneous increase at the nucleus. Using sucrose, a clathrin endocytic inhibitor, dominant-negative constructs, and siRNA against arrestin-3, we suggest that a clathrin-, arrestin-3, and Rab-5-dependent process mediated the internalization of CysLT(1R. Altering the CysLT(1R internalization process at either the clathrin or the arrestin-3 stage led to disruption of LTD(4-induced Erk1/2 activation and up-regulation of COX-2 mRNA levels. CONCLUSIONS/SIGNIFICANCE: Our data suggests that upon ligand activation, CysLT(1R is tyrosine-phosphorylated and released from heterodimers with CysLT(2R and, subsequently, internalizes from the plasma membrane to the nuclear membrane in a clathrin-, arrestin-3-, and Rab-5-dependent manner, thus, enabling Erk1/2 signaling and downstream transcription of the COX-2 gene.

  9. Met receptor tyrosine kinase signaling induces secretion of the angiogenic chemokine interleukin-8/CXCL8 in pancreatic cancer.

    Directory of Open Access Journals (Sweden)

    Kristen S Hill

    Full Text Available At diagnosis, the majority of pancreatic cancer patients present with advanced disease when curative resection is no longer feasible and current therapeutic treatments are largely ineffective. An improved understanding of molecular targets for effective intervention of pancreatic cancer is thus urgent. The Met receptor tyrosine kinase is one candidate implicated in pancreatic cancer. Notably, Met is over expressed in up to 80% of invasive pancreatic cancers but not in normal ductal cells correlating with poor overall patient survival and increased recurrence rates following surgical resection. However the functional role of Met signaling in pancreatic cancer remains poorly understood. Here we used RNA interference to directly examine the pathobiological importance of increased Met signaling for pancreatic cancer. We show that Met knockdown in pancreatic tumor cells results in decreased cell survival, cell invasion, and migration on collagen I in vitro. Using an orthotopic model for pancreatic cancer, we provide in vivo evidence that Met knockdown reduced tumor burden correlating with decreased cell survival and tumor angiogenesis, with minimal effect on cell growth. Notably, we report that Met signaling regulates the secretion of the pro-angiogenic chemokine interleukin-8/CXCL8. Our data showing that the interleukin-8 receptors CXCR1 and CXCR2 are not expressed on pancreatic tumor cells, suggests a paracrine mechanism by which Met signaling regulates interleukin-8 secretion to remodel the tumor microenvironment, a novel finding that could have important clinical implications for improving the effectiveness of treatments for pancreatic cancer.

  10. Aβ–mediated NMDA receptor endocytosis in Alzheimer's disease involves ubiquitination of the tyrosine phosphatase STEP61

    Science.gov (United States)

    Kurup, Pradeep; Zhang, Yongfang; Xu, Jian; Venkitaramani, Deepa V.; Haroutunian, Vahram; Greengard, Paul; Nairn, Angus C.; Lombroso, Paul J.

    2010-01-01

    Amyloid beta (Aβ) is involved in the etiology of Alzheimer's disease (AD) and may contribute to cognitive deficits by increasing internalization of ionotropic glutamate receptors. STriatal-Enriched protein tyrosine Phosphatase 61 (STEP61), which is targeted in part to the postsynaptic terminal, has been implicated in this process. Here we show that STEP61 levels are progressively increased in the cortex of Tg2576 mice over the first year, as well as in prefrontal cortex of human AD brains. The increased STEP61 was associated with greater STEP activity, dephosphorylation of phospho-tyr1472 of the NR2B subunit, and decreased NR1 and NR2B subunits on neuronal membranes. Treatment with Aβ~-enriched medium also increased STEP61 levels and decreased NR1/NR2B abundance in mouse cortical cultures as determined by biotinylation experiments. In STEP knock-out cultures, Aβ treatment failed to induce NMDAR internalization. The mechanism for the increase in STEP61 levels appears to involve the ubiquitin proteasome system (UPS). Blocking the proteasome resulted in elevated levels of STEP61. Moreover, STEP61-ubiquitin conjugates were increased in wild-type cortical slices upon Aβ treatment as well as in 12-month Tg2576 cortex. These findings reveal a novel mechanism by which Aβ-mediated accumulation of STEP61 results in increased internalization of NR1/NR2B receptor that may contribute to the cognitive deficits in AD. PMID:20427654

  11. Systems Analysis of Drug-Induced Receptor Tyrosine Kinase Reprogramming Following Targeted Mono- and Combination Anti-Cancer Therapy

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    Alexey Goltsov

    2014-06-01

    Full Text Available The receptor tyrosine kinases (RTKs are key drivers of cancer progression and targets for drug therapy. A major challenge in anti-RTK treatment is the dependence of drug effectiveness on co-expression of multiple RTKs which defines resistance to single drug therapy. Reprogramming of the RTK network leading to alteration in RTK co-expression in response to drug intervention is a dynamic mechanism of acquired resistance to single drug therapy in many cancers. One route to overcome this resistance is combination therapy. We describe the results of a joint in silico, in vitro, and in vivo investigations on the efficacy of trastuzumab, pertuzumab and their combination to target the HER2 receptors. Computational modelling revealed that these two drugs alone and in combination differentially suppressed RTK network activation depending on RTK co-expression. Analyses of mRNA expression in SKOV3 ovarian tumour xenograft showed up-regulation of HER3 following treatment. Considering this in a computational model revealed that HER3 up-regulation reprograms RTK kinetics from HER2 homodimerisation to HER3/HER2 heterodimerisation. The results showed synergy of the trastuzumab and pertuzumab combination treatment of the HER2 overexpressing tumour can be due to an independence of the combination effect on HER3/HER2 composition when it changes due to drug-induced RTK reprogramming.

  12. A receptor tyrosine kinase, UFO/Axl, and other genes isolated by a modified differential display PCR are overexpressed in metastatic prostatic carcinoma cell line DU145.

    Science.gov (United States)

    Jacob, A N; Kalapurakal, J; Davidson, W R; Kandpal, G; Dunson, N; Prashar, Y; Kandpal, R P

    1999-01-01

    We have used a modified differential display PCR protocol for isolating 3' restriction fragments of cDNAs specifically expressed or overexpressed in metastatic prostate carcinoma cell line DU145. Several cDNA fragments were identified that matched to milk fat globule protein, UFO/Axl, a receptor tyrosine kinase, human homologue of a Xenopus maternal transcript, laminin and laminin receptor, human carcinoma-associated antigen, and some expressed sequence tags. The transcript for milk fat globule protein, a marker protein shown to be overexpressed in breast tumors, was elevated in DU145 cells. The expression of UFO/Axl, a receptor tyrosine kinase, was considerably higher in DU145 cells as compared to normal prostate cells and prostatic carcinoma cell line PC-3. The overexpression of UFO oncogene in DU145 cells is discussed in the context of prostate cancer metastasis.

  13. Differential Utilization and Localization of ErbB Receptor Tyrosine Kinases in Skin Compared to Normal and Malignant Keratinocytes

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    Stefan W. Stoll

    2001-01-01

    Full Text Available Induction of heparin-binding epidermal growth factorlike growth factor (HB-EGF mRNA in mouse skin organ culture was blocked by two pan-ErbB receptor tyrosine kinase (RTK inhibitors but not by genetic ablation of ErbB1, suggesting involvement of multiple ErbB species in skin physiology. Human skin, cultured normal keratinocytes, and A431 skin carcinoma cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4. Skin and A431 cells expressed more ErbB3 than did keratinocytes. Despite strong expression of ErbB2 and ErbB3, heregulin was inactive in stimulating tyrosine phosphorylation in A431 cells. In contrast, it was highly active in MDA-MB-453 breast carcinoma cells. ErbB2 displayed punctate cytoplasmic staining in A431 and keratinocytes, compared to strong cell surface staining in MDA-MB-453. In skin, ErbB2 was cytoplasmic in basal keratinocytes, assuming a cell surface pattern in the upper suprabasal layers. In contrast, ErbB1 retained a cell surface distribution in all epidermal layers. Keratinocyte proliferation in culture was found to be ErbB1-RTK-dependent, using a selective inhibitor. These results suggest that in skin keratinocytes, ErbB2 transduces ligand-dependent differentiation signals, whereas ErbB1 transduces ligand-dependent proliferation/survival signals. Intracellular sequestration of ErbB2 may contribute to the malignant phenotype of A431 cells, by allowing them to respond to ErbB1dependent growth/survival signals, while evading ErbB2-dependent differentiation signals.

  14. Insulin Resistance Induced by Hyperinsulinemia Coincides with a Persistent Alteration at the Insulin Receptor Tyrosine Kinase Domain

    Science.gov (United States)

    Catalano, Karyn J.; Maddux, Betty A.; Szary, Jaroslaw; Youngren, Jack F.; Goldfine, Ira D.; Schaufele, Fred

    2014-01-01

    Insulin resistance, the diminished response of target tissues to insulin, is associated with the metabolic syndrome and a predisposition towards diabetes in a growing proportion of the worldwide population. Under insulin resistant states, the cellular response of the insulin signaling pathway is diminished and the body typically responds by increasing serum insulin concentrations to maintain insulin signaling. Some evidence indicates that the increased insulin concentration may itself further dampen insulin response. If so, insulin resistance would worsen as the level of circulating insulin increases during compensation, which could contribute to the transition of insulin resistance to more severe disease. Here, we investigated the consequences of excess insulin exposure to insulin receptor (IR) activity. Cells chronically exposed to insulin show a diminished the level of IR tyrosine and serine autophosphorylation below that observed after short-term insulin exposure. The diminished IR response did not originate with IR internalization since IR amounts at the cell membrane were similar after short- and long-term insulin incubation. Förster resonance energy transfer between fluorophores attached to the IR tyrosine kinase (TK) domain showed that a change in the TK domain occurred upon prolonged, but not short-term, insulin exposure. Even though the altered ‘insulin refractory’ IR TK FRET and IR autophosphorylation levels returned to baseline (non-stimulated) levels after wash-out of the original insulin stimulus, subsequent short-term exposure to insulin caused immediate re-establishment of the insulin-refractory levels. This suggests that some cell-based ‘memory’ of chronic hyperinsulinemic exposure acts directly at the IR. An improved understanding of that memory may help define interventions to reset the IR to full insulin responsiveness and impede the progression of insulin resistance to more severe disease states. PMID:25259572

  15. Catalytic and substrate promiscuity: distinct multiple chemistries catalysed by the phosphatase domain of receptor protein tyrosine phosphatase.

    Science.gov (United States)

    Srinivasan, Bharath; Marks, Hanna; Mitra, Sreyoshi; Smalley, David M; Skolnick, Jeffrey

    2016-07-15

    The presence of latent activities in enzymes is posited to underlie the natural evolution of new catalytic functions. However, the prevalence and extent of such substrate and catalytic ambiguity in evolved enzymes is difficult to address experimentally given the order-of-magnitude difference in the activities for native and, sometimes, promiscuous substrate/s. Further, such latent functions are of special interest when the activities concerned do not fall into the domain of substrate promiscuity. In the present study, we show a special case of such latent enzyme activity by demonstrating the presence of two mechanistically distinct reactions catalysed by the catalytic domain of receptor protein tyrosine phosphatase isoform δ (PTPRδ). The primary catalytic activity involves the hydrolysis of a phosphomonoester bond (C─O─P) with high catalytic efficiency, whereas the secondary activity is the hydrolysis of a glycosidic bond (C─O─C) with poorer catalytic efficiency. This enzyme also displays substrate promiscuity by hydrolysing diester bonds while being highly discriminative for its monoester substrates. To confirm these activities, we also demonstrated their presence on the catalytic domain of protein tyrosine phosphatase Ω (PTPRΩ), a homologue of PTPRδ. Studies on the rate, metal-ion dependence, pH dependence and inhibition of the respective activities showed that they are markedly different. This is the first study that demonstrates a novel sugar hydrolase and diesterase activity for the phosphatase domain (PD) of PTPRδ and PTPRΩ. This work has significant implications for both understanding the evolution of enzymatic activity and the possible physiological role of this new chemistry. Our findings suggest that the genome might harbour a wealth of such alternative latent enzyme activities in the same protein domain that renders our knowledge of metabolic networks incomplete. © 2016 The Author(s). published by Portland Press Limited on behalf of the

  16. Protein tyrosine kinase but not protein kinase C inhibition blocks receptor induced alveolar macrophage activation

    Directory of Open Access Journals (Sweden)

    K. Pollock

    1993-01-01

    Full Text Available The selective enzyme inhibitors genistein and Ro 31-8220 were used to assess the importance of protein tyrosine kinase (PTK and protein kinase C (PKC, respectively, in N-formyl-methionyl-leucyl-phenylalanine (FMLP induced generation of superoxide anion and thromboxane B2 (TXB2 in guinea-pig alveolar macrophages (AM. Genistein (3–100 μM dose dependently inhibited FMLP (3 nM induced superoxide generation in non-primed AM and TXB2 release in non-primed or in lipopolysaccharide (LPS (10 ng/ml primed AM to a level > 80% but had litle effect up to 100 μM on phorbol myristate acetate (PMA (10 nM induced superoxide release. Ro 31-8220 inhibited PMA induced superoxide generation (IC50 0.21 ± 0.10 μM but had no effect on or potentiated (at 3 and 10 μM FMLP responses in non-primed AM. In contrast, when present during LPS priming as well as during FMLP challenge Ro 31-8220 (10 μM inhibited primed TXB2 release by > 80%. The results indicate that PTK activation is required for the generation of these inflammatory mediators by FMLP in AM. PKC activation appears to be required for LPS priming but not for transducing the FMLP signal; rather, PKC activation may modulate the signal by a negative feedback mechanism.

  17. Oncogenic tyrosine kinase NPM-ALK induces expression of the growth-promoting receptor ICOS

    DEFF Research Database (Denmark)

    Zhang, Qian; Wang, HongYi; Kantekure, Kanchan

    2011-01-01

    Here we report that T-cell lymphoma cells carrying the NPM-ALK fusion protein (ALK(+) TCL) frequently express the cell-stimulatory receptor ICOS. ICOS expression in ALK(+) TCL is moderate and strictly dependent on the expression and enzymatic activity of NPM-ALK. NPM-ALK induces ICOS expression v...

  18. Cis and trans regulatory mechanisms control AP2-mediated B cell receptor endocytosis via select tyrosine-based motifs.

    Directory of Open Access Journals (Sweden)

    Kathleen Busman-Sahay

    Full Text Available Following antigen recognition, B cell receptor (BCR-mediated endocytosis is the first step of antigen processing and presentation to CD4+ T cells, a crucial component of the initiation and control of the humoral immune response. Despite this, the molecular mechanism of BCR internalization is poorly understood. Recently, studies of activated B cell-like diffuse large B cell lymphoma (ABC DLBCL have shown that mutations within the BCR subunit CD79b leads to increased BCR surface expression, suggesting that CD79b may control BCR internalization. Adaptor protein 2 (AP2 is the major mediator of receptor endocytosis via clathrin-coated pits. The BCR contains five putative AP2-binding YxxØ motifs, including four that are present within two immunoreceptor tyrosine-based activation motifs (ITAMs. Using a combination of in vitro and in situ approaches, we establish that the sole mediator of AP2-dependent BCR internalization is the membrane proximal ITAM YxxØ motif in CD79b, which is a major target of mutation in ABC DLBCL. In addition, we establish that BCR internalization can be regulated at a minimum of two different levels: regulation of YxxØ AP2 binding in cis by downstream ITAM-embedded DCSM and QTAT regulatory elements and regulation in trans by the partner cytoplasmic domain of the CD79 heterodimer. Beyond establishing the basic rules governing BCR internalization, these results illustrate an underappreciated role for ITAM residues in controlling clathrin-dependent endocytosis and highlight the complex mechanisms that control the activity of AP2 binding motifs in this receptor system.

  19. Cis and trans regulatory mechanisms control AP2-mediated B cell receptor endocytosis via select tyrosine-based motifs.

    Science.gov (United States)

    Busman-Sahay, Kathleen; Drake, Lisa; Sitaram, Anand; Marks, Michael; Drake, James R

    2013-01-01

    Following antigen recognition, B cell receptor (BCR)-mediated endocytosis is the first step of antigen processing and presentation to CD4+ T cells, a crucial component of the initiation and control of the humoral immune response. Despite this, the molecular mechanism of BCR internalization is poorly understood. Recently, studies of activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) have shown that mutations within the BCR subunit CD79b leads to increased BCR surface expression, suggesting that CD79b may control BCR internalization. Adaptor protein 2 (AP2) is the major mediator of receptor endocytosis via clathrin-coated pits. The BCR contains five putative AP2-binding YxxØ motifs, including four that are present within two immunoreceptor tyrosine-based activation motifs (ITAMs). Using a combination of in vitro and in situ approaches, we establish that the sole mediator of AP2-dependent BCR internalization is the membrane proximal ITAM YxxØ motif in CD79b, which is a major target of mutation in ABC DLBCL. In addition, we establish that BCR internalization can be regulated at a minimum of two different levels: regulation of YxxØ AP2 binding in cis by downstream ITAM-embedded DCSM and QTAT regulatory elements and regulation in trans by the partner cytoplasmic domain of the CD79 heterodimer. Beyond establishing the basic rules governing BCR internalization, these results illustrate an underappreciated role for ITAM residues in controlling clathrin-dependent endocytosis and highlight the complex mechanisms that control the activity of AP2 binding motifs in this receptor system.

  20. Tyrosine-610 in the Receptor Kinase BAK1 Does Not Play a Major Role in Brassinosteroid Signaling or Innate Immunity

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    Vijayata Singh

    2017-08-01

    Full Text Available The plasma membrane-localized BRI1-ASSOCIATED KINASE1 (BAK1 functions as a co-receptor with several receptor kinases including the brassinosteroid (BR receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1, which is involved in growth, and the receptors for bacterial flagellin and EF-Tu, FLAGELLIN-SENSING 2 (FLS2 and EF-TU RECEPTOR (EFR, respectively, which are involved in immunity. BAK1 is a dual specificity protein kinase that can autophosphorylate on serine, threonine and tyrosine residues. It was previously reported that phosphorylation of Tyr-610 in the carboxy-terminal domain of BAK1 is required for its function in BR signaling and immunity. However, the functional role of Tyr-610 in vivo has recently come under scrutiny. Therefore, we have generated new BAK1 (Y610F transgenic plants for functional studies. We first produced transgenic Arabidopsis lines expressing BAK1 (Y610F-Flag in the homozygous bak1-4 bkk1-1 double null background. In a complementary approach, we expressed untagged BAK1 and BAK1 (Y610F in the bak1-4 null mutant. Neither BAK1 (Y610F transgenic line had any obvious growth phenotype when compared to wild-type BAK1 expressed in the same background. In addition, the BAK1 (Y610F-Flag plants responded similarly to plants expressing BAK1-Flag in terms of brassinolide (BL inhibition of root elongation, and there were only minor changes in gene expression between the two transgenic lines as monitored by microarray analysis and quantitative real-time PCR. In terms of plant immunity, there were no significant differences between plants expressing BAK1 (Y610F-Flag and BAK1-Flag in the growth of the non-pathogenic hrpA- mutant of Pseudomonas syringae pv. tomato DC3000. Furthermore, untagged BAK1 (Y610F transgenic plants were as responsive as plants expressing BAK1 (in the bak1-4 background and wild-type Col-0 plants toward treatment with the EF-Tu- and flagellin-derived peptide epitopes elf18- and flg22, respectively, as measured by reactive

  1. Targeting Receptor Tyrosine Kinases Using Monoclonal Antibodies: The Most Specific Tools for Targeted-Based Cancer Therapy.

    Science.gov (United States)

    Shabani, Mahdi; Hojjat-Farsangi, Mohammad

    2016-01-01

    Receptor tyrosine kinases (RTKs) family is comprised of different cell surface glycoproteins. These enzymes participate in and regulate vital processes such as cell proliferation, polarity, differentiation, cell to cell interactions, signaling, and cell survival. Dysregulation of RTKs contributes to the development of different types of tumors. RTKs deregulation in different types of cancer has been reported for more than 30 RTKs. Due to their critical roles, the specific targeting of RTKs in malignancies is a promising approach. Targeted cellular and molecular therapies (personalized medicine) have been known as new types of therapeutics, which prevent tumor cell proliferation and invasion by interfering with molecules essential for tumor growth and survival. Specific targeting of RTKs using monoclonal antibodies (mAbs) in malignancies as well as in autoimmune disorders is of great interest. The growing number of mAbs approved by the authorities implies on the increasing attentions and applications of these therapeutic tools. Due to the high specificity, mAbs are the most promising substances that target RTKs expressed on the tumor cell surface. In this communication, we review the recent progresses in the development of mAbs targeting oncogenic RTKs for cancer treatment.

  2. Ecdysone-Induced Receptor Tyrosine Phosphatase PTP52F Regulates Drosophila Midgut Histolysis by Enhancement of Autophagy and Apoptosis

    Science.gov (United States)

    Santhanam, Abirami; Peng, Wen-Hsin; Yu, Ya-Ting; Sang, Tzu-Kang

    2014-01-01

    The rapid removal of larval midgut is a critical developmental process directed by molting hormone ecdysone during Drosophila metamorphosis. To date, it remains unclear how the stepwise events can link the onset of ecdysone signaling to the destruction of larval midgut. This study investigated whether ecdysone-induced expression of receptor protein tyrosine phosphatase PTP52F regulates this process. The mutation of the Ptp52F gene caused significant delay in larval midgut degradation. Transitional endoplasmic reticulum ATPase (TER94), a regulator of ubiquitin proteasome system, was identified as a substrate and downstream effector of PTP52F in the ecdysone signaling. The inducible expression of PTP52F at the puparium formation stage resulted in dephosphorylation of TER94 on its Y800 residue, ensuring the rapid degradation of ubiquitylated proteins. One of the proteins targeted by dephosphorylated TER94 was found to be Drosophila inhibitor of apoptosis 1 (DIAP1), which was rapidly proteolyzed in cells with significant expression of PTP52F. Importantly, the reduced level of DIAP1 in response to inducible PTP52F was essential not only for the onset of apoptosis but also for the initiation of autophagy. This study demonstrates a novel function of PTP52F in regulating ecdysone-directed metamorphosis via enhancement of autophagic and apoptotic cell death in doomed Drosophila midguts. PMID:24550005

  3. Oncogenic potential is related to activating effect of cancer single and double somatic mutations in receptor tyrosine kinases

    Science.gov (United States)

    Hashimoto, Kosuke; Rogozin, Igor B.; Panchenko, Anna R.

    2012-01-01

    Aberrant activation of receptor tyrosine kinases (RTKs) is a common feature of many cancer cells. It was previously suggested that the mechanisms of kinase activation in cancer might be linked to transitions between active and inactive states. Here we estimate the effects of single and double cancer mutations on the stability of active and inactive states of the kinase domains from different RTKs. We show that singleton cancer mutations destabilize active and inactive states, however inactive states are destabilized more than the active ones leading to kinase activation. We show that there exists a relationship between the estimate of oncogenic potential of cancer mutation and kinase activation. Namely, more frequent mutations have a higher activating effect, which might allow us to predict the activating effect of the mutations from the mutation spectra. Independent evolutionary analysis of mutation spectra complements this observation and finds the same frequency threshold defining mutation hot spots. We analyze double mutations and report a positive epistasis and additional advantage of doublets with respect to cancer cell fitness. The activation mechanisms of double mutations differ from those of single mutations and double mutation spectrum is found to be dissimilar to the mutation spectrum of singletons. PMID:22753356

  4. Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies.

    Directory of Open Access Journals (Sweden)

    Jiahui Yang

    Full Text Available Based on its selective cell surface expression in chronic lymphocytic leukemia (CLL and mantle cell lymphoma (MCL, receptor tyrosine kinase ROR1 has recently emerged as a promising target for therapeutic monoclonal antibodies (mAbs. To further assess the suitability of ROR1 for targeted therapy of CLL and MCL, a panel of mAbs was generated and its therapeutic utility was investigated.A chimeric rabbit/human Fab library was generated from immunized rabbits and selected by phage display. Chimeric rabbit/human Fab and IgG1 were investigated for their capability to bind to human and mouse ROR1, to mediate antibody-dependent cellular cytotoxicity (ADCC, complement-dependent cytotoxicity (CDC, and internalization, and to agonize or antagonize apoptosis using primary CLL cells from untreated patients as well as MCL cell lines. A panel of mAbs demonstrated high affinity and specificity for a diverse set of epitopes that involve all three extracellular domains of ROR1, are accessible on the cell surface, and mediate internalization. The mAb with the highest affinity and slowest rate of internalization was found to be the only mAb that mediated significant, albeit weak, ADCC. None of the mAbs mediated CDC. Alone, they did not enhance or inhibit apoptosis.Owing to its relatively low cell surface density, ROR1 may be a preferred target for armed rather than naked mAbs. Provided is a panel of fully sequenced and thoroughly characterized anti-ROR1 mAbs suitable for conversion to antibody-drug conjugates, immunotoxins, chimeric antigen receptors, and other armed mAb entities for preclinical and clinical studies.

  5. Heightened cleavage of Axl receptor tyrosine kinase by ADAM metalloproteases may contribute to disease pathogenesis in SLE.

    Science.gov (United States)

    Orme, Jacob J; Du, Yong; Vanarsa, Kamala; Mayeux, Jessica; Li, Li; Mutwally, Azza; Arriens, Cristina; Min, Soyoun; Hutcheson, Jack; Davis, Laurie S; Chong, Benjamin F; Satterthwaite, Anne B; Wu, Tianfu; Mohan, Chandra

    2016-08-01

    Systemic lupus erythematosus (SLE) is characterized by antibody-mediated chronic inflammation in the kidney, lung, skin, and other organs to cause inflammation and damage. Several inflammatory pathways are dysregulated in SLE, and understanding these pathways may improve diagnosis and treatment. In one such pathway, Axl tyrosine kinase receptor responds to Gas6 ligand to block inflammation in leukocytes. A soluble form of the Axl receptor ectodomain (sAxl) is elevated in serum from patients with SLE and lupus-prone mice. We hypothesized that sAxl in SLE serum originates from the surface of leukocytes and that the loss of leukocyte Axl contributes to the disease. We determined that macrophages and B cells are a source of sAxl in SLE and in lupus-prone mice. Shedding of the Axl ectodomain from the leukocytes of lupus-prone mice is mediated by the matrix metalloproteases ADAM10 and TACE (ADAM17). Loss of Axl from lupus-prone macrophages renders them unresponsive to Gas6-induced anti-inflammatory signaling in vitro. This phenotype is rescued by combined ADAM10/TACE inhibition. Mice with Axl-deficient macrophages develop worse disease than controls when challenged with anti-glomerular basement membrane (anti-GBM) sera in an induced model of nephritis. ADAM10 and TACE also mediate human SLE PBMC Axl cleavage. Collectively, these studies indicate that increased metalloprotease-mediated cleavage of leukocyte Axl may contribute to end organ disease in lupus. They further suggest dual ADAM10/TACE inhibition as a potential therapeutic modality in SLE. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. EphA4 receptor tyrosine kinase is a modulator of onset and disease severity of experimental autoimmune encephalomyelitis (EAE.

    Directory of Open Access Journals (Sweden)

    Kathryn M Munro

    Full Text Available The EphA4 receptor tyrosine kinase is a major regulator of axonal growth and astrocyte reactivity and is a possible inflammatory mediator. Given that multiple sclerosis (MS is primarily an inflammatory demyelinating disease and in mouse models of MS, such as experimental autoimmune encephalomyelitis (EAE, axonal degeneration and reactive gliosis are prominent clinical features, we hypothesised that endogenous EphA4 could play a role in modulating EAE. EAE was induced in EphA4 knockout and wildtype mice using MOG peptide immunisation and clinical severity and histological features of the disease were then compared in lumbar spinal cord sections. EphA4 knockout mice exhibited a markedly less severe clinical course than wildtype mice, with a lower maximum disease grade and a slightly later onset of clinical symptoms. Numbers of infiltrating T cells and macrophages, the number and size of the lesions, and the extent of astrocytic gliosis were similar in both genotypes; however, EphA4 knockout mice appeared to have decreased axonal pathology. Blocking of EphA4 in wildtype mice by administration of soluble EphA4 (EphA4-Fc as a decoy receptor following induction of EAE produced a delay in onset of clinical symptoms; however, most mice had clinical symptoms of similar severity by 22 days, indicating that EphA4 blocking treatment slowed early EAE disease evolution. Again there were no apparent differences in histopathology. To determine whether the role of EphA4 in modulating EAE was CNS mediated or due to an altered immune response, MOG primed T cells from wildtype and EphA4 knockout mice were passively transferred into naive recipient mice and both were shown to induce disease of equivalent severity. These results are consistent with a non-inflammatory, CNS specific, deleterious effect of EphA4 during neuroinflammation that results in axonal pathology.

  7. Stretch-induced mitogen-activated protein kinase activation in lung fibroblasts is independent of receptor tyrosine kinases.

    Science.gov (United States)

    Boudreault, Francis; Tschumperlin, Daniel J

    2010-07-01

    Lung growth and remodeling are modulated by mechanical stress, with fibroblasts thought to play a leading role. Little mechanistic information is available about how lung fibroblasts respond to mechanical stress. We exposed cultured lung fibroblasts to tonic stretch and measured changes in phosphorylation status of mitogen-activated protein kinases (MAPKs), selected receptor tyrosine kinases (RTKs), and phospholipase Cgamma1 (PLCgamma1) and activation of the small G-protein Ras. Human lung fibroblasts (LFs) were seeded on matrix-coated silicone membranes and exposed to equibiaxial 10 to 40% static stretch or 20% contraction. LFs were stimulated with EGF, FGF2, or PDGF-BB or exposed to stretch in the presence of inhibitors of EGFR (AG1478), FGFR (PD173074), and PDGFR (AG1296). Phospho-MAPK, phospho-RTK, and phospho-PLCgamma1 levels were measured by Western blotting. Active GTP-Ras was quantified by immunoblotting after pull-down with a glutathione S-transferase-Raf-RBD construct. Normalized p-ERK1/2, p-JNK, and p-p38 levels increased after stretch but not contraction. Ligands to RTKs broadly stimulated MAPKs, with the responses to EGF and PDGF most similar to stretch in terms of magnitude and rank order of MAPK responses. Stretching cells failed to elicit measurable activation of EGFR, FGFR (FRS2alpha phosphorylation), or PDGFR. Potent inhibitors of the kinase activity of each receptor failed to attenuate stretch-induced MAPK activation. PLCgamma1 and Ras, prominent effectors downstream of RTKs, were not activated by stretch. Our findings demonstrate that MAPKs are potently activated by stretch in lung fibroblasts, but, in contrast to stress responses observed in other cell types, RTKs are not necessary for stretch-induced MAPK activation in LFs.

  8. Biophysical Evidence for Intrinsic Disorder in the C-terminal Tails of the Epidermal Growth Factor Receptor (EGFR) and HER3 Receptor Tyrosine Kinases.

    Science.gov (United States)

    Keppel, Theodore R; Sarpong, Kwabena; Murray, Elisa M; Monsey, John; Zhu, Jian; Bose, Ron

    2017-01-13

    The epidermal growth factor receptor (EGFR)/ErbB family of receptor tyrosine kinases includes oncogenes important in the progression of breast and other cancers, and they are targets for many drug development strategies. Each member of the ErbB family possesses a unique, structurally uncharacterized C-terminal tail that plays an important role in autophosphorylation and signal propagation. To determine whether these C-terminal tails are intrinsically disordered regions, we conducted a battery of biophysical experiments on the EGFR and HER3 tails. Using hydrogen/deuterium exchange mass spectrometry, we measured the conformational dynamics of intracellular half constructs and compared the tails with the ordered kinase domains. The C-terminal tails demonstrate more rapid deuterium exchange behavior when compared with the kinase domains. Next, we expressed and purified EGFR and HER3 tail-only constructs. Results from circular dichroism spectroscopy, size exclusion chromatography with multiangle light scattering, dynamic light scattering, analytical ultracentrifugation, and small angle X-ray scattering each provide evidence that the EGFR and HER3 C-terminal tails are intrinsically disordered with extended, non-globular structure in solution. The intrinsic disorder and extended conformation of these tails may be important for their function by increasing the capture radius and reducing the thermodynamic barriers for binding of downstream signaling proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Cardiac Metabolic Deregulation Induced by the Tyrosine Kinase Receptor Inhibitor Sunitinib is rescued by Endothelin Receptor Antagonism

    Science.gov (United States)

    Sourdon, Joevin; Lager, Franck; Viel, Thomas; Balvay, Daniel; Moorhouse, Rebecca; Bennana, Evangeline; Renault, Gilles; Tharaux, Pierre-Louis; Dhaun, Neeraj; Tavitian, Bertrand

    2017-01-01

    The growing field of cardio-oncology addresses the side effects of cancer treatment on the cardiovascular system. Here, we explored the cardiotoxicity of the antiangiogenic therapy, sunitinib, in the mouse heart from a diagnostic and therapeutic perspective. We showed that sunitinib induces an anaerobic switch of cellular metabolism within the myocardium which is associated with the development of myocardial fibrosis and reduced left ventricular ejection fraction as demonstrated by echocardiography. The capacity of positron emission tomography with [18F]fluorodeoxyglucose to detect the changes in cardiac metabolism caused by sunitinib was dependent on fasting status and duration of treatment. Pan proteomic analysis in the myocardium showed that sunitinib induced (i) an early metabolic switch with enhanced glycolysis and reduced oxidative phosphorylation, and (ii) a metabolic failure to use glucose as energy substrate, similar to the insulin resistance found in type 2 diabetes. Co-administration of the endothelin receptor antagonist, macitentan, to sunitinib-treated animals prevented both metabolic defects, restored glucose uptake and cardiac function, and prevented myocardial fibrosis. These results support the endothelin system in mediating the cardiotoxic effects of sunitinib and endothelin receptor antagonism as a potential therapeutic approach to prevent cardiotoxicity. Furthermore, metabolic and functional imaging can monitor the cardiotoxic effects and the benefits of endothelin antagonism in a theranostic approach. PMID:28824714

  10. Effect of ghrelin receptor agonist and antagonist on the activity of arcuate nucleus tyrosine hydroxylase containing neurons in C57BL/6 male mice exposed to normal or high fat diet

    Czech Academy of Sciences Publication Activity Database

    Pirník, Z.; Majerčíková, Z.; Holubová, Martina; Pirník, R.; Železná, Blanka; Maletínská, Lenka; Kiss, A.

    2014-01-01

    Roč. 65, č. 4 (2014), s. 477-486 ISSN 0867-5910 Institutional support: RVO:61388963 Keywords : growth hormone secretagogue receptor * ghrelin receptor agonist * ghrelin receptor antagonist * high fat diet * tyrosine hydroxylase * arcuate nucleus * food intake Subject RIV: CE - Biochemistry Impact factor: 2.386, year: 2014

  11. 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.

  12. Cloning and partial characterization of the human tie-2 receptor tyrosine kinase gene promoter.

    Science.gov (United States)

    Hewett, P W; Daft, E L; Murray, J C

    1998-11-27

    The Tie-2 receptor plays a key role in vascular development, although little is known about the factors controlling its expression. Here we report the first cloning and characterisation of the 5' regulatory region of human tie-2. Multiple transcription start sites were identified between -414 and -265 bp upstream of the start codon using 5' RACE, fluorescent primer extension, and RNase protection assays. The human tie-2 promoter contains several transcription factor-binding sequences including ets, SP-1, AP-1, and GATA-1, but there are no canonical TATA or CCAAT initiation sequences proximal to the transcription start sites. Human tie-2 reporter constructs demonstrated approximately 10-fold greater activity in endothelial cells compared with fibroblasts. In endothelial cells the tie-2 promoter exhibited 5 and 16% of the activity of human tie-1 (830 bp) and KDR (1.1 kb) promoters, respectively. This promoter will be a useful tool for studying factors that regulate tie-2 expression and targeting the vasculature. Copyright 1998 Academic Press.

  13. Reduced levels of the tyrosine phosphatase STEP block β amyloid-mediated GluA1/GluA2 receptor internalization.

    Science.gov (United States)

    Zhang, Yongfang; Kurup, Pradeep; Xu, Jian; Anderson, George M; Greengard, Paul; Nairn, Angus C; Lombroso, Paul J

    2011-11-01

    Striatal-Enriched protein tyrosine Phosphatase of MW 61 kDa (STEP(61)) is a protein tyrosine phosphatase recently implicated in the pathophysiology of Alzheimer's disease (AD). STEP(61) is elevated in human AD prefrontal cortex and in the cortex of several AD mouse models. The elevated levels of active STEP(61) down-regulate surface expression of GluN1/GluN2B (formerly NR1/NR2B) receptor complexes, while genetically reducing STEP levels rescues both the biochemical and cognitive deficits in a triple transgenic AD mouse model (3xTg-AD). Here, we show that increased STEP(61) also plays a role in beta amyloid (Aβ)-mediated internalization of the α-amino-3-hydroxy-5-methyl-4-(AMPA) receptor (AMPAR) subunits GluA1/GluA2 (formerly GluR1/GluR2). We purified Aβ oligomers and determined that oligomers, but not monomers, lead to endocytosis of GluA1/GluA2 receptors in cortical cultures. The decrease in GluA1/GluA2 receptors is reversed in the progeny of STEP knock-out (KO) mice crossed with Tg2576 mice, despite elevated levels of Aβ. These results provide strong support for the hypothesis that STEP(61) is required for Aβ-mediated internalization of GluA1/GluA2 receptors. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  14. c-MET receptor tyrosine kinase as a molecular target in advanced hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Granito A

    2015-04-01

    Full Text Available Alessandro Granito,1 Elena Guidetti,1 Laura Gramantieri2,3 1Dipartimento di Scienze Mediche e Chirurgiche Università di Bologna, Bologna, Italy; 2Dipartimento dell'Apparato Digerente, Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 3Centro di Ricerca Biomedica Applicata (CRBA, Azienda Ospedaliero-Universitaria Policlinico S Orsola-Malpighi e Università di Bologna, Bologna, Italy Abstract: c-MET is the membrane receptor for hepatocyte growth factor (HGF, also known as scatter factor or tumor cytotoxic factor, a mitogenic growth factor for hepatocytes. HGF is mainly produced by cells of mesenchymal origin and it mainly acts on neighboring epidermal and endothelial cells, regulating epithelial growth and morphogenesis. HGF/MET signaling has been identified among the drivers of tumorigenesis in human cancers. As such, c-MET is a recognized druggable target, and against it, targeted agents are currently under clinical investigation. c-MET overexpression is a common event in a wide range of human malignancies, including gastric, lung, breast, ovary, colon, kidney, thyroid, and liver carcinomas. Despite c-MET overexpression being reported by a large majority of studies, no evidence for a c-MET oncogenic addiction exists in hepatocellular carcinoma (HCC. In particular, c-MET amplification is a rare event, accounting for 4%–5% of cases while no mutation has been identified in c-MET oncogene in HCC. Thus, the selection of patient subgroups more likely to benefit from c-MET inhibition is challenging. Notwithstanding, c-MET overexpression was reported to be associated with increased metastatic potential and poor prognosis in patients with HCC, providing a rationale for its therapeutic inhibition. Here we summarize the role of activated HGF/MET signaling in HCC, its prognostic relevance, and the implications for therapeutic approaches in HCC. Keywords: hepatocellular carcinoma, c-MET, clinical trials

  15. Prediction for response duration to epidermal growth factor receptor-tyrosine kinase inhibitors in EGFR mutated never smoker lung adenocarcinoma.

    Science.gov (United States)

    Kim, Hye Ryun; Cho, Byoung Chul; Shim, Hyo Sup; Lim, Sun Min; Kim, Se Kyu; Chang, Joon; Kim, Dae Joon; Kim, Joo Hang

    2014-03-01

    Among non-small cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations, ∼ 20-30% exhibit de novo resistance to EGFR-tyrosine kinase inhibitor (TKI). The aim of this study was to examine whether mutations in the EGFR-downstream genes may be associated with de novo resistance to EGFR-TKIs in EGFR mutation-positive patients. Sixty-eight never-smoker adenocarcinoma patients with an activating EGFR mutation were included in the mutational analysis and 55 patients treated with EGFR-TKIs were analyzed for the treatment outcomes to EGFR-TKIs. We concurrently analyzed mutations in PIK3CA, PTEN, AKT and STK11, which are all EGFR-downstream genes. Mutations in PIK3CA, PTEN, AKT, and STK11 were analyzed by polymerase chain reaction-based sequencing. PIK3CA mutations were detected in 4.4% (3/68) of patients, PTEN mutations in 16.1% (11/68), AKT mutations in 5.9% (4/68), and STK11 mutations in 13.2% (9/68). One patient with an activating exon 21 L858R mutation concomitantly had an exon 20 T790M mutation in EGFR. The proportion of patients who had mutations in EGFR-downstream genes was 32.4% (22/68). When we analyzed the treatment outcome of 55 patients treated with EGFR-TKI, the presence of mutations in EGFR-downstream genes correlated with a poor overall response rate to EGFR-TKIs (63.6 vs.14.5% in patients with mutation in EGFR-downstream gene, Padenocarcinoma patients with activating EGFR mutations. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. Epidermal growth factor receptor-tyrosine kinase inhibitors in advanced squamous cell carcinoma of the lung: a meta-analysis.

    Science.gov (United States)

    Ameratunga, Malaka; Pavlakis, Nick; Gebski, Val; Broad, Adam; Khasraw, Mustafa

    2014-09-01

    Tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) are well established in treating metastatic pulmonary adenocarcinoma, especially patients with activating EGFR mutations. EGFR mutations are rare in pulmonary squamous cell carcinomas (SCCs). There are conflicting data supporting the efficacy of EGFR-TKIs in advanced lung SCC. We analyzed the impact of EGFR-TKIs on progression-free survival (PFS) and overall survival (OS) in unselected patients with lung SCC. We searched for randomized controlled trials (RCTs) comparing EGFR-TKIs alone with placebo in patients with metastatic non-small cell lung cancer. RCTs in all settings (front line/maintenance/subsequent) were included. The primary outcome was OS in the SCC population. We used published hazard ratios (HRs), and when unavailable, unpublished data were sought. Pooled estimates of treatment effect on OS and PFS were calculated using the fixed-effects inverse variance weighted method. Eight eligible RCTs were included: 2 first-line, 6 second-line or beyond, evaluating 1781 patients. Data were available for OS in four studies (second-line; N=1420) and for PFS in four studies (3 second-line, 1 first-line; N=788). EGFR-TKIs significantly prolonged OS with a HR of 0.88 (95% confidence interval [CI] 0.78-1.00, P=0.04), and significantly prolonged PFS with a HR of 0.77 (95% CI 0.65-0.92, P=0.004). EGFR mutations are rare in lung SCC. However, EGFR-TKIs have a modest therapeutic effect compared to placebo in unselected patients with advanced pulmonary SCC, and can be considered in these patients. EGFR-mutation-independent mechanisms may explain efficacy of EGFR inhibitors in this setting. © 2014 Wiley Publishing Asia Pty Ltd.

  17. Dopaminergic Receptors and Tyrosine Hydroxylase Expression in Peripheral Blood Mononuclear Cells: A Distinct Pattern in Central Obesity.

    Science.gov (United States)

    Leite, Fernanda; Lima, Margarida; Marino, Franca; Cosentino, Marco; Ribeiro, Laura

    2016-01-01

    Dopamine (DA) may be involved in central obesity (CO), an inflammatory condition, through its role in the central nervous system and in periphery, where it may affect immune cell function through five different DA receptors (DR). Whether dopaminergic pathways in peripheral immune cells are implicated in the inflammatory condition linked to CO is however unknown. In a cohort of blood donors with and without CO, categorized by waist circumference (WC) (CO: WC ≥ 0.80 m in women and ≥ 0.94 m in men), we studied the expression of DR and tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of DA, in peripheral blood mononuclear cells (PBMCs) and their relation with anthropometric and metabolic/endocrine and inflammatory parameters. DR D1-5 and TH expression was assessed by semi quantitative real-time PCR. As inflammatory markers we investigated the immunophenotype of monocyte subsets by flow cytometry, staining for CD14, CD16, CD11b and CD36. CO individuals showed higher plasma levels of leptin and higher inflammatory pattern of monocytes compared with non-CO. PBMC expression of DR D2, DR D4 and DR D5 as well as of TH were lower in CO in comparison with non-CO. DR D2, and DR D5 expression correlated with lower WC and weight, and with lower inflammatory pattern of monocytes, and TH expression correlated with lower WC. DR D4 expression correlated with lower plasma levels of glycosylated hemoglobin, and DR D2 expression correlated with lower CO. Results show that CO is associated with peripheral inflammation and downregulation of dopaminergic pathways in PBMCs, possibly suggesting DR expressed on immune cells as pharmacological targets in obesity for better metabolic outcome.

  18. Dopaminergic Receptors and Tyrosine Hydroxylase Expression in Peripheral Blood Mononuclear Cells: A Distinct Pattern in Central Obesity.

    Directory of Open Access Journals (Sweden)

    Fernanda Leite

    Full Text Available Dopamine (DA may be involved in central obesity (CO, an inflammatory condition, through its role in the central nervous system and in periphery, where it may affect immune cell function through five different DA receptors (DR. Whether dopaminergic pathways in peripheral immune cells are implicated in the inflammatory condition linked to CO is however unknown.In a cohort of blood donors with and without CO, categorized by waist circumference (WC (CO: WC ≥ 0.80 m in women and ≥ 0.94 m in men, we studied the expression of DR and tyrosine hydroxylase (TH, the rate-limiting enzyme in the synthesis of DA, in peripheral blood mononuclear cells (PBMCs and their relation with anthropometric and metabolic/endocrine and inflammatory parameters. DR D1-5 and TH expression was assessed by semi quantitative real-time PCR. As inflammatory markers we investigated the immunophenotype of monocyte subsets by flow cytometry, staining for CD14, CD16, CD11b and CD36.CO individuals showed higher plasma levels of leptin and higher inflammatory pattern of monocytes compared with non-CO. PBMC expression of DR D2, DR D4 and DR D5 as well as of TH were lower in CO in comparison with non-CO. DR D2, and DR D5 expression correlated with lower WC and weight, and with lower inflammatory pattern of monocytes, and TH expression correlated with lower WC. DR D4 expression correlated with lower plasma levels of glycosylated hemoglobin, and DR D2 expression correlated with lower CO.Results show that CO is associated with peripheral inflammation and downregulation of dopaminergic pathways in PBMCs, possibly suggesting DR expressed on immune cells as pharmacological targets in obesity for better metabolic outcome.

  19. Expression pattern and function of tyrosine receptor kinase B isoforms in rat mesenteric arterial smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Otani, Kosuke; Okada, Muneyoshi; Yamawaki, Hideyuki, E-mail: yamawaki@vmas.kitasato-u.ac.jp

    2015-11-27

    Tyrosine receptor kinaseB (TrkB) is a high affinity receptor for brain-derived neurotrophic factor (BDNF). TrkB isoforms involve full length TrkB (TrkB FL) and truncated TrkB type1 (TrkB T1) and type 2 (TrkB T2) in rats. The aim of present study was to explore their expression pattern and function in mesenteric arterial smooth muscle cells (MASMCs). The expression of TrkB isoform protein and mRNA was examined by Western blotting, immunofluorescence and quantitative RT-PCR analyses. Cell proliferation was measured by a bromodeoxyuridine (BrdU) incorporation assay. Cell migration was measured by a Boyden chamber assay. Cell morphology was observed with a phase-contrast microscope. Protein and mRNA expression of BDNF and TrkB isoforms was confirmed in MASMCs. Expression level of TrkB FL was less, while that of TrkB T1 was the highest in MASMCs. Although BDNF increased phosphorylation of ERK, it had no influence on migration and proliferation of MASMCs. TrkB T1 gene knockdown by a RNA interference induced morphological changes and reduced expression level of α-smooth muscle actin (α-SMA) in MASMCs. Similar morphological changes and reduced α-SMA expression were induced in MASMCs by a Rho kinase inhibitor, Y-27632. In conclusion, we for the first time demonstrate that TrkB T1 expressed highly in MASMCs contributes to maintain normal cell morphology possibly via regulation of Rho activity. This study firstly defined expression level of TrkB isoforms and partly revealed their functions in peripheral vascular cells. - Highlights: • BDNF-TrkB axis mediates neurogenesis, growth, differentiation and survival. • Expression pattern and function of TrkB in vascular smooth muscle remain unclear. • Expression of TrkB FL is low, while that of TrkB T1 is the highest. • TrkB T1 contributes to maintain normal morphology possibly via activating Rho.

  20. The receptor-like protein-tyrosine phosphatase DEP-1 is constitutively associated with a 64-kDa protein serine/threonine kinase.

