WorldWideScience

Sample records for egf receptor phosphorylation

  1. ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES SRC-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)

    Science.gov (United States)

    ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES Src-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)Weidong Wu1, Lee M. Graves2, Gordon N. Gill3 and James M. Samet4 1Center for Environmental Medicine and Lung Biology; 2Department of Pharmacology, University o...

  2. 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 p...

  3. Nitric oxide generated by ionizing radiation and EGF is implicated in EGF receptor phosphorylation in A549 lung carcinoma cells

    International Nuclear Information System (INIS)

    Park, In Chul; Lee, Hyung Chahn; Rhee, Chang Hun; Hong, Seok Il

    2004-01-01

    Although it has been demonstrated that ionizing radiation (IR) control various cell functions in a different cell types, the mechanisms of its action via NO are not well understood. NO may potentially affect every type of mammalian cells, owing to its ubiquitous production and participate in the control of cell proliferation in a great variety of cell types. The epidermal growth factor (EGF) receptor is a transmembrane glycoprotein of Mr 170,000. When EGF binds to its receptor, the receptor is dimerized and autophosphorylated at the carboxyl-terminal tyrosine 992, 1608, 1086, 1148 and 1173. This phosphorylated receptor initiates a series of signal tranduction events through interacting proteins of SH2 family including Shc, Grb2 and Sos, which in turn trigger ativation of MAPK cascades. Although the number of signaling events mediated by IR-induced NO is growing, it is still unclear how NO activate cellular signaling events. Thus, we examined the effect of NO on cellular phosphorylation and found that NO was produced by ionizing radiation in A549 lung adenocarcinoma cells and enhances the unique tyrosine phosphorylation on EGF receptor

  4. Augmenter of liver regeneration causes different kinetics of ERK1/2 and Akt/PKB phosphorylation than EGF and induces hepatocyte proliferation in an EGF receptor independent and liver specific manner

    Energy Technology Data Exchange (ETDEWEB)

    Ilowski, Maren; Putz, Christine [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Weiss, Thomas S. [Department of Surgery, University of Regensburg Hospital, Regensburg (Germany); Brand, Stephan [Department of Internal Medicine II, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Jauch, Karl-Walter [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Hengstler, Jan G. [Leibniz Research Centre for Working Environment and Human Factors, TU Dortmund University, Dortmund (Germany); Thasler, Wolfgang Erwin, E-mail: wolfgang.thasler@med.uni-muenchen.de [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany)

    2010-04-16

    Background/Aim: Augmenter of liver regeneration (ALR) is a potent growth factor which supports liver regeneration in experimental animals. The aim of this study was to compare proliferation as well as the kinetics of ERK1/2 and Akt/PKB phosphorylation by recombinant human ALR (rhALR) and EGF in human hepatocytes and extrahepatic cells. Methods: Kinetics of ERK1/2 and Akt/PKB phosphorylation were determined in primary human hepatocytes (phh) after stimulation with rhALR and EGF. Induction of proliferation was analyzed in phh and several cell lines of hepatic and extrahepatic origin by the MTT and [{sup 3}H]-thymidine assay. Results: The kinetics of ERK phosphorylation showed clear differences, whereby rhALR caused a transient and EGF a permanent increase during the observation period of 60 min. For both, Akt and ERK phosphorylation, EGF caused a faster effect with maximal levels observed already after 2 min, whereas rhALR caused maximal phosphorylation between 10 and 15 min. Using the EGF receptor inhibitor AG1478 we provide evidence of an EGF receptor independent induction of proliferation by rhALR. Furthermore, rhALR induced proliferation only in phh and the human liver derived cell lines HepG2 and Chang. In contrast, EGF enhanced proliferation in all analyzed cell types including cell lines of colon, bronchial, pancreatic and gastric origin (SW480, BC1, L36PL and GC1). Conclusion: rhALR and EGF induce different kinetics of ERK and Akt phosphorylation in human hepatocytes. The mitogenic effect of rhALR is liver specific and seems to be at least partially independent from EGF receptor mediated signaling.

  5. CysLT1 receptor-induced human airway smooth muscle cells proliferation requires ROS generation, EGF receptor transactivation and ERK1/2 phosphorylation

    Directory of Open Access Journals (Sweden)

    Capra Valérie

    2006-03-01

    Full Text Available Abstract Background Cysteine-containing leukotrienes (cysteinyl-LTs are pivotal inflammatory mediators that play important roles in the pathophysiology of asthma, allergic rhinitis, and other inflammatory conditions. In particular, cysteinyl-LTs exert a variety of effects with relevance to the aetiology of asthma such as smooth muscle contraction, eosinophil recruitment, increased microvascular permeability, enhanced mucus secretion and decreased mucus transport and, finally, airway smooth muscle cells (ASMC proliferation. We used human ASMC (HASMC to identify the signal transduction pathway(s of the leukotriene D4 (LTD4-induced DNA synthesis. Methods Proliferation of primary HASMC was measured by [3H]thymidine incorporation. Phosphorylation of EGF receptor (EGF-R and ERK1/2 was assessed with a polyclonal anti-EGF-R or anti-phosphoERKl/2 monoclonal antibody. A Ras pull-down assay kit was used to evaluate Ras activation. The production of reactive oxygen species (ROS was estimated by measuring dichlorodihydrofluorescein (DCF oxidation. Results We demonstrate that in HASMC LTD4-stimulated thymidine incorporation and potentiation of EGF-induced mitogenic signaling mostly depends upon EGF-R transactivation through the stimulation of CysLT1-R. Accordingly, we found that LTD4 stimulation was able to trigger the increase of Ras-GTP and, in turn, to activate ERK1/2. We show here that EGF-R transactivation was sensitive to pertussis toxin (PTX and phosphoinositide 3-kinase (PI3K inhibitors and that it occurred independently from Src activity, despite the observation of a strong impairment of LTD4-induced DNA synthesis following Src inhibition. More interestingly, CysLT1-R stimulation increased the production of ROS and N-acetylcysteine (NAC abolished LTD4-induced EGF-R phosphorylation and thymidine incorporation. Conclusion Collectively, our data demonstrate that in HASMC LTD4 stimulation of a Gi/o coupled CysLT1-R triggers the transactivation of the EGF

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

    Science.gov (United States)

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

    2012-08-28

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

  7. SMOC Binds to Pro-EGF, but Does Not Induce Erk Phosphorylation via the EGFR.

    Science.gov (United States)

    Thomas, J Terrig; Chhuy-Hy, Lina; Andrykovich, Kristin R; Moos, Malcolm

    2016-01-01

    In an attempt to identify the cell-associated protein(s) through which SMOC (Secreted Modular Calcium binding protein) induces mitogen-activated protein kinase (MAPK) signaling, the epidermal growth factor receptor (EGFR) became a candidate. However, although in 32D/EGFR cells, the EGFR was phosphorylated in the presence of a commercially available human SMOC-1 (hSMOC-1), only minimal phosphorylation was observed in the presence of Xenopus SMOC-1 (XSMOC-1) or human SMOC-2. Analysis of the commercial hSMOC-1 product demonstrated the presence of pro-EGF as an impurity. When the pro-EGF was removed, only minimal EGFR activation was observed, indicating that SMOC does not signal primarily through EGFR and its receptor remains unidentified. Investigation of SMOC/pro-EGF binding affinity revealed a strong interaction that does not require the C-terminal extracellular calcium-binding (EC) domain of SMOC or the EGF domain of pro-EGF. SMOC does not appear to potentiate or inhibit MAPK signaling in response to pro-EGF, but the interaction could provide a mechanism for retaining soluble pro-EGF at the cell surface.

  8. Odin (ANKS1A modulates EGF receptor recycling and stability.

    Directory of Open Access Journals (Sweden)

    Jiefei Tong

    Full Text Available The ANKS1A gene product, also known as Odin, was first identified as a tyrosine-phosphorylated component of the epidermal growth factor receptor network. Here we show that Odin functions as an effector of EGFR recycling. In EGF-stimulated HEK293 cells tyrosine phosphorylation of Odin was induced prior to EGFR internalization and independent of EGFR-to-ERK signaling. Over-expression of Odin increased EGF-induced EGFR trafficking to recycling endosomes and recycling back to the cell surface, and decreased trafficking to lysosomes and degradation. Conversely, Odin knockdown in both HEK293 and the non-small cell lung carcinoma line RVH6849, which expresses roughly 10-fold more EGF receptors than HEK293, caused decreased EGFR recycling and accelerated trafficking to the lysosome and degradation. By governing the endocytic fate of internalized receptors, Odin may provide a layer of regulation that enables cells to contend with receptor cell densities and ligand concentration gradients that are physiologically and pathologically highly variable.

  9. EGF-induced stimualtion of EGF-receptor synthesis in human cytotrophoblasts and A431 cells

    International Nuclear Information System (INIS)

    DePalo, L.; Basu, A.; Das, M.

    1987-01-01

    EGF-receptor is a transmembrane glycoprotein whose intracellular degradation is known to be enhanced by EGF. The authors tested whether the receptor is replenished during this process by an enhanced rate of synthesis. Human A431 epidermoid carcinoma cells, and primary cultures of human placental cytotrophoblasts were used in these studies. Cells were labeled with 35 S-methionine, and EGF-receptor biosynthesis was quantitated by immunoprecipitation using a monoclonal anti-EGF-receptor antibody. EGF stimulated receptor biosynthesis at concentrations of 0.1-1 nM. The effect was seen within 2 h of EGF addition. The maximal stimulatory effect was modest in A431 (∼ 2-fold), but marked in the cytotrophoblasts (>5-fold). At EGF concentrations higher than 3 nM, the stimulatory effect was abolished. In contrast, the effect of EGF on receptor degradation is negligible at low subnanomolar concentrations, and is pronounced only at saturating concentrations. These results show that occupation of the cell surface EGF-receptor by its ligand can lead to production of more receptor protein, thus counterbalancing the negative effect on receptor degradation. At low subnanomolar (mitogenic) concentrations of EGF the stimulator effect on receptor synthesis is likely to predominate over the effect on receptor degradation

  10. Decreased autophosphorylation of EGF receptor in insulin-deficient diabetic rats

    International Nuclear Information System (INIS)

    Okamoto, M.; Kahn, C.R.; Maron, R.; White, M.F.

    1988-01-01

    The authors have previously reported that despite an increase in receptor concentration, there is a decrease in autophosphorylation and tyrosine kinase activity of the insulin receptor in insulin-deficient diabetic rats. To determine if other tyrosine kinases might be altered, they have studied the epidermal growth factor (EGF) receptor kinase in wheat germ agglutinin-purified, Triton X-100-solubilized liver membranes from streptozotocin (STZ)-induced diabetic rats and the insulin-deficient BB rat. They find that autophosphorylation of EGF receptor is decreased in proportion to the severity of the diabetic state in STZ rats with a maximal decrease of 67%. A similar decrease in autophosphorylation was observed in diabetic BB rats that was partially normalized by insulin treatment. Separation of tryptic phosphopeptides by reverse-phase high-performance liquid chromatography revealed a decrease in labeling at all sites of autophosphorylation. A parallel decrease in EGF receptor phosphorylation was also found by immunoblotting with an antiphosphotyrosine antibody. EGF receptor concentration, determined by Scatchard analysis of 125 I-labeled EGF binding, was decreased by 39% in the STZ rat and 27% in the diabetic BB rat. Thus autophosphorylation of EGF receptor, like that of the insulin receptor, is decreased in insulin-deficient rat liver. In the case of EGF receptor, this is due in part to a decrease in receptor number and in part to a decrease in the specific activity of the kinase

  11. Stat5 phosphorylation is responsible for the excessive potency of HB-EGF.

    Science.gov (United States)

    Heo, Jeongyeon; Kim, Jae Geun; Kim, Sunghwan; Kang, Hara

    2017-12-23

    Heparin-binding EGF-like growth factor (HB-EGF) is a potent growth factor involved in wound healing and tumorigenesis. Despite the sequence similarity between HB-EGF and EGF, HB-EGF induces cellular proliferation and migration more potently than EGF. However, the differential regulation by HB-EGF and EGF has not been thoroughly elucidated. In this study, we compared signaling pathways activated by HB-EGF and EGF to understand the details of the molecular mechanism of the high potency induced by HB-EGF. HB-EGF specifically induced the phosphorylation of EGFR-Y1045 and activated Stat5, which is responsible for promoting cell proliferation, and migration. The competition of phosphorylated EGFR-Y1045 inhibited Stat5 activation and consequently lowered the effect of HB-EGF on cell proliferation, suggesting that the phosphorylation of EGFR-Y1045 is essential for the activation of Stat5. The phosphorylation of EGFR-Y1045 and Stat5 induced by HB-EGF was prevented by sequestering the heparin-binding domain, suggesting that the heparin-binding domain is critical for HB-EGF-mediated signaling and cellular responses. In conclusion, the heparin-binding domain of HB-EGF was responsible for EGFR-mediated Stat5 activation, resulting in a more potent cellular proliferation, and migration than that mediated by EGF. This molecular mechanism is useful for understanding ligand-specific EGFR signaling and developing biomedicines for wound healing or cancer therapy. © 2017 Wiley Periodicals, Inc.

  12. EGF stimulates the activation of EGF receptors and the selective activation of major signaling pathways during mitosis.

    Science.gov (United States)

    Wee, Ping; Shi, Huaiping; Jiang, Jennifer; Wang, Yuluan; Wang, Zhixiang

    2015-03-01

    Mitosis and epidermal growth factor (EGF) receptor (EGFR) are both targets for cancer therapy. The role of EGFR signaling in mitosis has been rarely studied and poorly understood. The limited studies indicate that the activation of EGFR and downstream signaling pathways is mostly inhibited during mitosis. However, we recently showed that EGFR is phosphorylated in response to EGF stimulation in mitosis. Here we studied EGF-induced EGFR activation and the activation of major signaling pathways downstream of EGFR during mitosis. We showed that EGFR was strongly activated by EGF during mitosis as all the five major tyrosine residues including Y992, Y1045, Y1068, Y1086, and Y1173 were phosphorylated to a level similar to that in the interphase. We further showed that the activated EGFR is able to selectively activate some downstream signaling pathways while avoiding others. Activated EGFR is able to activate PI3K and AKT2, but not AKT1, which may be responsible for the observed effects of EGF against nocodazole-induced cell death. Activated EGFR is also able to activate c-Src, c-Cbl and PLC-γ1 during mitosis. However, activated EGFR is unable to activate ERK1/2 and their downstream substrates RSK and Elk-1. While it activated Ras, EGFR failed to fully activate Raf-1 in mitosis due to the lack of phosphorylation at Y341 and the lack of dephosphorylation at pS259. We conclude that contrary to the dogma, EGFR is activated by EGF during mitosis. Moreover, EGFR-mediated cell signaling is regulated differently from the interphase to specifically serve the needs of the cell in mitosis. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2012-07-30

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

  14. Autocrine EGF receptor activation mediates endothelial cell migration and vascular morphogenesis induced by VEGF under interstitial flow

    International Nuclear Information System (INIS)

    Semino, Carlos E.; Kamm, Roger D.; Lauffenburger, Douglas A.

    2006-01-01

    We show here that autocrine ligand activation of epidermal growth factor (EGF) receptor in combination with interstitial flow is critically involved in the morphogenetic response of endothelial cells to VEGF stimulation. Human umbilical vein endothelial cell (HUVEC) monolayers cultured on a collagen gel and exposed to low interstitial flow in the absence of EGF and VEGF remained viable and mitotic but exhibited little evidence of vascular morphogenesis. Addition of VEGF produced a flow-dependent morphogenetic response within 48 to 72 h, characterized by branched capillary-like structures. The response was substantially abolished by inhibitors related to the autocrine EGF receptor pathway including Galardin, AG1478, PD98059, and an EGF receptor-blocking antibody, indicating that regulation of the morphogenetic process operates via autocrine EGF receptor activation. Moreover, we observed that in our system the EGF receptor was always activated independently of the interstitial flow, and, in addition, the EGF receptor inhibitors used above reduced the phosphorylation state of the receptor, correlating with inhibition of capillary morphogenesis. Finally, 5'bromo-2'-deoxyuridine (BrdU) labeling identified dividing cells at the monolayer but not in the extending capillary-like structures. EGF pathway inhibitors Galardin and AG1478 did not reduce BrdU incorporation in the monolayer, indicating that the EGF-receptor-mediated morphogenetic behavior is mainly due to cell migration rather than proliferation. Based on these results, we propose a two-step model for in vitro capillary morphogenesis in response to VEGF stimulation with interstitial fluid flow: monolayer maintenance by mitotic activity independent of EGF receptors and a migratory response mediated by autocrine EGF receptor activation wherein cells establish capillary-like structures

  15. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    Directory of Open Access Journals (Sweden)

    George Kourouniotis

    2016-07-01

    Full Text Available The binding of epidermal growth factor (EGF to EGF receptor (EGFR stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ and tagged a green fluorescent protein (GFP at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc, extracellular signal-regulated kinase (ERK and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis.

  16. Cell model for the study of receptor and regulatory functions of human proHB-EGF

    Directory of Open Access Journals (Sweden)

    N. V. Korotkevych

    2014-08-01

    Full Text Available Developing of new models and approaches, particularly with fluorescent techniques, for investigation of intracellular transport of proHB-EGF and its ligand-receptor complexes is strongly required. In order to create a model for studying proHB-EGF functions the genetic construction pEGFP-N1-proHB-EGF, encoding proHB-EGF-EGFP which is fluorescent-labeled form of proHB-EGF with enhanced green fluorescent protein EGFP in the cytoplasmic terminus of the molecule, was obtained. Eukaryotic cells expressing fusion protein proHB-EGF-EGFP on the cell surface were obtained by transfection with pEGFP-N1-proHB-EGF. Expressed in the Vero cells proHB-EGF-EGFP could bind fluorescent derivative of nontoxic receptor-binding subunit B of diphtheria toxin mCherry-SubB. After stimulation of transfected cells with TPA (12-O-Tetradecanoylphorbol-13-acetate, proHB-EGF-EGFP formed a fluorescentl-labeled C-terminal fragment of the molecule – CTF-EGFP. Thus, the obtained genetic construction pEGFP-N1-proHB-EGF could be helpful in visualization of molecules proHB-EGF and CTF in cells, may open new possibilities for the studying of their functions, such as receptor function of proHB-EGF for diphtheria toxin, intracellular translocation of CTF and provide possibilities for natural proHB-EGF ligands search.

  17. Differences in human skin between the epidermal growth factor receptor distribution detected by EGF binding and monoclonal antibody recognition

    DEFF Research Database (Denmark)

    Green, M R; Couchman, J R

    1985-01-01

    , the eccrine sweat glands, capillary system, and the hair follicle outer root sheath, generally similar in pattern to that previously reported for full-thickness rat skin and human epidermis. The same areas also bound EGF-R1 but in addition the monoclonal antibody recognized a cone of melanin containing......Two methods have been used to examine epidermal growth factor (EGF) receptor distribution in human scalp and foreskin. The first employed [125I]EGF viable explants and autoradiography to determine the EGF binding pattern while the second used a monoclonal antibody to the human EGF receptor to map...... whether EGF-R1 could recognize molecules unrelated to the EGF receptor, the EGF binding and EGF-R1 recognition profiles were compared on cultures of SVK14 cells, a SV40 transformed human keratinocyte cell line. EGF binding and EGF-R1 monoclonal antibody distribution on these cells was found to be similar...

  18. Identifying novel targets of oncogenic EGF receptor signaling in lung cancer through global phosphoproteomics.

    Science.gov (United States)

    Zhang, Xu; Belkina, Natalya; Jacob, Harrys Kishore Charles; Maity, Tapan; Biswas, Romi; Venugopalan, Abhilash; Shaw, Patrick G; Kim, Min-Sik; Chaerkady, Raghothama; Pandey, Akhilesh; Guha, Udayan

    2015-01-01

    Mutations in the epidermal growth factor receptor (EGFR) kinase domain occur in 10-30% of lung adenocarcinoma and are associated with tyrosine kinase inhibitor (TKI) sensitivity. We sought to identify the immediate direct and indirect phosphorylation targets of mutant EGFRs in lung adenocarcinoma. We undertook SILAC strategy, phosphopeptide enrichment, and quantitative MS to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring EGFR(L858R) and EGFR(L858R/T790M) , the TKI-sensitive, and TKI-resistant mutations, respectively. Top canonical pathways that were inhibited upon erlotinib treatment in sensitive cells, but not in the resistant cells include EGFR, insulin receptor, hepatocyte growth factor, mitogen-activated protein kinase, mechanistic target of rapamycin, ribosomal protein S6 kinase beta 1, and Janus kinase/signal transducer and activator of transcription signaling. We identified phosphosites in proteins of the autophagy network, such as ULK1 (S623) that is constitutively phosphorylated in these lung adenocarcinoma cells; phosphorylation is inhibited upon erlotinib treatment in sensitive cells, but not in resistant cells. Finally, kinase-substrate prediction analysis from our data indicated that substrates of basophilic kinases from, AGC and Calcium and calmodulin-dependent kinase groups, as well as STE group kinases were significantly enriched and those of proline-directed kinases from, CMGC and Casein kinase groups were significantly depleted among substrates that exhibited increased phosphorylation upon EGF stimulation and reduced phosphorylation upon TKI inhibition. This is the first study to date to examine global phosphorylation changes upon erlotinib treatment of lung adenocarcinoma cells and results from this study provide new insights into signaling downstream of mutant EGFRs in lung adenocarcinoma. All MS data have been deposited in the ProteomeXchange with identifier PXD001101 (http

  19. Helicobacter pylori-Induced HB-EGF Upregulates Gastrin Expression via the EGF Receptor, C-Raf, Mek1, and Erk2 in the MAPK Pathway

    Directory of Open Access Journals (Sweden)

    Niluka Gunawardhana

    2018-01-01

    Full Text Available Helicobacter pylori is associated with hypergastrinemia, which has been linked to the development of gastric diseases. Although the molecular mechanism is not fully understood, H. pylori is known to modulate the Erk pathway for induction of gastrin expression. Herein we found that an epidermal growth factor (EGF receptor kinase inhibitor significantly blocked H. pylori-induced gastrin promoter activity, suggesting involvement of EGF receptor ligands. Indeed, H. pylori induced mRNA expression of EGF family members such as amphiregulin, EGF, heparin-binding EGF-like growth factor (HB-EGF, and transforming growth factor-α. Of these, specific siRNA targeting of HB-EGF significantly blocked H. pylori-induced gastrin expression. Moreover, H. pylori induced HB-EGF ectodomain shedding, which we found to be a critical process for H. pylori-induced gastrin expression. Thus, we demonstrate a novel role for human mature HB-EGF in stimulating gastrin promoter activity during H. pylori infection. Further investigation using specific siRNAs targeting each isoform of Raf, Mek, and Erk elucidated that the mechanism underlying H. pylori-induced gastrin expression can be delineated as the sequential activation of HB-EGF, the EGF receptor, C-Raf, Mek1, and the Erk2 molecules in the MAPK pathway. Surprisingly, whereas Erk2 acts as a potent activator of gastrin expression, siRNA knockdown of Erk1 induced gastrin promoter activity, suggesting that Erk1 typically acts as a repressor of gastrin expression. Elucidation of the mechanism of gastrin modulation by HB-EGF-mediated EGF receptor transactivation should facilitate the development of therapeutic strategies against H. pylori-related hypergastrinemia and consequently gastric disease development, including gastric cancers.

  20. Helicobacter pylori-Induced HB-EGF Upregulates Gastrin Expression via the EGF Receptor, C-Raf, Mek1, and Erk2 in the MAPK Pathway.

    Science.gov (United States)

    Gunawardhana, Niluka; Jang, Sungil; Choi, Yun Hui; Hong, Youngmin A; Jeon, Yeong-Eui; Kim, Aeryun; Su, Hanfu; Kim, Ji-Hye; Yoo, Yun-Jung; Merrell, D Scott; Kim, Jinmoon; Cha, Jeong-Heon

    2017-01-01

    Helicobacter pylori is associated with hypergastrinemia, which has been linked to the development of gastric diseases. Although the molecular mechanism is not fully understood, H. pylori is known to modulate the Erk pathway for induction of gastrin expression. Herein we found that an epidermal growth factor (EGF) receptor kinase inhibitor significantly blocked H. pylori -induced gastrin promoter activity, suggesting involvement of EGF receptor ligands. Indeed, H. pylori induced mRNA expression of EGF family members such as amphiregulin, EGF, heparin-binding EGF-like growth factor (HB-EGF), and transforming growth factor-α. Of these, specific siRNA targeting of HB-EGF significantly blocked H. pylori -induced gastrin expression. Moreover, H. pylori induced HB-EGF ectodomain shedding, which we found to be a critical process for H. pylori -induced gastrin expression. Thus, we demonstrate a novel role for human mature HB-EGF in stimulating gastrin promoter activity during H. pylori infection. Further investigation using specific siRNAs targeting each isoform of Raf, Mek, and Erk elucidated that the mechanism underlying H. pylori -induced gastrin expression can be delineated as the sequential activation of HB-EGF, the EGF receptor, C-Raf, Mek1, and the Erk2 molecules in the MAPK pathway. Surprisingly, whereas Erk2 acts as a potent activator of gastrin expression, siRNA knockdown of Erk1 induced gastrin promoter activity, suggesting that Erk1 typically acts as a repressor of gastrin expression. Elucidation of the mechanism of gastrin modulation by HB-EGF-mediated EGF receptor transactivation should facilitate the development of therapeutic strategies against H. pylori -related hypergastrinemia and consequently gastric disease development, including gastric cancers.

  1. Conformational stability of the epidermal growth factor (EGF) receptor as influenced by glycosylation, dimerization and EGF hormone binding.

    Science.gov (United States)

    Taylor, Eric S; Pol-Fachin, Laercio; Lins, Roberto D; Lower, Steven K

    2017-04-01

    The epidermal growth factor receptor (EGFR) is an important transmembrane glycoprotein kinase involved the initiation or perpetuation of signal transduction cascades within cells. These processes occur after EGFR binds to a ligand [epidermal growth factor (EGF)], thus inducing its dimerization and tyrosine autophosphorylation. Previous publications have highlighted the importance of glycosylation and dimerization for promoting proper function of the receptor and conformation in membranes; however, the effects of these associations on the protein conformational stability have not yet been described. Molecular dynamics simulations were performed to characterize the conformational preferences of the monomeric and dimeric forms of the EGFR extracellular domain upon binding to EGF in the presence and absence of N-glycan moieties. Structural stability analyses revealed that EGF provides the most conformational stability to EGFR, followed by glycosylation and dimerization, respectively. The findings also support that EGF-EGFR binding takes place through a large-scale induced-fitting mechanism. Proteins 2017; 85:561-570. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. PET imaging of EGF receptors using [{sup 18}F]FBEM-EGF in a head and neck squamous cell carcinoma model

    Energy Technology Data Exchange (ETDEWEB)

    Li, Weihua [Harbin Medical University, Department of Medical Imaging and Nuclear Medicine, Fourth Affiliated Hospital, Harbin (China); National Institutes of Health (NIH), Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), Bethesda, MD (United States); Niu, Gang; Lang, Lixin; Guo, Ning; Ma, Ying; Kiesewetter, Dale O.; Chen, Xiaoyuan [National Institutes of Health (NIH), Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), Bethesda, MD (United States); Backer, Joseph M. [SibTech Inc., Brookfield, CT (United States); Shen, Baozhong [Harbin Medical University, Department of Medical Imaging and Nuclear Medicine, Fourth Affiliated Hospital, Harbin (China)

    2012-02-15

    To prepare and evaluate a new radiotracer for molecular imaging of cell surface receptors for epidermal growth factor (EGF). Cys-tagged EGF (cEGF) was labeled with {sup 18}F by coupling the free thiol group of the Cys tag with N-[2-(4-[{sup 18}F]fluorobenzamido)ethyl]maleimide ([{sup 18}F]FBEM) to form [{sup 18}F]FBEM-cEGF. Cell uptake, internalization and efflux of [{sup 18}F]FBEM-cEGF were tested in human head and neck squamous carcinoma UM-SCC1 cells. In vivo tumor targeting and pharmacokinetics of the radiotracers were evaluated in UM-SCC1 tumor-bearing athymic nude mice by static and dynamic microPET imaging. Ex vivo biodistribution assays were performed to confirm the noninvasive imaging results. The radiolabeling yield for [{sup 18}F]FBEM-cEGF was over 60%, based on starting [{sup 18}F]FBEM. [{sup 18}F]FBEM-cEGF exhibited rapid blood clearance through both hepatobiliary and renal excretion. UM-SCC1 tumors were clearly visualized and showed modest tracer uptake of 2.60 {+-} 0.59 %ID/g at 30 min after injection. Significantly higher tumor uptake of [{sup 18}F]FBEM-cEGF (5.99 {+-} 1.61%ID/g at 30 min after injection, p < 0.01) and tumor/nontumor ratio were achieved by coinjection of 50 {mu}g of unlabeled EGF. Decreased liver uptake of [{sup 18}F]FBEM-cEGF was observed when unlabeled EGF was coadministered. With optimized liver blocking, [{sup 18}F]FBEM-cEGF has the potential to be used in a noninvasive and quantitative manner for detection of malignant lesions and evaluation of EGFR activity. (orig.)

  3. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.

    Science.gov (United States)

    Rozakis-Adcock, M; Fernley, R; Wade, J; Pawson, T; Bowtell, D

    1993-05-06

    Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.

  4. Phosphoproteomics identified Endofin, DCBLD2, and KIAA0582 as novel tyrosine phosphorylation targets of EGF signaling and Iressa in human cancer cells

    DEFF Research Database (Denmark)

    Chen, Yunhao; Low, Teck-Yew; Choong, Lee-Yee

    2007-01-01

    With the completion of the human genome project, analysis of enriched phosphotyrosyl proteins from epidermal growth factor (EGF)-induced phosphotyrosine proteome permits the identification of novel downstream substrates of the EGF receptor (EGFR). Using cICAT-based LC-MS/MS method, we identified...

  5. EGF receptor targeted tumor imaging with biotin-PEG-EGF linked to 99mTc-HYNIC labeled avidin and streptavidin

    International Nuclear Information System (INIS)

    Jung, Kyung-Ho; Park, Jin Won; Paik, Jin-Young; Quach, Cung Hoa Thien; Choe, Yearn Seong; Lee, Kyung-Han

    2012-01-01

    Introduction: As direct radiolabeled peptides suffer limitations for in vivo imaging, we investigated the usefulness of radioloabeled avidin and streptavidin as cores to link peptide ligands for targeted tumor imaging. Methods: Human epidermal growth factor (EGF) was site specifically conjugated with a single PEG-biotin molecule and linked to 99m Tc-HYNIC labeled avidin-FITC (Av) or streptavidin-Cy5.5 (Sav). Receptor targeting was verified in vitro, and in vivo pharmacokinetic and biodistribution profiles were studied in normal mice. Scintigraphic imaging was performed in MDA-MB-468 breast tumor xenografted nude mice. Results: Whereas both 99m Tc-Av-EGF and 99m Tc-Sav-EGF retained receptor-specific binding in vitro, the two probes substantially diverged in pharmacokinetic and biodistribution behavior in vivo. 99m Tc-Av-EGF was rapidly eliminated from the circulation with a T1/2 of 4.3 min, and showed intense hepatic accumulation but poor tumor uptake (0.6%ID/gm at 4 h). 99m Tc-Sav-EGF displayed favorable in vivo profiles of longer circulation (T1/2β, 51.5 min) and lower nonspecific uptake that resulted in higher tumor uptake (3.8 %ID/gm) and clear tumor visualization at 15 h. Conclusion: 99m Tc-HYNIC labeled streptavidin linked with growth factor peptides may be useful as a protein-ligand complex for targeted imaging of tumor receptors.

  6. In brown adipocytes, adrenergically induced β1-/β3-(Gs)-, α2-(Gi)- and α1-(Gq)-signalling to Erk1/2 activation is not mediated via EGF receptor transactivation

    International Nuclear Information System (INIS)

    Wang, Yanling; Fälting, Johanna M.; Mattsson, Charlotte L.; Holmström, Therése E.; Nedergaard, Jan

    2013-01-01

    Brown adipose tissue is unusual in that the neurotransmitter norepinephrine influences cell destiny in ways generally associated with effects of classical growth factors: regulation of cell proliferation, of apoptosis, and progression of differentiation. The norepinephrine effects are mediated through G-protein-coupled receptors; further mediation of such stimulation to e.g. Erk1/2 activation is in cell biology in general accepted to occur through transactivation of the EGF receptor (by external or internal pathways). We have examined here the significance of such transactivation in brown adipocytes. Stimulation of mature brown adipocytes with cirazoline (α 1 -adrenoceptor coupled via G q ), clonidine (α 2 via G i ) or CL316243 (β 3 via G s ) or via β 1 -receptors significantly activated Erk1/2. Pretreatment with the EGF receptor kinase inhibitor AG1478 had, remarkably, no significant effect on Erk1/2 activation induced by any of these adrenergic agonists (although it fully abolished EGF-induced Erk1/2 activation), demonstrating absence of EGF receptor-mediated transactivation. Results with brown preadipocytes (cells in more proliferative states) were not qualitatively different. Joint stimulation of all adrenoceptors with norepinephrine did not result in synergism on Erk1/2 activation. AG1478 action on EGF-stimulated Erk1/2 phosphorylation showed a sharp concentration–response relationship (IC 50 0.3 µM); a minor apparent effect of AG1478 on norepinephrine-stimulated Erk1/2 phosphorylation showed nonspecific kinetics, implying caution in interpretation of partial effects of AG1478 as reported in other systems. Transactivation of the EGF receptor is clearly not a universal prerequisite for coupling of G-protein coupled receptors to Erk1/2 signalling cascades. - Highlights: • In brown adipocytes, norepinephrine regulates proliferation, apoptosis, differentiation. • EGF receptor transactivation is supposed to mediate GPCR-induced Erk1/2 activation. •

  7. Phenobarbital Meets Phosphorylation of Nuclear Receptors.

    Science.gov (United States)

    Negishi, Masahiko

    2017-05-01

    Phenobarbital was the first therapeutic drug to be characterized for its induction of hepatic drug metabolism. Essentially at the same time, cytochrome P450, an enzyme that metabolizes drugs, was discovered. After nearly 50 years of investigation, the molecular target of phenobarbital induction has now been delineated to phosphorylation at threonine 38 of the constitutive androstane receptor (NR1I3), a member of the nuclear receptor superfamily. Determining this mechanism has provided us with the molecular basis to understand drug induction of drug metabolism and disposition. Threonine 38 is conserved as a phosphorylation motif in the majority of both mouse and human nuclear receptors, providing us with an opportunity to integrate diverse functions of nuclear receptors. Here, I review the works and accomplishments of my laboratory at the National Institutes of Health National Institute of Environmental Health Sciences and the future research directions of where our study of the constitutive androstane receptor might take us. U.S. Government work not protected by U.S. copyright.

  8. Regulation of EGF receptor signaling by the MARVEL domain-containing protein CKLFSF8.

    Science.gov (United States)

    Jin, Caining; Ding, Peiguo; Wang, Ying; Ma, Dalong

    2005-11-21

    It is known that chemokine-like factor superfamily 8 (CKLFSF8), a member of the CKLF superfamily, has four putative transmembrane regions and a MARVEL domain. Its structure is similar to TM4SF11 (plasmolipin) and widely distributed in normal tissue. However, its function is not yet known. We show here that CKLFSF8 is associated with the epidermal growth factor receptor (EGFR) and that ectopic expression of CKLFSF8 in several cell lines suppresses EGF-induced cell proliferation, whereas knockdown of CKLFSF8 by siRNA promotes cell proliferation. In cells overexpressing CKLFSF8, the initial activation of EGFR was not affected, but subsequent desensitization of EGF-induced signaling occurred rapidly. This attenuation was correlated with an increased rate of receptor endocytosis. In contrast, knockdown of CKLFSF8 by siCKLFSF8 delayed EGFR endocytosis. These results identify CKLFSF8 as a novel regulator of EGF-induced signaling and indicate that the association of EGFR with four transmembrane proteins is critical for EGFR desensitization.

  9. Dihydrotestosterone activates the MAPK pathway and modulates maximum isometric force through the EGF receptor in isolated intact mouse skeletal muscle fibres.

    Science.gov (United States)

    Hamdi, M M; Mutungi, G

    2010-02-01

    It is generally believed that steroid hormones have both genomic and non-genomic (rapid) actions. Although the latter form an important component of the physiological response of these hormones, little is known about the cellular signalling pathway(s) mediating these effects and their physiological functions in adult mammalian skeletal muscle fibres. Therefore, the primary aim of this study was to investigate the non-genomic actions of dihydrotestosterone (DHT) and their physiological role in isolated intact mammalian skeletal muscle fibre bundles. Our results show that treating the fibre bundles with physiological concentrations of DHT increases both twitch and tetanic contractions in fast twitch fibres. However, it decreases them in slow twitch fibres. These changes in force are accompanied by an increase in the phosphorylation of MAPK/ERK1/2 in both fibre types and that of regulatory myosin light chains in fast twitch fibres. Both effects were insensitive to inhibitors of Src kinase, androgen receptor, insulin-like growth factor 1 receptor and platelet-derived growth factor receptor. However, they were abolished by the MAPK/ERK1/2 kinase inhibitor PD98059 and the epidermal growth factor (EGF) receptor inhibitor tyrphostin AG 1478. In contrast, testosterone had no effect on force and increased the phosphorylation of ERK1/2 in slow twitch fibres only. From these results we conclude that sex steroids have non-genomic actions in isolated intact mammalian skeletal muscle fibres. These are mediated through the EGF receptor and one of their main physiological functions is the enhancement of force production in fast twitch skeletal muscle fibres.

  10. ZN-INDUCED EGF RECEPTOR REQUIRES SRC-MEDIATED PHOSPHORYLATION OF EGF RECEPTOR ON TYROSINE 845. (R829214)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  11. Internalization of EGF receptor following lipid rafts disruption in keratinocytes is delayed and dependent on p38 MAPK activation

    DEFF Research Database (Denmark)

    Lambert, S.; Ameels, H.; Gniadecki, R.

    2008-01-01

    The receptor for epidermal growth factor (EGF) plays an important role in epidermal keratinocytes and is known to move out of lipid raft after cholesterol depletion, leading to ligand-independent activation. Accumulation of evidence indicates the ability of EGF receptor (EGFR) to undergo internal......The receptor for epidermal growth factor (EGF) plays an important role in epidermal keratinocytes and is known to move out of lipid raft after cholesterol depletion, leading to ligand-independent activation. Accumulation of evidence indicates the ability of EGF receptor (EGFR) to undergo...... internalization without participation of the ligand under the control of p38 MAPK during stress conditions. Since cholesterol depletion using methyl-beta-cyclodextrin is known to induce ligand-independent activation of EGFR in keratinocytes, we investigated by confocal microscopy and ligand-binding tests...... the process of internalization, which can be considered as a protective response to stress. Moreover, cholesterol-depleted keratinocytes recover their ability to proliferate during the recovery period that follows lipid raft disruption Udgivelsesdato: 2008/12...

  12. PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response

    KAUST Repository

    Liao, Hsin-Wei

    2015-11-16

    Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment.

  13. PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response

    Science.gov (United States)

    Liao, Hsin-Wei; Hsu, Jung-Mao; Xia, Weiya; Wang, Hung-Ling; Wang, Ying-Nai; Chang, Wei-Chao; Arold, Stefan T.; Chou, Chao-Kai; Tsou, Pei-Hsiang; Yamaguchi, Hirohito; Fang, Yueh-Fu; Lee, Hong-Jen; Lee, Heng-Huan; Tai, Shyh-Kuan; Yang, Mhu-Hwa; Morelli, Maria P.; Sen, Malabika; Ladbury, John E.; Chen, Chung-Hsuan; Grandis, Jennifer R.; Kopetz, Scott; Hung, Mien-Chie

    2015-01-01

    Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment. PMID:26571401

  14. PRMT1-mediated methylation of the EGF receptor regulates signaling and cetuximab response

    KAUST Repository

    Liao, Hsin-Wei; Hsu, Jung-Mao; Xia, Weiya; Wang, Hung-Ling; Wang, Ying-Nai; Chang, Wei-Chao; Arold, Stefan T.; Chou, Chao-Kai; Tsou, Pei-Hsiang; Yamaguchi, Hirohito; Fang, Yueh-Fu; Lee, Hong-Jen; Lee, Heng-Huan; Tai, Shyh-Kuan; Yang, Mhu-Hwa; Morelli, Maria P.; Sen, Malabika; Ladbury, John E.; Chen, Chung-Hsuan; Grandis, Jennifer R.; Kopetz, Scott; Hung, Mien-Chie

    2015-01-01

    Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment.

  15. Dihydrotestosterone Potentiates EGF-Induced ERK Activation by Inducing SRC in Fetal Lung Fibroblasts

    Science.gov (United States)

    Smith, Susan M.; Murray, Sandy; Pham, Lucia D.; Minoo, Parviz; Nielsen, Heber C.

    2014-01-01

    Lung maturation is regulated by interactions between mesenchymal and epithelial cells, and is delayed by androgens. Fibroblast–Type II cell communications are dependent on extracellular signal-regulated kinases (ERK) 1/2 activation by the ErbB receptor ligands epidermal growth factor (EGF), transforming growth factor (TGF)-α, and neuregulin (Nrg). In other tissues, dihydrotestosterone (DHT) has been shown to activate SRC by a novel nontranscriptional mechanism, which phosphorylates EGF receptors to potentiate EGF-induced ERK1/2 activation. This study sought to determine if DHT potentiates EGFR signaling by a nontranscriptional mechanism. Embryonic day (E)17 fetal lung cells were isolated from dams treated with or without DHT since E12. Cells were exposed to 30 ng/ml DHT for periods of 30 minutes to 3 days before being stimulated with 100 ng/ml EGF, TGF-α, or Nrg for up to 30 minutes. Lysates were immunoblotted for ErbB and SRC pathway signaling intermediates. DHT increased ERK1/2 activation by EGF, TGF-α, and Nrg in fibroblasts and Type II cells. Characterization in fibroblasts showed that potentiation of the EGF pathway was significant after 60 minutes of DHT exposure and persisted in the presence of the translational inhibitor cycloheximide. SRC and EGF receptor phosphorylation was increased by DHT, as was EGF-induced SHC1 phosphorylation and subsequent association with GRB2. Finally, SRC silencing, SRC inhibition with PP2, and overexpression of a dominant-negative SRC each prevented DHT from increasing EGF-induced ERK1/2 phosphorylation. These results suggest that DHT activates SRC to potentiate the signaling pathway leading from the EGF receptor to ERK activation in primary fetal lung fibroblasts. PMID:24484548

  16. In brown adipocytes, adrenergically induced β{sub 1}-/β{sub 3}-(G{sub s})-, α{sub 2}-(G{sub i})- and α{sub 1}-(G{sub q})-signalling to Erk1/2 activation is not mediated via EGF receptor transactivation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yanling; Fälting, Johanna M.; Mattsson, Charlotte L.; Holmström, Therése E.; Nedergaard, Jan, E-mail: jan@metabol.su.se

    2013-10-15

    Brown adipose tissue is unusual in that the neurotransmitter norepinephrine influences cell destiny in ways generally associated with effects of classical growth factors: regulation of cell proliferation, of apoptosis, and progression of differentiation. The norepinephrine effects are mediated through G-protein-coupled receptors; further mediation of such stimulation to e.g. Erk1/2 activation is in cell biology in general accepted to occur through transactivation of the EGF receptor (by external or internal pathways). We have examined here the significance of such transactivation in brown adipocytes. Stimulation of mature brown adipocytes with cirazoline (α{sub 1}-adrenoceptor coupled via G{sub q}), clonidine (α{sub 2} via G{sub i}) or CL316243 (β{sub 3} via G{sub s}) or via β{sub 1}-receptors significantly activated Erk1/2. Pretreatment with the EGF receptor kinase inhibitor AG1478 had, remarkably, no significant effect on Erk1/2 activation induced by any of these adrenergic agonists (although it fully abolished EGF-induced Erk1/2 activation), demonstrating absence of EGF receptor-mediated transactivation. Results with brown preadipocytes (cells in more proliferative states) were not qualitatively different. Joint stimulation of all adrenoceptors with norepinephrine did not result in synergism on Erk1/2 activation. AG1478 action on EGF-stimulated Erk1/2 phosphorylation showed a sharp concentration–response relationship (IC{sub 50} 0.3 µM); a minor apparent effect of AG1478 on norepinephrine-stimulated Erk1/2 phosphorylation showed nonspecific kinetics, implying caution in interpretation of partial effects of AG1478 as reported in other systems. Transactivation of the EGF receptor is clearly not a universal prerequisite for coupling of G-protein coupled receptors to Erk1/2 signalling cascades. - Highlights: • In brown adipocytes, norepinephrine regulates proliferation, apoptosis, differentiation. • EGF receptor transactivation is supposed to mediate GPCR

  17. Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation

    Energy Technology Data Exchange (ETDEWEB)

    Massa, Fabienne; Tormo, Aurelie; Beraud-Dufour, Sophie; Coppola, Thierry [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France); Mazella, Jean, E-mail: mazella@ipmc.cnrs.fr [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France)

    2011-10-14

    Highlights: {yields} We compare intracellular pathways of NT and EGF in HT29 cells. {yields} NT does not transactivate EGFR. {yields} Transactivation of EGFR is not a general rule in cancer cell growth. -- Abstract: Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation in HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor. Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.

  18. Self-phosphorylation of epidermal growth factor receptor: evidence for a model of intermolecular allosteric activation

    International Nuclear Information System (INIS)

    Yarden, Y.; Schlessinger, J.

    1987-01-01

    The membrane receptor for epidermal growth factor (EGF) is a 170,000 dalton glycoprotein composed of an extracellular EGF-binding domain and a cytoplasmic kinase domain connected by a stretch of 23 amino acids traversing the plasma membrane. The binding of EGF to the extracellular domain activates the cytoplasmic kinase function even in highly purified preparations of EGF receptor, suggesting that the activation occurs exclusively within the EGF receptor moiety. Conceivably, kinase activation may require the transfer of a conformational change through the single transmembrane region from the ligand binding domain to the cytoplasmic kinase region. Alternatively, ligand-induced receptor-receptor interactions may activate the kinase and thus bypass this requirement. Both mechanisms were contrasted by employing independent experimental approaches. On the basis of these results, an allosteric aggregation model is formulated for the activation of the cytoplasmic kinase function of the receptor by EGF. This model may be relevant to the mechanism by which the mitogenic signal of EGF is transferred across the membrane

  19. Importance of tyrosine phosphorylation in receptor kinase complexes.

    Science.gov (United States)

    Macho, Alberto P; Lozano-Durán, Rosa; Zipfel, Cyril

    2015-05-01

    Tyrosine phosphorylation is an important post-translational modification that is known to regulate receptor kinase (RK)-mediated signaling in animals. Plant RKs are annotated as serine/threonine kinases, but recent work has revealed that tyrosine phosphorylation is also crucial for the activation of RK-mediated signaling in plants. These initial observations have paved the way for subsequent detailed studies on the mechanism of activation of plant RKs and the biological relevance of tyrosine phosphorylation for plant growth and immunity. In this Opinion article we review recent reports on the contribution of RK tyrosine phosphorylation in plant growth and immunity; we propose that tyrosine phosphorylation plays a major regulatory role in the initiation and transduction of RK-mediated signaling in plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. EGF receptor-targeted synthetic double-stranded RNA eliminates glioblastoma, breast cancer, and adenocarcinoma tumors in mice.

    Directory of Open Access Journals (Sweden)

    Alexei Shir

    2006-01-01

    Full Text Available BACKGROUND: Glioblastoma multiforme (GBM is the most lethal form of brain cancer. With the available treatments, survival does not exceed 12-14 mo from the time of diagnosis. We describe a novel strategy to selectively induce the death of glioblastoma cells and other cancer cells that over-express the EGF receptor. Using a non-viral delivery vector that homes to the EGF receptor, we target synthetic anti-proliferative dsRNA (polyinosine-cytosine [poly IC], a strong activator of apoptosis, selectively to cancer cells. METHODS AND FINDINGS: Poly IC was delivered by means of a non-viral vector: 25kDa polyethylenimine-polyethyleneglycol-EGF (PEI25-PEG-EGF. EGFR-targeted poly IC induced rapid apoptosis in the target cells in vitro and in vivo. Expression of several cytokines and "bystander killing" of untransfected tumor cells was detected in vitro and in vivo. Intra-tumoral delivery of the EGFR-targeted poly IC induced the complete regression of pre-established intracranial tumors in nude mice, with no obvious adverse toxic effects on normal brain tissue. A year after treatment completion the treated mice remain cancer-free and healthy. Similarly, non-viral delivery of poly IC completely eliminated pre-established breast cancer and adenocarcinoma xenografts derived from EGFR over-expressing cancer cell lines, suggesting that the strategy is applicable to other EGFR-over-expressing tumors. CONCLUSION: The strategy described has yielded an effective treatment of EGFR over-expressing GBM in an animal model. If this strategy is translated successfully to the clinical setting, it may actually offer help to GBM patients. Moreover the elimination of two additional EGFR over-expressing cancers in vivo suggests that in principle this strategy can be applied to treat other tumors that over-express EGFR.

  1. A basic peptide within the juxtamembrane region is required for EGF receptor dimerization.

    Science.gov (United States)

    Aifa, Sami; Aydin, Jan; Nordvall, Gunnar; Lundström, Ingemar; Svensson, Samuel P S; Hermanson, Ola

    2005-01-01

    The epidermal growth factor receptor (EGFR) is fundamental for normal cell growth and organ development, but has also been implicated in various pathologies, notably tumors of epithelial origin. We have previously shown that the initial 13 amino acids (P13) within the intracellular juxtamembrane region (R645-R657) are involved in the interaction with calmodulin, thus indicating an important role for this region in EGFR function. Here we show that P13 is required for proper dimerization of the receptor. We expressed either the intracellular domain of EGFR (TKJM) or the intracellular domain lacking P13 (DeltaTKJM) in COS-7 cells that express endogenous EGFR. Only TKJM was immunoprecipitated with an antibody directed against the extracellular part of EGFR, and only TKJM was tyrosine phosphorylated by endogenous EGFR. Using SK-N-MC cells, which do not express endogenous EGFR, that were stably transfected with either wild-type EGFR or recombinant full-length EGFR lacking P13 demonstrated that P13 is required for appropriate receptor dimerization. Furthermore, mutant EGFR lacking P13 failed to be autophosphorylated. P13 is rich in basic amino acids and in silico modeling of the EGFR in conjunction with our results suggests a novel role for the juxtamembrane domain (JM) of EGFR in mediating intracellular dimerization and thus receptor kinase activation and function.