    Science.gov (United States)

    Jallal, B; Mossie, K; Vasiloudis, G; Knyazev, P; Zachwieja, J; Clairvoyant, F; Schilling, J; Ullrich, A

    1997-05-02

    Protein-tyrosine phosphatases (PTPs) are involved in the regulation of diverse cellular processes and may function as positive effectors as well as negative regulators of intracellular signaling. Recent data demonstrate that malignant transformation of cells is frequently associated with changes in PTP expression or activity. Our analysis of PTP expression in mammary carcinoma cell lines resulted in the molecular cloning of a receptor-like PTP, also known as DEP-1. DEP-1 was found to be expressed at varying levels in mammary carcinoma cell lines and A431 cells. In all tumor cell lines analyzed, DEP-1 was constitutively phosphorylated on tyrosine residues. Phosphorylation of DEP-1 increased significantly after treatment of cells with the PTP inhibitor pervanadate. In A431 cells, tyrosine phosphorylation of DEP-1 was also observed after stimulation with epidermal growth factor, however, only after prolonged exposure of the cells to the ligand, suggesting an indirect mechanism of phosphorylation. In addition, DEP-1 coprecipitated with several tyrosine-phosphorylated proteins from pervanadate-treated cells. In vitro binding experiments using a glutathione S-transferase fusion protein containing the catalytically inactive PTP domain of DEP-1 (Gst-DEP-1-C/S) identify these proteins as potential substrates of DEP-1. In addition, we found a 64-kDa serine/threonine kinase to be constitutively associated with DEP-1 in all tumor cell lines tested. The 64-kDa kinase forms a stable complex with DEP-1 and phosphorylates DEP-1 and DEP-1-interacting proteins in vitro. These data suggest a possible mechanism of DEP-1 regulation in tumor cell lines involving serine/threonine and/or tyrosine phosphorylation.

  1. The Role of Dynamic in the Regulation of Signaling by the erbB Family of Receptor Tyrosine Kinases

    National Research Council Canada - National Science Library

    King, Megan C; Lemmon, Mark

    2005-01-01

    ... of mitogenic signaling cascades. The large GTPase dynamin is a key regulator both of transport of receptors to the plasma membrane after receptor biosynthesis and down-regulation of receptors via receptor-mediated endocytosis (RME...

  2. Janus kinase (Jak) subcellular localization revisited: the exclusive membrane localization of endogenous Janus kinase 1 by cytokine receptor interaction uncovers the Jak.receptor complex to be equivalent to a receptor tyrosine kinase.

    Science.gov (United States)

    Behrmann, Iris; Smyczek, Tanja; Heinrich, Peter C; Schmitz-Van de Leur, Hildegard; Komyod, Waraporn; Giese, Bernd; Müller-Newen, Gerhard; Haan, Serge; Haan, Claude

    2004-08-20

    The Janus kinases are considered to be cytoplasmic kinases that constitutively associate with the cytoplasmic region of cytokine receptors, and the Janus kinases (Jaks) are crucial for cytokine signal transduction. We investigated Jak1 localization using subcellular fractionation techniques and fluorescence microscopy (immunofluorescence and yellow fluorescent protein-tagged Jaks). In the different experimental approaches we found Jak1 (as well as Jak2 and Tyk2) predominantly located at membranes. In contrast to previous reports we did not observe Jak proteins in significant amounts within the nucleus or in the cytoplasm. The cytoplasmic localization observed for the Jak1 mutant L80A/Y81A, which is unable to associate with cytokine receptors, indicates that Jak1 does not have a strong intrinsic membrane binding potential and that only receptor binding is crucial for the membrane recruitment. Finally we show that Jak1 remains a membrane-localized protein after cytokine stimulation. These data strongly support the hypothesis that cytokine receptor.Janus kinase complexes can be regarded as receptor tyrosine kinases.

  3. Receptor tyrosine kinases activate canonical WNT/β-catenin signaling via MAP kinase/LRP6 pathway and direct β-catenin phosphorylation.

    Directory of Open Access Journals (Sweden)

    Pavel Krejci

    Full Text Available Receptor tyrosine kinase signaling cooperates with WNT/β-catenin signaling in regulating many biological processes, but the mechanisms of their interaction remain poorly defined. We describe a potent activation of WNT/β-catenin by FGFR2, FGFR3, EGFR and TRKA kinases, which is independent of the PI3K/AKT pathway. Instead, this phenotype depends on ERK MAP kinase-mediated phosphorylation of WNT co-receptor LRP6 at Ser1490 and Thr1572 during its Golgi network-based maturation process. This phosphorylation dramatically increases the cellular response to WNT. Moreover, FGFR2, FGFR3, EGFR and TRKA directly phosphorylate β-catenin at Tyr142, which is known to increase cytoplasmic β-catenin concentration via release of β-catenin from membranous cadherin complexes. We conclude that signaling via ERK/LRP6 pathway and direct β-catenin phosphorylation at Tyr142 represent two mechanisms used by various receptor tyrosine kinase systems to activate canonical WNT signaling.

  4. The strange connection between epidermal growth factor receptor tyrosine kinase inhibitors and dapsone: from rash mitigation to the increase in anti-tumor activity.

    Science.gov (United States)

    Boccellino, Mariarosaria; Quagliuolo, Lucio; Alaia, Concetta; Grimaldi, Anna; Addeo, Raffaele; Nicoletti, Giovanni Francesco; Kast, Richard Eric; Caraglia, Michele

    2016-11-01

    The presence of an aberrantly activated epidermal growth factor receptor (EGFR) in many epithelial tumors, due to its overexpression, activating mutations, gene amplification and/or overexpression of receptor ligands, represent the fundamental basis underlying the use of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Drugs inhibiting the EGFR have different mechanisms of action; while erlotinib and gefitinib inhibit the intracellular tyrosine kinase, monoclonal antibodies like cetuximab and panitumumab bind the extracellular domain of the EGFR both activating immunomediated anti-cancer effect and inhibiting receptor function. On the other hand, interleukin-8 has tumor promoting as well as neo-angiogenesis enhancing effects and several attempts have been made to inhibit its activity. One of these is based on the use of the old sulfone antibiotic dapsone that has demonstrated several interleukin-8 system inhibiting actions. Erlotinib typically gives a rash that has recently been proven to come out via up-regulated keratinocyte interleukin-8 synthesis with histological features reminiscent of typical neutrophilic dermatoses. In this review, we report experimental evidence that shows the use of dapsone to improve quality of life in erlotinib-treated patients by ameliorating rash as well as short-circuiting a growth-enhancing aspect of erlotinib based on increased interleukin-8 secretion.

  5. Defining the molecular basis of interaction between R3 receptor-type protein tyrosine phosphatases and VE-cadherin.

    Directory of Open Access Journals (Sweden)

    Olga Dorofejeva

    Full Text Available Receptor-type protein tyrosine phosphatases (RPTPs of the R3 subgroup play key roles in the immune, vascular and nervous systems. They are characterised by a large ectodomain comprising multiple FNIII-like repeats, a transmembrane domain, and a single intracellular phosphatase domain. The functional role of the extracellular region has not been clearly defined and potential roles in ligand interaction, dimerization, and regulation of cell-cell contacts have been reported. Here bimolecular fluorescence complementation (BiFC in live cells was used to examine the molecular basis for the interaction of VE-PTP with VE-cadherin, two proteins involved in endothelial cell contact and maintenance of vascular integrity. The potential of other R3-PTPs to interact with VE-cadherin was also explored using this method. Quantitative BiFC analysis, using a VE-PTP construct expressing only the ectodomain and transmembrane domain, revealed a specific interaction with VE-cadherin, when compared with controls. Controls were sialophorin, an unrelated membrane protein with a large ectodomain, and a membrane anchored C-terminal Venus-YFP fragment, lacking both ectodomain and transmembrane domains. Truncation of the first 16 FNIII-like repeats from the ectodomain of VE-PTP indicated that removal of this region is not sufficient to disrupt the interaction with VE-cadherin, although it occurs predominantly in an intracellular location. A construct with a deletion of only the 17th domain of VE-PTP was, in contrast to previous studies, still able to interact with VE-cadherin, although this also was predominantly intracellular. Other members of the R3-PTP family (DEP-1, GLEPP1 and SAP-1 also exhibited the potential to interact with VE-cadherin. The direct interaction of DEP-1 with VE-cadherin is likely to be of physiological relevance since both proteins are expressed in endothelial cells. Together the data presented in the study suggest a role for both the ectodomain and

  6. Pyridoxine improves hippocampal cognitive function via increases of serotonin turnover and tyrosine hydroxylase, and its association with CB1 cannabinoid receptor-interacting protein and the CB1 cannabinoid receptor pathway.

    Science.gov (United States)

    Jung, Hyo Young; Kim, Dae Won; Nam, Sung Min; Kim, Jong Whi; Chung, Jin Young; Won, Moo-Ho; Seong, Je Kyung; Yoon, Yeo Sung; Yoo, Dae Young; Hwang, In Koo

    2017-12-01

    In the present study, we investigated the effects of pyridoxine on hippocampal functions and changes in protein profiles based on the proteomic approach. Eight-week-old mice received intraperitoneal injections of physiological saline (vehicle) or 350mg/kg pyridoxine twice a day for 21days. Phosphoglycerate mutase 1 was up-regulated, while CB1 cannabinoid receptor-interacting protein 1 (CRIP1) was down-regulated, in the pyridoxine-treated group. Additionally, the serotonin and tyrosine hydroxylase was increased in the hippocampus of the pyridoxine-treated group than in that of the vehicle-treated group. Furthermore, discrimination indices based on the novel object recognition test were significantly higher in the pyridoxine-treated group than in the vehicle-treated group. Administration of CRIP1a siRNA significantly increases the discrimination index as well as cell proliferation and neuroblast differentiation in the dentate gyrus. In addition, the administration of rimonabant, a CB1 cannabinoid receptor antagonist, for 3weeks significantly decreased the novel object recognition memory, the tyrosine hydroxylase level, the amount of cell proliferation, and neuroblast differentiation in the dentate gyrus. Treatment with pyridoxine significantly increased novel object recognition memory, but slightly ameliorated rimonabant-induced reduction in serotonin, the tyrosine hydroxylase level, the amount of cell proliferation, and neuroblast differentiation in the dentate gyrus. These results suggest that pyridoxine promotes hippocampal functions by increasing serotonin and tyrosine hydroylase immunoreactivity in the hippocampus. This positive effect may be associated with CRIP1a and CB1 cannabinoid receptor function. Vitamin-B6 enhances hippocampal functions and this is closely associated with CRIP1a and CB1 cannabinoid receptors. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. The Caenorhabditis elegans matrix non-peptidase MNP-1 is required for neuronal cell migration and interacts with the Ror receptor tyrosine kinase CAM-1.

    Science.gov (United States)

    Craft, Teresa R; Forrester, Wayne C

    2017-04-01

    Directed cell migration is critical for metazoan development. During Caenorhabditis elegans development many neuronal, muscle and other cell types migrate. Multiple classes of proteins have been implicated in cell migration including secreted guidance cues, receptors for guidance cues and intracellular proteins that respond to cues to polarize cells and produce the forces that move them. In addition, cell surface and secreted proteases have been identified that may clear the migratory route and process guidance cues. We report here that mnp-1 is required for neuronal cell and growth cone migrations. MNP-1 is expressed by migrating cells and functions cell autonomously for cell migrations. We also find a genetic interaction between mnp-1 and cam-1, which encodes a Ror receptor tyrosine kinase required for some of the same cell migrations. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Dataset of mRNA levels for dopaminergic receptors, adrenoceptors and tyrosine hydroxylase in lymphocytes from subjects with clinically isolated syndromes

    Directory of Open Access Journals (Sweden)

    Marco Cosentino

    2016-12-01

    Full Text Available This data article presents a dataset of mRNA levels for dopaminergic receptors, adrenoceptors and for tyrosine hydoxylase, the rate-limiting enzyme in the synthesis of catecholamines, in peripheral blood mononuclear cells as well as in CD4+ T effector and regulatory cells from subjects with clinically isolated syndromes (CIS, which is a first episode of neurological disturbance(s suggestive of multiple sclerosis. CIS subjects are divided into two groups according to their eventual progression, after 12 months from CIS, to clinically established multiple sclerosis. The data reported are related to the article entitled "Dopaminergic receptors and adrenoceptors in circulating lymphocytes as putative biomarkers for the early onset and progression of multiple sclerosis" (M. Cosentino, M. Zaffaroni, M. Legnaro, R. Bombelli, L. Schembri, D. Baroncini, A. Bianchi, R. Clerici, M. Guidotti, P. Banfi, G. Bono, F. Marino, 2016 [1].

  9. Tyrosine-610 in the receptor kinase BAK1 does not play a major role in brassinosteroid signaling or innate immunity

    Science.gov (United States)

    The plasma membrane-localized BRI1-ASSOCIATED KINASE1 (BAK1) functions as a co-receptor with several receptor kinases including the brassinosteroid (BR) receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1), which is involved in growth, and the receptors for bacterial flagellin and EF-Tu, FLAGELLIN-SENSING ...

  10. Characterization of the specific interaction between the DNA aptamer sgc8c and protein tyrosine kinase-7 receptors at the surface of T-cells by biosensing AFM.

    Science.gov (United States)

    Leitner, Michael; Poturnayova, Alexandra; Lamprecht, Constanze; Weich, Sabine; Snejdarkova, Maja; Karpisova, Ivana; Hianik, Tibor; Ebner, Andreas

    2017-04-01

    We studied the interaction of the specific DNA aptamer sgc8c immobilized at the AFM tip with its corresponding receptor, the protein tyrosine kinase-7 (PTK7) embedded in the membrane of acute lymphoblastic leukemia (ALL) cells (Jurkat T-cells). Performing single molecule force spectroscopy (SMFS) experiments, we showed that the aptamer sgc8c bound with high probability (38.3 ± 7.48%) and high specificity to PTK7, as demonstrated by receptor blocking experiments and through comparison with the binding behavior of a nonspecific aptamer. The determined kinetic off-rate (koff = 5.16 s-1) indicates low dissociation of the sgc8c-PTK7 complex. In addition to the pulling force experiments, simultaneous topography and recognition imaging (TREC) experiments using AFM tips functionalized with sgc8c aptamers were realized on the outer regions surface of surface-immobilized Jurkat cells for the first time. This allowed determination of the distribution of PTK7 without any labeling and at near physiological conditions. As a result, we could show a homogeneous distribution of PTK7 molecules on the outer regions of ALL cells with a surface density of 325 ± 12 PTK7 receptors (or small receptor clusters) per μm2. Graphical Abstract The specific interaction of the DNA aptamer sgc8c and protein tyrosine kinase-7 (PTK7) on acute lymphoblastic leukemia (ALL) cells was characterized. AFM based single molecule force spectroscopy (SMFS) yielded a kinetic off-rate of 5.16 s-1 of the complex. Simultaneous topography and recognition imaging (TREC) revealed a PTK7 density of 325 ± 12 molecules or clusters per μm2 in the cell membrane.

  11. A protein-binding domain, EH, identified in the receptor tyrosine kinase substrate Eps15 and conserved in evolution

    DEFF Research Database (Denmark)

    Wong, W T; Schumacher, C; Salcini, A E

    1995-01-01

    In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several heteroge...... (for Eps15-related). Structural comparison of Eps15 and Eps15r defines a family of signal transducers possessing extensive networking abilities including EH-mediated binding and association with Src homology 3-containing proteins.......In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several...

  12. 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.

  13. 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 tyros......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...... these RPTPalpha-/- mice had impaired tyrosine kinase activity of both c-Src and Fyn, and this was accompanied by a concomitant increase in c-Src Tyr527 phosphorylation. RPTPalpha-/- fibroblasts also showed a reduction in the rate of spreading on fibronectin substrates, a trait that is a phenocopy of the effect...

  14. Synthesis and anti-tyrosine kinase activity of 3-(substituted-benzylidene)-1, 3-dihydro-indolin derivatives: investigation of their role against p60c-Src receptor tyrosine kinase with the application of receptor docking studies.

    Science.gov (United States)

    Olgen, Sureyya; Akaho, Eiichi; Nebioglu, Dogu

    2005-01-01

    A series of 3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-thione derivatives were synthesized as modified congeners of 3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-one series. All the synthesized compounds were examined for their in vitro anti-tyrosine kinase activity against p60c-Src. The activity results revealed that compounds (Z)-3-(4'-Dimethylamino-benzylidene)-1, 3-dihydro-indolin-2-thione (12) (E)-3-(2', 6'-Dichloro-benzylidene)-1, 3-dihydro-indolin-2-thione (13) and (E)-3-(3'-Hydroxy-4'-methoxy-benzylidene)-1, 3-dihydro-indolin-2-thione (19) exhibited anti-tyrosine kinase activity with IC50 value of 21.91, 21.20 and 30.92 microM, respectively. These results are comparable to PP1 [1-tert-Butyl-3-p-tolyl-1H-pyrazolo[3, 4-d]pyrimidine-4-yl-amine] (IC50=0.17 microM), which is reported as a potent and selective p60c-Src tyrosine kinase inhibitor. Some thio congeners are found to be more potent than oxo derivatives; however, no significant correlation was observed between the activity profiles of these two series. Docking program was used to investigate the docking mode of each compound at the active site. Among all of the compounds, only (Z)-3-(2'-Chloro-benzylidene)-1, 3-dihydro-indolin-2-one (8) and (E)-3-(3'-Nitro-benzylidene)-1, 3-dihydro-indolin-2-thione (16) were docked at the active site where the PP1 was embedded.

  15. Associations of mRNA:microRNA for the shared downstream molecules of EGFR and alternative tyrosine kinase receptors in Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Fengfeng Wang

    2016-10-01

    Full Text Available Lung cancer is the top cancer killer worldwide with high mortality rate. Majority belong to non-small cell lung cancers (NSCLCs. The epidermal growth factor receptor (EGFR has been broadly explored as a drug target for therapy. However, the drug responses are not durable due to the acquired resistance. MicroRNAs (miRNAs are small noncoding and endogenous molecules that can inhibit mRNA translation initiation and degrade mRNAs. We wonder if some downstream molecules shared by EGFR and the other tyrosine kinase receptors (TKRs further transduce the signals alternatively, and some miRNAs play the key roles in affecting the expression of these downstream molecules. In this study, we investigated the mRNA:miRNA associations for the direct EGFR downstream molecules in the EGFR signaling pathway shared with the other TKRs, including c-MET (hepatocyte growth factor receptor, Ron (a protein tyrosine kinase related to c-MET, PDGFR (platelet-derived growth factor receptor, and IGF-1R (insulin-like growth factor receptor-1. The multiple linear regression and support vector regression (SVR models were used to discover the statistically significant and the best weighted miRNAs regulating the mRNAs of these downstream molecules. These two models revealed the similar mRNA:miRNA associations. It was found that the miRNAs significantly affecting the mRNA expressions in the multiple regression model were also those with the largest weights in the SVR model. To conclude, we effectively identified a list of meaningful mRNA:miRNA associations: phospholipase C, gamma 1 (PLCG1 with miR-34a, phosphoinositide-3-kinase, regulatory subunit 2 (PIK3R2 with miR-30a-5p, growth factor receptor-bound protein 2 (GRB2 with miR-27a, and Janus kinase 1 (JAK1 with miR-302b and miR-520e. These associations could make great contributions to explore new mechanism in NSCLCs. These candidate miRNAs may be regarded as the potential drug targets for treating NSCLCs with acquired drug

  16. Environmental neurotoxic pesticide dieldrin activates a non receptor tyrosine kinase to promote PKCδ-mediated dopaminergic apoptosis in a dopaminergic neuronal cell model.

    Science.gov (United States)

    Saminathan, Hariharan; Asaithambi, Arunkumar; Anantharam, Vellareddy; Kanthasamy, Anumantha G; Kanthasamy, Arthi

    2011-10-01

    Oxidative stress and apoptosis are two key pathophysiological mechanisms underlying dopaminergic degeneration in Parkinson's disease (PD). Recently, we identified that proteolytic activation of protein kinase C-delta (PKCδ), a member of the novel PKC family, contributes to oxidative stress-induced dopaminergic degeneration and that phosphorylation of tyrosine residue 311 (tyr311) on PKCδ is a key event preceding the PKCδ proteolytic activation during oxidative damage. Herein, we report that a non-receptor tyrosine kinase Fyn is significantly expressed in a dopaminergic neuronal N27 cell model. Exposure of N27 cells to the dopaminergic toxicant dieldrin (60 μM) rapidly activated Fyn kinase, PKCδ-tyr311 phosphorylation and proteolytic cleavage. Fyn kinase activation precedes the caspase-3-mediated proteolytic activation of PKCδ. Pre-treatment with p60-tyrosine-specific kinase inhibitor (TSKI) almost completely attenuated dieldrin-induced phosphorylation of PKCδ-tyr311 and its proteolytic activation. Additionally, TSKI almost completely blocked dieldrin-induced apoptotic cell death. To further confirm Fyn's role in the pro-apoptotic function of PKCδ, we adopted the RNAi approach. siRNA-mediated knockdown of Fyn kinase also effectively attenuated dieldrin-induced phosphorylation of PKCδ-tyr311, caspase-3-mediated PKCδ proteolytic cleavage, and DNA fragmentation, suggesting that Fyn kinase regulates the pro-apoptotic function of PKCδ. Collectively, these results demonstrate for the first time that Fyn kinase is a pro-apoptotic kinase that regulates upstream signaling of the PKCδ-mediated apoptotic cell death pathway in neurotoxicity models of pesticide exposure. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. 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.

  18. Silencing of Receptor Tyrosine Kinase ROR1 Inhibits Tumor-Cell Proliferation via PI3K/AKT/mTOR Signaling Pathway in Lung Adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Yanchun Liu

    Full Text Available Receptor tyrosine kinase ROR1, an embryonic protein involved in organogenesis, is expressed in certain hematological malignancies and solid tumors, but is generally absent in adult tissues. This makes the protein an ideal drug target for cancer therapy. In order to assess the suitability of ROR1 as a cell surface antigen for targeted therapy of lung adenocarcinoma, we carried out a comprehensive analysis of ROR1 protein expression in human lung adenocarcinoma tissues and cell lines. Our data show that ROR1 protein is selectively expressed on lung adenocarcinoma cells, but do not support the hypothesis that expression levels of ROR1 are associated with aggressive disease. However silencing of ROR1 via siRNA treatment significantly down-regulates the activity of the PI3K/AKT/mTOR signaling pathway. This is associated with significant apoptosis and anti-proliferation of tumor cells. We found ROR1 protein expressed in lung adenocarcinoma but almost absent in tumor-adjacent tissues of the patients. The finding of ROR1-mediated proliferation signals in both tyrosine kinase inhibitor (TKI-sensitive and -resistant tumor cells provides encouragement to develop ROR1-directed targeted therapy in lung adenocarcinoma, especially those with TKI resistance.

  19. Radiotherapy of non-small-cell lung cancer in the era of EGFR gene mutations and EGF receptor tyrosine kinase inhibitors.

    Science.gov (United States)

    Moschini, Ilaria; Dell'Anna, Cristina; Losardo, Pier Luigi; Bordi, Paola; D'Abbiero, Nunziata; Tiseo, Marcello

    2015-01-01

    Non-small-cell lung cancer (NSCLC) occurs, approximately, in 80-85% of all cases of lung cancer. The majority of patients present locally advanced or metastatic disease when diagnosed, with poor prognosis. The discovery of activating mutations in the EGFR gene has started a new era of personalized treatment for NSCLC patients. To improve the treatment outcome in patients with unresectable NSCLC and, in particular, EGFR mutated, a combined strategy of radiotherapy and medical treatment can be undertaken. In this review we will discuss preclinical data regarding EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) and radiotherapy, available clinical trials investigating efficacy and toxicity of combined treatment (thoracic or whole brain radiotherapy and EGFR-TKIs) and, also, the role of local radiation in mutated EGFR patients who developed EGFR-TKI resistance.

  20. Frontal affinity chromatography with MS detection of EphB2 tyrosine kinase receptor. 2. Identification of small-molecule inhibitors via coupling with virtual screening.

    Science.gov (United States)

    Toledo-Sherman, Leticia; Deretey, Eugen; Slon-Usakiewicz, Jacek J; Ng, William; Dai, Jin-Rui; Foster, J Estelle; Redden, Peter R; Uger, Marni D; Liao, Linda C; Pasternak, Andrew; Reid, Neil

    2005-05-05

    We have integrated two complementary methods, high-throughput virtual screening with a "high-content" wet screening technique based on frontal affinity chromatography with mass spectrometry detection (FAC-MS), for identification of hits against the erythropoietin-producing hepatocellular B2 (EphB2) receptor tyrosine kinase domain. Both an EphB2-directed virtual screen combining docking and scoring and a kinase-directed pharmacophore search strategy were used to identify a compound set enriched in bioactive compounds against EphB2. The coupling of virtual screening methodologies with FAC-MS is a unique hybrid approach that can be used to increase the efficacy of both hit discovery and optimization efforts in drug discovery and has successfully identified hits, in particular 19a (36% shift, IC(50) = 5.2 microM, K(d) = 3.3 microM), as inhibitors for EphB2, a potential cancer target.

  1. 6-Phosphogluconate dehydrogenase regulates tumor cell migration in vitro by regulating receptor tyrosine kinase c-Met

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Barden, E-mail: cchan@bidmc.harvard.edu [Division of Interdisciplinary Medicine and Biotechnology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 (United States); VanderLaan, Paul A. [Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 (United States); Sukhatme, Vikas P. [Division of Interdisciplinary Medicine and Biotechnology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 (United States)

    2013-09-20

    Highlights: •Expression of 6PGD positively correlates with advancing stage of lung carcinoma. •Knockdown of 6PGD by shRNA potently inhibits c-Met tyrosine phosphorylation. •Exogenous HGF fails to restore c-Met phosphorylation in cells with 6PGD knocked down. •6PGD knockdown results in inhibition of cell migration in vitro. •Constitutively active TPR-cMet significantly restores migration of cells without 6PGD. -- Abstract: 6-Phosphogluconate dehydrogenase (6PGD) is the third enzyme in the oxidative pentose phosphate pathway (PPP). Recently, we reported that knockdown of 6PGD inhibited lung tumor growth in vitro and in a xenograft model in mice. In this study, we continued to examine the functional role of 6PGD in cancer. We show that 6PGD expression positively correlates with advancing stage of lung carcinoma. In search of functional signals related to 6PGD, we discovered that knockdown of 6PGD significantly inhibited phosphorylation of c-Met at tyrosine residues known to be critical for activity. This downregulation of c-Met phosphorylation correlated with inhibition of cell migration in vitro. Overexpression of a constitutively active c-Met specifically rescued the migration but not proliferation phenotype of 6PGD knockdown. Therefore, 6PGD appears to be required for efficient c-Met signaling and migration of tumor cells in vitro.

  2. The receptor tyrosine kinase inhibitor vandetanib activates Akt and increases side population in a salivary gland tumor cell line (A253).

    Science.gov (United States)

    Fujishiro, Yuka; Tonogi, Morio; Ochiai, Hiromi; Matsuzaka, Kenichi; Yamane, Gen-Yuki; Azuma, Toshifumi

    2012-07-01

    We and others have reported that cancer side population (SP) cells have self-renewal and multidrug resistance capabilities. These phenotypes are similar to those of cancer stem cells (CSCs), cancer stem-like cells and tumor-initiating cells (TICs). It has also been reported that upregulation of the epidermal growth factor receptor (EGFR) significantly increases the number of cancer SP cells, conversely, molecular targeting of EGFR tyrosine kinases using specific kinase inhibitors downregulates CSCs. Thus, we used flow cytometric analysis and cell sorting to examine cancer SP cells in the SCA9.cl-15, WR21 and A253 cell lines that originate from a salivary gland tumor (SGT). We successfully isolated cancer SP cells from all of these cell lines. SP cells were detected following treatment of these cell lines with the receptor tyrosine kinase inhibitors (RTKIs) lapatinib, erlotinib and vandetanib. Several studies reported that RTKIs mostly reduced the SP population in cancer cells. We did not observe any detectable morphological differences between SP cells and non-SP cells. We found that the EGF RTKI lapatinib decreased the number of cancer SP cells in all cell lines investigated; however, the EGF RTKI erlotinib did not cause significant differences in the frequency of cancer SP cells in these cell lines. Addition of vandetanib significantly increased the number of cancer SP cells and upregulated the phosphorylated Akt. As far as we know, this is the first report to show that one of the RTKIs, vandetanib, can activate Akt and increase the number of cancer SP cells. It has been reported that RTKIs could competitively inhibit ABC transporters and subsequently reduced the number of SP cells. However, our observation indicated that signaling changes induced by RTKIs could even activate Akt and induce the SP population. Investigation of the SP phenotype of SGTs is important for the establishment of optimal cancer therapy.

  3. The human cytomegalovirus UL11 protein interacts with the receptor tyrosine phosphatase CD45, resulting in functional paralysis of T cells.

    Directory of Open Access Journals (Sweden)

    Ildar Gabaev

    2011-12-01

    Full Text Available Human cytomegalovirus (CMV exerts diverse and complex effects on the immune system, not all of which have been attributed to viral genes. Acute CMV infection results in transient restrictions in T cell proliferative ability, which can impair the control of the virus and increase the risk of secondary infections in patients with weakened or immature immune systems. In a search for new immunomodulatory proteins, we investigated the UL11 protein, a member of the CMV RL11 family. This protein family is defined by the RL11 domain, which has homology to immunoglobulin domains and adenoviral immunomodulatory proteins. We show that pUL11 is expressed on the cell surface and induces intercellular interactions with leukocytes. This was demonstrated to be due to the interaction of pUL11 with the receptor tyrosine phosphatase CD45, identified by mass spectrometry analysis of pUL11-associated proteins. CD45 expression is sufficient to mediate the interaction with pUL11 and is required for pUL11 binding to T cells, indicating that pUL11 is a specific CD45 ligand. CD45 has a pivotal function regulating T cell signaling thresholds; in its absence, the Src family kinase Lck is inactive and signaling through the T cell receptor (TCR is therefore shut off. In the presence of pUL11, several CD45-mediated functions were inhibited. The induction of tyrosine phosphorylation of multiple signaling proteins upon TCR stimulation was reduced and T cell proliferation was impaired. We therefore conclude that pUL11 has immunosuppressive properties, and that disruption of T cell function via inhibition of CD45 is a previously unknown immunomodulatory strategy of CMV.

  4. [Application of the concetrations ratio of soluble receptor tyrosine kinase type 1, and placental growth factor for short-term prediction and diagnosis of preeclampsia].

    Science.gov (United States)

    Bubeníková, Š; Cíchová, A; Roubalová, L; Durdová, V; Vlk, R

    Bring a comprehensive overview of the available information about applications of the concetration ratio of soluble receptor tyrosine kinase type 1 (sFlt-1), and placental growth factor for short-term prediction and diagnosis of preeclampsia. Overview study. Department of Midwifery, Faculty of Health Sciences, Olomouc; Department of Clinical Biochemistry, University Hospital Olomouc; Department of Obstetrics and Gynecology, University Hospital Olomouc; Department of Obstetrics and Gynecology, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital. Analysis of literary sources and databases Ovid, Medline (2001-2016). Preeclampsia is a multisystem disease with not fully understood etiology. This disease occurs in 2-5% of pregnant women. Preeclampsia is one of the main causes of global maternal and perinatal morbidity and mortality. It manifests itself as a newborn hypertension and proteinuria after 20 weeks of pregnancy in previously normotensive women. The only effective treatment is the delivery of the child. Diagnosis of preeclampsia comprises measuring blood pressure and proteinuria. These indicators have low diagnostic sensitivity and specificity. In preeclampsia, there is a decrease of serum levels of placental growth factor (PlGF). Soluble receptor tyrosine kinase type 1 (sFlt-1) is an antagonist of PlGF. Increased levels of sFlt-1 in proportion to the reduced level of PlGF are associated with an increased risk of preeclampsia. The sFlt-1/PlGF ratio can be a better predictive marker in the diagnosis of pre-eclampsia after 20 weeks of gestation.

  5. 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

    In an effort to clone novel tyrosine-phosphorylated substrates of the epidermal growth factor receptor, we have initiated an approach coupling affinity purification using anti-phosphotyrosine antibodies to mass spectrometry-based identification. Here, we report the identification of a signaling m...

  6. A proximity ligation assay using transiently transfected, epitope-tagged proteins: application for in situ detection of dimerized receptor tyrosine kinases.

    Science.gov (United States)

    Gajadhar, Aaron; Guha, Abhijit

    2010-02-01

    The development of small molecule and antibody inhibitors targeting the interaction of receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR), is of high pharmacological and biological interest. Unfortunately, conventional biochemical techniques using cell or tissue lysates and co-immunoprecipitation experiments to investigate EGFR dimerization are not always conclusive. Here we describe a series of technical and biological validation experiments demonstrating the utility of a proximity ligation assay (PLA)-based methodology for in situ visualization and quantification of ligand-dependent EGFR receptor dimerization in intact cells. Using the PLA approach combined with a universally applicable epitope tagging strategy, we detected EGFR dimers in cells transiently co-expressing FLAG-tagged and MYC-tagged human EGFRs. Our data strongly suggest that PLA can be used to detect ligand-dependent EGFR dimerization and this signal is generated in a protein interaction-based manner, rather than solely due to proximity of target proteins. This application represents a generalized RTK expression strategy for protein-interaction analysis in a transient expression system where antibody epitopes are not known or not unique enough to discriminate between interaction partners. This assay also holds promise as a general RTK dimerization screening tool in tissue specimens to identify potential dimerization inhibitors with clinical relevance.

  7. Diverse roles for the ror-family receptor tyrosine kinases in neurons and glial cells during development and repair of the nervous system.

    Science.gov (United States)

    Endo, Mitsuharu; Minami, Yasuhiro

    2017-05-03

    The Ror-family of receptor tyrosine kinases (RTKs) are involved critically in tissue genesis and organogenesis during development. In mammals, Ror1 and Ror2, members of the Ror-family RTKs, have been shown to mediate cell polarity, migration, proliferation, and differentiation through the activation of noncanonical Wnt signaling by acting as receptors or co-receptors for Wnt5a. Nematodes bearing mutations within the cam-1 gene, encoding a Ror2 ortholog, exhibit defects in various developmental processes of the nervous system, including neuronal cell migration, polarization, axonal extension, and synaptic transmission. In mice, Ror2 and/or Ror1 are also shown to play roles in regulating neurite extension, synapse formation, and synaptic transmission of hippocampal neurons, indicating that the Ror-family RTKs have evolutionarily conserved functions at least in part in neurons during development. Furthermore, Ror2 and/or Ror1 are expressed in neural stem/progenitor cells of the developing brain and in astrocytes of the adult brain after injury, and they play important roles in regulating cell proliferation under these different contexts. In this article, we overview recent advances in our understanding of the roles of the Ror-family RTKs in the development and repair of the nervous system and discuss their potential for therapeutic targets to neurodegenerative diseases. Developmental Dynamics, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  8. Torso, a Drosophila receptor tyrosine kinase, plays a novel role in the larval fat body in regulating insulin signaling and body growth.

    Science.gov (United States)

    Jun, Jong Woo; Han, Gangsik; Yun, Hyun Myoung; Lee, Gang Jun; Hyun, Seogang

    2016-08-01

    Torso is a receptor tyrosine kinase whose localized activation at the termini of the Drosophila embryo is mediated by its ligand, Trunk. Recent studies have unveiled a second function of Torso in the larval prothoracic gland (PG) as the receptor for the prothoracicotropic hormone, which triggers pupariation. As such, inhibition of Torso in the PG prolongs the larval growth period, thereby increasing the final pupa size. Here, we report that Torso also acts in the larval fat body, regulating body size in a manner opposite from that of Torso in PG. We confirmed the expression of torso mRNA in the larval fat body and its reduction by RNA interference (RNAi). Fat body-specific knockdown of torso, by either of the two independent RNAi transgenes, significantly decreased the final pupal size. We found that torso knockdown suppresses insulin/target of rapamycin (TOR) signaling in the fat body, as confirmed by repression of Akt and S6K. Notably, the decrease in insulin/TOR signaling and decrease of pupal size induced by the knockdown of torso were rescued by the expression of a constitutively active form of the insulin receptor or by the knockdown of FOXO. Our study revealed a novel role for Torso in the fat body with respect to regulation of insulin/TOR signaling and body size. This finding exemplifies the contrasting effects of the same gene expressed in two different organs on organismal physiology.

  9. Identification of a neuregulin and protein-tyrosine phosphatase response element in the nicotinic acetylcholine receptor epsilon subunit gene: regulatory role of an Rts transcription factor.

    Science.gov (United States)

    Sapru, M K; Florance, S K; Kirk, C; Goldman, D

    1998-02-03

    At the neuromuscular synapse, innervation induces endplate-specific expression of adult-type nicotinic acetylcholine receptors by selective expression of their subunit-encoding genes (alpha2betaepsilondelta) in endplate-associated myonuclei. These genes are specifically regulated by protein-tyrosine phosphatase (PTPase) activity. In addition, neuregulin/acetylcholine-receptor-inducing activity, a nerve-derived factor that stimulates nicotinic acetylcholine receptor synthesis, induces adult-type specific epsilon subunit gene expression via activation of a Ras/mitogen-activated protein kinase pathway. However, the DNA regulatory elements and the binding proteins that mediate PTPase and neuregulin-dependent gene expression remain unknown. Herein we report that PTPase, neuregulin, and Ras-dependent regulation of the epsilon subunit gene map to a 15-bp promoter sequence. Interestingly, this same 15-bp sequence appears to be necessary for low epsilon subunit gene expression in extrajunctional regions of the muscle fiber. Site-directed mutagenesis of a putative Ets binding site located within this 15-bp sequence, reduced PTPase, neuregulin, and Ras-dependent regulation. Overexpression of the rat muscle Ets-2 transcription factor resulted in a sequence-specific induction of epsilon subunit promoter activity. Further, a dominant negative mutant of Ets-2 abolished neuregulin-dependent induction of epsilon subunit gene expression. Thus, these results indicate a crucial role for the 15-bp element in determining synapse-specific and neuregulin-mediated motor neuron control of epsilon subunit gene expression and suggest the participation of Ets transcription factor(s) in this control.

  10. The continuing role of epidermal growth factor receptor tyrosine kinase inhibitors in advanced squamous cell carcinoma of the lung.

    Science.gov (United States)

    Tan, Wan Ling; Ng, Quan-Sing

    2016-02-01

    Squamous cell carcinoma (SCC) of the lung represents about 20-30% of non-small cell lung cancers (NSCLC) and is associated with a poorer prognosis with limited treatment options. Erlotinib is an approved, standard second-line therapy in this setting, besides docetaxel. The LUX-Lung 8 study has shown superior overall survival (OS), progression-free survival (PFS), as well as disease control rates for treatment with afatinib compared to erlotinib in this head-to-head trial in patients with previously treated advanced SCC of the lung, with manageable side effect profile. This is the first and largest prospective phase III trial comparing two different tyrosine kinase inhibitors in patients with advanced SCC of the lung. Whether the results would be practice-changing remains to be seen, especially with the advent of novel immunotherapeutic agents such as nivolumab, which is recently approved for advanced lung SCC.

  11. Endometrial Cancers Harboring Mutated Fibroblast Growth Factor Receptor 2 Protein Are Successfully Treated With a New Small Tyrosine Kinase Inhibitor in an Orthotopic Mouse Model.

    Science.gov (United States)

    Taurin, Sebastien; Yang, Chieh-Hsiang; Reyes, Maria; Cho, Sungpil; Coombs, Demetrius M; Jarboe, Elke A; Werner, Theresa L; Peterson, C Matthew; Janát-Amsbury, Margit M

    2017-09-26

    AL3818 (anlotinib) is a receptor tyrosine kinase inhibitor targeting vascular endothelial growth factor receptors (VEGFR1, VEGFR2/KDR, and VEGFR3), stem cell factor receptor (C-kit), platelet-derived growth factor (PDGFβ), and fibroblast growth factor receptors (FGFR1, FGFR2, and FGFR3). This study evaluates the efficacy of AL3818 studying tumor regression in an orthotopic murine endometrial cancer model. We tested the cytotoxicity of AL3818 on a panel of 7 human endometrial cancer cell lines expressing either wild-type or mutant FGFR2 and also assessed the in vivo antitumor efficacy in a murine, orthotopic AN3CA endometrial cancer model. AL3818 was administered daily per os either alone or in combination with carboplatin and paclitaxel, which represent the current standard of adjuvant care for endometrial cancer. AL3818 significantly reduces AN3CA cell number in vitro, characterized by high expression of a mutated FGFR2 protein. Daily oral administration of AL3818 (5 mg/kg) resulted in a complete response in 55% of animals treated and in a reduced tumor volume, as well as decreased tumor weights of AN3CA tumors by 94% and 96%, respectively, following a 29-day treatment cycle. Whereas carboplatin and paclitaxel failed to alter tumor growth, the combination with AL3818 did not seem to exhibit a superior effect when compared with AL3818 treatment alone. AL3818 shows superior efficacy for the treatment of endometrial cancer irresponsive to conventional carboplatin and paclitaxel combination and warrants further investigation.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

  12. Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases*

    Science.gov (United States)

    Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J.; Nie, Guangjun

    2016-01-01

    Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. PMID:26895960

  13. Expression of the vitamin K-dependent proteins GAS6 and protein S and the TAM receptor tyrosine kinases in human atherosclerotic carotid plaques.

    Science.gov (United States)

    Hurtado, B; Muñoz, X; Recarte-Pelz, P; García, N; Luque, A; Krupinski, J; Sala, N; García de Frutos, P

    2011-05-01

    The GAS6/ProS-TAM system is composed of two vitamin K-dependent ligands (GAS6 and protein S) and their three protein tyrosine kinase receptors TYRO3, AXL and MERTK, known as the TAM receptors. The system plays a prominent role in conditions of injury, inflammation and repair. In murine models of atherosclerotic plaque formation, mutations in its components affect atherosclerosis severity. Here we used Taqman low-density arrays and immunoblotting to study mRNA and protein expression of GAS6, ProS and the TAM receptors in human carotid arteries with different degrees of atherosclerosis. The results show a clear down-regulation of the expression of AXL in atheroma plaques with respect to normal carotids that is matched by decreased abundance of AXL in protein extracts detected by immunoblotting. A similar decrease was observed in PROS1 mRNA expression in atherosclerotic carotids compared to the normal ones, but in this case protein S (ProS) was clearly increased in protein extracts of carotid arteries with increasing grade of atherosclerosis, suggesting that ProS is carried into the plaque. MERTK was also increased in atherosclerotic carotid arteries with respect to the normal ones, suggesting that the ProS-MERTK axis is functional in advanced human atherosclerotic plaques. MERTK was expressed in macrophages, frequently in association with ProS, while ProS was abundant also in the necrotic core. Our data suggest that the ProS-MERTK ligand-receptor pair was active in advanced stages of atherosclerosis, while AXL signalling is probably down-regulated.

  14. Screening of multi-targeted natural compounds for receptor tyrosine kinases inhibitors and biological evaluation on cancer cell lines, in silico and in vitro.

    Science.gov (United States)

    Singh, Pushpendra; Bast, Felix

    2015-09-01

    Receptors for growth factors encompass within the superfamily of receptor tyrosine kinases and are known to regulate numerous biological processes including cellular growth, proliferation, metabolism, survival, cell differentiation and apoptosis. These receptors have recently caught the attention of the researchers as an attractive target to combat cancer owing to the evidence suggesting their over-expression in cancer cells. Therefore, we studied receptor-based molecular docking of IR (PDB; 3ETA), IGF1R (PDB; 1K3A), EGFR (PDB; 1M17), VEGFIR (PDB; 3HNG), and VEGFIIR (PDB; 2OH4) against natural compounds. Further, in vitro investigation of the biological effect of lead molecules in an array of cancer cell lines was done. All selected natural compounds were docked with the X-ray crystal structure of selected protein by employing GLIDE (Grid-based Ligand Docking with Energetics) Maestro 9.6. InterBioScreen natural compounds docked with each selected protein molecules by using GLIDE high throughput virtual screening. On the basis of Gscore, we select 20 compounds along with 68 anticancer compounds for GLIDE extra precision molecular docking. It was discovered in this study that compound epigallocatechin gallate (EGCG) yielded magnificent Gscore with IGF1R (PDB; 1K3A) and VEGFIIR (PDB; 2OH4), and protein-ligand interactions are chart out. Effect of EGCG on biological activity such as mRNA expression of selected protein, cell proliferation, oxidative stress, and cell migration was reported after the 48 h treatments in cancer cell lines. The RT-PCR densitometric bands analysis showed that compound EGCG reduced the mRNA expression of IGF1R, VEGFIIR, and mTOR at 80 μM concentration. Moreover, EGCG significantly reduced cell proliferation and ROS generation after 48 h treatments. Our result also indicated a reduction in the potential for cell migration that might show in vivo anti-metastasis activity of EGCG.

  15. Good Tolerance to Full-Dose Crizotinib in a Patient with Anaplastic Lymphoma Receptor Tyrosine Kinase-Rearranged Lung Adenocarcinoma and Preexisting Renal Impairment

    Science.gov (United States)

    Rosoux, Adeline; Duplaquet, Fabrice; Ocak, Sebahat

    2017-01-01

    Background Crizotinib is an approved tyrosine kinase inhibitor in the treatment of advanced-stage non-small-cell lung cancer patients with anaplastic lymphoma receptor tyrosine kinase (ALK) rearrangement. Renal dysfunction after crizotinib administration was recently reported, but the physiopathological explanation and the safety in patients with preexisting renal dysfunction are still not clear. Case Presentation A 44-year-old female and current smoker was diagnosed with a stage IV lung adenocarcinoma and treated with five lines of chemotherapy during a 4-year period of time. While she developed symptomatic tumor progression with deterioration of her performance status and renal dysfunction after these five lines of treatment, we discovered that her lung cancer was ALK-rearranged. We therefore proposed a treatment with full-dose crizotinib despite the renal impairment (creatinine clearance: 33 mL/min/1.73 m2) of unknown origin. A renal function worsening occurred after the initiation of crizotinib but we did not reduce the dose as recommended and this did not induce further deterioration. During the 15 months under crizotinib, the patient had a good general status, no clinically noticeable side effect, and a stable renal dysfunction, which even improved after the initial worsening and almost returned to the baseline (pre-crizotinib) status. Conclusion This case report suggests that full-dose crizotinib may be continued even in patients with severe renal dysfunction and deterioration at treatment initiation, in parallel to careful follow-up of renal function and particular attention to avoid the use of concomitant nephrotoxic drugs. PMID:28868009

  16. Inhibition of the Receptor Tyrosine Kinase ROR1 by Anti-ROR1 Monoclonal Antibodies and siRNA Induced Apoptosis of Melanoma Cells

    Science.gov (United States)

    Hojjat-Farsangi, Mohammad; Ghaemimanesh, Fatemeh; Daneshmanesh, Amir Hossein; Bayat, Ali-Ahmad; Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Mellstedt, Hakan

    2013-01-01

    The receptor tyrosine kinase (RTK) ROR1 is overexpressed and of importance for the survival of various malignancies, including lung adenocarcinoma, breast cancer and chronic lymphocytic leukemia (CLL). There is limited information however on ROR1 in melanoma. In the present study we analysed in seven melanoma cell lines ROR1 expression and phosphorylation as well as the effects of anti-ROR1 monoclonal antibodies (mAbs) and ROR1 suppressing siRNA on cell survival. ROR1 was overexpressed at the protein level to a varying degree and phosphorylated at tyrosine and serine residues. Three of our four self-produced anti-ROR1 mAbs (clones 3H9, 5F1 and 1A8) induced a significant direct apoptosis of the ESTDAB049, ESTDAB112, DFW and A375 cell lines as well as cell death in complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC). The ESTDAB081 and 094 cell lines respectively were resistant to direct apoptosis of the four anti-ROR1 mAbs alone but not in CDC or ADCC. ROR1 siRNA transfection induced downregulation of ROR1 expression both at mRNA and protein levels proceeded by apoptosis of the melanoma cells (ESTDAB049, ESTDAB112, DFW and A375) including ESTDAB081, which was resistant to the direct apoptotic effect of the mAbs. The results indicate that ROR1 may play a role in the survival of melanoma cells. The surface expression of ROR1 on melanoma cells may support the notion that ROR1 might be a suitable target for mAb therapy. PMID:23593420

  17. Inhibition of the receptor tyrosine kinase ROR1 by anti-ROR1 monoclonal antibodies and siRNA induced apoptosis of melanoma cells.

    Directory of Open Access Journals (Sweden)

    Mohammad Hojjat-Farsangi

    Full Text Available The receptor tyrosine kinase (RTK ROR1 is overexpressed and of importance for the survival of various malignancies, including lung adenocarcinoma, breast cancer and chronic lymphocytic leukemia (CLL. There is limited information however on ROR1 in melanoma. In the present study we analysed in seven melanoma cell lines ROR1 expression and phosphorylation as well as the effects of anti-ROR1 monoclonal antibodies (mAbs and ROR1 suppressing siRNA on cell survival. ROR1 was overexpressed at the protein level to a varying degree and phosphorylated at tyrosine and serine residues. Three of our four self-produced anti-ROR1 mAbs (clones 3H9, 5F1 and 1A8 induced a significant direct apoptosis of the ESTDAB049, ESTDAB112, DFW and A375 cell lines as well as cell death in complement dependent cytotoxicity (CDC and antibody dependent cellular cytotoxicity (ADCC. The ESTDAB081 and 094 cell lines respectively were resistant to direct apoptosis of the four anti-ROR1 mAbs alone but not in CDC or ADCC. ROR1 siRNA transfection induced downregulation of ROR1 expression both at mRNA and protein levels proceeded by apoptosis of the melanoma cells (ESTDAB049, ESTDAB112, DFW and A375 including ESTDAB081, which was resistant to the direct apoptotic effect of the mAbs. The results indicate that ROR1 may play a role in the survival of melanoma cells. The surface expression of ROR1 on melanoma cells may support the notion that ROR1 might be a suitable target for mAb therapy.

  18. Cocaine-induced changes of synaptic transmission in the striatum are modulated by adenosine A2A receptors and involve the tyrosine phosphatase STEP.

    Science.gov (United States)

    Chiodi, Valentina; Mallozzi, Cinzia; Ferrante, Antonella; Chen, Jiang F; Lombroso, Paul J; Di Stasi, Anna Maria Michela; Popoli, Patrizia; Domenici, Maria Rosaria

    2014-02-01

    The striatum is a brain area implicated in the pharmacological action of drugs of abuse. Adenosine A2A receptors (A2ARs) are highly expressed in the striatum and mediate, at least in part, cocaine-induced psychomotor effects in vivo. Here we studied the synaptic mechanisms implicated in the pharmacological action of cocaine in the striatum and investigated the influence of A2ARs. We found that synaptic transmission was depressed in corticostriatal slices after perfusion with cocaine (10 μM). This effect was reduced by the A2AR antagonist ZM241385 and almost abolished in striatal A2AR-knockout mice (mice lacking A2ARs in striatal neurons, stA2ARKO). The effect of cocaine on synaptic transmission was also prevented by the protein tyrosine phosphatases (PTPs) inhibitor sodium orthovanadate (Na3VO4). In synaptosomes prepared from striatal slices, we found that the activity of striatal-enriched protein tyrosine phosphatase (STEP) was upregulated by cocaine, prevented by ZM241385, and absent in synaptosomes from stA2ARKO. The role played by STEP in cocaine modulation of synaptic transmission was investigated in whole-cell voltage clamp recordings from medium spiny neurons of the striatum. We found that TAT-STEP, a peptide that renders STEP enzymatically inactive, prevented cocaine-induced reduction in AMPA- and NMDA-mediated excitatory post-synaptic currents, whereas the control peptide, TAT-myc, had no effect. These results demonstrate that striatal A2ARs modulate cocaine-induced synaptic depression in the striatum and highlight the potential role of PTPs and specifically STEP in the effects of cocaine.

  19. Calpain and STriatal-Enriched protein tyrosine phosphatase (STEP) activation contribute to extrasynaptic NMDA receptor localization in a Huntington's disease mouse model.

    Science.gov (United States)

    Gladding, Clare M; Sepers, Marja D; Xu, Jian; Zhang, Lily Y J; Milnerwood, Austen J; Lombroso, Paul J; Raymond, Lynn A

    2012-09-01

    In Huntington's disease (HD), the mutant huntingtin (mhtt) protein is associated with striatal dysfunction and degeneration. Excitotoxicity and early synaptic defects are attributed, in part, to altered NMDA receptor (NMDAR) trafficking and function. Deleterious extrasynaptic NMDAR localization and signalling are increased early in yeast artificial chromosome mice expressing full-length mhtt with 128 polyglutamine repeats (YAC128 mice). NMDAR trafficking at the plasma membrane is regulated by dephosphorylation of the NMDAR subunit GluN2B tyrosine 1472 (Y1472) residue by STriatal-Enriched protein tyrosine Phosphatase (STEP). NMDAR function is also regulated by calpain cleavage of the GluN2B C-terminus. Activation of both STEP and calpain is calcium-dependent, and disruption of calcium homeostasis occurs early in the HD striatum. Here, we show increased calpain cleavage of GluN2B at both synaptic and extrasynaptic sites, and elevated extrasynaptic total GluN2B expression in the YAC128 striatum. Calpain inhibition significantly reduced extrasynaptic GluN2B expression in the YAC128 but not wild-type striatum. Furthermore, calpain inhibition reduced whole-cell NMDAR current and the surface/internal GluN2B ratio in co-cultured striatal neurons, without affecting synaptic GluN2B localization. Synaptic STEP activity was also significantly higher in the YAC128 striatum, correlating with decreased GluN2B Y1472 phosphorylation. A substrate-trapping STEP protein (TAT-STEP C-S) significantly increased VGLUT1-GluN2B colocalization, as well as increasing synaptic GluN2B expression and Y1472 phosphorylation. Moreover, combined calpain inhibition and STEP inactivation reduced extrasynaptic, while increasing synaptic GluN2B expression in the YAC128 striatum. These results indicate that increased STEP and calpain activation contribute to altered NMDAR localization in an HD mouse model, suggesting new therapeutic targets for HD.

  20. Mapping of the receptor protein-tyrosine kinase 10 to human chromosome 1q21-q23 and mouse chromosome 1H1-5 by fluorescence in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Edelhoff, S.; Disteche, C.M. [Univ. of Washington School of Medicine, Seattle, WA (United States); Lai, C. [Scripps Research Inst., LaJolla, CA (United States)

    1995-01-01

    Receptor protein-tyrosine kinases (PTKs) play a critical role in the transduction of signals important to cell growth, differentiation, and survival. Mutations affecting the expression of receptor PTK genes have been associated with a number of vertebrate and invertebrate developmental abnormalities, and the aberrant regulation of tyrosine phosphorylation is implicated in a variety of neoplasias. One estimate suggests that approximately 100 receptor PTK genes exist in the mammalian genome, about half of which have been identified. The tyro-10 receptor protein-tyrosine kinase, first identified in a PCR-based survey for novel tyrosine kinases in the rat nervous system, defines a new subfamily of PTKs. It exhibits a catalytic domain most closely related to those found in the trk PTK receptor subfamily, which transduces signals for nerve growth factor and the related molecules brain-derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4 (NT-3 and NT-4). Trk and the related PTK receptors trkB and trkC play a critical role in the neurotrophin-dependent survival of subsets of sensory and motor neurons. The predicted tyro-10 extracellular region is, however, distinct from that of the trk subfamily and is unique except for a domain shared with the blood coagulation factors V and VIII, thought to be involved in phospholipid binding. Although tyro-10 RNA is most abundant in heart and skeletal muscle in the adult rat, it is expressed in a wide variety of tissues, including the developing and mature brain. Tyro-10 appears identical to the murine TKT sequence reported by Karn et al. and exhibits a high degree of similarity with the CaK, DDR, and Nep PTKs. A ligand for tyro-10 has not yet been identified. 10 refs., 1 fig.

  1. Identification of the tyrosine-protein phosphatase non-receptor type 2 as a rheumatoid arthritis susceptibility locus in europeans.

    Directory of Open Access Journals (Sweden)

    Joanna E Cobb

    Full Text Available OBJECTIVES: Genome-wide association studies have facilitated the identification of over 30 susceptibility loci for rheumatoid arthritis (RA. However, evidence for a number of potential susceptibility genes have not so far reached genome-wide significance in studies of Caucasian RA. METHODS: A cohort of 4286 RA patients from across Europe and 5642 population matched controls were genotyped for 25 SNPs, then combined in a meta-analysis with previously published data. RESULTS: Significant evidence of association was detected for nine SNPs within the European samples. When meta-analysed with previously published data, 21 SNPs were associated with RA susceptibility. Although SNPs in the PTPN2 gene were previously reported to be associated with RA in both Japanese and European populations, we show genome-wide evidence for a different SNP within this gene associated with RA susceptibility in an independent European population (rs7234029, P = 4.4×10(-9. CONCLUSIONS: This study provides further genome-wide evidence for the association of the PTPN2 locus (encoding the T cell protein tyrosine phosphastase with Caucasian RA susceptibility. This finding adds to the growing evidence for PTPN2 being a pan-autoimmune susceptibility gene.

  2. Identification of the Tyrosine-Protein Phosphatase Non-Receptor Type 2 as a Rheumatoid Arthritis Susceptibility Locus in Europeans

    Science.gov (United States)

    Cobb, Joanna E.; Plant, Darren; Flynn, Edward; Tadjeddine, Meriem; Dieudé, Philippe; Cornélis, François; Ärlestig, Lisbeth; Dahlqvist, Solbritt Rantapää; Goulielmos, George; Boumpas, Dimitrios T.; Sidiropoulos, Prodromos; Krintel, Sophine B.; Ørnbjerg, Lykke M.; Hetland, Merete L.; Klareskog, Lars; Haeupl, Thomas; Filer, Andrew; Buckley, Christopher D.; Raza, Karim; Witte, Torsten; Schmidt, Reinhold E.; FitzGerald, Oliver; Veale, Douglas; Eyre, Stephen; Worthington, Jane

    2013-01-01

    Objectives Genome-wide association studies have facilitated the identification of over 30 susceptibility loci for rheumatoid arthritis (RA). However, evidence for a number of potential susceptibility genes have not so far reached genome-wide significance in studies of Caucasian RA. Methods A cohort of 4286 RA patients from across Europe and 5642 population matched controls were genotyped for 25 SNPs, then combined in a meta-analysis with previously published data. Results Significant evidence of association was detected for nine SNPs within the European samples. When meta-analysed with previously published data, 21 SNPs were associated with RA susceptibility. Although SNPs in the PTPN2 gene were previously reported to be associated with RA in both Japanese and European populations, we show genome-wide evidence for a different SNP within this gene associated with RA susceptibility in an independent European population (rs7234029, P = 4.4×10−9). Conclusions This study provides further genome-wide evidence for the association of the PTPN2 locus (encoding the T cell protein tyrosine phosphastase) with Caucasian RA susceptibility. This finding adds to the growing evidence for PTPN2 being a pan-autoimmune susceptibility gene. PMID:23840476

  3. 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 affinity...

  4. Cytokine responses to fungal pathogens in Kupffer Cells are Toll-like receptor 4 independent and mediated by tyrosine kinases.

    NARCIS (Netherlands)

    Overland, G.; Stuestol, J.F.; Dahle, M.K.; Myhre, A.E.; Netea, M.G.; Verweij, P.E.; Yndestad, A.; Aukrust, P.; Kullberg, B.J.; Warris, A.; Wang, J.; Aasen, A.O.

    2005-01-01

    Disseminated fungal infections are increasing. However, the interactions between the body's largest population of tissue macrophages, the Kupffer cells and the fungal pathogens are scarcely understood. The aim of this study was to examine the involvement of Toll-like receptor 4 (TLR4) signalling in

  5. The triple angiokinase inhibitor nintedanib directly inhibits tumor cell growth and induces tumor shrinkage via blocking oncogenic receptor tyrosine kinases.

    Science.gov (United States)

    Hilberg, Frank; Tontsch-Grunt, Ulrike; Baum, Anke; Le, Anh T; Doebele, Robert C; Lieb, Simone; Dianni, Davide; Voss, Tilman; Garin-Chesa, Pilar; Haslinger, Christian; Kraut, Norbert

    2017-12-20

    The triple angiokinase inhibitor nintedanib is an orally available, potent and selective inhibitor of tumor angiogenesis by blocking the tyrosine kinase activities of VEGFR 1-3, PDGFR α and β and FGFR 1-3. Nintedanib has received regulatory approval in second line adenocarcinoma NSCLC in combination with docetaxel. In addition, nintedanib has been approved for the treatment of idiopathic lung fibrosis. Here we report the results from a broad kinase screen that identified additional kinases as targets for nintedanib in the low nanomolar range. Several of these kinases are known to be mutated or overexpressed and are involved in tumor development (DDR1 and 2, TRKA and C, Ret) as well as in fibrotic diseases (eg. DDRs). In tumor cell lines displaying molecular alterations in potential nintedanib targets, the inhibitor demonstrates direct anti-proliferative effects: in the NSCLC cell line NCI-H1703 carrying a PDGFRα amplification; the gastric cancer cell line KatoIII and the breast cancer cell line MFM223 both driven by a FGFR2 amplification; AN3CA (endometrial carcinoma) bearing a mutated FGFR2; the AML cell lines MOLM-13 and MV-4-11-B with FLT3 mutations; the NSCLC adenocarcinoma LC-2/ad harboring a CCDC6-RET fusion. However, potent kinase inhibition does not strictly translate into anti-proliferative activity as demonstrated in the TRKA dependent cell lines CUTO-3 and KM-12. Importantly, nintedanib treatment of NCI-H1703 tumor xenografts triggered effective tumor shrinkage, indicating the direct effect on the tumor cells on top of the antiangiogenic effect on the tumor stroma. These findings will be instructive to guide future genome-based clinical trials with nintedanib. The American Society for Pharmacology and Experimental Therapeutics.

  6. Non-peptide fibrinogen receptor antagonists. 2. Optimization of a tyrosine template as a mimic for Arg-Gly-Asp.

    Science.gov (United States)

    Egbertson, M S; Chang, C T; Duggan, M E; Gould, R J; Halczenko, W; Hartman, G D; Laswell, W L; Lynch, J J; Lynch, R J; Manno, P D

    1994-08-05

    Inhibitors of platelet-fibrinogen binding offer an opportunity to interrupt the final, common pathway for platelet aggregation. Small molecule inhibitors of the platelet fibrinogen receptor GPIIb/IIIa were prepared and evaluated for their ability to prevent platelet aggregation. Compound 23m (L-700,462/MK-383) inhibited in vitro platelet aggregation with an IC50 of 9 nM and demonstrated a selectivity of > 24,000-fold between platelet and human umbilical vein endothelial cell fibrinogen receptors. Dose-dependent inhibition of ex vivo platelet aggregation induced by ADP was achieved with i.v. infusions of 0.1-10 micrograms/kg/min of 23m in anesthetized dogs, with 10 micrograms/kg/min completely inhibiting platelet aggregation during the entire 6 h infusion protocol. Platelet aggregatability returned rapidly after the termination of the 23m infusions. These features suggest that 23m may be useful in the treatment of arterial occlusive disorders.

  7. The tyrosine kinase receptor Tyro3 enhances lifespan and neuropeptide Y (Npy neuron survival in the mouse anorexia (anx mutation

    Directory of Open Access Journals (Sweden)

    Dennis Y. Kim

    2017-05-01

    Full Text Available Severe appetite and weight loss define the eating disorder anorexia nervosa, and can also accompany the progression of some neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS. Although acute loss of hypothalamic neurons that produce appetite-stimulating neuropeptide Y (Npy and agouti-related peptide (Agrp in adult mice or in mice homozygous for the anorexia (anx mutation causes aphagia, our understanding of the factors that help maintain appetite regulatory circuitry is limited. Here we identify a mutation (C19T that converts an arginine to a tryptophan (R7W in the TYRO3 protein tyrosine kinase 3 (Tyro3 gene, which resides within the anx critical interval, as contributing to the severity of anx phenotypes. Our observation that, like Tyro3−/− mice, anx/anx mice exhibit abnormal secondary platelet aggregation suggested that the C19T Tyro3 variant might have functional consequences. Tyro3 is expressed in the hypothalamus and other brain regions affected by the anx mutation, and its mRNA localization appeared abnormal in anx/anx brains by postnatal day 19 (P19. The presence of wild-type Tyro3 transgenes, but not an R7W-Tyro3 transgene, doubled the weight and lifespans of anx/anx mice and near-normal numbers of hypothalamic Npy-expressing neurons were present in Tyro3-transgenic anx/anx mice at P19. Although no differences in R7W-Tyro3 signal sequence function or protein localization were discernible in vitro, distribution of R7W-Tyro3 protein differed from that of Tyro3 protein in the cerebellum of transgenic wild-type mice. Thus, R7W-Tyro3 protein localization deficits are only detectable in vivo. Further analyses revealed that the C19T Tyro3 mutation is present in a few other mouse strains, and hence is not the causative anx mutation, but rather an anx modifier. Our work shows that Tyro3 has prosurvival roles in the appetite regulatory circuitry and could also provide useful insights towards the development of interventions

  8. Cetuximab Resistance in Squamous Carcinomas of the Upper Aerodigestive Tract Is Driven by Receptor Tyrosine Kinase Plasticity

    DEFF Research Database (Denmark)

    Kjær, Ida; Lindsted, Trine; Fröhlich, Camilla

    2016-01-01

    Squamous cell carcinomas (SCC) arising in upper parts of the aero-digestive tract (UAT) are among the leading causes of death worldwide. The epidermal growth factor receptor (EGFR) has been found to play an essential role in driving the malignancy of SCCUAT, but despite this, clinical results using...... by continuous selective pressure in vitro and in vivo. Our results show that resistant clones maintain partial dependency on EGFR and that RTK plasticity mediated by HER3 and IGF1R plays an essential role. A multi-target mAb mixture against EGFR, HER3, and IGF1R was able to overcome cetuximab resistance...

  9. Crystal Structure of the Agrin-Responsive Immunoglobulin-like Domains 1 and 2 of the Receptor Tyrosine Kinase MuSK

    Energy Technology Data Exchange (ETDEWEB)

    Stiegler,A.; Burden, S.; Hubbard, S.

    2006-01-01

    Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed exclusively in skeletal muscle, where it is required for formation of the neuromuscular junction. MuSK is activated by agrin, a neuron-derived heparan sulfate proteoglycan. Here, we report the crystal structure of the agrin-responsive first and second immunoglobulin-like domains (Ig1 and Ig2) of the MuSK ectodomain at 2.2 {angstrom} resolution. The structure reveals that MuSK Ig1 and Ig2 are Ig-like domains of the I-set subfamily, which are configured in a linear, semi-rigid arrangement. In addition to the canonical internal disulfide bridge, Ig1 contains a second, solvent-exposed disulfide bridge, which our biochemical data indicate is critical for proper folding of Ig1 and processing of MuSK. Two Ig1-2 molecules form a non-crystallographic dimer that is mediated by a unique hydrophobic patch on the surface of Ig1. Biochemical analyses of MuSK mutants introduced into MuSK{sup -/-} myotubes demonstrate that residues in this hydrophobic patch are critical for agrin-induced MuSK activation.

  10. Fibroblast growth factor receptor 3 interacts with and activates TGFβ-activated kinase 1 tyrosine phosphorylation and NFκB signaling in multiple myeloma and bladder cancer.

    Directory of Open Access Journals (Sweden)

    Lisa Salazar

    Full Text Available Cancer is a major public health problem worldwide. In the United States alone, 1 in 4 deaths is due to cancer and for 2013 a total of 1,660,290 new cancer cases and 580,350 cancer-related deaths are projected. Comprehensive profiling of multiple cancer genomes has revealed a highly complex genetic landscape in which a large number of altered genes, varying from tumor to tumor, impact core biological pathways and processes. This has implications for therapeutic targeting of signaling networks in the development of treatments for specific cancers. The NFκB transcription factor is constitutively active in a number of hematologic and solid tumors, and many signaling pathways implicated in cancer are likely connected to NFκB activation. A critical mediator of NFκB activity is TGFβ-activated kinase 1 (TAK1. Here, we identify TAK1 as a novel interacting protein and target of fibroblast growth factor receptor 3 (FGFR3 tyrosine kinase activity. We further demonstrate that activating mutations in FGFR3 associated with both multiple myeloma and bladder cancer can modulate expression of genes that regulate NFκB signaling, and promote both NFκB transcriptional activity and cell adhesion in a manner dependent on TAK1 expression in both cancer cell types. Our findings suggest TAK1 as a potential therapeutic target for FGFR3-associated cancers, and other malignancies in which TAK1 contributes to constitutive NFκB activation.

  11. Development of a 3D Tissue Culture-Based High-Content Screening Platform That Uses Phenotypic Profiling to Discriminate Selective Inhibitors of Receptor Tyrosine Kinases.

    Science.gov (United States)

    Booij, Tijmen H; Klop, Maarten J D; Yan, Kuan; Szántai-Kis, Csaba; Szokol, Balint; Orfi, Laszlo; van de Water, Bob; Keri, Gyorgy; Price, Leo S

    2016-10-01

    3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared with 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses invasive human prostate cancer cells cultured in 3D in standard 384-well assay plates to study the activity of potential therapeutic small molecules and antibody biologics. Image analysis tools were developed to process 3D image data to measure over 800 phenotypic parameters. Multiparametric analysis was used to evaluate the effect of compounds on tissue morphology. We applied this screening platform to measure the activity and selectivity of inhibitors of the c-Met and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases in 3D cultured prostate carcinoma cells. c-Met and EGFR activity was quantified based on the phenotypic profiles induced by their respective ligands, hepatocyte growth factor and EGF. The screening method was applied to a novel collection of 80 putative inhibitors of c-Met and EGFR. Compounds were identified that induced phenotypic profiles indicative of selective inhibition of c-Met, EGFR, or bispecific inhibition of both targets. In conclusion, we describe a fully scalable high-content screening platform that uses phenotypic profiling to discriminate selective and nonselective (off-target) inhibitors in a physiologically relevant 3D cell culture setting. © 2016 Society for Laboratory Automation and Screening.

  12. Application of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor as the First-line Therapy in Patients with Advanced Non-small Cell Lung Cancer

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    Chunsun LI

    2010-01-01

    Full Text Available Background and objective Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI has been widely used as the second- and third-line therapy in patients with advanced non-small cell lung cancer (NSCLC. However, its effect in the first-line treatment is unclear. The aim of this study was to evaluate the efficacy and safety of EGFRTKI as first-line therapy. Methods The clinical characteristics, responses rate, disease control rate and overall survival were retrospectively analyzed in 77 chemonaive patients with advanced NSCLC. All of the patients received oral gefitinib (250 mg/d or erlotinib (150 mg/d until disease progression or unacceptable toxicity occurrence. Results The overall response rate was 33.8% and the disease control rate was 68.8%. The median progression-free survival and the median survival time were 6.0 months and 8.9 months, respectively. One-year survival rate was 61.4%. Responses correlated significantly with histology, PS score, smoking history, skin rash, EGFR mutations and serum CEA. Histology and skin rash were the independent predictors of survival. Common toxicities were skin rash and mild diarrhea. EGFR-TKI could improve the clinical symptoms and the quality of life. Conclusion EGFR-TKI is effective and well tolerated as first-line therapy in patients with advanced NSCLC.

  13. Detection of K-ras Mutations in Predicting Efficacy of Epidermal Growth Factor Receptor Tyrosine Kinase (EGFR-TK Inhibitor in Patients with Metastatic Colorectal Cancer.

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    Ze Li

    Full Text Available Epidermal growth factor receptor tyrosine kinase (EGFR-TK inhibitors are useful in treating different advanced human cancers; however, their clinical efficacy varies. This study detected K-ras mutations to predict the efficacy of EGFR-TK inhibitor cetuximab treatment on Chinese patients with metastatic colorectal cancer (mCRC. A total of 87 patients with metastatic colorectal cancer were treated with cetuximab for 2-16 months, in combination with chemotherapy between August 2008 and July 2012, and tissue samples were used to detect K-ras mutations. The data showed that K-ras mutation occurred in 27/87 (31%. The objective response rates and disease control rate in K-ras wild type and mutant patients were 42% (25/60 versus 11% (3/27 (p<0.05 and 60% (36/60 versus 26% (7/27 (p<0.05, respectively. Patients with the wild-type K-ras had significantly higher median survival times and progression-free survival, than patients with mutated K-ras (21 months versus 17 months, p=0.017; 10 months versus 6 months, p=0.6. These findings suggest that a high frequency of K-ras mutations occurs in Chinese mCRC patients and that K-ras mutation is required to select patients for eligibility for cetuximab therapy. Further prospective studies using a large sample size are needed to confirm these preliminary findings.

  14. Kinome-wide shRNA Screen Identifies the Receptor Tyrosine Kinase AXL as a Key Regulator for Mesenchymal Glioblastoma Stem-like Cells

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    Peng Cheng

    2015-05-01

    Full Text Available Glioblastoma is a highly lethal cancer for which novel therapeutics are urgently needed. Two distinct subtypes of glioblastoma stem-like cells (GSCs were recently identified: mesenchymal (MES and proneural (PN. To identify mechanisms to target the more aggressive MES GSCs, we combined transcriptomic expression analysis and kinome-wide short hairpin RNA screening of MES and PN GSCs. In comparison to PN GSCs, we found significant upregulation and phosphorylation of the receptor tyrosine kinase AXL in MES GSCs. Knockdown of AXL significantly decreased MES GSC self-renewal capacity in vitro and inhibited the growth of glioblastoma patient-derived xenografts. Moreover, inhibition of AXL with shRNA or pharmacologic inhibitors also increased cell death significantly more in MES GSCs. Clinically, AXL expression was elevated in the MES GBM subtype and significantly correlated with poor prognosis in multiple cancers. In conclusion, we identified AXL as a potential molecular target for novel approaches to treat glioblastoma and other solid cancers.

  15. Extrinsic Protein Tyrosine Phosphatase Non-Receptor 22 Signals Contribute to CD8 T Cell Exhaustion and Promote Persistence of Chronic Lymphocytic Choriomeningitis Virus Infection

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    Tatiana Jofra

    2017-07-01

    Full Text Available A genetic variant of the protein tyrosine phosphatase non-receptor 22 (PTPN22 is associated with a wide range of autoimmune diseases; however, the reasons behind its prevalence in the general population remain not completely understood. Recent evidence highlights an important role of autoimmune susceptibility genetic variants in conferring resistance against certain pathogens. In this study, we examined the role of PTPN22 in persistent infection in mice lacking PTPN22 infected with lymphocytic choriomeningitis virus clone 13. We found that lack of PTPN22 in mice resulted in viral clearance 30 days after infection, which was reflected in their reduced weight loss and overall improved health. PTPN22−/− mice exhibited enhanced virus-specific CD8 and CD4 T cell numbers and functionality and reduced exhausted phenotype. Moreover, mixed bone marrow chimera studies demonstrated no differences in virus-specific CD8 T cell accumulation and function between the PTPN22+/+ and PTPN22−/− compartments, showing that the effects of PTPN22 on CD8 T cells are T cell-extrinsic. Together, these findings identify a CD8 T cell-extrinsic role for PTPN22 in weakening early CD8 T cell responses to collectively promote persistence of a chronic viral infection.

  16. Extrinsic Protein Tyrosine Phosphatase Non-Receptor 22 Signals Contribute to CD8 T Cell Exhaustion and Promote Persistence of Chronic Lymphocytic Choriomeningitis Virus Infection.

    Science.gov (United States)

    Jofra, Tatiana; Galvani, Giuseppe; Kuka, Mirela; Di Fonte, Roberta; Mfarrej, Bechara G; Iannacone, Matteo; Salek-Ardakani, Shahram; Battaglia, Manuela; Fousteri, Georgia

    2017-01-01

    A genetic variant of the protein tyrosine phosphatase non-receptor 22 (PTPN22) is associated with a wide range of autoimmune diseases; however, the reasons behind its prevalence in the general population remain not completely understood. Recent evidence highlights an important role of autoimmune susceptibility genetic variants in conferring resistance against certain pathogens. In this study, we examined the role of PTPN22 in persistent infection in mice lacking PTPN22 infected with lymphocytic choriomeningitis virus clone 13. We found that lack of PTPN22 in mice resulted in viral clearance 30 days after infection, which was reflected in their reduced weight loss and overall improved health. PTPN22(-/-) mice exhibited enhanced virus-specific CD8 and CD4 T cell numbers and functionality and reduced exhausted phenotype. Moreover, mixed bone marrow chimera studies demonstrated no differences in virus-specific CD8 T cell accumulation and function between the PTPN22(+/+) and PTPN22(-/-) compartments, showing that the effects of PTPN22 on CD8 T cells are T cell-extrinsic. Together, these findings identify a CD8 T cell-extrinsic role for PTPN22 in weakening early CD8 T cell responses to collectively promote persistence of a chronic viral infection.

  17. Novel frameshift and splice site mutations in the neurotrophic tyrosine kinase receptor type 1 gene (NTRK1) associated with hereditary sensory neuropathy type IV.

    Science.gov (United States)

    Verpoorten, Nathalie; Claeys, Kristl G; Deprez, Liesbet; Jacobs, An; Van Gerwen, Veerle; Lagae, Lieven; Arts, Willem Frans; De Meirleir, Linda; Keymolen, Kathelijn; Ceuterick-de Groote, Chantal; De Jonghe, Peter; Timmerman, Vincent; Nelis, Eva

    2006-01-01

    Congenital insensitivity to pain with anhidrosis or hereditary sensory and autonomic neuropathy type IV (HSAN IV) is the first human genetic disorder implicated in the neurotrophin signal transduction pathway. HSAN IV is characterized by absence of reaction to noxious stimuli, recurrent episodes of fever, anhidrosis, self-mutilating behavior and often mental retardation. Mutations in the neurotrophic tyrosine kinase, receptor, type 1 (NTRK1) are associated with this disorder. Here we report four homozygous mutations, two frameshift (p.Gln626fsX6 and p.Gly181fsX58), one missense (p.Arg761Trp) and one splice site (c.359+5G>T) mutation in four HSAN IV patients. The splice site mutation caused skipping of exons 2 and 3 in patient's mRNA resulting in an in-frame deletion of the second leucine-rich motif. NTRK1 mutations are only rarely reported in the European population. This report extends the spectrum of NTRK1 mutations observed in patients diagnosed with HSAN IV.

  18. Fibroblast Growth Factor Receptor 3 Interacts with and Activates TGFβ-Activated Kinase 1 Tyrosine Phosphorylation and NFκB Signaling in Multiple Myeloma and Bladder Cancer

    Science.gov (United States)

    Krejci, Pavel; Meyer, April N.; Casale, Malcolm; Hallowell, Matthew; Wilcox, William R.; Donoghue, Daniel J.; Thompson, Leslie Michels

    2014-01-01

    Cancer is a major public health problem worldwide. In the United States alone, 1 in 4 deaths is due to cancer and for 2013 a total of 1,660,290 new cancer cases and 580,350 cancer-related deaths are projected. Comprehensive profiling of multiple cancer genomes has revealed a highly complex genetic landscape in which a large number of altered genes, varying from tumor to tumor, impact core biological pathways and processes. This has implications for therapeutic targeting of signaling networks in the development of treatments for specific cancers. The NFκB transcription factor is constitutively active in a number of hematologic and solid tumors, and many signaling pathways implicated in cancer are likely connected to NFκB activation. A critical mediator of NFκB activity is TGFβ-activated kinase 1 (TAK1). Here, we identify TAK1 as a novel interacting protein and target of fibroblast growth factor receptor 3 (FGFR3) tyrosine kinase activity. We further demonstrate that activating mutations in FGFR3 associated with both multiple myeloma and bladder cancer can modulate expression of genes that regulate NFκB signaling, and promote both NFκB transcriptional activity and cell adhesion in a manner dependent on TAK1 expression in both cancer cell types. Our findings suggest TAK1 as a potential therapeutic target for FGFR3-associated cancers, and other malignancies in which TAK1 contributes to constitutive NFκB activation. PMID:24466111

  19. Breakpoint cluster region-c-abl oncogene 1, non-receptor tyrosine kinase signaling: Current patterns of the versatile regulator revisited

    Directory of Open Access Journals (Sweden)

    Aamir Rana

    2013-01-01

    Full Text Available Increasing sophisticated information suggests that cancer cells express constitutively active oncogenic kinases such as breakpoint cluster region- c-abl oncogene 1, non-receptor tyrosine kinase (BCR-ABL1 that promote carcinogenesis independent of extrinsic growth factors. It is a well-established fact that through the aberrant activation of BCR-ABL1 signal transduction cascade, the perception of cellular growth signals becomes disconnected from the processes promoting cell growth, and this underlies the pathophysiology of leukemia. In this particular review we discuss the oncogenes and tumor suppressors comprising the regulatory network upstream and downstream of BCR-ABL1 and dismantle how derailed BCR-ABL1 signaling provides cell a selective growth advantage. Besides, we discuss why activation of BCR-ABL1, as an outcome of distinct oncogenic events, results in miscellaneous clinical outcomes, and how the intricacy of the BCR-ABL1 signaling network might dictate therapeutic approaches. In this review, our current comprehension of BCR-ABL1 signaling will be summarized.

  20. Tyrosine kinase inhibitors directed against the vascular endothelial growth factor receptor (VEGFR) have distinct cutaneous toxicity profiles: a meta-analysis and review of the literature.

    Science.gov (United States)

    Massey, Paul R; Okman, Jonathan S; Wilkerson, Julia; Cowen, Edward W

    2015-06-01

    Inhibition of the vascular endothelial growth factor receptor (VEGFR) with tyrosine kinase inhibitors (TKIs) is associated with cutaneous adverse effects that increase patient morbidity. Our objective was to examine the skin toxicity profile of anti-VEGFR TKIs and determine the changing incidence in clinical trials. PubMed was queried for phase II or III trials of anti-VEGFR TKIs between 2000 and 2013 involving ≥50 patients. Adverse events were abstracted, with results presented in both fixed and random effects models. Odds ratios (OR) and 95 % confidence intervals (CIs) were estimated for studies with at least two arms. Across 82 included studies, all grades rash (OR, 2.68; 95 % CI, 2.45-2.94), hand-foot skin reaction (HFSR) (OR, 2.70; 95 % CI, 2.43-3.00), and pruritus (OR, 1.25; 95 % CI, 1.12-1.39) were associated with anti-VEGFR TKIs. Vandetanib had the highest incidence of rash (41 %), while sorafenib was most commonly associated with HFSR (37 %) and pruritus (14 %). The incidence of HFSR from 2000 to 2013 showed an upward trend (r (2) = 0.042, p = 0.10) and in sunitinib therapy increased significantly (r (2) = 0.237, p = 0.04). The incidence of HFSR, rash, and pruritus varies considerably by drug. Our data suggest a continued need to address skin toxicities and improve reporting strategies.

  1. Efficacy of chemotherapy in epidermal growth factor receptor (EGFR) mutated metastatic pulmonary adenocarcinoma patients who had acquired resistance to first-line EGFR tyrosine kinase inhibitor (TKI).

    Science.gov (United States)

    Tseng, Yen-Han; Hung, Hsiu-Ying; Sung, Yi-Chen; Tseng, Yen-Chiang; Lee, Yu-Chin; Whang-Peng, Jacqueline; Chen, Yuh-Min

    2016-01-01

    Salvage chemotherapy is frequently used when tumour epidermal growth factor receptor (EGFR) mutated patients experience disease progression with first-line EGFR-tyrosine kinase inhibitor (TKI) treatment. However, the efficacy of salvage chemotherapy is still unknown. We retrospectively reviewed the chart records of our pulmonary adenocarcinoma patients between 2010 and 2013. Five hundred and six of the 1240 stage IV adenocarcinoma patients had an EGFR mutation and 338 received first-line EGFR-TKI treatment. In all, 169 patients in this group received salvage chemotherapy after failure of EGFR-TKI, and 102 patients were eligible for this study. The chemotherapy response rate of these 102 patients was 24.5%, with a median progression-free survival (PFS) of 4.5?months, and median survival time was 14.6?months. Patients who received pemetrexed-based chemotherapy had longer PFS and overall survival (OS), although the extent was statistically insignificant. Progression-free survival and OS were longer for patients who received combination chemotherapy than single-agent chemotherapy. Pemetrexed-based combination chemotherapy is preferred before a more efficient treatment strategy is found.

  2. A Novel Occulta-Type Spina Bifida Mediated by Murine Double Heterozygotes EphA2 and EphA4 Receptor Tyrosine Kinases

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    Nor Linda Abdullah

    2017-12-01

    Full Text Available Members of the Eph receptor tyrosine kinase have previously been implicated in cranial neural tube development. Failure of neural tube closure leads to the devastating conditions known as anencephaly and spina bifida. EphA2 and EphA4 are expressed at the tips of the closing spinal neural folds prior and during neural tube closure. We investigated the possible role of murine EphA2 and EphA4 during the last step of primary neural tube closure, which is adhesion and fusion. The individual mouse knockouts of EphA2 and EphA4 per se do not exhibit neural tube defects (NTDs. The embryos generated by the crossing of double heterozygotes Epha2tm1Jrui/+Epha4rb-2J/+ displayed NTDs with a wide degree of severity including close exencephaly and close spina bifida (spina bifida occulta. Interestingly, mutants displaying NTDs had skin covering the underlying lesion. The tissue sections revealed the elevated neural folds had not adhered and fused. The phenotypes seen in Epha2tm1Jrui/+Epha4rb-2J/+ double heterozygous embryos suggest both genes play a compensatory role with each other in the adhesion and fusion of the neural tube. In this study, there exists a >50% penetrance of NTDs in the mouse mutants, which genetically have a single allele each of EphA2 and EphA4 absent.

  3. A Novel Occulta-Type Spina Bifida Mediated by Murine Double HeterozygotesEphA2andEphA4Receptor Tyrosine Kinases.

    Science.gov (United States)

    Abdullah, Nor Linda; Mohd-Zin, Siti W; Ahmad-Annuar, Azlina; Abdul-Aziz, Noraishah M

    2017-01-01

    Members of the Eph receptor tyrosine kinase have previously been implicated in cranial neural tube development. Failure of neural tube closure leads to the devastating conditions known as anencephaly and spina bifida. EphA2 and EphA4 are expressed at the tips of the closing spinal neural folds prior and during neural tube closure. We investigated the possible role of murine EphA2 and EphA4 during the last step of primary neural tube closure, which is adhesion and fusion. The individual mouse knockouts of EphA2 and EphA4 per se do not exhibit neural tube defects (NTDs). The embryos generated by the crossing of double heterozygotes Epha2 tm1Jrui/+ Epha4 rb-2J/+ displayed NTDs with a wide degree of severity including close exencephaly and close spina bifida (spina bifida occulta). Interestingly, mutants displaying NTDs had skin covering the underlying lesion. The tissue sections revealed the elevated neural folds had not adhered and fused. The phenotypes seen in Epha2 tm1Jrui/+ Epha4 rb-2J/+ double heterozygous embryos suggest both genes play a compensatory role with each other in the adhesion and fusion of the neural tube. In this study, there exists a >50% penetrance of NTDs in the mouse mutants, which genetically have a single allele each of EphA2 and EphA4 absent.

  4. Deletion of protein tyrosine phosphatase, non-receptor type 4 (PTPN4) in twins with a Rett syndrome-like phenotype

    Science.gov (United States)

    Williamson, Sarah L; Ellaway, Carolyn J; Peters, Greg B; Pelka, Gregory J; Tam, Patrick PL; Christodoulou, John

    2015-01-01

    Rett syndrome (RTT), a neurodevelopmental disorder that predominantly affects females, is primarily caused by variants in MECP2. Variants in other genes such as CDKL5 and FOXG1 are usually associated with individuals who manifest distinct phenotypes that may overlap with RTT. Individuals with phenotypes suggestive of RTT are typically screened for variants in MECP2 and then subsequently the other genes dependent on the specific phenotype. Even with this screening strategy, there are individuals in whom no causative variant can be identified, suggesting that there are other novel genes that contribute to the RTT phenotype. Here we report a de novo deletion of protein tyrosine phosphatase, non-receptor type 4 (PTPN4) in identical twins with a RTT-like phenotype. We also demonstrate the reduced expression of Ptpn4 in a Mecp2 null mouse model of RTT, as well as the activation of the PTPN4 promoter by MeCP2. Our findings suggest that PTPN4 should be considered for addition to the growing list of genes that warrant screening in individuals with a RTT-like phenotype. PMID:25424712

  5. Mutations of the EPHB6 receptor tyrosine kinase induce a pro-metastatic phenotype in non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Etmar Bulk

    Full Text Available Alterations of Eph receptor tyrosine kinases are frequent events in human cancers. Genetic variations of EPHB6 have been described but the functional outcome of these alterations is unknown. The current study was conducted to screen for the occurrence and to identify functional consequences of EPHB6 mutations in non-small cell lung cancer. Here, we sequenced the entire coding region of EPHB6 in 80 non-small cell lung cancer patients and 3 tumor cell lines. Three potentially relevant mutations were identified in primary patient samples of NSCLC patients (3.8%. Two point mutations led to instable proteins. An in frame deletion mutation (del915-917 showed enhanced migration and accelerated wound healing in vitro. Furthermore, the del915-917 mutation increased the metastatic capability of NSCLC cells in an in vivo mouse model. Our results suggest that EPHB6 mutations promote metastasis in a subset of patients with non-small cell lung cancer.

  6. Evaluation of the safety and pharmacokinetics of the multi-targeted receptor tyrosine kinase inhibitor sunitinib during embryo-fetal development in rats and rabbits.

    Science.gov (United States)

    Patyna, S; Haznedar, J; Morris, D; Freshwater, K; Peng, G; Sukbuntherng, J; Chmielewski, G; Matsumoto, D

    2009-06-01

    Angiogenesis plays a key role in embryo-fetal development and, based on nonclinical safety data, the majority of vascular endothelial growth factor (VEGF)-targeted antiangiogenic agents used in cancer therapy are not recommended during pregnancy. We investigated the effects of sunitinib (an oral inhibitor of multiple receptor tyrosine kinases [RTKs] including VEGF-receptors) on embryo-fetal development. Presumed-pregnant Sprague-Dawley rats and New Zealand White rabbits received repeated daily oral doses of sunitinib (0-30 mg/kg/day), during the major period of organogenesis. Clinical/physical examinations were performed throughout the gestation phase, and blood samples were collected to determine systemic exposure. Necropsy (including uterine examination) was performed on all animals and fetal morphology was examined. The no-observed-adverse-effect level was 1-5 mg/kg/day for maternal toxicity and 3 mg/kg/day for developmental toxicity in rats; 1 and 0.5 mg/kg/day, respectively, in rabbits. Embryo-fetal toxicity included decreases in the number of live fetuses and increases in the numbers of resorptions and post-implantation/complete litter losses; these were observed at doses of > or =5 mg/kg/day in rats and 5 mg/kg/day in rabbits. Malformations included fetal skeletal malformations (generally thoracic/lumbar vertebral alterations) in rats and cleft lip/palate in rabbits. These developmental effects were observed at approximately 5.5- (rats) and approximately 0.3-times (rabbits) the human systemic exposure at the approved sunitinib dose (50 mg/day). Similar effects have been reported with the prototype monoclonal antibody bevacizumab. As is typically observed for potent inhibitors of RTKs involved in angiogenesis, sunitinib was associated with embryo-fetal developmental toxicity in rats and rabbits at clinically relevant dose levels. 2009 Wiley-Liss, Inc.

  7. Thymus histology and concomitant autoimmune diseases in Japanese patients with muscle-specific receptor tyrosine kinase-antibody-positive myasthenia gravis.

    Science.gov (United States)

    Nakata, R; Motomura, M; Masuda, T; Shiraishi, H; Tokuda, M; Fukuda, T; Ando, T; Yoshimura, T; Tsujihata, M; Kawakami, A

    2013-09-01

    The differences in the characteristics of thymus histology, coexisting autoimmune diseases and related autoantibodies between anti-muscle-specific receptor tyrosine kinase (MuSK)-antibody (Ab)-positive myasthenia gravis (MG) patients, and anti-acetylcholine receptor (AChR)-Ab-positive MG patients are not clearly defined. The types of thymus histology, coexisting autoimmune diseases and associated Abs in 83 MuSK-Ab-positive patients nationwide were investigated and were compared with those in AChR-Ab-positive patients followed at our institute (n = 83). As for the autoantibodies associated with thymoma, titin Abs were measured. Thymoma was not present in any of the MuSK-Ab-positive patients but presented in 21 patients (25.3%) amongst the AChR-Ab-positive patients. Titin Abs were absent in MuSK-Ab-positive patients but positive in 25 (30.1%) of the AChR-Ab-positive patients. Concomitant autoimmune diseases were present in eight MuSK-Ab-positive patients (9.6%) amongst whom Hashimoto's thyroiditis and rheumatoid arthritis predominated, whereas 22 AChR-Ab-positive patients (26.5%) had one or more concomitant autoimmune diseases of which Graves' disease predominated. Differences in frequency of thymoma and thymic hyperplasia, coexisting autoimmune diseases and autoantibody positivity between MuSK-Ab-positive and AChR-Ab-positive MG were indicated, suggesting that, in contrast with AChR-Ab-positive MG, thymus does not seem to be involved in the pathogenic mechanisms of MuSK-Ab-positive MG. © 2013 The Author(s) European Journal of Neurology © 2013 EFNS.

  8. Histological transformation of adenocarcinoma to small cell carcinoma lung as a rare mechanism of resistance to epidermal growth factor receptor-tyrosine kinase inhibitors: Report of a case with review of literature.

    Science.gov (United States)

    Hui, Monalisa; Uppin, Shantveer G; Stalin, Bala Joseph; Sadashivudu, G

    2018-01-01

    A subset of non-small cell lung carcinoma (NSCC) harbor active mutations of epidermal growth factor receptor (EGFR). In these, EGFR tyrosine kinase inhibitors (EGFR-TKIs) are recommended as the first-line treatment. Though drug resistance is inevitable, histological transformation to small cell lung carcinoma (SCLC) is a rare mechanism for acquired resistance. Here we report one such rare case of histological transformation of pulmonary adenocarcinoma to small cell lung carcinoma in 46 year old male treated with Gefitinib.

  9. Glycomic analysis of gastric carcinoma cells discloses glycans as modulators of RON receptor tyrosine kinase activation in cancer

    DEFF Research Database (Denmark)

    Mereiter, Stefan; Magalhães, Ana; Adamczyk, Barbara

    2016-01-01

    gastric carcinoma cells transfected with the sialyltransferase ST3GAL4 were established as a model overexpressing sialylated terminal glycans. We have evaluated at the structural level the glycome and the sialoproteome of this gastric cancer cell line applying liquid chromatography and mass spectrometry...... known to be key players in malignancy. Further analysis of RON confirmed its modification with SLe(X) and the concomitant activation. SLe(X) and RON co-expression was validated in gastric tumors. CONCLUSION: The overexpression of ST3GAL4 interferes with the overall glycophenotype of cancer cells...... affecting a multitude of key proteins involved in malignancy. Aberrant glycosylation of the RON receptor was shown as an alternative mechanism of oncogenic activation. GENERAL SIGNIFICANCE: This study provides novel targets and points to an integrative tumor glycomic/proteomic-profiling for gastric cancer...

  10. Tyrosylprotein sulfotransferase-1 and tyrosine sulfation of chemokine receptor 4 are induced by Epstein-Barr virus encoded latent membrane protein 1 and associated with the metastatic potential of human nasopharyngeal carcinoma.

    Directory of Open Access Journals (Sweden)

    Juan Xu

    Full Text Available The latent membrane protein 1 (LMP1, which is encoded by the Epstein-Barr virus (EBV, is an important oncogenic protein that is closely related to carcinogenesis and metastasis of nasopharyngeal carcinoma (NPC, a prevalent cancer in China. We previously reported that the expression of the functional chemokine receptor CXCR4 is associated with human NPC metastasis. In this study, we show that LMP1 induces tyrosine sulfation of CXCR4 through tyrosylprotein sulfotransferase-1 (TPST-1, an enzyme that is responsible for catalysis of tyrosine sulfation in vivo, which is likely to contribute to the highly metastatic character of NPC. LMP1 could induce tyrosine sulfation of CXCR4 and its associated cell motility and invasiveness in a NPC cell culture model. In contrast, the expression of TPST-1 small interfering RNA reversed LMP1-induced tyrosine sulfation of CXCR4. LMP1 conveys signals through the epidermal growth factor receptor (EGFR pathway, and EGFR-targeted siRNA inhibited the induction of TPST-1 by LMP1. We used a ChIP assay to show that EGFR could bind to the TPST-1 promoter in vivo under the control of LMP1. A reporter gene assay indicated that the activity of the TPST-1 promoter could be suppressed by deleting the binding site between EGFR and TPST-1. Finally, in human NPC tissues, the expression of TPST-1 and LMP1 was directly correlated and clinically, the expression of TPST-1 was associated with metastasis. These results suggest the up-regulation of TPST-1 and tyrosine sulfation of CXCR4 by LMP1 might be a potential mechanism contributing to NPC metastasis.

  11. Tyrosine phosphatases such as SHP-2 act in a balance with Src-family kinases in stabilization of postsynaptic clusters of acetylcholine receptors

    Directory of Open Access Journals (Sweden)

    Rüegg Markus A

    2007-07-01

    Full Text Available Abstract Background Development of neural networks requires that synapses are formed, eliminated and stabilized. At the neuromuscular junction (NMJ, agrin/MuSK signaling, by triggering downstream pathways, causes clustering and phosphorylation of postsynaptic acetylcholine receptors (AChRs. Postnatally, AChR aggregates are stabilized by molecular pathways that are poorly characterized. Gain or loss of function of Src-family kinases (SFKs disassembles AChR clusters at adult NMJs in vivo, whereas AChR aggregates disperse rapidly upon withdrawal of agrin from cultured src-/-;fyn-/- myotubes. This suggests that a balance between protein tyrosine phosphatases (PTPs and protein tyrosine kinases (PTKs such as those of the Src-family may be essential in stabilizing clusters of AChRs. Results We have analyzed the role of PTPs in maintenance of AChR aggregates, by adding and then withdrawing agrin from cultured myotubes in the presence of PTP or PTK inhibitors and quantitating remaining AChR clusters. In wild-type myotubes, blocking PTPs with pervanadate caused enhanced disassembly of AChR clusters after agrin withdrawal. When added at the time of agrin withdrawal, SFK inhibitors destabilized AChR aggregates but concomitant addition of pervanadate rescued cluster stability. Likewise in src-/-;fyn-/- myotubes, in which agrin-induced AChR clusters form normally but rapidly disintegrate after agrin withdrawal, pervanadate addition stabilized AChR clusters. The PTP SHP-2, known to be enriched at the NMJ, associated and colocalized with MuSK, and agrin increased this interaction. Specific SHP-2 knockdown by RNA interference reduced the stability of AChR clusters in wild-type myotubes. Similarly, knockdown of SHP-2 in adult mouse soleus muscle by electroporation of RNA interference constructs caused disassembly of pretzel-shaped AChR-rich areas in vivo. Finally, we found that src-/-;fyn-/- myotubes contained elevated levels of SHP-2 protein. Conclusion Our data

  12. Receptor tyrosine kinase-like orphan receptor 1 (ROR-1): An emerging target for diagnosis and therapy of chronic lymphocytic leukemia.