  2. Vasopressin up-regulates the expression of growth-related immediate-early genes via two distinct EGF receptor transactivation pathways

    Science.gov (United States)

    Fuentes, Lida Q.; Reyes, Carlos E.; Sarmiento, José M.; Villanueva, Carolina I.; Figueroa, Carlos D.; Navarro, Javier; González, Carlos B.

    2008-01-01

    Activation of V1a receptor triggers the expression of growth-related immediate-early genes (IEGs), including c-Fos and Egr-1. Here we found that pre-treatment of rat vascular smooth muscle A-10 cell line with the EGF receptor inhibitor AG1478 or the over-expression of an EGFR dominant negative mutant (HEBCD533) blocked the vasopressin-induced expression of IEGs, suggesting that activation of these early genes mediated by V1a receptor is via transactivation of the EGF receptor. Importantly, the inhibition of the metalloproteinases, which catalyzed the shedding of the EGF receptor agonist HB-EGF, selectively blocked the vasopressin-induced expression c-Fos. On the other hand, the inhibition of c-Src selectively blocked the vasopressin-induced expression of Egr-1. Interestingly, in contrast to the expression of c-Fos, the expression of Egr-1 was mediated via the Ras/MEK/MAPK-dependent signalling pathway. Vasopressin-triggered expression of both genes required the release of intracellular calcium, activation of PKC and β-arrestin 2. These findings demonstrated that vasopressin up-regulated the expression of c-Fos and Erg-1 via transactivation of two distinct EGF receptor-dependent signalling pathways. PMID:18571897

  3. GABAB receptor phosphorylation regulates KCTD12-induced K+ current desensitization

    DEFF Research Database (Denmark)

    Adelfinger, L; Turecek, R; Ivankova, K

    2014-01-01

    released from the G-protein. Receptor-activated K+ currents desensitize in the sustained presence of agonist to avoid excessive effects on neuronal activity. Desensitization of K+ currents integrates distinct mechanistic underpinnings. GABAB receptor activity reduces protein kinase-A activity, which...... reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation. This form of desensitization operates on the time scale of several minutes to hours. A faster form of desensitization is induced by the auxiliary subunit KCTD12, which interferes with channel activation by binding to the G......-protein βγ subunits. Here we show that the two mechanisms of desensitization influence each other. Serine-892 phosphorylation in heterologous cells rearranges KCTD12 at the receptor and slows KCTD12-induced desensitization. Likewise, protein kinase-A activation in hippocampal neurons slows fast...

  4. Stromal cell-derived factor-1α (SDF-1α/CXCL12) stimulates ovarian cancer cell growth through the EGF receptor transactivation

    International Nuclear Information System (INIS)

    Porcile, Carola; Bajetto, Adriana; Barbieri, Federica; Barbero, Simone; Bonavia, Rudy; Biglieri, Marianna; Pirani, Paolo; Florio, Tullio; Schettini, Gennaro

    2005-01-01

    Ovarian cancer (OC) is the leading cause of death in gynecologic diseases in which there is evidence for a complex chemokine network. Chemokines are a family of proteins that play an important role in tumor progression influencing cell proliferation, angiogenic/angiostatic processes, cell migration and metastasis, and, finally, regulating the immune cells recruitment into the tumor mass. We previously demonstrated that astrocytes and glioblastoma cells express both the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1), and that SDF-1α treatment induced cell proliferation, supporting the hypothesis that chemokines may play an important role in tumor cells' growth in vitro. In the present study, we report that CXCR4 and SDF-1 are expressed in OC cell lines. We demonstrate that SDF-1α induces a dose-dependent proliferation in OC cells, by the specific interaction with CXCR4 and a biphasic activation of ERK1/2 and Akt kinases. Our results further indicate that CXCR4 activation induces EGF receptor (EGFR) phosphorylation that in turn was linked to the downstream intracellular kinases activation, ERK1/2 and Akt. In addition, we provide evidence for cytoplasmic tyrosine kinase (c-Src) involvement in the SDF-1/CXCR4-EGFR transactivation. These results suggest a possible important 'cross-talk' between SDF-1/CXCR4 and EGFR intracellular pathways that may link signals of cell proliferation in ovarian cancer

  5. Stress induces pain transition by potentiation of AMPA receptor phosphorylation.

    Science.gov (United States)

    Li, Changsheng; Yang, Ya; Liu, Sufang; Fang, Huaqiang; Zhang, Yong; Furmanski, Orion; Skinner, John; Xing, Ying; Johns, Roger A; Huganir, Richard L; Tao, Feng

    2014-10-08

    Chronic postsurgical pain is a serious issue in clinical practice. After surgery, patients experience ongoing pain or become sensitive to incident, normally nonpainful stimulation. The intensity and duration of postsurgical pain vary. However, it is unclear how the transition from acute to chronic pain occurs. Here we showed that social defeat stress enhanced plantar incision-induced AMPA receptor GluA1 phosphorylation at the Ser831 site in the spinal cord and greatly prolonged plantar incision-induced pain. Interestingly, targeted mutation of the GluA1 phosphorylation site Ser831 significantly inhibited stress-induced prolongation of incisional pain. In addition, stress hormones enhanced GluA1 phosphorylation and AMPA receptor-mediated electrical activity in the spinal cord. Subthreshold stimulation induced spinal long-term potentiation in GluA1 phosphomimetic mutant mice, but not in wild-type mice. Therefore, spinal AMPA receptor phosphorylation contributes to the mechanisms underlying stress-induced pain transition. Copyright © 2014 the authors 0270-6474/14/3413737-10$15.00/0.

  6. Phosphorylation site dynamics of early T-cell receptor signaling

    DEFF Research Database (Denmark)

    Chylek, Lily A; Akimov, Vyacheslav; Dengjel, Jörn

    2014-01-01

    In adaptive immune responses, T-cell receptor (TCR) signaling impacts multiple cellular processes and results in T-cell differentiation, proliferation, and cytokine production. Although individual protein-protein interactions and phosphorylation events have been studied extensively, we lack...... that diverse dynamic patterns emerge within seconds. We detected phosphorylation dynamics as early as 5 s and observed widespread regulation of key TCR signaling proteins by 30 s. Development of a computational model pointed to the presence of novel regulatory mechanisms controlling phosphorylation of sites...... a systems-level understanding of how these components cooperate to control signaling dynamics, especially during the crucial first seconds of stimulation. Here, we used quantitative proteomics to characterize reshaping of the T-cell phosphoproteome in response to TCR/CD28 co-stimulation, and found...

  7. Gβ promotes pheromone receptor polarization and yeast chemotropism by inhibiting receptor phosphorylation.

    Science.gov (United States)

    Ismael, Amber; Tian, Wei; Waszczak, Nicholas; Wang, Xin; Cao, Youfang; Suchkov, Dmitry; Bar, Eli; Metodiev, Metodi V; Liang, Jie; Arkowitz, Robert A; Stone, David E

    2016-04-12

    Gradient-directed cell migration (chemotaxis) and growth (chemotropism) are processes that are essential to the development and life cycles of all species. Cells use surface receptors to sense the shallow chemical gradients that elicit chemotaxis and chemotropism. Slight asymmetries in receptor activation are amplified by downstream signaling systems, which ultimately induce dynamic reorganization of the cytoskeleton. During the mating response of budding yeast, a model chemotropic system, the pheromone receptors on the plasma membrane polarize to the side of the cell closest to the stimulus. Although receptor polarization occurs before and independently of actin cable-dependent delivery of vesicles to the plasma membrane (directed secretion), it requires receptor internalization. Phosphorylation of pheromone receptors by yeast casein kinase 1 or 2 (Yck1/2) stimulates their internalization. We showed that the pheromone-responsive Gβγ dimer promotes the polarization of the pheromone receptor by interacting with Yck1/2 and locally inhibiting receptor phosphorylation. We also found that receptor phosphorylation is essential for chemotropism, independently of its role in inducing receptor internalization. A mathematical model supports the idea that the interaction between Gβγ and Yck1/2 results in differential phosphorylation and internalization of the pheromone receptor and accounts for its polarization before the initiation of directed secretion. Copyright © 2016, American Association for the Advancement of Science.

  8. Age-related loss of EGF-receptor related protein (ERRP) in the aging colon is a potential risk factor for colon cancer.

    Science.gov (United States)

    Schmelz, Eva M; Levi, Edi; Du, Jianhua; Xu, Hu; Majumdar, Adhip P N

    2004-12-01

    Although in Fischer-344 rats, aging is associated with increased activation of EGF-receptor (EGFR) in mucosa of much of the gastrointestinal tract, including the colon, regulation of this process is poorly understood. We hypothesize that loss of suppressor of EGFR may partly be responsible for this process. To test this hypothesis, we examined the expression of EGFR related protein (ERRP), a recently identified negative regulator of EGFR, in the colonic mucosa during aging and following administration of the colonic carcinogen dimethylhydrazine (DMH) that resulted in the formation of aberrant crypt foci (ACF), which are considered to be precursor of adenoma and carcinoma. In Fischer-344 rats, aging is associated with increased activation of EGFR in the colonic mucosa, as evidenced by 30-35% increase in the levels of tyrosine phosphorylated EGFR in the proximal and distal colon of aged (20-22 months old) than in young (4-6 months old) rats. In contrast, the levels of ERRP in both regions of the colon of aged rats were decreased by 50-60%, compared to their younger counterparts. Administration of DMH, which induced a greater number of ACF in the colon of aged rats than in young animals, resulted in a corresponding reduction in ERRP in the colon. These results suggest that loss of ERRP expression is a common event during aging and early stages of chemically induced colon cancer. We also suggest that loss of ERRP could be a risk factor for developing colorectal cancer in the older population.

  9. Tyrosine Phosphorylation in Toll-Like Receptor Signaling

    Science.gov (United States)

    Chattopadhyay, Saurabh; Sen, Ganes C.

    2014-01-01

    There is a wealth of knowledge about how different Ser/Thr protein kinases participate in Toll-like receptor (TLR) signaling. In many cases, we know the identities of the Ser/Thr residues of various components of the TLR-signaling pathways that are phosphorylated, the functional consequences of the phosphorylation and the responsible protein kinases. In contrast, the analysis of Tyr-phosphorylation of TLRs and their signaling proteins is currently incomplete, because several existing analyses are not systematic or they do not rely on robust experimental data. Nevertheless, it is clear that many TLRs require, for signaling, ligand-dependent phosphorylation of specific Tyr residues in their cytoplasmic domains; the list includes TLR2, TLR3, TLR4, TLR5, TLR8 and TLR9. In this article, we discuss the current status of knowledge on the effect of Tyr-phosphorylation of TLRs and their signaling proteins on their biochemical and biological functions, the possible identities of the relevant protein tyrosine kinases (PTKs) and the nature of regulations of PTK-mediated activation of TLR signaling pathways. PMID:25022196

  10. IL-1beta signals through the EGF receptor and activates Egr-1 through MMP-ADAM.

    Directory of Open Access Journals (Sweden)

    Estella Sanchez-Guerrero

    Full Text Available The immediate-early gene Egr-1 controls the inducible expression of many genes implicated in the pathogenesis of a range of vascular disorders, yet our understanding of the mechanisms controlling the rapid expression of this prototypic zinc finger transcription factor is poor. Here we show that Egr-1 expression induced by IL-1beta is dependent on metalloproteinases (MMP and a disintegrin and a metalloproteinase (ADAM. Pharmacologic MMP/ADAM inhibitors and siRNA knockdown prevent IL-1beta induction of Egr-1. Further, IL-1beta activates Egr-1 via the epidermal growth factor receptor (EGFR. This is blocked by EGFR tyrosine kinase inhibition and EGFR knockdown. IL-1beta induction of Egr-1 expression is reduced in murine embryonic fibroblasts (mEFs deficient in ADAM17 despite unbiased expression of EGFR and IL-1RI in ADAM17-deficient and wild-type mEFs. Finally, we show that IL-1beta-inducible wound repair after mechanical injury requires both EGFR and MMP/ADAM. This study reports for the first time that Egr-1 induction by IL-1beta involves EGFR and MMP/ADAM-dependent EGFR phosphorylation.

  11. Estradiol attenuates EGF-induced rapid uPAR mobilization and cell migration via the G-protein-coupled receptor 30 in ovarian cancer cells

    DEFF Research Database (Denmark)

    Henic, Emir; Noskova, Vera; Høyer-Hansen, Gunilla

    2009-01-01

    : rapid mobilization of uPAR from detergent-resistant domains, increased mRNA, and decreased degradation. G-protein-coupled receptor 30 (GPR30) is a newly identified membrane estrogen receptor (ER).The objective of this study was to explore the effects of 17beta-estradiol (E(2)) on uPAR expression...... for ERalpha, and quantitative polymerase chain reaction. Estradiol attenuates the stimulatory effect of EGF on cell migration and uPAR expression. Specifically, E(2) reduces the very rapid increase of detergent extractable uPAR, which occurs within minutes of EGF stimulation and probably represents...... agonist G1, mimicked the effect of E(2) on uPAR expression and cell migration. OVCAR-3 cells express mRNA for GPR30.Estradiol attenuates EGF-induced mobilization of ligated uPAR from detergent-resistant domains and subsequent migration in ovarian cancer cells. The response to various ER ligands indicates...

  12. TGF{beta} induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Ebi, Masahide [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Kataoka, Hiromi, E-mail: hkataoka@med.nagoya-cu.ac.jp [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Shimura, Takaya; Kubota, Eiji; Hirata, Yoshikazu; Mizushima, Takashi; Mizoshita, Tsutomu; Tanaka, Mamoru; Mabuchi, Motoshi; Tsukamoto, Hironobu; Tanida, Satoshi; Kamiya, Takeshi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan); Higashiyama, Shigeki [Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Ehime (Japan); Joh, Takashi [Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya (Japan)

    2010-11-19

    Research highlights: {yields} TGF{beta} induces EGFR transactivation through proHB-EGF shedding by activated ADAM members in gastric cancer cells. {yields} TGF{beta} induces nuclear translocation of HB-EGF-CTF cleaved by ADAM members. {yields} TGF{beta} enhances cell growth by EGFR transactivation and HB-EGF-CTF nuclear translocation and ADAM inhibitors block these effects. {yields} Silencing of ADAM17 also blocks EGFR transactivation, HB-EGF-CTF nuclear translocation and cancer cell growth by TGF{beta}. {yields} ADAM17 may play a crucial role in this TGF{beta}-HB-EGF signal transduction. -- Abstract: Background and aims: Transforming growth factor-beta (TGF{beta}) is known to potently inhibit cell growth. Loss of responsiveness to TGF{beta} inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGF{beta} and HB-EGF signal transduction via ADAM activation. Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGF{beta}. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGF{beta} was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGF{beta} was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. Result: TGF{beta}-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGF{beta} induced shedding of proHB-EGF allowing HB-EGF-CTF to

  13. TGFβ induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

    International Nuclear Information System (INIS)

    Ebi, Masahide; Kataoka, Hiromi; Shimura, Takaya; Kubota, Eiji; Hirata, Yoshikazu; Mizushima, Takashi; Mizoshita, Tsutomu; Tanaka, Mamoru; Mabuchi, Motoshi; Tsukamoto, Hironobu; Tanida, Satoshi; Kamiya, Takeshi; Higashiyama, Shigeki; Joh, Takashi

    2010-01-01

    Research highlights: → TGFβ induces EGFR transactivation through proHB-EGF shedding by activated ADAM members in gastric cancer cells. → TGFβ induces nuclear translocation of HB-EGF-CTF cleaved by ADAM members. → TGFβ enhances cell growth by EGFR transactivation and HB-EGF-CTF nuclear translocation and ADAM inhibitors block these effects. → Silencing of ADAM17 also blocks EGFR transactivation, HB-EGF-CTF nuclear translocation and cancer cell growth by TGFβ. → ADAM17 may play a crucial role in this TGFβ-HB-EGF signal transduction. -- Abstract: Background and aims: Transforming growth factor-beta (TGFβ) is known to potently inhibit cell growth. Loss of responsiveness to TGFβ inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGFβ and HB-EGF signal transduction via ADAM activation. Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGFβ. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGFβ was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGFβ was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. Result: TGFβ-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGFβ induced shedding of proHB-EGF allowing HB-EGF-CTF to translocate to the nucleus. ADAM inhibitors blocked this nuclear translocation. TGF

  14. Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase.

    Science.gov (United States)

    Shetty, Praveenkumar; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P; Liu, Ming C; Shetty, Sreerama

    2010-02-01

    The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1-100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.

  15. Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells

    International Nuclear Information System (INIS)

    Kosheverova, Vera V.; Kamentseva, Rimma S.; Gonchar, Ilya V.; Kharchenko, Marianna V.; Kornilova, Elena S.

    2016-01-01

    Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering. - Highlights: • EEA1 mobility was compared in serum-starved and EGF-stimulated interphase HeLa cells. • FRAP analysis revealed fast and slow components of EEA1 recovery in both cases. • Stimulation of EGFR endocytosis did not affect fast EEA1 turnover. • EGF stimulation significantly increased half-time of slowly exchanged EEA1 fraction.

  16. Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Kosheverova, Vera V., E-mail: kosheverova_vera@incras.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); Kamentseva, Rimma S., E-mail: rkamentseva@yandex.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); St. Petersburg State University, 7-9, Universitetskaya nab, St. Petersburg, 199034 (Russian Federation); Gonchar, Ilya V., E-mail: ample@mail.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); Kharchenko, Marianna V., E-mail: mariannakharchenko@gmail.com [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); Kornilova, Elena S., E-mail: lenkor@mail.cytspb.rssi.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); St. Petersburg State University, 7-9, Universitetskaya nab, St. Petersburg, 199034 (Russian Federation); Department of Medical Physics, Peter the Great St. Petersburg Polytechnic University, 29, Polytechnicheskaya, St.Petersburg, 195251 (Russian Federation)

    2016-04-22

    Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering. - Highlights: • EEA1 mobility was compared in serum-starved and EGF-stimulated interphase HeLa cells. • FRAP analysis revealed fast and slow components of EEA1 recovery in both cases. • Stimulation of EGFR endocytosis did not affect fast EEA1 turnover. • EGF stimulation significantly increased half-time of slowly exchanged EEA1 fraction.

  17. Icotinib, a selective EGF receptor tyrosine kinase inhibitor, for the treatment of non-small-cell lung cancer.

    Science.gov (United States)

    Tan, Fenlai; Shi, Yuankai; Wang, Yinxiang; Ding, Lieming; Yuan, Xiaobin; Sun, Yan

    2015-01-01

    Advanced non-small-cell lung cancer (NSCLC) is the main cause for cancer-related mortality. Treatments for advanced NSCLC are largely palliative and a benefit plateau appears to have reached with the platinum-based chemotherapy regimens. EGF receptor (EGFR) tyrosine kinase inhibitors gefitinib, erlotinib and afatinib came up with prolonged progression-free survival and improved quality of life, especially in EGFR-mutated patients. Icotinib is an oral selective EGFR tyrosine kinase, which was approved by China Food and Drug administration in June 2011 for treating advanced NSCLC. Its approval was based on the registered Phase III trial (ICOGEN), which showed icotinib is noninferior to gefitinib. This review will discuss the role of icotinib in NSCLC, and its potential application and ongoing investigations.

  18. Bg1II polymorphism of the epidermal growth factor receptor (EGF-R) gene

    Energy Technology Data Exchange (ETDEWEB)

    Biunno, I; Pozzi, M R; Radice, P; Mondini, P; Pierotti, M A; Porta, G D [Istituto Nazionale Tumori, Milan (Italy); Haley, J; Waterfield, M D [Ludwig Institute for Cancer Research, London (England)

    1988-08-11

    A 770 bp cDNA fragment was derived from the cytoplasmic portion of the EGF-R (ref. Libermann et al., 1985). Bg1II identifies 4 invariant bands of 7.0, 5.0, 3.5 and 1.2 kb and a two allele polymorphism with a band of either 10.6 kb (lane 1) or 9.4 kb (lane 3). An heterozygote individual is represented. The frequency was analyzed in 78 unrelated European Caucasians. Its chromosomal location was determined. Co-dominant segregation was demonstrated in three families of 12 individuals. A rare variant of 8.3 kb was seen in one chromosome out of the 144 examined. This allelic form has not yet been fully characterized.

  19. Inhibition of radiation induced migration of human head and neck squamous cell carcinoma cells by blocking of EGF receptor pathways

    International Nuclear Information System (INIS)

    Pickhard, Anja C; Schlegel, Jürgen; Arnold, Wolfgang; Reiter, Rudolf; Margraf, Johanna; Knopf, Andreas; Stark, Thomas; Piontek, Guido; Beck, Carolin; Boulesteix, Anne-Laure; Scherer, Elias Q; Pigorsch, Steffi

    2011-01-01

    Recently it has been shown that radiation induces migration of glioma cells and facilitates a further spread of tumor cells locally and systemically. The aim of this study was to evaluate whether radiotherapy induces migration in head and neck squamous cell carcinoma (HNSCC). A further aim was to investigate the effects of blocking the epidermal growth factor receptor (EGFR) and its downstream pathways (Raf/MEK/ERK, PI3K/Akt) on tumor cell migration in vitro. Migration of tumor cells was assessed via a wound healing assay and proliferation by a MTT colorimeritric assay using 3 HNSCC cell lines (BHY, CAL-27, HN). The cells were treated with increasing doses of irradiation (2 Gy, 5 Gy, 8 Gy) in the presence or absence of EGF, EGFR-antagonist (AG1478) or inhibitors of the downstream pathways PI3K (LY294002), mTOR (rapamycin) and MEK1 (PD98059). Biochemical activation of EGFR and the downstream markers Akt and ERK were examined by Western blot analysis. In absence of stimulation or inhibition, increasing doses of irradiation induced a dose-dependent enhancement of migrating cells (p < 0.05 for the 3 HNSCC cell lines) and a decrease of cell proliferation (p < 0.05 for the 3 HNSCC cell lines). The inhibition of EGFR or the downstream pathways reduced cell migration significantly (almost all p < 0.05 for the 3 HNSCC cell lines). Stimulation of HNSCC cells with EGF caused a significant increase in migration (p < 0.05 for the 3 HNSCC cell lines). After irradiation alone a pronounced activation of EGFR was observed by Western blot analysis. Our results demonstrate that the EGFR is involved in radiation induced migration of HNSCC cells. Therefore EGFR or the downstream pathways might be a target for the treatment of HNSCC to improve the efficacy of radiotherapy

  20. [INHIBITORS OF MAP-KINASE PATHWAY U0126 AND PD98059 DIFFERENTLY AFFECT ORGANIZATION OF TUBULIN CYTOSKELETON AFTER STIMULATION OF EGF RECEPTOR ENDOCYTOSIS].

    Science.gov (United States)

    Zlobina, M V; Steblyanko, Yu Yu; Shklyaeva, M A; Kharchenko, V V; Salova, A V; Kornilova, E S

    2015-01-01

    To confirm the hypothesis about the involvement of EGF-stimulated MAP-kinase ERK1/2 in the regulation of microtubule (MT) system, the influence of two widely used ERK1/2 inhibitors, U0126 and PD98059, on the organization of tubulin cytoskeleton in interphase HeLa cells during EGF receptor endocytosis has been investigated. We have found that addition of U0126 or PD98059 to not-stimulated with EGF ells for 30 min has no effect on radially organized MT system. However, in the case of U0126 addition before EGF endocytosis stimulation, the number of MT per cell decreased within 15 min after such stimulation and was followed by complete MT depolymerization by 60-90 min. Stimulation of EGF endocytosis in the presence of PD98059 resulted only in insignificant depolymerization of MT and it could be detected mainly from their minus-ends. At the same time, MT regions close to plasma membrane became stabilized, which was proved by increase in tubulin acetylation level. This situation was characteristic for all period of the experiment. It has been also found that the inhibitors affect endocytosis dynamics of EGF-receptor complexes. Quantitative analysis demonstrated that the stimulation of endocytosis in the presence of U0126 generated a greater number of endosomes compared to control cells, and their number did not change significantly during the experiment. All these endosomes were localized peripherally. Effect of PD98059 resulted in the formation of lower number of endosomes that in control, but they demonstrated very slow clusterization despite the presence of some intact MT. Both inhibitors decreased EGFR colocolization with early endosomal marker EEA1, which indicated a delay in endosome fusions and maturation. The inhibitors were also shown to affect differently phospho-ERK 1 and 2 forms: U0126 completely inhibited phospho-ERK1 and 2, white, in the presence of PD98059, the two ERK forms demonstrated sharp transient activation in 15 min after stimulation, but only

  1. Regulation of EGF Receptor Signaling by Histone Deacetylase 6 (HDAC6)-Mediated Reversible Acetylation

    National Research Council Canada - National Science Library

    Kovacs, Jeffrey J

    2005-01-01

    One of the hallmarks of cancer is uncontrolled cell growth and proliferation. In cells, a group of proteins called growth factor receptors are responsible for responding to the signals that trigger proliferation...

  2. An unusual protein kinase phosphorylates the chemotactic receptor of Dictystelium discoideum

    International Nuclear Information System (INIS)

    Meier, K.; Klein, C.

    1988-01-01

    The authors report the cAMP-dependent phosphorylation of the chemotactic receptor of Dictyostelium discoideum in partially purified plasma membranes. The protein kinase responsible for receptor phosphorylation is associated with this fraction and preferentially phosphorylates the ligand-occupied form of the receptor. 8-Azido[ 32 P]cAMP labeling of the cell surface has shown that the cAMP receptor exists in two forms. A 45-kDa protein is predominant on unstimulated cells. cAMP stimulation results in an increased receptor phosphorylation such that the receptor migrates on NaDodSO 4 /PAGE as a 47-kDa protein. Phosphorylation of the chemotactic receptor is not detected in membrane preparations unless cAMP is added to the incubation mixture. Only under those conditions is the phosphorylated 47-kDa form observed. The requirement for cAMP reflects the fact that the kinase involved preferentially uses the ligand-occupied receptor as a substrate. In vitro phosphorylation of the receptor does not involve tyrosine residues. The enzyme does not appear to be a cAMP- or cGMP-dependent protein kinase nor is it sensitive to guanine nucleotides, Ca 2+ /calmodulin, Ca 2+ /phospholipid, or EGTA. Similarities with the β-adrenergic receptor protein kinase are discussed

  3. Use of SLAM and PVRL4 and identification of pro-HB-EGF as cell entry receptors for wild type phocine distemper virus.

    Directory of Open Access Journals (Sweden)

    Mary M Melia

    Full Text Available Signalling lymphocyte activation molecule (SLAM has been identified as an immune cell receptor for the morbilliviruses, measles (MV, canine distemper (CDV, rinderpest and peste des petits ruminants (PPRV viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4, also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF,for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation.

  4. Epsin 1 is involved in recruitment of ubiquitinated EGF receptors into clathrin-coated pits

    DEFF Research Database (Denmark)

    Kazazic, Maja; Bertelsen, Vibeke; Pedersen, Ketil Winther

    2008-01-01

    . Furthermore, RNAi-mediated knock down of epsin 1 was found to inhibit internalization of the EGFR, while having no effect on endocytosis of the transferrin receptor. Additionally, upon knock down of epsin 1, translocation of the EGFR to central parts of clathrin-coated pits was inhibited. This supports...

  5. Altered [125I]epidermal growth factor binding and receptor distribution in psoriasis

    International Nuclear Information System (INIS)

    Nanney, L.B.; Stoscheck, C.M.; Magid, M.; King, L.E. Jr.

    1986-01-01

    Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that [ 125 I]EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers

  6. Direct visualization of epidermal growth factor receptors (EGFR) in A431 and placental cell membrane by western blot with 125I-EGF

    International Nuclear Information System (INIS)

    Lin, P.H.; Selinfreund, R.; Wharton, W.

    1986-01-01

    Using the western blot technique, they have devised a new procedure that allowed the direct visualization of both the 150KD and the 170KD forms of EGFR by its natural ligand, 125 I-EGF. A431, and placental plasmalemma were purified and solubilized in either SDS-PAGE buffer (without DTT, EDTA) or Triton X-100 (0.5%), resolved on PAGE and electrophoretically transferred onto nitrocellulose (NC) paper. In the absence of boiling, SDS did not denature the EGFR. Although EGER band can be detected after hybridization with 125 I-EGF, the receptor signal was considerably improved with the addition of 0.1% Tween-20. The binding of 125 I-EGF to the both the 150KD and the 170KD bands of the EGFR was specific, reversible and increased with the amount of membrane protein present. The direct visualization of the EGFR using its natural ligand eliminated the necessity for the time consuming antibody preparation. Presently, they are using this technique to identify specific receptors for other ligands

  7. Characterization of the epidermal growth factor receptor associated with cytoskeletons of A431 cells

    International Nuclear Information System (INIS)

    Roy, L.M.; Gittinger, C.K.; Landreth, G.E.

    1989-01-01

    Epidermal growth factor receptors (EGF-R) have been shown to be associated with the detergent-insoluble cytoskeleton of A431 cells, where they retained both a functional ligand-binding domain and tyrosine kinase activity. In the present study we have characterized the tyrosine kinase and ligand binding activities of this cytoskeletally associated EGF-R. The tyrosine kinase activity of the cytoskeletally associated EGF-R was stimulated by EGF treatment of intact cells as evidenced by increased autophosphorylation and phosphorylation of the exogenous substrate angiotensin II (AII). The kinetic behavior of the EGF-R associated with cytoskeletons of EGF-treated cells was similar to that of purified receptors. The stimulation of the receptor kinase activity required EGF treatment of intact cells prior to Triton extraction. If cytoskeletons were prepared from untreated cells and then incubated with EGF, there was no stimulation of the detergent-insoluble receptor kinase activity, indicating that the immobilized receptor was unable to undergo EGF-stimulated activation. Comparison of peptide maps from soluble and cytoskeletally associated EGF-R revealed qualitatively similar patterns; however, they are distinguished by a prominent 46 kD band in digests of the cytoskeletal EGF-R. Saturable binding of 125I-EGF to A431 cytoskeletons prepared from adherent and suspended cells demonstrated the presence of specific receptors on the cytoskeleton. High-affinity EGF-R were preferentially retained upon detergent extraction of adherent cells, whereas both low- and high-affinity receptors were solubilized from the cytoskeletons of suspended cells. Suspension of cells resulted in the solubilization of an additional 15% of the EGF-R to that solubilized in adherent cells, indicating that EGF-R can reversibly associate with the structural elements of the cell

  8. EGF Prevents the Neuroendocrine Differentiation of LNCaP Cells Induced By Serum Deprivation: The Modulator Role of P13K/Akt

    Directory of Open Access Journals (Sweden)

    Rosa M. Martín-Orozco

    2007-08-01

    Full Text Available The primary focus of this investigation was to study the relationship between neuroendocrine (NE differentiation, epidermal growth factor (EGF because both have been implicated in the progression of prostate cancer. For this purpose, we used gefitinib, trastuzumab, which are inhibitors of EGF receptor (EGFR, ErbB2, respectively. EGF prevents NE differentiation induced by androgen depletion. This effect is prevented by gefitinib, which blocks the activation of EGFR, ErbB2, stimulation of mitogen-activated protein kinase (MAPK, cell proliferation induced by EGF. Conversely, trastuzumab does not inhibit the effect of EGF on EGFR phosphorylation, MAPK activity, cell proliferation, NE differentiation, although it reduces ErbB2 levels specifically, suggesting that ErbB2 is not necessary to inhibit NE differentiation. Prevention of NE differentiation by EGF is mediated by a MAPK-dependent mechanism, requires constitutive Akt activation. The abrogation of the PI3K/Akt pathway changes the role of EGF from inhibitor to inductor of NE differentiation. We show that EGFR tyrosine kinase, MAPK, PI3K inhibitors inhibit the cell proliferation stimulated by EGF but induce the acquisition of NE phenotype. Altogether, the present data should be borne in mind when designing new clinical schedules for the treatment of prostate cancer, including the use of ErbB receptors, associated signaling pathway inhibitors.

  9. The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

    International Nuclear Information System (INIS)

    Hoskins, J.T.; Zhou, Z.; Harding, P.A.

    2008-01-01

    A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser 108/121 , HB-EGF-Cys/Ser 116/132 , and HB-EGF-Cys/Ser 134/143 ) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with M r of 6.5, 21 and 24 kDa were observed from lysates of HB-EGF and each HB-EGF disulfide analogue. HB-EGF immunohistochemical analyses of each HB-EGF stable cell line demonstrated ubiquitous protein expression except HB-EGF-Cys/Ser 108/121 and HB-EGF-Cys/Ser 116/132 stable cell lines which exhibited accumulated expression immediately outside the nucleus. rHB-EGF, HB-EGF, and HB-EGF 134/143 proteins competed with 125 I-EGF in an A431 competitive binding assay, whereas HB-EGF-Cys/Ser 108/121 and HB-EGF-Cys/Ser 116/132 failed to compete. Each HB-EGF disulfide analogue lacked the ability to stimulate tyrosine phosphorylation of the 170 kDa EGFR. These results suggest that HB-EGF-Cys/Ser 134/143 antagonizes EGFRs

  10. Distinct Phosphorylation Clusters Determine the Signaling Outcome of Free Fatty Acid Receptor 4/G Protein-Coupled Receptor 120

    DEFF Research Database (Denmark)

    Prihandoko, Rudi; Alvarez-Curto, Elisa; Hudson, Brian D

    2016-01-01

    of these phosphoacceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation. Rather, an inhibitor of Gq/11proteins completely prevented receptor signaling to ERK1/2. By contrast, the recruitment...... activation. These unique observations define differential effects on signaling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signaling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode...

  11. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    International Nuclear Information System (INIS)

    Berger, Christian; Madshus, Inger Helene; Stang, Espen

    2012-01-01

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: ► Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. ► Antibody combination causes internalization of EGFR by macropinocytosis. ► Antibody-induced internalization of EGFR is independent of EGFR kinase activity. ► Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  12. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Berger, Christian [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Madshus, Inger Helene [Institute of Pathology, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Stang, Espen, E-mail: espsta@rr-research.no [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway)

    2012-12-10

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: Black-Right-Pointing-Pointer Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. Black-Right-Pointing-Pointer Antibody combination causes internalization of EGFR by macropinocytosis. Black-Right-Pointing-Pointer Antibody-induced internalization of EGFR is independent of EGFR kinase activity. Black-Right-Pointing-Pointer Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  13. Wingless, decapentaplegic and EGF receptor signaling pathways interact to specify dorso-ventral pattern in the adult abdomen of Drosophila.

    Science.gov (United States)

    Kopp, A; Blackman, R K; Duncan, I

    1999-08-01

    Adult abdominal segments of Drosophila are subdivided along the dorso-ventral axis into a dorsal tergite, a ventral sternite and ventro-lateral pleural cuticle. We report that this pattern is largely specified during the pupal stage by Wingless (Wg), Decapentaplegic (Dpp) and Drosophila EGF Receptor (DER) signaling. Expression of wg and dpp is activated at the posterior edge of the anterior compartment by Hedgehog signaling. Within this region, wg and dpp are expressed in domains that are mutually exclusive along the dorso-ventral axis: wg is expressed in the sternite and medio-lateral tergite, whereas dpp expression is confined to the pleura and the dorsal midline. Neither gene is expressed in the lateral tergite. Shirras and Couso (1996, Dev. Biol. 175, 24-36) have shown that tergite and sternite cell fates are specified by Wg signaling. We find that DER acts synergistically with Wg to promote tergite and sternite identities, and that Wg and DER activities are opposed by Dpp signaling, which promotes pleural identity. Wg and Dpp interact antagonistically at two levels. First, their expression is confined to complementary domains by mutual transcriptional repression. Second, Wg and Dpp compete directly with one another by exerting opposite effects on cell fate. DER signaling does not affect the expression of wg or dpp, indicating that it interacts with Wg and Dpp at the level of cell fate determination. Within the tergite, the requirements for Wg and DER function are roughly complementary: Wg is required mainly in the medial region, whereas DER is most important laterally. Finally, we show that Dpp signaling at the dorsal midline controls dorso-ventral patterning within the tergite by promoting pigmentation in the medial region.

  14. 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

    Stimulation of GH receptors leads to rapid activation of Jak2 kinase and subsequent tyrosine phosphorylation of the GH receptor. Three specific tyrosines located in the C-terminal domain of the GH receptor have been identified as being involved in GH-stimulated transcription of the Spi 2.1 promoter....... 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...

  15. Autocrine production of TGF-β confers resistance to apoptosis after an epithelial-mesenchymal transition process in hepatocytes: Role of EGF receptor ligands

    International Nuclear Information System (INIS)

    Castillo, Gaelle del; Murillo, Miguel M.; Alvarez-Barrientos, Alberto; Bertran, Esther; Fernandez, Margarita; Sanchez, Aranzazu; Fabregat, Isabel

    2006-01-01

    Transforming growth factor-beta (TGF-β) induces apoptosis in fetal rat hepatocytes. However, a subpopulation of these cells survives, concomitant with changes in phenotype, reminiscent of an epithelial-mesenchymal transition (EMT). We have previously suggested that EMT might confer cell resistance to apoptosis (Valdes et al., Mol. Cancer Res., 1: 68-78, 2002). However, the molecular mechanisms responsible for this resistance are not explored yet. In this work, we have isolated and subcultured the population of hepatocytes that suffered the EMT process and are resistant to apoptosis (TGF-β-treated fetal hepatocytes: TβT-FH). We prove that they secrete mitogenic and survival factors, as analyzed by the proliferative and survival capacity of conditioned medium. Inhibition of the epidermal growth factor receptor (EGFR) sensitizes TβT-FH to die after serum withdrawal. TβT-FH expresses high levels of transforming growth factor-alpha (TGF-α) and heparin-binding EGF-like growth factor (HB-EGF) and shows constitutive activation of the EGFR pathway. A blocking anti-TGF-α antibody restores the capacity of cells to die. TGF-β, which is expressed by TβT-FH, mediates up-regulation of TGF-α and HB-EGF expression in those cells. In summary, results suggest that an autocrine loop of TGF-β confers resistance to apoptosis after an EMT process in hepatocytes, through the increase in the expression of EGFR ligands

  16. Structure of the EGF receptor transactivation circuit integrates multiple signals with cell context

    Energy Technology Data Exchange (ETDEWEB)

    Joslin, Elizabeth J.; Shankaran, Harish; Opresko, Lee K.; Bollinger, Nikki; Lauffenburger, Douglas A.; Wiley, H. S.

    2010-05-10

    Transactivation of the epidermal growth factor receptor (EGFR) has been proposed to be a mechanism by which a variety of cellular inputs can be integrated into a single signaling pathway, but the regulatory topology of this important system is unclear. To understand the transactivation circuit, we first created a “non-binding” reporter for ligand shedding. We then quantitatively defined how signals from multiple agonists were integrated both upstream and downstream of the EGFR into the extracellular signal regulated kinase (ERK) cascade in human mammary epithelial cells. We found that transactivation is mediated by a recursive autocrine circuit where ligand shedding drives EGFR-stimulated ERK that in turn drives further ligand shedding. The time from shedding to ERK activation is fast (<5 min) whereas the recursive feedback is slow (>15 min). Simulations showed that this delay in positive feedback greatly enhanced system stability and robustness. Our results indicate that the transactivation circuit is constructed so that the magnitude of ERK signaling is governed by the sum of multiple direct inputs, while recursive, autocrine ligand shedding controls signal duration.

  17. Endophilin, Lamellipodin, and Mena cooperate to regulate F-actin-dependent EGF-receptor endocytosis.

    Science.gov (United States)

    Vehlow, Anne; Soong, Daniel; Vizcay-Barrena, Gema; Bodo, Cristian; Law, Ah-Lai; Perera, Upamali; Krause, Matthias

    2013-10-16

    The epidermal growth factor receptor (EGFR) plays an essential role during development and diseases including cancer. Lamellipodin (Lpd) is known to control lamellipodia protrusion by regulating actin filament elongation via Ena/VASP proteins. However, it is unknown whether this mechanism supports endocytosis of the EGFR. Here, we have identified a novel role for Lpd and Mena in clathrin-mediated endocytosis (CME) of the EGFR. We have discovered that endogenous Lpd is in a complex with the EGFR and Lpd and Mena knockdown impairs EGFR endocytosis. Conversely, overexpressing Lpd substantially increases the EGFR uptake in an F-actin-dependent manner, suggesting that F-actin polymerization is limiting for EGFR uptake. Furthermore, we found that Lpd directly interacts with endophilin, a BAR domain containing protein implicated in vesicle fission. We identified a role for endophilin in EGFR endocytosis, which is mediated by Lpd. Consistently, Lpd localizes to clathrin-coated pits (CCPs) just before vesicle scission and regulates vesicle scission. Our findings suggest a novel mechanism in which Lpd mediates EGFR endocytosis via Mena downstream of endophilin.

  18. Protein kinase Cδ signaling downstream of the EGF receptor mediates migration and invasiveness of prostate cancer cells

    International Nuclear Information System (INIS)

    Kharait, Sourabh; Dhir, Rajiv; Lauffenburger, Douglas; Wells, Alan

    2006-01-01

    Tumor progression to the invasive phenotype occurs secondary to upregulated signaling from growth factor receptors that drive key cellular responses like proliferation, migration, and invasion. We hypothesized that Protein kinase Cδ (PKCδ)-mediated transcellular contractility is required for migration and invasion of prostate tumor cells. Two invasive human prostate cancer cell lines, DU145 cells overexpressing wildtype human EGFR (DU145WT) and PC3 cells, were studied. PKCδ is overexpressed in these cells relative to normal prostate epithelial cells, and is activated downstream of EGFR leading to cell motility via modulation of myosin light chain activity. Abrogation of PKCδ using Rottlerin and specific siRNA significantly decreased migration and invasion of both cell lines in vitro. Both PKCδ and phosphorylated PKCδ protein levels were higher in human prostate cancer tissue relative to normal donor prostate as assessed by Western blotting and immunohistochemistry. Thus, we conclude that PKCδ inhibition can limit migration and invasion of prostate cancer cells

  19. Inhibition of the Ras-ERK pathway in mitotic COS7 cells is due to the inability of EGFR/Raf to transduce EGF signaling to downstream proteins.

    Science.gov (United States)

    Shi, Huaiping; Zhang, Tianying; Yi, Yongqing; Ma, Yue

    2016-06-01

    Although previous studies have shown that Ras-ERK signaling in mitosis is closed due to the inhibition of signal transduction, the events involved in the molecular mechanisms are still unclear. In the present study, we investigated the Ras-ERK signaling pathway in mitotic COS7 cells. The results demonstrated that treatment with epidermal growth factor (EGF) failed to increase the endocytosis of EGF-EGFR (EGF receptor) complexes in mitotic COS7 cells, although a large amount of endosomes were found in asynchronous COS7 cells. Clathrin expression levels in mitotic COS7 cells were inhibited whereas caveolin expression levels in mitotic COS7 cells were almost unaffected. Y1068 and Y1086 residues of EGFR in the mitotic COS7 cells were activated. However, Grb2 and Shc in the mitotic COS7 cells did not bind to activated EGFR. Ras activity was inhibited in the mitotic COS7 cells whereas its downstream protein, Raf, was obviously phosphorylated by EGF in mitosis. Treatment with phorbol 12-myristate 13-acetate (PMA) also increased the phosphorylation levels of Raf in the mitotic COS7 cells. Nevertheless, Raf phosphorylation in mitosis was significantly inhibited by AG1478. Lastly, activation of EGF-mediated MEK and ERK in the mitotic COS7 cells was obviously inhibited. In summary, our results suggest that the Ras-ERK pathway is inhibited in mitotic COS7 cells which may be the dual result of the difficulty in the transduction of EGF signaling by EGFR or Raf to downstream proteins.

  20. G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs).

    Science.gov (United States)

    Li, Lingyong; Homan, Kristoff T; Vishnivetskiy, Sergey A; Manglik, Aashish; Tesmer, John J G; Gurevich, Vsevolod V; Gurevich, Eugenia V

    2015-04-24

    G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β2-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

    Science.gov (United States)

    Takayama, S; White, M F; Kahn, C R

    1988-03-05

    The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function

  2. Hemin inhibits internalization of transferrin by reticulocytes and promotes phosphorylation of the membrane transferrin receptor

    International Nuclear Information System (INIS)

    Cox, T.M.; O'Donnell, M.W.; Aisen, P.; London, I.M.

    1985-01-01

    Addition of hemin to reticulocytes inhibits incorporation of iron from transferrin. Heme also regulates protein synthesis in immature erythroid cells through its effects on phosphorylation of the initiation factor eIF-2. The authors have examined its effects on endocytosis of iron-transferrin and phosphorylation of the transferrin receptor. Hemin reduced iron transport but increased cell-associated transferrin. During uptake of 125 I-labeled transferrin in the steady state, the use of a washing technique to dissociate bound transferrin on the cell membrane showed that radioligand accumulated on the surface of hemin-treated cells. Receptor phosphorylation was investigated by immunoprecipitation of reticulocyte extracts after metabolic labeling with [ 32 P]P/sub i/. In the absence of ligand, phosphorylated receptor was chiefly localized on cell stroma. Exposure to transferrin increased cytosolic phosphorylated receptor from 15-30% to approximately 50% of the total, an effect overcome by hemin treatment. The findings suggest a possible relationship of phosphorylation to endocytosis of the transferrin receptor in reticulocytes

  3. Phorbol ester induced phosphorylation of the estrogen receptor in intact MCF-7 human breast cancer cells

    International Nuclear Information System (INIS)

    Knabbe, C.; Lippman, M.E.; Greene, G.L.; Dickson, R.B.

    1986-01-01

    Recent studies with a variety of cellular receptors have shown that phorbol ester induced phosphorylation modulates ligand binding and function. In this study the authors present direct evidence that the estrogen receptor in MCF-7 human breast cancer cells is a phosphoprotein whose phosphorylation state can be enhanced specifically by phorbol-12-myristate-13-acetate (PMA). Cells were cultured to 6h in the presence of [ 32 P]-orthophosphate. Whole cell extracts were immunoprecipitated with a monoclonal antibody (D58) against the estrogen receptor and subjected to SDS-polyacrylamide electrophoresis. Autoradiography showed a specific band in the region of 60-62 kDa which was significantly increased in preparations from PMA treated cells. Phospho-amino acid analysis demonstrated specific phosphorylation of serine and threonine residues. Cholera toxin or forskolin did not change the phosphorylation state of this protein. In a parallel binding analysis PMA led to a rapid decrease of estrogen binding sites. The estrogen induction of both progesterone receptors and growth in semisolid medium was blocked by PMA, whereas the estrogen induction of the 8kDa protein corresponding to the ps2 gene product and of the 52 kDa protein was not affected. In conclusion, phorbol esters can induce phosphorylation of the estrogen receptor. This process may be associated with the inactivation of certain receptor functions

  4. 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.

  5. Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation

    Directory of Open Access Journals (Sweden)

    Raj Kumar

    2008-08-01

    Full Text Available Raj Kumar1, William J Calhoun21Division of Gastroenterology; 2Division of Allergy, Pulmonary, Immunology, Critical Care, and Sleep (APICS, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, USAAbstract: Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade

  6. Phosphorylation of G Protein-Coupled Receptors: From the Barcode Hypothesis to the Flute Model.

    Science.gov (United States)

    Yang, Zhao; Yang, Fan; Zhang, Daolai; Liu, Zhixin; Lin, Amy; Liu, Chuan; Xiao, Peng; Yu, Xiao; Sun, Jin-Peng

    2017-09-01

    Seven transmembrane G protein-coupled receptors (GPCRs) are often phosphorylated at the C terminus and on intracellular loops in response to various extracellular stimuli. Phosphorylation of GPCRs by GPCR kinases and certain other kinases can promote the recruitment of arrestin molecules. The arrestins critically regulate GPCR functions not only by mediating receptor desensitization and internalization, but also by redirecting signaling to G protein-independent pathways via interactions with numerous downstream effector molecules. Accumulating evidence over the past decade has given rise to the phospho-barcode hypothesis, which states that ligand-specific phosphorylation patterns of a receptor direct its distinct functional outcomes. Our recent work using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance ( 19 F-NMR) spectroscopy led to the flute model, which provides preliminary insight into the receptor phospho-coding mechanism, by which receptor phosphorylation patterns are recognized by an array of phosphate-binding pockets on arrestin and are translated into distinct conformations. These selective conformations are recognized by various effector molecules downstream of arrestin. The phospho-barcoding mechanism enables arrestin to recognize a wide range of phosphorylation patterns of GPCRs, contributing to their diverse functions. Copyright © 2017 by The Author(s).

  7. Identification of steroid-binding and phosphorylated sites within the glucocorticoid receptor

    International Nuclear Information System (INIS)

    Smith, L.I.

    1989-01-01

    The primary goal of these studies was to localize the steroid-binding and phosphorylated sites of the glucocorticoid receptor. The synthetic steroid, dexamethasone 21-mesylate (DM) forms a covalent thioether bond via the sulfhydryl group of a cysteine residue in the receptor. To determine the covalent site of attachment of this ligand, receptors in WEHI-7 mouse thymoma cells were labeled with [ 3 H]DM and purified with a monoclonal antibody. The receptor was completely digested with trypsin and a single peptide covalently labeled with steroid identified by reversed-phase HPLC. This peptide was analyzed by automated Edman degradation to determine the location of the steroid-labeled residue. A similar analysis was performed on an overlapping peptide produced by Staphylococcus aureus protease digestion. Analysis of tryptic peptides from receptors labeled with both [ 3 H]DM and L-[ 35 S]methionine indicated that this peptide contained methionine. These analyses, coupled with the published amino acid sequence of the receptor, identified Cysteine-644 in the steroid-binding domain of the mouse glucocorticoid receptor as the residue involved in covalent steroid-binding. A synthetic peptide representing amino acids 640-650 of the mouse receptor was prepared and analyzed to confirm the identification. These biochemical studies represent a direct demonstration of an amino acid important in receptor function. It has been proposed that the receptor functions through a phosphorylation-dephosphorylation cycle to explain the dependence of hormone binding capacity upon cellular ATP. The glucocorticoid receptor has been shown to be a phosphoprotein. As an initial step to identifying a role of phosphorylation in receptor action, phosphorylated sites within the functional domains of the protein were identified

  8. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is increased in osteoarthritis and regulates chondrocyte catabolic and anabolic activities

    Science.gov (United States)

    Long, D.L.; Ulici, V.; Chubinskaya, S.; Loeser, R.F.