    Science.gov (United States)

    Aghebati-Maleki, Leili; Shabani, Mahdi; Baradaran, Behzad; Motallebnezhad, Morteza; Majidi, Jafar; Yousefi, Mehdi

    2017-04-01

    Chronic lymphocytic leukemia (CLL) is characterized by reposition of malignant B cells in the blood, bone marrow, spleen and lymph nodes. It remains the most common leukemia in the Western world. Within the recent years, major breakthroughs have been made to prolong the survival and improve the health of patients. Despite these advances, CLL is still recognized as a disease without definitive cure. New treatment approaches, based on unique targets and novel drugs, are highly desired for CLL therapy. The Identification and subsequent targeting of molecules that are overexpressed uniquely in malignant cells not normal ones play critical roles in the success of anticancer therapeutic strategies. In this regard, ROR family proteins are known as a subgroup of protein kinases which have gained huge popularity in the scientific community for the diagnosis and treatment of different cancer types. ROR1 as an antigen exclusively expressed on the surface of tumor cells can be a target for immunotherapy. ROR-1 targeting using different approaches such as siRNA, tyrosine kinase inhibitors, cell therapy and antibody induces tumor growth suppression in cancer cells. In the current review, we aim to present an overview of the efforts and scientific achievements in targeting ROR family, particularly ROR-1, for the diagnosis and treatment of chronic lymphocytic leukemia (CLL). Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  13. Effect of dioxins on regulation of tyrosine hydroxylase gene expression by aryl hydrocarbon receptor: a neurotoxicology study

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    Akahoshi Eiichi

    2009-06-01

    Full Text Available Abstract Background Dioxins and related compounds are suspected of causing neurological disruption. Epidemiological studies indicated that exposure to these compounds caused neurodevelopmental disturbances such as learning disability and attention deficit hyperactivity disorder, which are thought to be closely related to dopaminergic dysfunction. Although the molecular mechanism of their actions has not been fully investigated, a major participant in the process is aryl hydrocarbon receptor (AhR. This study focused on the effect of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD exposure on the regulation of TH, a rate-limiting enzyme of dopamine synthesis, gene expression by AhR. Methods N2a-Rβ cells were established by transfecting murine neuroblastoma Neuro2a with the rat AhR cDNA. TH expression induced by TCDD was assessed by RT-PCR and Western blotting. Participation of AhR in TCDD-induced TH gene expression was confirmed by suppressing AhR expression using the siRNA method. Catecholamines including dopamine were measured by high-performance liquid chromatography. A reporter gene assay was used to identify regulatory motifs in the promoter region of TH gene. Binding of AhR with the regulatory motif was confirmed by an electrophoretic mobility shift assay (EMSA. Results Induction of TH by TCDD through AhR activation was detected at mRNA and protein levels. Induced TH protein was functional and its expression increased dopamine synthesis. The reporter gene assay and EMSA indicated that AhR directly regulated TH gene expression. Regulatory sequence called aryl hydrocarbon receptor responsive element III (AHRE-III was identified upstream of the TH gene from -285 bp to -167 bp. Under TCDD exposure, an AhR complex was bound to AHRE-III as well as the xenobiotic response element (XRE, though AHRE-III was not identical to XRE, the conventional AhR-binding motif. Conclusion Our results suggest TCDD directly regulate the dopamine system by TH gene

  14. Ginsenoside-Rg{sub 1} induces angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a

    Energy Technology Data Exchange (ETDEWEB)

    Kwok, Hoi-Hin [Dr. Gilbert Hung Ginseng Laboratory, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Chan, Lai-Sheung [Department of Biology, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Poon, Po-Ying [Dr. Gilbert Hung Ginseng Laboratory, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Yue, Patrick Ying-Kit [Dr. Gilbert Hung Ginseng Laboratory, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Department of Biology, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Wong, Ricky Ngok-Shun, E-mail: rnswong@hkbu.edu.hk [Dr. Gilbert Hung Ginseng Laboratory, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China); Department of Biology, Faculty of Science, Hong Kong Baptist University, Hong Kong SAR (China)

    2015-09-15

    Therapeutic angiogenesis has been implicated in ischemic diseases and wound healing. Ginsenoside-Rg{sub 1} (Rg{sub 1}), one of the most abundant active components of ginseng, has been demonstrated as an angiogenesis-stimulating compound in different models. There is increasing evidence implicating microRNAs (miRNAs), a group of non-coding RNAs, as important regulators of angiogenesis, but the role of microRNAs in Rg{sub 1}-induced angiogenesis has not been fully explored. In this report, we found that stimulating endothelial cells with Rg{sub 1} could reduce miR-23a expression. In silico experiments predicted hepatocyte growth factor receptor (MET), a well-established mediator of angiogenesis, as the target of miR-23a. Transfection of the miR-23a precursor or inhibitor oligonucleotides validated the inverse relationship of miR-23a and MET expression. Luciferase reporter assays further confirmed the interaction between miR-23a and the MET mRNA 3′-UTR. Intriguingly, ginsenoside-Rg{sub 1} was found to increase MET protein expression in a time-dependent manner. We further demonstrated that ginsenoside-Rg{sub 1}-induced angiogenic activities were indeed mediated through the down-regulation of miR-23a and subsequent up-regulation of MET protein expression, as confirmed by gain- and loss-of-function angiogenic experiments. In summary, our results demonstrated that ginsenoside-Rg{sub 1} could induce angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a. This study has broadened our understanding of the non-genomic effects of ginsenoside-Rg{sub 1,} and provided molecular evidence that warrant further development of natural compound as novel angiogenesis-promoting therapy. - Highlights: • Therapeutic angiogenesis has been implicated in ischemic diseases and wound healing. • Ginsenoside-Rg{sub 1} (Rg{sub 1}) has been demonstrated as an angiogenesis-stimulating compound. • We found that Rg{sub 1} induces angiogenesis by

  15. Pigment Pattern Formation in the Guppy, Poecilia reticulata, Involves the Kita and Csf1ra Receptor Tyrosine Kinases

    Science.gov (United States)

    Kottler, Verena A.; Fadeev, Andrey; Weigel, Detlef; Dreyer, Christine

    2013-01-01

    Males of the guppy (Poecilia reticulata) vary tremendously in their ornamental patterns, which are thought to have evolved in response to a complex interplay between natural and sexual selection. Although the selection pressures acting on the color patterns of the guppy have been extensively studied, little is known about the genes that control their ontogeny. Over 50 years ago, two autosomal color loci, blue and golden, were described, both of which play a decisive role in the formation of the guppy color pattern. Orange pigmentation is absent in the skin of guppies with a lesion in blue, suggesting a defect in xanthophore development. In golden mutants, the development of the melanophore pattern during embryogenesis and after birth is affected. Here, we show that blue and golden correspond to guppy orthologs of colony-stimulating factor 1 receptor a (csf1ra; previously called fms) and kita. Most excitingly, we found that both genes are required for the development of the black ornaments of guppy males, which in the case of csf1ra might be mediated by xanthophore–melanophore interactions. Furthermore, we provide evidence that two temporally and genetically distinct melanophore populations contribute to the adult camouflage pattern expressed in both sexes: one early appearing and kita-dependent and the other late-developing and kita-independent. The identification of csf1ra and kita mutants provides the first molecular insights into pigment pattern formation in this important model species for ecological and evolutionary genetics. PMID:23666934

  16. [Experimental study of effect of epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 in combination with irinotecan].

    Science.gov (United States)

    Xu, Jian-ming; Li, Yue-min; Wang, Yan; Zhao, Chuan-hua; Yuan, Shou-jun; Yang, Wu-wei; Li, Zhi-qiang; Han, Yu; Azzariti, Amalia; Paradiso, Angelo

    2006-08-01

    To assess the optimal regimen and its mechanism of ZD1839 in combination with SN38, the active metabolite of irinotecan (CPT-11), in the colon cancer cell lines HT-29 and LoVo. Chou and Talalay method was used to analyze the combination effects of sequencing of ZD1839 and SN38. Western blotting and immunoprecipitation were used to determine the effects of ZD1839 and/or SN38 on their targeted enzymes and downstream markers. Apoptosis was assayed by analyzing histone-associated DNA fragment. Sequential SN38 followed by ZD1839 produced a synergistic effect. In contrast, SN38 following ZD1839 exhibited an antagonist effect. SN38 markedly inhibited topoisomerase I (Topo-I) activity. ZD1839 did not alter epidermal growth factor receptor (EGFR) expression, but resulted in a complete inhibition of EGFR phosphorylation. Sequential ZD1839 followed by SN38 did not show any enhanced inhibition effect on Topo-I activity, phosphorylation of EGFR and one of its downstream markers MAPK. However, simultaneous SN38 plus ZD1839, and sequential SN38 followed by ZD1839 administrations showed modest inhibition effect on EGFR's another downstream marker AKT. The combination schedules also showed prominent influence on cell cycle distribution. ZD1839 maintained SN38-induced DNA damage and apoptosis. Sequential SN38 followed by ZD1839 may be a favorable combination schedule.

  17. Receptor-Type Protein-Tyrosine Phosphatase ζ and Colony Stimulating Factor-1 Receptor in the Intestine: Cellular Expression and Cytokine- and Chemokine Responses by Interleukin-34 and Colony Stimulating Factor-1.

    Science.gov (United States)

    Zwicker, Stephanie; Bureik, Daniela; Bosma, Madeleen; Martinez, Gisele Lago; Almer, Sven; Boström, Elisabeth A

    2016-01-01

    Differential intestinal expression of the macrophage growth factors colony stimulating factor-1 (CSF-1), interleukin (IL)-34, and their shared CSF-1 receptor (CSF-1R) in inflammatory bowel disease (IBD) has been shown. Diverse expression between CSF-1 and IL-34, suggest that IL-34 may signal via an alternate receptor. Receptor-type protein-tyrosine phosphatase ζ (PTPRZ1, RPTP-ζ), an additional IL-34 receptor, was recently identified. Here, we aimed to assess PTPRZ1 expression in IBD and non-IBD intestinal biopsies. Further, we aimed to investigate cellular PTPRZ1 and CSF-1R expression, and cytokine- and chemokine responses by IL-34 and CSF-1. The expression of PTPRZ1 was higher in non-IBD colon compared to ileum. PTPRZ1 expression was not altered with inflammation in IBD, however, correlated to IL34, CSF1, and CSF1R. The expression patterns of PTPRZ1 and CSF-1R differed in peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and intestinal epithelial cell line. PBMCs and monocytes of the same donors responded differently to IL-34 and CSF-1 with altered expression of tumor-necrosis factor α (TNF-α), IL-1β, interferon γ (IFN-γ), IL-13, IL-8, and monocyte chemotactic protein-1 (MCP-1) levels. This study shows that PTPRZ1 was expressed in bowel tissue. Furthermore, CSF-1R protein was detected in an intestinal epithelial cell line and donor dependently in primary PBMCs, monocytes, and macrophages, and first hints also suggest an expression in these cells for PTPRZ1, which may mediate IL-34 and CSF-1 actions.

  18. 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.

  19. Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells

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    Minyong Kang

    2017-02-01

    Full Text Available Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1, chloroquine (CQ and 3-methyladenine (3-MA were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8 assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating

  20. Impact of epidermal growth factor receptor gene expression level on clinical outcomes in epidermal growth factor receptor mutant lung adenocarcinoma patients taking first-line epidermal growth factor receptor-tyrosine kinase inhibitors.

    Science.gov (United States)

    Chang, Huang-Chih; Chen, Yu-Mu; Tseng, Chia-Cheng; Huang, Kuo-Tung; Wang, Chin-Chou; Chen, Yung-Che; Lai, Chien-Hao; Fang, Wen-Feng; Kao, Hsu-Ching; Lin, Meng-Chih

    2017-03-01

    Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are first-choice treatments for advanced non-small-cell lung cancer patients harboring EGFR mutations. Although EGFR mutations are strongly predictive of patients' outcomes and their response to treatment with EGFR-TKIs, early failure of first-line therapy with EGFR-TKIs in patients with EGFR mutations is not rare. Besides several clinical factors influencing EGFR-TKI efficacies studied earlier such as the Eastern Cooperative Oncology Group performance status or uncommon mutation, we would like to see whether semi-quantify EGFR mutation gene expression calculated by 2(-ΔΔct) was a prognostic factor in EGFR-mutant non-small cell lung cancer patients receiving first-line EGFR-TKIs. This retrospective study reviews 926 lung cancer patients diagnosed from January 2011 to October 2013 at the Kaohsiung Chang Gung Memorial Hospital in Taiwan. Of 224 EGFR-mutant adenocarcinoma patients, 148 patients who had 2(-ΔΔct) data were included. The best cutoff values of 2(-ΔΔct) for in-frame deletions in exon 19 (19 deletion) and a position 858 substituted from leucine (L) to an arginine (R) in exon 21 (L858R) were determined using receiver operating characteristic curves. Patients were divided into high and low 2(-ΔΔct) expression based on the above cutoff level. The best cutoff point of 2(-ΔΔct) value of 19 deletion and L858R was 31.1 and 104.7, respectively. In all, 92 (62.1%) patients showed high 2(-ΔΔct) expression and 56 patients (37.9%) low 2(-ΔΔct) expression. The mean age was 65.6 years. Progression-free survival of 19 deletion mutant patients with low versus high expression level was 17.07 versus 12.04 months (P = 0.004), respectively. Progression-free survival of L858 mutant patients was 13.75 and 7.96 months (P = 0.008), respectively. EGFR-mutant lung adenocarcinoma patients with lower EGFR gene expression had longer progression-free survival duration without

  1. Cooperation of tyrosine kinase receptor TrkB and epidermal growth factor receptor signaling enhances migration and dispersal of lung tumor cells.

    Directory of Open Access Journals (Sweden)

    Rudolf Götz

    Full Text Available TrkB mediates the effects of brain-derived neurotrophic factor (BDNF in neuronal and nonnneuronal cells. Based on recent reports that TrkB can also be transactivated through epidermal growth-factor receptor (EGFR signaling and thus regulates migration of early neurons, we investigated the role of TrkB in migration of lung tumor cells. Early metastasis remains a major challenge in the clinical management of non-small cell lung cancer (NSCLC. TrkB receptor signaling is associated with metastasis and poor patient prognosis in NSCLC. Expression of this receptor in A549 cells and in another adenocarcinoma cell line, NCI-H441, promoted enhanced migratory capacity in wound healing assays in the presence of the TrkB ligand BDNF. Furthermore, TrkB expression in A549 cells potentiated the stimulatory effect of EGF in wound healing and in Boyden chamber migration experiments. Consistent with a potential loss of cell polarity upon TrkB expression, cell dispersal and de-clustering was induced in A549 cells independently of exogeneous BDNF. Morphological transformation involved extensive cytoskeletal changes, reduced E-cadherin expression and suppression of E-cadherin expression on the cell surface in TrkB expressing tumor cells. This function depended on MEK and Akt kinase activity but was independent of Src. These data indicate that TrkB expression in lung adenoma cells is an early step in tumor cell dissemination, and thus could represent a target for therapy development.

  2. Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines.

    Science.gov (United States)

    Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

    2016-07-01

    Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application.

  3. Improved plasma membrane expression of the trafficking defective P344R mutant of muscle, skeletal, receptor tyrosine kinase (MuSK) causing congenital myasthenic syndrome.

    Science.gov (United States)

    Milhem, Reham M; Al-Gazali, Lihadh; Ali, Bassam R

    2015-03-01

    Muscle, skeletal, receptor tyrosine kinase (MuSK) is a key organizer at the postsynaptic membrane and critical for proper development and maintenance of the neuromuscular junction. Mutations in MUSK result in congenital myasthenic syndrome (CMS). We hypothesized that the CMS-causing missense mutation (P344R), found within the cysteine-rich domain of the protein, will affect its conformational tertiary structure. Consequently, the protein will misfold, get retained in the endoplasmic reticulum (ER) and lose its biological function through degradation by the highly conserved ER associated degradation (ERAD) machinery. We report that P344R-MuSK mutant is trafficking-deficient when expressed at 37°C in HeLa, COS-7 and HEK293 cell lines. It colocalized with the ER marker calnexin in contrast to wild-type MuSK which localized to the plasma membrane. The N-glycosylation status of P344R-MuSK is that of an immature and not properly post-translationally modified protein. Inhibition of protein synthesis showed that the P344R mutant's half-life is shorter than wild-type MuSK protein. Proteasomal inhibition resulted in the stabilization of the mutant protein. The mutant protein is highly ubiquitinated compared to wild-type confirming targeting for proteasomal degradation. The mutant showed around 50% of its in vivo autophosphorylation activity. P344R-MuSK mutant's trafficking defect is correctable by culturing the expressing cells at 27°C. Moreover, chemical compounds namely 2.5% glycerol, 1% dimethyl sulfoxide, 10 μM thapsigargin and 1 μM curcumin improved the maturation and exit of the mutant protein from the ER. These findings open perspectives for potential therapeutic intervention for patients with CMS harboring the P344R-MuSK mutation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Phosphorylation of tyrosine receptor kinase B in the dorsal striatum and dorsal hippocampus is associated with response learning in a water plus maze.

    Science.gov (United States)

    Pahng, Amanda R; Colombo, Paul J

    2017-02-01

    The dorsal hippocampus and dorsal striatum have dissociable roles in learning and memory that are related to region-specific changes in proteins necessary for neuronal plasticity and memory formation. There is additional evidence that the hippocampus and striatum can interact during memory formation. Phosphorylation of tyrosine receptor kinase B is important for memory formation in the hippocampus, but whether or not it has a role in striatum-dependent learning, or in interactions between the hippocampus and striatum, has not been examined. In the present study, we tested the hypothesis that response training increases pTrkB in the dorsal striatum, but decreases pTrkB in dorsal hippocampus, due to an interaction between the systems during memory formation. Results show a significant decrease in pTrkB levels in the dorsal hippocampus of rats trained on the response task compared with swim controls. Response training did not increase pTrkB levels in the dorsal striatum. Positive correlations were found between response learning and the total area of cells expressing pTrkB in the dorsal striatum, while no correlations were found in swim controls. Our results partially support our hypothesis and indicate that response learning is associated with a decrease in hippocampal pTrkB, while phosphorylation of TrkB in the dorsal striatum remains constant. This indicates that suppression of hippocampal pTrkB during response learning may be involved in striatum-dependent memory formation. Additionally, our findings suggest that activation of TrkB in a sparse arrangement of cells may be associated with faster acquisition of a response task. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  5. Cytotoxic chemotherapy may overcome the development of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) therapy.

    Science.gov (United States)

    Kanda, Shintaro; Horinouchi, Hidehito; Fujiwara, Yutaka; Nokihara, Hiroshi; Yamamoto, Noboru; Sekine, Ikuo; Kunitoh, Hideo; Kubota, Kaoru; Tamura, Tomohide; Ohe, Yuichiro

    2015-09-01

    In the first-line treatment of non-small cell lung cancer (NSCLC) harboring EGFR mutations, epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) has been shown to yield a longer progression-free survival (PFS) rate than platinum-doublet chemotherapy; however, after the initial response, most patients develop resistance to the EGFR-TKIs. We hypothesized that the insertion of platinum-doublet chemotherapy after the initial response to EGFR-TKIs might prevent the emergence of acquired resistance to EGFR-TKIs and prolong survival. We carried out a phase II study of the following first-line treatment for patients with advanced NSCLC harboring EGFR mutations. Gefitinib (250 mg) was administered on days 1-56. Then, after a two-week drug-free period, three cycles of cisplatin (80 mg/m2) and docetaxel (60 mg/m2) were administered on days 71, 92, and 113. Thereafter, gefitinib was re-started on day 134 and continued until disease progression. The primary endpoint was the two-year PFS rate. A total of 34 patients were enrolled. Of the 33 eligible patients and 12 achieved a two-year PFS. Thus, this therapeutic strategy met the criterion for usefulness. The 1-, 2-, 3-, and 5-year PFS rates were 67.0%, 40.2%, 36.9%, and 22.0%, respectively, and the median PFS was 19.5 months. The 1-, 2-, 3- and 5-year survival rates were 90.6%, 71.9%, 64.8%, and 36.5% respectively, and the median survival time was 48.0 months. These results indicate that the insertion of platinum-doublet chemotherapy might prevent the development of acquired resistance to EGFR-TKIs in patients with advanced NSCLC harboring EGFR mutations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  6. Loss of the receptor tyrosine kinase Axl leads to enhanced inflammation in the CNS and delayed removal of myelin debris during Experimental Autoimmune Encephalomyelitis

    Directory of Open Access Journals (Sweden)

    Prieto Anne L

    2011-05-01

    Full Text Available Abstract Background Axl, together with Tyro3 and Mer, constitute the TAM family of receptor tyrosine kinases. In the nervous system, Axl and its ligand Growth-arrest-specific protein 6 (Gas6 are expressed on multiple cell types. Axl functions in dampening the immune response, regulating cytokine secretion, clearing apoptotic cells and debris, and maintaining cell survival. Axl is upregulated in various disease states, such as in the cuprizone toxicity-induced model of demyelination and in multiple sclerosis (MS lesions, suggesting that it plays a role in disease pathogenesis. To test for this, we studied the susceptibility of Axl-/- mice to experimental autoimmune encephalomyelitis (EAE, an animal model for multiple sclerosis. Methods WT and Axl-/- mice were immunized with myelin oligodendrocyte glycoprotein (MOG35-55 peptide emulsified in complete Freund's adjuvant and injected with pertussis toxin on day 0 and day 2. Mice were monitored daily for clinical signs of disease and analyzed for pathology during the acute phase of disease. Immunological responses were monitored by flow cytometry, cytokine analysis and proliferation assays. Results Axl-/- mice had a significantly more severe acute phase of EAE than WT mice. Axl-/- mice had more spinal cord lesions with larger inflammatory cuffs, more demyelination, and more axonal damage than WT mice during EAE. Strikingly, lesions in Axl-/- mice had more intense Oil-Red-O staining indicative of inefficient clearance of myelin debris. Fewer activated microglia/macrophages (Iba1+ were found in and/or surrounding lesions in Axl-/- mice relative to WT mice. In contrast, no significant differences were noted in immune cell responses between naïve and sensitized animals. Conclusions These data show that Axl alleviates EAE disease progression and suggests that in EAE Axl functions in the recruitment of microglia/macrophages and in the clearance of debris following demyelination. In addition, these data

  7. Fatigue associated with newly approved vascular endothelial growth factor receptor tyrosine kinase inhibitors in cancer patients: an up-to-date meta-analysis.

    Science.gov (United States)

    Li, Jing; Gu, Jian

    2017-10-01

    The fatigue associated with five newly approved vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKIs) (regorafenib, vandetanib, cabozantinib, lenvatinib, axitinib) is poorly understood. We conducted this systematic review to fully investigate the fatigue associated with these VEGFR-TKIs in cancer patients. Relevant studies of randomized controlled trials in cancer patients treated with the five VEGFR-TKIs were retrieved and a systematic evaluation was conducted. EMBASE, MEDLINE, and PubMed were searched for articles published until March 2017. Thirteen randomized controlled trials and 4395 patients were included. The current analysis suggested that the use of five newly approved VEGFR-TKIs increased the risk of all-grade fatigue (1.43; 95% CI 1.23-1.66; p < 0.00001) and high-grade (≥grade 3) fatigue (1.97; 95% CI1.44-2.70; p < 0.0001). On subgroup analysis, the risk ratio (RR) of all-grade fatigue varied significantly within drug type, but high-grade fatigue did not. The RR of all-grade and high-grade fatigue did not vary significantly according to cancer type, treatment line, and treatment duration. The risk of high-grade fatigue may vary with treatment duration, whereas all-grade fatigue may not. The available data suggest that the use of the five newly approved VEGFR-TKIs is associated with a significantly increased risk of fatigue in cancer patients. Physicians should be aware of this adverse effect and should monitor cancer patients receiving these drugs.

  8. Correlation of degree of hypothyroidism with survival outcomes in patients with metastatic renal cell carcinoma receiving vascular endothelial growth factor receptor tyrosine kinase inhibitors.

    Science.gov (United States)

    Bailey, Erin B; Tantravahi, Srinivas K; Poole, Austin; Agarwal, Archana M; Straubhar, Alli M; Batten, Julia A; Patel, Shiven B; Wells, Chesley E; Stenehjem, David D; Agarwal, Neeraj

    2015-06-01

    Hypothyroidism is a common adverse effect of vascular endothelial growth factor receptor tyrosine kinase inhibitor (VEGFR-TKI) therapy in patients with metastatic renal cell carcinoma (mRCC). Some studies have shown an association with improved survival. However, hypothyroidism severity has not been correlated with survival outcomes. We report the incidence and severity of VEGFR-TKI therapy-associated hypothyroidism in correlation with the survival outcomes of patients with mRCC. A retrospective analysis of patients with mRCC who received VEGFR-TKIs (2004 through 2013) was conducted from a single institutional database. Hypothyroidism, progression-free survival (PFS), and overall survival (OS) were assessed. Univariate and multivariate analyses were performed using the Kaplan-Meier method and Cox proportional hazard models. Of 125 patients with mRCC, 65 were eligible. Their median age was 59 years (range, 45-79 years), and 46 (70.8%) were male. Hypothyroidism occurred in 25 patients (38.5%), of whom 13 had a peak thyroid-stimulating hormone (TSH) level > 10 mIU/L during treatment. The median OS was significantly longer in patients with a peak TSH > 10 mIU/L than in patients with a peak TSH of ≤ 10 mIU/L (not reached vs. 21.4 months, P = .005). On multivariate analysis, risk criteria, number of previous therapies, and severe hypothyroidism (TSH > 10 mIU/L) during VEGFR-TKI therapy remained significant for improvements in PFS and OS. The severity of VEGFR-TKI therapy-associated hypothyroidism (TSH > 10 mIU/L) was associated with improved survival outcomes in patients with mRCC and should not necessitate a dose reduction or therapy discontinuation. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Outcome in advanced non-small cell lung cancer patients with successful rechallenge after recovery from epidermal growth factor receptor tyrosine kinase inhibitor-induced interstitial lung disease.

    Science.gov (United States)

    Kashiwabara, Kosuke; Semba, Hiroshi; Fujii, Shinji; Tsumura, Shinsuke

    2017-04-01

    Several non-small cell lung cancer (NSCLC) cases of successful rechallenge with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) after recovery from gefitinib or erlotinib-induced interstitial lung disease (ILD) have been reported, but it is not clear whether the rechallenge affects the outcome. We retrospectively evaluated the difference in the outcome between advanced NCLC patients with active EGFR mutations who received EGFR-TKI rechallenge after recovery from EGFR-TKI-induced ILD and those who did not. EGFR-TKI-induced ILD occurred in 11 (10%) of 110 patients receiving gefitinib, five (7%) of 73 patients receiving erlotinib and one (8%) of 13 patients receiving afatinib. Diffuse alveolar damage pattern ILD was observed in six cases, four of which had chemotherapy-related death. Five of 13 patients who had recovered from ILD received EGFR-TKI rechallenge with concurrent oral administration of prednisolone 0.5 mg/kg after the strict informed consent of the risk for the recurrence of severe ILD. All of the five patients achieved a partial response. The median overall survival from the occurrence of EGFR-TKI-induced ILD was longer in patients with EGFR-TKI rechallenge than that in patients without (15.5 vs. 3.5 months, p = 0.029). The adverse events of EGFR-TKI rechallenge were tolerable, but one case receiving EGFR-TKI rechallenge with the suspected drug exhibited the recurrence of grade 3 ILD after the discontinuation of prednisolone. EGFR-TKI rechallenge with concurrent prednisolone therapy might be salvage therapy in advanced NSCLC patients with active EGFR mutations after recovery from EGFR-TKI-induced ILD.

  10. Non-small-cell lung cancer cells combat epidermal growth factor receptor tyrosine kinase inhibition through immediate adhesion-related responses.

    Science.gov (United States)

    Wang, Hsian-Yu; Hsu, Min-Kung; Wang, Kai-Hsuan; Tseng, Ching-Ping; Chen, Feng-Chi; Hsu, John T-A

    2016-01-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib, and afatinib, have greatly improved treatment efficacy in non-small cell lung cancer (NSCLC) patients with drug-sensitive EGFR mutations. However, in some TKI responders, the benefits of such targeted therapies are limited by the rapid development of resistance, and strategies to overcome this resistance are urgently needed. Studies of drug resistance in cancer cells typically involve long term in vitro induction to obtain stably acquired drug-resistant cells followed by elucidation of resistance mechanisms, but the immediate responses of cancer cells upon drug treatment have been ignored. The aim of this study was to investigate the immediate responses of NSCLC cells upon treatment with EGFR TKIs. Both NSCLC cells, ie, PC9 and H1975, showed immediate enhanced adhesion-related responses as an apoptosis-countering mechanism upon first-time TKI treatment. By gene expression and pathway analysis, adhesion-related pathways were enriched in gefitinib-treated PC9 cells. Pathway inhibition by small-hairpin RNAs or small-molecule drugs revealed that within hours of EGFR TKI treatment, NSCLC cells used adhesion-related responses to combat the drugs. Importantly, we show here that the Src family inhibitor, dasatinib, dramatically inhibits cell adhesion-related response and greatly enhances the cell-killing effects of EGFR TKI (gefitinib for the PC9 cells; afatinib for the H1975 cells) in NSCLC cells, which would otherwise escape the TKI-induced apoptosis. Results from this study indicate that NSCLC cells can employ the adhesion response as a survival pathway to survive under EGFR-targeted therapy. Simultaneous targeting of EGFR signaling and adhesion pathways would further boost the efficacy of EGFR-targeted therapy in NSCLC.

  11. Debio 0617B Inhibits Growth of STAT3-Driven Solid Tumors through Combined Inhibition of JAK, SRC, and Class III/V Receptor Tyrosine Kinases.

    Science.gov (United States)

    Murone, Maximilien; Vaslin Chessex, Anne; Attinger, Antoine; Ramachandra, Raghuveer; Shetty, Shankar J; Daginakatte, Girish; Sengupta, Saumitra; Marappan, Sivapriya; Dhodheri, Samiulla; Rigotti, Stefania; Bachhav, Yogeshwar; Brienza, Silvano; Traxler, Peter; Lang, Marc; Aguet, Michel; Zoete, Vincent; Michielin, Olivier; Nicholas, Courtney; Johnson, Faye M; Ramachandra, Murali; McAllister, Andres

    2016-10-01

    Tumor survival, metastases, chemoresistance, and escape from immune responses have been associated with inappropriate activation of STAT3 and/or STAT5 in various cancers, including solid tumors. Debio 0617B has been developed as a first-in-class kinase inhibitor with a unique profile targeting phospho-STAT3 (pSTAT3) and/or pSTAT5 in tumors through combined inhibition of JAK, SRC, ABL, and class III/V receptor tyrosine kinases (RTK). Debio 0617B showed dose-dependent inhibition of pSTAT3 in STAT3-activated carcinoma cell lines; Debio 0617B also showed potent antiproliferative activity in a panel of cancer cell lines and in patient-derived tumor xenografts tested in an in vitro clonogenic assay. Debio 0617B showed in vivo efficacy by inhibiting tumor growth in several mouse xenograft models. To increase in vivo efficacy and STAT3 inhibition, Debio 0617B was tested in combination with the EGFR inhibitor erlotinib in a non-small cell lung cancer xenograft model. To evaluate the impact of in vivo STAT3 blockade on metastases, Debio 0617B was tested in an orthotopic tumor model. Measurement of primary tumor weight and metastatic counts in lung tissue demonstrated therapeutic efficacy of Debio 0617B in this model. These data show potent activity of Debio 0617B on a broad spectrum of STAT3-driven solid tumors and synergistic activity in combination with EGFR inhibition. Mol Cancer Ther; 15(10); 2334-43. ©2016 AACR. ©2016 American Association for Cancer Research.

  12. Cocaine-induced behavioral sensitization in adolescent rats endures until adulthood: lack of association with GluR1 and NR1 glutamate receptor subunits and tyrosine hydroxylase.

    Science.gov (United States)

    Marin, Marcelo T; Cruz, Fábio C; Planeta, Cleopatra S

    2008-11-01

    Exposure to repeated cocaine induces enduring behavioral sensitization, which has been implicated in the psychostimulant-induced craving and psychosis. Adaptations in dopamine and glutamate neurotransmission in the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) seem to mediate psychostimulant-induced behavioral sensitization. The abuse of drugs often begins during adolescence; however few studies have been devoted to study the effects of drugs of abuse at this age. The aim of our study was to examine whether repeated cocaine during adolescence could induce behavioral sensitization that endures into adulthood. Moreover, the protein levels of Tyrosine Hydroxylase (TH) and the glutamate receptor subunits GluR1 and NR1 in the NAc and mPFC were measured following the behavioral tests. Adolescent rats were treated with cocaine from postnatal day (PND) 30 to PND34 and behavioral sensitization was verified recording locomotor activity after cocaine challenge injection to adolescent (PND37) or adult (PND64 or 94) rats in separate groups at each time point. TH, GluR1, and NR1 protein levels were measured by Western blotting. Rats exposed to cocaine during adolescence expressed behavioral sensitization when tested on PND37 and PND64. In cocaine sensitized rats GluR1 protein was increased in the mPFC on PND37 but not in other ages. Thus, cocaine-induced behavioral sensitization during adolescence endures into early adulthood. However, cocaine pretreatment during adolescence induced a transient increase of GluR1 in the mPFC only when animals were challenged in the same age.

  13. Functional Analyses of Mutations in Receptor Tyrosine Kinase Genes in Non-Small Cell Lung Cancer: Double-Edged Sword of DDR2.

    Science.gov (United States)

    Terashima, Masato; Togashi, Yosuke; Sato, Katsuaki; Mizuuchi, Hiroshi; Sakai, Kazuko; Suda, Kenichi; Nakamura, Yu; Banno, Eri; Hayashi, Hidetoshi; De Velasco, Marco A; Fujita, Yoshihiko; Tomida, Shuta; Mitsudomi, Tetsuya; Nishio, Kazuto

    2016-07-15

    This study investigated whether mutations of receptor tyrosine kinase (RTK) genes detected using next-generation sequencing (NGS) are suitable therapeutic targets. Fifty surgically resected non-small cell lung cancer (NSCLC) samples were target resequenced using NGS. We then investigated the functions of the identified RTK gene mutations, including their oncogenic potential, in vitro Mutations in RTK genes were found in 20 samples (EGFR, 15; ERBB4, 1; ALK, 1; DDR2, 2; FGFR1, 1), mutations in MAPK pathway genes were found in nine samples (KRAS, 7; NRAS, 1; BRAF, 2), and mutations in PI3K pathway genes were found in three samples (PIK3CA, 1; PTEN, 3). Among the mutations in RTKs, the functions of four mutations were unclear (ERBB4 D245G; DDR2 H246R and E655K; FGFR1 A263V). These mutations did not exhibit any transformational activities. Neither the phosphorylation nor the protein expressions of RTKs were changed by the DDR2 H246R, ERBB4 D245G, and FGFR1 A263V mutations, although the expression level of the DDR2 protein harboring the E655K mutation was particularly low. Collagen stimulation decreased cellular proliferation through p38 activation in the DDR2 wild-type-overexpressed cell lines, whereas the growth-suppressive effect was weakened in DDR2 E655K-overexpressed cell lines. Furthermore, the DDR2 E655K protein strongly bound to ubiquitin ligase E3 (Cbl-b), and the mutant protein expression was increased after treatment with a proteasome inhibitor. Our experimental findings suggest that RTK mutations are not always suitable as therapeutic targets. The DDR2 E655K mutation can play a role in cancer progression by reducing the growth-inhibitory effect of collagen. Clin Cancer Res; 22(14); 3663-71. ©2016 AACR. ©2016 American Association for Cancer Research.

  14. Genetic Mapping and Functional Studies of a Natural Inhibitor of the Insulin Receptor Tyrosine Kinase: The Mouse Ortholog of Human α2-HS Glycoprotein

    Science.gov (United States)

    Cintrón, Vivian J.; Ko, Minoru S. H.; Chi, Kenneth D.; Gross, Jason P.; Srinivas, Pothur R.; Goustin, Anton Scott

    2000-01-01

    Fetuin/α2-HS glycoprotein (α2-HSG) homologs have been identified in several species including rat, sheep, pig, rabbit, guinea pig, cattle, mouse and human. Multiple physiological roles for these homologs have been suggested, including ability to bind to hydroxyapatite crystals and to specifically inhibit the tyrosine kinase (TK) activity of the insulin receptor (IR). In this study we report the identification, cloning, and characterization of the mouse Ahsg gene and its function as an IR-TK inhibitor. Genomic clones derived from a mouse Svj 129 genomic library were sequenced in order to characterize the intron–exon organization of the mouse Ahsg gene, including an 875 bp subclone containing 154 bp upstream from the transcription start site, the first exon, and part of the first intron. A second genomic subclone harboring a 3.45 kb Bgl II fragment contained exons 2, 3 and 4 in addition to two adjacent elements within the first intron-a repetitive element of the B1 family (92 bp) and a 271 bp tract of (T,C)n * (A,G)n. We have mapped mouse Ahsg at 16 cM adjacent to the Diacylglycerol kinase 3 (Dagk3) gene on chromosome 16 by genotyping interspecific backcross panels between C57BL/6J and Mus spretus. The position is syntenic with human chromosome 3q27, where the human AHSG gene resides. Using recombinant mouse α2-HSG expressed from a recombinant baculovirus, we demonstrate that mouse α2-HSG inhibits insulin–stimulated IR autophosphorylation and IR-TKA in vitro. In addition, mouse α2-HSG (25μg/ml) completely abolishes insulin-induced DNA synthesis in H-35 rat hepatoma cells. Based on the sequence data and functional analysis, we conclude that the mouse Ahsg gene is the true ortholog of the human AHSG gene. PMID:11467416

  15. Decreased expression of protein tyrosine phosphatase non-receptor type 12 is involved in the proliferation and recurrence of bladder transitional cell carcinoma

    Science.gov (United States)

    PIAO, YONGRUI; LIU, XIANKUI; LIN, ZHENHUA; JIN, ZHEHU; JIN, XUANSHUN; YUAN, KUICHANG; WU, WENYUAN

    2015-01-01

    Protein tyrosine phosphatase non-receptor type 12 (PTPN12) has been shown to be involved in the development of a number of types of carcinoma. However, the effect of PTPN12 on the proliferation and recurrence of human bladder transitional cell carcinoma (TCC) is unclear. The present study aimed to investigate the expression and function of PTPN12 in human TCC. Samples from 164 patients with TCC, in addition to 146 patients undergoing bladder surgery for indications other than TCC, were examined. PTPN12 protein expression was examined using immunohistochemistry and western blotting, and PTPN12 mRNA expression was examined using reverse transcription-quantitative polymerase chain reaction. PTPN12 expression was increased following transfection with the PTPN12-expressing, pcDEF3 vector, and PTPN12 expression was decreased by RNA interference, in four TCC cell lines. The proliferation of TCC cells was analyzed by a WST-1 assay and in xenografts on BALB/C nude mice. The effect of PTPN12 on tumor recurrence was analyzed by adhesion, migration and invasion assays in TCC cell lines. PTPN12 expression was significantly decreased in TCC tissues compared with that in normal urothelium, and the level of PTPN12 expression was negatively correlated with tumor size, pathological grade, clinical stage and tumor recurrence. Furthermore, decreased expression of PTPN12 significantly enhanced the proliferation of TCC cells in vitro and in vivo. TCC cells with lower levels of PTPN12 exhibited greater adhesion, migration and invasion. In conclusion, PTPN12 expression is downregulated in human TCC. Restoring PTPN12 activity may represent a novel therapeutic strategy for this disease. PMID:26622721

  16. ZEB1 Mediates Acquired Resistance to the Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer.

    Directory of Open Access Journals (Sweden)

    Takeshi Yoshida

    Full Text Available Epithelial-mesenchymal transition (EMT is one mechanism of acquired resistance to inhibitors of the epidermal growth factor receptor-tyrosine kinases (EGFR-TKIs in non-small cell lung cancer (NSCLC. The precise mechanisms of EMT-related acquired resistance to EGFR-TKIs in NSCLC remain unclear. We generated erlotinib-resistant HCC4006 cells (HCC4006ER by chronic exposure of EGFR-mutant HCC4006 cells to increasing concentrations of erlotinib. HCC4006ER cells acquired an EMT phenotype and activation of the TGF-β/SMAD pathway, while lacking both T790M secondary EGFR mutation and MET gene amplification. We employed gene expression microarrays in HCC4006 and HCC4006ER cells to better understand the mechanism of acquired EGFR-TKI resistance with EMT. At the mRNA level, ZEB1 (TCF8, a known regulator of EMT, was >20-fold higher in HCC4006ER cells than in HCC4006 cells, and increased ZEB1 protein level was also detected. Furthermore, numerous ZEB1 responsive genes, such as CDH1 (E-cadherin, ST14, and vimentin, were coordinately regulated along with increased ZEB1 in HCC4006ER cells. We also identified ZEB1 overexpression and an EMT phenotype in several NSCLC cells and human NSCLC samples with acquired EGFR-TKI resistance. Short-interfering RNA against ZEB1 reversed the EMT phenotype and, importantly, restored erlotinib sensitivity in HCC4006ER cells. The level of micro-RNA-200c, which can negatively regulate ZEB1, was significantly reduced in HCC4006ER cells. Our results suggest that increased ZEB1 can drive EMT-related acquired resistance to EGFR-TKIs in NSCLC. Attempts should be made to explore targeting ZEB1 to resensitize TKI-resistant tumors.

  17. Epithelial mesenchymal transition status is associated with anti-cancer responses towards receptor tyrosine-kinase inhibition by dovitinib in human bladder cancer cells.

    Science.gov (United States)

    Hänze, Jörg; Henrici, Marcus; Hegele, Axel; Hofmann, Rainer; Olbert, Peter J

    2013-12-11

    Dovitinib (TKI-258) is a receptor tyrosine kinase (RTK) inhibitor targeting fibroblast growth factor receptor (FGFR) and further related RTKs. TKI-258 is under investigation as anticancer drug for the treatment of various cancers including bladder cancer with aberrant RTK signaling. Here, we analyzed the responses of ten human bladder cancer cell lines towards TKI-258 treatment in relation to the epithelial mesenchymal transition (EMT) status of the cells. Expression of epithelial marker E-cadherin as well as mesenchymal markers N-cadherin and vimentin was determined by quantitative RT-PCR and Western-blot in RNA and protein extracts from the cultured cell lines. The cell responses were analyzed upon addition of TKI-258 by viability/proliferation (XTT assay) and colony formation assay for measurement of cell contact independent growth. The investigated bladder cancer cell lines turned out to display quite different EMT patterns as indicated by the abundance of E-cadherin or N-cadherin and vimentin. Protein and mRNA levels of the respective components strongly correlated. Based on E-cadherin and N-cadherin mRNA levels that were expressed approximately mutual exclusively, an EMT-score was calculated for each cell line. A high EMT-score indicated mesenchymal-like cells and a low EMT-score epithelial-like cells. Then, we determined the IC₅₀ values for TKI-258 by dose response curves (0-12 μM TKI-258) in XTT assays for each cell line. Also, we measured the clonogenic survival fraction after adding TKI-258 (1 μM) by colony formation assay. We observed significant correlations between EMT-score and IC₅₀ values (r = 0.637, p = 0.0474) and between EMT-score and clonogenic survival fraction (r = 0.635, p = 0.0483) as analyzed by linear regression analyses. In sum, we demonstrated that the EMT status based on E-cadherin and N-cadherin mRNA levels may be useful to predict responses towards TKI-258 treatment in bladder cancer.

  18. Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region

    DEFF Research Database (Denmark)

    Jiang, Y P; Wang, H; D'Eustachio, P

    1993-01-01

    We describe a new member of the receptor protein tyrosine phosphatase family, R-PTP-kappa, cDNA cloning predicts that R-PTP-kappa is synthesized from a precursor protein of 1,457 amino acids. Its intracellular domain displays the classical tandemly repeated protein tyrosine phosphatase homology......, separated from the transmembrane segment by an uncharacteristically large juxta-membrane region. The extracellular domain of the R-PTP-kappa precursor protein contains an immunoglobulin-like domain and four fibronectin type III-like repeats, preceded by a signal peptide and a region of about 150 amino acids...... processing, following which both cleavage products remain associated. By site-directed mutagenesis, the likely cleavage site was shown to be a consensus sequence for cleavage by the processing endopeptidase furin, located in the fourth fibronectin type III-like repeat. In situ hybridization analysis...

  19. JAK and Src tyrosine kinase signaling in asthma.

    Science.gov (United States)

    Tundwal, Kavita; Alam, Rafeul

    2012-06-01

    Tyrosine kinases play a critical role in transducing intracellular signals from the receptors. Many receptors do not have intrinsic tyrosine kinase activity, so they rely on cytosolic and/or membrane-associated tyrosine kinases for initial signal generation. The Src and JAK family kinases are frequently associated with receptors and generate the initial cytosolic signals. These signals are then transduced to other compartments of the cytosol and to the nucleus to elicit a specific cellular response. In this review we focus on these two families of tyrosine kinases and review their involvement in activation of cells that are involved in the pathogenesis of asthma. A Th2-type immune response dominates the processes that lead to the phenotype of asthma. For this reason we give special attention to the tyrosine kinases that are involved in a Th2 response. Further we examine the involvement of tyrosine kinases in activation of mast cells, eosinophils and other cells.

  20. Efficacy and safety of sequential use of everolimus in Japanese patients with advanced renal cell carcinoma after failure of first-line treatment with vascular endothelial growth factor receptor tyrosine kinase inhibitor: a multicenter phase II clinical trial.

    Science.gov (United States)

    Oyama, Masafumi; Sugiyama, Takayuki; Nozawa, Masahiro; Fujimoto, Kiyohide; Kishida, Takeshi; Kimura, Go; Tokuda, Noriaki; Hinotsu, Shiro; Shimozuma, Kojiro; Akaza, Hideyuki; Ozono, Seiichiro

    2017-06-01

    Many studies have shown the efficacy of everolimus after pretreatment with vascular endothelial growth factor receptor-tyrosine kinase inhibitors. We investigated the efficacy and safety of everolimus as a second-line treatment after the failure of vascular endothelial growth factor receptor-tyrosine kinase inhibitor therapy in Japanese patients with advanced renal cell carcinoma. This was an open-label, multicenter, phase II trial conducted in Japan through the central registration system. A total of 57  patients were enrolled. Patients were administered 10 mg of everolimus q.d. orally. The primary efficacy endpoint was progression-free survival achieved by administration of everolimus. The median progression-free survival of patients administered everolimus was 5.03 months (95% confidence interval: 3.70-6.20). The median overall survival was not reached. The objective response rate was 9.4% (95% confidence interval: 3.1-20.7). The progression-free survival in the group of <100% relative dose intensity was 6.70 months (95% confidence interval: 4.13-11.60), and that in the group of 100% relative dose intensity was 3.77 months (hazard ratio: 2.79, 95% confidence interval: 2.77-5.63). The commonly observed adverse events and laboratory abnormalities were stomatitis (49.1%), hypertriglyceridemia (26.4%), interstitial lung disease (26.4%), anemia (22.6%) and hypercholesterolemia (22.6%). The median progression-free survival was almost similar to that recorded in the RECORD-1 study, whereas prolongation of overall survival was observed in the present study compared with the RECORD-1 study. The treatment outcomes of first-line vascular endothelial growth factor receptor-tyrosine kinase inhibitor therapy and second-line everolimus treatment in Japanese patients were successfully established in the present study.

  1. Patients with acute-on-chronic liver failure have increased numbers of regulatory immune cells expressing the receptor tyrosine kinase MERTK.

    Science.gov (United States)

    Bernsmeier, Christine; Pop, Oltin T; Singanayagam, Arjuna; Triantafyllou, Evangelos; Patel, Vishal C; Weston, Christopher J; Curbishley, Stuart; Sadiq, Fouzia; Vergis, Nikhil; Khamri, Wafa; Bernal, William; Auzinger, Georg; Heneghan, Michael; Ma, Yun; Jassem, Wayel; Heaton, Nigel D; Adams, David H; Quaglia, Alberto; Thursz, Mark R; Wendon, Julia; Antoniades, Charalambos G

    2015-03-01

    Characteristics of decompensated cirrhosis and acute-on-chronic liver failure (ACLF) include susceptibility to infection, immuneparesis, and monocyte dysfunction. MER receptor tyrosine kinase (MERTK) is expressed by monocytes and macrophages and contributes to down-regulation of innate immune responses. We investigated whether MERTK expression is altered on monocytes from patients with liver failure. We analyzed blood and liver samples collected from patients admitted to the liver intensive therapy unit at King's College Hospital in London from December 2012 through July 2014. Patients had either ACLF (n = 41), acute decompensation of cirrhosis without ACLF (n = 9), cirrhosis without decompensation (n = 17), or acute liver failure (n = 23). We also analyzed samples from healthy individuals (controls, n = 29). We used flow cytometry to determine the level of innate immune function, and associated the findings with disease severity. We developed an assay to measure recruitment and migration of immune cells from the tissue parenchyma. Immunohistochemistry and confocal microscopy were used to determine levels of MERTK in bone marrow, liver, and lymph node tissues. We performed immunophenotype analyses and measured the production of tumor necrosis factor and interleukin 6 and intracellular killing of Escherichia coli by monocytes and peritoneal macrophages incubated with lipopolysaccharide, with or without an inhibitor of MERTK (UNC569). The number of monocytes and macrophages that expressed MERTK was greatly increased in the circulation, livers, and lymph nodes of patients with ACLF, compared with patients with stable cirrhosis and controls. MERTK expression (mean fluorescence intensity) correlated with the severity of hepatic and extrahepatic disease and systemic inflammatory responses. Based on immunophenotype, migration, and functional analyses, MERTK-expressing monocytes migrate across the endothelia to localize into tissue sites and regional lymph nodes

  2. Gene expression patterns that predict sensitivity to epidermal growth factor receptor tyrosine kinase inhibitors in lung cancer cell lines and human lung tumors

    Directory of Open Access Journals (Sweden)

    Haura Eric B

    2006-11-01

    Full Text Available Abstract Background Increased focus surrounds identifying patients with advanced non-small cell lung cancer (NSCLC who will benefit from treatment with epidermal growth factor receptor (EGFR tyrosine kinase inhibitors (TKI. EGFR mutation, gene copy number, coexpression of ErbB proteins and ligands, and epithelial to mesenchymal transition markers all correlate with EGFR TKI sensitivity, and while prediction of sensitivity using any one of the markers does identify responders, individual markers do not encompass all potential responders due to high levels of inter-patient and inter-tumor variability. We hypothesized that a multivariate predictor of EGFR TKI sensitivity based on gene expression data would offer a clinically useful method of accounting for the increased variability inherent in predicting response to EGFR TKI and for elucidation of mechanisms of aberrant EGFR signalling. Furthermore, we anticipated that this methodology would result in improved predictions compared to single parameters alone both in vitro and in vivo. Results Gene expression data derived from cell lines that demonstrate differential sensitivity to EGFR TKI, such as erlotinib, were used to generate models for a priori prediction of response. The gene expression signature of EGFR TKI sensitivity displays significant biological relevance in lung cancer biology in that pertinent signalling molecules and downstream effector molecules are present in the signature. Diagonal linear discriminant analysis using this gene signature was highly effective in classifying out-of-sample cancer cell lines by sensitivity to EGFR inhibition, and was more accurate than classifying by mutational status alone. Using the same predictor, we classified human lung adenocarcinomas and captured the majority of tumors with high levels of EGFR activation as well as those harbouring activating mutations in the kinase domain. We have demonstrated that predictive models of EGFR TKI sensitivity can

  3. Non-small-cell lung cancer cells combat epidermal growth factor receptor tyrosine kinase inhibition through immediate adhesion-related responses

    Directory of Open Access Journals (Sweden)

    Wang HY

    2016-05-01

    Full Text Available Hsian-Yu Wang,1,2 Min-Kung Hsu,3,4 Kai-Hsuan Wang,1 Ching-Ping Tseng,2,4 Feng-Chi Chen,3,4 John T-A Hsu1,4 1Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes (NHRI, Zhunan, Miaoli County, 2Institute of Molecular Medicine and Bioengineering, National Chiao Tung University (NCTU, Hsinchu, 3Division of Biostatistics and Bioinformatics, Institute of Population Health Sciences, National Health Research Institutes (NHRI, Zhunan, Miaoli County, 4Department of Biological Science and Technology, National Chiao Tung University (NCTU, Hsinchu, Taiwan, Republic of China Background: Epidermal growth factor receptor (EGFR tyrosine kinase inhibitors (TKIs, such as gefitinib, erlotinib, and afatinib, have greatly improved treatment efficacy in non-small cell lung cancer (NSCLC patients with drug-sensitive EGFR mutations. However, in some TKI responders, the benefits of such targeted therapies are limited by the rapid development of resistance, and strategies to overcome this resistance are urgently needed. Studies of drug resistance in cancer cells typically involve long term in vitro induction to obtain stably acquired drug-resistant cells followed by elucidation of resistance mechanisms, but the immediate responses of cancer cells upon drug treatment have been ignored. The aim of this study was to investigate the immediate responses of NSCLC cells upon treatment with EGFR TKIs.Results: Both NSCLC cells, ie, PC9 and H1975, showed immediate enhanced adhesion-related responses as an apoptosis-countering mechanism upon first-time TKI treatment. By gene expression and pathway analysis, adhesion-related pathways were enriched in gefitinib-treated PC9 cells. Pathway inhibition by small-hairpin RNAs or small-molecule drugs revealed that within hours of EGFR TKI treatment, NSCLC cells used adhesion-related responses to combat the drugs. Importantly, we show here that the Src family inhibitor, dasatinib, dramatically inhibits

  4. Brain metastases in non-small cell lung cancer patients on epidermal growth factor receptor tyrosine kinase inhibitors: symptom and economic burden.

    Science.gov (United States)

    Fernandes, Ancilla W; Wu, Bingcao; Turner, Ralph M

    2017-11-01

    This study describes the symptom and economic burden associated with brain metastases (BM) in patients with non-small cell lung cancer (NSCLC) receiving epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs). This retrospective study included adults with ≥2 medical claims, within 90 days, for lung cancer and ≥1 administration of EGFR-TKIs. Based on ICD-9 codes, patients were stratified into cohorts by type of metastases (BM, other metastases [OM], or no metastases [NM]), and by when the metastasis diagnosis occurred (synchronous or asynchronous). The population (synchronous BM [SBM] = 24, synchronous OM [SOM] = 23, asynchronous BM [ASBM] = 15, asynchronous OM [ASOM] = 49, NM = 85) was mostly female (57%), average age 69 years (SD = 11). SBM patients experienced more fatigue and nausea/vomiting compared with SOM and NM patients and more headaches and loss of appetite than NM patients. ASBM was associated with more fatigue, nausea/vomiting, headaches, pain/numbness, altered mental status, and seizures than NM, and more headaches and pain/numbness than ASOM. SBM patients experienced a greater increase in per-member-per-month all-cause total healthcare costs after diagnosis ($20,301) vs SOM ($9,131, p = .001) and NM ($2,493, p = .001). ASBM's cost increase between baseline and follow-up ($7,867) did not differ from ASOM's ($4,947, p = .195); both were larger than NM ($2,493, p = .001 and p = .009, respectively). EGFR mutation status was inferred based on EGFR-TKI treatment, not by molecular testing. Patients were from US commercial insurance plans; results may not be generalizable to other populations. Among patients with EGFR-TKI-treated NSCLC, patients with BM experienced more symptoms and, when diagnosed synchronously, had significant increases in total medical costs vs patients with OM and NM. Therapeutic options with central nervous system activity may offer advantages in symptomatology and

  5. Loss of mRor1 Enhances the Heart and Skeletal Abnormalities in mRor2-Deficient Mice: Redundant and Pleiotropic Functions of mRor1 and mRor2 Receptor Tyrosine Kinases

    OpenAIRE

    Nomi, Masashi; Oishi, Isao; Kani, Shuichi; Suzuki, Hiroaki; Matsuda, Takeru; Yoda, Akinori; Kitamura, Makiko; Itoh, Kyoko; Takeuchi, Shigeto; Takeda, Kiyoshi; Akira, Shizuo; Ikeya, Makoto; Takada, Shinji; Minami, Yasuhiro

    2001-01-01

    The mammalian Ror family of receptor tyrosine kinases consists of two structurally related proteins, Ror1 and Ror2. We have shown that mRor2-deficient mice exhibit widespread skeletal abnormalities, ventricular septal defects in the heart, and respiratory dysfunction, leading to neonatal lethality (S. Takeuchi, K. Takeda, I. Oishi, M. Nomi, M. Ikeya, K. Itoh, S. Tamura, T. Ueda, T. Hatta, H. Otani, T. Terashima, S. Takada, H. Yamamura, S. Akira, and Y. Minami, Genes Cells 5:71–78, 2000). Here...

  6. [Case report: tyrosine deposits].

    Science.gov (United States)

    Stephan, R; Tholen, R; Untermann, F

    1996-01-01

    Whitish precipitations in three samples of raw cured ham, which appeared in the stereomicroscope as piles of crystals, were confirmed as tyrosine crystals. Tyrosine is readily soluble in nitric acid (yellowish discoloration) and, after addition of potash lye, it precipitates as yellow-orange picrate. Factors that influence the formation of tyrosine crystals are largely unknown. In raw cured ham of Parma experience has shown that tyrosine crystals are found in ham stored for a very long time.

  7. In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship.

    Science.gov (United States)

    Mendel, Dirk B; Laird, A Douglas; Xin, Xiaohua; Louie, Sharianne G; Christensen, James G; Li, Guangmin; Schreck, Randall E; Abrams, Tinya J; Ngai, Theresa J; Lee, Leslie B; Murray, Lesley J; Carver, Jeremy; Chan, Emily; Moss, Katherine G; Haznedar, Joshua O; Sukbuntherng, Juthamas; Blake, Robert A; Sun, Li; Tang, Cho; Miller, Todd; Shirazian, Sheri; McMahon, Gerald; Cherrington, Julie M

    2003-01-01

    One challenging aspect in the clinical development of molecularly targeted therapies, which represent a new and promising approach to treating cancers, has been the identification of a biologically active dose rather than a maximum tolerated dose. The goal of the present study was to identify a pharmacokinetic/pharmacodynamic relationship in preclinical models that could be used to help guide selection of a clinical dose. SU11248, a novel small molecule receptor tyrosine kinase inhibitor with direct antitumor as well as antiangiogenic activity via targeting the vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), KIT, and FLT3 receptor tyrosine kinases, was used as the pharmacological agent in these studies. In mouse xenograft models, SU11248 exhibited broad and potent antitumor activity causing regression, growth arrest, or substantially reduced growth of various established xenografts derived from human or rat tumor cell lines. To predict the target SU11248 exposure required to achieve antitumor activity in mouse xenograft models, we directly measured target phosphorylation in tumor xenografts before and after SU11248 treatment and correlated this with plasma inhibitor levels. In target modulation studies in vivo, SU11248 selectively inhibited Flk-1/KDR (VEGF receptor 2) and PDGF receptor beta phosphorylation (in a time- and dose-dependent manner) when plasma concentrations of inhibitor reached or exceeded 50-100 ng/ml. Similar results were obtained in a functional assay of VEGF-induced vascular permeability in vivo. Constant inhibition of VEGFR2 and PDGF receptor beta phosphorylation was not required for efficacy; at highly efficacious doses, inhibition was sustained for 12 h of a 24-h dosing interval. The pharmacokinetic/pharmacodynamic relationship established for SU11248 in these preclinical studies has aided in the design, selection, and evaluation of dosing regimens being tested in human trials.

  8. 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

    with diabetes). Group 1 (19 subjects) underwent skeletal muscle biopsies for the measurement of basal and insulin-stimulated tyrosine phosphorylation of the IR (stimulated by 100 nmol/l insulin). The fold increase after insulin stimulation was calculated as the ratio between maximal and basal phosphorylation...

  9. Impact of clinical parameters and systemic inflammatory status on epidermal growth factor receptor-mutant non-small cell lung cancer patients readministration with epidermal growth factor receptor tyrosine kinase inhibitors.

    Science.gov (United States)

    Chen, Yu-Mu; Lai, Chien-Hao; Rau, Kun-Ming; Huang, Cheng-Hua; Chang, Huang-Chih; Chao, Tung-Ying; Tseng, Chia-Cheng; Fang, Wen-Feng; Chung, Yu-Hsiu; Wang, Yi-Hsi; Su, Mao-Chang; Huang, Kuo-Tung; Liu, Shih-Feng; Chen, Hung-Chen; Chang, Ya-Chun; Chang, Yu-Ping; Wang, Chin-Chou; Lin, Meng-Chih

    2016-11-08

    Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) readministration to lung cancer patients is common owing to the few options available. Impact of clinical factors on prognosis of EGFR-mutant non-small cell lung cancer (NSCLC) patients receiving EGFR-TKI readministration after first-line EGFR-TKI failure and a period of TKI holiday remains unclear. Through this retrospective study, we aimed to understand the impact of clinical factors in such patients. Of 1386 cases diagnosed between December 2010 and December 2013, 80 EGFR-mutant NSCLC patients who were readministered TKIs after failure of first-line TKIs and intercalated with at least one cycle of cytotoxic agent were included. We evaluated clinical factors that may influence prognosis of TKI readministration as well as systemic inflammatory status in terms of neutrophil-to-lymphocyte ratio (NLR) and lymphocyte-to-monocyte ratio (LMR). Baseline NLR and LMR were estimated at the beginning of TKI readministration and trends of NLR and LMR were change amount from patients receiving first-Line TKIs to TKIs readministration. Median survival time since TKI readministration was 7.0 months. In the univariable analysis, progression free survival (PFS) of first-line TKIs, baseline NLR and LMR, and trend of LMR were prognostic factors in patients receiving TKIs readministration. In the multivariate analysis, only PFS of first-line TKIs (p factors. Longer PFS of first-line TKIs, low baseline NLR, and high trend of LMR were good prognostic factors in EGFR-mutant NSCLC patients receiving TKI readministration.

  10. Staphylococcal enterotoxins modulate interleukin 2 receptor expression and ligand-induced tyrosine phosphorylation of the Janus protein-tyrosine kinase 3 (Jak3) and signal transducers and activators of transcription (Stat proteins)

    DEFF Research Database (Denmark)

    Nielsen, M; Svejgaard, A; Röpke, C

    1995-01-01

    . In addition, SE can induce an interleukin-2 (IL-2) nonresponsive state and apoptosis. Here, we show that SE induce dynamic changes in the expression of and signal transduction through the IL-2 receptor (IL-2R) beta and gamma chains (IL-2R beta and IL-2R gamma) in human antigen-specific CD4+ T-cell lines. Thus...

  11. A systematic review of economic evaluations of Tyrosine Kinase Inhibitors of Vascular Endothelial Growth Factor Receptors, mammalian target of rapamycin inhibitors and Programmed Death-1 inhibitors in metastatic renal cell cancer.

    Science.gov (United States)

    Petrou, Panagiotis

    2018-02-16

    The therapeutic categories of Tyrosine Kinase Inhibitors of Vascular Endothelial Growth Factor Receptors, mammalian target of rapamycin inhibitors and Programmed Death-1 inhibitors have transformed the treatment of metastatic renal cell cancer. Nevertheless, this comes at an increased cost, in tandem with similar fiscal pressures in the broader oncology sector, which may jeopardize the sustainability of health systems. Areas covered To this direction, the economic evaluation of these agents is essential for rational and efficient resource allocation. The aim of this study is to glean, assess and present an outline of the available cost-effectiveness studies of these agents in the management of metastatic renal cell cancer. Expert Commentary We concluded that the results of economic evaluation are pertinent, apart from the product under evaluation, to the country setting as well.

  12. Monitoring of epidermal growth factor receptor tyrosine kinase inhibitor-sensitizing and resistance mutations in the plasma DNA of patients with advanced non-small cell lung cancer during treatment with erlotinib

    DEFF Research Database (Denmark)

    Sorensen, Boe S; Wu, Lin; Wei, Wen

    2014-01-01

    BACKGROUND: The feasibility of monitoring epidermal growth factor receptor (EGFR) mutations in plasma DNA from patients with advanced non-small cell lung cancer (NSCLC) during treatment with erlotinib and its relation to disease progression was investigated. METHODS: The amount of EGFR-mutant DNA...... was tested in plasma DNA from patients with advanced NSCLC with allele-specific polymerase chain reaction assays. Blood samples from 23 patients with adenocarcinoma of NSCLC that carried tyrosine kinase inhibitor-sensitizing EGFR mutations were taken immediately before treatment with erlotinib. Additional...... blood samples were taken at timed intervals until erlotinib treatment was withdrawn. RESULTS: The amount of plasma DNA with sensitizing EGFR mutations was found to be reduced after the first cycle of erlotinib treatment in 22 of 23 patients (96%). No patients presented with the resistant T790M mutation...

  13. Synthesis and biological evaluation of pyrimidine-based dual inhibitors of human epidermal growth factor receptor 1 (HER-1) and HER-2 tyrosine kinases.

    Science.gov (United States)

    Cha, Mi Young; Lee, Kwang-Ok; Kang, Seok-Jong; Jung, Young Hee; Song, Ji Yeon; Choi, Kyung Jin; Byun, Joo Yun; Lee, Han-Jae; Lee, Gwan Sun; Park, Seung Bum; Kim, Maeng Sup

    2012-03-22

    A novel series of N(4)-(3-chlorophenyl)-5-(oxazol-2-yl)pyrimidine-4,6-diamines were synthesized and evaluated as dual inhibitors of HER-1/HER-2 tyrosine kinases. In contrast to the currently approved HER-2-targeted agent (lapatinib, 1), our irreversible HER-1/HER-2 inhibitors have the potential to overcome the clinically relevant and mutation-induced drug resistance. The selected compound (19a) showed excellent inhibitory activity toward HER-1/HER-2 tyrosine kinases with selectivity over 20 other kinases and inhibited the proliferation of both cancer cell types: lapatinib-sensitive cell lines (SK-Br3, MDA-MB-175, and N87) and lapatinib-resistant cell lines (MDA-MB-453, H1781, and H1975). The excellent pharmacokinetic profiles of 19a in mice and rats led us to further investigation of a novel therapeutic agent for HER-2-targeting treatment of solid tumors, especially HER-2-positive breast/gastric cancer and HER-2-mutated lung cancer.

  14. Cross Talk between inhibitory immunoreceptor Tyrosine-Based Activation Motif-Signaling and Toll-Like Receptor Pathways in Macrophages and Dendritic Cells

    Czech Academy of Sciences Publication Activity Database

    Hirsch, Ivan; Janovec, V.; Stranska, R.; Bendriss-Vermare, N.

    2017-01-01

    Roč. 8, Apr 7 (2017), č. článku 394. ISSN 1664-3224 Institutional support: RVO:61388963 Keywords : plasmacytoid dendritic cell * conventional dendritic cells * macrophage * toll-like receptors * regulatory receptors Subject RIV: EE - Microbiology, Virology Impact factor: 6.429, year: 2016 http://journal.frontiersin.org/article/10.3389/fimmu.2017.00394/full

  15. Effect of treatment with a colloidal oatmeal lotion on the acneform eruption induced by epidermal growth factor receptor and multiple tyrosine-kinase inhibitors.

    Science.gov (United States)

    Alexandrescu, D T; Vaillant, J G; Dasanu, C A

    2007-01-01

    Current treatment modalities for epidermal growth factor (EGFR)-positive cancers have recently included the use of antibodies and small-molecule tyrosine-kinase inhibitors (TKI). A significant limiting step in the use of these agents is dermatological toxicity, frequently in the form of an acneiform eruption. Present management modalities for this toxicity are largely ineffective. Colloidal oatmeal lotion demonstrates multiple anti-inflammatory properties with known effects on arachidonic acid, cytosolic phospholipase A2 and tumour necrosis factor-alpha pathways, along with an excellent side-effect profile. Treatment with colloidal oatmeal was applied to 11 patients with a rash induced by cetuximab, erlotinib, panitumumab and sorafenib. Of the 10 assessable patients, 6 had complete response and 4 partial response, giving a response rate of 100% with no associated toxicities. Treatment with colloidal oatmeal lotion is efficient in controlling the rash associated with EGFR and multiple TKI, and allows continuation of the antineoplastic treatment.

  16. Differential expression of dopamine D2 and D4 receptor and tyrosine hydroxylase mRNA in mice prone, or resistant, to chronic high-fat diet-induced obesity.

    Science.gov (United States)

    Huang, Xu-Feng; Yu, Yinghua; Zavitsanou, Katerina; Han, Mei; Storlien, Len

    2005-04-27

    The present study examined brain dopamine D2 and D4 receptor and tyrosine hydroxylase (TH) mRNA expression in chronic high-fat diet-induced obese (cDIO) and obese-resistant (cDR) mice. Twenty-eight mice were fed a high-fat diet (HF: 40% of calories from fat) for 6 weeks and then classified as cDIO (n = 8) or cDR (n = 8) mice according to the highest and lowest body weight gainers, respectively. Seven mice were fed a low-fat diet (LF: 10% of calories from fat) and used as controls. After 20 weeks of feeding, visceral fat per gram of initial body weight was significantly higher in the cDIO group (ratio: 0.25, 0.09, and 0.04; P AcbC, +16%) and ventral parts of caudate putamen (CPu, 21% and 24%) compared to the cDR and LF mice. The levels of D2 receptor mRNA expression in the AcbC and ventromedial part of the CPu were positively related to the final body weight. This study is the first to systematically examine the D4 mRNA expression in the mouse brain using in situ hybridization method. D4 receptor mRNA expression in the ventromedial hypothalamic nucleus (VMH) and the ventral part of the lateral septal nucleus were also significantly higher in the cDIO mice compared to the cDR and LF mice (+31% and +60%; P < 0.05). TH mRNA expression was significantly higher in the ventral tegmental area (+17%, P receptor and TH mRNA expression in specific brain regions of cDIO and cDR mice. It provides evidence that D4 receptors may play an important role influencing satiety via the mesohypothalamic pathway while the D2 receptor may regulate reward and motor centers via mesolimbic and nigrostriatal pathways. These findings contribute to the understanding of the role of these receptors in susceptibility, or resistance, to diet-induced obesity.

  17. Effect of hyperthyroidism on circulating prolactin and hypothalamic expression of tyrosine hydroxylase, prolactin signaling cascade members and estrogen and progesterone receptors during late pregnancy and lactation in the rat.

    Science.gov (United States)

    Pennacchio, Gisela E; Neira, Flavia J; Soaje, Marta; Jahn, Graciela A; Valdez, Susana R

    2017-02-15

    Hyperthyroidism (HyperT) compromises pregnancy and lactation, hindering suckling-induced PRL release. We studied the effect of HyperT on hypothalamic mRNA (RT-qPCR) and protein (Western blot) expression of tyrosine hydroxylase (TH), PRL receptor (PRLR) and signaling pathway members, estrogen-α (ERα) and progesterone (PR) receptors on late pregnancy (days G19, 20 and 21) and early lactation (L2) in rats. HyperT advanced pre-partum PRL release, reduced circulating PRL on L2 and increased TH mRNA (G21 and L2), p-TH, PRLR mRNA, STAT5 protein (G19 and L2), PRLR protein (G21) and CIS protein (G19). PRs mRNAs and protein decreased on G19 but afterwards PRA mRNA (G20), PRB mRNA (G21) and PRA mRNA and protein (L2) increased. ERα protein increased on G19 and decreased on G20. Thus, the altered hypothalamic PRLR, STAT5, PR and ERα expression in hyperthyroid rats may induce elevated TH expression and activation, that consequently, elevate dopaminergic tone during lactation, blunting suckling-induced PRL release and litter growth. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. The expression of a novel receptor-type tyrosine phosphatase suggests a role in morphogenesis and plasticity of the nervous system

    DEFF Research Database (Denmark)

    Canoll, P D; Barnea, G; Levy, J B

    1993-01-01

    . In the adult, high levels of RPTP-beta are seen in regions of the brain where there is continued neurogenesis and neurite outgrowth. The spatial and temporal patterns of RPTP-beta expression suggest that this receptor phosphatase plays a role in morphogenesis and plasticity of the nervous system....

  19. Identification of c-Src tyrosine kinase substrates using mass spectrometry and peptide microarrays

    DEFF Research Database (Denmark)

    Amanchy, Ramars; Zhong, Jun; Molina, Henrik

    2008-01-01

    c-Src tyrosine kinase plays a critical role in signal transduction downstream of growth factor receptors, integrins and G protein-coupled receptors. We used stable isotope labeling with amino acids in cell culture (SILAC) approach to identify additional substrates of c-Src tyrosine kinase in human...

  20. Risks of Proteinuria Associated with Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitors in Cancer Patients: A Systematic Review and Meta-Analysis

    Science.gov (United States)

    Liu, Li-Hua; Guo, Hui-Qin

    2014-01-01

    Background Vascular endothelial growth factor tyrosine-kinase inhibitors (VEGFR-TKIs) have emerged as an effective targeted therapy in the treatment of cancer patients, the overall incidence and risk of proteinuria associated these drugs is unclear. We performed a systematic review and meta-analysis of published clinical trials to quantify the incidence and risk of proteinuria associated with VEGFR-TKIs. Methodology Databases from PubMed, Web of Science and abstracts presented at ASCO meeting up to May 31, 2013 were searched to identify relevant studies. Eligible studies included prospective phase II and III trials evaluating VEGFR-TKIs in cancer patients with adequate data on proteinuria. Statistical analyses were conducted to calculate the summary incidence, Odds ratio (OR) and 95% confidence intervals (CIs) by using either random effects or fixed effect models according to the heterogeneity of included studies. Principal Findings A total of 6,882 patients with a variety of solid tumors from 33 clinical trials were included in our analysis. The incidence of all-grade and high-grade (grade 3 or higher) proteinuria was 18.7% (95% CI, 13.3%–25.6%) and 2.4% (95% CI, 1.6%–3.7%), respectively. Patients treated with VEGFR-TKIs had a significantly increased risk of all-grade (OR 2.92, 95%CI: 1.09–7.82, p = 0.033) and high-grade proteinuria (OR 1.97, 95%CI: 1.01–3.84, p = 0.046) when compared to patients treated with control medication. No evidence of publication bias was observed. Conclusions The use of VEGFR-TKIs is associated with a significant increased risk of developing proteinuria. Physicians should be aware of this adverse effect and should monitor cancer patients receiving VEGFR-TKIs. PMID:24621598

  1. A role for the non-receptor tyrosine kinase ACK1 in TNF-alpha-mediated apoptosis and proliferation in human intestinal epithelial caco-2 cells.

    Science.gov (United States)

    Zhao, Xinmei; Lv, Chaolan; Chen, Shengbo; Zhi, Fachao

    2017-09-16

    The roles of tumor necrosis factor alpha (TNF-alpha) and its mediators in cellular processes related to intestinal diseases remain elusive. In this study, we aimed to determine the biological role of activated Cdc42-associated kinase 1 (ACK1) in TNF-alpha-mediated apoptosis and proliferation in Caco-2 cells. ACK1 expression was knocked down using ACK1-specific siRNAs, and ACK1 activity was disrupted using a small molecule ACK1 inhibitor. The Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (TUNEL) and the BrdU incorporation assays were used to measure apoptosis and cell proliferation, respectively. ACK1-specific siRNA and the pharmacological ACK1 inhibitor significantly abrogated the TNF-alpha-mediated anti-apoptotic effects and proliferation of Caco-2 cells. Interestingly, TNF-alpha activated ACK1 at tyrosine 284 (Tyr284), and the ErbB family of proteins was implicated in ACK1 activation in Caco-2 cells. ACK1-Tyr284 was required for protein kinase B (AKT) activation, and ACK1 signaling was mediated through recruiting and phosphorylating the down-stream adaptor protein AKT, which likely promoted cell proliferation in response to TNF-alpha. Moreover, ACK1 activated AKT and Src enhanced nuclear factor-кB (NF-кB) activity, suggesting a correlation between NF-кB signaling and TNF-alpha-mediated apoptosis in Caco-2 cells. Our results demonstrate that ACK1 plays an important role in modulating TNF-alpha-induced aberrant cell proliferation and apoptosis, mediated in part by ACK1 activation. ACK1 and its down-stream effectors may hold promise as therapeutic targets in the prevention and treatment of gastrointestinal cancers, in particular, those induced by chronic intestinal inflammation. © 2017 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.

  2. Multilevel dysregulation of STAT3 activation in anaplastic lymphoma kinase-positive T/null-cell lymphoma

    DEFF Research Database (Denmark)

    Zhang, Qian; Raghunath, Puthryaveett N; Xue, Liquan

    2002-01-01

    , STAT3 was constitutively associated with NPM/ALK in the ALK+ TCL cell lines. Additional studies into the mechanisms of STAT3 activation revealed that the ALK+ TCL cells expressed a positive regulator of STAT3 activation, protein phosphatase 2A (PP2A), which was constitutively associated with STAT3....... Treatment with the PP2A inhibitor calyculin A abrogated tyrosine phosphorylation of STAT3. Finally, ALK+ T cells failed to express a negative regulator of activated STAT3, protein inhibitor of activated STAT3. These data indicate that NPM/ALK activates STAT3 and that PP2A and lack of protein inhibitor...

  3. 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...... metastases, by evaluating the expression of phosphorylated HER1, HER2, HER3, Erk, Akt and the total level of HER4 and HER2. METHODS: Immunohistochemical analysis was performed on 268 primary breast tumors and 30 paired metastatic lesions from postmenopausal women with hormone receptor-positive breast tumors...... 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...

  4. Regulation of cell adhesion by protein-tyrosine phosphatases: II. Cell-cell adhesion.

    Science.gov (United States)

    Sallee, Jennifer L; Wittchen, Erika S; Burridge, Keith

    2006-06-16

    Cell-cell adhesion is critical to the development and maintenance of multicellular organisms. The stability of many adhesions is regulated by protein tyrosine phosphorylation of cell adhesion molecules and their associated components, with high levels of phosphorylation promoting disassembly. The level of tyrosine phosphorylation reflects the balance between protein-tyrosine kinase and protein-tyrosine phosphatase activity. Many protein-tyrosine phosphatases associate with the cadherin-catenin complex, directly regulating the phosphorylation of these proteins, thereby affecting their interactions and the integrity of cell-cell junctions. Tyrosine phosphatases can also affect cell-cell adhesions indirectly by regulating the signaling pathways that control the activities of Rho family G proteins. In addition, receptor-type tyrosine phosphatases can mediate outside-in signaling through both ligand binding and dimerization of their extracellular domains. This review will discuss the role of protein-tyrosine phosphatases in cell-cell interactions, with an emphasis on cadherin-mediated adhesions.

  5. Dual regulation of receptor tyrosine kinase genes EGFR and c-Met by the tumor-suppressive microRNA-23b/27b cluster in bladder cancer.

    Science.gov (United States)

    Chiyomaru, Takeshi; Seki, Naohiko; Inoguchi, Satoru; Ishihara, Tomoaki; Mataki, Hiroko; Matsushita, Ryosuke; Goto, Yusuke; Nishikawa, Rika; Tatarano, Shuichi; Itesako, Toshihiko; Nakagawa, Masayuki; Enokida, Hideki

    2015-02-01

    Recent clinical trials of chemotherapeutics for advanced bladder cancer (BC) have shown limited benefits. Therefore, new prognostic markers and more effective treatment strategies are required. One approach to achieve these goals is through the analysis of RNA networks. Our recent studies of microRNA (miRNA) expression signatures revealed that the microRNA-23b/27b (miR-23b/27b) cluster is frequently downregulated in various types of human cancers. However, the functional role of the miR-23b/27b cluster in BC cells is still unknown. Thus, the aim of the present study was to investigate the functional significance of the miR-23b/27b cluster and its regulated molecular targets, with an emphasis on its contributions to BC oncogenesis and metastasis. The expression levels of the miR-23b/27b cluster were significantly reduced in BC clinical specimens. Restoration of mature miR-23b or miR-27b miRNAs significantly inhibited cancer cell migration and invasion, suggesting that these clustered miRNAs function as tumor suppressors. Gene expression data and in silico analysis demonstrated that the genes coding for the epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met) were potential targets of the miR-23b/27b cluster. Luciferase reporter assays and western blotting demonstrated that EGFR and c-Met receptor trypsine kinases were directly regulated by these clustered miRNAs. We conclude that the decreased expression of the tumor-suppressive miR-23b/27b cluster enhanced cancer cell proliferation, migration and invasion in BC through direct regulation of EGFR and c-Met signaling pathways. Our data on RNA networks regulated by tumor-suppressive miR-23b/27b provide new insights into the potential mechanisms of BC oncogenesis and metastasis.

  6. Synthesis and evaluation of 5-(arylthio)-9H-pyrimido[4,5-b]indole-2,4-diamines as receptor tyrosine kinase and thymidylate synthase inhibitors and as antitumor agents.

    Science.gov (United States)

    Zaware, Nilesh; Kisliuk, Roy; Bastian, Anja; Ihnat, Michael A; Gangjee, Aleem

    2017-04-01

    In an effort to optimize the structural requirements for combined cytostatic and cytotoxic effects in single agents, a series of 5-(arylthio)-9H-pyrimido[4,5-b]indole-2,4-diamines 3-7 were synthesized and evaluated as inhibitors of receptor tyrosine kinases (RTKs) as well as thymidylate synthase (TS). The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-bromo/5-chloro-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate aryl thiols. A novel four step synthetic scheme to the common intermediate was developed which is more efficient relative to the previously reported six-step sequence. Biological evaluation of these compounds indicated dual activity in RTKs and human TS (hTS). In the VEGFR-2 assay, compound 5 was equipotent to the standard compound semaxanib and was better than standard TS inhibitor pemetrexed, in the hTS assay. Compounds 3, 6 and 7 were nanomolar inhibitors of hTS and were several fold better than pemetrexed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Editor’s Pick: TYRO3, AXL, and MERTK Receptor Tyrosine Kinases: Is There Evidence of Direct Involvement in Development and Onset of Sjögren’s Syndrome?

    Directory of Open Access Journals (Sweden)

    Arun Wanchoo

    2016-07-01

    Full Text Available Sjögren’s syndrome (SjS is a chronic, progressive, systemic, human autoimmune disease in which an auto-inflammatory process within the salivary and lacrimal glands results in loss of saliva and tear production, respectively. In-depth analyses of the autoimmune process in humans and animal models of SjS substantiates one of the more important pathoaetiological pathways: an increased level of glandular apoptosis and/or cell lysis. We have hypothesised that failure in clearance of dying cells by macrophages, dendritic cells, and neighbouring tissues results in a sustained innate inflammatory response that transitions to autoimmunity. Since the intrinsic inhibition of inflammation following phagocytosis of dying cells is a function of a family of three receptor tyrosine kinases (RTKs known as the TAM (Tyro3, Axl, and Mertk, we put forward the following hypothesis: based on published information and analysis of our public microarray data, the failure of TAM RTK signalling, specifically in activating suppressor of cytokine signalling (SOCS 1 and SOCS3 (which are inhibitors of immune responses, may lead to autoimmunity, and specifically, to SjS-like disease.

  8. Targeted exome sequencing for the identification of a protective variant against Internet gaming disorder at rs2229910 of neurotrophic tyrosine kinase receptor, type 3 (NTRK3): A pilot study.

    Science.gov (United States)

    Kim, Jeong-Yu; Jeong, Jo-Eun; Rhee, Je-Keun; Cho, Hyun; Chun, Ji-Won; Kim, Tae-Min; Choi, Sam-Wook; Choi, Jung-Seok; Kim, Dai-Jin

    2016-12-01

    Background and aims Internet gaming disorder (IGD) has gained recognition as a potential new diagnosis in the fifth revision of the Diagnostic and Statistical Manual of Mental Disorders, but genetic evidence supporting this disorder remains scarce. Methods In this study, targeted exome sequencing was conducted in 30 IGD patients and 30 control subjects with a focus on genes linked to various neurotransmitters associated with substance and non-substance addictions, depression, and attention deficit hyperactivity disorder. Results rs2229910 of neurotrophic tyrosine kinase receptor, type 3 (NTRK3) was the only single nucleotide polymorphism (SNP) that exhibited a significantly different minor allele frequency in IGD subjects compared to controls (p = .01932), suggesting that this SNP has a protective effect against IGD (odds ratio = 0.1541). The presence of this potentially protective allele was also associated with less time spent on Internet gaming and lower scores on the Young's Internet Addiction Test and Korean Internet Addiction Proneness Scale for Adults. Conclusions The results of this first targeted exome sequencing study of IGD subjects indicate that rs2229910 of NTRK3 is a genetic variant that is significantly related to IGD. These findings may have significant implications for future research investigating the genetics of IGD and other behavioral addictions.

  9. Polymeric Immunoglobulin Receptor-mediated Invasion of Streptococcus pneumoniae into Host Cells Requires a Coordinate Signaling of SRC Family of Protein-tyrosine Kinases, ERK, and c-Jun N-terminal Kinase*

    Science.gov (United States)

    Agarwal, Vaibhav; Asmat, Tauseef M.; Dierdorf, Nina I.; Hauck, Christof R.; Hammerschmidt, Sven

    2010-01-01

    Streptococcus pneumoniae are commensals of the human nasopharynx with the capacity to invade mucosal respiratory cells. PspC, a pneumococcal surface protein, interacts with the human polymeric immunoglobulin receptor (pIgR) to promote bacterial adherence to and invasion into epithelial cells. Internalization of pneumococci requires the coordinated action of actin cytoskeleton rearrangements and the retrograde machinery of pIgR. Here, we demonstrate the involvement of Src protein-tyrosine kinases (PTKs), focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinases (MAPK) in pneumococcal invasion via pIgR. Pharmacological inhibitors of PTKs and MAPKs and genetic interference with Src PTK and FAK functions caused a significant reduction of pIgR-mediated pneumococcal invasion but did not influence bacterial adhesion to host cells. Furthermore, pneumococcal ingestion by host cells induces activation of ERK1/2 and JNK. In agreement with activated JNK, its target molecule and DNA-binding protein c-Jun was phosphorylated. We also show that functionally active Src PTK is essential for activation of ERK1/2 upon pneumococcal infections. In conclusion, these data illustrate the importance of a coordinated signaling between Src PTKs, ERK1/2, and JNK during PspC-pIgR-mediated uptake of pneumococci by host epithelial cells. PMID:20829350

  10. Polymeric immunoglobulin receptor-mediated invasion of Streptococcus pneumoniae into host cells requires a coordinate signaling of SRC family of protein-tyrosine kinases, ERK, and c-Jun N-terminal kinase.

    Science.gov (United States)

    Agarwal, Vaibhav; Asmat, Tauseef M; Dierdorf, Nina I; Hauck, Christof R; Hammerschmidt, Sven

    2010-11-12

    Streptococcus pneumoniae are commensals of the human nasopharynx with the capacity to invade mucosal respiratory cells. PspC, a pneumococcal surface protein, interacts with the human polymeric immunoglobulin receptor (pIgR) to promote bacterial adherence to and invasion into epithelial cells. Internalization of pneumococci requires the coordinated action of actin cytoskeleton rearrangements and the retrograde machinery of pIgR. Here, we demonstrate the involvement of Src protein-tyrosine kinases (PTKs), focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinases (MAPK) in pneumococcal invasion via pIgR. Pharmacological inhibitors of PTKs and MAPKs and genetic interference with Src PTK and FAK functions caused a significant reduction of pIgR-mediated pneumococcal invasion but did not influence bacterial adhesion to host cells. Furthermore, pneumococcal ingestion by host cells induces activation of ERK1/2 and JNK. In agreement with activated JNK, its target molecule and DNA-binding protein c-Jun was phosphorylated. We also show that functionally active Src PTK is essential for activation of ERK1/2 upon pneumococcal infections. In conclusion, these data illustrate the importance of a coordinated signaling between Src PTKs, ERK1/2, and JNK during PspC-pIgR-mediated uptake of pneumococci by host epithelial cells.

  11. Recombinant adeno-associated virus type 2-mediated gene transfer into human keratinocytes is influenced by both the ubiquitin/proteasome pathway and epidermal growth factor receptor tyrosine kinase.

    Science.gov (United States)

    Braun-Falco, Markus; Eisenried, Angelika; Büning, Hildegard; Ring, Johannes

    2005-05-01

    Efficient gene delivery into keratinocytes is a prerequisite for successful skin gene therapy. Vectors based on recombinant adeno-associated virus type 2 (rAAV-2) offer several promising features that make them attractive for cutaneous applications. However, highly efficient gene delivery may be hampered by different cellular factors, including lack of viral receptors, impairment of cytoplasmic trafficking or limitations in viral second-strand synthesis. This study was undertaken to find factors that influence rAAV-2-mediated in vitro gene transfer into human keratinocytes and, consequently, ways to optimize gene delivery. Transduction experiments using rAAV-2 vectors expressing green fluorescent protein (GFP) demonstrated that impaired cellular trafficking of vector particles and high levels of autophosphorylation at epidermal growth factor receptor tyrosine kinase (EGF-R TK) have a negative influence on gene transfer into keratinocytes. Treatment of keratinocytes with proteasome inhibitor MG132 resulted in a transient augmentation of GFP expression in up to 37% of cells. Treatment with EGF-R TK inhibitors (quinazoline type) enhanced transgene expression in 10-14.5% of the cells. Gene expression was stable for more than 10 weeks and persisted until proliferative senescence occurred. This stable gene expression allows speculation that keratinocyte stem cells have initially been transduced. These findings might have relevance for the use of rAAV-2 vectors in skin gene therapy: transient enhancement of rAAV-2 transduction with proteasome inhibitors might be useful for genetic promotion of wound healing or skin-directed vaccination. Treatment with quinazolines may increase rAAV-2 transduction of keratinocyte stem cells, which is important for gene therapy approaches to inherited diseases.

  12. The Transcriptional Response of Neurotrophins and Their Tyrosine Kinase Receptors in Lumbar Sensorimotor Circuits to Spinal Cord Contusion is Affected by Injury Severity and Survival Time

    Science.gov (United States)

    Hougland, M. Tyler; Harrison, Benjamin J.; Magnuson, David S. K.; Rouchka, Eric C.; Petruska, Jeffrey C.

    2013-01-01

    Traumatic spinal cord injury (SCI) results in changes to the anatomical, neurochemical, and physiological properties of cells in the central and peripheral nervous system. Neurotrophins, acting by binding to their cognate Trk receptors on target cell membranes, contribute to modulation of anatomical, neurochemical, and physiological properties of neurons in sensorimotor circuits in both the intact and injured spinal cord. Neurotrophin signaling is associated with many post-SCI changes including maladaptive plasticity leading to pain and autonomic dysreflexia, but also therapeutic approaches such as training-induced locomotor improvement. Here we characterize expression of mRNA for neurotrophins and Trk receptors in lumbar dorsal root ganglia (DRG) and spinal cord after two different severities of mid-thoracic injury and at 6 and 12 weeks post-SCI. There was complex regulation that differed with tissue, injury severity, and survival time, including reversals of regulation between 6 and 12 weeks, and the data suggest that natural regulation of neurotrophins in the spinal cord may continue for months after birth. Our assessments determined that a coordination of gene expression emerged at the 12-week post-SCI time point and bioinformatic analyses address possible mechanisms. These data can inform studies meant to determine the role of the neurotrophin signaling system in post-SCI function and plasticity, and studies using this signaling system as a therapeutic approach. PMID:23316162

  13. Suppressor of Cytokine Signaling (SOCS) 1 Inhibits Type I Interferon (IFN) Signaling via the Interferon α Receptor (IFNAR1)-associated Tyrosine Kinase Tyk2*

    Science.gov (United States)

    Piganis, Rebecca A. R.; De Weerd, Nicole A.; Gould, Jodee A.; Schindler, Christian W.; Mansell, Ashley; Nicholson, Sandra E.; Hertzog, Paul J.

    2011-01-01

    Type I IFNs are critical players in host innate and adaptive immunity. IFN signaling is tightly controlled to ensure appropriate immune responses as imbalance could result in uncontrolled inflammation or inadequate responses to infection. It is therefore important to understand how type I IFN signaling is regulated. Here we have investigated the mechanism by which suppressor of cytokine signaling 1 (SOCS1) inhibits type I IFN signaling. We have found that SOCS1 inhibits type I IFN signaling not via a direct interaction with the IFN α receptor 1 (IFNAR1) receptor component but through an interaction with the IFNAR1-associated kinase Tyk2. We have characterized the residues/regions involved in the interaction between SOCS1 and Tyk2 and found that SOCS1 associates via its SH2 domain with conserved phosphotyrosines 1054 and 1055 of Tyk2. The kinase inhibitory region of SOCS1 is also essential for its interaction with Tyk2 and inhibition of IFN signaling. We also found that Tyk2 is preferentially Lys-63 polyubiquitinated and that this activation reaction is inhibited by SOCS1. The consequent effect of SOCS1 inhibition of Tyk2 not only results in a reduced IFN response because of inhibition of Tyk2 kinase-mediated STAT signaling but also negatively impacts IFNAR1 surface expression, which is stabilized by Tyk2. PMID:21757742

  14. Suppressor of cytokine signaling (SOCS) 1 inhibits type I interferon (IFN) signaling via the interferon alpha receptor (IFNAR1)-associated tyrosine kinase Tyk2.

    Science.gov (United States)

    Piganis, Rebecca A R; De Weerd, Nicole A; Gould, Jodee A; Schindler, Christian W; Mansell, Ashley; Nicholson, Sandra E; Hertzog, Paul J

    2011-09-30

    Type I IFNs are critical players in host innate and adaptive immunity. IFN signaling is tightly controlled to ensure appropriate immune responses as imbalance could result in uncontrolled inflammation or inadequate responses to infection. It is therefore important to understand how type I IFN signaling is regulated. Here we have investigated the mechanism by which suppressor of cytokine signaling 1 (SOCS1) inhibits type I IFN signaling. We have found that SOCS1 inhibits type I IFN signaling not via a direct interaction with the IFN α receptor 1 (IFNAR1) receptor component but through an interaction with the IFNAR1-associated kinase Tyk2. We have characterized the residues/regions involved in the interaction between SOCS1 and Tyk2 and found that SOCS1 associates via its SH2 domain with conserved phosphotyrosines 1054 and 1055 of Tyk2. The kinase inhibitory region of SOCS1 is also essential for its interaction with Tyk2 and inhibition of IFN signaling. We also found that Tyk2 is preferentially Lys-63 polyubiquitinated and that this activation reaction is inhibited by SOCS1. The consequent effect of SOCS1 inhibition of Tyk2 not only results in a reduced IFN response because of inhibition of Tyk2 kinase-mediated STAT signaling but also negatively impacts IFNAR1 surface expression, which is stabilized by Tyk2.

  15. The expression of inflammatory cytokines, TAM tyrosine kinase receptors and their ligands is upregulated in venous leg ulcer patients: a novel insight into chronic wound immunity.

    Science.gov (United States)

    Filkor, Kata; Németh, Tibor; Nagy, István; Kondorosi, Éva; Urbán, Edit; Kemény, Lajos; Szolnoky, Győző

    2016-08-01

    The systemic host defence mechanisms, especially innate immunity, in venous leg ulcer patients are poorly investigated. The aim of the current study was to measure Candida albicans killing activity and gene expressions of pro- and anti-inflammatory cytokines and innate immune response regulators, TAM receptors and ligands of peripheral blood mononuclear cells separated from 69 venous leg ulcer patients and 42 control probands. Leg ulcer patients were stratified into responder and non-responder groups on the basis of wound healing properties. No statistical differences were found in Candida killing among controls, responders and non-responders. Circulating blood mononuclear cells of patients overexpress pro-inflammatory (IL-1α, TNFα, CXCL-8) and anti-inflammatory (IL-10) cytokines as well as TAM receptors (Tyro, Axl, MerTK) and their ligands Gas6 and Protein S compared with those of control individuals. IL-1α is notably overexpressed in venous leg ulcer treatment non-responders; in contrast, Axl gene expression is robustly stronger among responders. These markers may be considered as candidates for the prediction of treatment response among venous leg ulcer patients. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

  16. A Case of Orbital Metastasis as Disease Progression of Anaplastic Lymphoma Kinase-Positive Lung Cancer Treated with Crizotinib

    Directory of Open Access Journals (Sweden)

    Yoshihiko Sakata

    2015-01-01

    Full Text Available Orbital metastasis of lung cancer is rare. It often causes visual disorder. To date, there are only a few case reports. Crizotinib is an anaplastic lymphoma kinase (ALK tyrosine kinase inhibitor that leads to responses in most patients with ALK-positive non-small-cell lung cancer. Visual disorder is one of the popular adverse events of crizotinib, but the symptom almost decreases over time. We report a case of orbital metastasis as the disease progression of ALK-positive lung cancer treated with crizotinib. It should be kept in mind that orbital metastasis can be the disease progression of lung adenocarcinoma with ALK translocation treated with crizotinib. When physicians encounter a patient receiving crizotinib with visual disorder, we must distinguish between adverse events and orbital metastasis.

  17. A Src-family-tyrosine kinase, Lyn, is required for efficient IFN-β expression in pattern recognition receptor, RIG-I, signal pathway by interacting with IPS-1.

    Science.gov (United States)

    Lim, Young Ju; Koo, Jung Eun; Hong, Eun-Hee; Park, Zee-Yong; Lim, Kyung-Min; Bae, Ok-Nam; Lee, Joo Young

    2015-03-01

    Retinoic acid-inducible gene I (RIG-I) plays an important role in antiviral immunity as a cytosolic receptor recognizing invading viruses. The activation of downstream signaling pathways led by IFN-β promoter stimulator-1 (IPS-1), an adaptor, is known to culminate in the activation of IRFs and the expression of type I interferons. However, the role of Src-family-tyrosine kinases (STKs) in the RIG-I signaling pathway has not been fully evaluated. Through a combined approach of immunoprecipitation and micro reversed phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) analysis, we established that Lyn, one of the STKs, is associated with RIG-I in macrophages. The association of Lyn and RIG-I was confirmed by co-immunoprecipitation study with 293T cells overexpressing Lyn and RIG-I. Suppression of Lyn by siRNA knockdown or a pharmacological inhibitor (PP2) resulted in the attenuation of IRF3 activation and IFN-β expression induced by short poly I:C, a RIG-I agonist, in macrophages. Lyn activation, as determined by phosphorylation of Tyr396 residue, was observed upon short poly I:C stimulation in the mitochondria of macrophages. Short poly I:C induced the formation of speckle-like aggregates of Lyn, which are prominent in mitochondria. Lyn associated with IPS-1, an adaptor protein of RIG-I, which resides on mitochondria membrane. Helicase domain of RIG-I and CARD of IPS-1 are responsible for the interaction with Lyn while SH3 and SH2 domains in Lyn are required for the association with RIG-I and IPS-1. Collectively, our results indicate that Lyn plays a positive regulatory role in RIG-I-mediated interferon expression as a downstream component of IPS-1. They provide further information as to how tyrosine kinases such as STKs play a role in the regulation of antiviral immunity. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Distinct saturable pathways for the endocytosis of different tyrosine motifs.