    2015-01-01

    Objective We determined if the epidermal growth factor receptor ligand HB-EGF is produced in cartilage and if it regulates chondrocyte anabolic or catabolic activity. Methods HB-EGF expression was measured by quantitative PCR using RNA isolated from mouse knee joint tissues and from normal and OA human chondrocytes. Immunohistochemistry was performed on normal and OA human cartilage and meniscus sections. Cultured chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus and osteogenic protein 1 (OP-1) as an anabolic stimulus. Effects of HB-EGF on cell signaling were analyzed by immunoblotting of selected signaling proteins. MMP-13 was measured in conditioned media, proteoglycan synthesis was measured by sulfate incorporation, and matrix gene expression by quantitative PCR. Results HB-EGF expression was increased in 12-month old mice at 8 weeks after surgery to induce OA and increased amounts of HB-EGF were noted in human articular cartilage from OA knees. FN-f stimulated chondrocyte HB-EGF expression and HB-EGF stimulated chondrocyte MMP-13 production. However, HB-EGF was not required for FN-f stimulation of MMP-13 production. HB-EGF activated the ERK and p38 MAP kinases and stimulated phosphorylation of Smad1 at an inhibitory serine site which was associated with inhibition of OP-1 mediated proteoglycan synthesis and reduced aggrecan (ACAN) but not COL2A1 expression. Conclusion HB-EGF is a new factor identified in OA cartilage that promotes chondrocyte catabolic activity while inhibiting anabolic activity suggesting it could contribute to the catabolic-anabolic imbalance seen in OA cartilage. PMID:25937027

  9. Kinome analysis of receptor-induced phosphorylation in human natural killer cells.

    Directory of Open Access Journals (Sweden)

    Sebastian König

    Full Text Available BACKGROUND: Natural killer (NK cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244 and DNAM-1 (CD226, act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome are involved in NK cell activation. RESULTS: A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2, FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated. CONCLUSIONS: The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses.

  10. 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.

  11. The role of glucocorticoid receptor phosphorylation in Mcl-1 and NOXA gene expression

    Directory of Open Access Journals (Sweden)

    Demonacos Constantinos

    2010-02-01

    Full Text Available Abstract Background The cyclin-dependent kinase (CDK and mitogen-activated protein kinase (MAPK mediated phosphorylation of glucocorticoid receptor (GR exerts opposite effects on GR transcriptional activity and affects other posttranslational modifications within this protein. The major phosphorylation site of human GR targeted by MAPK family is the serine 226 and multiple kinase complexes phosphorylate receptor at the serine 211 residue. We hypothesize that GR posttranslational modifications are involved in the determination of the cellular fate in human lymphoblastic leukemia cells. We investigated whether UV signalling through alternative GR phosphorylation determined the cell type specificity of glucocorticoids (GCs mediated apoptosis. Results We have identified putative Glucocorticoid Response Elements (GREs within the promoter regulatory regions of the Bcl-2 family members NOXA and Mcl-1 indicating that they are direct GR transcriptional targets. These genes were differentially regulated in CEM-C7-14, CEM-C1-15 and A549 cells by glucocorticoids and JNK pathway. In addition, our results revealed that the S211 phosphorylation was dominant in CEM-C7-14, whereas the opposite was the case in CEM-C1-15 where prevalence of S226 GR phosphorylation was observed. Furthermore, multiple GR isoforms with cell line specific patterns were identified in CEM-C7-14 cells compared to CEM-C1-15 and A549 cell lines with the same antibodies. Conclusions GR phosphorylation status kinetics, and site specificity as well as isoform variability differ in CEM-C7-14, CEM-C1-15, and A549 cells. The positive or negative response to GCs induced apoptosis in these cell lines is a consequence of the variable equilibrium of NOXA and Mcl-1 gene expression potentially mediated by alternatively phosphorylated GR, as well as the balance of MAPK/CDK pathways controlling GR phosphorylation pattern. Our results provide molecular base and valuable knowledge for improving the GC

  12. Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation

    Energy Technology Data Exchange (ETDEWEB)

    Faria, Jerusa A.Q.A.; Andrade, Carolina de; Goes, Alfredo M. [Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil); Rodrigues, Michele A. [Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil); Department of General Pathology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil); Gomes, Dawidson A., E-mail: dawidson@ufmg.br [Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil)

    2016-09-09

    The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. - Highlights: • EGF, HB-EGF, TGF-α, β-Cellulin are involved in the EGFR nuclear translocation. • Amphiregulin and epiregulin did not promote nuclear translocation of EGFR. • EGF, HB-EGF, TGF-α and β-Cellulin have a role in SkHep-1 cells migration. • EGFR ligands associated with better prognosis don't stimulate EGFR translocation.

  13. Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation

    International Nuclear Information System (INIS)

    Faria, Jerusa A.Q.A.; Andrade, Carolina de; Goes, Alfredo M.; Rodrigues, Michele A.; Gomes, Dawidson A.

    2016-01-01

    The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. - Highlights: • EGF, HB-EGF, TGF-α, β-Cellulin are involved in the EGFR nuclear translocation. • Amphiregulin and epiregulin did not promote nuclear translocation of EGFR. • EGF, HB-EGF, TGF-α and β-Cellulin have a role in SkHep-1 cells migration. • EGFR ligands associated with better prognosis don't stimulate EGFR translocation.

  14. The biological activity of the human epidermal growth factor receptor is positively regulated by its C-terminal tyrosines

    DEFF Research Database (Denmark)

    Helin, K; Velu, T; Martin, P

    1991-01-01

    mutants in the full length receptor. EGF-dependent transforming ability of the single point mutants is similar to that of the wild type, while that of double mutants is decreased and an even lower activity is present in the triple mutant. In each bioassay, including EGF-dependent focal transformation...... biologically. The EGF-R kinase activity is affected by tyrosine substitution since in vitro phosphorylation of exogenous substrates is reduced in the double and triple mutants. Autophosphorylation, in vivo and in vitro, is also reduced, but not totally abolished in the triple point mutant and Dc123 indicating......The epidermal growth factor receptor (EGF-R) C-terminus contains three conserved tyrosines (Y-1068, Y-1148, Y-1173) which are phosphorylated upon EGF activation. To clarify the functional role of these tyrosines, each has been mutated to phenylalanine and studied as single, double and triple...

  15. Interaction of the phosphorylated DNA-binding domain in nuclear receptor CAR with its ligand-binding domain regulates CAR activation.

    Science.gov (United States)

    Shizu, Ryota; Min, Jungki; Sobhany, Mack; Pedersen, Lars C; Mutoh, Shingo; Negishi, Masahiko

    2018-01-05

    The nuclear protein constitutive active/androstane receptor (CAR or NR1I3) regulates several liver functions such as drug and energy metabolism and cell growth or death, which are often involved in the development of diseases such as diabetes and hepatocellular carcinoma. CAR undergoes a conversion from inactive homodimers to active heterodimers with retinoid X receptor α (RXRα), and phosphorylation of the DNA-binding domain (DBD) at Thr-38 in CAR regulates this conversion. Here, we uncovered the molecular mechanism by which this phosphorylation regulates the intramolecular interaction between CAR's DBD and ligand-binding domain (LBD), enabling the homodimer-heterodimer conversion. Phosphomimetic substitution of Thr-38 with Asp increased co-immunoprecipitation of the CAR DBD with CAR LBD in Huh-7 cells. Isothermal titration calorimetry assays also revealed that recombinant CAR DBD-T38D, but not nonphosphorylated CAR DBD, bound the CAR LBD peptide. This DBD-LBD interaction masked CAR's dimer interface, preventing CAR homodimer formation. Of note, EGF signaling weakened the interaction of CAR DBD T38D with CAR LBD, converting CAR to the homodimer form. The DBD-T38D-LBD interaction also prevented CAR from forming a heterodimer with RXRα. However, this interaction opened up a CAR surface, allowing interaction with protein phosphatase 2A. Thr-38 dephosphorylation then dissociated the DBD-LBD interaction, allowing CAR heterodimer formation with RXRα. We conclude that the intramolecular interaction of phosphorylated DBD with the LBD enables CAR to adapt a transient monomer configuration that can be converted to either the inactive homodimer or the active heterodimer.

  16. Phosphorylation and Internalization of Lysophosphatidic Acid Receptors LPA1, LPA2, and LPA3.

    Directory of Open Access Journals (Sweden)

    Rocío Alcántara-Hernández

    Full Text Available The lysophosphatidic acid receptors LPA1, LPA2, and LPA3 were individually expressed in C9 cells and their signaling and regulation were studied. Agonist-activation increases intracellular calcium concentration in a concentration-dependent fashion. Phorbol myristate acetate markedly inhibited LPA1- and LPA3-mediated effect, whereas that mediated by LPA2 was only partially diminished; the actions of the phorbol ester were inhibited by bisindolylmaleimide I and by overnight incubation with the protein kinase C activator, which leads to down regulation of this protein kinase. Homologous desensitization was also observed for the three LPA receptors studied, with that of LPA2 receptors being consistently of lesser magnitude; neither inhibition nor down-regulation of protein kinase C exerted any effect on homologous desensitization. Activation of LPA1-3 receptors induced ERK 1/2 phosphorylation; this effect was markedly attenuated by inhibition of epidermal growth factor receptor tyrosine kinase activity, suggesting growth factor receptor transactivation in this effect. Lysophosphatidic acid and phorbol myristate acetate were able to induce LPA1-3 phosphorylation, in time- and concentration-dependent fashions. It was also clearly observed that agonists and protein kinase C activation induced internalization of these receptors. Phosphorylation of the LPA2 subtype required larger concentrations of these agents and its internalization was less intense than that of the other subtypes.Our data show that these three LPA receptors are phosphoproteins whose phosphorylation state is modulated by agonist-stimulation and protein kinase C-activation and that differences in regulation and cellular localization exist, among the subtypes.

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

    DEFF Research Database (Denmark)

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

    1995-01-01

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

  18. Understanding the Differential Selectivity of Arrestins toward the Phosphorylation State of the Receptor

    NARCIS (Netherlands)

    Sensoy, Ozge; de Sousa Moreira, Irina; Morra, Giulia

    2016-01-01

    Proteins in the arrestin family exhibit a conserved structural fold that nevertheless allows for significant differences in their selectivity for G-protein coupled receptors (GPCRs) and their phosphorylation states. To reveal the mechanism of activation that prepares arrestin for selective

  19. Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.

    Science.gov (United States)

    Chen, Chongguang; Chiu, Yi-Ting; Wu, Wenman; Huang, Peng; Mann, Anika; Schulz, Stefan; Liu-Chen, Lee-Yuan

    2016-02-15

    Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated

  20. Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion

    International Nuclear Information System (INIS)

    Shimura, Takaya; Higashiyama, Shigeki; Joh, Takashi; Yoshida, Michihiro; Fukuda, Shinji; Ebi, Masahide; Hirata, Yoshikazu; Mizoshita, Tsutomu; Tanida, Satoshi; Kataoka, Hiromi; Kamiya, Takeshi

    2012-01-01

    Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR) ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF) after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C), translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF) and mutated HB-EGF (HB-EGF-mC), which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 %) and in the cytoplasm only in 25 cases (26.0 %). The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P < 0.01). The growth of wt-HB-EGF- and HB-EGF-mC-expressing cells significantly increased compared with control cells, but the growth of HB-EGF-mC-expressing cells was significantly decreased compared with wt-HB-EGF-expressing cells. Gastric cancer cell invasion obviously increased in wt-HB-EGF-expressing cells, but invasion in HB-EGF-mC-expressing cells showed a slight increase compared with control cells. Moreover, wt-HB-EGF overexpression increased the effectiveness of wound healing, but had no significant effect in HB-EGF-mC-expressing cells. Both the function of HB-EGF as an EGFR ligand and a novel signal for

  1. Phosphorylation inhibits DNA-binding of alternatively spliced aryl hydrocarbon receptor nuclear translocator

    International Nuclear Information System (INIS)

    Kewley, Robyn J.; Whitelaw, Murray L.

    2005-01-01

    The basic helix-loop-helix/PER-ARNT-SIM homology (bHLH/PAS) transcription factor ARNT (aryl hydrocarbon receptor nuclear translocator) is a key component of various pathways which induce the transcription of cytochrome P450 and hypoxia response genes. ARNT can be alternatively spliced to express Alt ARNT, containing an additional 15 amino acids immediately N-terminal to the DNA-binding basic region. Here, we show that ARNT and Alt ARNT proteins are differentially phosphorylated by protein kinase CKII in vitro. Phosphorylation had an inhibitory effect on DNA-binding to an E-box probe by Alt ARNT, but not ARNT, homodimers. This inhibitory phosphorylation occurs through Ser77. Moreover, a point mutant, Alt ARNT S77A, shows increased activity on an E-box reporter gene, consistent with Ser77 being a regulatory site in vivo. In contrast, DNA binding by an Alt ARNT/dioxin receptor heterodimer to the xenobiotic response element is not inhibited by phosphorylation with CKII, nor does Alt ARNT S77A behave differently from wild type Alt ARNT in the context of a dioxin receptor heterodimer

  2. Developmental defects in zebrafish for classification of EGF pathway inhibitors

    International Nuclear Information System (INIS)

    Pruvot, Benoist; Curé, Yoann; Djiotsa, Joachim; Voncken, Audrey; Muller, Marc

    2014-01-01

    One of the major challenges when testing drug candidates targeted at a specific pathway in whole animals is the discrimination between specific effects and unwanted, off-target effects. Here we used the zebrafish to define several developmental defects caused by impairment of Egf signaling, a major pathway of interest in tumor biology. We inactivated Egf signaling by genetically blocking Egf expression or using specific inhibitors of the Egf receptor function. We show that the combined occurrence of defects in cartilage formation, disturbance of blood flow in the trunk and a decrease of myelin basic protein expression represent good indicators for impairment of Egf signaling. Finally, we present a classification of known tyrosine kinase inhibitors according to their specificity for the Egf pathway. In conclusion, we show that developmental indicators can help to discriminate between specific effects on the target pathway from off-target effects in molecularly targeted drug screening experiments in whole animal systems. - Highlights: • We analyze the functions of Egf signaling on zebrafish development. • Genetic blocking of Egf expression causes cartilage, myelin and circulatory defects. • Chemical inhibition of Egf receptor function causes similar defects. • Developmental defects can reveal the specificity of Egf pathway inhibitors

  3. Developmental defects in zebrafish for classification of EGF pathway inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Pruvot, Benoist; Curé, Yoann; Djiotsa, Joachim; Voncken, Audrey; Muller, Marc, E-mail: m.muller@ulg.ac.be

    2014-01-15

    One of the major challenges when testing drug candidates targeted at a specific pathway in whole animals is the discrimination between specific effects and unwanted, off-target effects. Here we used the zebrafish to define several developmental defects caused by impairment of Egf signaling, a major pathway of interest in tumor biology. We inactivated Egf signaling by genetically blocking Egf expression or using specific inhibitors of the Egf receptor function. We show that the combined occurrence of defects in cartilage formation, disturbance of blood flow in the trunk and a decrease of myelin basic protein expression represent good indicators for impairment of Egf signaling. Finally, we present a classification of known tyrosine kinase inhibitors according to their specificity for the Egf pathway. In conclusion, we show that developmental indicators can help to discriminate between specific effects on the target pathway from off-target effects in molecularly targeted drug screening experiments in whole animal systems. - Highlights: • We analyze the functions of Egf signaling on zebrafish development. • Genetic blocking of Egf expression causes cartilage, myelin and circulatory defects. • Chemical inhibition of Egf receptor function causes similar defects. • Developmental defects can reveal the specificity of Egf pathway inhibitors.

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  5. High epiregulin expression in human U87 glioma cells relies on IRE1α and promotes autocrine growth through EGF receptor

    International Nuclear Information System (INIS)

    Auf, Gregor; Vajkoczy, Peter; Seno, Masaharu; Bikfalvi, Andreas; Minchenko, Dmitri; Minchenko, Oleksandr; Moenner, Michel; Jabouille, Arnaud; Delugin, Maylis; Guérit, Sylvaine; Pineau, Raphael; North, Sophie; Platonova, Natalia; Maitre, Marlène; Favereaux, Alexandre

    2013-01-01

    Epidermal growth factor (EGF) receptors contribute to the development of malignant glioma. Here we considered the possible implication of the EGFR ligand epiregulin (EREG) in glioma development in relation to the activity of the unfolded protein response (UPR) sensor IRE1α. We also examined EREG status in several glioblastoma cell lines and in malignant glioma. Expression and biological properties of EREG were analyzed in human glioma cells in vitro and in human tumor xenografts with regard to the presence of ErbB proteins and to the blockade of IRE1α. Inactivation of IRE1α was achieved by using either the dominant-negative strategy or siRNA-mediated knockdown. EREG was secreted in high amounts by U87 cells, which also expressed its cognate EGF receptor (ErbB1). A stimulatory autocrine loop mediated by EREG was evidenced by the decrease in cell proliferation using specific blocking antibodies directed against either ErbB1 (cetuximab) or EREG itself. In comparison, anti-ErbB2 antibodies (trastuzumab) had no significant effect. Inhibition of IRE1α dramatically reduced EREG expression both in cell culture and in human xenograft tumor models. The high-expression rate of EREG in U87 cells was therefore linked to IRE1α, although being modestly affected by chemical inducers of the endoplasmic reticulum stress. In addition, IRE1-mediated production of EREG did not depend on IRE1 RNase domain, as neither the selective dominant-negative invalidation of the RNase activity (IRE1 kinase active) nor the siRNA-mediated knockdown of XBP1 had significant effect on EREG expression. Finally, chemical inhibition of c-Jun N-terminal kinases (JNK) using the SP600125 compound reduced the ability of cells to express EREG, demonstrating a link between the growth factor production and JNK activation under the dependence of IRE1α. EREG may contribute to glioma progression under the control of IRE1α, as exemplified here by the autocrine proliferation loop mediated in U87 cells by the

  6. Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers.

    Science.gov (United States)

    Iwamoto, Ryo; Takagi, Mika; Akatsuka, Jun-Ichi; Ono, Ken-Ichiro; Kishi, Yoshiro; Mekada, Eisuke

    2016-04-01

    Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.

  7. Exploring in vivo cholesterol-mediated interactions between activated EGF receptors in plasma membrane with single-molecule optical tracking

    International Nuclear Information System (INIS)

    Lin, Chien Y.; Huang, Jung Y.; Lo, Leu-Wei

    2016-01-01

    The first step in many cellular signaling processes occurs at various types of receptors in the plasma membrane. Membrane cholesterol can alter these signaling pathways of living cells. However, the process in which the interaction of activated receptors is modulated by cholesterol remains unclear. In this study, we measured single-molecule optical trajectories of epidermal growth factor receptors moving in the plasma membranes of two cancerous cell lines and one normal endothelial cell line. A stochastic model was developed and applied to identify critical information from single-molecule trajectories. We discovered that unliganded epidermal growth factor receptors may reside nearby cholesterol-riched regions of the plasma membrane and can move into these lipid domains when subjected to ligand binding. The amount of membrane cholesterol considerably affects the stability of correlated motion of activated epidermal growth factor receptors. Our results provide single-molecule evidence of membrane cholesterol in regulating signaling receptors. Because the three cell lines used for this study are quite diverse, our results may be useful to shed light on the mechanism of cholesterol-mediated interaction between activated receptors in live cells

  8. A Role for Estrogen Receptor Phosphorylation in the Resistance to Tamoxifen

    International Nuclear Information System (INIS)

    De Leeuw, R.; Neefjes, J.; Michalides, R.

    2011-01-01

    About two thirds of all human breast cancer cases are estrogen receptor positive. The drug of first choice for these patients is tamoxifen. However, about half of the recurrences after removal of the primary tumor are or become resistant to this drug. While many mechanisms have been identified for tamoxifen resistance in the lab, at present only a few have been translated to the clinic. This paper highlights the role in tamoxifen resistance of phosphorylation by different kinases on different sites of the estrogen receptor. We will discuss the molecular pathways and kinases that are involved in phosphorylation of ERa and how these affect tamoxifen resistance. Finally, we will elaborate on the clinical translation of these observations and the possibility to predict tamoxifen responses in patient tumor samples before treatment onset. The findings made originally on the bench may translate into a better and personalized treatment of breast cancer patients using an old and safe anticancer drug: tamoxifen

  9. Epidermal growth factor treatment of A431 cells alters the binding capacity and electrophoretic mobility of the cytoskeletally associated epidermal growth factor receptor

    International Nuclear Information System (INIS)

    Roy, L.M.; Gittinger, C.K.; Landreth, G.E.

    1991-01-01

    Epidermal growth factor receptor interacts with structural elements of A431 cells and remains associated with the cytoskeleton following extraction with nonionic detergents. Extraction of cells with 0.15% Triton X-100 resulted in detection of only approximately 40% of the EGF binding sites on the cytoskeleton. If the cells were exposed to EGF prior to extraction, approximately twofold higher levels of low-affinity EGF binding sites were detected. The difference in number of EGF binding sites was not a consequence of differences in numbers of EGF receptors associated with the cytoskeleton; equal amounts of 35S-labeled receptor were immunoprecipitated from the cytoskeletons of both control and EGF-treated cells. The effect of EGF pretreatment on binding activity was coincident with a change in the mobility of the receptor from a doublet of Mr approximately 160-180 kDa to a single sharp band at 180 kDa. The alteration in receptor mobility was not a simple consequence of receptor phosphorylation in that the alteration was not reversed by alkaline phosphatase treatment, nor was the shift produced by treatment of the cells with phorbol ester. The two EGF receptor species demonstrated differential susceptibility to V8 proteinase digestion. The EGF-induced 180 kDa species was preferentially digested by the proteinase relative to the 160 kDa species, indicating that EGF binding results in a conformational change in the receptor. The EGF-mediated preservation of binding activity and altered conformation may be related to receptor oligomerization

  10. Noradrenaline, oxymetazoline and phorbol myristate acetate induce distinct functional actions and phosphorylation patterns of α1A-adrenergic receptors.

    Science.gov (United States)

    Alcántara-Hernández, Rocío; Hernández-Méndez, Aurelio; Romero-Ávila, M Teresa; Alfonzo-Méndez, Marco A; Pupo, André S; García-Sáinz, J Adolfo

    2017-12-01

    In LNCaP cells that stably express α 1A -adrenergic receptors, oxymetazoline increased intracellular calcium and receptor phosphorylation, however, this agonist was a weak partial agonist, as compared to noradrenaline, for calcium signaling. Interestingly, oxymetazoline-induced receptor internalization and desensitization displayed greater effects than those induced by noradrenaline. Phorbol myristate acetate induced modest receptor internalization and minimal desensitization. α 1A -Adrenergic receptor interaction with β-arrestins (colocalization/coimmunoprecipitation) was induced by noradrenaline and oxymetazoline and, to a lesser extent, by phorbol myristate acetate. Oxymetazoline was more potent and effective than noradrenaline in inducing ERK 1/2 phosphorylation. Mass spectrometric analysis of immunopurified α 1A -adrenergic receptors from cells treated with adrenergic agonists and the phorbol ester clearly showed that phosphorylated residues were present both at the third intracellular loop and at the carboxyl tail. Distinct phosphorylation patterns were observed under the different conditions. The phosphorylated residues were: a) Baseline and all treatments: T233; b) noradrenaline: S220, S227, S229, S246, S250, S389; c) oxymetazoline: S227, S246, S381, T384, S389; and d) phorbol myristate acetate: S246, S250, S258, S351, S352, S401, S402, S407, T411, S413, T451. Our novel data, describing the α 1A -AR phosphorylation sites, suggest that the observed different phosphorylation patterns may participate in defining adrenoceptor localization and action, under the different conditions examined. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. SRC-mediated EGF Receptor Activation Regulates Ozone-induced Interleukin 8 Expression in Human Bronchial Epithelial Cells

    Science.gov (United States)

    BACKGROUND: Human exposure to ozone (03) results in pulmonary function decrements and airway inflammation. The mechanisms underlying these adverse effects remain unclear. Epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of lung inflammation. ...

  12. Phosphorylation of the dimeric cytoplasmic domain of the phytosulfokine receptor, PSKR1

    KAUST Repository

    Muleya, V.; Marondedze, Claudius; Wheeler, J. I.; Thomas, Ludivine; Mok, Y.-F.; Griffin, M. D. W.; Manallack, D. T.; Kwezi, L.; Lilley, K. S.; Gehring, Christoph A; Irving, H. R.

    2016-01-01

    Phytosulfokines (PSKs) are plant peptide hormones that co-regulate plant growth, differentiation and defense responses. PSKs signal through a plasma membrane localized leucine-rich repeat receptor-like kinase (phytosulfokine receptor 1, PSKR1) that also contains a functional cytosolic guanylate cyclase with its cyclase catalytic center embedded within the kinase domain. To functionally characterize this novel type of overlapping dual catalytic function, we investigated the phosphorylation of PSKR1 in vitro Tandem mass spectrometry of the cytoplasmic domain of PSKR1 (PSKR1cd) revealed at least 11 phosphorylation sites (8 serines, 2 threonines and 1 tyrosine) within the PSKR1cd. Phosphomimetic mutations of three serine residues (Ser686, Ser696 and Ser698) in tandem at the juxta-membrane position resulted in enhanced kinase activity in the on-mutant that was suppressed in the off-mutant, but both mutations reduced guanylate cyclase activity. Both the on and off phosphomimetic mutations of the phosphotyrosine (Tyr888) residue in the activation loop suppressed kinase activity, while neither mutation affected guanylate cyclase activity. Size exclusion and analytical ultracentrifugation analysis of the PSKR1cd suggest that it is reversibly dimeric in solution, which was further confirmed by biflourescence complementation. Taken together, these data suggest that in this novel type of receptor domain architecture, specific phosphorylation and dimerization are possibly essential mechanisms for ligand-mediated catalysis and signaling.

  13. Phosphorylation of the dimeric cytoplasmic domain of the phytosulfokine receptor, PSKR1

    KAUST Repository

    Muleya, V.

    2016-08-04

    Phytosulfokines (PSKs) are plant peptide hormones that co-regulate plant growth, differentiation and defense responses. PSKs signal through a plasma membrane localized leucine-rich repeat receptor-like kinase (phytosulfokine receptor 1, PSKR1) that also contains a functional cytosolic guanylate cyclase with its cyclase catalytic center embedded within the kinase domain. To functionally characterize this novel type of overlapping dual catalytic function, we investigated the phosphorylation of PSKR1 in vitro Tandem mass spectrometry of the cytoplasmic domain of PSKR1 (PSKR1cd) revealed at least 11 phosphorylation sites (8 serines, 2 threonines and 1 tyrosine) within the PSKR1cd. Phosphomimetic mutations of three serine residues (Ser686, Ser696 and Ser698) in tandem at the juxta-membrane position resulted in enhanced kinase activity in the on-mutant that was suppressed in the off-mutant, but both mutations reduced guanylate cyclase activity. Both the on and off phosphomimetic mutations of the phosphotyrosine (Tyr888) residue in the activation loop suppressed kinase activity, while neither mutation affected guanylate cyclase activity. Size exclusion and analytical ultracentrifugation analysis of the PSKR1cd suggest that it is reversibly dimeric in solution, which was further confirmed by biflourescence complementation. Taken together, these data suggest that in this novel type of receptor domain architecture, specific phosphorylation and dimerization are possibly essential mechanisms for ligand-mediated catalysis and signaling.

  14. Endocytosis of G protein-coupled receptors is regulated by clathrin light chain phosphorylation.

    Science.gov (United States)

    Ferreira, Filipe; Foley, Matthew; Cooke, Alex; Cunningham, Margaret; Smith, Gemma; Woolley, Robert; Henderson, Graeme; Kelly, Eamonn; Mundell, Stuart; Smythe, Elizabeth

    2012-08-07

    Signaling by transmembrane receptors such as G protein-coupled receptors (GPCRs) occurs at the cell surface and throughout the endocytic pathway, and signaling from the cell surface may differ in magnitude and downstream output from intracellular signaling. As a result, the rate at which signaling molecules traverse the endocytic pathway makes a significant contribution to downstream output. Modulation of the core endocytic machinery facilitates differential uptake of individual cargoes. Clathrin-coated pits are a major entry portal where assembled clathrin forms a lattice around invaginating buds that have captured endocytic cargo. Clathrin assembles into triskelia composed of three clathrin heavy chains and associated clathrin light chains (CLCs). Despite the identification of clathrin-coated pits at the cell surface over 30 years ago, the functions of CLCs in endocytosis have been elusive. In this work, we identify a novel role for CLCs in the regulated endocytosis of specific cargoes. Small interfering RNA-mediated knockdown of either CLCa or CLCb inhibits the uptake of GPCRs. Moreover, we demonstrate that phosphorylation of Ser204 in CLCb is required for efficient endocytosis of a subset of GPCRs and identify G protein-coupled receptor kinase 2 (GRK2) as a kinase that can phosphorylate CLCb on Ser204. Overexpression of CLCb(S204A) specifically inhibits the endocytosis of those GPCRs whose endocytosis is GRK2-dependent. Together, these results indicate that CLCb phosphorylation acts as a discriminator for the endocytosis of specific GPCRs. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Integrated regulation of AMPA glutamate receptor phosphorylation in the striatum by dopamine and acetylcholine.

    Science.gov (United States)

    Xue, Bing; Chen, Elton C; He, Nan; Jin, Dao-Zhong; Mao, Li-Min; Wang, John Q

    2017-01-01

    Dopamine (DA) and acetylcholine (ACh) signals converge onto protein kinase A (PKA) in medium spiny neurons of the striatum to control cellular and synaptic activities of these neurons, although underlying molecular mechanisms are less clear. Here we measured phosphorylation of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) at a PKA site (S845) as an indicator of AMPAR responses in adult rat brains in vivo to explore how DA and ACh interact to modulate AMPARs. We found that subtype-selective activation of DA D1 receptors (D1Rs), D2 receptors (D2Rs), or muscarinic M4 receptors (M4Rs) induced specific patterns of GluA1 S845 responses in the striatum. These defined patterns support a local multitransmitter interaction model in which D2Rs inhibited an intrinsic inhibitory element mediated by M4Rs to enhance the D1R efficacy in modulating AMPARs. Consistent with this, selective enhancement of M4R activity by a positive allosteric modulator resumed the cholinergic inhibition of D1Rs. In addition, D1R and D2R coactivation recruited GluA1 and PKA preferentially to extrasynaptic sites. In sum, our in vivo data support an existence of a dynamic DA-ACh balance in the striatum which actively modulates GluA1 AMPAR phosphorylation and trafficking. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Coordinate phosphorylation of insulin-receptor kinase and its 175,000-Mr endogenous substrate in rat hepatocytes

    International Nuclear Information System (INIS)

    Okamoto, M.; Karasik, A.; White, M.F.; Kahn, C.R.

    1991-01-01

    To investigate the early events in insulin signal transmission in liver, isolated rat hepatocytes were labeled with 32 P, and proteins phosphorylated in response to insulin were detected by immunoprecipitation with anti-phosphotyrosine and anti-receptor antibodies and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and autoradiography. In these cells, insulin rapidly stimulated tyrosine phosphorylation of the 95,000-Mr beta-subunit of the insulin receptor and a 175,000-Mr phosphoprotein (pp175). Both proteins were precipitated by anti-phosphotyrosine antibody, whereas only the insulin receptor was recognized with anti-insulin-receptor antibody. In the insulin-stimulated state, both pp175 and the receptor beta-subunit were found to be phosphorylated on tyrosine and serine residues. Based on precipitation by the two antibodies, receptor phosphorylation was biphasic with an initial increase in tyrosine phosphorylation followed by a more gradual increase in serine phosphorylation over the first 30 min of stimulation. The time course of phosphorylation of pp175 was rapid and paralleled that of the beta-subunit of the insulin receptor. The pp175 was clearly distinguished from the insulin receptor, because it was detected only when boiling SDS was used to extract cellular phosphoproteins, whereas the insulin receptor was extracted with either Triton X-100 or SDS. In addition, the tryptic peptide maps of the two proteins were distinct. The dose-response curve for insulin stimulation was shifted slightly to the left of the insulin receptor, suggesting some signal amplification at this step. These data suggest that pp175 is a major endogenous substrate of the insulin receptor in liver and may be a cytoskeletal-associated protein

  17. Microfluidic generated EGF-gradients induce chemokinesis of transplantable retinal progenitor cells via the JAK/STAT and PI3kinase signaling pathways.

    Directory of Open Access Journals (Sweden)

    Uchenna J Unachukwu

    Full Text Available A growing number of studies are evaluating retinal progenitor cell (RPC transplantation as an approach to repair retinal degeneration and restore visual function. To advance cell-replacement strategies for a practical retinal therapy, it is important to define the molecular and biochemical mechanisms guiding RPC motility. We have analyzed RPC expression of the epidermal growth factor receptor (EGFR and evaluated whether exposure to epidermal growth factor (EGF can coordinate motogenic activity in vitro. Using Boyden chamber analysis as an initial high-throughput screen, we determined that RPC motility was optimally stimulated by EGF concentrations in the range of 20-400 ng/ml, with decreased stimulation at higher concentrations, suggesting concentration-dependence of EGF-induced motility. Using bioinformatics analysis of the EGF ligand in a retina-specific gene network pathway, we predicted a chemotactic function for EGF involving the MAPK and JAK-STAT intracellular signaling pathways. Based on targeted inhibition studies, we show that ligand binding, phosphorylation of EGFR and activation of the intracellular STAT3 and PI3kinase signaling pathways are necessary to drive RPC motility. Using engineered microfluidic devices to generate quantifiable steady-state gradients of EGF coupled with live-cell tracking, we analyzed the dynamics of individual RPC motility. Microfluidic analysis, including center of mass and maximum accumulated distance, revealed that EGF induced motility is chemokinetic with optimal activity observed in response to low concentration gradients. Our combined results show that EGFR expressing RPCs exhibit enhanced chemokinetic motility in the presence of low nanomole levels of EGF. These findings may serve to inform further studies evaluating the extent to which EGFR activity, in response to endogenous ligand, drives motility and migration of RPCs in retinal transplantation paradigms.

  18. Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function.

    Science.gov (United States)

    Bubeck Wardenburg, J; Fu, C; Jackman, J K; Flotow, H; Wilkinson, S E; Williams, D H; Johnson, R; Kong, G; Chan, A C; Findell, P R

    1996-08-16

    Two families of tyrosine kinases, the Src and Syk families, are required for T-cell receptor activation. While the Src kinases are responsible for phosphorylation of receptor-encoded signaling motifs and for up-regulation of ZAP-70 activity, the downstream substrates of ZAP-70 are unknown. Evidence is presented herein that the Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is a substrate of ZAP-70. Phosphorylation of SLP-76 is diminished in T cells that express a catalytically inactive ZAP-70. Moreover, SLP-76 is preferentially phosphorylated by ZAP-70 in vitro and in heterologous cellular systems. In T cells, overexpression of wild-type SLP-76 results in a hyperactive receptor, while expression of a SLP-76 molecule that is unable to be tyrosine-phosphorylated attenuates receptor function. In addition, the SH2 domain of SLP-76 is required for T-cell receptor function, although its role is independent of the ability of SLP-76 to undergo tyrosine phosphorylation. As SLP-76 interacts with both Grb2 and phospholipase C-gamma1, these data indicate that phosphorylation of SLP-76 by ZAP-70 provides an important functional link between the T-cell receptor and activation of ras and calcium pathways.

  19. Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion.

    Science.gov (United States)

    Shimura, Takaya; Yoshida, Michihiro; Fukuda, Shinji; Ebi, Masahide; Hirata, Yoshikazu; Mizoshita, Tsutomu; Tanida, Satoshi; Kataoka, Hiromi; Kamiya, Takeshi; Higashiyama, Shigeki; Joh, Takashi

    2012-05-30

    Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR) ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF) after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C), translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF) and mutated HB-EGF (HB-EGF-mC), which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 %) and in the cytoplasm only in 25 cases (26.0 %). The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P Gastric cancer cell invasion obviously increased in wt-HB-EGF-expressing cells, but invasion in HB-EGF-mC-expressing cells showed a slight increase compared with control cells. Moreover, wt-HB-EGF overexpression increased the effectiveness of wound healing, but had no significant effect in HB-EGF-mC-expressing cells. Both the function of HB-EGF as an EGFR ligand and a novel signal for HB-EGF-C nuclear translocation induce gastric cancer growth, whereas HB-EGF-C nuclear translocation independently plays a critical role in gastric cancer invasion. The present study demonstrated that HB-EGF-C nuclear translocation

  20. Heparin-Binding EGF-like Growth Factor (HB-EGF) stimulates the proliferation of Müller glia-derived progenitor cells in avian and murine retinas

    Science.gov (United States)

    Todd, Levi; Volkov, Leo I.; Zelinka, Chris; Squires, Natalie; Fischer, Andy J.

    2015-01-01

    Müller glia can be stimulated to de-differentiate, proliferate and form Müller glia-derived progenitor cells (MGPCs) that regenerate retinal neurons. In the zebrafish retina, Heparin-Binding EGF-like Growth Factor (HB-EGF) may be one of the key factors that stimulate the formation of proliferating MGPCs. Currently nothing is known about the influence of HB-EGF on the proliferative potential of Müller glia in retinas of birds and rodents. In the chick retina, we found that levels of both hb-egf and egf-receptor are rapidly and transiently up-regulated following NMDA-induced damage. Although intraocular injections of HB-EGF failed to stimulate cell-signaling or proliferation of Müller glia in normal retinas, HB-EGF stimulated proliferation of MGPCs in damaged retinas. By comparison, inhibition of the EGF-receptor (EGFR) decreased the proliferation of MGPCs in damaged retinas. HB-EGF failed to act synergistically with FGF2 to stimulate the formation of MGPCs in the undamaged retina and inhibition of EGF-receptor did not suppress FGF2-mediated formation of MGPCs. In the mouse retina, HB-EGF stimulated the proliferation of Müller glia following NMDA-induced damage. Furthermore, HB-EGF stimulated not only MAPK-signaling in Müller glia/MGPCs, but also activated mTor- and Jak/Stat-signaling. We propose that levels of expression of EGFR are rate-limiting to the responses of Müller glia to HB-EGF and the expression of EGFR can be induced by retinal damage, but not by FGF2-treatment. We conclude that HB-EGF is mitogenic to Müller glia in both chick and mouse retinas, and HB-EGF is an important player in the formation of MGPCs in damaged retinas. PMID:26500021

  1. Homologous desensitization of adenylate cyclase: the role of β-adrenergic receptor phosphorylation and dephosphorylation

    International Nuclear Information System (INIS)

    Sibley, D.R.; Strasser, R.H.; Daniel, K.; Lefkowitz, R.J.

    1986-01-01

    The authors utilized the frog erythrocyte (FE) as a β-adreneric receptor (βAR) model system in which to study homologous desensitization. Preincubation with isoproterenol (ISO) leads to a 50% decline in ISO-stimulated adenylate cyclase (AC) activity without significant changes in basal, PGE 1 -, NaF-, GppNHp-, forskolin-, or MnCl 2 -stimulated AC activities. ISO treatment also induces the sequestration of βAR from the cell surface as evidenced by a 35% decline in [ 3 H]CGP-12177 binding sites on the surface of intact FE. Treatment of intact FE with ISO also promotes βAR phosphorylation to 2 mol PO 4 /mol of βAR. At 25 0 C, the time courses of ISO-induced AC desensitization, βAR sequestration and βAR phosphorylation are identical occurring without a lag and exhibiting a t 1/2 of 30 min and a maximal response at 2.5 hrs. The sequestered βAR can be partially recovered upon cell lysis in a light membrane fraction (LMF), separable from the plasma membranes using sucrose gradients or differential centrifugation. βAR phosphorylation is reversed in the sequestered LMF exhibiting a PO 4 /βAR stoichiometry of 0.7 mol/mol - similar to that observed under basal conditions. These data suggest that phosphorylation of βAR in the plasma membrane promotes their translocation away from the cell surface into a sequestered membrane domain where the phosphorylation is reversed, thus, enabling the return of βAR back to the cell surface and recoupling with AC

  2. EGF receptor targeted lipo-oligocation polyplexes for antitumoral siRNA and miRNA delivery

    Science.gov (United States)

    Müller, Katharina; Klein, Philipp M.; Heissig, Philipp; Roidl, Andreas; Wagner, Ernst

    2016-11-01

    Antitumoral siRNA and miRNA delivery was demonstrated by epidermal growth factor receptor (EGFR) targeted oligoaminoamide polyplexes. For this purpose, the T-shaped lipo-oligomer 454 was used to complex RNA into a core polyplex, which was subsequently functionalized with the targeting peptide ligand GE11 via a polyethylene glycol (PEG) linker. To this end, free cysteines on the surface of 454 polyplex were coupled with a maleimide-PEG-GE11 reagent (Mal-GE11). Resulting particles with sizes of 120-150 nm showed receptor-mediated uptake into EGFR-positive T24 bladder cancer cells, MDA-MB 231 breast cancer cells and Huh7 liver cancer cells. Furthermore, these formulations led to ligand-dependent gene silencing. RNA interference (RNAi) triggered antitumoral effects were observed for two different therapeutic RNAs, a miRNA-200c mimic or EG5 siRNA. Using polyplexes modified with a ratio of 0.8 molar equivalents of Mal-GE11, treatment of T24 or MDA-MB 231 cancer cells with miR-200c led to the expected decreased proliferation and migration, changes in cell cycle and enhanced sensitivity towards doxorubicin. Delivery of EG5 siRNA into Huh7 cells resulted in antitumoral activity with G2/M arrest, triggered by loss of mitotic spindle separation and formation of mono-astral spindles. These findings demonstrate the potential of GE11 ligand-containing RNAi polyplexes for cancer treatment.

  3. Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium.

    LENUS (Irish Health Repository)

    Dooley, Ruth

    2012-02-01

    BACKGROUND: Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation. RESULTS: Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells. CONCLUSION: Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

  4. Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium

    LENUS (Irish Health Repository)

    Dooley, Ruth

    2011-08-22

    Abstract Background Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation. Results Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells. Conclusion Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

  5. Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium

    Directory of Open Access Journals (Sweden)

    Hatt Hanns

    2011-08-01

    Full Text Available Abstract Background Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation. Results Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells. Conclusion Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

  6. Lapatinib Plasma and Tumor Concentrations and Effects on HER Receptor Phosphorylation in Tumor.

    Directory of Open Access Journals (Sweden)

    Neil L Spector

    Full Text Available The paradigm shift in cancer treatment from cytotoxic drugs to tumor targeted therapies poses new challenges, including optimization of dose and schedule based on a biologically effective dose, rather than the historical maximum tolerated dose. Optimal dosing is currently determined using concentrations of tyrosine kinase inhibitors in plasma as a surrogate for tumor concentrations. To examine this plasma-tumor relationship, we explored the association between lapatinib levels in tumor and plasma in mice and humans, and those effects on phosphorylation of human epidermal growth factor receptors (HER in human tumors.Mice bearing BT474 HER2+ human breast cancer xenografts were dosed once or twice daily (BID with lapatinib. Drug concentrations were measured in blood, tumor, liver, and kidney. In a randomized phase I clinical trial, 28 treatment-naïve female patients with early stage HER2+ breast cancer received lapatinib 1000 or 1500 mg once daily (QD or 500 mg BID before evaluating steady-state lapatinib levels in plasma and tumor.In mice, lapatinib levels were 4-fold higher in tumor than blood with a 4-fold longer half-life. Tumor concentrations exceeded the in vitro IC90 (~ 900 nM or 500 ng/mL for inhibition of HER2 phosphorylation throughout the 12-hour dosing interval. In patients, tumor levels were 6- and 10-fold higher with QD and BID dosing, respectively, compared to plasma trough levels. The relationship between tumor and plasma concentration was complex, indicating multiple determinants. HER receptor phosphorylation varied depending upon lapatinib tumor concentrations, suggestive of changes in the repertoire of HER homo- and heterodimers.Plasma lapatinib concentrations underestimated tumor drug levels, suggesting that optimal dosing should be focused on the site of action to avoid to inappropriate dose escalation. Larger clinical trials are required to determine optimal dose and schedule to achieve tumor concentrations that maximally

  7. The EGF receptor and notch signaling pathways control the initiation of the morphogenetic furrow during Drosophila eye development.

    Science.gov (United States)

    Kumar, J P; Moses, K

    2001-07-01

    The onset of pattern formation in the developing Drosophila retina begins with the initiation of the morphogenetic furrow, the leading edge of a wave of retinal development that transforms a uniform epithelium, the eye imaginal disc into a near crystalline array of ommatidial elements. The initiation of this wave of morphogenesis is under the control of the secreted morphogens Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg). We show that the Epidermal Growth Factor Receptor and Notch signaling cascades are crucial components that are also required to initiate retinal development. We also show that the initiation of the morphogenetic furrow is the sum of two genetically separable processes: (1) the 'birth' of pattern formation at the posterior margin of the eye imaginal disc; and (2) the subsequent 'reincarnation' of retinal development across the epithelium.

  8. Molecular mechanisms of the synergy between cysteinyl-leukotrienes and receptor tyrosine kinase growth factors on human bronchial fibroblast proliferation

    Directory of Open Access Journals (Sweden)

    Hajime Yoshisue

    2006-12-01

    Full Text Available We have reported that cysteinyl-leukotrienes (cys-LTs synergise not only with epidermal growth factor (EGF but also with platelet-derived growth factor (PDGF and fibroblast growth factor (FGF to induce mitogenesis in human bronchial fibroblasts. We now describe the molecular mechanisms underlying this synergism. Mitogenesis was assessed by incorporation of [3H]thymidine into DNA and changes in protein phosphorylation by Western blotting. Surprisingly, no CysLT receptor antagonists (MK-571, montelukast, BAY u9773 prevented the synergistic mitogenesis. LTD4 did not cause phosphorylation of EGFR nor did it augment EGF-induced phosphorylation of EGFR, and the synergy between LTD4 and EGF was not blocked by the metalloproteinase inhibitor GM6001 or by an HB-EGF neutralising antibody. The EGFR-selective kinase inhibitor, AG1478, suppressed the synergy by LTD4 and EGF, but had no effect on the synergy with PDGF and FGF. While inhibitors of mitogen-activated protein kinase, phosphatidylinositol 3-kinase and protein kinase C (PKC prevented the synergy, these drugs also inhibited mitogenesis elicited by EGF alone. In contrast, pertussis toxin (PTX efficiently inhibited the potentiating effect of LTD4 on EGF-induced mitogenesis, as well as that provoked by PDGF or FGF, but had no effect on mitogenesis elicited by the growth factors alone. Whereas LTD4 alone did not augment phosphorylation of extracellular signal-regulated kinase (Erk-1/2 and Akt, it increased phosphorylation of PKC in a Gi-dependent manner. Addition of LTD4 prolonged the duration of EGF-induced phosphorylation of Erk-1/2 and Akt, both of which were sensitive to PTX. The effect of cys-LTs involves a PTX-sensitive and PKC-mediated intracellular pathway leading to sustained growth factor-dependent phosphorylation of Erk-1/2 and Akt.

  9. Nonpolar interactions between trans-membrane helical EGF peptide and phosphatidylcholines, sphingomyelins and cholesterol. Molecular dynamics simulation studies

    NARCIS (Netherlands)

    Róg, T.; Murzyn, K.; Karttunen, M.E.J.; Pasenkiewicz-Gierula, M.

    2008-01-01

    A molecular dynamics simulation study of four lipid bilayers with inserted trans-membrane helical fragment of epithelial growth factor (EGF) receptor (EGF peptide) was performed. The lipid bilayers differ in their lipid composition and consist of (i) unsaturated phosphatidylcholine

  10. Carboxyl-terminal multi-site phosphorylation regulates internalization and desensitization of the human sst2 somatostatin receptor.

    Science.gov (United States)

    Lehmann, Andreas; Kliewer, Andrea; Schütz, Dagmar; Nagel, Falko; Stumm, Ralf; Schulz, Stefan

    2014-04-25

    The somatostatin receptor 2 (sst2) is the pharmacological target of somatostatin analogs that are widely used in the diagnosis and treatment of human neuroendocrine tumors. We have recently shown that the stable somatostatin analogs octreotide and pasireotide (SOM230) stimulate distinct patterns of sst2 receptor phosphorylation and internalization. Like somatostatin, octreotide promotes the phosphorylation of at least six carboxyl-terminal serine and threonine residues namely S341, S343, T353, T354, T356 and T359, which in turn leads to a robust receptor endocytosis. Unlike somatostatin, pasireotide stimulates a selective phosphorylation of S341 and S343 of the human sst2 receptor followed by a partial receptor internalization. Here, we show that exchange of S341 and S343 by alanine is sufficient to block pasireotide-driven internalization, whereas mutation of T353, T354, T356 and T359 to alanine is required to strongly inhibited both octreotide- and somatostatin-induced internalization. Yet, combined mutation of T353, T354, T356 and T359 is not sufficient to prevent somatostatin-driven β-arrestin mobilization and receptor desensitization. Replacement of all fourteen carboxyl-terminal serine and threonine residues by alanine completely abrogates sst2 receptor internalization and β-arrestin mobilization in HEK293 cells. Together, our findings demonstrate for the first time that agonist-selective sst2 receptor internalization is regulated by multi-site phosphorylation of its carboxyl-terminal tail. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  11. Fluoride-induced IL-8 release in human epithelial lung cells: Relationship to EGF-receptor-, SRC- and MAP-kinase activation

    International Nuclear Information System (INIS)

    Refsnes, Magne; Skuland, Tonje; Schwarze, Per E.; Ovrevik, Johan; Lag, Marit

    2008-01-01

    Exposure of human epithelial lung cells to fluorides is known to induce a marked increase in the release of interleukin (IL)-8, a chemokine involved in neutrophil recruitment. In the present study, the involvement of mitogen-activating protein kinases (MAPKs), the role of upstream activation of Src family kinases (SFKs), epidermal growth factor receptor (EGFR) activation and the interrelationships between these pathways in fluoride-induced IL-8 were examined in a human epithelial lung cell line (A549). Sodium fluoride strongly activated MAPK, in particular JNK1/2 and p38. The ERK1/2-inhibitor PD98059, the p38-inhibitor SB202190 and the JNK1/2-inhibitor SP600125 partially inhibited the fluoride-induced IL-8 response. Combinations of these inhibitors reduced the responses nearly to basal levels. Treatment with siRNA against JNK2 also reduced the IL-8 response to fluoride. Furthermore, fluoride activated SFKs, which was abolished by the SFK-inhibitor PP2. PP2 substantially inhibited the increased levels of IL-8, and partially reduced the fluoride-induced activation of ERK1/2, p38 and JNK1/2. Fluoride exposure also led to a phosphorylation of the EGFR, that was partially inhibited by PP2. AG1478, an EGFR-inhibitor, partially reduced the fluoride-induced IL-8 response and the phosphorylation of JNK1/2 and ERK1/2, but less the phosphorylation of p38. The effects of AG1478 were less than that of PP2. In conclusion, our findings suggest that the fluoride-induced IL-8 release involves the combined activation of ERK1/2, JNK1/2 and p38, and that the phosphorylation of these kinases, and in particular JNK1/2 and ERK1/2, partly, is mediated via a SFK-dependent EGFR-linked pathway. SFK-dependent, but EGFR-independent mechanisms seem important, and especially for phosphorylation of p38

  12. 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

  13. Biodistribution of 99mTc-labeled anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody h-R3 in a xenograft model of human lung adenocarcinoma

    International Nuclear Information System (INIS)

    Morales-Morales, Alejo; Duconge, Jorge; Caballero-Torres, Idania; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Iznaga-Escobar, Normando

    1999-01-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody (MAb) h-R3 is an (IgG 1 ), which binds to an extracellular domain of EGF-R. It was used to evaluate the biodistribution on nude mice xenografted with H-125 human lung adenocarcinoma cell line. Results were compared with its murine version of the MAb ior-egf/r3. Twenty-one athymic female 4NMRI nu/nu mice were injected intraperitoneally with 10 μg/100 μCi of 99m Tc-labeled MAbs. Immunoreactivity of 99m Tc-labeled MAbs were measured by enzyme-linked immunosorbent assay (ELISA) on H-125 cell line and the immunoreactive fractions was determined by the Lindmo method. Among all organs, significant accumulation was found in serum (27.05 ± 2.08 %ID/g) and tumor (3.903 ± 0.89 %ID/g) at 4 h after injection. These values decreased to 5.03 ± 0.50 %ID/g and 2.19 ± 0.56 %ID/g for serum and tumor, respectively. The immunoreactive fraction was found to be 0.70, with a correlation coefficient r=0.9984. With the good biodistribution and tumor uptake of the 99m Tc-labeled humanized antibody h-R3, a phase I diagnostic clinical trial of tumor with epithelial origin should be pursued

  14. Biodistribution of {sup 99m}Tc-labeled anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody h-R3 in a xenograft model of human lung adenocarcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Morales-Morales, Alejo; Duconge, Jorge; Caballero-Torres, Idania; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Iznaga-Escobar, Normando E-mail: normando@ict.cim.sld.cu

    1999-04-01

    The anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody (MAb) h-R3 is an (IgG{sub 1}), which binds to an extracellular domain of EGF-R. It was used to evaluate the biodistribution on nude mice xenografted with H-125 human lung adenocarcinoma cell line. Results were compared with its murine version of the MAb ior-egf/r3. Twenty-one athymic female 4NMRI nu/nu mice were injected intraperitoneally with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled MAbs. Immunoreactivity of {sup 99m}Tc-labeled MAbs were measured by enzyme-linked immunosorbent assay (ELISA) on H-125 cell line and the immunoreactive fractions was determined by the Lindmo method. Among all organs, significant accumulation was found in serum (27.05 {+-} 2.08 %ID/g) and tumor (3.903 {+-} 0.89 %ID/g) at 4 h after injection. These values decreased to 5.03 {+-} 0.50 %ID/g and 2.19 {+-} 0.56 %ID/g for serum and tumor, respectively. The immunoreactive fraction was found to be 0.70, with a correlation coefficient r=0.9984. With the good biodistribution and tumor uptake of the {sup 99m}Tc-labeled humanized antibody h-R3, a phase I diagnostic clinical trial of tumor with epithelial origin should be pursued.

  15. Neutron capture therapy of an Egf receptor positive glioma using boronated cetuximab alone or in combination with boronophenylalanine

    International Nuclear Information System (INIS)

    Wu, Gong; Yang, Weilian; Barth, Rolf F.

    2006-01-01

    The purpose of the present study was to evaluate the monoclonal antibody cetuximab (IMC-C225), which is directed against EGFR, as a boron delivery agent for NCT of a human EGFR gene transfected rat glioma, designated F98 EGFR . A heavily boronated polyamidoamine (PAMAM) dendrimer (BD) was chemically linked to cetuximab by means of heterobifunctional reagents. In vitro, the bioconjugate (BD-C225) was specifically taken up by F98 EGFR glioma cells (41.8 μg/g) compared to receptor (-) F98 WT cells (9.1 μg/g). Glioma cells were stereotactically implanted into the brains of Fischer rats and biodistribution studies were initiated 14 d later. The amount of boron retained by F98 EGFR gliomas 24 h following either convection enhanced delivery (CED) or intratumoral (i.t.) injection were 77.2 and 50.8 μg/g, respectively, and normal brain and blood values were 180 d) compared to 40 d for i.v. BPA alone and 31 d and 26 d for irradiated and untreated controls, respectively. Our data convincingly demonstrate the therapeutic efficacy of molecular targeting of EGFR using either boronated cetuximab alone or in combination with BPA and should provide a platform for the future development of combinations of high and low molecular weight delivery agents for BNCT of brain tumors. (author)

  16. SP600125 Induces Src and Type I IGF Receptor Phosphorylation Independent of JNK

    Directory of Open Access Journals (Sweden)

    Qingbin Kong

    2014-09-01

    Full Text Available c-Jun N-terminal kinases (JNK are members of the mitogen-activated protein kinase (MAPK family that have important roles in signal transduction. The small molecule SP600125 is widely used in biochemical studies as a JNK inhibitor. However, recent studies indicate that SP600125 may also act independent of JNK. Here, we report that SP600125 can induce Src, type I insulin-like growth factor receptor (IGF-IR, Akt and Erk1/2 phosphorylation. Notably, these effects are independent of its inhibition of JNK. Inhibition of Src abrogates the stimulation of IGF-IR, Akt and Erk1/2 phosphorylation. IGF-IR knockdown blunts the induction of both Akt and Erk1/2 phosphorylation by SP600125. Moreover, combination of SP600125 and the Src inhibitor saracatinib synergistically inhibits cell proliferation. We conclude that SP600125 can activate Src-IGF-IR-Akt/Erk1/2 signaling pathways independent of JNK.

  17. Apoptosis inducer NGFI-B is degraded by the proteasome and stabilized by treatment with EGF

    Energy Technology Data Exchange (ETDEWEB)

    Strom, Bjorn O. [Department of Pharmaceutical Biosciences, University of Oslo, P.O. Box 1068 Blindern, N-0316 Oslo (Norway); Paulsen, Ragnhild E., E-mail: r.e.paulsen@farmasi.uio.no [Department of Pharmaceutical Biosciences, University of Oslo, P.O. Box 1068 Blindern, N-0316 Oslo (Norway)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer NGFI-B is a molecular target for some anti-cancer drugs. Black-Right-Pointing-Pointer NGFI-B turnover may be important for their anti-cancer action. Black-Right-Pointing-Pointer NGFI-B is degraded by the proteasome. Black-Right-Pointing-Pointer NGFI-B is stabilized by treatment with EGF. Black-Right-Pointing-Pointer Mimicking the EGF-induced phosphorylation also stabilizes the protein. -- Abstract: NGFI-B is a nuclear receptor and immediate early gene that is upregulated in many different tumour cell lines. As it is involved in cell death and survival, it has been suggested as a target for anti-cancer drugs. The protein level of NGFI-B is important for its functions and may be regulated through induction or stabilization. NGFI-B protein stability was studied using the protein synthesis inhibitor cycloheximide in CV1 cells transiently transfected with NGFI-B. Inhibiting the proteasome with MG132 stabilized NGFI-B, indicating that the proteasome is responsible for break-down of NGFI-B, as it is for many nuclear receptors. In order to determine regions responsible for the break-down of NGFI-B two N-terminal regions with high PEST-scores were deleted. Deletion of amino acids 122-195 containing a PEST-sequence which includes an ERK2 phosphorylation target, gave a more stable protein. In addition, treatment of the cells with the ERK2 activator EGF increased the stability of wild type NGFI-B. We then tested whether a mutation at threonine 142 influenced the stability of NGFI-B. We found that the phosphorylation-mimicking mutant NGFI-B T142E had an increased stability, while the non-phosphorylable mutant (T142A) showed similar stability to the wild type. Thus, EGF-stimulation of cells may be a mechanism for priming the cells for effects of NGFI-B by increasing its stability.

  18. Clinical significance of proliferation, apoptosis and senescence of nasopharyngeal cells by the simultaneously blocking EGF, IGF-1 receptors and Bcl-xl genes

    International Nuclear Information System (INIS)

    Dai, Guodong; Peng, Tao; Zhou, Xuhong; Zhu, Jun; Kong, Zhihua; Ma, Li; Xiong, Zhi; Yuan, Yulin

    2013-01-01

    Highlight: •Construction of shRNA segments expression vectors is valid by the investigation of RT-PCR for IGF1R, EGFR and Bcl-xl mRNA and protein expression. •Studies have suggested that the vectors in blocking these genes of the growth factor receptors and anti- apoptosis is capable of breaking the balance of tumor growth so that tumor trend apoptosis and senescence. •Simultaneously blocking multiple genes that are abnormally expressed may be more effective in treating cancer cells than silencing a single gene. -- Abstract: Background: In previous work, we constructed short hairpin RNA (shRNA) expression plasmids that targeted human EGF and IGF-1 receptors messenger RNA, respectively, and demonstrated that these vectors could induce apoptosis of human nasopharyngeal cell lines (CNE2) and inhibit ligand-induced pAkt and pErk activation. Method: We have constructed multiple shRNA expression vectors of targeting EGFR, IGF1R and Bcl-xl, which were transfected to the CNE2 cells. The mRNA expression was assessed by RT-PCR. The growth of the cells, cell cycle progression, apoptosis of the cells, senescent tumor cells and the proteins of EGFR, IGF1R and Bcl-xl were analyzed by MTT, flow cytometry, cytochemical therapy or Western blot. Results: In group of simultaneously blocking EGFR, IGF1R and Bcl-xl genes, the mRNA of EGFR, IGF1R and Bcl-xl expression was decreased by (66.66 ± 3.42)%, (73.97 ± 2.83)% and (64.79 ± 2.83)%, and the protein expressions was diminished to (67.69 ± 4.02)%, (74.32 ± 2.30)%, and (60.00 ± 3.34)%, respectively. Meanwhile, the cell apoptosis increased by 65.32 ± 0.18%, 65.16 ± 0.25% and 55.47 ± 0.45%, and senescent cells increased by 1.42 ± 0.15%, 2.26 ± 0.15% and 3.22 ± 0.15% in the second, third and fourth day cultures, respectively. Conclusions: Simultaneously blocking EGFR, IGF1R and Bcl-xl genes is capable of altering the balance between proliferating versus apoptotic and senescent cells in the favor of both of apoptosis and

  19. Clinical significance of proliferation, apoptosis and senescence of nasopharyngeal cells by the simultaneously blocking EGF, IGF-1 receptors and Bcl-xl genes

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Guodong [Anatomy and Embryology, Wuhan University School of Medicine, Wuhan, Hubei 430071 (China); Peng, Tao; Zhou, Xuhong [Department of Otolaryngology-Head and Neck Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Zhu, Jun; Kong, Zhihua; Ma, Li; Xiong, Zhi [Anatomy and Embryology, Wuhan University School of Medicine, Wuhan, Hubei 430071 (China); Yuan, Yulin, E-mail: yuanyulin19620120@126.com [Anatomy and Embryology, Wuhan University School of Medicine, Wuhan, Hubei 430071 (China)

    2013-11-01

    Highlight: •Construction of shRNA segments expression vectors is valid by the investigation of RT-PCR for IGF1R, EGFR and Bcl-xl mRNA and protein expression. •Studies have suggested that the vectors in blocking these genes of the growth factor receptors and anti- apoptosis is capable of breaking the balance of tumor growth so that tumor trend apoptosis and senescence. •Simultaneously blocking multiple genes that are abnormally expressed may be more effective in treating cancer cells than silencing a single gene. -- Abstract: Background: In previous work, we constructed short hairpin RNA (shRNA) expression plasmids that targeted human EGF and IGF-1 receptors messenger RNA, respectively, and demonstrated that these vectors could induce apoptosis of human nasopharyngeal cell lines (CNE2) and inhibit ligand-induced pAkt and pErk activation. Method: We have constructed multiple shRNA expression vectors of targeting EGFR, IGF1R and Bcl-xl, which were transfected to the CNE2 cells. The mRNA expression was assessed by RT-PCR. The growth of the cells, cell cycle progression, apoptosis of the cells, senescent tumor cells and the proteins of EGFR, IGF1R and Bcl-xl were analyzed by MTT, flow cytometry, cytochemical therapy or Western blot. Results: In group of simultaneously blocking EGFR, IGF1R and Bcl-xl genes, the mRNA of EGFR, IGF1R and Bcl-xl expression was decreased by (66.66 ± 3.42)%, (73.97 ± 2.83)% and (64.79 ± 2.83)%, and the protein expressions was diminished to (67.69 ± 4.02)%, (74.32 ± 2.30)%, and (60.00 ± 3.34)%, respectively. Meanwhile, the cell apoptosis increased by 65.32 ± 0.18%, 65.16 ± 0.25% and 55.47 ± 0.45%, and senescent cells increased by 1.42 ± 0.15%, 2.26 ± 0.15% and 3.22 ± 0.15% in the second, third and fourth day cultures, respectively. Conclusions: Simultaneously blocking EGFR, IGF1R and Bcl-xl genes is capable of altering the balance between proliferating versus apoptotic and senescent cells in the favor of both of apoptosis and

  20. Phosphorylation of the Transient Receptor Potential Ankyrin 1 by Cyclin-dependent Kinase 5 affects Chemo-nociception

    OpenAIRE

    Hall, Bradford E.; Prochazkova, Michaela; Sapio, Matthew R.; Minetos, Paul; Kurochkina, Natalya; Binukumar, B. K.; Amin, Niranjana D.; Terse, Anita; Joseph, John; Raithel, Stephen J.; Mannes, Andrew J.; Pant, Harish C.; Chung, Man-Kyo; Iadarola, Michael J.; Kulkarni, Ashok B.

    2018-01-01

    Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase that is upregulated during inflammation, and can subsequently modulate sensitivity to nociceptive stimuli. We conducted an in silico screen for Cdk5 phosphorylation sites within proteins whose expression was enriched in nociceptors and identified the chemo-responsive ion channel Transient Receptor Potential Ankyrin 1 (TRPA1) as a possible Cdk5 substrate. Immunoprecipitated full length TRPA1 was shown to be phosphorylated by Cdk5 and th...

  1. A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes

    International Nuclear Information System (INIS)

    Itoh, Reina E.; Kurokawa, Kazuo; Fujioka, Aki; Sharma, Alok; Mayer, Bruce J.; Matsuda, Michiyuki

    2005-01-01

    Epidermal growth factor (EGF) receptor plays a pivotal role in a variety of cellular functions, such as proliferation, differentiation, and migration. To monitor the EGF receptor (EGFR) activity in living cells, we developed a probe for EGFR activity based on the principle of fluorescence resonance energy transfer (FRET). Previously, we developed a probe designated as Picchu (Phosphorylation indicator of the CrkII chimeric unit), which detects the tyrosine phosphorylation of the CrkII adaptor protein. We used a pair of synthetic amphipathic helixes, WinZipA2 and WinZipB1, to bind Picchu non-covalently to the carboxyl-terminus of the EGFR. Using this modified probe named Picchu-Z, the activity of EGFR was followed in EGF-stimulated Cos7 cells. We found that a high level of tyrosine phosphorylation of Picchu-Z probe remained after endocytosis until the point when the EGFR was translocated to the perinuclear region. These findings are in agreement with the previously reported 'signaling endosome' model. Furthermore, by pulse stimulation with EGF and by acute ablation of EGFR activity with AG1478, it was suggested that the phosphorylation of Picchu-Z probe, and probably the phosphorylation of EGFR also, underwent a rapid equilibrium (τ 1/2 < 2 min) between the phosphorylated and dephosphorylated states in the presence of EGF

  2. Characterization of phosphorylation sites in the cytoplasmic domain of the 300 kDa mannose-6-phosphate receptor

    DEFF Research Database (Denmark)

    Rosorius, O; Mieskes, G; Issinger, O G

    1993-01-01

    The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation and the phosphorylat......The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation...... and the phosphorylation sites the entire coding sequence of the cytoplasmic tail was expressed in Escherichia coli. The isolated cytoplasmic domain was used as a substrate for four purified serine/threonine kinases [casein kinase II (CK II), protein kinase A (PKA), protein kinase C and Ca2+/calmodulin kinase]. All...... kinases phosphorylate the cytoplasmic tail exclusively on serine residues. Inhibition studies using synthetic peptides, partial sequencing of isolated tryptic phosphopeptides and co-migration with tryptic phosphopeptides from MPR 300 labelled in vivo showed that (i) PKA phosphorylates the cytoplasmic MPR...

  3. Phosphorylation of chloroform soluble compounds in plasma membranes of human epidermoid carcinoma A431 cells

    International Nuclear Information System (INIS)

    Brautigan, D.L.; Randazzo, P.; Shriner, C.; Fain, J.N.

    1985-01-01

    This study investigated a possible role for the epidermal growth factor (EGF) receptor protein tyrosine kinase in phosphoinositide metabolism with plasma membrane vesicles from human epidermoid carcinoma (A431) cells. The authors found a novel chloroform-soluble product radiolabeled with [gamma- 32 P]ATP that did not migrate from the origin in the thin layer system designed to separate the phosphoinositides, appeared as a single band of Mr = 3500 on polyacrylamide gels in the presence of dodecyl sulfate, had an ultraviolet absorbance spectrum with a maximum at 275 nm and stained with Coomassie dye. Based on these properties this phosphorylation product is referred to as a proteolipid. The 32 P label was not detected in phosphotyrosine [Tyr(P)], phosphoserine [Ser(P)] or phosphothreonine [Thr(P)] and was lost during acid or base hydrolysis. Phosphorylation of proteolipid was increased significantly by EGF, whereas phosphorylation of phosphatidic acid was decreased and labeling of phosphoinositides was unaffected. Thus, it appears that in A431 membranes the EGF receptor/kinase does not utilize phosphatidylinositol as a substrate, but does phosphorylate a membrane proteolipid

  4. Characterization of phosphorylated beta-adrenergic receptors from desensitized turkey erythrocytes

    International Nuclear Information System (INIS)

    Rebar, R.; Crooke, S.T.; Stadel, J.M.

    1986-01-01

    Catecholamine-induced desensitization of turkey erythrocyte (TE) adenylate cyclase results in a 40-50 percent decrease in agonist stimulated cyclase activity. Desensitization is accompanied by decreased mobility on SDS-PAGE of beta-adrenergic receptor (BAR) proteins photoaffinity labeled with [ 125 I]-p-azidobenzylcarazolol compared to control. Using a low crosslinked gel, the M/sub r/ = 42,000 band of BAR from desensitized TE was further resolved into a doublet compared to a single M/sub r/ = 38,000 band for control. The formation of the doublet appears to correlate with the amount of adenylate cyclase desensitization. Preincubating TE for 20 hr at 37 0 C with 32 P-/sub i/ labels BAR. 32 P-BAR was partially purified by affinity chromatography over alprenolol-Sepharose. Limited digest peptide maps of 32 P-BAR using papain identified a unique peptide (M/sub r/ = 2800) from BAR of desensitized TE which was absent in control. This unique 32 P-peptide was found only in the upper band of the doublet of BAR from desensitized TE. These data indicate that BAR is not uniformly phosphorylated following agonist-induced desensitization of TE and identify a peptide of BAR which is a site of phosphorylation correlating with desensitization of TE adenylate cyclase

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

    DEFF Research Database (Denmark)

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

    2000-01-01

    phosphorylation. Consistent with the effects on STAT5B phosphorylation, tyrosine-to-phenylalanine mutation of tyrosine 595 prolongs the duration of tyrosyl phosphorylation of GHR and JAK2. These data suggest that tyrosine 595 is a major site of interaction of GHR with SHP-2, and that GHR-bound SHP-2 negatively......Binding of GH to GH receptor (GHR) rapidly and transiently activates multiple signal transduction pathways that contribute to the growth-promoting and metabolic effects of GH. While the events that initiate GH signal transduction, such as activation of the Janus tyrosine kinase JAK2, are beginning...

  6. Heterologous activation of protein kinase C stimulates phosphorylation of delta-opioid receptor at serine 344, resulting in beta-arrestin- and clathrin-mediated receptor internalization

    DEFF Research Database (Denmark)

    Xiang, B; Yu, G H; Guo, J

    2001-01-01

    The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our result showed that removing the C terminus of delta opioid receptor (DOR......) containing six Ser/Thr residues abolished both DPDPE- and phorbol 12-myristate 13-acetate (PMA)-induced DOR phosphorylation. The phosphorylation levels of DOR mutants T352A, T353A, and T358A/T361A/S363S were comparable to that of the wild-type DOR, whereas S344G substitution blocked PMA-induced receptor......, and ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative beta-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a beta-arrestin- and clathrin-dependent mechanism...

  7. Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion

    Directory of Open Access Journals (Sweden)

    Shimura Takaya

    2012-05-01

    Full Text Available Abstract Background Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C, translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. Methods We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF and mutated HB-EGF (HB-EGF-mC, which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Results Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 % and in the cytoplasm only in 25 cases (26.0 %. The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P  Conclusions Both the function of HB-EGF as an EGFR ligand and a novel signal for HB-EGF-C nuclear translocation induce gastric cancer growth, whereas HB-EGF-C nuclear translocation independently plays a critical role in gastric cancer invasion. The present study demonstrated that HB-EGF-C nuclear translocation might be crucial in gastric cancer invasion. HB-EGF-C nuclear translocation may offer a prognostic marker and a new molecular target for gastric cancer therapy.

  8. In vitro phosphorylation of insulin receptor substrate 1 by protein kinase C-zeta: functional analysis and identification of novel phosphorylation sites.

    Science.gov (United States)

    Sommerfeld, Mark R; Metzger, Sabine; Stosik, Magdalene; Tennagels, Norbert; Eckel, Jürgen

    2004-05-18

    Protein kinase C-zeta (PKC-zeta) participates both in downstream insulin signaling and in the negative feedback control of insulin action. Here we used an in vitro approach to identify PKC-zeta phosphorylation sites within insulin receptor substrate 1 (IRS-1) and to characterize the functional implications. A recombinant IRS-1 fragment (rIRS-1(449)(-)(664)) containing major tyrosine motifs for interaction with phosphatidylinositol (PI) 3-kinase strongly associated to the p85alpha subunit of PI 3-kinase after Tyr phosphorylation by the insulin receptor. Phosphorylation of rIRS-1(449)(-)(664) by PKC-zeta induced a prominent inhibition of this process with a mixture of classical PKC isoforms being less effective. Both PKC-zeta and the classical isoforms phosphorylated rIRS-1(449)(-)(664) on Ser(612). However, modification of this residue did not reduce the affinity of p85alpha binding to pTyr-containing peptides (amino acids 605-615 of rat IRS-1), as determined by surface plasmon resonance. rIRS-1(449)(-)(664) was then phosphorylated by PKC-zeta using [(32)P]ATP and subjected to tryptic phosphopeptide mapping based on two-dimensional HPLC coupled to mass spectrometry. Ser(498) and Ser(570) were identified as novel phosphoserine sites targeted by PKC-zeta. Both sites were additionally confirmed by phosphopeptide mapping of the corresponding Ser --> Ala mutants of rIRS-1(449)(-)(664). Ser(570) was specifically targeted by PKC-zeta, as shown by immunoblotting with a phosphospecific antiserum against Ser(570) of IRS-1. Binding of p85alpha to the S570A mutant was less susceptible to inhibition by PKC-zeta, when compared to the S612A mutant. In conclusion, our in vitro data demonstrate a strong inhibitory action of PKC-zeta at the level of IRS-1/PI 3-kinase interaction involving multiple serine phosphorylation sites. Whereas Ser(612) appears not to participate in the negative control of insulin signaling, Ser(570) may at least partly contribute to this process.

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  10. Glycoprotein VI/Fc receptor γ chain-independent tyrosine phosphorylation and activation of murine platelets by collagen

    OpenAIRE

    Jarvis, Gavin E.; Best, Denise; Watson, Steve P.

    2004-01-01

    We have investigated the ability of collagen to induce signalling and functional responses in suspensions of murine platelets deficient in the FcRγ (Fc receptor γ) chain, which lack the collagen receptor GPVI (glycoprotein VI). In the absence of the FcRγ chain, collagen induced a unique pattern of tyrosine phosphorylation which was potentiated by the thromboxane analogue U46619. Immunoprecipitation studies indicated that neither collagen alone nor the combination of collagen plus U46619 induc...

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

    Science.gov (United States)

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

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

    DEFF Research Database (Denmark)

    Su, J; Batzer, A; Sap, J

    1994-01-01

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

  13. Epidermal growth factor (EGF) as a potential targeting agent for delivery of boron to malignant gliomas

    International Nuclear Information System (INIS)

    Capala, J.; Barth, R.F.; Adams, D.M.; Bailey, M.Q.; Soloway, A.H.; Carlsson, J.

    1994-01-01

    The majority of high grade gliomas express an amplified epidermal growth factor receptor (EGFR) gene, and this often is associated with an increase in cell surface receptor expression. The rapid internalization and degradation of EGF-EGFR complexes, as well as their high affinity make EGF a potential targeting agent for delivery of 10 B to tumor cells with an amplified number of EGFR. Human glioma cells can expresses as many as 10 5 -10 6 EGF receptors per cell, and if these could be saturated with boronated EGF, then > 10 8 boron atoms would be delivered per cell. Since EGF has a comparatively low molecular weight (∼ 6 kD), this has allowed us to construct relatively small bioconjugates containing ∼ 900 boron atoms per EGF molecule 3 , which also had high affinity for EGFR on tumor cells. In the present study, the feasibility of using EGF receptors as a potential target for therapy of gliomas was investigated by in vivo scintigraphic studies using 131 I- or 99m T c -labeled EGF in a rat brain tumor model. Our results indicate that intratumorally delivered boron- EGF conjugates might be useful for targeting EGFR on glioma cells if the boron containing moiety of the conjugates persisted intracellularly. Further studies are required, however, to determine if this approach can be used for BNCT of the rat glioma

  14. A targetable HB-EGF-CITED4 axis controls oncogenesis in lung cancer.

    Science.gov (United States)

    Hsieh, C-H; Chou, Y-T; Kuo, M-H; Tsai, H-P; Chang, J-L; Wu, C-W

    2017-05-25

    Aberrant epidermal growth factor (EGF) receptor (EGFR) signaling contributes to neoplastic initiation and progression in lung. Mutated EGFR has become as an important therapeutic target in lung cancer, whereas targeted treatment is not available for wild-type EGFR or its ligands. In this study, we found that heparin-binding (HB)-EGF, a member of the EGF family, was highly expressed in a subset of lung cancer, proliferation of which was dependent on HB-EGF signaling. Silencing of HB-EGF with RNA interference inhibited cell cycle progression in lung cancer cells. We observed that, upon HB-EGF induction, CITED4 was induced through a signal transducer and activator of transcription 3 (STAT3)-dependent pathway, regulating cell proliferation. CITED4 interacted with MYC and potentiated MYC-mediated transactivation of the CCND1 promoter, leading to cell cycle progression. Correlation analysis revealed that HB-EGF and CITED4 were significantly positively associated in primary lung tumors, and expression of HB-EGF predicted a poor survival outcome in patients. In vitro and in vivo experiments revealed that pharmacological inhibition of HB-EGF with CRM197 significantly attenuated tumor cell growth. Thus, CITED4 functions as a molecular switch in HB-EGF-induced growth control, and HB-EGF provides a novel therapeutic target for lung cancer intervention.

  15. Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion

    Science.gov (United States)

    2012-01-01

    Background Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR) ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF) after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C), translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. Methods We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF) and mutated HB-EGF (HB-EGF-mC), which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Results Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 %) and in the cytoplasm only in 25 cases (26.0 %). The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P HB-EGF- and HB-EGF-mC-expressing cells significantly increased compared with control cells, but the growth of HB-EGF-mC-expressing cells was significantly decreased compared with wt-HB-EGF-expressing cells. Gastric cancer cell invasion obviously increased in wt-HB-EGF-expressing cells, but invasion in HB-EGF-mC-expressing cells showed a slight increase compared with control cells. Moreover, wt-HB-EGF overexpression increased the effectiveness of wound healing, but had no significant effect in HB-EGF-mC-expressing cells. Conclusions Both the function of HB-EGF as an EGFR ligand

  16. EGF Functionalized Polymer-Coated Gold Nanoparticles Promote EGF Photostability and EGFR Internalization for Photothermal Therapy.

    Directory of Open Access Journals (Sweden)

    Catarina Oliveira Silva

    Full Text Available The application of functionalized nanocarriers on photothermal therapy for cancer ablation has wide interest. The success of this application depends on the therapeutic efficiency and biocompatibility of the system, but also on the stability and biorecognition of the conjugated protein. This study aims at investigating the hypothesis that EGF functionalized polymer-coated gold nanoparticles promote EGF photostability and EGFR internalization, making these conjugated particles suitable for photothermal therapy. The conjugated gold nanoparticles (100-200 nm showed a plasmon absorption band located within the near-infrared range (650-900 nm, optimal for photothermal therapy applications. The effects of temperature, of polymer-coated gold nanoparticles and of UVB light (295nm on the fluorescence properties of EGF have been investigated with steady-state and time-resolved fluorescence spectroscopy. The fluorescence properties of EGF, including the formation of Trp and Tyr photoproducts, is modulated by temperature and by the intensity of the excitation light. The presence of polymeric-coated gold nanoparticles reduced or even avoided the formation of Trp and Tyr photoproducts when EGF is exposed to UVB light, protecting this way the structure and function of EGF. Cytotoxicity studies of conjugated nanoparticles carried out in normal-like human keratinocytes showed small, concentration dependent decreases in cell viability (0-25%. Moreover, conjugated nanoparticles could activate and induce the internalization of overexpressed Epidermal Growth Factor Receptor in human lung carcinoma cells. In conclusion, the gold nanoparticles conjugated with Epidermal Growth Factor and coated with biopolymers developed in this work, show a potential application for near infrared photothermal therapy, which may efficiently destroy solid tumours, reducing the damage of the healthy tissue.

  17. EGF Functionalized Polymer-Coated Gold Nanoparticles Promote EGF Photostability and EGFR Internalization for Photothermal Therapy

    Science.gov (United States)

    Silva, Catarina Oliveira; Petersen, Steffen B.; Reis, Catarina Pinto; Rijo, Patrícia; Molpeceres, Jesús; Fernandes, Ana Sofia; Gonçalves, Odete; Gomes, Andreia C.; Correia, Isabel; Vorum, Henrik; Neves-Petersen, Maria Teresa

    2016-01-01

    The application of functionalized nanocarriers on photothermal therapy for cancer ablation has wide interest. The success of this application depends on the therapeutic efficiency and biocompatibility of the system, but also on the stability and biorecognition of the conjugated protein. This study aims at investigating the hypothesis that EGF functionalized polymer-coated gold nanoparticles promote EGF photostability and EGFR internalization, making these conjugated particles suitable for photothermal therapy. The conjugated gold nanoparticles (100–200 nm) showed a plasmon absorption band located within the near-infrared range (650–900 nm), optimal for photothermal therapy applications. The effects of temperature, of polymer-coated gold nanoparticles and of UVB light (295nm) on the fluorescence properties of EGF have been investigated with steady-state and time-resolved fluorescence spectroscopy. The fluorescence properties of EGF, including the formation of Trp and Tyr photoproducts, is modulated by temperature and by the intensity of the excitation light. The presence of polymeric-coated gold nanoparticles reduced or even avoided the formation of Trp and Tyr photoproducts when EGF is exposed to UVB light, protecting this way the structure and function of EGF. Cytotoxicity studies of conjugated nanoparticles carried out in normal-like human keratinocytes showed small, concentration dependent decreases in cell viability (0–25%). Moreover, conjugated nanoparticles could activate and induce the internalization of overexpressed Epidermal Growth Factor Receptor in human lung carcinoma cells. In conclusion, the gold nanoparticles conjugated with Epidermal Growth Factor and coated with biopolymers developed in this work, show a potential application for near infrared photothermal therapy, which may efficiently destroy solid tumours, reducing the damage of the healthy tissue. PMID:27788212

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

    DEFF Research Database (Denmark)

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

    1995-01-01

    ), the principle substrate of the insulin receptor. Tyrosyl phosphorylation of IRS-1 is a critical step in insulin signaling and provides binding sites for proteins with the appropriate Src homology 2 domains, including the 85-kDa regulatory subunit of phosphatidylinositol (PI) 3'-kinase. In 3T3-F442A fibroblasts......., Campbell, G. S., Allevato, G., Billestrup, N., Norstedt, G., and Carter-Su, C. (1994) J. Biol. Chem. 269, 21709-21717). When other cytokines that activate JAK2 were tested for the ability to stimulate the tyrosyl phosphorylation of IRS-1, stimulation was detected with interferon-gamma and leukemia...... to JAK2. GH is also shown to stimulate binding of IRS-1 to the 85-kDa regulatory subunit of PI 3'-kinase. The ability of GH to stimulate tyrosyl phosphorylation of IRS-1 and its association with PI 3'-kinase provides a biochemical basis for responses shared by insulin and GH including the well...

  19. Bisphenol-A rapidly enhanced passive avoidance memory and phosphorylation of NMDA receptor subunits in hippocampus of young rats

    International Nuclear Information System (INIS)

    Xu Xiaohong; Li Tao; Luo Qingqing; Hong Xing; Xie Lingdan; Tian Dong

    2011-01-01

    Bisphenol-A (BPA), an endocrine disruptor, is found to influence development of brain and behaviors in rodents. The previous study indicated that perinatal exposure to BPA impaired learning-memory and inhibited N-methyl-D-aspartate receptor (NMDAR) subunits expressions in hippocampus during the postnatal development in rats; and in cultured hippocampal neurons, BPA rapidly promotes dynamic changes in dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDAR subunit NR2B. In the present study, we examined the rapid effect of BPA on passive avoidance memory and NMDAR in the developing hippocampus of Sprague-Dawley rats at the age of postnatal day 18. The results showed that BPA or estradiol benzoate (EB) rapidly extended the latency to step down from the platform 1 h after footshock and increased the phosphorylation levels of NR1, NR2B, and mitogen-activated extracellular signal-regulated kinase (ERK) in hippocampus within 1 h. While 24 h after BPA or EB treatment, the improved memory and the increased phosphorylation levels of NR1, NR2B, ERK disappeared. Furthermore, pre-treatment with an estrogen receptors (ERs) antagonist, ICI182,780, or an ERK-activating kinase inhibitor, U0126, significantly attenuated EB- or BPA-induced phosphorylations of NR1, NR2B, and ERK within 1 h. These data suggest that BPA rapidly enhanced short-term passive avoidance memory in the developing rats. A non-genomic effect via ERs may mediate the modulation of the phosphorylation of NMDAR subunits NR1 and NR2B through ERK signaling pathway. - Highlights: → BPA rapidly extended the latency to step down from platform 1 h after footshock. → BPA rapidly increased pNR1, pNR2B, and pERK in hippocampus within 1 h. → ERs antagonist or MEK inhibitor attenuated BPA-induced pNR1, pNR2B, and pERK.

  20. Cellular retention of radioactivity and increased radiation dose. Model experiments with EGF-dextran

    International Nuclear Information System (INIS)

    Sundberg, Aasa Liljegren; Blomquist, Erik; Carlsson, Joergen; Steffen, Ann-Charlott; Gedda, Lars

    2003-01-01

    Targeting of tumor cells with radiolabeled biomolecules is a possible approach to inactivate disseminated tumor cells. However, rapid degradation of the biomolecules after cellular internalization and subsequent excretion of the radioactivity is a problem. We studied the possibility of using dextran as a carrier of radionuclides to improve the intracellular retention. An EGF-dextran conjugate, aimed for targeting of tumor cells overexpressing the EGF-receptor, was used as model. Retention tests were performed with 125 I on different parts: [ 125 I]-EGF-dextran-[ 125 I], [ 125 I]-EGF-dextran and EGF-dextran-[ 125 I]. Comparisons were made with [ 125 I]-EGF. The radiolabeled compounds were incubated with cultured glioma cells for different times. The cellular retention of radioactivity was then measured for up to 24 h. Expected radiation doses at the cellular level were calculated assuming that 131 I, instead of 125 I, was coupled to EGF and EGF-dextran. The results indicated that the EGF-part of the conjugate was degraded and the EGF-attached radioactivity was rapidly excreted, whereas radioactivity on dextran was retained intracellularly to a high degree, i.e. 70-80% of the radioactivity bound to dextran was still cell-associated after 24 h. The retention after 24 h was significantly higher (p < 0.001) when the radioactivity was on the dextran instead of the EGF-part. The radiolabeled EGF-dextran had a notably high specific radioactivity; up to 11 MBq/μg. There was potential for at least hundred times increased radiation dose per receptor interaction when the radioactivity was on the dextran part. The advantage with radioactivity on the dextran part was the high cellular retention and the high specific radioactivity (higher than previously reported for other residualizing labels) without severe loss of receptor specific binding. Thus, dextran seems suitable as a carrier of radionuclides aimed for therapy and gives potential for a highly increased radiation dose

  1. Towards the systematic discovery of signal transduction networks using phosphorylation dynamics data

    Directory of Open Access Journals (Sweden)

    Yachie Nozomu

    2010-05-01

    Full Text Available Abstract Background Phosphorylation is a ubiquitous and fundamental regulatory mechanism that controls signal transduction in living cells. The number of identified phosphoproteins and their phosphosites is rapidly increasing as a result of recent mass spectrometry-based approaches. Results We analyzed time-course phosphoproteome data obtained previously by liquid chromatography mass spectrometry with the stable isotope labeling using amino acids in cell culture (SILAC method. This provides the relative phosphorylation activities of digested peptides at each of five time points after stimulating HeLa cells with epidermal growth factor (EGF. We initially calculated the correlations between the phosphorylation dynamics patterns of every pair of peptides and connected the strongly correlated pairs to construct a network. We found that peptides extracted from the same intracellular fraction (nucleus vs. cytoplasm tended to be close together within this phosphorylation dynamics-based network. The network was then analyzed using graph theory and compared with five known signal-transduction pathways. The dynamics-based network was correlated with known signaling pathways in the NetPath and Phospho.ELM databases, and especially with the EGF receptor (EGFR signaling pathway. Although the phosphorylation patterns of many proteins were drastically changed by the EGF stimulation, our results suggest that only EGFR signaling transduction was both strongly activated and precisely controlled. Conclusions The construction of a phosphorylation dynamics-based network provides a useful overview of condition-specific intracellular signal transduction using quantitative time-course phosphoproteome data under specific experimental conditions. Detailed prediction of signal transduction based on phosphoproteome dynamics remains challenging. However, since the phosphorylation profiles of kinase-substrate pairs on the specific pathway were localized in the dynamics

  2. Selective inhibition reveals cyclin-dependent kinase 2 as another kinase that phosphorylates the androgen receptor at serine 81

    Czech Academy of Sciences Publication Activity Database

    Jorda, Radek; Bučková, Zuzana; Řezníčková, Eva; Bouchal, J.; Kryštof, Vladimír

    2018-01-01

    Roč. 1865, č. 2 (2018), s. 354-363 ISSN 0167-4889 R&D Projects: GA MŠk(CZ) LO1204; GA MŠk(CZ) LO1304 Institutional support: RVO:61389030 Keywords : Androgen receptor * Cyclin-dependent kinase * Inhibitor * Phosphorylation * Serine 81 Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 4.521, year: 2016

  3. Identification of diphtheria toxin R domain mutants with enhanced inhibitory activity against HB-EGF.

    Science.gov (United States)

    Suzuki, Keisuke; Mizushima, Hiroto; Abe, Hiroyuki; Iwamoto, Ryo; Nakamura, Haruki; Mekada, Eisuke

    2015-05-01

    Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a ligand of EGF receptor, is involved in the growth and malignant progression of cancers. Cross-reacting material 197, CRM197, a non-toxic mutant of diphtheria toxin (DT), specifically binds to the EGF-like domain of HB-EGF and inhibits its mitogenic activity, thus CRM197 is currently under evaluation in clinical trials for cancer therapy. To develop more potent DT mutants than CRM197, we screened various mutant proteins of R domain of DT, the binding site for HB-EGF. A variety of R-domain mutant proteins fused with maltose-binding protein were produced and their inhibitory activity was evaluated in vitro. We found four R domain mutants that showed much higher inhibitory activity against HB-EGF than wild-type (WT) R domain. These R domain mutants suppressed HB-EGF-dependent cell proliferation more effectively than WT R domain. Surface plasmon resonance revealed their higher affinity to HB-EGF than WT R domain. CRM197(R460H) carrying the newly identified mutation showed increased cell proliferation inhibitory activity and affinity to HB-EGF. These results suggest that CRM197(R460H) or other recombinant proteins carrying newly identified mutation(s) in the R domain are potential therapeutics targeting HB-EGF. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  4. Targeting hepatic heparin-binding EGF-like growth factor (HB-EGF) induces anti-hyperlipidemia leading to reduction of angiotensin II-induced aneurysm development.

    Science.gov (United States)

    Kim, Seonwook; Yang, Lihua; Kim, Seongu; Lee, Richard G; Graham, Mark J; Berliner, Judith A; Lusis, Aldons J; Cai, Lei; Temel, Ryan E; Rateri, Debra L; Lee, Sangderk

    2017-01-01

    The upregulated expression of heparin binding EGF-like growth factor (HB-EGF) in the vessel and circulation is associated with risk of cardiovascular disease. In this study, we tested the effects of HB-EGF targeting using HB-EGF-specific antisense oligonucleotide (ASO) on the development of aortic aneurysm in a mouse aneurysm model. Low-density lipoprotein receptor (LDLR) deficient mice (male, 16 weeks of age) were injected with control and HB-EGF ASOs for 10 weeks. To induce aneurysm, the mice were fed a high fat diet (22% fat, 0.2% cholesterol; w/w) at 5 week point of ASO administration and infused with angiotensin II (AngII, 1,000ng/kg/min) for the last 4 weeks of ASO administration. We confirmed that the HB-EGF ASO administration significantly downregulated HB-EGF expression in multiple tissues including the liver. Importantly, the HB-EGF ASO administration significantly suppressed development of aortic aneurysms including thoracic and abdominal types. Interestingly, the HB-EGF ASO administration induced a remarkable anti-hyperlipidemic effect by suppressing very low density lipoprotein (VLDL) level in the blood. Mechanistically, the HB-EGF targeting suppressed hepatic VLDL secretion rate without changing heparin-releasable plasma triglyceride (TG) hydrolytic activity or fecal neutral cholesterol excretion rate. This result suggested that the HB-EGF targeting induced protection against aneurysm development through anti-hyperlipidemic effects. Suppression of hepatic VLDL production process appears to be a key mechanism for the anti-hyperlipidemic effects by the HB-EGF targeting.

  5. Transmembrane signalling at the epidermal growth factor receptor. Positive regulation by the C-terminal phosphotyrosine residues

    DEFF Research Database (Denmark)

    Magni, M; Pandiella, A; Helin, K

    1991-01-01

    a positive role in the regulation of transmembrane signalling at the EGF receptor. The stepwise decrease in signal generation observed in single, double and triple point mutants suggest that the role of phosphotyrosine residues is not in the participation in specific amino acid sequences, but rather...... in the double and the triple mutants. In the latter mutant, expression of the EGF-receptor-activated lipolytic enzyme phospholipase C gamma was unchanged, whereas its tyrosine phosphorylation induced by the growth factor was lowered to approx. 25% of that in the controls. In all of the cell clones employed......, the accumulation of inositol phosphates induced by treatment with fetal calf serum varied only slightly, whereas the same effect induced by EGF was consistently lowered in those lines expressing mutated receptors. This decrease was moderate for those receptors missing only the distal tyrosine (point and deletion...

  6. A highly phosphorylated subpopulation of insulin-like growth factor II/mannose 6-phosphate receptors is concentrated in a clathrin-enriched plasma membrane fraction

    International Nuclear Information System (INIS)

    Corvera, S.; Folander, K.; Clairmont, K.B.; Czech, M.P.

    1988-01-01

    Insulin-like growth factor II (IGF-II)/mannose 6-phosphate (Man-6-P) receptors immunoprecipitated from purified plasma membranes of 32 P-labeled rat adipocytes are markedly heterogenous in their phosphorylation state. Approximately 80% of the plasma membrane receptors are solubilized in 1% (vol/vol) Triton X-100 and are phosphorylated on serine residues at a stoichiometry of ∼ 0.1-0.2 mol of phosphate per mol of receptor. In contrast, 15-20% of the receptors are Triton X-100-insoluble and are phosphorylated on serine and threonine residues at ∼ 4 or 5 mol of phosphate per mol of receptor. This Triton X-100-insoluble membrane subfraction contains only 5% of the total plasma membrane protein and yet contains all of the clathrin heavy chain associated with plasma membrane. Based on the relative yields of protein in the detergent-insoluble material, IGF-II/Man-6-P receptors are concentrated ∼ 3-fold in this clathrin-enriched subfraction. Taken together, these results indicate that insulin decreases the phosphorylation state of a highly phosphorylated subpopulation of IGF-II/Man-6-P receptors on the plasma membrane. In addition, insulin action may prevent the concentration of these receptors in a clathrin-enriched membrane subfraction

  7. Phosphorylation of the cytoplasmic tail of the 300-kDa mannose 6-phosphate receptor is required for the interaction with a cytosolic protein

    DEFF Research Database (Denmark)

    Rosorius, O; Issinger, O G; Braulke, T

    1993-01-01

    The cytoplasmic tail of the human 300-kDa mannose 6-phosphate receptor (MPR 300-CT) is an excellent substrate for casein kinase II in vitro. The phosphorylated MPR 300-CT was cross-linked by means of bis(sulfosuccinimidyl)suberate mainly to a cytosolic protein of 35 kDa (referred to as TIP 35...... with TIP 35 is phosphorylation-specific. Furthermore, TIP 35 was only cross-linked to the MPR 300-CT phosphorylated by casein kinase II whereas the MPR 300-CT phosphorylated by protein kinase A failed to cross-link to TIP 35. These results indicate that the cytoplasmic tail of the MPR 300 interacts...

  8. Receptor-type Protein Tyrosine Phosphatase β Regulates Met Phosphorylation and Function in Head and Neck Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Yiru Xu

    2012-11-01

    Full Text Available Head and neck squamous cell carcinoma (HNSCC is the sixth most common cancer and has a high rate of mortality. Emerging evidence indicates that hepatocyte growth factor receptor (or Met pathway plays a pivotal role in HNSCC metastasis and resistance to chemotherapy. Met function is dependent on tyrosine phosphorylation that is under direct control by receptor-type protein tyrosine phosphatase β (RPTP-β. We report here that RPTP-β expression is significantly downregulated in HNSCC cells derived from metastatic tumors compared to subject-matched cells from primary tumors. Knockdown of endogenous RPTP-β in HNSCC cells from primary tumor potentiated Met tyrosine phosphorylation, downstream mitogen-activated protein (MAP kinase pathway activation, cell migration, and invasion. Conversely, restoration of RPTP-β expression in cells from matched metastatic tumor decreased Met tyrosine phosphorylation and downstream functions. Furthermore, we observed that six of eight HNSCC tumors had reduced levels of RPTP-β protein in comparison with normal oral tissues. Collectively, the results demonstrate the importance of RPTP-β in tumor biology of HNSCC through direct dephosphorylation of Met and regulation of downstream signal transduction pathways. Reduced RPTP-β levels, with or without Met overexpression, could promote Met activation in HNSCC tumors.

  9. Changes in proHB-EGF expression after functional activation of the immune system cells

    Directory of Open Access Journals (Sweden)

    T. O. Chudina

    2017-12-01

    Full Text Available The level of proHB-EGF expression on J774, Raji, KG-1 cells derived from different types of human and mouse immune system cells under the standard in vitro culture conditions and during functional activation of these cells was investigated. Changes in the proHB-EGF expression on the cell surface were found to depend on the density of cell population, the content of fetal bovine serum in the culture medium, the effect of mitogenic factors – bacterial lipopolysaccharide, an inactive full-size form of diphtheria toxin (CRM197 and recombinant soluble HB-EGF – rsHB-EGF. The results obtained are important for the understanding of the functional role of proHB-EGF receptor on the surface of macrophage-like cells and B lymphocytes and indicate the involvement of this receptor in immune response regulation in an organism.

  10. SMAD4 loss enables EGF, TGF?1 and S100A8/A9 induced activation of critical pathways to invasion in human pancreatic adenocarcinoma cells

    OpenAIRE

    Moz, Stefania; Basso, Daniela; Bozzato, Dania; Galozzi, Paola; Navaglia, Filippo; Negm, Ola H.; Arrigoni, Giorgio; Zambon, Carlo-Federico; Padoan, Andrea; Tighe, Paddy; Todd, Ian; Franchin, Cinzia; Pedrazzoli, Sergio; Punzi, Leonardo; Plebani, Mario

    2016-01-01

    Epidermal Growth Factor (EGF) receptor overexpression, KRAS, TP53, CDKN2A and SMAD4 mutations characterize pancreatic ductal adenocarcinoma. This mutational landscape might influence cancer cells response to EGF, Transforming Growth Factor ?1 (TGF?1) and stromal inflammatory calcium binding proteins S100A8/A9. We investigated whether chronic exposure to EGF modifies in a SMAD4-dependent manner pancreatic cancer cell signalling, proliferation and invasion in response to EGF, TGF?1 and S100A8/A...