    Science.gov (United States)

    Warren, R A; Green, F A; Stenberg, P E; Enns, C A

    1998-07-03

    Endocytosis of surface proteins through clathrin-coated pits requires an internalization signal in the cytoplasmic domain. Two types of internalization signal have been described: one requiring a tyrosine as the critical residue (tyrosine-based motif), and the other consisting of either two consecutive leucines or an isoleucine and leucine (dileucine motif). Although it seems that these signals are necessary and sufficient for endocytic targeting, the mechanism of recognition is not well understood. To examine this question, tetracycline-repressible cell lines were used to overexpress one of several receptors bearing a tyrosine-based internalization signal. By measuring the rates of endocytosis for either the overexpressed receptor, or that of other endogenous receptors, we were able to show that the endocytosis of identical receptors could be saturated, but a complete lack of competition exists between the transferrin receptor (TfR), the low-density lipoprotein receptor, and the epidermal growth factor receptor. Overexpression of any one of these receptors resulted in its redistribution toward the cell surface, implying that entry into coated pits is limited. During high levels of TfR expression, however, a significant increase in the amount of surface Lamp1, but not low-density lipoprotein receptor, epidermal growth factor receptor, or Lamp2, is detected. This suggests that Lamp1 and TfR compete for the same endocytic sites. Together, these results support the idea that there are at least three distinct saturable components involved in clathrin-mediated endocytosis.

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

    Science.gov (United States)

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

  20. 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...

  1. Postnatal Development of Brain-Derived Neurotrophic Factor (BDNF) and Tyrosine Protein Kinase B (TrkB) Receptor Immunoreactivity in Multiple Brain Stem Respiratory-Related Nuclei of the Rat

    Science.gov (United States)

    Liu, Qiuli; Wong-Riley, Margaret T.T.

    2013-01-01

    Previously, we found a transient imbalance between suppressed excitation and enhanced inhibition in the respiratory network of the rat around postnatal days (P) 12–13, a critical period when the hypoxic ventilatory response is at its weakest. The mechanism underlying the imbalance is poorly understood. Brain-derived neurotrophic factor (BDNF) and its tyrosine protein kinase B (TrkB) receptors are known to potentiate glutamatergic and attenuate gamma-aminobutyric acid (GABA)ergic neurotransmission, and BDNF is essential for respiratory development. We hypothesized that the excitation-inhibition imbalance during the critical period stemmed from a reduced expression of BDNF and TrkB at that time within respiratory-related nuclei of the brain stem. An in-depth, semiquantitative immunohistochemical study was undertaken in seven respiratory-related brain stem nuclei and one nonrespiratory nucleus in P0–21 rats. The results indicate that the expressions of BDNF and TrkB: 1) in the pre-Bötzinger complex, nucleus ambiguus, commissural and ventrolateral subnuclei of solitary tract nucleus, and retrotrapezoid nucleus/parafacial respiratory group were significantly reduced at P12, but returned to P11 levels by P14; 2) in the lateral paragigantocellular nucleus and parapyramidal region were increased from P0 to P7, but were strikingly reduced at P10 and plateaued thereafter; and 3) in the nonrespiratory cuneate nucleus showed a gentle plateau throughout the first 3 post-natal weeks, with only a slight decline of BDNF expression after P11. Thus, the significant downregulation of both BDNF and TrkB in respiratory-related nuclei during the critical period may form the basis of, or at least contribute to, the inhibitory-excitatory imbalance within the respiratory network during this time. PMID:22678720

  2. Melatonin sensitizes H1975 non-small-cell lung cancer cells harboring a T790M-targeted epidermal growth factor receptor mutation to the tyrosine kinase inhibitor gefitinib.

    Science.gov (United States)

    Yun, Miyong; Kim, Eun-Ok; Lee, Duckgue; Kim, Ji-Hyun; Kim, Jaekwang; Lee, Hyemin; Lee, Jihyun; Kim, Sung-Hoon

    2014-01-01

    The use of tyrosine kinase inhibitors (TKIs) to target active epidermal growth factor receptor (EGFR)-harbouring mutations has been effective in patients with advanced non-small-cell lung cancer (NSCLC). However, the use of TKIs in NSCLS patients with somatic EGFR mutations, particularly T790M, causes drug resistance. Thus, in the present study, we investigated overcoming resistance against the TKI gefitinib by combination treatment with melatonin in H1975 NSCLC cells harbouring the T790M somatic mutation. H1975 and HCC827 cells were treated with melatonin in combination with gefitinib, and cell viability, cell cycle progression, apoptosis, and EGFR, AKT, p38, Bcl-2, Bcl-xL, caspase 3 and Bad protein levels were examined. Treatment with melatonin dose-dependently decreased the viability of H1975 cells harbouring the T790M somatic mutation compared to HCC827 cells with an EGFR active mutation. Melatonin-mediated cell death resulted in decreased phosphorylation of EGFR and Akt, leading to attenuated expression of survival proteins, such as Bcl-2, Bcl-xL and survivin, and activated caspase 3 in H1975 cells, but not in HCC827 cells. However, we did not observe a significant change in expression of cell cycle proteins, such as cyclin D, cyclin A, p21 and CDK4 in H1975 cells. Surprisingly, co-treatment of gefitinib with melatonin effectively decreased the viability of H1975 cells, but not HCC827 cells. Moreover, co-treatment of H1975 cells caused consistent down-regulation of EGFR phosphorylation and induced apoptosis compared to treatment with gefitinib or melatonin alone. Our findings demonstrate that melatonin acts as a potent chemotherapeutic agent by sensitising to gefitinib TKI-resistant H1975 cells that harbour a EGFR T790M mutation. © 2014 S. Karger AG, Basel.

  3. Melatonin Sensitizes H1975 Non-Small-Cell Lung Cancer Cells Harboring a T790M-Targeted Epidermal Growth Factor Receptor Mutation to the Tyrosine Kinase Inhibitor Gefitinib

    Directory of Open Access Journals (Sweden)

    Miyong Yun

    2014-08-01

    Full Text Available Background/Aims: The use of tyrosine kinase inhibitors (TKIs to target active epidermal growth factor receptor (EGFR-harbouring mutations has been effective in patients with advanced non-small-cell lung cancer (NSCLC. However, the use of TKIs in NSCLS patients with somatic EGFR mutations, particularly T790M, causes drug resistance. Thus, in the present study, we investigated overcoming resistance against the TKI gefitinib by combination treatment with melatonin in H1975 NSCLC cells harbouring the T790M somatic mutation. Methods: H1975 and HCC827 cells were treated with melatonin in combination with gefitinib, and cell viability, cell cycle progression, apoptosis, and EGFR, AKT, p38, Bcl-2, Bcl-xL, caspase 3 and Bad protein levels were examined. Results: Treatment with melatonin dose-dependently decreased the viability of H1975 cells harbouring the T790M somatic mutation compared to HCC827 cells with an EGFR active mutation. Melatonin-mediated cell death resulted in decreased phosphorylation of EGFR and Akt, leading to attenuated expression of survival proteins, such as Bcl-2, Bcl-xL and survivin, and activated caspase 3 in H1975 cells, but not in HCC827 cells. However, we did not observe a significant change in expression of cell cycle proteins, such as cyclin D, cyclin A, p21 and CDK4 in H1975 cells. Surprisingly, co-treatment of gefitinib with melatonin effectively decreased the viability of H1975 cells, but not HCC827 cells. Moreover, co-treatment of H1975 cells caused consistent down-regulation of EGFR phosphorylation and induced apoptosis compared to treatment with gefitinib or melatonin alone. Conclusions: Our findings demonstrate that melatonin acts as a potent chemotherapeutic agent by sensitising to gefitinib TKI-resistant H1975 cells that harbour a EGFR T790M mutation.

  4. Phase I/II trial evaluating the anti-vascular endothelial growth factor monoclonal antibody bevacizumab in combination with the HER-1/epidermal growth factor receptor tyrosine kinase inhibitor erlotinib for patients with recurrent non-small-cell lung cancer.

    Science.gov (United States)

    Herbst, Roy S; Johnson, David H; Mininberg, Eric; Carbone, David P; Henderson, Ted; Kim, Edward S; Blumenschein, George; Lee, Jack J; Liu, Diane D; Truong, Mylene T; Hong, Waun K; Tran, Hai; Tsao, Anne; Xie, Dong; Ramies, David A; Mass, Robert; Seshagiri, Somasekar; Eberhard, David A; Kelley, Sean K; Sandler, Alan

    2005-04-10

    Bevacizumab (Avastin; Genentech, South San Francisco, CA) is a recombinant, humanized anti-vascular endothelial growth factor monoclonal antibody. Erlotinib HCl (Tarceva, OSI-774; OSI Pharmaceuticals, New York, NY) is a potent, reversible, highly selective and orally available HER-1/epidermal growth factor receptor tyrosine kinase inhibitor. Preclinical data in various xenograft models produced greater growth inhibition than with either agent alone. Additionally, both agents have demonstrated benefit in patients with previously treated non-small-cell lung cancer (NSCLC). A phase I/II study in two centers examined erlotinib and bevacizumab (A+T) in patients with nonsquamous stage IIIB/IV NSCLC with > or = one prior chemotherapy. In phase I, erlotinib 150 mg/day orally plus bevacizumab 15 mg/kg intravenously every 21 days was established as the phase II dose, although no dose-limiting toxicities were observed. Phase II assessed the efficacy and tolerability of A+T at this dose. Pharmacokinetic parameters were evaluated. ResultsForty patients were enrolled and treated in this study (34 patients at phase II dose); the median age was 59 years (range, 36 to 72 years), 21 were female, 30 had adenocarcinoma histology, nine were never-smokers, and 22 had > or = two prior regimens (three patients had > or = four prior regimens). The most common adverse events were mild to moderate rash, diarrhea, and proteinuria. Preliminary data showed no pharmacokinetic interaction between A + T. Eight patients (20.0%; 95% CI, 7.6% to 32.4%) had partial responses and 26 (65.0%; 95% CI, 50.2% to 79.8%) had stable disease as their best response. The median overall survival for the 34 patients treated at the phase II dose was 12.6 months, with progression-free survival of 6.2 months. Encouraging antitumor activity and safety of A + T support further development of this combination for patients with advanced NSCLC and other solid tumors.

  5. Impact of Specific Epidermal Growth Factor Receptor (EGFR) Mutations and Clinical Characteristics on Outcomes After Treatment With EGFR Tyrosine Kinase Inhibitors Versus Chemotherapy in EGFR-Mutant Lung Cancer: A Meta-Analysis.

    Science.gov (United States)

    Lee, Chee Khoon; Wu, Yi-Long; Ding, Pei Ni; Lord, Sarah J; Inoue, Akira; Zhou, Caicun; Mitsudomi, Tetsuya; Rosell, Rafael; Pavlakis, Nick; Links, Matthew; Gebski, Val; Gralla, Richard J; Yang, James Chih-Hsin

    2015-06-10

    We examined the impact of different epidermal growth factor receptor (EGFR) mutations and clinical characteristics on progression-free survival (PFS) in patients with advanced EGFR-mutated non-small-cell lung cancer treated with EGFR tyrosine kinase inhibitors (TKIs) as first-line therapy. This meta-analysis included randomized trials comparing EGFR TKIs with chemotherapy. We calculated hazard ratios (HRs) and 95% CIs for PFS for the trial population and prespecified subgroups and calculated pooled estimates of treatment efficacy using the fixed-effects inverse-variance-weighted method. All statistical tests were two sided. In seven eligible trials (1,649 patients), EGFR TKIs, compared with chemotherapy, significantly prolonged PFS overall (HR, 0.37; 95% CI, 0.32 to 0.42) and in all subgroups. For tumors with exon 19 deletions, the benefit was 50% greater (HR, 0.24; 95% CI, 0.20 to 0.29) than for tumors with exon 21 L858R substitution (HR, 0.48; 95% CI, 0.39 to 0.58; Pinteraction < .001). Never-smokers had a 36% greater benefit (HR, 0.32; 95% CI, 0.27 to 0.37) than current or former smokers (HR, 0.50; 95% CI, 0.40 to 0.63; Pinteraction < .001). Women had a 27% greater benefit (HR, 0.33; 95% CI, 0.28 to 0.38) than men (HR, 0.45; 95% CI, 0.36 to 0.55; treatment-sex interaction P = .02). Performance status, age, ethnicity, and tumor histology did not significantly predict additional benefit from EGFR TKIs. Although EGFR TKIs significantly prolonged PFS overall and in all subgroups, compared with chemotherapy, greater benefits were observed in those with exon 19 deletions, never-smokers, and women. These findings should enhance drug development and economic analyses, as well as the design and interpretation of clinical trials. © 2015 by American Society of Clinical Oncology.

  6. Exploring oxidative modifications of tyrosine

    DEFF Research Database (Denmark)

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

    2015-01-01

    Protein oxidation is increasingly recognised as an important modulator of biochemical pathways controlling both physiological and pathological processes. While much attention has focused on cysteine modifications in reversible redox signalling, there is increasing evidence that other protein...... 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...

  7. Effect of Gingerol on Cisplatin-Induced Pica Analogous to Emesis Via Modulating Expressions of Dopamine 2 Receptor, Dopamine Transporter and Tyrosine Hydroxylase in the Vomiting Model of Rats.

    Science.gov (United States)

    Qian, Weibin; Cai, Xinrui; Wang, Yingying; Zhang, Xinying; Zhao, Hongmin; Qian, Qiuhai; Yang, Zhihong; Liu, Zhantao; Hasegawa, Junichi

    2016-06-01

    Gingerol, the generic term for pungent constituents in ginger, has been used for treating vomiting in China. We are going to investigate the mechanisms of inhibitive effect of gingerol on cisplatin-induced pica behaviour by studying on both peripheral and central levels, and the effects of gingerol on homeostasis of dopamine (DA) transmission: dopamine D2 receptor (D2R), dopamine transporter (DAT) and tyrosine hydroxylase (TH). The antiemetic effect of gingerol was investigated on a vomiting model in rats induced by cisplatin 3 mg·kg(-1) intraperitoneal injection (i.p.). Rats were randomly divided into the normal control group (C), simple gingerol control group (CG), cisplatin control group (V), cisplatin + metoclopramide group (M), cisplatin + low-dose gingerol group (GL), cisplatin + middle-dose gingerol group (GM) and cisplatin + high-dose gingerol group (GH). In observation period, rats in Groups C and V were pretreated with sterile saline 3 mL i.g.; rats in Group CG were pretreated with gingerol 40 mg·kg(-1) i.g.; rats in Group M were pretreated with metoclopramide 2.5 mg·kg(-1) i.g.; rats in Groups GL, GM and GH were pretreated with gingerol 10, 20 and 40 mg·kg(-1) i.g. for 3 days, respectively. Cisplatin (3 mg·kg(-1), i.p.) was administered one time after each treatment with the antiemetic agent or its vehicle except the Groups C and CG. The distribution of D2R, DAT and TH in the area postrema and ileum were measured by immunohistochemistry and quantitated based on the image analysis, and the expression of DAT and TH in the area postrema and ileum were measured by RT-PCR. The weights of kaolin eaten of the remaining rats were observed in every 6 h continuously for 72 h. The weight of kaolin eaten in rats induced by cisplatin was significantly reduced by pretreatment with gingerol in a dose-dependent manner during the 0-24 h and 24-72 h periods (P Gingerol markedly improved gastric emptying induced by cisplatin in a dose-dependent manner (P Gingerol is

  8. The Efficacy of Single-Agent Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Therapy in Biologically Selected Patients with Non-Small-Cell Lung Cancer: A Meta-Analysis of 19 Randomized Controlled Trials.

    Science.gov (United States)

    Li, Guifang; Gao, Shunji; Sheng, Zhixin; Li, Bin

    2016-01-01

    To determine the efficacy of first-generation single-agent epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy in advanced non-small-cell lung cancer patients with known EGFR mutation status, we undertook this pooled analysis. We searched for randomized controlled trials (RCTs) in Medline, Embase, the Cochrane Controlled Trials Register, the Science Citation Index, and the American Society of Clinical Oncology annual meetings. Out of 2,129 retrieved articles, 19 RCTs enrolling 2,016 patients with wild-type EGFR tumors and 1,034 patients with mutant EGFR tumors were identified. For these EGFR mutant patients, single-agent EGFR-TKI therapy improved progression-free survival (PFS) over chemotherapy: the summary hazard ratios (HRs) were 0.41 (p chemotherapy in the first-line setting (HR = 1.65, p = 0.03) and in the second-/third-line setting (HR = 1.27, p = 0.006). No statistically significant difference was observed in terms of overall survival (OS). Using platinum-based doublet chemotherapy as a common comparator, indirect comparison showed the superior efficacy of single-agent EGFR-TKI therapy over EGFR-TKIs added to chemotherapy in PFS [HR = 1.35 (1.03, 1.77), p = 0.03]. Additionally, a marginal trend towards the same direction was found in the OS analysis [HR = 1.16 (0.99, 1.35), p = 0.06]. Interestingly, for those EGFR wild-type tumors, single-agent EGFR-TKI therapy was inferior to EGFR-TKIs added to chemotherapy in PFS [HR = 0.38 (0.33, 0.44), p chemotherapy. However, single-agent EGFR-TKI therapy was inferior to chemotherapy in PFS for those EGFR wild-type patients. Single-agent EGFR-TKI therapy could improve PFS over the combination of EGFR-TKIs and chemotherapy in these EGFR mutant patients. However, EGFR-TKIs combined with chemotherapy could provide additive PFS and OS benefit over single-agent EGFR-TKI therapy in those EGFR wild-type patients. © 2016 S. Karger AG, Basel.

  9. 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.

  10. 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.

  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. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten

    2010-01-01

    enzymes that are unique in exploiting the ATP/GTP-binding Walker motif to catalyze phosphorylation of protein tyrosine residues. Characterized for the first time only a decade ago, BY-kinases have now come to the fore. Important regulatory roles have been linked with these enzymes, via their involvement......Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine...

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

    Science.gov (United States)

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

    2016-01-01

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

  14. Jak2 tyrosine kinase: a true jak of all trades?

    Science.gov (United States)

    Sandberg, Eric M; Wallace, Tiffany A; Godeny, Michael D; VonDerLinden, Danielle; Sayeski, Peter P

    2004-01-01

    Discovered roughly 10 yr ago, Jak2 tyrosine kinase has emerged as a critical molecule in mammalian development, physiology, and disease. Here, we review the early history of Jak2 and its role in health and disease. We will also review its critical role in mediating cytokine-dependent signal transduction. Additionally, more recent work demonstrating the importance of Jak2 in G protein-coupled receptor and tyrosine kinase growth factor receptor signal transduction will be discussed. The cellular and biochemical mechanisms by which Jak2 tyrosine kinase is activated and regulated within the cell also will be reviewed. Finally, structure-function and pharmacological-based studies that identified structural motifs and amino acids within Jak2 that are critical for its function will be examined. By reviewing the biology of Jak2 tyrosine kinase at the molecular, cellular, and physiological levels, we hope to advance the understanding of how a single gene can have such a profound impact on development, physiology, and disease.

  15. Wnt signaling through the Ror receptor in the nervous system

    NARCIS (Netherlands)

    Petrova, Iveta M; Malessy, Martijn J; Verhaagen, J.; Fradkin, Lee G; Noordermeer, Jasprina N

    The receptor tyrosine kinase-like orphan receptor (Ror) proteins are conserved tyrosine kinase receptors that play roles in a variety of cellular processes that pattern tissues and organs during vertebrate and invertebrate development. Ror signaling is required for skeleton and neuronal development

  16. 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

  17. Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis

    DEFF Research Database (Denmark)

    Confalonieri, S; Salcini, A E; Puri, C

    2000-01-01

    Membrane receptors are internalized either constitutively or upon ligand engagement. Whereas there is evidence for differential regulation of the two processes, little is known about the molecular machinery involved. Previous studies have shown that an unidentified kinase substrate is required fo...... determinant, other than those contained in the receptors themselves, which is involved in the differential regulation of constitutive vs. regulated endocytosis....... for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function...... of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular...

  18. 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

  19. Non-Targeted Metabolomics Analysis of the Effects of Tyrosine Kinase Inhibitors Sunitinib and Erlotinib on Heart, Muscle, Liver and Serum Metabolism In Vivo

    OpenAIRE

    Jensen, Brian C; Parry, Traci L; Wei Huang; Amro Ilaiwy; Bain, James R; Muehlbauer, Michael J.; O’Neal, Sara K.; Cam Patterson; Johnson, Gary L; Monte S. Willis

    2017-01-01

    Background: More than 90 tyrosine kinases have been implicated in the pathogenesis of malignant transformation and tumor angiogenesis. Tyrosine kinase inhibitors (TKIs) have emerged as effective therapies in treating cancer by exploiting this kinase dependency. The TKI erlotinib targets the epidermal growth factor receptor (EGFR), whereas sunitinib targets primarily vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR).TKIs that impact the fun...

  20. Marine sponge polyketide inhibitors of protein tyrosine kinase.

    Science.gov (United States)

    Lee, R H; Slate, D L; Moretti, R; Alvi, K A; Crews, P

    1992-04-30

    The marine polyketide natural product, halenaquinone, was shown to be an irreversible inhibitor of pp60v-src, the oncogenic protein tyrosine kinase encoded by the Rous sarcoma virus. This compound had an IC50 of approximately 1.5 microM against pp60v-src and also inhibited the ligand-stimulated kinase activity of the human epidermal growth factor receptor with an IC50 of approximately 19 microM. Halenaquinone blocked the proliferation of a number of cultured cell lines, including several transformed by oncogenic protein tyrosine kinases. Halenaquinol, xestoquinone, halenaquinol sulfate, and several simple synthetic quinone analogs were also shown to inhibit pp60v-src.

  1. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti

    2006-01-01

    Full Text Available Se trata de un estudio etnográfico, que tuvo lo objetivo de interpretar el sistema de conocimiento y del significado atribuidos a la sangre referente a la transfusión sanguínea por los donadores y receptores de un banco de sangre. Para la colecta de las informaciones se observaron los participantes y la entrevista etnográfica se realizó el análisis de dominio, taxonómicos y temáticos. Los dominios culturales fueron: la sangre es vida: fuente de vida y alimento valioso; creencias religiosas: fuentes simbólicas de apoyos; donación sanguínea: un gesto colaborador que exige cuidarse, gratifica y trae felicidad; donación sanguínea: fuente simbólica de inseguridad; estar enfermo es una condición para realizar transfusión sanguínea; transfusión sanguínea: esperanza de vida; Creencias populares: transfusión sanguínea como riesgo para la salud; donadores de sangre: personas benditas; donar y recibir sangre: como significado de felicidad. Temática: “líquido precioso que origina, sostiene, modifica la vida, provoca miedo e inseguridad”.

  2. Structure-based network analysis of activation mechanisms in the ErbB family of receptor tyrosine kinases: the regulatory spine residues are global mediators of structural stability and allosteric interactions.

    Directory of Open Access Journals (Sweden)

    Kevin A James

    Full Text Available The ErbB protein tyrosine kinases are among the most important cell signaling families and mutation-induced modulation of their activity is associated with diverse functions in biological networks and human disease. We have combined molecular dynamics simulations of the ErbB kinases with the protein structure network modeling to characterize the reorganization of the residue interaction networks during conformational equilibrium changes in the normal and oncogenic forms. Structural stability and network analyses have identified local communities integrated around high centrality sites that correspond to the regulatory spine residues. This analysis has provided a quantitative insight to the mechanism of mutation-induced "superacceptor" activity in oncogenic EGFR dimers. We have found that kinase activation may be determined by allosteric interactions between modules of structurally stable residues that synchronize the dynamics in the nucleotide binding site and the αC-helix with the collective motions of the integrating αF-helix and the substrate binding site. The results of this study have pointed to a central role of the conserved His-Arg-Asp (HRD motif in the catalytic loop and the Asp-Phe-Gly (DFG motif as key mediators of structural stability and allosteric communications in the ErbB kinases. We have determined that residues that are indispensable for kinase regulation and catalysis often corresponded to the high centrality nodes within the protein structure network and could be distinguished by their unique network signatures. The optimal communication pathways are also controlled by these nodes and may ensure efficient allosteric signaling in the functional kinase state. Structure-based network analysis has quantified subtle effects of ATP binding on conformational dynamics and stability of the EGFR structures. Consistent with the NMR studies, we have found that nucleotide-induced modulation of the residue interaction networks is not

  3. Crizotinib for Untreated Anaplastic Lymphoma Kinase-Positive Non-Small-Cell Lung Cancer: An Evidence Review Group Perspective of a NICE Single Technology Appraisal.

    Science.gov (United States)

    Morgan, Philip; Woolacott, Nerys; Biswas, Mousumi; Mebrahtu, Teumzghi; Harden, Melissa; Hodgson, Robert

    2017-09-01

    As part of the National Institute for Health and Care Excellence (NICE) single technology appraisal process, the manufacturer of crizotinib submitted evidence on the clinical and cost effectiveness of crizotinib in untreated anaplastic lymphoma kinase-positive (ALK-positive) non-small-cell lung cancer (NSCLC). Crizotinib has previously been assessed by NICE for patients with previously treated ALK-positive NSCLC (TA 296). It was not approved in this previous appraisal, but had been made available through the cancer drugs fund. As part of this new appraisal, the company included a price discount patient access scheme (PAS). The Centre for Reviews and Dissemination and Centre for Health Economics Technology Appraisal Group at the University of York was commissioned to act as the independent Evidence Review Group (ERG). This article provides a description of the company's submission and the ERG's review and summarises the resulting NICE guidance issued in August 2016. The main clinical-effectiveness data were derived from a multicentre randomised controlled trial-PROFILE 1014-that compared crizotinib with pemetrexed chemotherapy in combination with carboplatin or cisplatin in patients with untreated non-squamous ALK-positive NSCLC. In the trial, crizotinib demonstrated improvements in progression-free survival (PFS) and overall survival (OS). The company's economic model was a three-state 'area under the curve' Markov model. The base-case incremental cost-effectiveness ratio (ICER) was estimated to be greater than £50,000 per quality-adjusted life-year (QALY) gained (excluding the PAS discount). The ERG assessment of the evidence submitted by the company raised a number of concerns. In terms of the clinical evidence, the OS benefit was highly uncertain due to the cross-over permitted in the trial and the immaturity of the data; only 26% of events had occurred by the data cut-off point. In the economic modelling, the most significant concerns related to the analysis

  4. Epidermal growth factor receptor and cancer: control of oncogenic signalling by endocytosis

    DEFF Research Database (Denmark)

    Grandal, Michael Vibo; Madshus, I.H.

    2008-01-01

    The epidermal growth factor receptor (EGFR) and other members of the EGFR/ErbB receptor family of receptor tyrosine kinases (RTKs) are important regulators of proliferation, angiogenesis, migration, tumorigenesis and metastasis. Overexpression, mutations, deletions and production of autocrine lig...

  5. Dose-Dependent Effects of Oral Tyrosine Administration on Plasma Tyrosine Levels and Cognition in Aging

    Directory of Open Access Journals (Sweden)

    Ondine van de Rest

    2017-11-01

    Full Text Available The effects of tyrosine on plasma response and cognition in aging are unknown. We assessed the dose-dependent response to tyrosine administration in older adults in both plasma tyrosine concentrations and working memory performance. In this double blind randomized cross-over trial 17 older adults (aged 60–75 years received a single administration of 100, 150, or 200 mg/kg body weight of tyrosine. For comparison, 17 young adults (aged 18–35 years received a dose of 150 mg/kg body weight of tyrosine. Tyrosine plasma concentrations were determined before and 90, 120, 150, 180, 210, and 240 min after tyrosine intake. Working memory was assessed using the N-back task at 90 min after tyrosine administration. Older adults showed a dose-dependent increase in plasma tyrosine concentrations (p < 0.001, and the plasma response was higher than for young adults with the same dose (p < 0.001. Load-dependent working memory performance decreased with higher doses of tyrosine (p = 0.048, especially in older adults with greater dose-dependent plasma tyrosine responses (p = 0.035. Our results show an age-related increase in plasma tyrosine response, which was associated with a dose-dependent decline in cognitive functioning in older adults.

  6. Dose-dependent effects of oral tyrosine administration on plasma tyrosine levels and cognition in aging

    NARCIS (Netherlands)

    Rest, van de Ondine; Bloemendaal, Mirjam; Heus, De Rianne; Aarts, Esther

    2017-01-01

    The effects of tyrosine on plasma response and cognition in aging are unknown. We assessed the dose-dependent response to tyrosine administration in older adults in both plasma tyrosine concentrations and working memory performance. In this double blind randomized cross-over trial 17 older adults

  7. [Research progress of several protein tyrosine phosphatases in diabetes].

    Science.gov (United States)

    Chen, Ming; Sun, Jin-Peng; Liu, Jing; Yu, Xiao

    2010-04-25

    Diabetes mellitus is caused by deficiency of insulin secretion from the pancreatic islet beta cells and/or insulin resistance in liver, muscle and adipocytes, resulting in glucose intolerance and hyperglycemia. Several protein tyrosine phosphatases, such as PTP1B (PTPN1), TCPTP (PTPN2), LYP (PTPN22), PTPIA-2, PTPMEG2 (PTPN9) or OSTPTP are involved in insulin signaling pathway, insulin secretion and autoreactive attack to pancreatic beta cells. Genetic mutation or overexpression of these phosphotases has been found to cause or increase the risk of diabetes mellitus. Some population with high risk for type 2 diabetes has overexpressed PTP1B, a prototypical tyrosine phosphatase which down-regulates insulin and leptin signal transduction. Animal PTP1B knockout model and PTP1B specific inhibitor cellular studies indicate PTP1B may serve as a therapeutic target for type 2 diabetes. TCPTP shares more than 70% sequence identity with PTP1B in their catalytic domain. TCPTP dephosphorylates tyrosine phosphorylated substrates overlapping with PTP1B but also has its own distinct dephosphorylation sites and functions. Recent research indicates TCPTP may have role in type 1 diabetes via dysregultaion of cytokine-mediated immune responses or pancreatic beta cell apoptosis. The tyrosine phosphatase LYP, which down-regulates LCK activity in T cell response, can become mutated as R620W which is highly correlated to type 1 diabetes. LYP R620W may be a gain of function mutation which suppresses TCR signaling. Patients bearing the R620W mutant have impaired T cell responses and increased populations of (CD45RO+CD45RA-) CD4+ T cells. A detailed elucidation of mechanism of R620W in type 1 diabetes and specific LYP inhibitor development will help characterize LYP R620W as a therapeutic target. A receptor tyrosine phosphatase, PTPIA-2/beta is a major autoantigen of type 1 diabetes. A diagnosis kit identifying PTPIA-2/beta autoantibodies is valuable in early detection and prevention of type

  8. The Effects of Four Different Tyrosine Kinase Inhibitors on Medullary and Papillary Thyroid Cancer Cells

    NARCIS (Netherlands)

    Verbeek, Hans H. G.; Alves, Maria M.; de Groot, Jan-Willem B.; Osinga, Jan; Plukker, John T. M.; Links, Thera P.; Hofstra, Robert M. W.

    Context: Medullary and papillary thyroid carcinoma (MTC and PTC) are two types of thyroid cancer that can originate from activating mutations or rearrangements in the RET gene. Therapeutic options are limited in recurrent disease, but because RET is a tyrosine kinase (TK) receptor involved in

  9. Interleukin-2 induces tyrosine phosphorylation and nuclear translocation of stat3 in human T lymphocytes

    DEFF Research Database (Denmark)

    Nielsen, M; Svejgaard, A; Skov, S

    1994-01-01

    An early biochemical event associated with T cell activation through the interleukin-2 receptor (IL-2R) is tyrosine phosphorylation of several intracellular substrates. The exact mechanism by which IL-2 regulates transcription of different genes is presently unknown. Here, we report that stimulat...

  10. Tyrosine kinase inhibitors in hematological malignancies

    Directory of Open Access Journals (Sweden)

    Kamila Kosior

    2011-12-01

    Full Text Available Recently novel treatment modalities has focused on targeted therapies. Tyrosine kinases represent a good target for cancer treatment since they are involved in transferring phosphate groups from ATP to tyrosine residues in specific substrate proteins transducing intracellular signals engaged in the many mechanisms, playing an important role in the modulation of growth factors signaling that are strongly related to carcinogenesis. Deregulation of tyrosine kinases activity was also found in hematological malignancies, particularly overexpression of tyrosine kinases was observed in chronic myeloid leukemia or acute lymphoblastic leukemia. Herein we show that tyrosine kinase inhibitors have revolutionized hematology malignancies therapy in a very short period of time and they still remain one of the most interesting anticancer compounds that could give a hope for cure and not only long-lasting complete remission. This manuscript summarizes current view on the first generation tyrosine kinase inhibititor – imatinib, second generation – dasatinib, nilotinib and bosutnib as well as new generation tyrosine kinase inhibititors – ponatinib and danusertib in hematooncology.

  11. 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...... PTP1B mediates the cessation of the leptin signal transduction. Leptin-induced activation of a STAT3 responsive reporter was dose-dependently inhibited by co-transfection with PTP1B. No inhibition was observed when a catalytically inactive mutant of PTP1B was used or when other PTPs were co...

  12. The molecular basis for RET tyrosine-kinase inhibitors in thyroid cancer.

    Science.gov (United States)

    De Falco, Valentina; Carlomagno, Francesca; Li, Hong-Yu; Santoro, Massimo

    2017-06-01

    RET receptor tyrosine kinase acts as a mutated oncogenic driver in several human malignancies and it is over-expressed in other cancers. Small molecule compounds with RET tyrosine kinase inhibitory activity are being investigated for the targeted treatment of these malignancies. Multi-targeted compounds with RET inhibitory concentration in the nanomolar range have entered clinical practice. This review summarizes mechanisms of RET oncogenic activity and properties of new compounds that, at the preclinical stage, have demonstrated promising anti-RET activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Tyrosine Phosphorylation Modulates the Vascular Responses of Mesenteric Arteries from Human Colorectal Tumors

    Directory of Open Access Journals (Sweden)

    Eduardo Ferrero

    2013-01-01

    Full Text Available The aim of this study was to analyze whether tyrosine phosphorylation in tumoral arteries may modulate their vascular response. To do this, mesenteric arteries supplying blood flow to colorectal tumors or to normal intestine were obtained during surgery and prepared for isometric tension recording in an organ bath. Increasing tyrosine phosphorylation with the phosphatase inhibitor, sodium orthovanadate produced arterial contraction which was lower in tumoral than in control arteries, whereas it reduced the contraction to noradrenaline in tumoral but not in control arteries and reduced the relaxation to bradykinin in control but not in tumoral arteries. Protein expression of VEGF-A and of the VEGF receptor FLT1 was similar in control and tumoral arteries, but expression of the VEGF receptor KDR was increased in tumoral compared with control arteries. This suggests that tyrosine phosphorylation may produce inhibition of the contraction in tumoral mesenteric arteries, which may increase blood flow to the tumor when tyrosine phosphorylation is increased by stimulation of VEGF receptors.

  14. Expression of protein-tyrosine phosphatases in the major insulin target tissues

    DEFF Research Database (Denmark)

    Norris, K; Norris, F; Kono, D H

    1997-01-01

    Protein-tyrosine phosphatases (PTPs) are key regulators of the insulin receptor signal transduction pathway. We have performed a detailed analysis of PTP expression in the major human insulin target tissues or cells (liver, adipose tissue, skeletal muscle and endothelial cells). To obtain...... a representative picture, all tissues were analyzed by PCR using three different primer sets corresponding to conserved regions of known PTPs. A total of 24 different PTPs were identified. A multiprobe RNase protection assay was developed to obtain a semiquantitative measure of the expression levels of selected...... PTPs. Surprisingly, PTP-LAR, previously suggested to be a major regulator of the insulin receptor tyrosine kinase, was expressed in extremely low levels in skeletal muscle, whereas the related receptor-type PTP-sigma and PTP-alpha were expressed in relatively high levels in all four tissues. The low...

  15. In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.

    Science.gov (United States)

    Grecco, Hernán E; Roda-Navarro, Pedro; Girod, Andreas; Hou, Jian; Frahm, Thomas; Truxius, Dina C; Pepperkok, Rainer; Squire, Anthony; Bastiaens, Philippe I H

    2010-06-01

    Extracellular stimuli are transduced inside the cell by posttranslational modifications (PTMs), such as phosphorylation, of proteins in signaling networks. Insight into the structure of these networks requires quantification of PTM levels in individual cells. Fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to image PTM levels in situ. FLIM on cell arrays that express fluorescent protein fusions can quantify tyrosine phosphorylation patterns in large networks in individual cells. We identified tyrosine kinase substrates by imaging their phosphorylation levels after inhibition of protein tyrosine phosphatases. Analysis of the correlation between protein phosphorylation and expression levels at single cell resolution allowed us to identify positive feedback motifs. Using FLIM on cell arrays (CA-FLIM), we uncovered components that transduce signals from epidermal growth factor receptor.

  16. Protein tyrosine phosphatases as potential therapeutic targets.

    Science.gov (United States)

    He, Rong-Jun; Yu, Zhi-Hong; Zhang, Ruo-Yu; Zhang, Zhong-Yin

    2014-10-01

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

  17. 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

  18. Striatal-enriched protein-tyrosine phosphatase (STEP) regulates Pyk2 kinase activity.

    Science.gov (United States)

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

    2012-06-15

    Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family and is highly expressed in brain and hematopoietic cells. Pyk2 plays diverse functions in cells, including the regulation of cell adhesion, migration, and cytoskeletal reorganization. In the brain, it is involved in the induction of long term potentiation through regulation of N-methyl-d-aspartate receptor trafficking. This occurs through the phosphorylation and activation of Src family tyrosine kinase members, such as Fyn, that phosphorylate GluN2B at Tyr(1472). Phosphorylation at this site leads to exocytosis of GluN1-GluN2B receptors to synaptic membranes. Pyk2 activity is modulated by phosphorylation at several critical tyrosine sites, including Tyr(402). In this study, we report that Pyk2 is a substrate of striatal-enriched protein-tyrosine phosphatase (STEP). STEP binds to and dephosphorylates Pyk2 at Tyr(402). STEP KO mice showed enhanced phosphorylation of Pyk2 at Tyr(402) and of the Pyk2 substrates paxillin and ASAP1. Functional studies indicated that STEP opposes Pyk2 activation after KCl depolarization of cortical slices and blocks Pyk2 translocation to postsynaptic densities, a key step required for Pyk2 activation and function. This is the first study to identify Pyk2 as a substrate for STEP.

  19. Mice lacking leukocyte common antigen-related (LAR) protein tyrosine phosphatase domains demonstrate spatial learning impairment in the two-trial water maze and hyperactivity in multiple behavioural tests.

    NARCIS (Netherlands)

    Kolkman, M.J.M.; Streijger, F.; Linkels, M.; Bloemen, M.; Heeren, D.J.; Hendriks, W.J.A.J.; Zee, C.E.E.M. van der

    2004-01-01

    Leukocyte common antigen-related (LAR) protein is a cell adhesion molecule-like receptor-type protein tyrosine phosphatase. We previously reported that in LAR tyrosine phosphatase-deficient (LAR-Delta P) mice the number and size of basal forebrain cholinergic neurons as well as their innervation of

  20. Erkitinib, a novel EGFR tyrosine kinase inhibitor screened using a ProteoChip system from a phytochemical library

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Eung-Yoon; Choi, Young-Jin [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Innopharmascreen, Inc., Asan 336-795 (Korea, Republic of); Park, Chan-Won [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Dept. of Biological Science, Hoseo University, Asan 336-795 (Korea, Republic of); Kang, In-Cheol, E-mail: ickang@hoseo.edu [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Dept. of Biological Science, Hoseo University, Asan 336-795 (Korea, Republic of); Innopharmascreen, Inc., Asan 336-795 (Korea, Republic of)

    2009-11-20

    Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer. Therefore PTK inhibitors are currently under intensive investigation as potential drug candidates. Herein, we report on a ProteoChip-based screening of an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor, Erkitinibs, from phytochemical libraries. PLC-{gamma}-1 was used as a substrate immobilized on a ProteoChip and incubated with an EGFR kinase to phosphorylate tyrosine residues of the substrate, followed by a fluorescence detection of the substrate recognized by a phospho-specific monoclonal antibody. Erkitinibs inhibited HeLa cell proliferation in a dose-dependent manner. In conclusion, these data suggest that Erkitinibs can be a specific inhibitor of an EGFR kinase and can be further developed as a potent anti-tumor agent.

  1. HER2-targeted therapy in breast cancer. Monoclonal antibodies and tyrosine kinase inhibitors

    DEFF Research Database (Denmark)

    Nielsen, Dorte Lisbet; Andersson, Michael; Kamby, Claus

    2008-01-01

    There is strong clinical evidence that trastuzumab, a monoclonal antibody targeting the human epidermal growth factor receptor (HER) two tyrosine kinase receptor, is an important component of first-line treatment of patients with HER2-positive metastatic breast cancer. In particular the combination...... with taxanes and vinorelbine has been established. In the preoperative setting inclusion of trastuzumab has significantly increased the pathological complete response rate. Results from large phase III trials evaluating adjuvant therapy in HER2-positive early breast cancer indicate that the addition...... of trastuzumab to chemotherapy improves disease-free and overall survival. The use of lapatinib, a dual tyrosine kinase inhibitor of both HER1 and HER2, in combination with capecitabine in the second-line treatment of HER2-positive patients with metastatic breast cancer previously treated with trastuzumab has...

  2. Receptor tyrosine kinase signaling: a view from quantitative proteomics

    DEFF Research Database (Denmark)

    Dengjel, Joern; Kratchmarova, Irina; Blagoev, Blagoy

    2009-01-01

    RTKs. In recent years proteomic approaches have yielded detailed descriptions of cellular signaling events. Quantitative proteomics is able to characterize the exact position and strength of post-translational modifications (PTMs) providing essential information for understanding the molecular basis...

  3. UV ACTIVATION OF RECEPTOR TYROSINE KINASE-ACTIVITY

    NARCIS (Netherlands)

    COFFER, PJ; BURGERING, BMT; PEPPELENBOSCH, MP; BOS, JL; KRUIJER, W

    1995-01-01

    The exposure of mammalian cells to ultraviolet radiation (UV) may lead to DNA damage resulting in mutation and thus possibly cancer, while irradiation can further act as a potent tumour promoter. In addition UV induces p21ras-mediated signalling leading to activation of transcription factors such as

  4. EphB4 Receptor Tyrosine Kinase in Prostate Cancer

    Science.gov (United States)

    2011-09-01

    S,  Reddy  R,  Krasnoperov  V,  Quinn  DI,  Henshall  SM,   Sutherland  RL,  Pinski  JK,  Daneshmand  S,  Buscarini...N. Hay, Genes Dev. 20 (2006) 1569. [13] J. Blando, M. Portis, F. Benavides, A. Alexander, G. Mills, B. Dave , C.J. Conti, J. Kim, C.L. Walker, Am. J

  5. Retinitis pigmentosa: mutations in a receptor tyrosine kinase gene ...

    Indian Academy of Sciences (India)

    RP is characterized by abnormalities of photoreceptors (rods and cones) or the retinal pigment epithelium (RPE) leading to progressive loss of vision. It starts with the night blindness or defective dark adaptation in patients followed by constriction of the peripheral visual fields. Eventually, late in the course of the disease, the.

  6. 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

  7. Role of protein tyrosine phosphatase 1B in cardiovascular diseases.

    Science.gov (United States)

    Thiebaut, Pierre-Alain; Besnier, Marie; Gomez, Elodie; Richard, Vincent

    2016-12-01

    Protein Tyrosine Phosphatase 1B (PTP1B) is mostly involved in negative regulation of signaling mediated by Tyrosine Kinase Receptors, especially the insulin and leptin receptors. This enzyme thus plays a major role in the development of diseases associated with insulin resistance, such as obesity and diabetes. PTP1B inhibition is currently considered as an attractive treatment of insulin resistance and associated metabolic disorders. In parallel, emerging evidence also suggests that PTP1B is widely expressed in cardiovascular tissues, notably in the heart and the endothelium, and that it could also be a potential treatment of several cardiovascular diseases. PTP1B is especially present in endothelial cells, and appears to contribute to endothelial dysfunction. Indeed, preclinical evidence shows that pharmacological inhibition of gene deletion of PTP1B reduces endothelial dysfunction in various cardiovascular diseases associated or not with insulin resistance. In parallel, because PTP1B also negatively modulates VEGF signaling, inhibition of this enzyme also tends to favor cardiac angiogenesis. Importantly, blocking PTP1B also results in beneficial effects on cardiac dysfunction and remodeling not only in metabolic diseases but also in the context of heart failure, thus this enzyme represents an attractive new target for the treatment of this disease. This beneficial effect in heart failure may to a large extent result from the endothelial protective and/or proangiogenic effects of PTP1B blockade. Finally, PTP1B inhibition also reduces cardiac dysfunction, but also systemic inflammation and mortality in experimental models of septic shock, and thus may also constitute a new treatment of this disease. Altogether, accumulating preclinical evidence suggests that PTP1B represents an interesting molecular target to treat both cardiovascular and metabolic diseases, which often share the same risk factors. This concept now deserves to be tested in clinical studies that

  8. 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...... 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...

  9. The c-Met tyrosine kinase inhibitor JNJ-38877605 causes renal toxicity through species-specific insoluble metabolite formation

    NARCIS (Netherlands)

    M.P. Lolkema (Martijn); H.H. Bohets (Hilde H.); H.-T. Arkenau (H.); A. Lampo (Ann); E. Barale (Erio); M.J.A. de Jonge (Maja); L. van Doorn (Leni); P. Hellemans (Peter); J.S. de Bono (Johann); F.A.L.M. Eskens (Ferry)

    2015-01-01

    textabstractPurpose: The receptor tyrosine kinase c-Met plays an important role in tumorigenesis and is a novel target for anticancer treatment. This phase I, first-in-human trial, explored safety, pharmacokinetics, pharmacodynamics, and initial antitumor activity of JNJ-38877605, a potent and

  10. 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.

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

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-10-01

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

  12. 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.

  13. Role of nonreceptor protein tyrosine kinases during phospholipase C-gamma 1-related uterine contractions in the rat.

    Science.gov (United States)

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

    2009-03-01

    Activated phospholipase C1, produced in response to tyrosine phosphorylation, appears to play an important role during uterine contractions. These studies sought to determine which non-receptor protein tyrosine kinases are involved in the activation of phospholipase C1 in rat uterine tissue. In vitro contraction studies were performed utilizing isoform specific protein tyrosine kinase inhibitors. Western blots were performed utilizing antibodies to phosphotyrosine-phospholipase C1, total phospholipase C1, c-Src kinase and Lck kinase. Spontaneous, stretch-stimulated, and bpV(phen) (tyrosine phosphatase inhibitor) enhanced uterine contractions were significantly suppressed in response to Damnacanthal (Lck kinase inhibitor) and PP1 (c-Src kinase inhibitor). Damnacanthal and PP1 also significantly suppressed bpV(phen)-enhanced tyrosine phosphorylation of phospholipase C1. Western blots confirmed expression of Lck kinase and c-Src kinase in uterine tissue. In conclusion, the Lck and c-Src kinases appear to play an important role in regulating tyrosine phosphorylation of phospholipase C1 and contractile activity in the rat uterus.

  14. Ethyl p-methoxycinnamate from Kaempferia galanga inhibits angiogenesis through tyrosine kinase

    Directory of Open Access Journals (Sweden)

    Juni Ekowati

    2015-04-01

    Full Text Available Background Many tumors express on their receptor tyrosine kinases vascular endothelial growth factor activity associated with angiogenesis. Inhibition of angiogenesis through reduction of tyrosine kinase activity is a promising strategy for cancer therapy. The present study aimed to determine the mechanism and potency of ethyl p-methoxycinnamate (EPMC isolated from Kaempferia galanga as angiogenesis inhibitor. Methods A laboratory experimental study was conducted using chorio-allantoic membranes (CAMs of nine-day old chicken eggs induced by 60ng basic fibroblast growth factor (bFGF. Ethyl p-methoxycinnamate (EPMC potency was determined at dosages of 30, 60, 90 and 120 mg and compared with celecoxib 60 mg as reference drug and one negative bFGF-induced control group. Neovascularization and endothelial cell count in CAM blood vessels were evaluated. To predict the antiangiogenic mechanism of EPMC, a docking study was performed with the Molegro Virtual Docker program on tyrosine kinase as receptor (PDB 1XKK. Results Angiogenesis stimulation by bFGF was prevented significantly (p<0.05 by EPMC at dosages of 30, 60, 90 and 120 mg and this activity was dose dependent. Molecular docking showed interaction between EPMC functional groups and tyrosine kinase amino acids at Met766, Met793, Thr854, Thr790, Gln791 and Ala743. There was an association between EPMC antiangiogenic activity and docking study results. Conclusions Ethyl p-methoxycinnamate is a potential new angiogenesis inhibitor through interaction with tyrosine kinase. EPMC could be a promising therapeutic agent for treatment of angiogenesis-related diseases.