  11. PEST Motif Serine and Tyrosine Phosphorylation Controls Vascular Endothelial Growth Factor Receptor 2 Stability and Downregulation ▿

    Science.gov (United States)

    Meyer, Rosana D.; Srinivasan, Srimathi; Singh, Amrik J.; Mahoney, John E.; Gharahassanlou, Kobra Rezazadeh; Rahimi, Nader

    2011-01-01

    The internalization and degradation of vascular endothelial growth factor receptor 2 (VEGFR-2), a potent angiogenic receptor tyrosine kinase, is a central mechanism for the regulation of the coordinated action of VEGF in angiogenesis. Here, we show that VEGFR-2 is ubiquitinated in response to VEGF, and Lys 48-linked polyubiquitination controls its degradation via the 26S proteosome. The degradation and ubiquitination of VEGFR-2 is controlled by its PEST domain, and the phosphorylation of Ser1188/Ser1191 is required for the ubiquitination of VEGFR-2. F-box-containing β-Trcp1 ubiquitin E3 ligase is recruited to S1188/S1191 VEGFR-2 and mediates the ubiquitination and degradation of VEGFR-2. The PEST domain also controls the activation of p38 mitogen-activated protein kinase (MAPK) through phospho-Y1173. The activation of p38 stabilizes VEGFR-2, and its inactivation accelerates VEGFR-2 downregulation. The VEGFR-2-mediated activation of p38 is established through the protein kinase A (PKA)/MKK6 pathway. PKA is recruited to VEGFR-2 through AKAP1/AKAP149, and its phosphorylation requires Y1173 of VEGFR-2. The study has identified a unique mechanism in which VEGFR-2 stability and degradation is modulated. The PEST domain acts as a dual modulator of VEGFR-2; the phosphorylation of S1188/S1191 controls ubiquitination and degradation via β-Trcp1, where the phosphorylation of Y1173 through PKA/p38 MAPK controls the stability of VEGFR-2. PMID:21402774

  12. The heparin-binding domain of HB-EGF mediates localization to sites of cell-cell contact and prevents HB-EGF proteolytic release

    Energy Technology Data Exchange (ETDEWEB)

    Prince, Robin N.; Schreiter, Eric R.; Zou, Peng; Wiley, H. S.; Ting, Alice Y.; Lee, Richard T.; Lauffenburger, Douglas A.

    2010-07-01

    Heparin-binding EGF-like growth factor (HB-EGF) is a ligand for EGF receptor (EGFR) and possesses the ability to signal in juxtacrine, autocrine and/or paracrine mode, with these alternatives being governed by the degree of proteolytic release of the ligand. Although the spatial range of diffusion of released HB-EGF is restricted by binding heparan-sulfate proteoglycans (HSPGs) in the extracellular matrix and/or cellular glycocalyx, ascertaining mechanisms governing non-released HB-EGF localization is also important for understanding its effects. We have employed a new method for independently tracking the localization of the extracellular EGFlike domain of HB-EGF and the cytoplasmic C-terminus. A striking observation was the absence of the HB-EGF transmembrane proform from the leading edge of COS-7 cells in a wound-closure assay; instead, this protein localized in regions of cell-cell contact. A battery of detailed experiments found that this localization derives from a trans interaction between extracellular HSPGs and the HBEGF heparin-binding domain, and that disruption of this interaction leads to increased release of soluble ligand and a switch in cell phenotype from juxtacrine-induced growth inhibition to autocrine-induced proliferation. Our results indicate that extracellular HSPGs serve to sequester the transmembrane pro-form of HB-EGF at the point of cell-cell contact, and that this plays a role in governing the balance between juxtacrine versus autocrine and paracrine signaling.

  13. Phosphorylation of the dimeric cytoplasmic domain of the phytosulfokine receptor, PSKR1

    CSIR Research Space (South Africa)

    Muleya, V

    2016-10-01

    Full Text Available Phytosulfokines (PSKs) are plant peptide hormones that co-regulate plant growth, differentiation and defense responses. PSKs signal through a plasma membrane localized leucine-rich repeat receptor-like kinase (phytosulfokine receptor 1, PSKR1...

  14. Reduced phosphorylation of brain insulin receptor substrate and Akt proteins in apolipoprotein-E4 targeted replacement mice.

    Science.gov (United States)

    Ong, Qi-Rui; Chan, Elizabeth S; Lim, Mei-Li; Cole, Gregory M; Wong, Boon-Seng

    2014-01-17

    Human ApoE4 accelerates memory decline in ageing and in Alzheimer's disease. Although intranasal insulin can improve cognition, this has little effect in ApoE4 subjects. To understand this ApoE genotype-dependent effect, we examined brain insulin signaling in huApoE3 and huApoE4 targeted replacement (TR) mice. At 32 weeks, lower insulin receptor substrate 1 (IRS1) at S636/639 and Akt phosphorylation at T308 were detected in fasting huApoE4 TR mice as compared to fasting huApoE3 TR mice. These changes in fasting huApoE4 TR mice were linked to lower brain glucose content and have no effect on plasma glucose level. However, at 72 weeks of age, these early changes were accompanied by reduction in IRS2 expression, IRS1 phosphorylation at Y608, Akt phosphorylation at S473, and MAPK (p38 and p44/42) activation in the fasting huApoE4 TR mice. The lower brain glucose was significantly associated with higher brain insulin in the aged huApoE4 TR mice. These results show that ApoE4 reduces brain insulin signaling and glucose level leading to higher insulin content.

  15. Acyclic retinoid in chemoprevention of hepatocellular carcinoma: Targeting phosphorylated retinoid X receptor-α for prevention of liver carcinogenesis

    Directory of Open Access Journals (Sweden)

    Masahito Shimizu

    2012-01-01

    Full Text Available One of the key features of hepatocellular carcinoma (HCC is the high rate of intrahepatic recurrence that correlates with poor prognosis. Therefore, in order to improve the clinical outcome for patients with HCC, development of a chemopreventive agent that can decrease or delay the incidence of recurrence is a critical issue for urgent investigation. Acyclic retinoid (ACR, a synthetic retinoid, successfully improves HCC patient survival by preventing recurrence and the formation of secondary tumors. A malfunction of the retinoid X receptor-α (RXRα due to phosphorylation by the Ras-MAPK signaling pathway plays a critical role in liver carcinogenesis, and ACR exerts chemopreventive effects on HCC development by inhibiting RXRα phosphorylation. Here, we review the relationship between retinoid signaling abnormalities and liver disease, the mechanisms of how RXRα phosphorylation contributes to liver carcinogenesis, and the detailed effects of ACR on preventing HCC development, especially based on the results of our basic and clinical research. We also outline the concept of "clonal deletion and inhibition" therapy, which is defined as the removal and inhibition of latent malignant clones from the liver before they expand into clinically detectable HCC, because ACR prevents the development of HCC by implementing this concept. Looking toward the future, we discuss "combination chemoprevention" using ACR as a key drug since it can generate a synergistic effect, and may thus be an effective new strategy for the prevention of HCC.

  16. NADPH oxidase 1 deficiency alters caveolin phosphorylation and angiotensin II-receptor localization in vascular smooth muscle.

    Science.gov (United States)

    Basset, Olivier; Deffert, Christine; Foti, Michelangelo; Bedard, Karen; Jaquet, Vincent; Ogier-Denis, Eric; Krause, Karl-Heinz

    2009-10-01

    The superoxide-generating NADPH oxidase NOX1 is thought to be involved in signaling by the angiotensin II-receptor AT1R. However, underlying signaling steps are poorly understood. In this study, we investigated the effect of AngII on aortic smooth muscle from wild-type and NOX1-deficient mice. NOX1-deficient cells showed decreased basal ROS generation and did not produce ROS in response to AngII. Unexpectedly, AngII-dependent Ca(2+) signaling was markedly decreased in NOX1-deficient cells. Immunostaining demonstrated that AT1R was localized on the plasma membrane in wild-type, but intracellularly in NOX1-deficient cells. Immunohistochemistry and immunoblotting showed a decreased expression of AT1R in the aorta of NOX1-deficient mice. To investigate the basis of the abnormal AT1R targeting, we studied caveolin expression and phosphorylation. The amounts of total caveolin and of caveolae were not different in NOX1-deficient mice, but a marked decrease occurred in the phosphorylated form of caveolin. Exogenous H(2)O(2) or transfection of a NOX1 plasmid restored AngII responses in NOX1-deficient cells. Based on these findings, we propose that NOX1-derived reactive oxygen species regulate cell-surface expression of AT1R through mechanisms including caveolin phosphorylation. The lack cell-surface AT1R expression in smooth muscle could be involved in the decreased blood pressure in NOX1-deficient mice.

  17. The phosphorylation state of CD3gamma influences T cell responsiveness and controls T cell receptor cycling

    DEFF Research Database (Denmark)

    Dietrich, J; Bäckström, T; Lauritsen, J P

    1998-01-01

    The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3gamma. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR and the ......The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3gamma. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR...... the phosphorylation state of CD3gamma and T cell responsiveness. Based on these observations a physiological role of CD3gamma and TCR cycling is proposed....

  18. Tyrosine phosphorylation of platelet derived growth factor β receptors in coronary artery lesions: implications for vascular remodelling after directional coronary atherectomy and unstable angina pectoris

    Science.gov (United States)

    Abe, J; Deguchi, J; Takuwa, Y; Hara, K; Ikari, Y; Tamura, T; Ohno, M; Kurokawa, K

    1998-01-01

    Background—Growth factors such as platelet derived growth factor (PDGF) have been postulated to be important mediators of neointimal proliferation observed in atherosclerotic plaques and restenotic lesions following coronary interventions. Binding of PDGF to its receptor results in intrinsic receptor tyrosine kinase activation and subsequent cellular migration, proliferation, and vascular contraction.
Aims—To investigate whether the concentration of PDGF β receptor tyrosine phosphorylation obtained from directional coronary atherectomy (DCA) samples correlate with atherosclerotic plaque burden, the ability of diseased vessels to remodel, coronary risk factors, and clinical events.
Methods—DCA samples from 59 patients and 15 non-atherosclerotic left internal thoracic arteries (LITA) were analysed for PDGF β receptor tyrosine phosphorylation content by receptor immunoprecipitation and antiphosphotyrosine western blot. The amount of PDGF β receptor phosphorylation was analysed in relation to angiographic follow up data and clinical variables.
Results—PDGF β receptor tyrosine phosphorylation in the 59 DCA samples was greater than in the 15 non-atherosclerotic LITA (mean (SD) 0.84 (0.67) v 0.17 (0.08) over a control standard, p atherectomy;  restenosis PMID:9616351

  19. AMPA receptor phosphorylation and recognition memory: learning-related, time-dependent changes in the chick brain following filial imprinting.

    Science.gov (United States)

    Solomonia, Revaz O; Meparishvili, Maia; Mikautadze, Ekaterine; Kunelauri, Nana; Apkhazava, David; McCabe, Brian J

    2013-04-01

    There is strong evidence that a restricted part of the chick forebrain, the intermediate medial mesopallium (IMM), stores information acquired through the learning process of visual imprinting. We have previously demonstrated that at 1 h but not 24 h after imprinting training, a learning-specific increase in the amount of membrane Thr286-autophosphorylated α-calcium/calmodulin-dependent protein kinase II (αCaMKII), and in the proportion of total αCaMKII that is phosphorylated, occurs in the IMM but not in a control brain region, the posterior pole of the nidopallium (PPN). αCaMKII directly phosphorylates Ser831 in the GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. In the present study we have inquired whether the learning-related increase in αCaMKII autophosphorylation is followed by changes in the Ser831 phosphorylation of GluA1 (P-GluA1) and in the total amount of this subunit (T-GluA1). Trained chicks together with untrained control chicks were killed either 1 or 24 h after training. Tissue was removed from the IMM together with tissue from the PPN as a control. Amounts of P-GluA1 and T-GluA1 were measured. In the left IMM of the 1 h group the P-GluA1/T-GluA1 ratio increased in a learning-specific way. No learning-related changes were observed in other brain regions at 1 h or in any region 24 h after training. The results indicate that a time- and regionally-dependent, learning-specific increase in GluA1 phosphorylation occurs early in recognition memory formation.

  20. Mutation of androgen receptor N-terminal phosphorylation site Tyr-267 leads to inhibition of nuclear translocation and DNA binding.

    Directory of Open Access Journals (Sweden)

    Mehmet Karaca

    Full Text Available Reactivation of androgen receptor (AR may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.

  1. Deficiency of Lipoprotein Lipase in Neurons Decreases AMPA Receptor Phosphorylation and Leads to Neurobehavioral Abnormalities in Mice.

    Directory of Open Access Journals (Sweden)

    Tian Yu

    Full Text Available Alterations in lipid metabolism have been found in several neurodegenerative disorders, including Alzheimer's disease. Lipoprotein lipase (LPL hydrolyzes triacylglycerides in lipoproteins and regulates lipid metabolism in multiple organs and tissues, including the central nervous system (CNS. Though many brain regions express LPL, the functions of this lipase in the CNS remain largely unknown. We developed mice with neuron-specific LPL deficiency that became obese on chow by 16 wks in homozygous mutant mice (NEXLPL-/- and 10 mo in heterozygous mice (NEXLPL+/-. In the present study, we show that 21 mo NEXLPL+/- mice display substantial cognitive function decline including poorer learning and memory, and increased anxiety with no difference in general motor activities and exploratory behavior. These neurobehavioral abnormalities are associated with a reduction in the 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl propanoic acid (AMPA receptor subunit GluA1 and its phosphorylation, without any alterations in amyloid β accumulation. Importantly, a marked deficit in omega-3 and omega-6 polyunsaturated fatty acids (PUFA in the hippocampus precedes the development of the neurobehavioral phenotype of NEXLPL+/- mice. And, a diet supplemented with n-3 PUFA can improve the learning and memory of NEXLPL+/- mice at both 10 mo and 21 mo of age. We interpret these findings to indicate that LPL regulates the availability of PUFA in the CNS and, this in turn, impacts the strength of synaptic plasticity in the brain of aging mice through the modification of AMPA receptor and its phosphorylation.

  2. Sigma-1 receptor regulates Tau phosphorylation and axon extension by shaping p35 turnover via myristic acid.

    Science.gov (United States)

    Tsai, Shang-Yi A; Pokrass, Michael J; Klauer, Neal R; Nohara, Hiroshi; Su, Tsung-Ping

    2015-05-26

    Dysregulation of cyclin-dependent kinase 5 (cdk5) per relative concentrations of its activators p35 and p25 is implicated in neurodegenerative diseases. P35 has a short t½ and undergoes rapid proteasomal degradation in its membrane-bound myristoylated form. P35 is converted by calpain to p25, which, along with an extended t½, promotes aberrant activation of cdk5 and causes abnormal hyperphosphorylation of tau, thus leading to the formation of neurofibrillary tangles. The sigma-1 receptor (Sig-1R) is an endoplasmic reticulum chaperone that is implicated in neuronal survival. However, the specific role of the Sig-1R in neurodegeneration is unclear. Here we found that Sig-1Rs regulate proper tau phosphorylation and axon extension by promoting p35 turnover through the receptor's interaction with myristic acid. In Sig-1R-KO neurons, a greater accumulation of p35 is seen, which results from neither elevated transcription of p35 nor disrupted calpain activity, but rather to the slower degradation of p35. In contrast, Sig-1R overexpression causes a decrease of p35. Sig-1R-KO neurons exhibit shorter axons with lower densities. Myristic acid is found here to bind Sig-1R as an agonist that causes the dissociation of Sig-1R from its cognate partner binding immunoglobulin protein. Remarkably, treatment of Sig-1R-KO neurons with exogenous myristic acid mitigates p35 accumulation, diminishes tau phosphorylation, and restores axon elongation. Our results define the involvement of Sig-1Rs in neurodegeneration and provide a mechanistic explanation that Sig-1Rs help maintain proper tau phosphorylation by potentially carrying and providing myristic acid to p35 for enhanced p35 degradation to circumvent the formation of overreactive cdk5/p25.

  3. Lefty Blocks a Subset of TGFβ Signals by Antagonizing EGF-CFC Coreceptors

    Science.gov (United States)

    Cheng, Simon K; Olale, Felix; Brivanlou, Ali H

    2004-01-01

    Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFβ signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFβ signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFβ agonists and antagonists and suggest that subtle sequence variations in TGFβ signals result in greater ligand diversity. PMID:14966532

  4. Lefty blocks a subset of TGFbeta signals by antagonizing EGF-CFC coreceptors.

    Directory of Open Access Journals (Sweden)

    Simon K Cheng

    2004-02-01

    Full Text Available Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFbeta signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFbeta signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFbeta agonists and antagonists and suggest that subtle sequence variations in TGFbeta signals result in greater ligand diversity.

  5. ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor

    Energy Technology Data Exchange (ETDEWEB)

    Inagaki, Junko; Takahashi, Katsuyuki; Ogawa, Hiroko; Asano, Keiichi; Faruk Hatipoglu, Omer; Zeynel Cilek, Mehmet; Obika, Masanari; Ohtsuki, Takashi [Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama (Japan); Hofmann, Matthias [Department of Dermatology, Venereology and Allergology, Goethe University, Frankfurt (Germany); Kusachi, Shozo [Department of Medical Technology, Okayama University Graduate School of Health Sciences, Okayama (Japan); Ninomiya, Yoshifumi [Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama (Japan); Hirohata, Satoshi, E-mail: hirohas@cc.okayama-u.ac.jp [Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama (Japan); International Center, Okayama University, Okayama (Japan)

    2014-05-01

    Angiogenesis and lymphangiogenesis play roles in malignant tumor progression, dissemination, and metastasis. ADAMTS1, a member of the matrix metalloproteinase family, is known to inhibit angiogenesis. Recombinant ADAMTS1 was shown to strongly inhibit angiogenesis. We investigated whether ADAMTS1 inhibited lymphangiogenesis in the present study. We examined cell proliferation and cell migration in normal human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) transduced with or without adenoviral human ADAMTS1 gene therapy. We then examined the VEGFC/VEGFR3 signal transduction pathway in ADAMTS1-transduced HMVEC-dLy. Cell proliferation and tube formation in Matrigel were significantly lower with transduced ADAMTS1 than with control (non-transduced HMVEC-dLy). The phosphorylation of VEGFR3 was also attenuated by ADAMTS1 gene therapy in HMVEC-dLy. Immunoprecipitation assays revealed that ADAMTS1 formed a complex with VEGFC. Our results demonstrated that ADAMTS1 inhibited lymphangiogenesis in vitro. The data highlight the new function of ADAMTS1 in the regulation of lymphangiogenesis and the therapeutic potential of ADAMTS1 in cancer therapy. - Highlights: • ADAMTS1 significantly inhibited tube formation and cell proliferation in HMVEC-dLy. • Reduced lymph endothelial cell migration in ADAMTS1 transduced co-culture systems. • VEGFC-stimulated phosphorylation of VEGFR3 is attenuated by ADAMTS1. • Reduced phosphorylation of Akt and ERK1/2 in ADAMTS1 treated HMVEC-dLy. • ADAMTS1 binds directly to VEGFC.

  6. ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor

    International Nuclear Information System (INIS)

    Inagaki, Junko; Takahashi, Katsuyuki; Ogawa, Hiroko; Asano, Keiichi; Faruk Hatipoglu, Omer; Zeynel Cilek, Mehmet; Obika, Masanari; Ohtsuki, Takashi; Hofmann, Matthias; Kusachi, Shozo; Ninomiya, Yoshifumi; Hirohata, Satoshi

    2014-01-01

    Angiogenesis and lymphangiogenesis play roles in malignant tumor progression, dissemination, and metastasis. ADAMTS1, a member of the matrix metalloproteinase family, is known to inhibit angiogenesis. Recombinant ADAMTS1 was shown to strongly inhibit angiogenesis. We investigated whether ADAMTS1 inhibited lymphangiogenesis in the present study. We examined cell proliferation and cell migration in normal human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) transduced with or without adenoviral human ADAMTS1 gene therapy. We then examined the VEGFC/VEGFR3 signal transduction pathway in ADAMTS1-transduced HMVEC-dLy. Cell proliferation and tube formation in Matrigel were significantly lower with transduced ADAMTS1 than with control (non-transduced HMVEC-dLy). The phosphorylation of VEGFR3 was also attenuated by ADAMTS1 gene therapy in HMVEC-dLy. Immunoprecipitation assays revealed that ADAMTS1 formed a complex with VEGFC. Our results demonstrated that ADAMTS1 inhibited lymphangiogenesis in vitro. The data highlight the new function of ADAMTS1 in the regulation of lymphangiogenesis and the therapeutic potential of ADAMTS1 in cancer therapy. - Highlights: • ADAMTS1 significantly inhibited tube formation and cell proliferation in HMVEC-dLy. • Reduced lymph endothelial cell migration in ADAMTS1 transduced co-culture systems. • VEGFC-stimulated phosphorylation of VEGFR3 is attenuated by ADAMTS1. • Reduced phosphorylation of Akt and ERK1/2 in ADAMTS1 treated HMVEC-dLy. • ADAMTS1 binds directly to VEGFC

  7. 15-Deoxy-Δ12,14-prostaglandin J2 and thiazolidinediones transactivate epidermal growth factor and platelet-derived growth factor receptors in vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Ichiki, Toshihiro; Tokunou, Tomotake; Fukuyama, Kae; Iino, Naoko; Masuda, Satoko; Takeshita, Akira

    2004-01-01

    Proliferation of vascular smooth muscle cells (VSMCs) is induced by various mitogens through activation of extracellular signal-regulated protein kinase (ERK) pathway. We recently reported that peroxisome proliferator-activated receptor (PPAR)γ activators such as 15-deoxy-Δ 12,14 -prostaglandin J2 (15-d-PGJ2) and thiazolidinediones (TZDs) activated MEK/ERK pathway through phosphatidylinositol 3-kinase (PI3-K) and induced proliferation of VSMCs. However, the precise mechanisms of PPARγ activators-induced activation of PI3-K/ERK pathway have not been determined. We examined whether transactivation of growth factor receptor is involved in this process. Stimulation of VSMCs with 15-d-PGJ2 or TZDs for 15 min induced phosphorylation of ERK1/2 and Akt. 15-d-PGJ2- or TZDs-induced phosphorylation of ERK1/2 and Akt was inhibited by AG1478, an inhibitor of epidermal growth factor receptor (EGF-R) as well as AG1295, an inhibitor of platelet derived growth factor receptor (PDGF-R). 15-d-PGJ2-induced phosphorylation of both EGF-R and PDGF-R. GM6001, a matrix metalloproteinase inhibitor, and PP2, a Src family protein kinase inhibitor, suppressed 15-d-PGJ2- and TZDs-induced phosphorylation of EGF-R and PDGFβ-R as well as activation of ERK1/2 and Akt. PDGFβ-R was co-immunoprecipitated with EGF-R, regardless of the presence or absence of 15-d-PGJ2. These data suggest that 15-d-PGJ2 and TZDs activate PI3-K/ERK pathway through Src family kinase- and matrix metalloproteinase-dependent transactivation of EGF-R and PDGF-R. Both receptors seemed to associate constitutively. This novel signaling mechanisms may contribute to diverse biological functions of PPARγ activators

  8. Calcium regulation of EGF-induced ERK5 activation: role of Lad1-MEKK2 interaction.

    Directory of Open Access Journals (Sweden)

    Zhong Yao

    Full Text Available The ERK5 cascade is a MAPK pathway that transmits both mitogenic and stress signals, yet its mechanism of activation is not fully understood. Using intracellular calcium modifiers, we found that ERK5 activation by EGF is inhibited both by the depletion and elevation of intracellular calcium levels. This calcium effect was found to occur upstream of MEKK2, which is the MAP3K of the ERK5 cascade. Co-immunoprecipitation revealed that EGF increases MEKK2 binding to the adaptor protein Lad1, and this interaction was reduced by the intracellular calcium modifiers, indicating that a proper calcium concentration is required for the interactions and transmission of EGF signals to ERK5. In vitro binding assays revealed that the proper calcium concentration is required for a direct binding of MEKK2 to Lad1. The binding of these proteins is not affected by c-Src-mediated phosphorylation on Lad1, but slightly affects the Tyr phosphorylation of MEKK2, suggesting that the interaction with Lad1 is necessary for full Tyr phosphorylation of MEKK2. In addition, we found that changes in calcium levels affect the EGF-induced nuclear translocation of MEKK2 and thereby its effect on the nuclear ERK5 activity. Taken together, these findings suggest that calcium is required for EGF-induced ERK5 activation, and this effect is probably mediated by securing proper interaction of MEKK2 with the upstream adaptor protein Lad1.

  9. Interaction of epidermal growth factor receptors with the cytoskeleton is related to receptor clustering

    NARCIS (Netherlands)

    van Belzen, N.; Spaargaren, M.; Verkleij, A. J.; Boonstra, J.

    1990-01-01

    Recently it has been established that cytoskeleton-associated epidermal growth factor (EGF) receptors are predominantly of the high-affinity class and that EGF induces a recruitment of low-affinity receptors to the cytoskeleton. The nature of this EGF-induced receptor-cytoskeleton interaction,

  10. GABA(B) receptor phosphorylation regulates KCTD12-induced K+ current desensitization

    Czech Academy of Sciences Publication Activity Database

    Adelfinger, L.; Tureček, Rostislav; Ivankova, K.; Jensen, A. A.; Moss, S. J.; Gassmann, M.; Bettler, B.

    2014-01-01

    Roč. 91, č. 3 (2014), s. 369-379 ISSN 0006-2952 Institutional support: RVO:68378041 Keywords : GABA-B * G-protein coupled receptor * GPCR Subject RIV: FH - Neurology Impact factor: 5.009, year: 2014

  11. Pregnenolone sulfate modulation of N-methyl-d-aspartate receptors is phosphorylation dependent

    Czech Academy of Sciences Publication Activity Database

    Petrovič, Miloš; Sedláček, Miloslav; Cais, Ondřej; Horák, Martin; Chodounská, Hana; Vyklický ml., Ladislav

    2009-01-01

    Roč. 160, č. 3 (2009), s. 616-628 ISSN 0306-4522 R&D Projects: GA ČR(CZ) GA309/07/0271; GA ČR(CZ) GA203/08/1498; GA MŠk(CZ) LC554; GA MŠk(CZ) 1M0517 Grant - others:EC(XE) LSHM-CT-2007-037765 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z40550506 Keywords : NMDA * patch-clamp * phosphorylation Subject RIV: ED - Physiology Impact factor: 3.292, year: 2009

  12. Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-Kinase Inhibitors.

    Science.gov (United States)

    Marlowe, Timothy A; Lenzo, Felicia L; Figel, Sheila A; Grapes, Abigail T; Cance, William G

    2016-12-01

    Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms that drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTK) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAK's critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein-protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. In addition, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: (i) the rapid phosphorylation and activation of RTK signaling pathways in RTK High cells and (ii) the long-term acquisition of RTKs novel to the parental cell line in RTK Low cells. Finally, HER2 +: cancer cells displayed resistance to FAK-kinase inhibition in 3D growth assays using a HER2 isogenic system and HER2 + cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. Mol Cancer Ther; 15(12); 3028-39. ©2016 AACR. ©2016 American Association for Cancer Research.

  13. Genistein and tyrphostin AG556 decrease ultra-rapidly activating delayed rectifier K+ current of human atria by inhibiting EGF receptor tyrosine kinase.

    Science.gov (United States)

    Xiao, Guo-Sheng; Zhang, Yan-Hui; Wu, Wei; Sun, Hai-Ying; Wang, Yan; Li, Gui-Rong

    2017-03-01

    The ultra-rapidly activating delayed rectifier K + current I Kur (encoded by K v 1.5 or KCNA5) plays an important role in human atrial repolarization. The present study investigates the regulation of this current by protein tyrosine kinases (PTKs). Whole-cell patch voltage clamp technique and immunoprecipitation and Western blotting analysis were used to investigate whether the PTK inhibitors genistein, tyrphostin AG556 (AG556) and PP2 regulate human atrial I Kur and hKv1.5 channels stably expressed in HEK 293 cells. Human atrial I Kur was decreased by genistein (a broad-spectrum PTK inhibitor) and AG556 (a highly selective EGFR TK inhibitor) in a concentration-dependent manner. Inhibition of I Kur induced by 30 μM genistein or 10 μM AG556 was significantly reversed by 1 mM orthovanadate (a protein tyrosine phosphatase inhibitor). Similar results were observed in HEK 293 cells stably expressing hK v 1.5 channels. On the other hand, the Src family kinase inhibitor PP2 (1 μM) slightly enhanced I Kur and hK v 1.5 current, and the current increase was also reversed by orthovanadate. Immunoprecipitation and Western blotting analysis showed that genistein, AG556, and PP2 decreased tyrosine phosphorylation of hK v 1.5 channels and that the decrease was countered by orthovanadate. The PTK inhibitors genistein and AG556 decrease human atrial I Kur and cloned hK v 1.5 channels by inhibiting EGFR TK, whereas the Src kinase inhibitor PP2 increases I Kur and hK v 1.5 current. These results imply that EGFR TK and the soluble Src kinases may have opposite effects on human atrial I Kur . © 2017 The British Pharmacological Society.

  14. The phosphorylation state of CD3gamma influences T cell responsiveness and controls T cell receptor cycling

    DEFF Research Database (Denmark)

    Dietrich, J; Backstrom, T; Lauritsen, JP

    1998-01-01

    The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3gamma. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR...... the phosphorylation state of CD3gamma and T cell responsiveness. Based on these observations a physiological role of CD3gamma and TCR cycling is proposed....... and the mechanisms involved in the sorting events following PKC-induced internalization are not known. In this study, we demonstrated that following PKC-induced internalization, the TCR is recycled back to the cell surface in a functional state. TCR recycling was dependent on dephosphorylation of CD3gamma, probably...

  15. Increased expression of heparin binding EGF (HB-EGF), amphiregulin, TGF alpha and epiregulin in androgen-independent prostate cancer cell lines.

    DEFF Research Database (Denmark)

    Tørring, Niels; Sørensen, Boe Sandahl; Nexø, Ebba

    2000-01-01

    BACKGROUND: The proliferation of androgen-independent prostate cancer cell lines has previously been shown to be influenced by an autocrine loop of the epidermal growth factor (EGF) system. This observation has alerted us to study the expression of ligands and receptors from the EGF......-system in prostate cell lines. METHODS: The expression of the EGF system was determined by quantitative RT-PCR and ELISA in the normal prostate epithelial cell line (PNT1A), in the androgen sensitive-(LNCaP), and the androgen-independent (DU145 and PC3) prostate cancer cell lines. RESULTS: The expression of m...... which exhibit low expression of HER1. Similar results were obtained by ELISA. CONCLUSIONS: The data indicates a selective up-regulation of a subclass of ligands of the EGF-system in androgen-independent prostate cancer cell lines. We suggest this could be a mechanism to escape androgen dependence...

  16. Spinal Tolerance and Dependence: Some Observations on the Role of Spinal N-Methyl-D-Aspartate Receptors and Phosphorylation in the Loss of Opioid Analgesic Responses

    Directory of Open Access Journals (Sweden)

    Tony L Yaksh

    2000-01-01

    Full Text Available The continuous delivery of opiates can lead to a reduction in analgesic effects. In humans, as in other animals, some component of this change in sensitivity seems likely to have a strong pharmacodynamic component. Such loss of effect, deemed to be tolerance in the present article, can be readily demonstrated in animals with repeated bolus and continuous intrathecal infusion of mu and delta opioids and alpha-2 adrenergic agonists. Research has shown that this loss of effect can be diminished by concurrent treatment with N-methyl-D-aspartate (NMDA receptor antagonists and by the suppression of the activity of spinal protein kinase C (PKC. This suggests in part the probable role of PKC-mediated phosphorylation in the right shift in the dose-effect curves observed with continuous opiate or adrenergic exposure. Importantly, this right shift is seen to occur in parallel with an increase in the phosphorylating activity in the dorsal horn and in the expression of several PKC isozymes. The target of this phosphorylation is not certain. Phosphorylation of the NMDA receptor enhances its functionality, while phosphorylation of the opioid receptor or associated channels seems to diminish their activity or to enhance internalization. While the focus is on several specific components, the accumulating data emphasize the biological complexity of these changes in spinal drug reactivity.

  17. A neuroligin-1-derived peptide stimulates phosphorylation of the NMDA receptor NR1 subunit and rescues MK-801-induced decrease in long-term potentiation and memory impairment

    DEFF Research Database (Denmark)

    Korshunova, Irina; Gjørlund, Michelle D; Jacobsen, Sylwia Owczarek

    2015-01-01

    neurolide-1 effects on short- and long-term social and spatial memory in social recognition, Morris water-maze, and Y-maze tests. We found that subcutaneous neurolide-1 administration, restored hippocampal LTP compromised by NMDA receptor inhibitor MK-801. It counteracted MK-801-induced memory deficit...... in the water-maze and Y-maze tests after long-term treatment (24 h and 1-2 h before the test), but not after short-term exposure (1-2 h). Long-term exposure to neurolide-1 also facilitated social recognition memory. In addition, neurolide-1-induced phosphorylation of the NMDA receptor NR1 subunit on a site...... receptor phosphorylation after treatment with NL1 or a mimetic peptide, neurolide-1, was quantified by immunoblotting. Subsequently, we investigated effects of neurolide-1 on long-term potentiation (LTP) induction in hippocampal slices compromised by NMDA receptor inhibitor MK-801. Finally, we investigated...

  18. Both Autocrine Signaling and Paracrine Signaling of HB-EGF Enhance Ocular Neovascularization.

    Science.gov (United States)

    Inoue, Yuki; Shimazawa, Masamitsu; Nakamura, Shinsuke; Takata, Shinsuke; Hashimoto, Yuhei; Izawa, Hiroshi; Masuda, Tomomi; Tsuruma, Kazuhiro; Sakaue, Tomohisa; Nakayama, Hironao; Higashiyama, Shigeki; Hara, Hideaki

    2018-01-01

    The incidence of blindness is increasing because of the increase in abnormal ocular neovascularization. Anti-VEGF (vascular endothelial growth factor) therapies have led to good results, although they are not a cure for the blindness. The purpose of this study was to determine what role HB-EGF (heparin-binding epidermal growth factor-like growth factor) plays in ocular angiogenesis. We examined the role played by HB-EGF in ocular neovascularization in 2 animal models of neovascularization: laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy. We also studied human retinal microvascular endothelial cells in culture. Our results showed that the neovascularization was decreased in both the CNV and oxygen-induced retinopathy models in HB-EGF conditional knockout mice compared with that in wild-type mice. Moreover, the expressions of HB-EGF and VEGF were increased after laser-induced CNV and oxygen-induced retinopathy, and their expression sites were located around the neovascular areas. Exposure of human retinal microvascular endothelial cells to HB-EGF and VEGF increased their proliferation and migration, and CRM-197 (cross-reactive material-197), an HB-EGF inhibitor, decreased the HB-EGF-induced and VEGF-induced cell proliferation and migration. VEGF increased the expression of HB-EGF mRNA. VEGF-dependent activation of EGFR (epidermal growth factor receptor)/ERK1/2 (extracellular signal-regulated kinase 1/2) signaling and cell proliferation of endothelial cells required stimulation of the ADAM17 (a disintegrin and metalloprotease) and ADAM12. CRM-197 decreased the grades of the fluorescein angiograms and size of the CNV areas in marmoset monkeys. These findings suggest that HB-EGF plays an important role in the development of CNV. Therefore, further investigations of HB-EGF are needed as a potential therapeutic target in the treatment of exudative age-related macular degeneration. © 2017 American Heart Association, Inc.

  19. Concomitant action of structural elements and receptor phosphorylation determines arrestin-3 interaction with the free fatty acid receptor FFA4

    DEFF Research Database (Denmark)

    Butcher, Adrian J; Hudson, Brian D; Shimpukade, Bharat

    2014-01-01

    In addition to being nutrients, free fatty acids act as signaling molecules by activating a family of G protein-coupled receptors. Among these is FFA4, previously called GPR120, which responds to medium and long chain fatty acids, including health-promoting ω-3 fatty acids, which have been implic...

  20. Phosphorylation of threonine residues on Shc promotes ligand binding and mediates crosstalk between MAPK and Akt pathways in breast cancer cells

    KAUST Repository

    Suen, K.M.

    2017-12-06

    Scaffold proteins play important roles in regulating signalling network fidelity, the absence of which is often the basis for diseases such as cancer. In the present work, we show that the prototypical scaffold protein Shc is phosphorylated by the extracellular signal-regulated kinase, Erk. In addition, Shc threonine phosphorylation is specifically up-regulated in two selected triple-negative breast cancer (TNBC) cell lines. To explore how Erk-mediated threonine phosphorylation on Shc might play a role in the dysregulation of signalling events, we investigated how Shc affects pathways downstream of EGF receptor. Using an in vitro model and biophysical analysis, we show that Shc threonine phosphorylation is responsible for elevated Akt and Erk signalling, potentially through the recruitment of the 14-3-3 ζ and Pin-1 proteins.

  1. Phosphorylation of threonine residues on Shc promotes ligand binding and mediates crosstalk between MAPK and Akt pathways in breast cancer cells

    KAUST Repository

    Suen, K.M.; Lin, C.C.; Seiler, C.; George, R.; Poncet-Montange, G.; Biter, A.B.; Ahmed, Z.; Arold, Stefan T.; Ladbury, J.E.

    2017-01-01

    Scaffold proteins play important roles in regulating signalling network fidelity, the absence of which is often the basis for diseases such as cancer. In the present work, we show that the prototypical scaffold protein Shc is phosphorylated by the extracellular signal-regulated kinase, Erk. In addition, Shc threonine phosphorylation is specifically up-regulated in two selected triple-negative breast cancer (TNBC) cell lines. To explore how Erk-mediated threonine phosphorylation on Shc might play a role in the dysregulation of signalling events, we investigated how Shc affects pathways downstream of EGF receptor. Using an in vitro model and biophysical analysis, we show that Shc threonine phosphorylation is responsible for elevated Akt and Erk signalling, potentially through the recruitment of the 14-3-3 ζ and Pin-1 proteins.

  2. Chronic cerebral hypoperfusion-induced impairment of Aβ clearance requires HB-EGF-dependent sequential activation of HIF1α and MMP9.

    Science.gov (United States)

    Ashok, Anushruti; Rai, Nagendra Kumar; Raza, Waseem; Pandey, Rukmani; Bandyopadhyay, Sanghamitra

    2016-11-01

    Chronic cerebral hypoperfusion (CCH) manifests Alzheimer's Disease (AD) neuropathology, marked by increased amyloid beta (Aβ). Besides, hypoxia stimulates Heparin-binding EGF-like growth factor (HB-EGF) mRNA expression in the hippocampus. However, involvement of HB-EGF in CCH-induced Aβ pathology remains unidentified. Here, using Bilateral Common Carotid Artery Occlusion mouse model, we explored the mechanism of HB-EGF regulated Aβ induction in CCH. We found that HB-EGF inhibition suppressed, while exogenous-HB-EGF triggered hippocampal Aβ, proving HB-EGF-dependent Aβ increase. We also detected that HB-EGF affected the expression of primary Aβ transporters, receptor for advanced glycation end-products (RAGE) and lipoprotein receptor-related protein-1 (LRP-1), indicating impaired Aβ clearance across the blood-brain barrier (BBB). An HB-EGF-dependent loss in BBB integrity supported impaired Aβ clearance. The effect of HB-EGF on Amyloid Precursor Protein pathway was relatively insignificant, suggesting a lesser effect on Aβ generation. Delving into BBB disruption mechanism demonstrated HB-EGF-mediated stimulation of Matrix metalloprotease-9 (MMP9), which affected BBB via HB-EGF-ectodomain shedding and epidermal growth factor receptor activation. Examining the intersection of HB-EGF-regulated pathway and hypoxia revealed HB-EGF-dependent increase in transcription factor, Hypoxia-inducible factor-1alpha (HIF1α). Further, via binding to hypoxia-responsive elements in MMP9 gene, HIF1α stimulated MMP9 expression, and therefore appeared as a prominent intermediary in HB-EGF-induced BBB damage. Overall, our study reveals the essential role of HB-EGF in triggering CCH-mediated Aβ accumulation. The proposed mechanism involves an HB-EGF-dependent HIF1α increase, generating MMP9 that stimulates soluble-HB-EGF/EGFR-induced BBB disintegration. Consequently, CCH-mediated hippocampal RAGE and LRP-1 deregulation together with BBB damage impair Aβ transport and clearance

  3. The modulation of radiosensitivity by combined treatment of selective COX-2 inhibitor, NS 398 and EGF receptor blocker AG 1478 in HeLa cell line

    Energy Technology Data Exchange (ETDEWEB)

    Youn, Seon Min; Oh, Young Kee; Kim, Joo Heon; Park, Mi Ja; Seong, In Ock [Eulji University School of Medicine, Daejeon (Korea, Republic of); Kang, Ki Mun; Chai, Gyu Yong [Gyeongsang National University College of Medicine, Jinju (Korea, Republic of)

    2005-03-15

    Selective inhibition of multiple molecular targets may improve the antitumor activity of radiation. Two specific inhibitors of selective cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) were combined with radiation on the HeLa cell line. To investigate cooperative mechanism with selective COX-2 inhibitor and EGFR blocker, in vitro experiments were done. Antitumor effect was obtained by growth inhibition and apoptosis analysis by annexin V-Flous method. Radiation modulation effects were determined by the clonogenic cell survival assay. Surviving fractions at 2 Gy (SF{sub 2}) and dose enhancement radio at a surviving fraction of 0.25 were evaluated. To investigate the mechanism of the modulation of radiosensitivity, the cell cycle analyses were done by flow cytometry. The bcl-2 and bax expressions were analyzed by western blot. A cooperative effect were observed on the apoptosis of the HeLa cell line when combination of the two drugs, AG 1478 and NS 398 with radiation at the lowest doses, apoptosis of 22.70% compare with combination of the one drug with radiation, apoptosis of 8.49%. In cell cycle analysis, accumulation of cell on G{sub 0}/G{sub 1} phase and decrement of S phase fraction was observed from 24 hours to 72 hours after treatment with radiation, AG 1478 and NS 398. The combination of NS 398 and AG 1478 enhanced radiosensitivity in a concentration-dependent manner in HeLa cells with dose enhancement ratios of 3.00 and SF{sub 2} of 0.12 but the combination of one drug with radiation was not enhanced radiosensitivity with dose enhancement ratios of 1.12 and SF2 of 0.68 ({rho} = 0.005). The expression levels of bcl-2 and bax were reduced when combined with AG 1478 and NS 398. Our results indicate that the selective COX-2 inhibitor and EGFR blocker combined with radiation have potential additive or cooperative effects on radiation treatment and may act through various mechanisms including direct inhibition of tumor cell proliferation

  4. The modulation of radiosensitivity by combined treatment of selective COX-2 inhibitor, NS 398 and EGF receptor blocker AG 1478 in HeLa cell line

    International Nuclear Information System (INIS)

    Youn, Seon Min; Oh, Young Kee; Kim, Joo Heon; Park, Mi Ja; Seong, In Ock; Kang, Ki Mun; Chai, Gyu Yong

    2005-01-01

    Selective inhibition of multiple molecular targets may improve the antitumor activity of radiation. Two specific inhibitors of selective cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) were combined with radiation on the HeLa cell line. To investigate cooperative mechanism with selective COX-2 inhibitor and EGFR blocker, in vitro experiments were done. Antitumor effect was obtained by growth inhibition and apoptosis analysis by annexin V-Flous method. Radiation modulation effects were determined by the clonogenic cell survival assay. Surviving fractions at 2 Gy (SF 2 ) and dose enhancement radio at a surviving fraction of 0.25 were evaluated. To investigate the mechanism of the modulation of radiosensitivity, the cell cycle analyses were done by flow cytometry. The bcl-2 and bax expressions were analyzed by western blot. A cooperative effect were observed on the apoptosis of the HeLa cell line when combination of the two drugs, AG 1478 and NS 398 with radiation at the lowest doses, apoptosis of 22.70% compare with combination of the one drug with radiation, apoptosis of 8.49%. In cell cycle analysis, accumulation of cell on G 0 /G 1 phase and decrement of S phase fraction was observed from 24 hours to 72 hours after treatment with radiation, AG 1478 and NS 398. The combination of NS 398 and AG 1478 enhanced radiosensitivity in a concentration-dependent manner in HeLa cells with dose enhancement ratios of 3.00 and SF 2 of 0.12 but the combination of one drug with radiation was not enhanced radiosensitivity with dose enhancement ratios of 1.12 and SF2 of 0.68 (ρ = 0.005). The expression levels of bcl-2 and bax were reduced when combined with AG 1478 and NS 398. Our results indicate that the selective COX-2 inhibitor and EGFR blocker combined with radiation have potential additive or cooperative effects on radiation treatment and may act through various mechanisms including direct inhibition of tumor cell proliferation, suppression of tumor cell

  5. Disruption of δ-opioid receptor phosphorylation at threonine 161 attenuates morphine tolerance in rats with CFA-induced inflammatory hypersensitivity.

    Science.gov (United States)

    Chen, Hai-Jing; Xie, Wei-Yan; Hu, Fang; Zhang, Ying; Wang, Jun; Wang, Yun

    2012-04-01

    Our previous study identified Threonine 161 (Thr-161), located in the second intracellular loop of the δ-opioid receptor (DOR), as the only consensus phosphorylation site for cyclin-dependent kinase 5 (Cdk5). The aim of this study was to assess the function of DOR phosphorylation by Cdk5 in complete Freund's adjuvant (CFA)-induced inflammatory pain and morphine tolerance. Dorsal root ganglion (DRG) neurons of rats with CFA-induced inflammatory pain were acutely dissociated and the biotinylation method was used to explore the membrane localization of phosphorylated DOR at Thr-161 (pThr-161-DOR), and paw withdrawal latency was measured after intrathecal delivery of drugs or Tat-peptide, using a radiant heat stimulator in rats with CFA-induced inflammatory pain. Both the total amount and the surface localization of pThr-161-DOR were significantly enhanced in the ipsilateral DRG following CFA injection. Intrathecal delivery of the engineered Tat fusion-interefering peptide corresponding to the second intracellular loop of DOR (Tat-DOR-2L) increased inflammatory hypersensitivity, and inhibited DOR- but not µ-opioid receptor-mediated spinal analgesia in CFA-treated rats. However, intrathecal delivery of Tat-DOR-2L postponed morphine antinociceptive tolerance in rats with CFA-induced inflammatory pain. Phosphorylation of DOR at Thr-161 by Cdk5 attenuates hypersensitivity and potentiates morphine tolerance in rats with CFA-induced inflammatory pain, while disruption of the phosphorylation of DOR at Thr-161 attenuates morphine tolerance.

  6. Ghrelin upregulates the phosphorylation of the GluN2B subunit of the NMDA receptor by activating GHSR1a and Fyn in the rat hippocampus.

    Science.gov (United States)

    Berrout, Liza; Isokawa, Masako

    2018-01-01

    Ghrelin and its receptor GHSR1a have been shown to exert numerous physiological functions in the brain, in addition to the well-established orexigenic role in the hypothalamus. Earlier work indicated that ghrelin stimulated the phosphorylation of the GluN1 subunit of the NMDA receptor (NMDAR) and enhanced synaptic transmission in the hippocampus. In the present study, we report that the exogenous application of ghrelin increased GluN2B phosphorylation. This increase was independent of GluN2B subunit activity or NMDAR channel activity. However, it depended on the activation of GHSR1a and Fyn as it was blocked by D-Lys3-GHRP-6 and PP2, respectively. Inhibitors for G-protein-regulated second messengers, such as Rp-cAMP, H89, TBB, ryanodine, and thapsigargin, unexpectedly enhanced GluN2B phosphorylation, suggesting that cAMP, PKA, casein kinase II, and cytosolic calcium signaling may oppose to the effect of ghrelin on the phosphorylation of GluN2B. Our findings suggest that 1) GluN2B is likely a molecular target of ghrelin and GHSR1a-driven signaling cascades, and 2) the ghrelin-mediated phosphorylation of GluN2B depends on Fyn activation under complex negative regulation by other second messengers. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. 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...... of Akt and Erk were quite uniformly expressed in the categories; negative, moderate or strong. In univariate analysis, expression of total HER2, pHER1, pHER2 and pHER3 was significantly associated with poor disease-free survival. Strong HER4 expression was associated with prolonged disease-free as well...

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

    OpenAIRE

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

    2016-01-01

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

  9. Expression of FLNa in human melanoma cells regulates the function of integrin α1β1 and phosphorylation and localisation of PKB/AKT/ERK1/2 kinases.

    Science.gov (United States)

    Krebs, Kristi; Ruusmann, Anu; Simonlatser, Grethel; Velling, Teet

    2015-12-01

    FLNa is a ubiquitous cytoskeletal protein that links transmembrane receptors, including integrins, to F-actin and functions as a signalling intermediate. We investigated FLNa's role in the function of integrin-type collagen receptors, EGF-EGFR signalling and regulation of PKB/Akt and ERK1/2. Using FLNa-deficient M2 human melanoma cells, and same cells expressing EGFP-FLNa (M2F) or its Ig-like repeats 1-8+24, 8-15+24 and 16-24, we found that in M2F and M2 8-15+24 cells, EGF induced the increased phosphorylation of PKB/Akt and ERK1/2. In M2F cells EGF induced the localisation of these kinases to cell nucleus and lamellipodia, respectively, and the ERK1/2 phosphorylation-dependent co-immunoprecipitation of FLNa with ERK1/2. Only M2F and M2 8-15+24 cells adhered to and spread on type I collagen whereas on fibronectin all cells behaved similarly. α1β1 and α2β1 were the integrin-type collagen receptors expressed on these cells with primarily α1β1 localising to focal contacts and affecting cell adhesion and migration in a manner dependent on FLNa or its Ig-like repeats 8-15. Our results suggest a role for FLNa repeats 8-15 in the α1-subunit-dependent regulation of integrin α1β1 function, EGF-EGFR signalling to PKB/Akt and ERK1/2, identify ERK1/2 in EGF-induced FLNa-associated protein complexes, and show that the function of different integrins is subjected to differential regulation by FLNa. Copyright © 2015. Published by Elsevier GmbH.

  10. IGF-I Stimulates Cooperative Interaction between the IGF-I Receptor and CSK Homologous Kinase that Regulates SHPS-1 Phosphorylation in Vascular Smooth Muscle Cells

    Science.gov (United States)

    Radhakrishnan, Yashwanth; Shen, Xinchun; Maile, Laura A.; Xi, Gang

    2011-01-01

    IGF-I plays an important role in smooth muscle cell proliferation and migration. In vascular smooth muscle cells cultured in 25 mm glucose, IGF-I stimulated a significant increase in Src homology 2 domain containing protein tyrosine phosphatase substrate-1 (SHPS-1) phosphorylation compared with 5 mm glucose and this increase was required for smooth muscle cell proliferation. A proteome-wide screen revealed that carboxyl-terminal SRC kinase homologous kinase (CTK) bound directly to phosphotyrosines in the SHPS-1 cytoplasmic domain. Because the kinase(s) that phosphorylates these tyrosines in response to IGF-I is unknown, we determined the roles of IGF-I receptor (IGF-IR) and CTK in mediating SHPS-1 phosphorylation. After IGF-I stimulation, CTK was recruited to IGF-IR and subsequently to phospho-SHPS-1. Expression of an IGF-IR mutant that eliminated CTK binding reduced CTK transfer to SHPS-1, SHPS-1 phosphorylation, and cell proliferation. IGF-IR phosphorylated SHPS-1, which provided a binding site for CTK. CTK recruitment to SHPS-1 resulted in a further enhancement of SHPS-1 phosphorylation. CTK knockdown also impaired IGF-I-stimulated SHPS-1 phosphorylation and downstream signaling. Analysis of specific tyrosines showed that mutation of tyrosines 428/452 in SHPS-1 to phenylalanine reduced SHPS-1 phosphorylation but allowed CTK binding. In contrast, the mutation of tyrosines 469/495 inhibited IGF-IR-mediated the phosphorylation of SHPS-1 and CTK binding, suggesting that IGF-IR phosphorylated Y469/495, allowing CTK binding, and that CTK subsequently phosphorylated Y428/452. Based on the above findings, we conclude that after IGF-I stimulation, CTK is recruited to IGF-IR and its recruitment facilitates CTK's subsequent association with phospho-SHPS-1. This results in the enhanced CTK transfer to SHPS-1, and the two kinases then fully phosphorylate SHPS-1, which is necessary for IGF-I stimulated cellular proliferation. PMID:21799000

  11. 9-Hydroxystearic acid interferes with EGF signalling in a human colon adenocarcinoma

    International Nuclear Information System (INIS)

    Calonghi, Natalia; Pagnotta, Eleonora; Parolin, Carola; Tognoli, Cristina; Boga, Carla; Masotti, Lanfranco

    2006-01-01

    The epidermal growth factor has long been known to be strictly correlated with the highly proliferating activities of cancer cells and primary tumors. Moreover, in the nucleus, the epidermal growth factor/epidermal growth factor receptor complex (EGF/EGFR) functions as a transcriptional regulator that activates the cyclin D1 gene. 9-hydroxystearic acid (9-HSA) induces cell proliferation arrest and differentiation in HT29 colon cancer cells by inhibiting histone deacetylase 1 (HDAC1). 9-HSA-treated HT29, when stimulated with EGF, are not responsive and surprisingly undergo a further arrest. In order to understand the mechanisms of this effect, we analyzed the degree of internalization of the EGF/EGFR complex and its interactions with HDAC1. It appears that HDAC1, as modified by 9-HSA, is unable to associate with cyclin D1, interfering with the cell proliferation program, and sequesters the EGF/EGFR complex interrupting the transduction of the mitogenic signal

  12. Intense resistance exercise induces early and transient increases in ryanodine receptor 1 phosphorylation in human skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Sebastian Gehlert

    Full Text Available BACKGROUND: While ryanodine receptor 1 (RyR1 critically contributes to skeletal muscle contraction abilities by mediating Ca²⁺ion oscillation between sarcoplasmatic and myofibrillar compartments, AMP-activated protein kinase (AMPK senses contraction-induced energetic stress by phosphorylation at Thr¹⁷². Phosphorylation of RyR1 at serine²⁸⁴³ (pRyR1Ser²⁸⁴³ results in leaky RyR1 channels and impaired Ca²⁺homeostasis. Because acute resistance exercise exerts decreased contraction performance in skeletal muscle, preceded by high rates of Ca²⁺-oscillation and energetic stress, intense myofiber contractions may induce increased RyR1 and AMPK phosphorylation. However, no data are available regarding the time-course and magnitude of early RyR1 and AMPK phosphorylation in human myofibers in response to acute resistance exercise. PURPOSE: Determine the effects and early time-course of resistance exercise on pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² in type I and II myofibers. METHODS: 7 male subjects (age 23±2 years, height: 185±7 cm, weight: 82±5 kg performed 3 sets of 8 repetitions of maximum eccentric knee extensions. Muscle biopsies were taken at rest, 15, 30 and 60 min post exercise. pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² levels were determined by western blot and semi-quantitative immunohistochemistry techniques. RESULTS: While total RyR1 and total AMPK levels remained unchanged, RyR1 was significantly more abundant in type II than type I myofibers. pRyR1Ser²⁸⁴³ increased 15 min and peaked 30 min (p<0.01 post exercise in both myofiber types. Type I fibers showed relatively higher increases in pRyR1Ser²⁸⁴³ levels than type II myofibers and remained elevated up to 60 min post resistance exercise (p<0.05. pAMPKThr¹⁷² also increased 15 to 30 min post exercise (p<0.01 in type I and II myofibers and in whole skeletal muscle. CONCLUSION: Resistance exercise induces acutely increased pRyR1Ser²⁸⁴³ and

  13. Receptor for advanced glycation end products mediates sepsis-triggered amyloid-β accumulation, Tau phosphorylation, and cognitive impairment.

    Science.gov (United States)

    Gasparotto, Juciano; Girardi, Carolina S; Somensi, Nauana; Ribeiro, Camila T; Moreira, José C F; Michels, Monique; Sonai, Beatriz; Rocha, Mariane; Steckert, Amanda V; Barichello, Tatiana; Quevedo, João; Dal-Pizzol, Felipe; Gelain, Daniel P

    2018-01-05

    Patients recovering from sepsis have higher rates of CNS morbidities associated with long-lasting impairment of cognitive functions, including neurodegenerative diseases. However, the molecular etiology of these sepsis-induced impairments is unclear. Here, we investigated the role of the receptor for advanced glycation end products (RAGE) in neuroinflammation, neurodegeneration-associated changes, and cognitive dysfunction arising after sepsis recovery. Adult Wistar rats underwent cecal ligation and perforation (CLP), and serum and brain (hippocampus and prefrontal cortex) samples were obtained at days 1, 15, and 30 after the CLP. We examined these samples for systemic and brain inflammation; amyloid-β peptide (Aβ) and Ser-202-phosphorylated Tau (p-Tau Ser-202 ) levels; and RAGE, RAGE ligands, and RAGE intracellular signaling. Serum markers associated with the acute proinflammatory phase of sepsis (TNFα, IL-1β, and IL-6) rapidly increased and then progressively decreased during the 30-day period post-CLP, concomitant with a progressive increase in RAGE ligands (S100B, N ϵ-[carboxymethyl]lysine, HSP70, and HMGB1). In the brain, levels of RAGE and Toll-like receptor 4, glial fibrillary acidic protein and neuronal nitric-oxide synthase, and Aβ and p-Tau Ser-202 also increased during that time. Of note, intracerebral injection of RAGE antibody into the hippocampus at days 15, 17, and 19 post-CLP reduced Aβ and p-Tau Ser-202 accumulation, Akt/mechanistic target of rapamycin signaling, levels of ionized calcium-binding adapter molecule 1 and glial fibrillary acidic protein, and behavioral deficits associated with cognitive decline. These results indicate that brain RAGE is an essential factor in the pathogenesis of neurological disorders following acute systemic inflammation. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Social isolation stress induces ATF-7 phosphorylation and impairs silencing of the 5-HT 5B receptor gene

    Science.gov (United States)

    Maekawa, Toshio; Kim, Seungjoon; Nakai, Daisuke; Makino, Chieko; Takagi, Tsuyoshi; Ogura, Hiroo; Yamada, Kazuyuki; Chatton, Bruno; Ishii, Shunsuke

    2010-01-01

    Many symptoms induced by isolation rearing of rodents may be relevant to neuropsychiatric disorders, including depression. However, identities of transcription factors that regulate gene expression in response to chronic social isolation stress remain elusive. The transcription factor ATF-7 is structurally related to ATF-2, which is activated by various stresses, including inflammatory cytokines. Here, we report that Atf-7-deficient mice exhibit abnormal behaviours and increased 5-HT receptor 5B (Htr5b) mRNA levels in the dorsal raphe nuclei. ATF-7 silences the transcription of Htr5B by directly binding to its 5′-regulatory region, and mediates histone H3-K9 trimethylation via interaction with the ESET histone methyltransferase. Isolation-reared wild-type (WT) mice exhibit abnormal behaviours that resemble those of Atf-7-deficient mice. Upon social isolation stress, ATF-7 in the dorsal raphe nucleus is phosphorylated via p38 and is released from the Htr5b promoter, leading to the upregulation of Htr5b. Thus, ATF-7 may have a critical role in gene expression induced by social isolation stress. PMID:19893493

  15. Follicle-stimulating hormone (FSH) activates extracellular signal-regulated kinase phosphorylation independently of beta-arrestin- and dynamin-mediated FSH receptor internalization

    Science.gov (United States)

    Piketty, Vincent; Kara, Elodie; Guillou, Florian; Reiter, Eric; Crepieux, Pascale

    2006-01-01

    Background The follicle-stimulating hormone receptor (FSH-R) is a seven transmembrane spanning receptor (7TMR) which plays a crucial role in male and female reproduction. Upon FSH stimulation, the FSH-R activates the extracellular signal-regulated kinases (ERK). However, the mechanisms whereby the agonist-stimulated FSH-R activates ERK are poorly understood. In order to activate ERK, some 7 TMRs require beta-arrestin-and dynamin-dependent internalization to occur, whereas some others do not. In the present study, we examined the ability of the FSH-activated FSH-R to induce ERK phosphorylation, in conditions where its beta-arrestin- and dynamin-mediated internalization was impaired. Methods Human embryonic kidney (HEK) 293 cells were transiently transfected with the rat FSH-R. Internalization of the FSH-R was manipulated by co-expression of either a beta-arrestin (319–418) dominant negative peptide, either an inactive dynamin K44A mutant or of wild-type beta-arrestin 1 or 2. The outcomes on the FSH-R internalization were assayed by measuring 125I-FSH binding at the cell surface when compared to internalized 125I-FSH binding. The resulting ERK phosphorylation level was visualized by Western blot analysis. Results In HEK 293 cells, FSH stimulated ERK phosphorylation in a dose-dependent manner. Co-transfection of the beta- arrestin (319–418) construct, or of the dynamin K44A mutant reduced FSH-R internalization in response to FSH, without affecting ERK phosphorylation. Likewise, overexpression of wild-type beta-arrestin 1 or 2 significantly increased the FSH-R internalization level in response to FSH, without altering FSH-induced ERK phosphorylation. Conclusion From these results, we conclude that the FSH-R does not require beta-arrestin- nor dynamin-mediated internalization to initiate ERK phosphorylation in response to FSH. PMID:16787538

  16. EGF-CFC proteins are essential coreceptors for the TGF-β signals Vg1 and GDF1

    Science.gov (United States)

    Cheng, Simon K.; Olale, Felix; Bennett, James T.; Brivanlou, Ali H.; Schier, Alexander F.

    2003-01-01

    The TGF-β signals Nodal, Activin, GDF1, and Vg1 have been implicated in mesoderm induction and left-right patterning. Nodal and Activin both activate Activin receptors, but only Nodal requires EGF-CFC coreceptors for signaling. We report that Vg1 and GDF1 signaling in zebrafish also depends on EGF-CFC proteins, but not on Nodal signals. Correspondingly, we find that in Xenopus Vg1 and GDF1 bind to and signal through Activin receptors only in the presence of EGF-CFC proteins. These results establish that multiple TGF-β signals converge on Activin receptor/EGF-CFC complexes and suggest a more widespread requirement for coreceptors in TGF-β signaling than anticipated previously. PMID:12514096

  17. Diagnostic of tumours of epithelial origin with the monoclonal antibody IOR EGF/R3 murino

    International Nuclear Information System (INIS)

    Ramos, M.

    1997-01-01

    Despite of the advantages on anti tumoral therapy, the cancer of epithelial origin constitutes one of the first causes of death worldwide. That kind of tumors have a 10-30-fold overexpression of the epidermal growth factor receptor (EGFr). Monoclonal antibody ior egf/r3 is a lgG2a, which recognizes the epidermal growth factor receptor. The aim of the present work was the evaluate the diagnostic efficacy of the 99m Tc-labelled ior egf/r3 for the detection of epithelial derived tumors, its metastasis and its recurrences

  18. NK cell cytotoxicity mediated by 2B4 and NTB-A is dependent on SAP acting downstream of receptor phosphorylation

    Directory of Open Access Journals (Sweden)

    Stephan eMeinke

    2013-01-01

    Full Text Available 2B4 (CD244 and NK-T-B-antigen (NTB-A, CD352 are activating receptors on human NK cells and belong to the family of SLAM-related receptors. Engagement of these receptors leads to phosphorylation of their cytoplasmic tails and recruitment of the adapter proteins SAP and EAT-2. X-linked lymphoproliferative syndrome (XLP is a severe immunodeficiency that results from mutations in the SAP gene. 2B4 and NTB-A-mediated cytotoxicity are abrogated in XLP NK cells. To elucidate the molecular basis for this defect we analyzed early signaling events in SAP knockdown cells. Similar to XLP NK cells, knockdown of SAP in primary human NK cells leads to a reduction of 2B4 and NTB-A-mediated cytotoxicity. We found that early signaling events such as raft recruitment and receptor phosphorylation are not affected by the absence of SAP, indicating the defect in the absence of SAP is downstream of these events. In addition, knockdown of EAT-2 does not impair 2B4 or NTB-A-mediated cytotoxicity. Surprisingly, EAT-2 recruitment to both receptors is abrogated in the absence of SAP, revealing a novel cooperativity between these adapters.

  19. The A2b adenosine receptor antagonist PSB-603 promotes oxidative phosphorylation and ROS production in colorectal cancer cells via adenosine receptor-independent mechanism.

    Science.gov (United States)

    Mølck, Christina; Ryall, James; Failla, Laura M; Coates, Janine L; Pascussi, Jean-Marc; Heath, Joan K; Stewart, Gregory; Hollande, Frédéric

    2016-12-01

    Adenosine is a multifaceted regulator of tumor progression. It modulates immune cell activity as well as acting directly on tumor cells. The A 2b adenosine receptor (A 2b -AR) is thought to be an important mediator of these effects. In this study we sought to analyze the contribution of the A 2b -AR to the behavior of colorectal cancer cells. The A 2b -AR antagonist PSB-603 changed cellular redox state without affecting cellular viability. Quantification of cellular bioenergetics demonstrated that PSB-603 increased basal oxygen consumption rates, indicative of enhanced mitochondrial oxidative phosphorylation. Unexpectedly, pharmacological and genetic approaches to antagonize AR-related signalling of PSB-603 did not abolish the response, suggesting that it was AR-independent. PSB-603 also induced acute increases in reactive oxygen species, and PSB-603 synergized with chemotherapy treatment to increase colorectal cancer cell death, consistent with the known link between cellular metabolism and chemotherapy response. PSB-603 alters cellular metabolism in colorectal cancer cells and increases their sensitivity to chemotherapy. Although requiring more mechanistic insight into its A 2b -AR-independent activity, our results show that PSB-603 may have clinical value as an anti-colorectal cancer therapeutic. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  20. Development of Cu-64 labeled EGF for In Vivo PET Imaging of EGFR Expression

    Energy Technology Data Exchange (ETDEWEB)

    Backer, Joseph M.

    2009-07-12

    In this project we proposed to establish feasibility of the development of targeted tracers for radionuclide imaging of epidermal growth factor receptors (EGFR) in cancer patients. The significance and impact of the proposed radiotracers are determined by the crucial role that EGFR plays in many cancers and by the rapid entrance of EGFR-inhibiting drugs into clinic. Clinical experience, however, revealed that only 10-25% of patients that are defined as EGFR-positive by immunohistochemical analysis respond to EGFR-directed therapeutics and there is poor correlation between EGFR immunohistochemistry and treatment. Therefore, for more efficacious use of EGFR-targeting therapeutics, there is a need for information about EGFR activity in patients. We hypothesized that radionuclide imaging of functionally active EGFR will provide such information and would allow for 1) rational patient stratification, 2) rapid monitoring of responses to therapy, and 3) development of personalized treatment regimens. We hypothesized that tracers based epidermal growth factor (EGF), a natural EGFR ligand, as a targeting vector would be particularly advantageous. First, only functionally active and therefore critical for disease progression EGFRs will bind and internalize an EGF-based tracer. Second, continuous internalization of EGF-based tracers by recyclable EGFR would lead to intracellular accumulation of radionuclide and improved signal-to-background ratio. Third, small size of EGF relative to antibodies would facilitate tumor penetration with vastly better non-specific soft tissue and blood clearance rates. Fourth, as a human protein, EGF is not expected to be immunogenic. Finally, at the beginning of this project, we have already engineered and expressed functionally active EGF with an N-terminal Cys-tag for site-specific conjugation of various payloads, including radionuclide chelators. In the Phase I of this project, in collaboration with Dr. Blankenberg’s group at Stanford

  1. Development of Cu-64 labeled EGF for In Vivo PET Imaging of EGFR Expression

    International Nuclear Information System (INIS)

    Backer, Joseph M.

    2009-01-01

    In this project we proposed to establish feasibility of the development of targeted tracers for radionuclide imaging of epidermal growth factor receptors (EGFR) in cancer patients. The significance and impact of the proposed radiotracers are determined by the crucial role that EGFR plays in many cancers and by the rapid entrance of EGFR-inhibiting drugs into clinic. Clinical experience, however, revealed that only 10-25% of patients that are defined as EGFR-positive by immunohistochemical analysis respond to EGFR-directed therapeutics and there is poor correlation between EGFR immunohistochemistry and treatment. Therefore, for more efficacious use of EGFR-targeting therapeutics, there is a need for information about EGFR activity in patients. We hypothesized that radionuclide imaging of functionally active EGFR will provide such information and would allow for (1) rational patient stratification, (2) rapid monitoring of responses to therapy, and (3) development of personalized treatment regimens. We hypothesized that tracers based epidermal growth factor (EGF), a natural EGFR ligand, as a targeting vector would be particularly advantageous. First, only functionally active and therefore critical for disease progression EGFRs will bind and internalize an EGF-based tracer. Second, continuous internalization of EGF-based tracers by recyclable EGFR would lead to intracellular accumulation of radionuclide and improved signal-to-background ratio. Third, small size of EGF relative to antibodies would facilitate tumor penetration with vastly better non-specific soft tissue and blood clearance rates. Fourth, as a human protein, EGF is not expected to be immunogenic. Finally, at the beginning of this project, we have already engineered and expressed functionally active EGF with an N-terminal Cys-tag for site-specific conjugation of various payloads, including radionuclide chelators. In the Phase I of this project, in collaboration with Dr. Blankenberg's group at Stanford

  2. Relationship between tyrosine phosphorylation and protein expression of insulin receptor and insulin resistance in gestational diabetes mellitus.

    Science.gov (United States)

    Chu, Yong-li; Gong, Yu-dian; Su, Zhi-hui; Yu, Hong-na; Cui, Qing; Jiang, Hai-yang; Qu, Hong-mei

    2014-06-01

    The relationship between tyrosine phosphorylation (TP) and protein expression of insulin receptor (InsR) and insulin resistance (IR) in patients with gestational diabetes mellitus (GDM) was investigated. The InsR expression and TP in skeleton muscle tissue were determined by Western blotting and immunoprecipitation in women with GDM (GDM group, n=22), normal pregnant women (normal pregnancy group, n=22) and normal non-pregnant women (normal non-pregnant group, n=13). Fasting plasma glucose (FPG) and fasting insulin (FINS) were measured by oxidase assay and immunoradioassay. The results showed that the levels of FPG (5.61±0.78 mmol/L), FINS (15.42±5.13 mU/L) and Homeostasis model assessment-IR (HOMA-IR) (1.21±0.52) in GDM group were significantly higher than those in normal pregnancy group (4.43±0.46 mmol/L, 10.56±3.07 mU/L and 0.80±0.31 respectively) (Ppregnant group (7.56±2.31 mU/L and 0.47±0.26 respectively) (P0.05). TP of InsR with insulin stimulation was significantly decreased in GDM group (0.20±0.05) as compared with normal pregnancy group (0.26±0.06) (Pinsulin stimulation in normal pregnancy group was lower than that in normal non-pregnant group (0.31±0.06) (Pinsulin stimulation was negatively related with HOMA-IR in GDM group (r=-0.525, P0.05). It was suggested that there is no significant correlation between the protein expression of InsR in skeletal muscle and IR in GDM, but changes in TP of InsR are associated with IR in GDM.

  3. The influences of reproductive status and acute stress on the levels of phosphorylated mu opioid receptor immunoreactivity in rat hippocampus

    Directory of Open Access Journals (Sweden)

    Keith L. Gonzales

    2011-08-01

    Full Text Available Opioids play a critical role in hippocampally dependent behavior and plasticity. In the hippocampal formation, mu opioid receptors (MOR are prominent in parvalbumin (PARV containing interneurons. Previously we found that gonadal hormones modulate the trafficking of MORs in PARV interneurons. Although sex differences in response to stress are well documented, the point at which opioids, sex and stress interact to influence hippocampal function remains elusive. Thus, we used quantitative immunocytochemistry in combination with light and electron microscopy for the phosphorylated MOR at the SER375 carboxy-terminal residue (pMOR in male and female rats to assess these interactions. In both sexes, pMOR-immunoreactivity (ir was prominent in axons and terminals and in a few neuronal somata and dendrites, some of which contained PARV in the mossy fiber pathway region of the dentate gyrus (DG hilus and CA3 stratum lucidum. In unstressed rats, the levels of pMOR-ir in the DG or CA3 were not affected by sex or estrous cycle stage. However, immediately following 30 minutes of acute immobilization stress (AIS, males had higher levels of pMOR-ir whereas females at proestrus and estrus (high estrogen stages had lower levels of pMOR-ir within the DG. In contrast, the number and types of neuronal profiles with pMOR-ir were not altered by AIS in either males or proestrus females. These data demonstrate that although gonadal steroids do not affect pMOR levels at resting conditions, they are differentially activated both pre- and post-synaptic MORs following stress. These interactions may contribute to the reported sex differences in hippocampally dependent behaviors in stressed animals.

  4. The IkappaB kinase family phosphorylates the Parkinson's disease kinase LRRK2 at Ser935 and Ser910 during Toll-like receptor signaling.

    Directory of Open Access Journals (Sweden)

    Nicolas Dzamko

    Full Text Available Mutations in leucine-rich repeat kinase 2 (LRRK2 are strongly associated with late-onset autosomal dominant Parkinson's disease. LRRK2 is highly expressed in immune cells and recent work points towards a link between LRRK2 and innate immunity. Here we demonstrate that stimulation of the Toll-Like Receptor (TLR pathway by MyD88-dependent agonists in bone marrow-derived macrophages (BMDMs or RAW264.7 macrophages induces marked phosphorylation of LRRK2 at Ser910 and Ser935, the phosphorylation sites that regulate the binding of 14-3-3 to LRRK2. Phosphorylation of these residues is prevented by knock-out of MyD88 in BMDMs, but not the alternative TLR adaptor protein TRIF. Utilising both pharmacological inhibitors, including a new TAK1 inhibitor, NG25, and genetic models, we provide evidence that both the canonical (IKKα and IKKβ and IKK-related (IKKε and TBK1 kinases mediate TLR agonist induced phosphorylation of LRRK2 in vivo. Moreover, all four IKK members directly phosphorylate LRRK2 at Ser910 and Ser935 in vitro. Consistent with previous work describing Ser910 and Ser935 as pharmacodynamic biomarkers of LRRK2 activity, we find that the TLR independent basal phosphorylation of LRRK2 at Ser910 and Ser935 is abolished following treatment of macrophages with LRRK2 kinase inhibitors. However, the increased phosphorylation of Ser910 and Ser935 induced by activation of the MyD88 pathway is insensitive to LRRK2 kinase inhibitors. Finally, employing LRRK2-deficient BMDMs, we present data indicating that LRRK2 does not play a major role in regulating the secretion of inflammatory cytokines induced by activation of the MyD88 pathway. Our findings provide the first direct link between LRRK2 and the IKKs that mediate many immune responses. Further work is required to uncover the physiological roles that phosphorylation of LRRK2 by IKKs play in controlling macrophage biology and to determine how phosphorylation of LRRK2 by IKKs impacts upon the use of Ser

  5. Catalase reverses tumorigenicity in a malignant cell line by an epidermal growth factor receptor pathway.

    Science.gov (United States)

    Finch, Joanne S; Tome, Margaret E; Kwei, Kevin A; Bowden, G Tim

    2006-03-01

    We have used a keratinocyte in vivo/in vitro cell model to test the hypothesis that hydrogen peroxide acts as a signaling molecule, contributing to proliferation and tumorigenesis. A cell line, 6M90, that produces squamous cell carcinoma (SCC), has high levels of ROS and low levels of catalase. A new cell line, MTOC2, generated from parental 6M90 cells by introduction of a Tet-responsive catalase transgene, effectively expressed higher peroxisomal catalase. Increased catalase expression diminished constitutive ROS and enhanced viability after treatment with hydrogen peroxide. Protein tyrosine phosphatase activity was higher in the MTOC2 cells with high catalase, consistent with detection of a lower level of phosphorylation at tyrosine 1068 of the epidermal growth factor receptor (EGF-R). Transcription of downstream c-fos, AP-1 transactivation and cell proliferation were higher in the low catalase cells. An EGF-R inhibitor, AG1478, blocks the higher AP-1 transactivation and cell proliferation of the low catalase 6M90 cells. Tumorigenesis in SCID mice was greatly diminished in the high catalase cells. Our data suggest that hydrogen peroxide functions as a signaling molecule that can modulate activity of a protein tyrosine phosphatase/(s) resulting in phosphorylation of tryrosine/(s) on the EGF-R. Therefore, catalase acts as a tumor-suppressor gene in part by decreasing EGF-R signaling.

  6. 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.

  7. Epidermal growth factor receptor coexpression modulates susceptibility to Herceptin in HER2/neu overexpressing breast cancer cells via specific erbB-receptor interaction and activation

    International Nuclear Information System (INIS)

    Diermeier, Simone; Horvath, Gabor; Knuechel-Clarke, Ruth; Hofstaedter, Ferdinand; Szoellosi, Janos; Brockhoff, Gero

    2005-01-01

    Background: Growth factors and Herceptin specifically and differentially modulate cell proliferation of tumor cells. However, the mechanism of action on erbB-receptor level is incompletely understood. We evaluated Herceptin's capacity to modulate erbB-receptor activation and interaction on the cell surface level and thereby potentially impair cell proliferation of HER2/neu (c-erbB2) overexpressing breast cancer cells, both in the presence and absence of relevant growth factors. Methods: BT474 and SK-BR-3 breast cancer cell lines were treated with Epidermal Growth Factor (EGF), Heregulin, and with Herceptin in different combinations. Kinetics of cell proliferation were evaluated flow cytometrically based on BrdU-labeling. Fluorescence Resonance Energy Transfer, ELISAs and phosphorylation site specific Western Blotting was performed to investigate erbB-receptor interaction and activation. Results: EGF induced EGFR/EGFR and EGFR/c-erbB2 interactions correlate with stimulation of cell proliferation in BT474 cells. Both homo- and heterodimerization are considerably less pronounced in SK-BR-3 cells and heterointeraction is additionally reduced by EGF treatment, causing inhibition of cell proliferation. Heregulin stimulates cell proliferation extensively in both cell lines. Herceptin drives BT474 cells more efficiently into quiescence than it does with SK-BR-3 cells and thereby blocks cell cycle progress. In SK-BR-3 Herceptin treatment causes c-erbB2 phosphorylation of Y877 and Y1248, EGF induces Y877 and Y1112 phosphorylation. The Y1112 phosphorylation site, activated by EGF in SK-BR-3 cell, is bypassed in BT474. In addition the inhibitory capacity of Herceptin on BT474 and SK-BR-3 cell proliferation depends on the presence and absence of growth factors to a various extent. Conclusion: The growth inhibitory effect of Herceptin on c-erbB2 overexpressing breast cancer cells is considerably modulated by EGFR coexpression and consequently EGFR/c-erbB2 homo- and

  8. C1qTNF-related protein 1 improve insulin resistance by reducing phosphorylation of serine 1101 in insulin receptor substrate 1.

    Science.gov (United States)

    Xin, Yaping; Zhang, Dongming; Fu, Yanqin; Wang, Chongxian; Li, Qingju; Tian, Chenguang; Zhang, Suhe; Lyu, Xiaodong

    2017-08-30

    C1qTNF-related protein 1 (CTRP1) is independently associated with type 2 diabetes. However, the relationship between CTRP1 and insulin resistance is still not established. This study aimed to explore the role of CTRP1 under the situation of insulin resistance in adipose tissue. Plasma CTRP1 level was investigated in type 2 diabetic subjects (n = 35) and non-diabetic subjects (n = 35). The relationship between CTRP1 and phosphorylation of multi insulin receptor substrate 1 (IRS-1) serine (Ser) sites was further explored. Our data showed that Plasma CTRP1 was higher and negative correlation with insulin resistance in diabetic subjects (r = -0.283, p = 0.018). Glucose utilisation test revealed that the glucose utilisation rate of mature adipocytes was improved by CTRP1 in the presence of insulin. CTRP1 was not only related to IRS-1 protein, but also negatively correlated with IRS-1 Ser1101 phosphorylation (r = -0.398, p = 0.031). Furthermore, Phosphorylation levels of IRS-1 Ser1101 were significantly lower after incubation with 40 ng/mL CTRP1 in mature adipocytes than those with no intervention (p insulin resistance by reducing the phosphorylation of IRS-1 Ser1101, induced in the situation of insulin resistance as a feedback adipokine.

  9. Distinct phosphorylation sites on the ghrelin receptor, GHSR1a, establish a code that determines the functions of ß-arrestins

    Science.gov (United States)

    Bouzo-Lorenzo, Monica; Santo-Zas, Icía; Lodeiro, Maria; Nogueiras, Rubén; Casanueva, Felipe F.; Castro, Marian; Pazos, Yolanda; Tobin, Andrew B; Butcher, Adrian J.; Camiña, Jesús P.

    2016-01-01

    The growth hormone secretagogue receptor, GHSR1a, mediates the biological activities of ghrelin, which includes the secretion of growth hormone, as well as the stimulation of appetite, food intake and maintenance of energy homeostasis. Mapping phosphorylation sites on GHSR1a and knowledge of how these sites control specific functional consequences unlocks new strategies for the development of therapeutic agents targeting individual functions. Herein, we have identified the phosphorylation of different sets of sites within GHSR1a which engender distinct functionality of ß-arrestins. More specifically, the Ser362, Ser363 and Thr366 residues at the carboxyl-terminal tail were primarily responsible for ß-arrestin 1 and 2 binding, internalization and ß-arrestin-mediated proliferation and adipogenesis. The Thr350 and Ser349 are not necessary for ß-arrestin recruitment, but are involved in the stabilization of the GHSR1a-ß-arrestin complex in a manner that determines the ultimate cellular consequences of ß-arrestin signaling. We further demonstrated that the mitogenic and adipogenic effect of ghrelin were mainly dependent on the ß-arrestin bound to the phosphorylated GHSR1a. In contrast, the ghrelin function on GH secretion was entirely mediated by G protein signaling. Our data is consistent with the hypothesis that the phosphorylation pattern on the C terminus of GHSR1a determines the signaling and physiological output. PMID:26935831

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

    Science.gov (United States)

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

    1995-12-01

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

  11. Identification of serine 348 on the apelin receptor as a novel regulatory phosphorylation site in apelin-13-induced G protein-independent biased signaling.

    Science.gov (United States)

    Chen, Xiaoyu; Bai, Bo; Tian, Yanjun; Du, Hui; Chen, Jing

    2014-11-07

    Phosphorylation plays vital roles in the regulation of G protein-coupled receptor (GPCR) functions. The apelin and apelin receptor (APJ) system is involved in the regulation of cardiovascular function and central control of body homeostasis. Here, using tandem mass spectrometry, we first identified phosphorylated serine residues in the C terminus of APJ. To determine the role of phosphorylation sites in APJ-mediated G protein-dependent and -independent signaling and function, we induced a mutation in the C-terminal serine residues and examined their effects on the interaction between APJ with G protein or GRK/β-arrestin and their downstream signaling. Mutation of serine 348 led to an elimination of both GRK and β-arrestin recruitment to APJ induced by apelin-13. Moreover, APJ internalization and G protein-independent ERK signaling were also abolished by point mutation at serine 348. In contrast, this mutant at serine residues had no demonstrable impact on apelin-13-induced G protein activation and its intracellular signaling. These findings suggest that mutation of serine 348 resulted in inactive GRK/β-arrestin. However, there was no change in the active G protein thus, APJ conformation was biased. These results provide important information on the molecular interplay and impact of the APJ function, which may be extrapolated to design novel drugs for cardiac hypertrophy based on this biased signal pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Increased Level of Phosphorylated ShcA Measured by Chemiluminescence-Linked Immunoassay Is a Predictor of Good Prognosis in Primary Breast Cancer Expressing Low Levels of Estrogen Receptor

    Directory of Open Access Journals (Sweden)

    Serenella Eppenberger-Castori

    2010-03-01

    Full Text Available The SH2 domain-containing adaptor protein ShcA is a proto-oncogene involved in growth factor receptor signaling. The role of phosphorylated ShcA is to link receptor tyrosine kinases with the SH2-containing adaptor protein Grb2, thus facilitating signal transduction from receptor tyrosine kinases to Ras, leading to MAPK activation. The present study was designed to investigate the prognostic significance of phosphorylated ShcA in primary breast cancer and its association in the interactions between the ER and ErbB2 pathways. Using a two-site chemiluminescence-linked immunosorbent assay, we detected the quantitative expression levels of total tyrosine- and threonine-phosphorylated ShcA in cytosol fractions obtained from fresh frozen tissue samples of 153 selected primary breast cancer patients. ShcA phosphorylation was not associated with nodal status, estrogen receptor (ER status or grading. High levels of both tyrosine (pYShcA and serine (pSShcA phosphorylated ShcA correlated with good prognosis (p < 0.01, with respect to both disease-free (DFS and overall survival (OS. In addition, pShcA levels were found to correlate with threonine-phosphorylated ErbB2 and inversely with phosphorylated Akt (pAkt, as well as ErbB2 and ER expression levels. Our findings demonstrate that ShcA activation in primary breast cancer patients correlates with low levels of ER, and is associated with good prognosis.

  13. Increased Level of Phosphorylated ShcA Measured by Chemiluminescence-Linked Immunoassay Is a Predictor of Good Prognosis in Primary Breast Cancer Expressing Low Levels of Estrogen Receptor

    International Nuclear Information System (INIS)

    Cicenas, Jonas; Küng, Willy; Eppenberger, Urs; Eppenberger-Castori, Serenella

    2010-01-01

    The SH2 domain-containing adaptor protein ShcA is a proto-oncogene involved in growth factor receptor signaling. The role of phosphorylated ShcA is to link receptor tyrosine kinases with the SH2-containing adaptor protein Grb2, thus facilitating signal transduction from receptor tyrosine kinases to Ras, leading to MAPK activation. The present study was designed to investigate the prognostic significance of phosphorylated ShcA in primary breast cancer and its association in the interactions between the ER and ErbB2 pathways. Using a two-site chemiluminescence-linked immunosorbent assay, we detected the quantitative expression levels of total tyrosine- and threonine-phosphorylated ShcA in cytosol fractions obtained from fresh frozen tissue samples of 153 selected primary breast cancer patients. ShcA phosphorylation was not associated with nodal status, estrogen receptor (ER) status or grading. High levels of both tyrosine (pYShcA) and serine (pSShcA) phosphorylated ShcA correlated with good prognosis (p < 0.01), with respect to both disease-free (DFS) and overall survival (OS). In addition, pShcA levels were found to correlate with threonine-phosphorylated ErbB2 and inversely with phosphorylated Akt (pAkt), as well as ErbB2 and ER expression levels. Our findings demonstrate that ShcA activation in primary breast cancer patients correlates with low levels of ER, and is associated with good prognosis

  14. H2O2 attenuates IGF-1R tyrosine phosphorylation and its survival signaling properties in neuronal cells via NR2B containing NMDA receptor.

    Science.gov (United States)

    Zeng, Zhiwen; Wang, Dejun; Gaur, Uma; Rifang, Liao; Wang, Haitao; Zheng, Wenhua

    2017-09-12

    Impairment of insulin-like growth factor I (IGF-I) signaling plays an important role in the development of neurodegeneration. In the present study, we investigated the effect of H 2 O 2 on the survival signaling of IGF-1 and its underlying mechanisms in human neuronal cells SH-SY5Y. Our results showed that IGF-1 promoted cell survival and stimulated phosphorylation of IGF-1R as well as its downstream targets like AKT and ERK1/2 in these cells. Meanwhile, these effects of IGF-1 were abolished by H 2 O 2 at 200μM concentration which did not cause any significant toxicity to cells itself in our experiments. Moreover, studies using various glutamate receptor subtype antagonists displayed that N-methyl-D -aspartate (NMDA) receptor antagonist dizocilpine maleate (MK-801) blocked the effects of H 2 O 2 , whereas other glutamate receptor subtype antagonists, such as non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), metabolic glutamate receptor antagonists LY341495 and CPCCOEt, had no effect. Further studies revealed that NR2B-containing NMDARs are responsible for these effects as its effects were blocked by pharmacological inhibitor Ro25-698 or specific siRNA for NR2B, but not NR2A. Finally, our data also showed that Ca 2+ influx contributes to the effects of H 2 O 2 . Similar results were obtained in primary cultured cortical neurons. Taken together, the results from the present study suggested that H 2 O 2 attenuated IGF-1R tyrosine phosphorylation and its survival signaling properties via NR2B containing NMDA receptors and Ca 2+ influx in SH-SY5Y cells. Therefore, NMDAR antagonists, especially NR2B-selective ones, combined with IGF-1 may serve as an alternative therapeutic agent for oxidative stress related neurodegenerative disease.

  15. The SRC homology 2 domain of Rin1 mediates its binding to the epidermal growth factor receptor and regulates receptor endocytosis.

    Science.gov (United States)

    Barbieri, M Alejandro; Kong, Chen; Chen, Pin-I; Horazdovsky, Bruce F; Stahl, Philip D

    2003-08-22

    Activated epidermal growth factor receptors (EGFRs) recruit intracellular proteins that mediate receptor signaling and endocytic trafficking. Rin1, a multifunctional protein, has been shown to regulate EGFR internalization (1). Here we show that EGF stimulation induces a specific, rapid, and transient membrane recruitment of Rin1 and that recruitment is dependent on the Src homology 2 (SH2) domain of Rin1. Immunoprecipitation of EGFR is accompanied by co-immunoprecipitation of Rin1 in a time- and ligand-dependent manner. Association of Rin1 and specifically the SH2 domain of Rin1 with the EGFR was dependent on tyrosine phosphorylation of the intracellular domain of the EGFR. The recruitment of Rin1, observed by light microscopy, indicated that although initially cytosolic, Rin1 was recruited to both plasma membrane and endosomes following EGF addition. Moreover, the expression of the SH2 domain of Rin1 substantially impaired the internalization of EGF without affecting internalization of transferrin. Finally, we found that Rin1 co-immunoprecipitated with a number of tyrosine kinase receptors but not with cargo endocytic receptors. These results indicate that Rin1 provides a link via its SH2 domain between activated tyrosine kinase receptors and the endocytic pathway through the recruitment and activation of Rab5a.

  16. Activation of PKA, p38 MAPK and ERK1/2 by gonadotropins in cumulus cells is critical for induction of EGF-like factor and TACE/ADAM17 gene expression during in vitro maturation of porcine COCs

    Directory of Open Access Journals (Sweden)

    Yamashita Yasuhisa

    2009-12-01

    Full Text Available Abstract Objectives During ovulation, it has been shown that LH stimulus induces the expression of numerous genes via PKA, p38 MAPK, PI3K and ERK1/2 in cumulus cells and granulosa cells. Our recent study showed that EGF-like factor and its protease (TACE/ADAM17 are required for the activation of EGF receptor (EGFR, cumulus expansion and oocyte maturation of porcine cumulus-oocyte complexes (COCs. In the present study, we investigated which signaling pathways are involved in the gene expression of EGF-like factor and in Tace/Adam17 expression in cumulus cells of porcine COC during in vitro maturation. Methods Areg, Ereg, Tace/Adam17, Has2, Tnfaip6 and Ptgs2 mRNA expressions were detected in cumulus cells of porcine COCs by RT-PCR. Protein level of ERK1/2 phosphorylation in cultured cumulus cells was analyzed by westernblotting. COCs were visualized using a phase-contrast microscope. Results When COCs were cultured with FSH and LH up to 2.5 h, Areg, Ereg and Tace/Adam17 mRNA were expressed in cumulus cells of COCs. Areg, Ereg and Tace/Adam17 gene expressions were not suppressed by PI3K inhibitor (LY294002, whereas PKA inhibitor (H89, p38 MAPK inhibitor (SB203580 and MEK inhibitor (U0126 significantly suppressed these gene expressions. Phosphorylation of ERK1/2, and the gene expression of Has2, Tnfaip6 and Ptgs2 were also suppressed by H89, SB203580 and U0126, however, these negative effects were overcome by the addition of EGF to the medium, but not in the U0126 treatment group. Conclusion The results showed that PKA, p38 MAPK and ERK1/2 positively controlled the expression of EGF-like factor and TACE/ADMA17, the latter of which impacts the cumulus expansion and oocyte maturation of porcine COCs via the EGFR-ERK1/2 pathway in cumulus cells.

  17. The O-β-linked N-acetylglucosaminylation of the Lamin B receptor and its impact on DNA binding and phosphorylation.

    Science.gov (United States)

    Smet-Nocca, Caroline; Page, Adeline; Cantrelle, François-Xavier; Nikolakaki, Eleni; Landrieu, Isabelle; Giannakouros, Thomas

    2018-04-01

    Lamin B Receptor (LBR) is an integral protein of the interphase inner nuclear membrane that is implicated in chromatin anchorage to the nuclear envelope. Phosphorylation of a stretch of arginine-serine (RS) dipeptides in the amino-terminal nucleoplasmic domain of LBR regulates the interactions of the receptor with other nuclear proteins, DNA and RNA and thus modulates tethering of heterochromatin to the nuclear envelope. While phosphorylation has been extensively studied, very little is known about other post-translational modifications of the protein. There is only one report on the O-β-linked N-acetyl-glucosaminylation (O-GlcNAcylation) of a serine residue downstream of the RS domain of rat LBR. In the present study we identify additional O-GlcNAcylation sites by using as substrates of O-β-N-acetylglucosaminyltransferase (OGT) a set of peptides containing the entire LBR RS domain or parts of it as well as flanking sequences. The in vitro activity of OGT was assessed by tandem mass spectrometry and NMR spectroscopy. Furthermore, we provide evidence that O-GlcNAcylation hampers DNA binding while it marginally affects RS domain phosphorylation mediated by SRPK1, Akt2 and cdk1 kinases. Our methodology providing a quantitative description of O-GlcNAc patterns based on a combination of mass spectrometry and high resolution NMR spectroscopy on short peptide substrates allows subsequent functional analyses. Hence, our approach is of general interest to a wide audience of biologists aiming at deciphering the functional role of O-GlcNAc glycosylation and its crosstalk with phosphorylation. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Insulin receptors mediate growth effects in cultured fetal neurons. II. Activation of a protein kinase that phosphorylates ribosomal protein S6

    International Nuclear Information System (INIS)

    Heidenreich, K.A.; Toledo, S.P.

    1989-01-01

    As an initial attempt to identify early steps in insulin action that may be involved in the growth responses of neurons to insulin, we investigated whether insulin receptor activation increases the phosphorylation of ribosomal protein S6 in cultured fetal neurons and whether activation of a protein kinase is involved in this process. When neurons were incubated for 2 h with 32Pi, the addition of insulin (100 ng/ml) for the final 30 min increased the incorporation of 32Pi into a 32K microsomal protein. The incorporation of 32Pi into the majority of other neuronal proteins was unaltered by the 30-min exposure to insulin. Cytosolic extracts from insulin-treated neurons incubated in the presence of exogenous rat liver 40S ribosomes and [gamma-32P]ATP displayed a 3- to 8-fold increase in the phosphorylation of ribosomal protein S6 compared to extracts from untreated cells. Inclusion of cycloheximide during exposure of the neurons to insulin did not inhibit the increased cytosolic kinase activity. Activation of S6 kinase activity by insulin was dose dependent (seen at insulin concentration as low as 0.1 ng/ml) and reached a maximum after 20 min of incubation. Addition of phosphatidylserine, diolein, and Ca2+ to the in vitro kinase reaction had no effect on the phosphorylation of ribosomal protein S6. Likewise, treatment of neurons with (Bu)2cAMP did not alter the phosphorylation of ribosomal protein S6 by neuronal cytosolic extracts. We conclude that insulin activates a cytosolic protein kinase that phosphorylates ribosomal S6 in neurons and is distinct from protein kinase-C and cAMP-dependent protein kinase. Stimulation of this kinase may play a role in insulin signal transduction in neurons

  19. EFFECTS OF EPIDERMAL GROWTH FACTOR (EGF), TRANSFORMING GROWTH FACTOR- (TGF), AND 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN ON FUSION OF EMBRYONIC PALATES IN SERUM-FREE ORGAN CULTURE USING WILD-TYPE, EGF KNOCKOUT, AND TGF KNOCKOUT MOUSE STRAINS

    Science.gov (United States)

    Backround: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in mice, producing cleft palate (CP). TCDD exposure disrupts expression of epidermal growth factor (EGF) receptor, EGF, and transforming growth factor- (TGF) in the palate and affects proliferation and different...

  20. Sphingosine-1-Phosphate and the S1P3 Receptor Initiate Neuronal Retraction via RhoA/ROCK Associated with CRMP2 Phosphorylation

    Science.gov (United States)

    Quarta, Serena; Camprubí-Robles, Maria; Schweigreiter, Rüdiger; Matusica, Dusan; Haberberger, Rainer V.; Proia, Richard L.; Bandtlow, Christine E.; Ferrer-Montiel, Antonio; Kress, Michaela

    2017-01-01

    The bioactive lipid sphingosine-1-phosphate (S1P) is an important regulator in the nervous system. Here, we explored the role of S1P and its receptors in vitro and in preclinical models of peripheral nerve regeneration. Adult sensory neurons and motor neuron-like cells were exposed to S1P in an in vitro assay, and virtually all neurons responded with a rapid retraction of neurites and growth cone collapse which were associated with RhoA and ROCK activation. The S1P1 receptor agonist SEW2871 neither activated RhoA or neurite retraction, nor was S1P-induced neurite retraction mitigated in S1P1-deficient neurons. Depletion of S1P3 receptors however resulted in a dramatic inhibition of S1P-induced neurite retraction and was on the contrary associated with a significant elongation of neuronal processes in response to S1P. Opposing responses to S1P could be observed in the same neuron population, where S1P could activate S1P1 receptors to stimulate elongation or S1P3 receptors and retraction. S1P was, for the first time in sensory neurons, linked to the phosphorylation of collapsin response-mediated protein-2 (CRMP2), which was inhibited by ROCK inhibition. The improved sensory recovery after crush injury further supported the relevance of a critical role for S1P and receptors in fine-tuning axonal outgrowth in peripheral neurons. PMID:29066950

  1. Cannabinoid receptor expression and phosphorylation are differentially regulated between male and female cerebellum and brain stem after repeated stress: implication for PTSD and drug abuse.

    Science.gov (United States)

    Xing, Guoqiang; Carlton, Janis; Zhang, Lei; Jiang, Xiaolong; Fullerton, Carol; Li, He; Ursano, Robert

    2011-09-08

    Recent study demonstrated a close relationship between cerebellum atrophy and symptom severity of pediatric maltreatment-related posttraumatic stress disorder (PTSD). It has also been known that females are more vulnerable than males in developing anxiety disorders after exposure to traumatic stress. The mechanisms are unknown. Because cannabinoid receptors (CB₁ and CB₂) are neuroprotective and highly expressed in the cerebellum, we investigated cerebellar CB expression in stressed rats. Young male and female Sprague-Dawley rats were given 40 unpredictable electric tail-shocks for 2h daily on 3 consecutive days. CB₁ and CB₂ mRNA and protein levels in rat cerebellum and brain stem were determined using quantitative real-time PCR and Western blot, respectively. Two-way ANOVA revealed significant gender and stress effects on cerebellar CB₁ mRNA expression, with females and non-stressed rats exhibiting higher CB₁ mRNA levels than the males (3 fold, pstressed rats (30%, pstress increased the level of phosphorylated CB₁ receptors, the inactivated CB₁, in rat cerebellum (pstress interaction. Thus, repeated severe stress caused greater CB₁ mRNA suppression and CB₁ receptor phosphorylation in female cerebellum that could lead to increased susceptibility to stress-related anxiety disorders including PTSD. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  2. Protein kinase A mediates adenosine A2a receptor modulation of neurotransmitter release via synapsin I phosphorylation in cultured cells from medulla oblongata.