  15. 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

  16. 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 ...

  17. 21 CFR 582.5920 - Tyrosine.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Tyrosine. 582.5920 Section 582.5920 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

  18. Protein tyrosine phosphatases in health and disease

    NARCIS (Netherlands)

    Hendriks, W.J.; Elson, A.; Harroch, S.; Pulido, R.; Stoker, A.; den Hertog, J.

    2013-01-01

    Protein tyrosine phosphatases (PTPs) represent a super-family of enzymes that play essential roles in normal development and physiology. In this review, we will discuss the PTPs that have a causative role in hereditary diseases in humans. In addition, recent progress in the development and analysis

  19. Studying Protein-Tyrosine Phosphatases in Zebrafish

    NARCIS (Netherlands)

    Hale, Alexander James; den Hertog, Jeroen

    2016-01-01

    Protein-tyrosine phosphatases (PTPs) are a large family of signal transduction regulators that have an essential role in normal development and physiology. Aberrant activation or inactivation of PTPs is at the basis of many human diseases. The zebrafish, Danio rerio, is being used extensively to

  20. morphological features of tyrosine hydroxylase immunoreactive cells ...

    African Journals Online (AJOL)

    Mgina

    2Department of Cell Biology and Functional Morphology, Iwate Medical University, School of. Medicine,. Uchimaru 19-1, Morioka 020-8505, Japan. ABSTRACT. The current immunohistochemical study used the antibody against tyrosine hydroxylase (TH) to observe the immunoreactive elements in the mouse pancreas.

  1. 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...

  2. 1-tert-butyl-3-[6-(3,5-dimethoxy-phenyl)-2-(4-diethylamino-butylamino)-pyrido[2,3-d]pyrimidin-7-yl]-urea (PD173074), a selective tyrosine kinase inhibitor of fibroblast growth factor receptor-3 (FGFR3), inhibits cell proliferation of bladder cancer carrying the FGFR3 gene mutation along with up-regulation of p27/Kip1 and G1/G0 arrest.

    Science.gov (United States)

    Miyake, Makito; Ishii, Masazumi; Koyama, Naoki; Kawashima, Kiyotaka; Kodama, Tetsuro; Anai, Satoshi; Fujimoto, Kiyohide; Hirao, Yoshihiko; Sugano, Kokichi

    2010-03-01

    Activating mutation of the fibroblast growth factor receptor-3 (FGFR3) gene is known as a key molecular event in both oncogenesis and cell proliferation of low-grade noninvasive human bladder urothelial carcinoma (UC), which is characterized by frequent intravesical recurrence. In this study, we investigated the antitumor potentiality of 1-tert-butyl-3-[6-(3,5-dimethoxy-phenyl)-2-(4-diethylamino-butylamino)-pyrido[2,3-d]pyrimidin-7-yl]-urea (PD173074), a small-molecule FGFR3-selective tyrosine kinase inhibitor (TKI), as a therapeutic modality using eight UC cell lines. In our in vitro cell proliferation assay, PD173074 suppressed cell proliferation remarkably in two cell lines, namely, UM-UC-14 and MGHU3, which expressed mutated FGFR3 protein. In contrast, the other six cell lines expressing wild-type FGFR3 or without FGFR3 expression were resistant to PD173074 treatment. Cell cycle analysis revealed the growth inhibitory effect of PD173074 was associated with arrest at G(1)-S transition in a dose-depending manner. Furthermore, we observed an inverse relationship between Ki-67 and p27/Kip1 expression after PD173074 treatment, suggesting that up-regulation of p27 recruited UC cells harboring activating FGFR3 mutations in G(1) that was analogous with the other receptor TKIs acting on the epidermal growth factor receptors. In the mouse xenograft models using subcutaneously transplanted UM-UC-14 and MGHU3, orally administered PD173074 suppressed tumor growth and induced apoptotic changes comparable with the results of our in vitro assay. These findings elucidated the effectiveness of molecular targeted approach for bladder UC harboring FGFR3 mutations and the potential utility to decrease the intravesical recurrence of nonmuscle invasive bladder UC after transurethral surgical resection.

  3. Facile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click Reaction

    OpenAIRE

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

    2013-01-01

    The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivatives in peptide and protein chemistry, we synthesized PTAD derivatives possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivatives and peptides of increasing complexity. With proteins we studied the compatibility of the tyrosine click reaction ...

  4. Striatal-enriched protein tyrosine phosphatase regulates the PTPα/Fyn signaling pathway.

    Science.gov (United States)

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

    2015-08-01

    The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr(420)) or inhibit (Tyr(531)) Fyn activity. Within the central nervous system, two protein tyrosine phosphatases (PTPs) target these regulatory tyrosines in Fyn. PTPα dephosphorylates Tyr(531) and activates Fyn, while STEP (STriatal-Enriched protein tyrosine Phosphatase) dephosphorylates Tyr(420) and inactivates Fyn. Thus, PTPα and STEP have opposing functions in the regulation of Fyn; however, whether there is cross talk between these two PTPs remains unclear. Here, we used molecular techniques in primary neuronal cultures and in vivo to demonstrate that STEP negatively regulates PTPα by directly dephosphorylating PTPα at its regulatory Tyr(789). Dephosphorylation of Tyr(789) prevents the translocation of PTPα to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction in STEP61 activity increased the phosphorylation of PTPα at Tyr(789), as well as increased translocation of PTPα to synaptic membranes. Activation of PTPα and Fyn and trafficking of GluN2B to synaptic membranes are necessary for ethanol (EtOH) intake behaviors in rodents. We tested the functional significance of STEP61 in this signaling pathway by EtOH administration to primary cultures as well as in vivo, and demonstrated that the inactivation of STEP61 by EtOH leads to the activation of PTPα, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP61 regulates PTPα and suggest that STEP and PTPα coordinate the regulation of Fyn. STEP61 , PTPα, Fyn, and NMDA receptor (NMDAR) have been implicated in ethanol intake behaviors in the dorsomedial striatum (DMS) in rodents. Here, we report that PTPα is a novel substrate for STEP61. Upon ethanol exposure, STEP61 is phosphorylated and inactivated by protein kinase A (PKA) signaling in the DMS. As a result of STEP61

  5. Identification of tyrosine-9 of MAVS as critical target for inducible phosphorylation that determines activation.

    Directory of Open Access Journals (Sweden)

    Chaoyang Wen

    Full Text Available BACKGROUND: Innate immunity to viruses involves receptors such as RIG-I, which senses viral RNA and triggers an IFN-β signaling pathway involving the outer mitochondrial membrane protein MAVS. However, the functional status of MAVS phosphorylation remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate for the first time that MAVS undergoes extensive tyrosine phosphorylation upon viral infection, indicating that MAVS phosphorylation might play an important role in MAVS function. A tyrosine-scanning mutational analysis revealed that MAVS tyrosine-9 (Y9 is a phosphorylation site that is required for IFN-β signaling. Indeed, MAVS Y9F mutation severely impaired TRAF3/TRAF6 recruitment and displayed decreased tyrosine phosphorylation in response to VSV infection compared to wild type MAVS. Functionally, MAVS Y9 phosphorylation contributed to MAVS antiviral function without interfering with its apoptosis property. CONCLUSIONS/SIGNIFICANCE: These experiments identify a novel residue of MAVS that is crucially involved in the recruitment of TRAF3/TRAF6 and in downstream propagation of MAVS signaling.

  6. 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.

  7. Tyrosine phosphorylation of Munc18c on residue 521 abrogates binding to Syntaxin 4

    Directory of Open Access Journals (Sweden)

    Bryant Nia J

    2011-05-01

    Full Text Available Abstract Background Insulin stimulates exocytosis of GLUT4 from an intracellular store to the cell surface of fat and muscle cells. Fusion of GLUT4-containing vesicles with the plasma membrane requires the SNARE proteins Syntaxin 4, VAMP2 and the regulatory Sec1/Munc18 protein, Munc18c. Syntaxin 4 and Munc18c form a complex that is disrupted upon insulin treatment of adipocytes. Munc18c is tyrosine phosphorylated in response to insulin in these cells. Here, we directly test the hypothesis that tyrosine phosphorylation of Munc18c is responsible for the observed insulin-dependent abrogation of binding between Munc18c and Syntaxin 4. Results We show that Munc18c is directly phosphorylated by recombinant insulin receptor tyrosine kinase in vitro. Using pull-down assays, we show that phosphorylation abrogates binding of Munc18c to both Syntaxin 4 and the v-SNARE VAMP2, as does the introduction of a phosphomimetic mutation into Munc18c (Y521E. Conclusion Our data indicate that insulin-stimulated tyrosine phosphorylation of Munc18c impairs the ability of Munc18c to bind its cognate SNARE proteins, and may therefore represent a regulatory step in GLUT4 traffic.

  8. Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

    DEFF Research Database (Denmark)

    Pandey, A; Podtelejnikov, A V; Blagoev, B

    2000-01-01

    Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2...

  9. Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion

    Science.gov (United States)

    Davy, Alice; Gale, Nicholas W.; Murray, Elizabeth W.; Klinghoffer, Richard A.; Soriano, Philippe; Feuerstein, Claude; Robbins, Stephen M.

    1999-01-01

    Eph receptor tyrosine kinases and their corresponding surface-bound ligands, the ephrins, provide cues to the migration of cells and growth cones during embryonic development. Here we show that ephrin-A5, which is attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidylinositol-anchor, induces compartmentalized signaling within a caveolae-like membrane microdomain when bound to the extracellular domain of its cognate Eph receptor. The physiological response induced by this signaling event is concomitant with a change in the cellular architecture and adhesion of the ephrin-A5-expressing cells and requires the activity of the Fyn protein tyrosine kinase. This study stresses the relevance of bidirectional signaling involving the ephrins and Eph receptors during brain development. PMID:10601038

  10. A novel activating mutation in the RET tyrosine kinase domain mediates neoplastic transformation.

    Science.gov (United States)

    Cranston, Aaron; Carniti, Cristiana; Martin, Sam; Mondellini, Piera; Hooks, Yvette; Leyland, Jean; Hodgson, Shirley; Clarke, Sue; Pierotti, Marco; Ponder, Bruce A J; Bongarzone, Italia

    2006-07-01

    We report the finding of a novel missense mutation at codon 833 in the tyrosine kinase of the RET proto-oncogene in a patient with a carcinoma of the thyroid. In vitro experiments demonstrate that the R833C mutation induces transformed foci only when present in the long 3' splice isoform and, in keeping with a model in which the receptor has to dimerize to be completely activated, glial cell line-derived neurotrophic factor stimulation leads the RET(R833C) receptor to a higher level of activation. Tyrosine kinase assays show that the RET(R833C) long isoform has weak intrinsic kinase activity and phosphorylation of an exogenous substrate is not elevated even in the presence of glial cell line-derived neurotrophic factor. Furthermore, the R833C mutation is capable of sustaining the transformed phenotype in vivo but does not confer upon the transformed cells the ability to degrade the basement membrane in a manner analogous to metastasis. Our functional characterization of the R833C substitution suggests that, like the V804M and S891A mutations, this tyrosine kinase mutation confers a weak activating potential upon RET. This is the first report demonstrating that the introduction of an intracellular cysteine can activate RET. However, this does not occur via dimerization in a manner analogous to the extracellular cysteine mutants.

  11. The tyrosine phosphatase STEP constrains amygdala-dependent memory formation and neuroplasticity.

    Science.gov (United States)

    Olausson, P; Venkitaramani, D V; Moran, T D; Salter, M W; Taylor, J R; Lombroso, P J

    2012-12-06

    STriatal-Enriched protein tyrosine Phosphatase (STEP; PTPN5) is expressed in brain regions displaying adult neuroplasticity. STEP modulates neurotransmission by dephosphorylating regulatory tyrosine residues on its substrates. In this way, STEP inactivates extracellular-signal-regulated kinase 1/2 (ERK1/2), limiting the duration and spatial distribution of ERK signaling. Two additional substrates, the tyrosine kinase Fyn and the NR2B subunit of the N-methyl-d-aspartic acid receptor, link STEP to glutamate receptor internalization in the synapse. Thus, STEP may act through parallel pathways to oppose the development of experience-dependent synaptic plasticity. We examined the hypothesis that the absence of STEP facilitates amygdala-dependent behavioral and synaptic plasticity (i.e., fear conditioning and long-term potentiation) using STEP-deficient mice (STEP KO). These mice show no detectable expression of STEP in the brain along with increases in Tyr phosphorylation of STEP substrates. Here we demonstrate that STEP KO mice also display augmented fear conditioning as measured by an enhancement in conditioned suppression of instrumental response when a fear-associated conditioned stimulus was presented. Deletion of STEP also increases long-term potentiation and ERK phosphorylation in the lateral amygdala. The current experiments demonstrate that deletion of STEP can enhance experience-induced neuroplasticity and memory formation and identifies STEP as a target for pharmacological treatment aimed at improving the formation of long-term memories. Copyright © 2012. Published by Elsevier Ltd.

  12. 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...

  13. Covalent inhibition of the lymphoid tyrosine phosphatase.

    Science.gov (United States)

    Ahmed, Vanessa F; Bottini, Nunzio; Barrios, Amy M

    2014-02-01

    Covalent inhibitors of lymphoid tyrosine phosphatase (LYP) were identified from a screen of the NIH Molecular Libraries Small Molecules Repository (MLSMR). Both of the two lead compounds identified have phosphotyrosine-mimetic benzoic acid moieties as well as electrophilic acrylonitrile groups. Inhibition kinetics of both compounds are consistent with covalent modification of the enzyme, with nanomolar KI and reciprocal millisecond kinact values, representing the best efficiency ratios (kinact /KI ) among currently reported covalent LYP inhibitors. Covalent inhibitors can provide longer efficacy and better selectivity than more conventional noncovalent inhibitors, and these lead compounds are an important step toward the development of protein tyrosine phosphatase (PTP)-targeted covalent therapeutic compounds. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Tyrosine kinases in inflammatory dermatologic disease.

    Science.gov (United States)

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

    2011-08-01

    Tyrosine kinases (TKs) are enzymes that catalyze the phosphorylation of tyrosine residues on protein substrates. They are key components of signaling pathways that drive an array of cellular responses including proliferation, differentiation, migration, and survival. Specific TKs have recently been identified as critical to the pathogenesis of several autoimmune and inflammatory diseases. Small-molecule inhibitors of TKs are emerging as a novel class of therapy that may provide benefit in certain patient subsets. In this review, we highlight TK signaling implicated in inflammatory dermatologic diseases, evaluate strategies aimed at inhibiting these aberrant signaling pathways, and discuss prospects for future drug development. Copyright © 2010 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.

  15. Venus Kinase Receptors at the Crossroads of Insulin Signaling: Their Role in Reproduction for Helminths and Insects

    OpenAIRE

    Colette eDissous

    2015-01-01

    Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) first discovered in the human parasite Schistosoma. They contain an extracellular Venus FlyTrap (VFT) module similar to the ligand-binding domain of G protein-coupled receptors of class C and an intracellular tyrosine kinase domain similar to that of insulin receptors. VKRs are present from cnidarians to echinoderms. They were shown to be activated by amino-acids, to induce insulin-like intracellular pathways and ...

  16. Tyrosine Phosphorylation of Botulinum Neurotoxin Protease Domains

    Science.gov (United States)

    2012-06-01

    phosphorylated tyro - sine indicated by an asterisk (*). LcA− and LcA+ represent Src reaction mixtures that were incubated without and with (0.2mM...CONCLUSION In vitro reaction of LcA, LcB, LcC1, LcD, LcE, and LcG with Tyrosine kinase Src resulted in phosphorylation of several tyro - sine residues

  17. Mechanisms of Peroxynitrite Mediated Nitration of Tyrosine

    OpenAIRE

    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. Th...

  18. Differential turnover of tyrosinated and detyrosinated microtubules.

    OpenAIRE

    Webster, D. R.; Gundersen, G G; Bulinski, J C; Borisy, G G

    1987-01-01

    Turnover of tyrosinated and detyrosinated microtubules ([Tyr]MTs and [Glu]MTs, respectively) was analyzed by the combined use of hapten-mediated immunocytochemistry and peptide-specific antibodies. Cells were microinjected with hapten-labeled tubulin and then processed for triple-label immunofluorescence to determine the pattern of incorporation of the injected subunits into [Tyr]- and [Glu]-MTs. Within 2 min of microinjection, hapten-labeled domains were present at the ends of virtually all ...

  19. Influence of dopamine synthesis on methamphetamine-induced changes in striatal and adrenal tyrosine hydroxylase activity.

    Science.gov (United States)

    Gibb, J W; Kogan, F J

    1979-12-01

    Methamphetamine in large doses decreases striatal tyrosine hydroxylase activity. This effect is prevented by neuroleptic agents such as chlorpromazine and haloperidol which would suggest that released dopamine may be involved in the response. To test this hypothesis, we have altered dopamine synthesis with alpha-methyl-p-tyrosine and L-Dopa and found that dopamine synthesis is necessary for the observed depression of striatal TH activity by methamphetamine. In the adrenal gland, however, the increase in TH activity by methamphetamine is not prevented by inhibition of catecholamine synthesis. It is possible that released dopamine may be inhibiting TH activity by activation of pre- or postsynaptic dopamine receptors in the neostriatum resulting in activation of the neuronal feedback pathway or released dopamine may act on dendrodendritic autoreceptors in the substantia nigra.

  20. How tyrosine kinase inhibitors impair metabolism and endocrine system function: a systematic updated review.

    Science.gov (United States)

    Breccia, Massimo; Molica, Matteo; Alimena, Giuliana

    2014-12-01

    Tyrosine kinase inhibitors (TKIs) advent has deeply changed the outcome of chronic myeloid leukemia (CML) patients, with improved rates of response and overall survival. However, for this success some patients paid the price of a number of peculiar side effects, the so-called off-target side effects, specific for each one TKI. These effects are due to non-selective inhibition of other tyrosine kinase receptors, such as PDGFR, c-KIT, Src, VEGF. Consequences of this inhibition, some metabolic changes during the treatment with TKIs are reported. Aim of present review is to report metabolic changes and potential mechanisms involved in the pathogenesis related to imatinib, second (nilotinib and dasatinib) and third generation (bosutinib and ponatinib) TKIs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Tyrosine phosphatase STEP is a tonic brake on induction of long-term potentiation.

    Science.gov (United States)

    Pelkey, Kenneth A; Askalan, Rand; Paul, Surojit; Kalia, Lorraine V; Nguyen, Tri Hung; Pitcher, Graham M; Salter, Michael W; Lombroso, Paul J

    2002-03-28

    The functional roles of protein tyrosine phosphatases (PTPs) in the developed CNS have been enigmatic. Here we show that striatal enriched tyrosine phosphatase (STEP) is a component of the N-methyl-D-aspartate receptor (NMDAR) complex. Functionally, exogenous STEP depressed NMDAR single-channel activity in excised membrane patches. STEP also depressed NMDAR-mediated synaptic currents whereas inhibiting endogenous STEP enhanced these currents. In hippocampal slices, administering STEP into CA1 neurons did not affect basal glutamatergic transmission evoked by Schaffer collateral stimulation but prevented tetanus-induced long-term potentiation (LTP). Conversely, inhibiting STEP in CA1 neurons enhanced transmission and occluded LTP induction through an NMDAR-, Src-, and Ca(2+)-dependent mechanism. Thus, STEP acts as a tonic brake on synaptic transmission by opposing Src-dependent upregulation of NMDARs.

  2. The tyrosine kinase Tyk2 controls IFNAR1 cell surface expression.

    Science.gov (United States)

    Ragimbeau, Josiane; Dondi, Elisabetta; Alcover, Andrés; Eid, Pierre; Uzé, Gilles; Pellegrini, Sandra

    2003-02-03

    The four mammalian Jak tyrosine kinases are non-covalently associated with cell surface receptors binding helical bundled cytokines. In the type I interferon receptor, Tyk2 associates with the IFNAR1 receptor subunit and positively influences ligand binding to the receptor complex. Here, we report that Tyk2 is essential for stable cell surface expression of IFNAR1. In the absence of Tyk2, mature IFNAR1 is weakly expressed on the cell surface. Rather, it is localized into a perinuclear endosomal compartment which overlaps with that of recycling transferrin receptors and with early endosomal antigen-1 (EEA1) positive vesicles. Conversely, co-expressed Tyk2 greatly enhances surface IFNAR1 expression. Importantly, we demonstrate that Tyk2 slows down IFNAR1 degradation and that this is due, at least in part, to inhibition of IFNAR1 endocytosis. In addition, Tyk2 induces plasma membrane relocalization of the R2 subunit of the interleukin-10 receptor. These results reveal a novel function of a Jak protein on internalization of a correctly processed cytokine receptor. This function is distinct from the previously reported effect of other Jak proteins on receptor exit from the endoplasmic reticulum.

  3. Human ether-à-go-go gene potassium channels are regulated by EGFR tyrosine kinase.

    Science.gov (United States)

    Wu, Wei; Dong, Ming-Qing; Wu, Xing-Gang; Sun, Hai-Ying; Tse, Hung-Fat; Lau, Chu-Pak; Li, Gui-Rong

    2012-02-01

    Human ether á-go-go gene potassium channels (hEAG1 or Kv10.1) are expressed in brain and various human cancers and play a role in neuronal excitement and tumor progression. However, the functional regulation of hEAG channels by signal transduction is not fully understood. The present study was therefore designed to investigate whether hEAG1 channels are regulated by protein tyrosine kinases (PTKs) in HEK 293 cells stably expressing hEAG1 gene using whole-cell patch voltage-clamp, immunoprecipitation, Western blot, and mutagenesis approaches. We found that the selective epidermal growth factor receptor (EGFR) kinase inhibitor AG556 (10 μM), but not the platelet growth factor receptor (PDGFR) kinase inhibitor AG1295 (10 μM) or the Src-family inhibitor PP2 (10 μM), can inhibit hEAG1 current, and the inhibitory effect can be reversed by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hEAG1 channels was reduced by AG556, and the reduction was significantly countered by orthovanadate. The hEAG1 mutants Y90A, Y344A and Y485A, but not Y376A and Y479A, exhibited reduced response to AG556. Interestingly, the inhibition effect of AG556 was lost in triple mutant hEAG1 channels at Y90, Y344, and Y485 with alanine. These results demonstrate for the first time that hEAG1 channel activity is regulated by EGFR kinase at the tyrosine residues Tyr90, Try344, and Try485. This effect is likely involved in regulating neuronal activity and/or tumor growth. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. 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...

  5. Small Molecule Tyrosine Kinase Inhibitors of ErbB2/HER2/Neu in the Treatment of Aggressive Breast Cancer

    Directory of Open Access Journals (Sweden)

    Richard L. Schroeder

    2014-09-01

    Full Text Available The human epidermal growth factor receptor 2 (HER2 is a member of the erbB class of tyrosine kinase receptors. These proteins are normally expressed at the surface of healthy cells and play critical roles in the signal transduction cascade in a myriad of biochemical pathways responsible for cell growth and differentiation. However, it is widely known that amplification and subsequent overexpression of the HER2 encoding oncogene results in unregulated cell proliferation in an aggressive form of breast cancer known as HER2-positive breast cancer. Existing therapies such as trastuzumab (Herceptin® and lapatinib (Tyverb/Tykerb®, a monoclonal antibody inhibitor and a dual EGFR/HER2 kinase inhibitor, respectively, are currently used in the treatment of HER2-positive cancers, although issues with high recurrence and acquired resistance still remain. Small molecule tyrosine kinase inhibitors provide attractive therapeutic targets, as they are able to block cell signaling associated with many of the proposed mechanisms for HER2 resistance. In this regard we aim to present a review on the available HER2 tyrosine kinase inhibitors, as well as those currently in development. The use of tyrosine kinase inhibitors as sequential or combinatorial therapeutic strategies with other HER family inhibitors is also discussed.

  6. 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.

  7. [The insuline receptor: structure and function].

    Science.gov (United States)

    De Meyts, P

    2005-01-01

    About 35 years after the first in vitro studies of the insulin receptor, considerable progress has been accomplished in the structural biology of the insulin-receptor interaction, and of the receptor tyrosine kinase family in general. This brief review attempts to assemble the various pieces of the puzzle, despite the lack of a detailed crystallographic structure of the insulin-receptor complex, and to establish a model that explains the mechanism of receptor activation, its negative cooperativity, and the triggering of intracellular signalling pathways.

  8. Chlorinated tyrosine derivatives in insect cuticle

    DEFF Research Database (Denmark)

    Andersen, Svend Olav

    2004-01-01

    A method for quantitative measurement of 3-monochlorotyrosine and 3,5-dichlorotyrosine in insect cuticles is described, and it is used for determination of their distribution in various cuticular regions in nymphs and adults of the desert locust, Schistocerca gregaria. The two chlorinated tyrosine...... during sample hydrolysis. Mono- and dichlorotyrosine are also present in cuticular samples from other insect species, such as the beetle, Tenebrio molitor, the moth Hyalophora cecropia, the cockroach Blaberus craniifer, and the bug Rhodnius prolixus, but not in the sclerotized puparial cuticle...

  9. 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.

  10. 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

    Superoxide reacts rapidly with other radicals, but these reactions have received little attention in the context of oxidative stress. For tyrosyl radicals, reaction with superoxide is 3-fold faster than dimerization, and forms the addition product tyrosine hydroperoxide. We have explored structural...... 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...

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

    Science.gov (United States)

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

    2017-07-01

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

  12. Inhibitor of the tyrosine phosphatase STEP reverses cognitive deficits in a mouse model of Alzheimer's disease.

    Science.gov (United States)

    Xu, Jian; Chatterjee, Manavi; Baguley, Tyler D; Brouillette, Jonathan; Kurup, Pradeep; Ghosh, Debolina; Kanyo, Jean; Zhang, Yang; Seyb, Kathleen; Ononenyi, Chimezie; Foscue, Ethan; Anderson, George M; Gresack, Jodi; Cuny, Gregory D; Glicksman, Marcie A; Greengard, Paul; Lam, TuKiet T; Tautz, Lutz; Nairn, Angus C; Ellman, Jonathan A; Lombroso, Paul J

    2014-08-01

    STEP (STriatal-Enriched protein tyrosine Phosphatase) is a neuron-specific phosphatase that regulates N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking, as well as ERK1/2, p38, Fyn, and Pyk2 activity. STEP is overactive in several neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease (AD). The increase in STEP activity likely disrupts synaptic function and contributes to the cognitive deficits in AD. AD mice lacking STEP have restored levels of glutamate receptors on synaptosomal membranes and improved cognitive function, results that suggest STEP as a novel therapeutic target for AD. Here we describe the first large-scale effort to identify and characterize small-molecule STEP inhibitors. We identified the benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (known as TC-2153) as an inhibitor of STEP with an IC50 of 24.6 nM. TC-2153 represents a novel class of PTP inhibitors based upon a cyclic polysulfide pharmacophore that forms a reversible covalent bond with the catalytic cysteine in STEP. In cell-based secondary assays, TC-2153 increased tyrosine phosphorylation of STEP substrates ERK1/2, Pyk2, and GluN2B, and exhibited no toxicity in cortical cultures. Validation and specificity experiments performed in wild-type (WT) and STEP knockout (KO) cortical cells and in vivo in WT and STEP KO mice suggest specificity of inhibitors towards STEP compared to highly homologous tyrosine phosphatases. Furthermore, TC-2153 improved cognitive function in several cognitive tasks in 6- and 12-mo-old triple transgenic AD (3xTg-AD) mice, with no change in beta amyloid and phospho-tau levels.

  13. Inhibitor of the tyrosine phosphatase STEP reverses cognitive deficits in a mouse model of Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Jian Xu

    2014-08-01

    Full Text Available STEP (STriatal-Enriched protein tyrosine Phosphatase is a neuron-specific phosphatase that regulates N-methyl-D-aspartate receptor (NMDAR and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR trafficking, as well as ERK1/2, p38, Fyn, and Pyk2 activity. STEP is overactive in several neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease (AD. The increase in STEP activity likely disrupts synaptic function and contributes to the cognitive deficits in AD. AD mice lacking STEP have restored levels of glutamate receptors on synaptosomal membranes and improved cognitive function, results that suggest STEP as a novel therapeutic target for AD. Here we describe the first large-scale effort to identify and characterize small-molecule STEP inhibitors. We identified the benzopentathiepin 8-(trifluoromethyl-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (known as TC-2153 as an inhibitor of STEP with an IC50 of 24.6 nM. TC-2153 represents a novel class of PTP inhibitors based upon a cyclic polysulfide pharmacophore that forms a reversible covalent bond with the catalytic cysteine in STEP. In cell-based secondary assays, TC-2153 increased tyrosine phosphorylation of STEP substrates ERK1/2, Pyk2, and GluN2B, and exhibited no toxicity in cortical cultures. Validation and specificity experiments performed in wild-type (WT and STEP knockout (KO cortical cells and in vivo in WT and STEP KO mice suggest specificity of inhibitors towards STEP compared to highly homologous tyrosine phosphatases. Furthermore, TC-2153 improved cognitive function in several cognitive tasks in 6- and 12-mo-old triple transgenic AD (3xTg-AD mice, with no change in beta amyloid and phospho-tau levels.

  14. Regulation of Ack-Family Nonreceptor Tyrosine Kinases

    Directory of Open Access Journals (Sweden)

    Victoria Prieto-Echagüe

    2011-01-01

    Full Text Available Ack family non-receptor tyrosine kinases are unique with regard to their domain composition and regulatory properties. Human Ack1 (activated Cdc42-associated kinase is ubiquitously expressed and is activated by signals that include growth factors and integrin-mediated cell adhesion. Stimulation leads to Ack1 autophosphorylation and to phosphorylation of additional residues in the C-terminus. The N-terminal SAM domain is required for full activation. Ack1 exerts some of its effects via protein-protein interactions that are independent of its kinase activity. In the basal state, Ack1 activity is suppressed by an intramolecular interaction between the catalytic domain and the C-terminal region. Inappropriate Ack1 activation and signaling has been implicated in the development, progression, and metastasis of several forms of cancer. Thus, there is increasing interest in Ack1 as a drug target, and studies of the regulatory properties of the enzyme may reveal features that can be exploited in inhibitor design.

  15. Role of Striatal-Enriched Tyrosine Phosphatase in Neuronal Function.

    Science.gov (United States)

    Kamceva, Marija; Benedict, Jessie; Nairn, Angus C; Lombroso, Paul J

    2016-01-01

    Striatal-enriched protein tyrosine phosphatase (STEP) is a CNS-enriched protein implicated in multiple neurologic and neuropsychiatric disorders. STEP regulates key signaling proteins required for synaptic strengthening as well as NMDA and AMPA receptor trafficking. Both high and low levels of STEP disrupt synaptic function and contribute to learning and behavioral deficits. High levels of STEP are present in human postmortem samples and animal models of Alzheimer's disease, Parkinson's disease, and schizophrenia and in animal models of fragile X syndrome. Low levels of STEP activity are present in additional disorders that include ischemia, Huntington's chorea, alcohol abuse, and stress disorders. Thus the current model of STEP is that optimal levels are required for optimal synaptic function. Here we focus on the role of STEP in Alzheimer's disease and the mechanisms by which STEP activity is increased in this illness. Both genetic lowering of STEP levels and pharmacological inhibition of STEP activity in mouse models of Alzheimer's disease reverse the biochemical and cognitive abnormalities that are present. These findings suggest that STEP is an important point for modulation of proteins required for synaptic plasticity.

  16. Role of Striatal-Enriched Tyrosine Phosphatase in Neuronal Function

    Directory of Open Access Journals (Sweden)

    Marija Kamceva

    2016-01-01

    Full Text Available Striatal-enriched protein tyrosine phosphatase (STEP is a CNS-enriched protein implicated in multiple neurologic and neuropsychiatric disorders. STEP regulates key signaling proteins required for synaptic strengthening as well as NMDA and AMPA receptor trafficking. Both high and low levels of STEP disrupt synaptic function and contribute to learning and behavioral deficits. High levels of STEP are present in human postmortem samples and animal models of Alzheimer’s disease, Parkinson’s disease, and schizophrenia and in animal models of fragile X syndrome. Low levels of STEP activity are present in additional disorders that include ischemia, Huntington’s chorea, alcohol abuse, and stress disorders. Thus the current model of STEP is that optimal levels are required for optimal synaptic function. Here we focus on the role of STEP in Alzheimer’s disease and the mechanisms by which STEP activity is increased in this illness. Both genetic lowering of STEP levels and pharmacological inhibition of STEP activity in mouse models of Alzheimer’s disease reverse the biochemical and cognitive abnormalities that are present. These findings suggest that STEP is an important point for modulation of proteins required for synaptic plasticity.

  17. Striatal-enriched protein tyrosine phosphatase in Alzheimer's disease.

    Science.gov (United States)

    Xu, Jian; Kurup, Pradeep; Nairn, Angus C; Lombroso, Paul J

    2012-01-01

    Alzheimer's disease (AD) is the most common form of dementia among the elderly, affecting millions of people worldwide and representing a substantial economic burden. AD is a progressive disease associated with memory loss and impaired cognitive function. The neuropathology is characterized by cortical accumulation of amyloid plaques and neurofibrillary tangles (NFTs). Amyloid plaques are small, aggregated peptides called beta amyloid (Aβ) and NFTs are aggregates of hyperphosphorylated Tau protein. Because Aβ disrupts multiple intracellular signaling pathways, resulting in some of the clinical symptoms of AD, understanding the underlying molecular mechanisms has implications for the diagnosis and treatment of AD. Recent studies have demonstrated that Aβ regulates striatal-enriched protein tyrosine phosphatase (STEP) (PTPN5). Aβ accumulation is associated with increases in STEP levels and activity that in turn disrupts glutamate receptor trafficking to and from the neuronal membrane. These findings indicate that modulating STEP levels or inhibiting its activity may have beneficial effects for patients with AD, making it an important target for drug discovery. This article reviews the biology of STEP and its role in AD as well as the potential clinical applications. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Substrate-based fragment identification for the development of selective, nonpeptidic inhibitors of striatal-enriched protein tyrosine phosphatase.

    Science.gov (United States)

    Baguley, Tyler D; Xu, Hai-Chao; Chatterjee, Manavi; Nairn, Angus C; Lombroso, Paul J; Ellman, Jonathan A

    2013-10-10

    High levels of striatal-enriched protein tyrosine phosphatase (STEP) activity are observed in a number of neuropsychiatric disorders such as Alzheimer's disease. Overexpression of STEP results in the dephosphorylation and inactivation of many key neuronal signaling molecules, including ionotropic glutamate receptors. Moreover, genetically reducing STEP levels in AD mouse models significantly reversed cognitive deficits and decreased glutamate receptor internalization. These results support STEP as a potential target for drug discovery for the treatment of Alzheimer's disease. Herein, a substrate-based approach for the discovery and optimization of fragments called substrate activity screening (SAS) has been applied to the development of low molecular weight (<450 Da) and nonpeptidic, single-digit micromolar mechanism-based STEP inhibitors with greater than 20-fold selectivity across multiple tyrosine and dual specificity phosphatases. Significant levels of STEP inhibition in rat cortical neurons are also observed.

  19. Therapeutic Innovations: Tyrosine Kinase Inhibitors in Cancer

    Directory of Open Access Journals (Sweden)

    Nikolaos Dervisis

    2016-01-01

    Full Text Available Conventional cytotoxic chemotherapy involving DNA-interacting agents and indiscriminate cell death is no longer the future of cancer management. While chemotherapy is not likely to completely disappear from the armamentarium; the use of targeted therapies in combination with conventional treatment is becoming the standard of care in human medicine. Tyrosine kinases are pivotal points of functional cellular pathways and have been implicated in malignancy, inflammatory, and immune-mediated diseases. Pharmaceutical interventions targeting aberrant tyrosine kinase signaling has exploded and is the second most important area of drug development. The “Valley of Death” between drug discovery and approval threatens to blunt the enormous strides in cancer management seen thus far. Kinase inhibitors, as targeted small molecules, hold promise in the treatment and diagnosis of cancer. However, there are still many unanswered questions regarding the use of kinase inhibitors in the interpretation and management of cancer. Comparative oncology has the potential to address restrictions and limitations in the advancement in kinase inhibitor therapy.

  20. The venus kinase receptor (VKR) family: structure and evolution.

    OpenAIRE

    Vanderstraete, Mathieu; Gouignard, Nadège; Ahier, Arnaud; Morel, Marion; Vicogne, Jérôme; Dissous, Colette

    2013-01-01

    International audience; BACKGROUND: Receptor tyrosine kinases (RTK) form a family of transmembrane proteins widely conserved in Metazoa, with key functions in cell-to-cell communication and control of multiple cellular processes. A new family of RTK named Venus Kinase Receptor (VKR) has been described in invertebrates. The VKR receptor possesses a Venus Fly Trap (VFT) extracellular module, a bilobate structure that binds small ligands to induce receptor kinase activity. VKR was shown to be hi...

  1. Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib.

    Science.gov (United States)

    Woyach, Jennifer A; Furman, Richard R; Liu, Ta-Ming; Ozer, Hatice Gulcin; Zapatka, Marc; Ruppert, Amy S; Xue, Ling; Li, Daniel Hsieh-Hsin; Steggerda, Susanne M; Versele, Matthias; Dave, Sandeep S; Zhang, Jenny; Yilmaz, Ayse Selen; Jaglowski, Samantha M; Blum, Kristie A; Lozanski, Arletta; Lozanski, Gerard; James, Danelle F; Barrientos, Jacqueline C; Lichter, Peter; Stilgenbauer, Stephan; Buggy, Joseph J; Chang, Betty Y; Johnson, Amy J; Byrd, John C

    2014-06-12

    Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and is effective in chronic lymphocytic leukemia (CLL). Resistance to irreversible kinase inhibitors and resistance associated with BTK inhibition have not been characterized. Although only a small proportion of patients have had a relapse during ibrutinib therapy, an understanding of resistance mechanisms is important. We evaluated patients with relapsed disease to identify mutations that may mediate ibrutinib resistance. We performed whole-exome sequencing at baseline and the time of relapse on samples from six patients with acquired resistance to ibrutinib therapy. We then performed functional analysis of identified mutations. In addition, we performed Ion Torrent sequencing for identified resistance mutations on samples from nine patients with prolonged lymphocytosis. We identified a cysteine-to-serine mutation in BTK at the binding site of ibrutinib in five patients and identified three distinct mutations in PLCγ2 in two patients. Functional analysis showed that the C481S mutation of BTK results in a protein that is only reversibly inhibited by ibrutinib. The R665W and L845F mutations in PLCγ2 are both potentially gain-of-function mutations that lead to autonomous B-cell-receptor activity. These mutations were not found in any of the patients with prolonged lymphocytosis who were taking ibrutinib. Resistance to the irreversible BTK inhibitor ibrutinib often involves mutation of a cysteine residue where ibrutinib binding occurs. This finding, combined with two additional mutations in PLCγ2 that are immediately downstream of BTK, underscores the importance of the B-cell-receptor pathway in the mechanism of action of ibrutinib in CLL. (Funded by the National Cancer Institute and others.).

  2. 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

    Cutaneous T cell lymphomas (CTCLs) often show abnormal interleukin-2 (IL-2) receptor signaling. In this study, we investigated the role of Gab2, a recently identified adaptor molecule involved in IL-2 receptor signaling in CTCLs. We show that Gab2 was transiently phosphorylated by tyrosine in human...... mycosis fungoides (MF) tumor T cells upon IL-2 stimulation and that SHP2 as well as Stat5a associated inducibly with Gab2. IL-15, but not IL-4, also induced tyrosine phosphorylation of Gab2, suggesting that the IL-2 receptor beta-chain is important for IL-2-induced Gab2 phosphorylation. Preincubation...... 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...

  3. TNK2 Tyrosine Kinase as a Novel Therapeutic Target in Triple Negative Breast Cancer

    Science.gov (United States)

    2016-10-01

    protein phosphorylation levels with cellular oncogenic phenotypes, we observed a novel non-receptor tyrosine kinase, TNK2, to be hyperphosphorylated...phosphorylation is observed upon (R)-9bMS treatment (Figure 6A). Inhibition of TNK2 activation is also reflected in significant decrease in AKT Tyr176...Triple-negative breast cancer: a short review. American journal of clinical oncology . 2010;33(6):637-45. doi: 10.1097/COC.0b013e3181b8afcf. PubMed

  4. A combination of tyrosine kinase inhibitors, crizotinib and dasatinib for the treatment of glioblastoma multiforme.

    Science.gov (United States)

    Nehoff, Hayley; Parayath, Neha N; McConnell, Melanie J; Taurin, Sebastien; Greish, Khaled

    2015-11-10

    Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Despite the advances in surgery, radiotherapy and chemotherapy, patient survival averages only 14.6 months. In most GBM tumors, tyrosine kinases show increased activity and/or expression and actively contribute to the development, recurrence and onset of treatment resistance; making their inhibition an appealing therapeutic strategy. We compared the cytotoxicity of 12 tyrosine kinase inhibitors in vitro. A combination of crizotinib and dasatinib emerged as the most cytotoxic across established and primary human GBM cell lines. The combination treatment induced apoptotic cell death and polyploidy. Furthermore, the combination treatment led to the altered expression and localization of several tyrosine kinase receptors such as Met and EGFR and downstream effectors as such as SRC. Furthermore, the combination treatment reduced the migration and invasion of GBM cells and prevented endothelial cell tube formation in vitro. Overall, our study demonstrated the broad specificity of a combination of crizotinib and dasatinib across multiple GBM cell lines. These findings provide insight into the development of alternative therapy for the treatment of GBM.

  5. TRAP Induces More Intense Tyrosine Phosphorylation than Thrombin with Differential Ultrastructural Features

    Science.gov (United States)

    Fusté, Berta; Díaz-Ricart, Maribel; Jensen, Morten Krogh; Ordinas, Antonio; Escolar, Ginés; White, James G.

    2002-01-01

    We have analyzed modifications on platelet ultrastructural morphology, cytoskeletal assembly, and tyrosine phosphorylation developing in platelets activated by both thrombin and the thrombin receptor-activating peptide (TRAP). Washed platelets exposed to various concentrations of thrombin or TRAP, for different periods, were: fixed and examined by electron microscopy, or lysed and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under similar activating conditions, thrombin and TRAP induced different sequences of activation causing distinctive morphological and biochemical changes. Platelets exposed to thrombin showed centralized organelles encircled by constricted microtubule coils and granules secreting their contents through narrow channels of the open canalicular system. In contrast, activation by TRAP induced swelling of the open canalicular system with organelles remaining randomly dispersed and microtubules peripherally distributed. Compared to thrombin activation, TRAP induced higher rates of actin polymerization; increased association of actin-binding protein, myosin, and α-actinin; and higher association of tyrosine-phosphorylated proteins with the insoluble cytoskeletal fraction. Secretion of intragranule substances, measured as expression of P-selectin and lysosomal integral membrane protein at the surface level, were similar for both agonists at equivalent concentrations. Our biochemical observations indicate that TRAP causes more intense changes in signaling through tyrosine phosphorylation of proteins associated with the cytoskeletal fraction than thrombin. However, as derived from ultrastructural observations, TRAP seems to be less efficient in triggering cytoskeletal assembly and internal contraction in an organized manner in contrast with the natural protease. PMID:12057926

  6. Ret function in muscle stem cells points to tyrosine kinase inhibitor therapy for facioscapulohumeral muscular dystrophy.

    Science.gov (United States)

    Moyle, Louise A; Blanc, Eric; Jaka, Oihane; Prueller, Johanna; Banerji, Christopher Rs; Tedesco, Francesco Saverio; Harridge, Stephen Dr; Knight, Robert D; Zammit, Peter S

    2016-11-14

    Facioscapulohumeral muscular dystrophy (FSHD) involves sporadic expression of DUX4, which inhibits myogenesis and is pro-apoptotic. To identify target genes, we over-expressed DUX4 in myoblasts and found that the receptor tyrosine kinase Ret was significantly up-regulated, suggesting a role in FSHD. RET is dynamically expressed during myogenic progression in mouse and human myoblasts. Constitutive expression of either RET9 or RET51 increased myoblast proliferation, whereas siRNA-mediated knockdown of Ret induced myogenic differentiation. Suppressing RET activity using Sunitinib, a clinically-approved tyrosine kinase inhibitor, rescued differentiation in both DUX4-expressing murine myoblasts and in FSHD patient-derived myoblasts. Importantly, Sunitinib also increased engraftment and differentiation of FSHD myoblasts in regenerating mouse muscle. Thus, DUX4-mediated activation of Ret prevents myogenic differentiation and could contribute to FSHD pathology by preventing satellite cell-mediated repair. Rescue of DUX4-induced pathology by Sunitinib highlights the therapeutic potential of tyrosine kinase inhibitors for treatment of FSHD.

  7. 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.

  8. Evaluation of placental growth factor and soluble Fms-like tyrosine kinase 1 as predictors of all-cause and cardiovascular mortality in patients with Type 1 diabetes with and without diabetic nephropathy

    DEFF Research Database (Denmark)

    Theilade, S; Lajer, Maria Stenkil; Jorsal, Anders

    2012-01-01

    Placental growth factor is a vascular endothelial growth factor involved in angiogenesis, vascular inflammation and plaque formation. Soluble Fms-like tyrosine kinase 1 is a decoy receptor for placental growth factor, reducing its activity. The aim of this study is to evaluate the predictive valu...... of placental growth factor and soluble Fms-like tyrosine kinase 1 in relation to all-cause and cardiovascular mortality and decline in kidney function in Type 1 diabetes....

  9. ECM-Stimulated Actin Bundle Formation in Embryonic Corneal Epithelia is Tyrosine Phosphorylation Dependent

    Science.gov (United States)

    SVOBODA, KATHY K.H.; ORLOW, DANIEL L.; CHU, CHIA LIN; REENSTRA, WENDE R.

    2009-01-01

    Previous studies demonstrated that corneal epithelial cells isolated without basal lamina respond to extracellular matrix (ECM) in an actin dependent manner; the basal cell surface flattens and the actin cortical mat reorganizes. We hypothesize that the actin reorganization is initiated by intracellular signaling mechanisms that includes tyrosine phoshporylation and activation of the Rho, MAP kinase, and PI3 kinase signal transduction pathways. Our goals were to develop a morphological assay to test this hypothesis by answering the following questions: 1) Do the actin bundle formations in the cortical mat have the same configuration in response to different ECM molecules? 2) What is the minimum time ECM molecules need to be in contact with the tissue for the actin to reorganize? 3) Will blocking tyrosine phosphorylation inhibit reorganization of the actin? 4) Are known signal transduction proteins phosphorylated in response to soluble matrix molecules? The actin cortical mat demonstrated distinct bundle configurations in the presence of different ECM molecules. Soluble fibronectin accumulated at the basal cell surfaces 75-fold over 30 min in a clustered pattern. The cells need contact with ECM for a minimum of 10 min to reform the actin bundles at 2 hr. In contrast, two substances that bind to heptahelical receptors to stimulate the Rho pathway, bombesin and lysophosphatidic acid, reorganized the actin bundles in 15–30 min. Focal adhesion kinase, p190 Rho-GAP, tensin, and paxillin were tyrosine phosphorylated in response to soluble fibronectin, type I collagen, or laminin 1. Erk-1, erk-2, and PI3 kinase were activated after 1 hr stimulation by type I collagen. Herbimycin A blocked actin reorganization induced by ECM molecules. In conclusion, we have developed two morphological assays to examine the response of corneal epithelial cells to ECM molecules. In addition, actin bundle reorganization involved tyrosine phosphorylation, MAP kinase, and PI3 kinase

  10. Vascular endothelial-cadherin tyrosine phosphorylation in angiogenic and quiescent adult tissues.

    Science.gov (United States)

    Lambeng, Nathalie; Wallez, Yann; Rampon, Christine; Cand, Francine; Christé, Georges; Gulino-Debrac, Danielle; Vilgrain, Isabelle; Huber, Philippe

    2005-02-18

    Vascular endothelial-cadherin (VE-cadherin) plays a key role in angiogenesis and in vascular permeability. The regulation of its biological activity may be a central mechanism in normal or pathological angiogenesis. VE-cadherin has been shown to be phosphorylated on tyrosine in vitro under various conditions, including stimulation by VEGF. In the present study, we addressed the question of the existence of a tyrosine phosphorylated form of VE-cadherin in vivo, in correlation with the quiescent versus angiogenic state of adult tissues. Phosphorylated VE-cadherin was detected in mouse lung, uterus, and ovary but not in other tissues unless mice were injected with peroxovanadate to block protein phosphatases. Remarkably, VE-cadherin tyrosine phosphorylation was dramatically increased in uterus and ovary, and not in other organs, during PMSG/hCG-induced angiogenesis. In parallel, we observed an increased association of VE-cadherin with Flk1 (VEGF receptor 2) during hormonal angiogenesis. Additionally, Src kinase was constitutively associated with VE-cadherin in both quiescent and angiogenic tissues and increased phosphorylation of VE-cadherin-associated Src was detected in uterus and ovary after hormonal treatment. Src-VE-cadherin association was detected in cultured endothelial cells, independent of VE-cadherin phosphorylation state and Src activation level. In this model, Src inhibition impaired VEGF-induced VE-cadherin phosphorylation, indicating that VE-cadherin phosphorylation was dependent on Src activation. We conclude that VE-cadherin is a substrate for tyrosine kinases in vivo and that its phosphorylation, together with that of associated Src, is increased by angiogenic stimulation. Physical association between Flk1, Src, and VE-cadherin may thus provide an efficient mechanism for amplification and perpetuation of VEGF-stimulated angiogenic processes.