    Science.gov (United States)

    Matsumoto, Joao Paulo Pontes; Almeida, Marina Gomes; Castilho-Martins, Emerson Augusto; Costa, Maisa Aparecida; Fior-Chadi, Debora Rejane

    2014-08-01

    Synaptic transmission is an essential process for neuron physiology. Such process is enabled in part due to modulation of neurotransmitter release. Adenosine is a synaptic modulator of neurotransmitter release in the Central Nervous System, including neurons of medulla oblongata, where several nuclei are involved with neurovegetative reflexes. Adenosine modulates different neurotransmitter systems in medulla oblongata, specially glutamate and noradrenaline in the nucleus tractussolitarii, which are involved in hypotensive responses. However, the intracellular mechanisms involved in this modulation remain unknown. The adenosine A2a receptor modulates neurotransmitter release by activating two cAMP protein effectors, the protein kinase A and the exchange protein activated by cAMP. Therefore, an in vitro approach (cultured cells) was carried out to evaluate modulation of neurotransmission by adenosine A2a receptor and the signaling intracellular pathway involved. Results show that the adenosine A2a receptor agonist, CGS 21680, increases neurotransmitter release, in particular, glutamate and noradrenaline and such response is mediated by protein kinase A activation, which in turn increased synapsin I phosphorylation. This suggests a mechanism of A2aR modulation of neurotransmitter release in cultured cells from medulla oblongata of Wistar rats and suggest that protein kinase A mediates this modulation of neurotransmitter release via synapsin I phosphorylation. Copyright © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

  3. P2X7, NMDA and BDNF receptors converge on GSK3 phosphorylation and cooperate to promote survival in cerebellar granule neurons.

    Science.gov (United States)

    Ortega, Felipe; Pérez-Sen, Raquel; Morente, Verónica; Delicado, Esmerilda G; Miras-Portugal, Maria Teresa

    2010-05-01

    Glycogen synthase kinase-3 (GSK3) is a key player in the regulation of neuronal survival. Herein, we report evidence of an interaction between P2X7 receptors with NMDA and BDNF receptors at the level of GSK3 signalling and neuroprotection. The activation of these receptors in granule neurons led to a sustained pattern of GSK3 phosphorylation that was mainly PKC-dependent. BDNF was the most potent at inducing GSK3 phosphorylation, which was also dependent on PI3K. The P2X7 agonist, BzATP, exhibited additive effects with both NMDA and BDNF to rescue granule neurons from cell death induced by PI3K inhibition. This survival effect was mediated by the PKC-dependent GSK3 pathway. In addition, ERK1/2 proteins were also involved in BDNF protective effect. These results show the function of ATP in amplifying neuroprotective actions of glutamate and neurotrophins, and support the role of GSK3 as an important convergence point for these survival promoting factors in granule neurons.

  4. Ezrin/NF-kB activation regulates epithelial- mesenchymal transition induced by EGF and promotes metastasis of colorectal cancer.

    Science.gov (United States)

    Li, Yingru; Lin, Zhaoyu; Chen, Bin; Chen, Shuang; Jiang, Zhipeng; Zhou, Taicheng; Hou, Zehui; Wang, Youyuan

    2017-08-01

    There is growing evidence that epithelial mesenchymal-transition (EMT) plays significant roles in terms of tumor metastasis. There are a lot of cytokines inducing EMT of tumor cells, EGF is one of the important cytokines.Ezrin is a connexin between the cytoskeleton and the cell membrane, which is closely related to the morphological movement and metastasis of tumor cells.EGF can activate Ezrin and affects cell motility. In recent years, many studies have shown that NF-kB acts as an important transcription factor, involving in the process of EMT. However, does Ezrin participate in the regulation of EGF-induced EMT through the NF-kB pathway? This question needs us to discuss.In the present study, we found that EGF could induce colorectal cancer cells to develop EMT,enhance their ability to invade and migrate and promotes phosphorylation of Ezrin Tyr353.On the other hand, inhibition of Ezrin could reverse EGF-induced EMT and inhibit NF-kB P65 translocating into the nucleus. Finally, knockout of Ezrin inhibited EGF-induced lung metastasis of colorectal cancer xenografts and abnormal activation of Ezrin and NF-kB were related with colorectal cancer metastasis and poor prognosis. Our present results suggest that Ezrin/NF-kB pathway may provide experimental evidence for new targeted drugs for colorectal cancer metastasis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  5. Coordinated Regulation Among Progesterone, Prostaglandins, and EGF-Like Factors in Human Ovulatory Follicles.

    Science.gov (United States)

    Choi, Yohan; Wilson, Kalin; Hannon, Patrick R; Rosewell, Katherine L; Brännström, Mats; Akin, James W; Curry, Thomas E; Jo, Misung

    2017-06-01

    In animal models, the luteinizing hormone surge increases progesterone (P4) and progesterone receptor (PGR), prostaglandins (PTGs), and epidermal growth factor (EGF)-like factors that play essential roles in ovulation. However, little is known about the expression, regulation, and function of these key ovulatory mediators in humans. To determine when and how these key ovulatory mediators are induced after the luteinizing hormone surge in human ovaries. Timed periovulatory follicles were obtained from cycling women. Granulosa/lutein cells were collected from in vitro fertilization patients. The in vivo and in vitro expression of PGR, PTG synthases and transporters, and EGF-like factors were examined at the level of messenger RNA and protein. PGR binding to specific genes was assessed. P4 and PTGs in conditioned media were measured. PGR, PTGS2, and AREG expressions dramatically increased in ovulatory follicles at 12 to 18 hours after human chorionic gonadotropin (hCG). In human granulosa/lutein cell cultures, hCG increased P4 and PTG production and the expression of PGR, specific PTG synthases and transporters, and EGF-like factors, mimicking in vivo expression patterns. Inhibitors for P4/PGR and EGF-signaling pathways reduced hCG-induced increases in PTG production and the expression of EGF-like factors. PGR bound to the PTGS2, PTGES, and SLCO2A1 genes. This report demonstrated the time-dependent induction of PGR, AREG, and PTGS2 in human periovulatory follicles. In vitro studies indicated that collaborative actions of P4/PGR and EGF signaling are required for hCG-induced increases in PTG production and potentiation of EGF signaling in human periovulatory granulosa cells. Copyright © 2017 Endocrine Society

  6. EGF signalling pathway regulates colon cancer stem cell proliferation and apoptosis.

    Science.gov (United States)

    Feng, Y; Dai, X; Li, X; Wang, H; Liu, J; Zhang, J; Du, Y; Xia, L

    2012-10-01

    Cancer stem cells (CSCs) compose a subpopulation of cells within a tumour that can self-renew and proliferate. Growth factors such as epidermal growth factor (EGF) and basic fibroblast growth factor (b-FGF) promote cancer stem cell proliferation in many solid tumours. This study assesses whether EGF, bFGF and IGF signalling pathways are essential for colon CSC proliferation and self-renewal. Colon CSCs were cultured in serum-free medium (SFM) with one of the following growth factors: EGF, bFGF or IGF. Characteristics of CSC gene expression were evaluated by real time PCR. Tumourigenicity of CSCs was determined using a xenograft model in vivo. Effects of EGF receptor inhibitors, Gefitinib and PD153035, on CSC proliferation, apoptosis and signalling were evaluated using fluorescence-activated cell sorting and western blotting. Colon cancer cell HCT116 transformed to CSCs in SFM. Compared to other growth factors, EGF was essential to support proliferation of CSCs that expressed higher levels of progenitor genes (Musashi-1, LGR5) and lower levels of differential genes (CK20). CSCs promoted more rapid tumour growth than regular cancer cells in xenografts. EGFR inhibitors suppressed proliferation and induced apoptosis of CSCs by inhibiting autophosphorylation of EGFR and downstream signalling proteins, such as Akt kinase, extracellular signal-regulated kinase 1/2 (ERK 1/2). This study indicates that EGF signalling was essential for formation and maintenance of colon CSCs. Inhibition of the EGF signalling pathway may provide a useful strategy for treatment of colon cancer. © 2012 Blackwell Publishing Ltd.

  7. The tumor suppressor PTEN inhibits EGF-induced TSP-1 and TIMP-1 expression in FTC-133 thyroid carcinoma cells

    International Nuclear Information System (INIS)

    Soula-Rothhut, Mahdhia; Coissard, Cyrille; Sartelet, Herve; Boudot, Cedric; Bellon, Georges; Martiny, Laurent; Rothhut, Bernard

    2005-01-01

    Thrombospondin-1 (TSP-1) is a multidomain extracellular macromolecule that was first identified as natural modulator of angiogenesis and tumor growth. In the present study, we found that epidermal growth factor (EGF) up-regulated TSP-1 expression in FTC-133 (primary tumor) but not in FTC-238 (lung metastasis) thyroid cancer cells. Both EGF and TSP-1 induced expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In FTC-133 cells, EGF induced proliferation in a TSP-1- and TIMP-1-dependent manner. In addition, we determined that re-expression of the tumor suppressor protein PTEN induced cell death, an effect that correlated with a block of Akt kinase phosphorylation. EGF-induced TSP-1 and TIMP-1 promoter activity and protein expression were inhibited in FTC-133 cells stably expressing wtPTEN but not in cells expressing mutant PTEN. Furthermore, we found that wtPTEN inhibited EGF-but not TSP-1-stimulated FTC-133 cell migration and also inhibited invasion induced by EGF and by TSP-1. Finally, an antibody against TSP-1 reversed EGF-stimulated FTC-133 cell invasion as well as the constitutive invasive potential of FTC-238 cells. Overall, our results suggest that PTEN can function as an important modulator of extracellular matrix proteins in thyroid cancer. Therefore, analyzing differential regulation of TSP-1 by growth factors such as EGF can be helpful in understanding thyroid cancer development

  8. Vascular endothelin ET(B) receptor-mediated contraction requires phosphorylation of ERK1/2 proteins

    DEFF Research Database (Denmark)

    Luo, Guogang; Jamali, Roya; Cao, Yong-Xiao

    2006-01-01

    In cardiovascular diseases, endothelin type B (ET(B)) receptors in arterial smooth muscle cells are upregulated. The present study revealed that organ culture of rat mesenteric artery segments enhanced endothelin ET(B) receptor-mediated contraction paralleled with increase in the receptor mRNA an...

  9. Tyrosine phosphorylation of the BRI1 receptor kinase occurs via a posttranslational modification and is activated by the juxtamembrane domain

    Science.gov (United States)

    In metazoans, receptor kinases control many essential processes related to growth and development and response to the environment. The receptor kinases in plants and animals are structurally similar but evolutionarily distinct from one another, and thus while most animal receptor kinases are tyrosin...

  10. Rational design of an EGF-IL18 fusion protein: Implication for developing tumor therapeutics

    International Nuclear Information System (INIS)

    Lu Jianxin; Peng Ying; Meng Zhefeng; Jin Liqin; Lu Yongsui; Guan Minxin

    2005-01-01

    Interleukin-18 (IL-18) is a proinflammatory cytokine. This protein has a role in regulating immune responses and exhibits significant anti-tumor activities. Epidermal growth factor (EGF) is an important growth factor that plays a central role in the regulation of cell cycle and differentiation. It was proposed that a targeted delivery of IL-18 by generation of IL-18-EGF fusion protein might decrease adverse effects and result in enhancing cytotoxic and antitumor activities. In the present study, a fusion protein, consisting of EGFR binding domain fused to human IL-18 mature peptide via a linker peptide of (Gly 4 Ser) 3, was constructed and expressed in the insect cell line Sf9 using Bac-to-Bac baculovirus expression system. We showed that the purified recombinant fusion protein induced similar levels of IFN-γ to that of native IL-18 protein in human PBMC in the presence of ConA. Furthermore, EGF receptor competitive test in human epithelial cancer A431 cell line showed that EGF-IL18 fusion protein can specifically bind with EGFR by competing with native EGF protein. These suggest that this rationally designed protein can be further developed as novel tumor therapeutics

  11. Insulin receptor substrate proteins create a link between the tyrosine phosphorylation cascade and the Ca2+-ATPases in muscle and heart.

    Science.gov (United States)

    Algenstaedt, P; Antonetti, D A; Yaffe, M B; Kahn, C R

    1997-09-19

    Following phosphorylation by the insulin receptor kinase, the insulin receptor substrates (IRS)-1 and IRS-2 bind to and activate several Src homology 2 (SH2) domain proteins. To identify novel proteins that interact with IRS proteins in muscle, a human skeletal muscle cDNA expression library was created in the lambdaEXlox system and probed with baculovirus-produced and tyrosine-phosphorylated human IRS-1. One clone of the 10 clones which was positive through three rounds of screening represented the C terminus of the human homologue of the adult fast twitch skeletal muscle Ca2+-ATPase (SERCA1) including the cytoplasmic tail and part of transmembrane region 10. Western blot analysis of extracts of rat muscle demonstrated co-immunoprecipitation of both IRS-1 and IRS-2 with the skeletal muscle Ca2+-ATPase (SERCA1) and the cardiac muscle isoform (SERCA2). In both cases, injection of insulin stimulated a 2- to 6-fold increase in association of which was maximal within 5 min. In primary cultures of aortic smooth muscle cells and C2C12 cells, the insulin-stimulated interaction between IRS proteins and SERCA1 and -2 was dose-dependent with a maximum induction at 100 nM insulin. This interaction was confirmed in a "pull down" experiment using a glutathione S-transferase fusion protein containing the C terminus of the human SERCA isoform and phosphorylated IRS-1 in vitro and could be blocked by a FLVRES-like domain peptide present in the human SERCA sequence. Affinity chromatography of phosphopeptide libraries using the glutathione S-transferase fusion protein of the C terminus of SERCA1 indicated a consensus sequence for binding of XpYGSS; this is identical to potential tyrosine phosphorylation sites at position 431 of human IRS-1 and at position 500 of human IRS-2. In streptozotocin diabetic rats the interaction between IRS proteins and SERCA1 in skeletal muscle and SERCA2 in cardiac muscle was significantly reduced. Taken together, these results indicate that the IRS

  12. Gene Expression of the EGF System-a Prognostic Model in Non-Small Cell Lung Cancer Patients Without Activating EGFR Mutations

    DEFF Research Database (Denmark)

    Sandfeld-Paulsen, Birgitte; Folkersen, Birgitte Holst; Rasmussen, Torben Riis

    2016-01-01

    OBJECTIVES: Contradicting results have been demonstrated for the expression of the epidermal growth factor receptor (EGFR) as a prognostic marker in non-small cell lung cancer (NSCLC). The complexity of the EGF system with four interacting receptors and more than a dozen activating ligands is a l.......17-6.47], P model that takes the complexity of the EGF system into account and shows that this model is a strong prognostic marker in NSCLC patients.......OBJECTIVES: Contradicting results have been demonstrated for the expression of the epidermal growth factor receptor (EGFR) as a prognostic marker in non-small cell lung cancer (NSCLC). The complexity of the EGF system with four interacting receptors and more than a dozen activating ligands...... is a likely explanation. The aim of this study is to demonstrate that the combined network of receptors and ligands from the EGF system is a prognostic marker. MATERIAL AND METHODS: Gene expression of the receptors EGFR, HER2, HER3, HER4, and the ligands AREG, HB-EGF, EPI, TGF-α, and EGF was measured...

  13. Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Yogi, Alvaro; Callera, Glaucia E. [Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario (Canada); Mecawi, André S. [Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP (Brazil); Batalhão, Marcelo E.; Carnio, Evelin C. [Department of General and Specialized Nursing, College of Nursing of Ribeirão Preto, USP, São Paulo (Brazil); Antunes-Rodrigues, José [Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP (Brazil); Queiroz, Regina H. [Department of Clinical, Toxicological and Food Science Analysis, Faculty of Pharmaceutical Sciences, USP, São Paulo (Brazil); Touyz, Rhian M. [Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario (Canada); Tirapelli, Carlos R., E-mail: crtirapelli@eerp.usp.br [Department of Psychiatric Nursing and Human Sciences, Laboratory of Pharmacology, College of Nursing of Ribeirão Preto, USP, Ribeirão Preto, SP (Brazil)

    2012-11-01

    Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin–angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective AT{sub 1} receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)H oxidase‐mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT{sub 1}-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. -- Highlights: ► Acute ethanol intake stimulates RAS activity and vascular oxidative stress. ► RAS plays a role in acute ethanol-induced oxidative damage via AT{sub 1} receptor activation.

  14. Phosphorylation of ERK in neurokinin 1 receptor-expressing neurons in laminae III and IV of the rat spinal dorsal horn following noxious stimulation

    Directory of Open Access Journals (Sweden)

    Watanabe Masahiko

    2007-02-01

    Full Text Available Abstract Background There is a population of large neurons with cell bodies in laminae III and IV of the spinal dorsal horn which express the neurokinin 1 receptor (NK1r and have dendrites that enter the superficial laminae. Although it has been shown that these are all projection neurons and that they are innervated by substance P-containing (nociceptive primary afferents, we know little about their responses to noxious stimuli. In this study we have looked for phosphorylation of extracellular signal-regulated kinases (ERKs in these neurons in response to different types of noxious stimulus applied to one hindlimb of anaesthetised rats. The stimuli were mechanical (repeated pinching, thermal (immersion in water at 52°C or chemical (injection of 2% formaldehyde. Results Five minutes after each type of stimulus we observed numerous cells with phosphorylated ERK (pERK in laminae I and IIo, together with scattered positive cells in deeper laminae. We found that virtually all of the lamina III/IV NK1r-immunoreactive neurons contained pERK after each of these stimuli and that in the great majority of cases there was internalisation of the NK1r on the dorsal dendrites of these cells. In addition, we also saw neurons in lamina III that were pERK-positive but lacked the NK1r, and these were particularly evident in animals that had had the pinch stimulus. Conclusion Our results demonstrate that lamina III/IV NK1r-immunoreactive neurons show receptor internalisation and ERK phosphorylation after mechanical, thermal or chemical noxious stimuli.

  15. Fasciola hepatica Surface Coat Glycoproteins Contain Mannosylated and Phosphorylated N-glycans and Exhibit Immune Modulatory Properties Independent of the Mannose Receptor.

    Directory of Open Access Journals (Sweden)

    Alessandra Ravidà

    2016-04-01

    Full Text Available Fascioliasis, caused by the liver fluke Fasciola hepatica, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. Like other helminths, F. hepatica employs mechanisms of immune suppression in order to evade its host immune system. In this study the N-glycosylation of F. hepatica's tegumental coat (FhTeg and its carbohydrate-dependent interactions with bone marrow derived dendritic cells (BMDCs were investigated. Mass spectrometric analysis demonstrated that FhTeg N-glycans comprised mainly of oligomannose and to a lesser extent truncated and complex type glycans, including a phosphorylated subset. The interaction of FhTeg with the mannose receptor (MR was investigated. Binding of FhTeg to MR-transfected CHO cells and BMDCs was blocked when pre-incubated with mannan. We further elucidated the role played by MR in the immunomodulatory mechanism of FhTeg and demonstrated that while FhTeg's binding was significantly reduced in BMDCs generated from MR knockout mice, the absence of MR did not alter FhTeg's ability to induce SOCS3 or suppress cytokine secretion from LPS activated BMDCs. A panel of negatively charged monosaccharides (i.e. GlcNAc-4P, Man-6P and GalNAc-4S were used in an attempt to inhibit the immunoregulatory properties of phosphorylated oligosaccharides. Notably, GalNAc-4S, a known inhibitor of the Cys-domain of MR, efficiently suppressed FhTeg binding to BMDCs and inhibited the expression of suppressor of cytokine signalling (SOCS 3, a negative regulator the TLR and STAT3 pathway. We conclude that F. hepatica contains high levels of mannose residues and phosphorylated glycoproteins that are crucial in modulating its host's immune system, however the role played by MR appears to be limited to the initial binding event suggesting that other C-type lectin receptors are involved in the immunomodulatory mechanism of FhTeg.

  16. A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival.

    Science.gov (United States)

    Vyas, Falguni S; Hargreaves, Alan J; Bonner, Philip L R; Boocock, David J; Coveney, Clare; Dickenson, John M

    2016-05-01

    The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells. H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Internalization of the human CRF receptor 1 is independent of classical phosphorylation sites and of beta-arrestin 1 recruitment

    DEFF Research Database (Denmark)

    Rasmussen, Trine N; Novak, Ivana; Nielsen, Søren M

    2004-01-01

    The corticotropin releasing factor receptor 1 (CRFR1) belongs to the superfamily of G-protein coupled receptors. Though CRF is involved in the aetiology of several stress-related disorders, including depression and anxiety, details of CRFR1 regulation such as internalization remain uncharacterized...

  18. Basal Levels of AMPA Receptor GluA1 Subunit Phosphorylation at Threonine 840 and Serine 845 in Hippocampal Neurons

    Science.gov (United States)

    Babiec, Walter E.; Guglietta, Ryan; O'Dell, Thomas J.

    2016-01-01

    Dephosphorylation of AMPA receptor (AMPAR) GluA1 subunits at two sites, serine 845 (S845) and threonine 840 (T840), is thought to be involved in NMDA receptor-dependent forms of long-term depression (LTD). Importantly, the notion that dephosphorylation of these sites contributes to LTD assumes that a significant fraction of GluA1 subunits are…

  19. Technetium-99m direct radiolabeling of monoclonal antibody ior egf/r3

    Energy Technology Data Exchange (ETDEWEB)

    Morales, Alejo A. Morales; Crespo, Francisco Zayas; Gandolff, Gilda Nunez; Escobar, Normando Iznaga; Perez, Niuvis Perez; Hernandez, Juan C. Izquierdo

    1998-01-01

    Monoclonal antibodies (MAbs) are being widely used for imaging studies, coupled mainly with {sup 99m}Tc. The antibody ior egf/r3 is a MAb against human epidermal growth factor receptor (hEGF-r), and we have developed a method for optimum labeling of this MAb with {sup 99m}Tc. The reduction was performed with 2-mercaptoethanol (2-ME) at a molar ratio of 2000:1 (2-ME:MAb) and methylene diphosphonate as transchelant. The integrity of reduced MAb was checked by mean of native polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography on Superose 12 (purity >99%). Radio colloids remained lower than 2%, and the labeling efficiency was 98.5%. The number of sulfhydryl groups generated was quantified using Ellman's reagent and was found to be 6.65 {+-} 0.69 per antibody molecule. In vitro stability studies in several challenging conditions (DTPA, human serum albumin and human serum) were performed, and no significant loss in binding percentage was seen. Radio receptor assay was used to test immunoreactivity of the reduced MAb. Both labeled and unlabeled MAbs were able to compete for binding to the hEGF-r with radioiodinated EGF. Biodistribution studies in BALB/c mice are reported.

  20. Technetium-99m direct radiolabeling of monoclonal antibody ior egf/r3

    International Nuclear Information System (INIS)

    Morales, Alejo A. Morales; Crespo, Francisco Zayas; Gandolff, Gilda Nunez; Escobar, Normando Iznaga; Perez, Niuvis Perez; Hernandez, Juan C. Izquierdo

    1998-01-01

    Monoclonal antibodies (MAbs) are being widely used for imaging studies, coupled mainly with 99m Tc. The antibody ior egf/r3 is a MAb against human epidermal growth factor receptor (hEGF-r), and we have developed a method for optimum labeling of this MAb with 99m Tc. The reduction was performed with 2-mercaptoethanol (2-ME) at a molar ratio of 2000:1 (2-ME:MAb) and methylene diphosphonate as transchelant. The integrity of reduced MAb was checked by mean of native polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography on Superose 12 (purity >99%). Radio colloids remained lower than 2%, and the labeling efficiency was 98.5%. The number of sulfhydryl groups generated was quantified using Ellman's reagent and was found to be 6.65 ± 0.69 per antibody molecule. In vitro stability studies in several challenging conditions (DTPA, human serum albumin and human serum) were performed, and no significant loss in binding percentage was seen. Radio receptor assay was used to test immunoreactivity of the reduced MAb. Both labeled and unlabeled MAbs were able to compete for binding to the hEGF-r with radioiodinated EGF. Biodistribution studies in BALB/c mice are reported

  1. 131I-recombinant human EGF has antitumor effects against MCF-7 human breast cancer xenografts with low levels of EGFR

    International Nuclear Information System (INIS)

    Li Y.-C.; Xu, W.-Y.; Tan, T.-Z.; He Sheng

    2004-01-01

    This study investigated the inhibitory action of 131 I-recombinant human EGF ( 131 I-rhEGF) on MCF-7 human breast cancer tumor development in nude mice. The activity and tumor uptake of 131 I-rhEGF was measured by tissue distribution assay, and its effect on tumor growth was measured by monitoring tumor size after treatment with 131 I-rhEGF. Changes in tumor cell ultrastructure were observed by transmission electron microscopy (TEM), and pathological changes in tumor tissue were observed by light microscopy. The tissue distribution assay revealed that 131 I-rhEGF was markedly absorbed by the tumor and reached its maximal uptake rate (16.73%ID · g -1 ) at 120 hours at which point the drug concentration in the tumor was 11.1-fold, 8.1-fold, and 6.6-fold higher than that in blood, liver, and kidneys, respectively. Tumor size measurements showed that tumor development was significantly inhibited by intravenously and intratumorally injected 131 I-rhEGF. Tumor inhibition rates (82.0% and 80.7%, respectively) were significantly higher than those of tumors treated with 131 I (7.49%) and 131 I-HSA (6.91%; P 131 I-rhEGF could significantly damage and ultimately kill tumor cells. Our results suggest that 131 I-rhEGF suppresses development of xenografted breast cancer cells in nude mice, providing a novel candidate for receptor-mediated targeted radiotherapy

  2. Inhibition of Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Phosphorylation on Tumor-Associated Endothelial Cells Leads to Treatment of Orthotopic Human Colon Cancer in Nude Mice

    Directory of Open Access Journals (Sweden)

    Takamitsu Sasaki

    2007-12-01

    Full Text Available The purpose of our study was to determine whether the dual inhibition of epidermal growth factor receptor (EGFR and vascular endothelial growth factor receptor (VEGFR signaling pathways in tumor-associated endothelial cells can inhibit the progressive growth of human colon carcinoma in the cecum of nude mice. SW620CE2 human colon cancer cells growing in culture and orthotopically in the cecum of nude mice expressed a high level of transforming growth factor alpha (TGF-α and vascular endothelial growth factor (VEGF but were negative for EGFR, human epidermal growth factor receptor 2 (HER2, VEGFR. Double immunofluorescence staining revealed that tumorassociated endothelial cells expressed EGFR, VEGFR2, phosphorylated EGFR (pEGFR, phosphorylated VEGFR (pVEGFR. Treatment of mice with either 7H-pyrrolo [2,3-d]-pyrimidine lead scaffold (AEE788; an inhibitor of EGFR and VEGFR tyrosine kinase or CPT-11 as single agents significantly inhibited the growth of cecal tumors (P < .01; this decrease was even more pronounced with AEE788 combined with CPT-11 (P < .001. AEE788 alone or combined with CPT-11 also inhibited the expression of pEGFR and pVEGFR on tumor-associated endothelial cells, significantly decreased vascularization and tumor cell proliferation, increased the level of apoptosis in both tumorassociated endothelial cells and tumor cells. These data demonstrate that targeting EGFR and VEGFR signaling on tumor-associated endothelial cells provides a viable approach for the treatment of colon cancer.

  3. Adaptor Protein Complex-2 (AP-2) and Epsin-1 Mediate Protease-activated Receptor-1 Internalization via Phosphorylation- and Ubiquitination-dependent Sorting Signals*

    Science.gov (United States)

    Chen, Buxin; Dores, Michael R.; Grimsey, Neil; Canto, Isabel; Barker, Breann L.; Trejo, JoAnn

    2011-01-01

    Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs. PMID:21965661

  4. Mechanical stretch augments insulin-induced vascular smooth muscle cell proliferation by insulin-like growth factor-1 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Gang [Department of Pharmacology, Faculty of Medicine, Kagawa University, Kagawa (Japan); Department of Anesthesiology, First Affiliated Hospital of China Medical University, Shenyang (China); Hitomi, Hirofumi, E-mail: hitomi@kms.ac.jp [Department of Pharmacology, Faculty of Medicine, Kagawa University, Kagawa (Japan); Hosomi, Naohisa [Department of Cardiorenal and Cerebrovascular Medicine, Faculty of Medicine, Kagawa University, Kagawa (Japan); Lei, Bai; Nakano, Daisuke [Department of Pharmacology, Faculty of Medicine, Kagawa University, Kagawa (Japan); Deguchi, Kazushi; Mori, Hirohito; Masaki, Tsutomu [Department of Gastroenterology and Neurology, Faculty of Medicine, Kagawa University, Kagawa (Japan); Ma, Hong [Department of Anesthesiology, First Affiliated Hospital of China Medical University, Shenyang (China); Griendling, Kathy K. [Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta, GA (United States); Nishiyama, Akira [Department of Pharmacology, Faculty of Medicine, Kagawa University, Kagawa (Japan)

    2011-10-15

    Insulin resistance and hypertension have been implicated in the pathogenesis of cardiovascular disease; however, little is known about the roles of insulin and mechanical force in vascular smooth muscle cell (VSMC) remodeling. We investigated the contribution of mechanical stretch to insulin-induced VSMC proliferation. Thymidine incorporation was stimulated by insulin in stretched VSMCs, but not in un-stretched VSMCs. Insulin increased 2-deoxy-glucose incorporation in both stretched and un-stretched VSMCs. Mechanical stretch augmented insulin-induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation. Inhibitors of epidermal growth factor (EGF) receptor tyrosine kinase and Src attenuated insulin-induced ERK and Akt phosphorylation, as well as thymidine incorporation, whereas 2-deoxy-glucose incorporation was not affected by these inhibitors. Moreover, stretch augmented insulin-like growth factor (IGF)-1 receptor expression, although it did not alter the expression of insulin receptor and insulin receptor substrate-1. Insulin-induced ERK and Akt activation, and thymidine incorporation were inhibited by siRNA for the IGF-1 receptor. Mechanical stretch augments insulin-induced VSMC proliferation via upregulation of IGF-1 receptor, and downstream Src/EGF receptor-mediated ERK and Akt activation. Similar to in vitro experiment, IGF-1 receptor expression was also augmented in hypertensive rats. These results provide a basis for clarifying the molecular mechanisms of vascular remodeling in hypertensive patients with hyperinsulinemia. -- Highlights: {yields} Mechanical stretch augments insulin-induced VSMC proliferation via IGF-1 receptor. {yields} Src/EGFR-mediated ERK and Akt phosphorylation are augmented in stretched VSMCs. {yields} Similar to in vitro experiment, IGF-1 receptor is increased in hypertensive rats. {yields} Results provide possible mechanisms of vascular remodeling in hypertension with DM.

  5. Epidermal growth factor receptor signalling in human breast cancer cells operates parallel to estrogen receptor α signalling and results in tamoxifen insensitive proliferation

    International Nuclear Information System (INIS)

    Moerkens, Marja; Zhang, Yinghui; Wester, Lynn; Water, Bob van de; Meerman, John HN

    2014-01-01

    Tamoxifen resistance is a major problem in the treatment of estrogen receptor (ER) α -positive breast cancer patients. Although the mechanisms behind tamoxifen resistance are still not completely understood, clinical data suggests that increased expression of receptor tyrosine kinases is involved. Here, we studied the estrogen and anti-estrogen sensitivity of human breast cancer MCF7 cells that have a moderate, retroviral-mediated, ectopic expression of epidermal growth factor receptor (MCF7-EGFR). Proliferation of MCF7-EGFR and parental cells was induced by 17β-estradiol (E2), epidermal growth factor (EGF) or a combination of these. Inhibition of proliferation under these conditions was investigated with 4-hydroxy-tamoxifen (TAM) or fulvestrant at 10 -12 to 10 -6 M. Cells were lysed at different time points to determine the phosphorylation status of EGFR, MAPK 1/3 , AKT and the expression of ERα. Knockdown of target genes was established using smartpool siRNAs. Transcriptomics analysis was done 6 hr after stimulation with growth factors using Affymetrix HG-U133 PM array plates. While proliferation of parental MCF7 cells could only be induced by E2, proliferation of MCF7-EGFR cells could be induced by either E2 or EGF. Treatment with TAM or fulvestrant did significantly inhibit proliferation of MCF7-EGFR cells stimulated with E2 alone. EGF treatment of E2/TAM treated cells led to a marked cell proliferation thereby overruling the anti-estrogen-mediated inhibition of cell proliferation. Under these conditions, TAM however did still inhibit ERα- mediated transcription. While siRNA-mediated knock-down of EGFR inhibited the EGF- driven proliferation under TAM/E2/EGF condition, knock down of ERα did not. The TAM resistant cell proliferation mediated by the conditional EGFR-signaling may be dependent on the PI3K/Akt pathway but not the MEK/MAPK pathway, since a MEK inhibitor (U0126), did not block the proliferation. Transcriptomic analysis under the various E2/TAM/EGF

  6. Decreased phosphorylation of δ and ε subunits of the acetylcholine receptor coincides with delayed postsynaptic maturation in PKC θ deficient mouse.

    Science.gov (United States)

    Lanuza, Maria A; Besalduch, Núria; González, Carmen; Santafé, Manel M; Garcia, Neus; Tomàs, Marta; Nelson, Phillip G; Tomàs, Josep

    2010-09-01

    Protein kinase C (PKC) activity is involved in the nicotinic acetylcholine receptor (nAChR) redistribution at the neuromuscular junction in vivo during postnatal maturation. Here we studied, in PKC theta (PKCtheta) deficient mice (KO), how the theta isoform of PKC is involved in the nAChR cluster maturation that is accompanied by the developmental activity-dependent neuromuscular synapse elimination process. We found that axonal elimination and dispersion of nAChR from the postsynaptic plaques and its redistribution to form the mature postsynaptic apparatus were delayed but not totally suppressed in PKCtheta deficient mice. Moreover, the delay in the maturation of the morphology of the nAChR clusters during the early postnatal synapse elimination period in the PKCtheta deficient mice coincides with a reduction in the PKCtheta-mediated phosphorylation on the delta subunit of the nAChR. In addition, we show evidence for PKCtheta regulation of PKA in normally phosphorylating the epsilon subunit of nAChR. We have also found that the theta isoform of PKC is located on the postsynaptic component of the neuromuscular junction but is also expressed by motoneurons in the spinal cord and in the motor nerve terminals. The results allow us to hypothesize that a spatially specific and opposing action of PKCtheta and PKA may result in activity-dependent alterations to synaptic connectivity at both the nerve inputs and the postsynaptic nAChR clusters. Copyright 2010 Elsevier Inc. All rights reserved.

  7. Hyperinsulinemia enhances interleukin-17-induced inflammation to promote prostate cancer development in obese mice through inhibiting glycogen synthase kinase 3-mediated phosphorylation and degradation of interleukin-17 receptor

    Science.gov (United States)

    Chen, Chong; Ge, Dongxia; Qu, Yine; Chen, Rongyi; Fan, Yi-Ming; Li, Nan; Tang, Wendell W.; Zhang, Wensheng; Zhang, Kun; Wang, Alun R.; Rowan, Brian G.; Hill, Steven M.; Sartor, Oliver; Abdel, Asim B.; Myers, Leann; Lin, Qishan; You, Zongbing

    2016-01-01

    Interleukin-17 (IL-17) plays important roles in inflammation, autoimmune diseases, and some cancers. Obese people are in a chronic inflammatory state with increased serum levels of IL-17, insulin, and insulin-like growth factor 1 (IGF1). How these factors contribute to the chronic inflammatory status that promotes development of aggressive prostate cancer in obese men is largely unknown. We found that, in obese mice, hyperinsulinemia enhanced IL-17-induced expression of downstream proinflammatory genes with increased levels of IL-17 receptor A (IL-17RA), resulting in development of more invasive prostate cancer. Glycogen synthase kinase 3 (GSK3) constitutively bound to and phosphorylated IL-17RA at T780, leading to ubiquitination and proteasome-mediated degradation of IL-17RA, thus inhibiting IL-17-mediated inflammation. IL-17RA phosphorylation was reduced, while the IL-17RA levels were increased in the proliferative human prostate cancer cells compared to the normal cells. Insulin and IGF1 enhanced IL-17-induced inflammatory responses through suppressing GSK3, which was shown in the cultured cell lines in vitro and obese mouse models of prostate cancer in vivo. These findings reveal a mechanism underlying the intensified inflammation in obesity and obesity-associated development of aggressive prostate cancer, suggesting that targeting GSK3 may be a potential therapeutic approach to suppress IL-17-mediated inflammation in the prevention and treatment of prostate cancer, particularly in obese men. PMID:26871944

  8. SNT-2 interacts with ERK2 and negatively regulates ERK2 signaling in response to EGF stimulation

    International Nuclear Information System (INIS)

    Huang Lin; Gotoh, Noriko; Zhang Shengliang; Shibuya, Masabumi; Yamamoto, Tadashi; Tsuchida, Nobuo

    2004-01-01

    The control of cellular responses with fibroblast growth factors and neurotrophins is mediated through membrane-linked docking proteins, SNT (suc1-binding neurotrophic target)-1/FRS2α and SNT-2/FRS2β. ERK1/2 are members of the mitogen-activated protein kinase family that regulate diverse cellular activities in response to various stimuli. Here, we demonstrate that SNT-2 does not become tyrosine phosphorylated significantly in response to EGF but forms a complex with ERK2 via the region of 186-252 amino acid residues, and the complex formation is enhanced upon EGF stimulation. SNT-2 downregulates ERK2 phosphorylation, suppresses and delays ERK2 nuclear accumulation which occurs following EGF stimulation. In contrast, the mutant SNT-2 which carries deletion of 186-252 amino acids and lacks ERK2 binding does not have these effects. These observations suggest that SNT-2 negatively regulates ERK2 signaling activated via EGF stimulation through direct binding to ERK2

  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. Recycling of epidermal growth factor-receptor complexes in A431 cells: Identification of dual pathways

    International Nuclear Information System (INIS)

    Sorkin, A.; Krolenko, S.; Kudrjavtceva, N.; Lazebnik, J.; Teslenko, L.; Soderquist, A.M.; Nikolsky, N.

    1991-01-01

    The intracellular sorting of EGF-receptor complexes (EGF-RC) has been studied in human epidermoid carcinoma A431 cells. Recycling of EGF was found to occur rapidly after internalization at 37 degrees C. The initial rate of EGF recycling was reduced at 18 degrees C. A significant pool of internalized EGF was incapable of recycling at 18 degrees C but began to recycle when cells were warmed to 37 degrees C. The relative rate of EGF outflow at 37 degrees C from cells exposed to an 18 degrees C temperature block was slower (t1/2 approximately 20 min) than the rate from cells not exposed to a temperature block (t1/2 approximately 5-7 min). These data suggest that there might be both short- and long-time cycles of EGF recycling in A431 cells. Examination of the intracellular EGF-RC dissociation and dynamics of short- and long-time recycling indicated that EGF recycled as EGF-RC. Moreover, EGF receptors that were covalently labeled with a photoactivatable derivative of 125 I-EGF recycled via the long-time pathway at a rate similar to that of 125 I-EGF. Since EGF-RC degradation was also blocked at 18 degrees C, we propose that sorting to the lysosomal and long-time recycling pathway may occur after a highly temperature-sensitive step, presumably in the late endosomes

  11. Insulin and insulin-like growth factor-I (IGF-I) receptor phosphorylation in µ-calpain knockout mice

    Science.gov (United States)

    Numerous cellular processes are controlled by insulin and IGF-I signaling pathways. Due to previous work in our laboratories, we hypothesized that insulin (IR) and type 1 IGF-I (IGF-IR) receptor signaling is decreased due to increased protein tyrosine phosphatase 1B (PTP1B) activity. C57BL/6J mice...

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

    DEFF Research Database (Denmark)

    Parhamifar, Ladan; Sime, Wondossen; Yudina, Yuliana

    2010-01-01

    Leukotriene D(4) (LTD(4)) belongs to the bioactive lipid group known as eicosanoids and has implications in pathological processes such as inflammation and cancer. Leukotriene D(4) exerts its effects mainly through two different G-protein-coupled receptors, CysLT(1) and CysLT(2). The high affinit...

  13. Parainfluenza virus 3 blocks antiviral mediators downstream of the interferon lambda receptor by modulating stat1 phosphorylation

    Science.gov (United States)

    Paramyxoviruses are known to inhibit type I interferon (IFN) production, however there is a lack of information regarding the type III IFN response during infection. Type III IFNs signal through a unique heterodimeric receptor, the IFN-'R1/IL-10R2, which is primarily expressed by epithelial cells. ...

  14. Ascofuranone suppresses EGF-induced HIF-1α protein synthesis by inhibition of the Akt/mTOR/p70S6K pathway in MDA-MB-231 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Yun-Jeong; Cho, Hyun-Ji [Research Institute of Biomedical Engineering and Department of Medicine, Catholic University of Daegu School of Medicine, Daegu 705-718 (Korea, Republic of); Magae, Junji [Magae Bioscience Institute, 49-4 Fujimidai, Tsukuba 300-1263 (Japan); Lee, In-Kyu [Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu 700-721 (Korea, Republic of); Park, Keun-Gyu, E-mail: kpark@knu.ac.kr [Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu 700-721 (Korea, Republic of); Chang, Young-Chae, E-mail: ycchang@cu.ac.kr [Research Institute of Biomedical Engineering and Department of Medicine, Catholic University of Daegu School of Medicine, Daegu 705-718 (Korea, Republic of)

    2013-12-15

    Hypoxia-inducible factor (HIF)-1 plays an important role in tumor progression, angiogenesis and metastasis. In this study, we investigated the potential molecular mechanisms underlying the anti-angiogenic effect of ascofuranone, an isoprenoid antibiotic from Ascochyta viciae, in epidermal growth factor (EGF)-1 responsive human breast cancer cells. Ascofuranone significantly and selectively suppressed EGF-induced HIF-1α protein accumulation, whereas it did not affect the expression of HIF-1β. Furthermore, ascofuranone inhibited the transcriptional activation of vascular endothelial growth factor (VEGF) by reducing protein HIF-1α. Mechanistically, we found that the inhibitory effects of ascofuranone on HIF-1α protein expression are associated with the inhibition of synthesis HIF-1α through an EGF-dependent mechanism. In addition, ascofuranone suppressed EGF-induced phosphorylation of Akt/mTOR/p70S6 kinase, but the phosphorylation of ERK/JNK/p38 kinase was not affected by ascofuranone. These results suggest that ascofuranone suppresses EGF-induced HIF-1α protein translation through the inhibition of Akt/mTOR/p70S6 kinase signaling pathways and plays a novel role in the anti-angiogenic action. - Highlights: • Inhibitory effect of ascofuranone on HIF-1α expression is EGF-specific regulation. • Ascofuranone decreases HIF-1α protein synthesis through Akt/mTOR pathways. • Ascofuranone suppresses EGF-induced VEGF production and tumor angiogenesis.

  15. Topoisomerase I tyrosine phosphorylation site and the DNA-interactive site

    International Nuclear Information System (INIS)

    Roll, D.; Durban, E.

    1986-01-01

    Phosphorylation of topoisomerase I (topo I) at serine by NII kinase is accompanied by stimulation of enzymatic activity. In contrast, phosphorylation at tyrosine by tyrosine kinase seems to inhibit enzymatic activity. This inhibition may be caused by interference of the phosphorylated tyrosine residue with the interaction of topo I with DNA. To test this, topo I was labeled with crude membrane fraction enriched for EGF-receptor kinase in presence of γ-P32-ATP and electrophoresed on SDS-polyacrylamide gels. Stained topo I bands were excised, dried, digested with trypsin and analyzed on a C18 reverse-phase HPLC column. One major peak of radioactivity eluted at fraction 23 with 20% acetonitrile. To obtain the DNA-interactive site, topo I was incubated with pBR322 DNA labeled by nick-translation followed by DNase I treatment, and electrophoresis on SDS-polyacrylamide gels. Tryptic peptides were generated and analyzed by reverse-phase HPLC. A major peak of radioactivity eluted at fraction 16-18 with 15.5-17% acetonitrile. Studies are in progress to resolve whether (a) the two peptides are different, i.e. the tyrosine-P site and DNA-tyrosine interactive site are localized at different regions of the topo I or (b) the peptide sequences are identical but the covalent attachment of deoxynucleotides altered the peptide's elution from the HPLC column

  16. Characterizing tyrosine phosphorylation signaling in lung cancer using SH2 profiling.

    Directory of Open Access Journals (Sweden)

    Kazuya Machida

    2010-10-01

    Full Text Available Tyrosine kinases drive the proliferation and survival of many human cancers. Thus profiling the global state of tyrosine phosphorylation of a tumor is likely to provide a wealth of information that can be used to classify tumors for prognosis and prediction. However, the comprehensive analysis of tyrosine phosphorylation of large numbers of human cancer specimens is technically challenging using current methods.We used a phosphoproteomic method termed SH2 profiling to characterize the global state of phosphotyrosine (pTyr signaling in human lung cancer cell lines. This method quantifies the phosphorylated binding sites for SH2 domains, which are used by cells to respond to changes in pTyr during signaling. Cells could be grouped based on SH2 binding patterns, with some clusters correlated with EGF receptor (EGFR or K-RAS mutation status. Binding of specific SH2 domains, most prominently RAS pathway activators Grb2 and ShcA, correlated with EGFR mutation and sensitivity to the EGFR inhibitor erlotinib. SH2 binding patterns also reflected MET activation and could identify cells driven by multiple kinases. The pTyr responses of cells treated with kinase inhibitors provided evidence of distinct mechanisms of inhibition.This study illustrates the potential of modular protein domains and their proteomic binding profiles as powerful molecular diagnostic tools for tumor classification and biomarker identification.

  17. Normalization of TAM post-receptor signaling reveals a cell invasive signature for Axl tyrosine kinase.

    Science.gov (United States)

    Kimani, Stanley G; Kumar, Sushil; Davra, Viralkumar; Chang, Yun-Juan; Kasikara, Canan; Geng, Ke; Tsou, Wen-I; Wang, Shenyan; Hoque, Mainul; Boháč, Andrej; Lewis-Antes, Anita; De Lorenzo, Mariana S; Kotenko, Sergei V; Birge, Raymond B

    2016-09-06

    Tyro3, Axl, and Mertk (TAMs) are a family of three conserved receptor tyrosine kinases that have pleiotropic roles in innate immunity and homeostasis and when overexpressed in cancer cells can drive tumorigenesis. In the present study, we engineered EGFR/TAM chimeric receptors (EGFR/Tyro3, EGFR/Axl, and EGF/Mertk) with the goals to interrogate post-receptor functions of TAMs, and query whether TAMs have unique or overlapping post-receptor activation profiles. Stable expression of EGFR/TAMs in EGFR-deficient CHO cells afforded robust EGF inducible TAM receptor phosphorylation and activation of downstream signaling. Using a series of unbiased screening approaches, that include kinome-view analysis, phosphor-arrays, RNAseq/GSEA analysis, as well as cell biological and in vivo readouts, we provide evidence that each TAM has unique post-receptor signaling platforms and identify an intrinsic role for Axl that impinges on cell motility and invasion compared to Tyro3 and Mertk. These studies demonstrate that TAM show unique post-receptor signatures that impinge on distinct gene expression profiles and tumorigenic outcomes.

  18. Phosphorylation of Ser1928 mediates the enhanced activity of the L-type Ca2+ channel Cav1.2 by the β2-adrenergic receptor in neurons.

    Science.gov (United States)

    Qian, Hai; Patriarchi, Tommaso; Price, Jennifer L; Matt, Lucas; Lee, Boram; Nieves-Cintrón, Madeline; Buonarati, Olivia R; Chowdhury, Dhrubajyoti; Nanou, Evanthia; Nystoriak, Matthew A; Catterall, William A; Poomvanicha, Montatip; Hofmann, Franz; Navedo, Manuel F; Hell, Johannes W

    2017-01-24

    The L-type Ca 2+ channel Ca v 1.2 controls multiple functions throughout the body including heart rate and neuronal excitability. It is a key mediator of fight-or-flight stress responses triggered by a signaling pathway involving β-adrenergic receptors (βARs), cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA). PKA readily phosphorylates Ser 1928 in Ca v 1.2 in vitro and in vivo, including in rodents and humans. However, S1928A knock-in (KI) mice have normal PKA-mediated L-type channel regulation in the heart, indicating that Ser 1928 is not required for regulation of cardiac Ca v 1.2 by PKA in this tissue. We report that augmentation of L-type currents by PKA in neurons was absent in S1928A KI mice. Furthermore, S1928A KI mice failed to induce long-term potentiation in response to prolonged theta-tetanus (PTT-LTP), a form of synaptic plasticity that requires Ca v 1.2 and enhancement of its activity by the β 2 -adrenergic receptor (β 2 AR)-cAMP-PKA cascade. Thus, there is an unexpected dichotomy in the control of Ca v 1.2 by PKA in cardiomyocytes and hippocampal neurons. Copyright © 2017, American Association for the Advancement of Science.

  19. Spatial relationship of phosphorylated epidermal growth factor receptor and activated AKT in head and neck squamous cell carcinoma

    International Nuclear Information System (INIS)

    Nijkamp, Monique M.; Hoogsteen, Ilse J.; Span, Paul N.; Takes, Robert P.; Lok, Jasper; Rijken, Paul F.; Kogel, Albert J. van der; Bussink, Johan; Kaanders, Johannes H.A.M.

    2011-01-01

    Background: Overexpression of EGFR correlates with decreased survival after radiotherapy in head and neck squamous cell carcinoma (HNSCC). However, the contribution of the activated form, pEGFR, and its downstream signaling (PI3-K/AKT) pathway is not clear yet. Methods: Fifty-eight patients with HNSCC were included in the study. pEGFR, pAKT, hypoxia, and vessels were visualized using immunohistochemistry. Fractions (defined as the tumor area positive for the respective markers relative to the total tumor area) were calculated by automated image analysis and related to clinical outcome. Results: Both pEGFR (median 0.6%, range 0–34%) and pAKT (median 1.8%, range 0–16%) expression differed between tumors. Also, a large variation in hypoxia was found (median pimonidazole fraction 3.9% 0–20%). A significant correlation between pEGFR and pAKT (r s 0.44, p = 0.004) was seen, however, analysis revealed that this was not always based on spatial coexpression. Low pAKT expression was associated with increased risk of regional recurrence (p < 0.05, log-rank) and distant metastasis (p = 0.04). Conclusion: The correlation between expression of pEGFR and pAKT is indicative of activation of the PI3-K/AKT pathway through phosphorylation of EGFR. Since not all tumors show coexpression to the same extent, other factors must be involved in the activation of this pathway as well.

  20. Inhibition of EGF processing in responsive and nonresponsive human fibroblasts

    International Nuclear Information System (INIS)

    Schaudies, R.P.; Wray, H.L.