  11. Behavioral and cognitive effects of tyrosine intake in healthy human adults

    NARCIS (Netherlands)

    Hase, Adrian; Jung, Sophie E.; aan het Rot, Marije

    2015-01-01

    The amino acid tyrosine is the precursor to the catecholamine neurotransmitters dopamine and norepinephrine. Increasing tyrosine uptake may positively influence catecholamine-related psychological functioning. We conducted a systematic review to examine the effects of tyrosine on behavior and

  12. Jak2 tyrosine kinase residues glutamic acid 1024 and arginine 1113 form a hydrogen bond interaction that is essential for Jak-STAT signal transduction.

    Science.gov (United States)

    Sandberg, Eric M; VonDerLinden, Dannielle; Ostrov, David A; Sayeski, Peter P

    2004-10-01

    Angiotensin II is a well-known vasoactive peptide, but it can also act as a potent growth factor, partially through activation of the tyrosine kinase Jak2. Activated Jak2 tyrosine phosphorylates and activates members of the Signal Transducers and Activators of Transcription (STAT) family of cytoplasmic transcription factors. Recently, we demonstrated that tryptophan 1020 and glutamic acid 1024 within the Jak2 activation loop are required for Jak2 tyrosine kinase activity. Here, we sought to elucidate the requirement of glutamic acid 1024 for Jak2 function. Using molecular modeling algorithms of the Jak2 kinase domain, we identified a putative interaction between glutamic acid 1024 and an arginine at position 1113. We generated a series of charge-based substitution mutations at position 1113 and found that conversion of arginine 1113 to glutamic acid, alanine, or lysine prevented Jak2 autophosphorylation. Furthermore, mutation of arginine 1113 prevented the following angiotensin II-dependent processes from occurring: (1) Jak2 tyrosine phosphorylation, (2) Jak2/AT1receptor co-association, (3) STAT1 recruitment to the Jak2/AT1receptor complex, (4) STAT1 tyrosine phosphorylation, and (5) STAT-mediated gene expression. We determined that the interaction between glutamic acid 1024 and arginine 1113 consists of two distinct hydrogen bonds. We conclude that these hydrogen bond interactions are critical for Jak2 kinase function and subsequent angiotensin II-dependent activation of the Jak/STAT signaling pathway.

  13. Redox-dependent regulation of epidermal growth factor receptor signaling

    Directory of Open Access Journals (Sweden)

    David E. Heppner

    2016-08-01

    Full Text Available Tyrosine phosphorylation-dependent cell signaling represents a unique feature of multicellular organisms, and is important in regulation of cell differentiation and specialized cell functions. Multicellular organisms also contain a diverse family of NADPH oxidases (NOXs that have been closely linked with tyrosine kinase-based cell signaling and regulate tyrosine phosphorylation via reversible oxidation of cysteine residues that are highly conserved within many proteins involved in this signaling pathway. An example of redox-regulated tyrosine kinase signaling involves the epidermal growth factor receptor (EGFR, a widely studied receptor system with diverse functions in normal cell biology as well as pathologies associated with oxidative stress such as cancer. The purpose of this Graphical Redox Review is to highlight recently emerged concepts with respect to NOX-dependent regulation of this important signaling pathway.

  14. Food for creativity: tyrosine promotes deep thinking.

    Science.gov (United States)

    Colzato, Lorenza S; de Haan, Annelies M; Hommel, Bernhard

    2015-09-01

    Anecdotal evidence suggests that creative people sometimes use food to overcome mental blocks and lack of inspiration, but empirical support for this possibility is still lacking. In this study, we investigated whether creativity in convergent- and divergent-thinking tasks is promoted by the food supplement L-Tyrosine (TYR)-a biochemical precursor of dopamine, which is assumed to drive cognitive control and creativity. We found no evidence for an impact of TYR on divergent thinking ("brainstorming") but it did promote convergent ("deep") thinking. As convergent thinking arguably requires more cognitive top-down control, this finding suggests that TYR can facilitate control-hungry creative operations. Hence, the food we eat may affect the way we think.

  15. Tyrosine Phosphorylation of Botulinum Neurotoxin Protease Domains

    Directory of Open Access Journals (Sweden)

    Stephen eToth

    2012-06-01

    Full Text Available Botulinum neurotoxins are most potent of all toxins. Their N-terminal light chain domain (Lc translocates into peripheral cholinergic neurons to exert its endoproteolytic action leading to muscle paralysis. Therapeutic development against these toxins is a major challenge due to their in vitro and in vivo structural differences. Although three-dimensional structures and reaction mechanisms are very similar, the seven serotypes designated A through G vastly vary in their intracellular catalytic stability. To investigate if protein phosphorylation could account for this difference, we employed Src-catalyzed tyrosine phosphorylation of the Lc of six serotypes namely LcA, LcB, LcC1, LcD, LcE, and LcG. Very little phosphorylation was observed with LcD and LcE but LcA, LcB and LcG were maximally phosphorylated by Src. Phosphorylation of LcA, LcB, and LcG did not affect their secondary and tertiary structures and thermostability significantly. Phosphorylation of Y250 and Y251 made LcA resistant to autocatalysis and drastically reduced its kcat/Km for catalysis. A tyrosine residue present near the essential cysteine at the C-terminal tail of LcA, LcB and LcG was readily phosphorylated in vitro. Inclusion of a competitive inhibitor protected this Y426 of LcA from phosphorylation, shedding light on the role of the C-terminus in the enzyme’s substrate or product binding.

  16. 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

  17. Quantitation of multisite EGF receptor phosphorylation using mass spectrometry and a novel normalization approach

    DEFF Research Database (Denmark)

    Erba, Elisabetta Boeri; Matthiesen, Rune; Bunkenborg, Jakob

    2007-01-01

    Using stable isotope labeling and mass spectrometry, we performed a sensitive, quantitative analysis of multiple phosphorylation sites of the epidermal growth factor (EGF) receptor. Phosphopeptide detection efficiency was significantly improved by using the tyrosine phosphatase inhibitor sodium...

  18. 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

  19. Striatal-enriched protein tyrosine phosphatase (STEP) knockout mice have enhanced hippocampal memory.

    Science.gov (United States)

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

    2011-06-01

    Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase that opposes synaptic strengthening by the regulation of key synaptic signaling proteins. Previous studies suggest a possible role for STEP in learning and memory. To demonstrate the functional importance of STEP in learning and memory, we generated STEP knockout (KO) mice and examined the effect of deletion of STEP on behavioral performance, as well as the phosphorylation and expression of its substrates. Here we report that loss of STEP leads to significantly enhanced performance in hippocampal-dependent learning and memory tasks. In addition, STEP KO mice displayed greater dominance behavior, although they were normal in their motivation, motor coordination, visual acuity and social interactions. STEP KO mice displayed enhanced tyrosine phosphorylation of extracellular-signal regulated kinase 1/2 (ERK1/2), the NR2B subunit of the N-methyl-D-aspartate receptor (NMDAR) and proline-rich tyrosine kinase (Pyk2), as well as an increased phosphorylation of ERK1/2 substrates. Concomitant with the increased phosphorylation of NR2B, synaptosomal expression of NR1/NR2B NMDARs was increased in STEP KO mice, as was the GluR1/GluR2 containing α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPARs), providing a potential molecular mechanism for the improved cognitive performance. The data support a role for STEP in the regulation of synaptic strengthening. The absence of STEP improves cognitive performance, and may do so by the regulation of downstream effectors necessary for synaptic transmission. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  20. Facile and stabile linkages through tyrosine: bioconjugation strategies with the tyrosine-click reaction.

    Science.gov (United States)

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

    2013-04-17

    The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivatives in peptide and protein chemistry, we synthesized PTAD derivatives possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivatives and peptides of increasing complexity. With proteins we studied the compatibility of the tyrosine click reaction with cysteine and lysine-targeted labeling approaches and demonstrate that chemoselective trifunctionalization of proteins is readily achieved. In particular cases, we noted that PTAD decomposition resulted in formation of a putative isocyanate byproduct that was promiscuous in labeling. This side reaction product, however, was readily scavenged by the addition of a small amount of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) to the reaction medium. To study the potential of the tyrosine click reaction to introduce poly(ethylene glycol) chains onto proteins (PEGylation), we demonstrate that this novel reagent provides for the selective PEGylation of chymotrypsinogen, whereas traditional succinimide-based PEGylation targeting lysine residues provided a more diverse range of PEGylated products. Finally, we applied the tyrosine click reaction to create a novel antibody-drug conjugate. For this purpose, we synthesized a PTAD derivative linked to the HIV entry inhibitor aplaviroc. Labeling of the antibody trastuzumab with this reagent provided a labeled antibody conjugate that demonstrated potent HIV-1 neutralization activity demonstrating the potential of this reaction in creating protein conjugates with small molecules. The tyrosine click linkage demonstrated stability to extremes of pH, temperature, and exposure to human blood plasma indicating that this linkage is significantly more robust than maleimide-type linkages that are commonly employed in bioconjugations. These

  1. Analysis of Cellular Tyrosine Phosphorylation via Chemical Rescue of Conditionally Active Abl Kinase.

    Science.gov (United States)

    Wang, Zhihong; Kim, Min-Sik; Martinez Ferrando, Isabel; Koleske, Anthony John; Pandey, Akhilesh; Cole, Philip Arthur

    2018-01-17

    Identifying direct substrates targeted by protein kinases is important in understanding cellular physiology and intracellular signal transduction. Mass-spectrometry based quantitative proteomics provides a powerful tool for comprehensively characterizing the downstream substrates of protein kinases. This approach is efficiently applied to receptor kinases which can be precisely, directly, and rapidly activated by some agent, such as a growth factor. However, non-receptor tyrosine kinase Abl lacks the experimental advantage of extracellular growth factors as immediate and direct stimuli. To circumvent this limitation, we combine a chemical rescue approach with quantitative phosphoproteomics to identify targets of Abl and their phosphorylation sites with enhanced temporal resolution. Both known and novel putative substrates are identified, presenting opportunities for studying unanticipated functions of Abl under physiological and pathological conditions.

  2. The tyrosine phosphatase STEP: implications in schizophrenia and the molecular mechanism underlying antipsychotic medications.

    Science.gov (United States)

    Carty, N C; Xu, J; Kurup, P; Brouillette, J; Goebel-Goody, S M; Austin, D R; Yuan, P; Chen, G; Correa, P R; Haroutunian, V; Pittenger, C; Lombroso, P J

    2012-07-10

    Glutamatergic signaling through N-methyl-D-aspartate receptors (NMDARs) is required for synaptic plasticity. Disruptions in glutamatergic signaling are proposed to contribute to the behavioral and cognitive deficits observed in schizophrenia (SZ). One possible source of compromised glutamatergic function in SZ is decreased surface expression of GluN2B-containing NMDARs. STEP(61) is a brain-enriched protein tyrosine phosphatase that dephosphorylates a regulatory tyrosine on GluN2B, thereby promoting its internalization. Here, we report that STEP(61) levels are significantly higher in the postmortem anterior cingulate cortex and dorsolateral prefrontal cortex of SZ patients, as well as in mice treated with the psychotomimetics MK-801 and phencyclidine (PCP). Accumulation of STEP(61) after MK-801 treatment is due to a disruption in the ubiquitin proteasome system that normally degrades STEP(61). STEP knockout mice are less sensitive to both the locomotor and cognitive effects of acute and chronic administration of PCP, supporting the functional relevance of increased STEP(61) levels in SZ. In addition, chronic treatment of mice with both typical and atypical antipsychotic medications results in a protein kinase A-mediated phosphorylation and inactivation of STEP(61) and, consequently, increased surface expression of GluN1/GluN2B receptors. Taken together, our findings suggest that STEP(61) accumulation may contribute to the pathophysiology of SZ. Moreover, we show a mechanistic link between neuroleptic treatment, STEP(61) inactivation and increased surface expression of NMDARs, consistent with the glutamate hypothesis of SZ.

  3. Therapeutic implications for striatal-enriched protein tyrosine phosphatase (STEP) in neuropsychiatric disorders.

    Science.gov (United States)

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

    2012-01-01

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

  4. NMDA-mediated activation of the tyrosine phosphatase STEP regulates the duration of ERK signaling.

    Science.gov (United States)

    Paul, Surojit; Nairn, Angus C; Wang, Ping; Lombroso, Paul J

    2003-01-01

    The intracellular mechanism(s) by which a cell determines the duration of extracellular signal-regulated kinase (ERK) activation is not well understood. We have investigated the role of STEP, a striatal-enriched tyrosine phosphatase, in the regulation of ERK activity in rat neurons. Glutamate-mediated activation of NMDA receptors leads to the rapid but transient phosphorylation of ERK in cultured neurons. Here we show that activation of NMDA receptors led to activation of STEP, which limited the duration of ERK activity as well as its translocation to the nucleus and its subsequent downstream nuclear signaling. In neurons, STEP is phosphorylated and inactive under basal conditions. NMDA-mediated influx of Ca(2+), but not increased intracellular Ca(2+) from other sources, leads to activation of the Ca(2+)-dependent phosphatase calcineurin and the dephosphorylation and activation of STEP. We have identified an important mechanism involved in the regulation of ERK activity in neurons that highlights the complex interplay between serine/threonine and tyrosine kinases and phosphatases.

  5. The human tyrosine hydroxylase gene promoter.

    Science.gov (United States)

    Kessler, Mark A; Yang, Ming; Gollomp, Kandace L; Jin, Hao; Iacovitti, Lorraine

    2003-04-10

    13.329 kilobases of the single copy human tyrosine hydroxylase (hTH) gene were isolated from a genomic library. The 5' flanking 11 kilobases fused to the reporter green fluorescent protein (GFP) drove high level expression in TH+ cells of the substantia nigra of embryonic and adult transgenic mice as determined by double label fluorescence microscopy. To provide a basis for future analysis of polymorphisms and structure-function studies, the previously unreported distal 10.5 kilobases of the hTH promoter were sequenced with an average coverage of 20-fold, the remainder with 4-fold coverage. Sequence features identified included four perfect matches to the bicoid binding element (BBE, consensus: BBTAATCYV) all of which exhibited specific binding by electrophoretic mobility shift assay (EMSA). Comparison to published sequences of mouse and rat TH promoters revealed five areas of exceptional homology shared by these species in the upstream TH promoter region -2 kb to -9 kb relative to the transcription start site. Within these conserved regions (CRs I-V), potential recognition sites for NR4A2 (Nurr1), HNF-3beta, HOXA4, and HOXA5 were shared across human, mouse, and rat TH promoters.

  6. Electrode Potentials of l-Tryptophan, l-Tyrosine, 3-Nitro-l-tyrosine, 2,3-Difluoro-l-tyrosine, and 2,3,5-Trifluoro-l-tyrosine.

    Science.gov (United States)

    Mahmoudi, Leila; Kissner, Reinhard; Nauser, Thomas; Koppenol, Willem H

    2016-05-24

    Electrode potentials for aromatic amino acid radical/amino acid couples were deduced from cyclic voltammograms and pulse radiolysis experiments. The amino acids investigated were l-tryptophan, l-tyrosine, N-acetyl-l-tyrosine methyl ester, N-acetyl-3-nitro-l-tyrosine ethyl ester, N-acetyl-2,3-difluoro-l-tyrosine methyl ester, and N-acetyl-2,3,5-trifluoro-l-tyrosine methyl ester. Conditional potentials were determined at pH 7.4 for all compounds listed; furthermore, Pourbaix diagrams for l-tryptophan, l-tyrosine, and N-acetyl-3-nitro-l-tyrosine ethyl ester were obtained. Electron transfer accompanied by proton transfer is reversible, as confirmed by detailed analysis of the current waves, and because the slopes of the Pourbaix diagrams obey Nernst's law. E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH) at pH 7 are 0.99 ± 0.01 and 0.97 ± 0.01 V, respectively. Pulse radiolysis studies of two dipeptides that contain both amino acids indicate a difference in E°' of approximately 0.06 V. Thus, in small peptides, we recommend values of 1.00 and 0.96 V for E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH), respectively. The electrode potential of N-acetyl-3-nitro-l-tyrosine ethyl ester is higher, while because of mesomeric stabilization of the radical, those of N-acetyl-2,3-difluoro-l-tyrosine methyl ester and N-acetyl-2,3,5-trifluoro-l-tyrosine methyl ester are lower than that of tyrosine. Given that the electrode potentials at pH 7 of E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH) are nearly equal, they would be, in principle, interchangeable. Proton-coupled electron transfer pathways in proteins that use TrpH and TyrOH are thus nearly thermoneutral.

  7. Direct observation of spin-injection in tyrosinate-functionalized single-wall carbon nanotubes

    NARCIS (Netherlands)

    Tsoufis, Theodoros; Ampoumogli, Asem; Gournis, Dimitrios; Georgakilas, Vasilios; Jankovic, Lubos; Christoforidis, Konstantinos C.; Deligiannakis, Yiannis; Mavrandonakis, Andreas; Froudakis, George E.; Maccallini, Enrico; Rudolf, Petra; Mateo-Alonso, Aurelio; Prato, Maurizio

    In this work, we report on the interaction of a tyrosinate radical with single wall carbon nanotubes (CNT). The tyrosinate radical was formed from tyrosine (ester) by Fenton's reagent and, reacted in situ with carbon nanotubes resulting in novel tyrosinated carbon nanotube derivatives. The covalent

  8. ERBB receptors in cancer: signaling from the inside.

    Science.gov (United States)

    Arteaga, Carlos L

    2011-03-16

    ERBB receptor tyrosine kinases are activated by ligand-induced dimerization followed by activation and transphosphorylation of their intracellular kinase domains. A recent study by Bill and colleagues demonstrates that receptor transphosphorylation can be regulated from inside the cell by members of the cytohesin protein family. These data highlight a novel mechanism of amplification of ERBB receptor signaling output that may contribute to embryogenesis and cancer progression.

  9. Chemical Inhibition of Bacterial Protein Tyrosine Phosphatase Suppresses Capsule Production

    OpenAIRE

    Standish, Alistair J.; Salim, Angela A; Hua Zhang; Capon, Robert J.; Renato Morona

    2012-01-01

    Capsule polysaccharide is a major virulence factor for a wide range of bacterial pathogens, including Streptococcus pneumoniae. The biosynthesis of Wzy-dependent capsules in both gram-negative and -positive bacteria is regulated by a system involving a protein tyrosine phosphatase (PTP) and a protein tyrosine kinase. However, how the system functions is still controversial. In Streptococcus pneumoniae, a major human pathogen, the system is present in all but 2 of the 93 serotypes found to dat...

  10. Intramolecular Photogeneration of a Tyrosine Radical in a Designed Protein

    Science.gov (United States)

    Tebo, Alison G.; Quaranta, Annamaria; Herrero, Christian; Pecoraro, Vincent L.; Aukauloo, Ally

    2017-01-01

    Long-distance biological electron transfer occurs through a hopping mechanism and often involves tyrosine as a high potential intermediate, for example in the early charge separation steps during photosynthesis. Protein design allows for the development of minimal systems to study the underlying principles of complex systems. Herein, we report the development of the first ruthenium-linked designed protein for the photogeneration of a tyrosine radical by intramolecular electron transfer. PMID:29046892

  11. The role of protein-tyrosine phosphorylation and gelatinase production in the migration and proliferation of smooth muscle cells.

    Science.gov (United States)

    Uzui, H; Lee, J D; Shimizu, H; Tsutani, H; Ueda, T

    2000-03-01

    It has been reported that matrix metalloproteinase (MMP) was expressed in coronary arterial atherosclerotic lesions. However, not much is known about the relationship between the production of MMP and the progression of atherosclerosis. To demonstrate the association between the protein-tyrosine phosphorylation (PTP) and the activation of extracellular MMP in the proliferation and migration of vascular smooth muscle cells (VSMCs), the effect of platelet-derived growth factor (PDGF) and vanadate (an inhibitor of protein-tyrosine phosphatase and an activator of certain protein-tyrosine kinases) on mitogenesis ([3H]thymidine incorporation after 24 hours), migration, PTP (Western blot analysis using anti-phosphotyrosine antibodies), and production of MMP (gelatin zymography) was examined in cultured VSMCs. Both vanadate (1-5 micromol/l) and PDGF (1-10 ng/ml) caused a dose-dependent increase in thymidine incorporation and migration and produced 72-kDa type IV gelatinase (MMP-2) in VSMCs. The combination of vanadate and PDGF resulted in a dose-dependent synergistic effect on thymidine incorporation and MMP-2 production. Western blot analysis revealed that PDGF caused an increase in PTP, extracellular signal-regulated kinases (ERK1, ERK2) and PDGF receptor in VSMCs. Vanadate given together with PDGF induced a marked increase in the intensity of tyrosine phosphorylation in these proteins. Tyrosine kinase inhibitors (genistein and herbimycin A) and a synthetic inhibitor of MMP (1,10-phenanthroline) and an anti-MMP-2 neutralizing antibody inhibited the mitogenic effect induced by vanadate and/or PDGF. The data suggest that the proliferation and migration of cultured VSMCs was closely related to the stimulation of MMP-2 production that was induced through activation of PTK.

  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...... in the protein were analyzed using the essential dynamics technique. Our results indicate that the predominately internal motions in PTP1B occur in a subspace of only a few degrees of freedom. Upon substrate binding, the flexibility of the protein is reduced by similar to 10%. The largest effect is found......Activity of enzymes, such as protein tyrosine phosphatases (PTPs), is often associated with structural changes in the enzyme, resulting in selective and stereospecific reactions with the substrate. To investigate the effect of a substrate on the motions occurring in PTPs, we have performed...

  13. Proteinase-activated receptors (PARs) – focus on receptor-receptor-interactions and their physiological and pathophysiological impact

    Science.gov (United States)

    2013-01-01

    Proteinase-activated receptors (PARs) are a subfamily of G protein-coupled receptors (GPCRs) with four members, PAR1, PAR2, PAR3 and PAR4, playing critical functions in hemostasis, thrombosis, embryonic development, wound healing, inflammation and cancer progression. PARs are characterized by a unique activation mechanism involving receptor cleavage by different proteinases at specific sites within the extracellular amino-terminus and the exposure of amino-terminal “tethered ligand“ domains that bind to and activate the cleaved receptors. After activation, the PAR family members are able to stimulate complex intracellular signalling networks via classical G protein-mediated pathways and beta-arrestin signalling. In addition, different receptor crosstalk mechanisms critically contribute to a high diversity of PAR signal transduction and receptor-trafficking processes that result in multiple physiological effects. In this review, we summarize current information about PAR-initiated physical and functional receptor interactions and their physiological and pathological roles. We focus especially on PAR homo- and heterodimerization, transactivation of receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RSTKs), communication with other GPCRs, toll-like receptors and NOD-like receptors, ion channel receptors, and on PAR association with cargo receptors. In addition, we discuss the suitability of these receptor interaction mechanisms as targets for modulating PAR signalling in disease. PMID:24215724

  14. 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...

  15. Overexpression of protein tyrosine phosphatase 1B impairs glucose-stimulated insulin secretion in INS-1 cells.

    Science.gov (United States)

    Lu, Bin; Gu, Ping; Xu, Yixin; Ye, Xiaozhen; Wang, Yingzhijie; DU, Hong; Shao, Jiaqing

    2016-03-01

    Protein tyrosine phosphatase 1B (PTP1B) has been implicated as a negative regulator of insulin signaling. We reported previously that impaired glucose-stimulated insulin secretion (GSIS) in rats fed high-fat diet was associated with higher PTP1B protein levels in islets. The aim of the present study was to investigate the effect of increasing PTP1B on insulin secretion in β-cells. INS-1 cells were transduced with recombinant adenoviruses containing human PTP1B cDNA (Ad-PTP1B), or no exogenous gene (Ad-ctrl). The expression levels of PTP1B, insulin receptor (IR), insulin receptor substrate-1(IRS-1), glucokinase and glucose transporter-2 were evaluated by Western blot. Then insulin-stimulated IR and IRS tyrosine phosphorylation, and Akt pathway activation were measured. GSIS was also performed to evaluate INS-1 cells function. PTP1B expression level was increased 5.9-fold at 48h post-transduction. The overexpression of PTP1B had no effect on proliferation and apoptosis of INS-1 cells. Compared with control cells, INS-1 cells overexpressing PTP1B showed decrease in insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR) and insulin receptor substrate-1(IRS-1) by 56.4% and 53.1%, respectively. In addition, Akt phosphorylation was reduced 59.6%. Moreover, in Ad-PTP1B-transduced cells, 16.7mM glucose caused a 1.6±0.2 fold increase (vs. 3.9±0.7 fold in nontransduced cells) in insulin secretion relative to secretion at 2.8mM glucose. Further analysis determined that overexpression of PTP1B induced down-regulated expression of glucokinase (42%) and glucose transporter-2 (48%). Our findings suggested that overexpression of PTP1B can inhibit GSIS in INS-1 cells through negatively regulating insulin signaling.

  16. Novel Mutations in the Tyrosine Hydroxylase Gene in the First Czech Patient with Tyrosine Hydroxylase Deficiency

    Directory of Open Access Journals (Sweden)

    K. Szentiványi

    2012-01-01

    Full Text Available Tyrosine hydroxylase deficiency manifests mainly in early childhood and includes two clinical phenotypes: an infantile progressive hypokinetic-rigid syndrome with dystonia (type A and a neonatal complex encephalopathy (type B. The biochemical diagnostics is exclusively based on the quantitative determination of the neurotransmitters or their metabolites in cerebrospinal fluid (CSF. The implementation of neurotransmitter analysis in clinical praxis is necessary for early diagnosis and adequate treatment. Neurotransmitter metabolites in CSF were analyzed in 82 children (at the age 1 month to 17 years with clinical suspicion for neurometabolic disorders using high performance liquid chromatography (HPLC with electrochemical detection. The CSF level of homovanillic acid (HVA was markedly decreased in three children (64, 79 and 94 nmol/l in comparison to age related controls (lower limit 218–450 nmol/l. Neurological findings including severe psychomotor retardation, quadruspasticity and microcephaly accompanied with marked dystonia, excessive sweating in the first patient was compatible with the diagnosis of tyrosine hydroxylase (TH deficiency (type B and subsequent molecular analysis revealed two novel heterozygous mutations c.636A>C and c.1124G>C in the TH gene. The treatment with L-DOPA/carbidopa resulted in the improvement of dystonia. Magnetic resonance imaging studies in two other patients with microcephaly revealed postischaemic brain damage, therefore secondary HVA deficit was considered in these children. Diagnostic work-up in patients with neurometabolic disorders should include analysis of neurotransmitter metabolites in CSF.

  17. A novel role of protein tyrosine kinase2 in mediating chloride secretion in human airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Lihua Liang

    Full Text Available Ca(2+ activated Cl(- channels (CaCC are up-regulated in cystic fibrosis (CF airway surface epithelia. The presence and functional properties of CaCC make it a possible therapeutic target to compensate for the deficiency of Cl(- secretion in CF epithelia. CaCC is activated by an increase in cytosolic Ca(2+, which not only activates epithelial CaCCs, but also inhibits epithelial Na(+ hyperabsorption, which may also be beneficial in CF. Our previous study has shown that spiperone, a known antipsychotic drug, activates CaCCs and stimulates Cl(- secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro, and in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR knockout mice in vivo. Spiperone activates CaCC not by acting in its well-known role as an antagonist of either 5-HT2 or D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Moreover, spiperone independently activates CFTR through a novel mechanism. Herein, we performed a mass spectrometry analysis and identified the signaling molecule that mediates the spiperone effect in activating chloride secretion through CaCC and CFTR. Proline-rich tyrosine kinase 2 (PYK2 is a non-receptor protein tyrosine kinase, which belongs to the focal adhesion kinase family. The inhibition of PYK2 notably reduced the ability of spiperone to increase intracellular Ca(2+ and Cl(- secretion. In conclusion, we have identified the tyrosine kinase, PYK2, as the modulator, which plays a crucial role in the activation of CaCC and CFTR by spiperone. The identification of this novel role of PYK2 reveals a new signaling pathway in human airway epithelial cells.

  18. 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.

  19. Estrogen-Related Receptor Alpha (ERRa)-Coactivator Interactions as Targets for Discovery of New Anti-Breast Cancer Therapeutics

    National Research Council Canada - National Science Library

    Burgess, Richard R

    2006-01-01

    .... ERalpha-negative breast cancers do not respond to anti-estrogen treatment; instead, current therapeutics, such as Herceptin, have focused on the transmembrane tyrosine kinase receptor ErbB2 (HER2...

  20. The insulin and IGF1 receptor kinase domains are functional dimers in the activated state

    Science.gov (United States)

    Cabail, M. Zulema; Li, Shiqing; Lemmon, Eric; Bowen, Mark E.; Hubbard, Stevan R.; Miller, W. Todd

    2015-03-01

    The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

  1. Downsizing treatment with tyrosine kinase inhibitors in patients with advanced gastrointestinal stromal tumors improved resectability.

    Science.gov (United States)

    Sjölund, Katarina; Andersson, Anna; Nilsson, Erik; Nilsson, Ola; Ahlman, Håkan; Nilsson, Bengt

    2010-09-01

    Gastrointestinal stromal tumors (GISTs) express the receptor tyrosine kinase KIT. Most GISTs have mutations in the KIT or PDGFRA gene, causing activation of tyrosine kinase. Imatinib, a tyrosine kinase inhibitor (TKI), is the first-line palliative treatment for advanced GISTs. Sunitinib was introduced for patients with mutations not responsive to imatinib. The aim was to compare the survival of patients with high-risk resected GISTs treated with TKI prior to surgery with historical controls and to determine if organ-preserving surgery was facilitated. Ten high-risk GIST-patients had downsizing/adjuvant TKI treatment: nine with imatinib and one with sunitinib. The patients were matched with historical controls (n = 89) treated with surgery alone, from our population-based series (n = 259). Mutational analysis of KIT and PDGFRA was performed in all cases. The progression-free survival was calculated. The primary tumors decreased in mean diameter from 20.4 cm to 10.5 cm on downsizing imatinib. Four patients with R0 resection and a period of adjuvant imatinib had no recurrences versus 67% in the historical control group. Four patients with residual liver metastases have stable disease on continuous imatinib treatment after surgery. One patient has undergone reoperation with liver resection. The downsizing treatment led to organ-preserving surgery in nine patients and improved preoperative nutritional status in one patient. Downsizing TKI is recommended for patients with bulky tumors with invasion of adjacent organs. Sunitinib can be used for patients in case of imatinib resistance (e.g., wild-type GISTs), underlining the importance of mutational analysis for optimal surgical planning.

  2. Tyrosine sulfation of the amino terminus of PSGL-1 is critical for enterovirus 71 infection.

    Directory of Open Access Journals (Sweden)

    Yorihiro Nishimura

    Full Text Available Enterovirus 71 (EV71 is one of the major causative agents of hand, foot, and mouth disease, a common febrile disease in children; however, EV71 has been also associated with various neurological diseases including fatal cases in large EV71 outbreaks particularly in the Asia Pacific region. Recently we identified human P-selectin glycoprotein ligand-1 (PSGL-1 as a cellular receptor for entry and replication of EV71 in leukocytes. PSGL-1 is a sialomucin expressed on the surface of leukocytes, serves as a high affinity counterreceptor for selectins, and mediates leukocyte rolling on the endothelium. The PSGL-1-P-selectin interaction requires sulfation of at least one of three clustered tyrosines and an adjacent O-glycan expressing sialyl Lewis x in an N-terminal region of PSGL-1. To elucidate the molecular basis of the PSGL-1-EV71 interaction, we generated a series of PSGL-1 mutants and identified the post-translational modifications that are critical for binding of PSGL-1 to EV71. We expressed the PSGL-1 mutants in 293T cells and the transfected cells were assayed for their abilities to bind to EV71 by flow cytometry. We found that O-glycosylation on T57, which is critical for PSGL-1-selectin interaction, is not necessary for PSGL-1 binding to EV71. On the other hand, site-directed mutagenesis at one or more potential tyrosine sulfation sites in the N-terminal region of PSGL-1 significantly impaired PSGL-1 binding to EV71. Furthermore, an inhibitor of sulfation, sodium chlorate, blocked the PSGL-1-EV71 interaction and inhibited PSGL-1-mediated viral replication of EV71 in Jurkat T cells in a dose-dependent manner. Thus, the results presented in this study reveal that tyrosine sulfation, but not O-glycosylation, in the N-terminal region of PSGL-1 may facilitate virus entry and replication of EV71 in leukocytes.

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

    Energy Technology Data Exchange (ETDEWEB)

    Li, Feifei; Jiang, Yinan [Department of Pathophysiology, Anhui Medical University, Hefei 230032 (China); Zheng, Qiping [Department of Anatomy and Cell Biology, Rush University Medical Center, Chicago, IL 60612 (United States); Yang, Xiaoming [Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850 (China); Wang, Siying, E-mail: sywang@ahmu.edu.cn [Department of Pathophysiology, Anhui Medical University, Hefei 230032 (China)

    2011-01-07

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

  4. Identification of tyrosine 806 as a molecular determinant of RET kinase sensitivity to ZD6474.

    Science.gov (United States)

    Carlomagno, Francesca; Guida, Teresa; Anaganti, Suresh; Provitera, Livia; Kjaer, Svend; McDonald, Neil Q; Ryan, Anderson J; Santoro, Massimo

    2009-03-01

    ZD6474 (vandetanib, Zactima, Astra Zeneca) is an anilinoquinazoline used to target the receptor tyrosine kinase RET in familial and sporadic thyroid carcinoma (IC(50): 100 nM). The aim of this study was to identify molecular determinants of RET sensitivity to ZD6474. Here, we show that mutation of RET tyrosine 806 to cysteine (Y806C) induced RET kinase resistance to ZD6474 (IC(50): 933 nM). Y806 maps close to the gate-keeper position at the RET kinase nucleotide-binding pocket. Although tyrosine 806 is a RET auto-phosphorylation site, its substitution to phenylalanine (Y806F) did not markedly affect RET susceptibility to ZD6474 (IC(50): 87 nM), suggesting that phosphorylation of Y806 is not required for compound binding. Accordingly, the introduction of a phosphomimetic residue (Y806E) also caused resistance to ZD6474, albeit of a lesser degree (IC(50): 512 nM) than the cysteine mutation. Y806C/E RET mutants were also resistant to ZD6474 with respect to intracellular signalling and activation of an AP1-responsive promoter. We conclude that Y806 is a molecular determinant of RET sensitivity to ZD6474. Y806C is a natural RET mutation identified in a patient affected by multiple endocrine neoplasia type 2B. Based on its rare occurrence, it is unlikely that Y806C will be a frequent cause of refractoriness to ZD6474; however, it may be envisaged that mutations at this site can mediate secondary resistance formation in patients treated with the compound.

  5. Modulation of catalytic activity in multi-domain protein tyrosine phosphatases.

    Directory of Open Access Journals (Sweden)

    Lalima L Madan

    Full Text Available Signaling mechanisms involving protein tyrosine phosphatases govern several cellular and developmental processes. These enzymes are regulated by several mechanisms which include variation in the catalytic turnover rate based on redox stimuli, subcellular localization or protein-protein interactions. In the case of Receptor Protein Tyrosine Phosphatases (RPTPs containing two PTP domains, phosphatase activity is localized in their membrane-proximal (D1 domains, while the membrane-distal (D2 domain is believed to play a modulatory role. Here we report our analysis of the influence of the D2 domain on the catalytic activity and substrate specificity of the D1 domain using two Drosophila melanogaster RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of Drosophila Leukocyte antigen Related (DLAR and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A. While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A reveal a conformational rationale for the experimental observations. These studies reveal that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain.

  6. Co-conserved features associated with cis regulation of ErbB tyrosine kinases.

    Directory of Open Access Journals (Sweden)

    Amar Mirza

    Full Text Available BACKGROUND: The epidermal growth factor receptor kinases, or ErbB kinases, belong to a large sub-group of receptor tyrosine kinases (RTKs, which share a conserved catalytic core. The catalytic core of ErbB kinases have functionally diverged from other RTKs in that they are activated by a unique allosteric mechanism that involves specific interactions between the kinase core and the flanking Juxtamembrane (JM and COOH-terminal tail (C-terminal tail. Although extensive studies on ErbB and related tyrosine kinases have provided important insights into the structural basis for ErbB kinase functional divergence, the sequence features that contribute to the unique regulation of ErbB kinases have not been systematically explored. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we use a Bayesian approach to identify the selective sequence constraints that most distinguish ErbB kinases from other receptor tyrosine kinases. We find that strong ErbB kinase-specific constraints are imposed on residues that tether the JM and C-terminal tail to key functional regions of the kinase core. A conserved RIxKExE motif in the JM-kinase linker region and a glutamine in the inter-lobe linker are identified as two of the most distinguishing features of the ErbB family. While the RIxKExE motif tethers the C-terminal tail to the N-lobe of the kinase domain, the glutamine tethers the C-terminal tail to hinge regions critical for inter-lobe movement. Comparison of the active and inactive crystal structures of ErbB kinases indicates that the identified residues are conformationally malleable and can potentially contribute to the cis regulation of the kinase core by the JM and C-terminal tail. ErbB3, and EGFR orthologs in sponges and parasitic worms, diverge from some of the canonical ErbB features, providing insights into sub-family and lineage-specific functional specialization. CONCLUSION/SIGNIFICANCE: Our analysis pinpoints key residues for mutational analysis, and

  7. Antibacterial and EGFR-Tyrosine Kinase Inhibitory Activities of Polyhydroxylated Xanthones from Garcinia succifolia

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    Susawat Duangsrisai

    2014-11-01

    Full Text Available Chemical investigation of the methanol extract of the wood of Garcinia succifolia Kurz (Clusiaceae led to the isolation of 1,5-dihydroxyxanthone (1, 1,7-dihydroxyxanthone (2, 1,3,7-trihydroxyxanthone (3, 1,5,6-trihydroxyxanthone (4, 1,6,7-trihydroxyxanthone (5, and 1,3,6,7-tetrahydroxyxanthone (6. All of the isolated xanthones were evaluated for their antibacterial activity against bacterial reference strains, two Gram-positive (Staphylococcus aureus ATTC 25923, Bacillus subtillis ATCC 6633 and two Gram-negative (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, and environmental drug-resistant isolates (S. aureus B1, Enteroccoccus faecalis W1, and E. coli G1, as well as for their epidermal growth factor receptor (EGFR of tyrosine kinase inhibitory activity. Only 1,5,6-trihydroxy-(4, 1,6,7-trihydroxy-(5, and 1,3,6,7-tetrahydroxyxanthones (6 exhibited antibacterial activity against Gram-positive bacteria, however none was active against vancomycin-resistant E. faecalis. Additionally, 1,7-dihydroxyxanthone (2 showed synergism with oxacillin, but not with ampicillin. On the other hand, only 1,5-dihydroxyxanthone (1 and 1,7-dihydroxyxanthone (2 were found to exhibit the EGFR-tyrosine kinase inhibitory activity, with IC50 values of 90.34 and 223 nM, respectively.

  8. Tyrosine kinase inhibitors in pulmonary arterial hypertension: a double-edge sword?

    Science.gov (United States)

    Godinas, Laurent; Guignabert, Christophe; Seferian, Andrei; Perros, Frederic; Bergot, Emmanuel; Sibille, Yves; Humbert, Marc; Montani, David

    2013-10-01

    New treatments for pulmonary arterial hypertension (PAH) are a crucial need. The increased proliferation, migration, and survival of pulmonary vascular cells within the pulmonary artery wall in PAH have allowed successful transposition of pathophysiological elements from oncologic researches. Next steps will require translation of these biological advances in PAH therapeutic arsenal and guidelines. This review synthesizes recent data concerning the role of receptor tyrosine kinases and their inhibitors in PAH, with implications in animal models and humans. Results of clinical trials are now accumulating to establish beneficial role of tyrosine kinase inhibitors (TKIs) in PAH and further findings are expected in the near future. Beside this curative approach, evidences of a possible TKI-induced cardiotoxicity are emerging. These safety issues raise concern about a potential amplified harmful effect in PAH, a pathology characterized by an underlying cardiac dysfunction. In addition, analyses of PAH registries shed light on a selective pulmonary vascular toxicity triggered by TKIs, especially dasatinib. These possible dual effects of the TKIs in PAH need to be taken in account for future pharmacological development of this therapeutic class in PAH. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

  9. Molecular mechanism of T-cell protein tyrosine phosphatase (TCPTP) activation by mitoxantrone.

    Science.gov (United States)

    Ylilauri, Mikko; Mattila, Elina; Nurminen, Elisa M; Käpylä, Jarmo; Niinivehmas, Sanna P; Määttä, Juha A; Pentikäinen, Ulla; Ivaska, Johanna; Pentikäinen, Olli T

    2013-10-01

    T-cell protein tyrosine phosphatase (TCPTP) is a ubiquitously expressed non-receptor protein tyrosine phosphatase. It is involved in the negative regulation of many cellular signaling pathways. Thus, activation of TCPTP could have important therapeutic applications in diseases such as cancer and inflammation. We have previously shown that the α-cytoplasmic tail of integrin α1β1 directly binds and activates TCPTP. In addition, we have identified in a large-scale high-throughput screen six small molecules that activate TCPTP. These small molecule activators include mitoxantrone and spermidine. In this study, we have investigated the molecular mechanism behind agonist-induced TCPTP activation. By combining several molecular modeling and biochemical techniques, we demonstrate that α1-peptide and mitoxantrone activate TCPTP via direct binding to the catalytic domain, whereas spermidine does not interact with the catalytic domain of TCPTP in vitro. Furthermore, we have identified a hydrophobic groove surrounded by negatively charged residues on the surface of TCPTP as a putative binding site for the α1-peptide and mitoxantrone. Importantly, these data have allowed us to identify a new molecule that binds to TCPTP, but interestingly cannot activate its phosphatase activity. Accordingly, we describe here mechanism of TCPTP activation by mitoxantrone, the cytoplasmic tail of α1-integrin, and a mitoxantrone-like molecule at the atomic level. These data provide invaluable insight into the development of novel TCPTP activators, and may facilitate the rational discovery of small-molecule cancer therapeutics. © 2013.

  10. Wnt signaling through the Ror receptor in the nervous system.

    Science.gov (United States)

    Petrova, Iveta M; Malessy, Martijn J; Verhaagen, Joost; Fradkin, Lee G; Noordermeer, Jasprina N

    2014-02-01

    The receptor tyrosine kinase-like orphan receptor (Ror) proteins are conserved tyrosine kinase receptors that play roles in a variety of cellular processes that pattern tissues and organs during vertebrate and invertebrate development. Ror signaling is required for skeleton and neuronal development and modulates cell migration, cell polarity, and convergent extension. Ror has also been implicated in two human skeletal disorders, brachydactyly type B and Robinow syndrome. Rors are widely expressed during metazoan development including domains in the nervous system. Here, we review recent progress in understanding the roles of the Ror receptors in neuronal migration, axonal pruning, axon guidance, and synaptic plasticity. The processes by which Ror signaling execute these diverse roles are still largely unknown, but they likely converge on cytoskeletal remodeling. In multiple species, Rors have been shown to act as Wnt receptors signaling via novel non-canonical Wnt pathways mediated in some tissues by the adapter protein disheveled and the non-receptor tyrosine kinase Src. Rors can either activate or repress Wnt target expression depending on the cellular context and can also modulate signal transduction by sequestering Wnt ligands away from their signaling receptors. Future challenges include the identification of signaling components of the Ror pathways and bettering our understanding of the roles of these pleiotropic receptors in patterning the nervous system.

  11. Tyrosine sulfation modulates activity of tick-derived thrombin inhibitors

    Science.gov (United States)

    Thompson, Robert E.; Liu, Xuyu; Ripoll-Rozada, Jorge; Alonso-García, Noelia; Parker, Benjamin L.; Pereira, Pedro José Barbosa; Payne, Richard J.

    2017-09-01

    Madanin-1 and chimadanin are two small cysteine-free thrombin inhibitors that facilitate blood feeding in the tick Haemaphysalis longicornis. Here, we report a post-translational modification—tyrosine sulfation—of these two proteins that is critical for potent anti-thrombotic and anticoagulant activity. Inhibitors produced in baculovirus-infected insect cells displayed heterogeneous sulfation of two tyrosine residues within each of the proteins. One-pot ligation-desulfurization chemistry enabled access to homogeneous samples of all possible sulfated variants of the proteins. Tyrosine sulfation of madanin-1 and chimadanin proved crucial for thrombin inhibitory activity, with the doubly sulfated variants three orders of magnitude more potent than the unmodified inhibitors. The three-dimensional structure of madanin-1 in complex with thrombin revealed a unique mode of inhibition, with the sulfated tyrosine residues binding to the basic exosite II of the protease. The importance of tyrosine sulfation within this family of thrombin inhibitors, together with their unique binding mode, paves the way for the development of anti-thrombotic drug leads based on these privileged scaffolds.

  12. Regulation of hERG and hEAG channels by Src and by SHP-1 tyrosine phosphatase via an ITIM region in the cyclic nucleotide binding domain.

    Directory of Open Access Journals (Sweden)

    Lyanne C Schlichter

    Full Text Available Members of the EAG K(+ channel superfamily (EAG/Kv10.x, ERG/Kv11.x, ELK/Kv12.x subfamilies are expressed in many cells and tissues. In particular, two prototypes, EAG1/Kv10.1/KCNH1 and ERG1/Kv11.1/KCNH2 contribute to both normal and pathological functions. Proliferation of numerous cancer cells depends on hEAG1, and in some cases, hERG. hERG is best known for contributing to the cardiac action potential, and for numerous channel mutations that underlie 'long-QT syndrome'. Many cells, particularly cancer cells, express Src-family tyrosine kinases and SHP tyrosine phosphatases; and an imbalance in tyrosine phosphorylation can lead to malignancies, autoimmune diseases, and inflammatory disorders. Ion channel contributions to cell functions are governed, to a large degree, by post-translational modulation, especially phosphorylation. However, almost nothing is known about roles of specific tyrosine kinases and phosphatases in regulating K(+ channels in the EAG superfamily. First, we show that tyrosine kinase inhibitor, PP1, and the selective Src inhibitory peptide, Src40-58, reduce the hERG current amplitude, without altering its voltage dependence or kinetics. PP1 similarly reduces the hEAG1 current. Surprisingly, an 'immuno-receptor tyrosine inhibitory motif' (ITIM is present within the cyclic nucleotide binding domain of all EAG-superfamily members, and is conserved in the human, rat and mouse sequences. When tyrosine phosphorylated, this ITIM directly bound to and activated SHP-1 tyrosine phosphatase (PTP-1C/PTPN6/HCP; the first report that a portion of an ion channel is a binding site and activator of a tyrosine phosphatase. Both hERG and hEAG1 currents were decreased by applying active recombinant SHP-1, and increased by the inhibitory substrate-trapping SHP-1 mutant. Thus, hERG and hEAG1 currents are regulated by activated SHP-1, in a manner opposite to their regulation by Src. Given the widespread distribution of these channels, Src and SHP

  13. Efficacy of HER2-targeted therapy in metastatic breast cancer. Monoclonal antibodies and tyrosine kinase inhibitors

    DEFF Research Database (Denmark)

    Nielsen, Dorte L; Kümler, Iben; Palshof, Jesper Andreas

    2013-01-01

    Therapies targeting the human epidermal growth factor receptor (HER) 2 are effective in metastatic breast cancer (MBC). We review the efficacy of HER2-directed therapies, focussing on monoclonal antibodies and tyrosine kinase inhibitors targeting HER2 that have been tested in phase II-III studies...... in MBC. Trastuzumab is an important component of first-line treatment of HER2-positive MBC. New anti-HER2 drugs have the potential to change clinical practice. The potential role of the different drugs and regimens is yet to be determined. The response rate for trastuzumab-DM1 of 26-64% is comparable...... to those obtained for capecitabine plus lapatinib (48%), continuing trastuzumab in combination with capecitabine (48%), pertuzumab plus trastuzumab (24%), and neratinib (24%). Strategies combining multiple HER2-directed therapies might yield additive or synergistic effects and lead to improved outcome...

  14. Evaluation of Brachypodium distachyon L-Tyrosine Decarboxylase Using L-Tyrosine Over-Producing Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Shuhei Noda

    Full Text Available To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC, which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.

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

    Science.gov (United States)

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

    2017-03-01

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

  16. Phenol red-silk tyrosine cross-linked hydrogels.

    Science.gov (United States)

    Sundarakrishnan, Aswin; Herrero Acero, Enrique; Coburn, Jeannine; Chwalek, Karolina; Partlow, Benjamin; Kaplan, David L

    2016-09-15

    Phenol red is a cytocompatible pH sensing dye that is commonly added to cell culture media, but removed from some media formulations due to its structural mimicry of estrogen. Phenol red free media is also used during live cel