    1988-01-01

    We have examined the proteolytic processing of radiolabeled epidermal growth factor (EGF) in EGF growth-responsive human foreskin fibroblasts (HFF) versus EGF nonresponsive human fetal lung fibroblasts (HFL). Previous studies have shown that both cell lines demonstrate similar binding affinities and numbers of binding sites, as well as similar rates of internalization and degradation of the bound, radiolabeled hormone. We have used nondenaturing electrophoresis to compare how these two cell lines process EGF at its carboxy terminus. EGF lacking either one [des-(53)-EGF] or six [des (48-53)-EGF] carboxy terminal amino acids could be distinguished by this method. Chloroquine or leupeptin were added to the incubation system in an attempt to accentuate potential differences in hormonal processing between the responsive and nonresponsive cell lines. In the absence of inhibitors, the responsive and nonresponsive cells generated similar distributions of processed forms of EGF after 30-minutes incubation. However, after 4-hours incubation in the constant presence of 125I-EGF, the electrophoretic profiles of extracted hormone were substantially different. The radiolabel within the responsive cells, as well as that released from them, migrated predominantly at the dye front, indicating complete degradation of EGF. In contrast, the majority of the radiolabel within the nonresponsive cells migrated as partially processed forms of hormone, while the released radiolabel migrated at the dye front. Addition of chloroquine to either cell line inhibited processing of EGF beyond removal of the carboxyl terminal arginine residue. Both intact 125I-EGF, and 125I-EGF lacking the carboxyl terminal arginine were released from chloroquine-treated cells in a ratio equal to that present in the intact cells

  1. Prenatal stress affects insulin-like growth factor-1 (IGF-1) level and IGF-1 receptor phosphorylation in the brain of adult rats.

    Science.gov (United States)

    Basta-Kaim, Agnieszka; Szczesny, Ewa; Glombik, Katarzyna; Stachowicz, Katarzyna; Slusarczyk, Joanna; Nalepa, Irena; Zelek-Molik, Agnieszka; Rafa-Zablocka, Katarzyna; Budziszewska, Boguslawa; Kubera, Marta; Leskiewicz, Monika; Lason, Wladyslaw

    2014-09-01

    It has been shown that stressful events occurring in early life have a powerful influence on the development of the central nervous system. Insulin-like growth factor-1 (IGF-1) promotes the growth, differentiation and survival of both neurons and glial cells and is thought to exert antidepressant-like activity. Thus, it is possible that disturbances in the function of the IGF-1 system may be responsible for disturbances observed over the course of depression. Prenatal stress was used as a valid model of depression. Adult male offspring of control and stressed rat dams were subjected to behavioural testing (forced swim test). The level of IGF-1 in the blood and the expression of IGF-1, IGF-1R, and IRS-1/2 in the hippocampus and frontal cortex using RT-PCR, ELISA and western blotting were measured. In addition the effect of intracerebroventricularly administered IGF-1 and/or the IGF-1R receptor antagonist JB1 in the forced swim test was studied. Prenatally stressed rats showed depressive like behaviour, including increased immobility time as well as decreased mobility and climbing. Intracerebroventricular administration of IGF-1 reversed these effects in stressed animals, whereas concomitant administration of the IGF-1R antagonist JB1 completely blocked the effects. Biochemical analysis of homogenates from the hippocampus and frontal cortex revealed decreases in IGF-1 level and IGF-1R phosphorylation along with disturbances in IRS-1 phosphorylation. These findings reveal that prenatal stress alters IGF-1 signalling, which may contribute to the behavioural changes observed in depression. Copyright © 2014 Elsevier B.V. and ECNP. All rights reserved.

  2. Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.

    Science.gov (United States)

    Waraky, Ahmed; Lin, Yingbo; Warsito, Dudi; Haglund, Felix; Aleem, Eiman; Larsson, Olle

    2017-11-03

    We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases ( e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G 1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Epidermal growth factor and its receptors in human pancreatic carcinoma

    International Nuclear Information System (INIS)

    Chen, Y.F.; Pan, G.Z.; Hou, X.; Liu, T.H.; Chen, J.; Yanaihara, C.; Yanaihara, N.

    1990-01-01

    The role of epidermal growth factor (EGF) in oncogenesis and progression of malignant tumors is a subject of vast interest. In this study, radioimmunoassay and radioreceptor assay of EGF were established. EGF contents in malignant and benign pancreatic tumors, in normal pancreas tissue, and in culture media of a human pancreatic carcinoma cell line were determined. EGF receptor binding studies were performed. It was shown that EGF contents in pancreatic carcinomas were significantly higher than those in normal pancreas or benign pancreatic tumors. EGF was also detected in the culture medium of a pancreatic carcinoma cell line. The binding of 125I-EGF to the pancreatic carcinoma cells was time and temperature dependent, reversible, competitive, and specific. Scatchard analysis showed that the dissociation constant of EGF receptor was 2.1 X 10(-9) M, number of binding sites was 1.3 X 10(5) cell. These results indicate that there is an over-expression of EGF/EGF receptors in pancreatic carcinomas, and that an autocrine regulatory mechanism may exist in the growth-promoting effect of EGF on tumor cells

  4. Interdependency of EGF and GLP-2 Signaling in Attenuating Mucosal Atrophy in a Mouse Model of Parenteral Nutrition

    DEFF Research Database (Denmark)

    Feng, Yongjia; Demehri, Farok R; Xiao, Weidong

    2017-01-01

    BACKGROUND & AIMS: Total parenteral nutrition (TPN), a crucial treatment for patients who cannot receive enteral nutrition, is associated with mucosal atrophy, barrier dysfunction, and infectious complications. Glucagon-like peptide-2 (GLP-2) and epidermal growth factor (EGF) improve intestinal...... deprived of enteral nutrition. METHODS: Adult C57BL/6J, IEC-Egfr(knock out (KO)) and IEC-pik3r1(KO) mice receiving TPN or enteral nutrition were treated with EGF or GLP-2 alone or in combination with reciprocal receptor inhibitors, GLP-2(3-33) or gefitinib. Jejunum was collected and mucosal atrophy and IEC...

  5. Gallic acid-capped gold nanoparticles inhibit EGF-induced MMP-9 expression through suppression of p300 stabilization and NFκB/c-Jun activation in breast cancer MDA-MB-231 cells

    International Nuclear Information System (INIS)

    Chen, Ying-Jung; Lee, Yuan-Chin; Huang, Chia-Hui; Chang, Long-Sen

    2016-01-01

    Triple-negative breast cancers (TNBCs) are highly invasive and have a higher rate of distant metastasis. Matrix metalloproteinase-9 (MMP-9) plays a crucial role in EGF/EGFR-mediated malignant progression and metastasis of TNBCs. Various studies have revealed that treatment with gallic acid down-regulates MMP-9 expression in cancer cells, and that conjugation of phytochemical compounds with gold nanoparticles (AuNPs) increases the anti-tumor activity of the phytochemical compounds. Thus, the effect of gallic acid-capped AuNPs (GA-AuNPs) on MMP-9 expression in EGF-treated TNBC MDA-MB-231 cells was analyzed in the present study. The so-called green synthesis of AuNPs by means of gallic acid was performed at pH 10, and the resulting GA-AuNPs had spherical shape with an average diameter of approximately 50 nm. GA-AuNPs notably suppressed migration and invasion of EGF-treated cells, and inhibited EGF-induced MMP-9 up-regulation. GA-AuNPs abrogated EGF-induced Akt/p65 and ERK/c-Jun phosphorylation, leading to down-regulation of MMP-9 mRNA and protein expression in EGF-treated cells. Meanwhile, EGF-induced p300 stabilization was found to be involved in MMP-9 expression, whereas GA-AuNPs inhibited the EGF-promoted stability of the p300 protein. Although GA-AuNPs and gallic acid suppressed EGF-induced MMP-9 up-regulation via the same signaling pathway, the effective concentration of gallic acid was approximately 100-fold higher than that of GA-AuNPs for inhibition of MMP-9 expression in EGF-treated cells to a similar extent. Collectively, our data indicate that, in comparison with gallic acid, GA-AuNPs have a superior ability to inhibit EGF/EGFR-mediated MMP-9 expression in TNBC MDA-MB-231 cells. Our findings also point to a way to improve the anti-tumor activity of gallic acid. - Highlights: • Gallic acid-capped gold nanoparticles inhibit EGF-induced MMP-9 expression. • EGF-induced MMP-9 expression via p300 stabilization and NFκB/c-Jun activation. • Gallic acid

  6. Gallic acid-capped gold nanoparticles inhibit EGF-induced MMP-9 expression through suppression of p300 stabilization and NFκB/c-Jun activation in breast cancer MDA-MB-231 cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying-Jung; Lee, Yuan-Chin; Huang, Chia-Hui [Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Chang, Long-Sen, E-mail: lschang@mail.nsysu.edu.tw [Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China)

    2016-11-01

    Triple-negative breast cancers (TNBCs) are highly invasive and have a higher rate of distant metastasis. Matrix metalloproteinase-9 (MMP-9) plays a crucial role in EGF/EGFR-mediated malignant progression and metastasis of TNBCs. Various studies have revealed that treatment with gallic acid down-regulates MMP-9 expression in cancer cells, and that conjugation of phytochemical compounds with gold nanoparticles (AuNPs) increases the anti-tumor activity of the phytochemical compounds. Thus, the effect of gallic acid-capped AuNPs (GA-AuNPs) on MMP-9 expression in EGF-treated TNBC MDA-MB-231 cells was analyzed in the present study. The so-called green synthesis of AuNPs by means of gallic acid was performed at pH 10, and the resulting GA-AuNPs had spherical shape with an average diameter of approximately 50 nm. GA-AuNPs notably suppressed migration and invasion of EGF-treated cells, and inhibited EGF-induced MMP-9 up-regulation. GA-AuNPs abrogated EGF-induced Akt/p65 and ERK/c-Jun phosphorylation, leading to down-regulation of MMP-9 mRNA and protein expression in EGF-treated cells. Meanwhile, EGF-induced p300 stabilization was found to be involved in MMP-9 expression, whereas GA-AuNPs inhibited the EGF-promoted stability of the p300 protein. Although GA-AuNPs and gallic acid suppressed EGF-induced MMP-9 up-regulation via the same signaling pathway, the effective concentration of gallic acid was approximately 100-fold higher than that of GA-AuNPs for inhibition of MMP-9 expression in EGF-treated cells to a similar extent. Collectively, our data indicate that, in comparison with gallic acid, GA-AuNPs have a superior ability to inhibit EGF/EGFR-mediated MMP-9 expression in TNBC MDA-MB-231 cells. Our findings also point to a way to improve the anti-tumor activity of gallic acid. - Highlights: • Gallic acid-capped gold nanoparticles inhibit EGF-induced MMP-9 expression. • EGF-induced MMP-9 expression via p300 stabilization and NFκB/c-Jun activation. • Gallic acid

  7. A role for the epidermal growth factor receptor signaling in development of intestinal serrated polyps in mice and humans.

    Science.gov (United States)

    Bongers, Gerold; Muniz, Luciana R; Pacer, Michelle E; Iuga, Alina C; Thirunarayanan, Nanthakumar; Slinger, Erik; Smit, Martine J; Reddy, E Premkumar; Mayer, Lloyd; Furtado, Glaucia C; Harpaz, Noam; Lira, Sergio A

    2012-09-01

    Epithelial cancers can be initiated by activating mutations in components of the mitogen-activated protein kinase signaling pathway such as v-raf murine sarcoma viral oncogene homolog B1 (BRAF), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), or epidermal growth factor receptor (EGFR). Human intestinal serrated polyps are a heterogeneous group of benign lesions, but some progress to colorectal cancer. Tumors that arise from these polyps frequently contain activating mutations in BRAF or KRAS, but little is known about the role of EGFR activation in their development. Polyp samples were obtained from adults during screening colonoscopies at Mount Sinai Hospital in New York. We measured levels of EGFR protein and phosphorylation in human serrated polyps by immunohistochemical and immunoblot analyses. We generated transgenic mice that express the ligand for EGFR, Heparin-binding EGF-like growth factor (HB-EGF), in the intestine. EGFR and the extracellular-regulated kinases (ERK)1/2 were phosphorylated in serrated areas of human hyperplastic polyps (HPPs), sessile serrated adenomas, and traditional serrated adenomas. EGFR and ERK1/2 were phosphorylated in the absence of KRAS or BRAF activating mutations in a subset of HPP. Transgenic expression of the EGFR ligand HB-EGF in the intestines of mice promoted development of small cecal serrated polyps. Mice that expressed a combination of HB-EGF and US28 (a constitutively active, G-protein-coupled receptor that increases processing of HB-EGF from the membrane) rapidly developed large cecal serrated polyps. These polyps were similar to HPPs and had increased phosphorylation of EGFR and ERK1/2 within the serrated epithelium. Administration of pharmacologic inhibitors of EGFR or MAPK to these transgenic mice significantly reduced polyp development. Activation of EGFR signaling in the intestine of mice promotes development of serrated polyps. EGFR signaling also is activated in human HPPs, sessile serrated adenomas

  8. EGF-Induced VEGF Exerts a PI3K-Dependent Positive Feedback on ERK and AKT through VEGFR2 in Hematological In Vitro Models.

    Directory of Open Access Journals (Sweden)

    Lilian Saryeddine

    Full Text Available EGFR and VEGFR pathways play major roles in solid tumor growth and progression, however, little is known about these pathways in haematological tumors. This study investigated the crosstalk between EGFR and VEGFR2 signaling in two hematological in vitro models: THP1, a human monocytic leukemia, and Raji, a Burkitt's lymphoma, cell lines. Results showed that both cell lines express EGFR and VEGFR2 and responded to EGF stimulation by activating EGFR, triggering VEGF production and phosphorylating ERK, AKT, and p38 very early, with a peak of expression at 10-20min. Blocking EGFR using Tyrphostin resulted in inhibiting EGFR induced activation of ERK, AKT, and p38. In addition, EGF stimulation caused a significant and immediate increase, within 1min, in pVEGFR2 in both cell lines, which peaked at ~5-10 min after treatment. Selective inhibition of VEGFR2 by DMH4, anti-VEGFR2 antibody or siRNA diminished EGF-induced pAKT and pERK, indicating a positive feedback exerted by EGFR-induced VEGF. Similarly, the specific PI3K inhibitor LY294002, suppressed AKT and ERK phosphorylation showing that VEGF feedback is PI3K-dependent. On the other hand, phosphorylation of p38, initiated by EGFR and independent of VEGF feedback, was diminished using PLC inhibitor U73122. Moreover, measurement of intracellular [Ca2+] and ROS following VEGFR2 inhibition and EGF treatment proved that VEGFR2 is not implicated in EGF-induced Ca2+ release whereas it boosts EGF-induced ROS production. Furthermore, a significant decrease in pAKT, pERK and p-p38 was shown following the addition of the ROS inhibitor NAC. These results contribute to the understanding of the crosstalk between EGFR and VEGFR in haematological malignancies and their possible combined blockade in therapy.

  9. Expression of epidermal growth factor receptors in human endometrial carcinoma

    DEFF Research Database (Denmark)

    Nyholm, H C; Nielsen, Anette Lynge; Ottesen, B

    1993-01-01

    Little data exist on the expression of epidermal growth factor receptors (EGF-Rs) in human endometrial cancer. EGF-R status was studied in 65 patients with endometrial carcinomas and in 26 women with nonmalignant postmenopausal endometria, either inactive/atrophic endometrium or adenomatous...... hyperplasia. EGF-R was identified on frozen tissue sections by means of an indirect immunoperoxidase technique with a monoclonal antibody against the external domain of the EGF-R. Seventy-one percent of the carcinomas expressed positive EGF-R immunoreactivity. In general, staining was most prominent...

  10. Improved expression of recombinant plant-made hEGF.

    Science.gov (United States)

    Thomas, David Rhys; Walmsley, Amanda Maree

    2014-11-01

    The yield of recombinant hEGF was increased approximately tenfold through a range of optimisations. Further, the recombinant protein was found to have biological activity comparable to commercial hEGF. Human epidermal growth factor (hEGF) is a powerful mitogen that can enhance the healing of a wide range of injuries, including burns, cuts, diabetic ulcers and gastric ulcers. However, despite its clinical value, hEGF is only consistently used for the treatment of chronic diabetic ulcers due to its high cost. In this study, hEGF was transiently expressed in Nicotiana benthamiana plants and targeted to the apoplast, ER and vacuole. Several other approaches were also included in a stepwise fashion to identify the optimal conditions for the expression of recombinant hEGF. Expression was found to be highest in the vacuole, while targeting hEGF to the ER caused a decrease in total soluble protein (TSP). Using a codon optimised sequence was found to increase vacuolar targeted hEGF yield by ~34 %, while it was unable to increase the yield of ER targeted hEGF. The use of the P19 silencing inhibitor was able to further increase expression by over threefold, and using 5-week-old plants significantly increased expression compared to 4- or 6-week-old-plants. The combined effect of these optimisations increased expression tenfold over the initial apoplast targeted construct to an average yield of 6.24 % of TSP. The plant-made hEGF was then shown to be equivalent to commercial E. coli derived hEGF in its ability to promote the proliferation of mouse keratinocytes. This study supports the potential for plants to be used for the commercial production of hEGF, and identifies a potential limitation for the further improvement of recombinant protein yields.

  11. Tc-99m direct radiolabeling of monoclonal antibody ior egf/r3: quality control and image studies in mice

    International Nuclear Information System (INIS)

    Dias, Carla Roberta; Marczewski, Barbara; Moraes, Vanessa; Barboza, Marycel Figols de; Osso Junior, Joao Alberto

    2005-01-01

    Monoclonal antibodies (Mabs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in the practice of nuclear medicine. The ior egf/r3 (Centis, Cuba) is a murine monoclonal antibody against epidermal growth factor receptor (EGF-R) and has been widely used in the radioimmunodiagnosis of tumors of epithelial origin. Labeled with 99m Tc, its main application in Nuclear Medicine is the follow up, detection and evaluation of tumor recurrences. The objective of this work is to describe the preparation of a lyophilized formulation (kit) for radiolabeling the Mab ior egf/r3 with 99m Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, image studies and stability of the formulation are reported. The study demonstrated that the kit formulation can be labeled with 99m Tc at high yields and can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies.(author)

  12. 131I-Recombinant human EGF has anti-tumor effects against MCF-7 human breast cancer xenografts with low levels of EGFR

    International Nuclear Information System (INIS)

    Li Yunchun; Tan Tianzhi; Xu Weiyun; He Sheng

    2004-01-01

    Purpose: This study investigated the inhibitory action of 131 I-recombinant human EGF ( 131 I-rhEGF) on MCF-7 human breast cancer tumor development in nude mice. Methods: The activity and tumor uptake of 131 I-rhEGF was measured by tissue distribution assay, and its effect on tumor growth was measured by monitoring tumor size after treatment with 131 I-rhEGF, Changes in tumor cell ultrastructure were observed by transmission electron microscopy (TEM), and pathological changes in tumor tissue were observed by light microscopy. Results: The tissue distribution assay revealed that 131 I-rhEGF was markedly absorbed by the tumor and reached its maximal uptake rate (16.73% ID·g-l) at 120 h, at which point the drug concentration in the tumor was 11.1-fold, 8.1-fold, 6.6-fold higher than that in blood, liver, kidneys, respectively. The tumor size measurements showed that tumor development was significantly inhibited by intravenously and intratumorally injected 131 I-rhEGF. The extent of tumor inhibition rates (82.0% and 80.7%, respectively) were significantly higher than those of tumors treated with 131 I (7.49%) and 131 I-HSA (6.91%; P 131 I-rhEGF could significantly damage and ultimately kill tumor cells. Conclusions: Our results suggest that 131 I-rhEGF suppresses development of xenografted breast cancer cells in nude mice, providing a novel candidate for receptor-mediated targeted radiotherapy. Key words. Iodine-131 rhEGF Breast cancer Therapy. (authors)

  13. Areca nut components stimulate ADAM17, IL-1α, PGE2 and 8-isoprostane production in oral keratinocyte: role of reactive oxygen species, EGF and JAK signaling.

    Science.gov (United States)

    Chang, Mei-Chi; Chan, Chiu-Po; Chen, Yi-Jane; Hsien, Hsiang-Chi; Chang, Ya-Ching; Yeung, Sin-Yuet; Jeng, Po-Yuan; Cheng, Ru-Hsiu; Hahn, Liang-Jiunn; Jeng, Jiiang-Huei

    2016-03-29

    Betel quid (BQ) chewing is an etiologic factor of oral submucous fibrosis (OSF) and oral cancer. There are 600 million BQ chewers worldwide. The mechanisms for the toxic and inflammatory responses of BQ are unclear. In this study, both areca nut (AN) extract (ANE) and arecoline stimulated epidermal growth factor (EGF) and interleukin-1α (IL-1α) production of gingival keratinocytes (GKs), whereas only ANE can stimulate a disintegrin and metalloproteinase 17 (ADAM17), prostaglandin E2 (PGE2) and 8-isoprostane production. ANE-induced EGF production was inhibited by catalase. Addition of anti-EGF neutralizing antibody attenuated ANE-induced cyclooxygenase-2 (COX-2), mature ADAM9 expression and PGE2 and 8-isoprostane production. ANE-induced IL-1α production was inhibited by catalase, anti-EGF antibody, PD153035 (EGF receptor antagonist) and U0126 (MEK inhibitor) but not by α-naphthoflavone (cytochrome p450-1A1 inhibitor). ANE-induced ADAM17 production was inhibited by pp2 (Src inhibitor), U0126, α-naphthoflavone and aspirin. AG490 (JAK inhibitor) prevented ANE-stimulated ADAM17, IL-1α, PGE2 production, COX-2 expression, ADAM9 maturation, and the ANE-induced decline in keratin 5 and 14, but showed little effect on cdc2 expression and EGF production. Moreover, ANE-induced 8-isoprostane production by GKs was inhibited by catalase, anti-EGF antibody, AG490, pp2, U0126, α-naphthoflavone, Zinc protoporphyrin (ZnPP) and aspirin. These results indicate that AN components may involve in BQ-induced oral cancer by induction of reactive oxygen species, EGF/EGFR, IL-1α, ADAMs, JAK, Src, MEK/ERK, CYP1A1, and COX signaling pathways, and the aberration of cell cycle and differentiation. Various blockers against ROS, EGF, IL-1α, ADAM, JAK, Src, MEK, CYP1A1, and COX can be used for prevention or treatment of BQ chewing-related diseases.

  14. Distribution of epidermal growth factor receptors in rat tissues during embryonic skin development, hair formation, and the adult hair growth cycle

    DEFF Research Database (Denmark)

    Green, M R; Couchman, J R

    1984-01-01

    on the binding distribution of [125I]EGF, representing the tissue localization of available EGF receptors, during embryonic rat skin development including hair follicle formation and the adult hair growth cycle. At 16 days embryonic development a relatively low receptor density is seen over all the epidermal...... condensates marking the first stage of hair follicle development. This restricted and temporary loss of EGF receptors above these specialized mesenchymal condensates implies a role for the EGF receptor and possibly EGF or an EGF-like ligand in stimulating the epithelial downgrowth required for hair follicle...... development. In the anagen hair bulb, receptors for EGF are detected over the outer root sheath and the epithelial cell layers at the base of the follicle and show a correlation with the areas of epithelial proliferation in the hair bulb. During the catagen and telogen phases of the hair cycle, receptors...

  15. Ibrutinib (ImbruvicaTM) potently inhibits ErbB receptor phosphorylation and cell viability of ErbB2-positive breast cancer cells.

    Science.gov (United States)

    Grabinski, Nicole; Ewald, Florian

    2014-12-01

    Ibrutinib (formerly PCI-32765) is a specific, irreversible, and potent inhibitor of Burton's tyrosine kinase (BTK) developed for the treatment of several forms of blood cancer. It is now an FDA-approved drug marketed under the name Imbruvica(TM) (Pharmacyclics, Inc.) and successfully used as an orally administered second-line drug in the treatment of mantle cell lymphoma. Since BTK is predominantly expressed in hematopoietic cells, the sensitivity of solid tumor cells to Ibrutinib has not been analyzed. In this study, we determined the effect of Ibrutinib on breast cancer cells. We demonstrate that Ibrutinib efficiently reduces the phosphorylation of the receptor tyrosine kinases ErbB1, ErbB2 and ErbB3, thereby suppressing AKT and MAPK signaling in ErbB2-positive (ErbB2+) breast cancer cell lines. Treatment with Ibrutinib significantly reduced the viability of ErbB2+ cell lines with IC50 values at nanomolar concentrations, suggesting therapeutic potential of Ibrutinib in breast cancer. Combined treatment with Ibrutinib and the dual PI3K/mTOR inhibitor BEZ235 synergistically reduces cell viability of ErbB2+ breast cancer cells. Combination indices below 0.25 at 50% inhibition of cell viability were determined by the Chou-Talalay method. Therefore, the combination of Ibrutinib and canonical PI3K pathway inhibitors could be a new and effective approach in the treatment of breast cancer with activated ErbB receptors. Ibrutinib could thus become a valuable component of targeted therapy in aggressive ErbB2+ breast cancer.

  16. Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

    Directory of Open Access Journals (Sweden)

    Ahnen Dennis

    2005-01-01

    Full Text Available Abstract Background Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs is associated with a decreased mortality from colorectal cancer (CRC. NSAIDs induce apoptotic cell death in colon cancer cells in vitro and inhibit growth of neoplastic colonic mucosa in vivo however, the biochemical mechanisms required for these growth inhibitory effects are not well defined. We previously reported that metabolites of the NSAID sulindac downregulate extracellular-signal regulated kinase 1/2 (ERK1/2 signaling and that this effect is both necessary and sufficient for the apoptotic effects of these drugs. The goal of this project was to specifically test the hypothesis that sulindac metabolites block activation and/or expression of the epidermal growth factor (EGF receptor (EGFR. Methods HT29 human colon cancer cells were treated with EGF, alone, or in the presence of sulindac sulfide or sulindac sulfone. Cells lysates were assayed by immunoblotting for phosphorylated EGFR (pEGFR, pY1068, total EGFR, phosphorylated ERK1/2 (pERK1/2, total ERK1/2, activated caspase-3, and α-tubulin. Results EGF treatment rapidly induced phosphorylation of both EGFR and ERK1/2 in HT29 colon cancer cells. Pretreatment with sulindac metabolites for 24 h blocked EGF-induced phosphorylation of both EGFR and ERK1/2 and decreased total EGFR protein expression. Under basal conditions, downregulation of pEGFR and total EGFR was detected as early as 12 h following sulindac sulfide treatment and persisted through at least 48 h. Sulindac sulfone induced downregulation of pEGFR and total EGFR was detected as early as 1 h and 24 h, respectively, following drug treatment, and persisted through at least 72 h. EGFR downregulation by sulindac metabolites was observed in three different CRC cell lines, occurred prior to the observed downregulation of pERK1/2 and induction of apoptosis by these drugs, and was not dependent of caspase activation. Conclusion These results suggest that

  17. Nitric oxide from inflammatory origin impairs neural stem cell proliferation by inhibiting epidermal growth factor receptor signaling

    Directory of Open Access Journals (Sweden)

    Bruno Pereira Carreira

    2014-10-01

    Full Text Available Neuroinflammation is characterized by activation of microglial cells, followed by production of nitric oxide (NO, which may have different outcomes on neurogenesis, favoring or inhibiting this process. In the present study, we investigated how the inflammatory mediator NO can affect proliferation of neural stem cells (NSC, and explored possible mechanisms underlying this effect. We investigated which mechanisms are involved in the regulation of NSC proliferation following treatment with an inflammatory stimulus (LPS plus IFN-γ, using a culture system of subventricular zone (SVZ-derived NSC mixed with microglia cells obtained from wild-type mice (iNOS+/+ or from iNOS knockout mice (iNOS-/-. We found an impairment of NSC cell proliferation in iNOS+/+ mixed cultures, which was not observed in iNOS-/- mixed cultures. Furthermore, the increased release of NO by activated iNOS+/+ microglial cells decreased the activation of the ERK/MAPK signaling pathway, which was concomitant with an enhanced nitration of the EGF receptor. Preventing nitrogen reactive species formation with MnTBAP, a scavenger of peroxynitrite, or using the peroxynitrite degradation catalyst FeTMPyP, cell proliferation and ERK signaling were restored to basal levels in iNOS+/+ mixed cultures. Moreover, exposure to the NO donor NOC-18 (100 µM, for 48 h, inhibited SVZ-derived NSC proliferation. Regarding the antiproliferative effect of NO, we found that NOC-18 caused the impairment of signaling through the ERK/MAPK pathway, which may be related to increased nitration of the EGF receptor in NSC. Using MnTBAP nitration was prevented, maintaining ERK signaling, rescuing NSC proliferation. We show that NO from inflammatory origin leads to a decreased function of the EGF receptor, which compromised proliferation of NSC. We also demonstrated that NO-mediated nitration of the EGF receptor caused a decrease in its phosphorylation, thus preventing regular proliferation signaling through the

  18. Potent homocysteine-induced ERK phosphorylation in cultured neurons depends on self-sensitization via system Xc-

    International Nuclear Information System (INIS)

    Gu Li; Hu Xiaoling; Xue Zhanxia; Yang Jun; Wan Lishu; Ren Yan; Hertz, Leif; Peng Liang

    2010-01-01

    Homocysteine is increased during pathological conditions, endangering vascular and cognitive functions, and elevated homocysteine during pregnancy may be correlated with an increased incidence of schizophrenia in the offspring. This study showed that millimolar homocysteine concentrations in saline medium cause phosphorylation of extracellular-signal regulated kinases 1 and 2 (ERK 1/2 ) in cerebellar granule neurons, inhibitable by metabotropic but not ionotropic glutamate receptor antagonists. These findings are analogous to observations by , that similar concentrations cause neuronal death. However, these concentrations are much higher than those occurring clinically during hyperhomocysteinemia. It is therefore important that a ∼ 10-fold increase in potency occurred in the presence of the glutamate precursor glutamine, when ERK 1/2 phosphorylation became inhibitable by NMDA or non-NMDA antagonists and dependent upon epidermal growth factor (EGF) receptor transactivation. However, glutamate release to the medium was reduced, suggesting that reversal of the cystine/glutamate antiporter, system X c - could be involved in potentiation of the response by causing a localized release of initially accumulated homocysteine. In agreement with this hypothesis further enhancement of ERK 1/2 phosphorylation occurred in the additional presence of cystine. Pharmacological inhibition of system X c - prevented the effect of micromolar homocysteine concentrations, and U0126-mediated inhibition of ERK 1/2 phosphorylation enhanced homocysteine-induced death. In conclusion, homocysteine interacts with system X c - like quisqualate (Venkatraman et al. 1994), by 'self-sensitization' with initial accumulation and subsequent release in exchange with cystine and/or glutamate, establishing high local homocysteine concentrations, which activate adjacent ionotropic glutamate receptors and cause neurotoxicity.

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

    Science.gov (United States)

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

    2009-04-01

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

  20. Radioimmunoscintigraphy with monoclonal antibody Technetium-99m-Anti-EGF-Receptor (R3-MAB) for the detection of head and neck tumours, metastasis and recurrence. Final report for the period 15 April 1995 - 15 April 1997

    International Nuclear Information System (INIS)

    Oliva Gonzalez, J.P.

    1998-03-01

    A clinical study was carried out to determine the sensitivity of radioimmunoscintigraphy (RIS) using indigenously produced mouse monoclonal antibody (MAB) against epidermal growth factor receptor in the detection of primary, recurrent and metastatic malignant epithelial tumours of the head and neck region in 13 patients. The MAB was labelled with 99m Tc and imaging was carried out using gamma camera and SPECT. The results were correlated with histopathological findings. RIS gave a sensitivity of 76.9%. This study showed that the indigenously produced MAB can be used for the detection of malignant epithelial tumours in the head and neck region but the MAB will be further characterized to improve its sensitivity in the detection of the neoplasia. (author)

  1. γ-aminobutyric acidA (GABAA) receptor regulates ERK1/2 phosphorylation in rat hippocampus in high doses of Methyl Tert-Butyl Ether (MTBE)-induced impairment of spatial memory

    International Nuclear Information System (INIS)

    Zheng Gang; Zhang Wenbin; Zhang Yun; Chen Yaoming; Liu Mingchao; Yao Ting; Yang Yanxia; Zhao Fang; Li Jingxia; Huang Chuanshu; Luo Wenjing; Chen Jingyuan

    2009-01-01

    Experimental and occupational exposure to Methyl Tert-Butyl Ether (MTBE) has been reported to induce neurotoxicological and neurobehavioral effects, such as headache, nausea, dizziness, and disorientation, etc. However, the molecular mechanisms involved in MTBE-induced neurotoxicity are still not well understood. In the present study, we investigated the effects of MTBE on spatial memory and the expression and function of GABA A receptor in the hippocampus. Our results demonstrated that intraventricular injection of MTBE impaired the performance of the rats in a Morris water maze task, and significantly increased the expression of GABA A receptor α1 subunit in the hippocampus. The phosphorylation of ERK1/2 decreased after the MTBE injection. Furthermore, the decreased ability of learning and the reduction of phosphorylated ERK1/2 level of the MTBE-treated rats was partly reversed by bicuculline injected 30 min before the training. These results suggested that MTBE exposure could result in impaired spatial memory. GABA A receptor may play an important role in the MTBE-induced impairment of learning and memory by regulating the phosphorylation of ERK in the hippocampus.

  2. Advanced computational biology methods identify molecular switches for malignancy in an EGF mouse model of liver cancer.

    Directory of Open Access Journals (Sweden)

    Philip Stegmaier

    Full Text Available The molecular causes by which the epidermal growth factor receptor tyrosine kinase induces malignant transformation are largely unknown. To better understand EGFs' transforming capacity whole genome scans were applied to a transgenic mouse model of liver cancer and subjected to advanced methods of computational analysis to construct de novo gene regulatory networks based on a combination of sequence analysis and entrained graph-topological algorithms. Here we identified transcription factors, processes, key nodes and molecules to connect as yet unknown interacting partners at the level of protein-DNA interaction. Many of those could be confirmed by electromobility band shift assay at recognition sites of gene specific promoters and by western blotting of nuclear proteins. A novel cellular regulatory circuitry could therefore be proposed that connects cell cycle regulated genes with components of the EGF signaling pathway. Promoter analysis of differentially expressed genes suggested the majority of regulated transcription factors to display specificity to either the pre-tumor or the tumor state. Subsequent search for signal transduction key nodes upstream of the identified transcription factors and their targets suggested the insulin-like growth factor pathway to render the tumor cells independent of EGF receptor activity. Notably, expression of IGF2 in addition to many components of this pathway was highly upregulated in tumors. Together, we propose a switch in autocrine signaling to foster tumor growth that was initially triggered by EGF and demonstrate the knowledge gain form promoter analysis combined with upstream key node identification.

  3. Antibody-induced dimerization activates the epidermal growth factor receptor tyrosine kinase

    NARCIS (Netherlands)

    Spaargaren, M.; Defize, L. H.; Boonstra, J.; de Laat, S. W.

    1991-01-01

    The relationship between epidermal growth factor receptor (EGF-R) protein tyrosine kinase activation and ligand-induced receptor dimerization was investigated using several bivalent anti-EGF-R antibodies directed against various receptor epitopes. In A431 membrane preparations and permeabilized

  4. Early nongenomic events in aldosterone action in renal collecting duct cells: PKCalpha activation, mineralocorticoid receptor phosphorylation, and cross-talk with the genomic response.

    Science.gov (United States)

    Le Moëllic, Cathy; Ouvrard-Pascaud, Antoine; Capurro, Claudia; Cluzeaud, Francoise; Fay, Michel; Jaisser, Frederic; Farman, Nicolette; Blot-Chabaud, Marcel

    2004-05-01

    Effects of aldosterone on its target cells have long been considered to be mediated exclusively through the genomic pathway; however, evidence has been provided for rapid effects of the hormone that may involve nongenomic mechanisms. Whether an interaction exists between these two signaling pathways is not yet established. In this study, the authors show that aldosterone triggers both early nongenomic and late genomic increase in sodium transport in the RCCD(2) rat cortical collecting duct cell line. In these cells, the early (up to 2.5 h) aldosterone-induced increase in short-circuit current (Isc) is not blocked by the mineralocorticoid receptor (MR) antagonist RU26752, it does not require mRNA or protein synthesis, and it involves the PKCalpha signaling pathway. In addition, this early response is reproduced by aldosterone-BSA, which acts at the cell surface and presumably does not enter the cells (aldo-BSA is unable to trigger the late response). The authors also show that MR is rapidly phosphorylated on serine and threonine residues by aldosterone or aldosterone-BSA. In contrast, the late (4 to 24 h) aldosterone-induced increase in ion transport occurs through activation of the MR and requires mRNA and protein synthesis. Interestingly, nongenomic and genomic aldosterone actions appear to be interdependent. Blocking the PKCalpha pathway results in the inhibition of the late genomic response to aldosterone, as demonstrated by the suppression of aldosterone-induced increase in MR transactivation activity, alpha1 Na(+)/K(+)/ATPase mRNA, and Isc. These data suggest cross-talk between the nongenomic and genomic responses to aldosterone in renal cells and suggest that the aldosterone-MR mediated increase in mRNA/protein synthesis and ion transport depends, at least in part, upon PKCalpha activation. E-mail: marcel.blot-chabaud@pharmacie.univ-mrs.fr

  5. The Frog Skin-Derived Antimicrobial Peptide Esculentin-1a(1-21)NH2 Promotes the Migration of Human HaCaT Keratinocytes in an EGF Receptor-Dependent Manner: A Novel Promoter of Human Skin Wound Healing?

    Science.gov (United States)

    Di Grazia, Antonio; Cappiello, Floriana; Imanishi, Akiko; Mastrofrancesco, Arianna; Picardo, Mauro; Paus, Ralf; Mangoni, Maria Luisa

    2015-01-01

    One of the many functions of skin is to protect the organism against a wide range of pathogens. Antimicrobial peptides (AMPs) produced by the skin epithelium provide an effective chemical shield against microbial pathogens. However, whereas antibacterial/antifungal activities of AMPs have been extensively characterized, much less is known regarding their wound healing-modulatory properties. By using an in vitro re-epithelialisation assay employing special cell-culture inserts, we detected that a derivative of the frog-skin AMP esculentin-1a, named esculentin-1a(1-21)NH2, significantly stimulates migration of immortalized human keratinocytes (HaCaT cells) over a wide range of peptide concentrations (0.025-4 μM), and this notably more efficiently than human cathelicidin (LL-37). This activity is preserved in primary human epidermal keratinocytes. By using appropriate inhibitors and an enzyme-linked immunosorbent assay we found that the peptide-induced cell migration involves activation of the epidermal growth factor receptor and STAT3 protein. These results suggest that esculentin-1a(1-21)NH2 now deserves to be tested in standard wound healing assays as a novel candidate promoter of skin re-epithelialisation. The established ability of esculentin-1a(1-21)NH2 to kill microbes without harming mammalian cells, namely its high anti-Pseudomonal activity, makes this AMP a particularly attractive candidate wound healing promoter, especially in the management of chronic, often Pseudomonas-infected, skin ulcers.

  6. The Frog Skin-Derived Antimicrobial Peptide Esculentin-1a(1-21NH2 Promotes the Migration of Human HaCaT Keratinocytes in an EGF Receptor-Dependent Manner: A Novel Promoter of Human Skin Wound Healing?

    Directory of Open Access Journals (Sweden)

    Antonio Di Grazia

    Full Text Available One of the many functions of skin is to protect the organism against a wide range of pathogens. Antimicrobial peptides (AMPs produced by the skin epithelium provide an effective chemical shield against microbial pathogens. However, whereas antibacterial/antifungal activities of AMPs have been extensively characterized, much less is known regarding their wound healing-modulatory properties. By using an in vitro re-epithelialisation assay employing special cell-culture inserts, we detected that a derivative of the frog-skin AMP esculentin-1a, named esculentin-1a(1-21NH2, significantly stimulates migration of immortalized human keratinocytes (HaCaT cells over a wide range of peptide concentrations (0.025-4 μM, and this notably more efficiently than human cathelicidin (LL-37. This activity is preserved in primary human epidermal keratinocytes. By using appropriate inhibitors and an enzyme-linked immunosorbent assay we found that the peptide-induced cell migration involves activation of the epidermal growth factor receptor and STAT3 protein. These results suggest that esculentin-1a(1-21NH2 now deserves to be tested in standard wound healing assays as a novel candidate promoter of skin re-epithelialisation. The established ability of esculentin-1a(1-21NH2 to kill microbes without harming mammalian cells, namely its high anti-Pseudomonal activity, makes this AMP a particularly attractive candidate wound healing promoter, especially in the management of chronic, often Pseudomonas-infected, skin ulcers.

  7. Acetylcholine receptor (AChR) clustering is regulated both by glycogen synthase kinase 3β (GSK3β)-dependent phosphorylation and the level of CLIP-associated protein 2 (CLASP2) mediating the capture of microtubule plus-ends.

    Science.gov (United States)

    Basu, Sreya; Sladecek, Stefan; Pemble, Hayley; Wittmann, Torsten; Slotman, Johan A; van Cappellen, Wiggert; Brenner, Hans-Rudolf; Galjart, Niels

    2014-10-31

    The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2-9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2-8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Diverse phosphorylation patterns of B cell receptor-associated signaling in naïve and memory human B cells revealed by phosphoflow, a powerful technique to study signaling at the single cell level

    Directory of Open Access Journals (Sweden)

    Franklin R Toapanta

    2012-10-01

    Full Text Available Following interaction with cognate antigens, B cells undergo cell activation, proliferation and differentiation. Ligation of the B cell receptor (BCR leads to the phosphorylation of BCR-associated signaling proteins within minutes of antigen binding, a process with profound consequences for the fate of the cells and development of effector immunity. Phosphoflow allows a rapid evaluation of various signaling pathways in complex heterogenous cell subsets. This novel technique was used in combination with multi-chromatic flow cytometry and fluorescent-cell barcoding to study phosphorylation of BCR-associated signaling pathways in naïve and memory human B cell subsets. Proteins of the initiation (Syk, propagation (Btk, Akt and integration (p38MAPK and Erk1/2 signaling units were studied. Switched memory (Sm CD27+ and Sm CD27- phosphorylation patterns were similar when stimulated with anti-IgA or -IgG. In contrast, naïve and unswitched memory (Um cells showed significant differences following IgM stimulation. Enhanced phosphorylation of Syk was observed in Um cells, suggesting a lower activation threshold. This is likely the result of higher amounts of IgM on the cell surface, higher pan-Syk levels and enhanced susceptibility to phosphatase inhibition. All other signaling proteins evaluated also showed some degree of enhanced phosphorylation in Um cells. Furthermore, both the PLC-γ2 and PI3K pathways were activated in Um cells, while only the PI3K pathway was activated on naïve cells. Um cells were the only ones that activated signaling pathways when stimulated with fluorescently-labeled S. Typhi and S. pneumoniae. Finally, simultaneous evaluation of signaling proteins at the single cell level (multi-phosphorylated cells revealed that interaction with gram positive and negative bacteria resulted in complex and diverse signaling patterns. Phosphoflow holds great potential to accelerate vaccine development by identifying signaling profiles in good

  9. 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

    with receptors in which 1, 2, or all 3 tyrosines were changed to phenylalanines. The triple point mutant EGF-R, expressed in NIH-3T3, exhibited low autophosphorylation in vivo, low biological and reduced kinase activities. Single and double point mutants were down-regulated, as well as wild type EGF......-R in response to EGF showing a half-life of about 1 h. Degradation of the triple point mutant, however, was impaired and resulted in a half-life of 4 h in the presence of EGF. EGF-dependent down-regulation of surface receptors was decreased in the triple point mutant EGF-R as was internalization and degradation...... 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...

  10. SMAD4 loss enables EGF, TGFβ1 and S100A8/A9 induced activation of critical pathways to invasion in human pancreatic adenocarcinoma cells.

    Science.gov (United States)

    Moz, Stefania; Basso, Daniela; Bozzato, Dania; Galozzi, Paola; Navaglia, Filippo; Negm, Ola H; Arrigoni, Giorgio; Zambon, Carlo-Federico; Padoan, Andrea; Tighe, Paddy; Todd, Ian; Franchin, Cinzia; Pedrazzoli, Sergio; Punzi, Leonardo; Plebani, Mario

    2016-10-25

    Epidermal Growth Factor (EGF) receptor overexpression, KRAS, TP53, CDKN2A and SMAD4 mutations characterize pancreatic ductal adenocarcinoma. This mutational landscape might influence cancer cells response to EGF, Transforming Growth Factor β1 (TGFβ1) and stromal inflammatory calcium binding proteins S100A8/A9. We investigated whether chronic exposure to EGF modifies in a SMAD4-dependent manner pancreatic cancer cell signalling, proliferation and invasion in response to EGF, TGFβ1 and S100A8/A9. BxPC3, homozigously deleted (HD) for SMAD4, and BxPC3-SMAD4+ cells were or not stimulated with EGF (100 ng/mL) for three days. EGF pre-treated and non pretreated cells were stimulated with a single dose of EGF (100 ng/mL), TGFβ1 (0,02 ng/mL), S100A8/A9 (10 nM). Signalling pathways (Reverse Phase Protein Array and western blot), cell migration (Matrigel) and cell proliferation (XTT) were evaluated. SMAD4 HD constitutively activated ERK and Wnt/β-catenin, while inhibiting PI3K/AKT pathways. These effects were antagonized by chronic EGF, which increased p-BAD (anti-apoptotic) in response to combined TGFβ1 and S100A8/A9 stimulation. SMAD4 HD underlied the inhibition of NF-κB and PI3K/AKT in response to TGFβ1 and S100A8/A9, which also induced cell migration. Chronic EGF exposure enhanced cell migration of both BxPC3 and BxPC3-SMAD4+, rendering the cells less sensitive to the other inflammatory stimuli. In conclusion, SMAD4 HD is associated with the constitutive activation of the ERK and Wnt/β-catenin signalling pathways, and favors the EGF-induced activation of multiple signalling pathways critical to cancer proliferation and invasion. TGFβ1 and S100A8/A9 mainly inhibit NF-κB and PI3K/AKT pathways and, when combined, sinergize with EGF in enhancing anti-apoptotic p-BAD in a SMAD4-dependent manner.

  11. Lipoxin A4 regulates expression of the estrogen receptor and inhibits 17β-estradiol induced p38 mitogen-activated protein kinase phosphorylation in human endometriotic stromal cells.

    Science.gov (United States)

    Chen, Shuo; Wu, Rong-Feng; Su, Lin; Zhou, Wei-Dong; Zhu, Mao-Bi; Chen, Qiong-Hua

    2014-07-01

    To study the role of lipoxin A4 (LXA4) in endometriosis. Molecular analysis in human samples and primary human endometriotic stromal cells (ESCs). University hospital. Forty-nine premenopausal women (30 patients with endometriosis and 19 controls). Normal and ectopic endometrial biopsies obtained during surgery performed during the proliferative phase of the menstrual cycle; ESCs used for in vitro studies. Levels of LXA4 measured by enzyme-linked immunosorbent assay (ELISA); mRNA levels of the estrogen receptor (ER), progestogen receptor (PR), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) quantified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR); and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation evaluated by Western blotting. The LXA4 expression level decreased in ectopic tissue as well as ERα and PR, although the expression of ERβ increased in ectopic endometrium compared with the controls. Investigations with correlation analysis revealed the expression of LXA4 was positively correlated with ERα and negatively correlated with ERβ in vivo. Moreover, administering LXA4 could augment ERβ expression in ESCs and inhibit the 17β-estradiol-induced phosphorylation of p38 MAPK very likely through ERβ. Our findings indicate that LXA4 regulates ERβ expression and inhibits 17β-estradiol-induced phosphorylation of p38 MAPK, very likely through ERβ in ESCs. Copyright © 2014. Published by Elsevier Inc.

  12. Overcoming acquired drug resistance in colorectal cancer cells by targeted delivery of 5-FU with EGF grafted hollow mesoporous silica nanoparticles

    Science.gov (United States)

    Chen, Lijue; She, Xiaodong; Wang, Tao; He, Li; Shigdar, Sarah; Duan, Wei; Kong, Lingxue

    2015-08-01

    Acquired drug resistance (ADR) can be developed in colorectal cancer cells after 5-fluorouracil (5-FU) treatment and diminish the effectiveness of chemotherapy. In this work, acquired 5-FU resistance in the colorectal cancer cell line SW480 was obtained with the up-regulation of dihydropyrimidine dehydrogenase (DPYD) gene expression which can convert 5-FU to its inactive metabolite. To overcome ADR in colorectal cancer, hollow mesoporous silica nanoparticles (HMSNs) grafted with epidermal growth factor (EGF) were used as nanocarriers to deliver 5-FU to colorectal cancer cells with acquired drug resistance. The effect and mechanism of 5-FU loaded EGF grafted HMSNs (EGF-HMSNs-5-FU) in overcoming acquired drug resistance in SW480/ADR cells were studied. The EGF-HMSNs were demonstrated to be specifically internalized in EGFR overexpressed SW480/ADR cells via a receptor-mediated endocytosis and can escape from endo-lysosomes. The EGF-HMSNs-5-FU exhibited much higher cytotoxicity on SW480/ADR cells than HMSNs-5-FU and free 5-FU while the plain HMSNs did not show significant cytotoxicity. The mechanism of EGF-HMSNs-5-FU in overcoming drug resistance in SW480/ADR cells could be attributed to the specific internalization of EGF-HMSNs-5-FU in EGFR overexpressed cells which can lead to high intracellular drug accumulation and cause cell death through S phase arrest.Acquired drug resistance (ADR) can be developed in colorectal cancer cells after 5-fluorouracil (5-FU) treatment and diminish the effectiveness of chemotherapy. In this work, acquired 5-FU resistance in the colorectal cancer cell line SW480 was obtained with the up-regulation of dihydropyrimidine dehydrogenase (DPYD) gene expression which can convert 5-FU to its inactive metabolite. To overcome ADR in colorectal cancer, hollow mesoporous silica nanoparticles (HMSNs) grafted with epidermal growth factor (EGF) were used as nanocarriers to deliver 5-FU to colorectal cancer cells with acquired drug resistance. The

  13. Transcription, Signaling Receptor Activity, Oxidative Phosphorylation, and Fatty Acid Metabolism Mediate the Presence of Closely Related Species in Distinct Intertidal and Cold-Seep Habitats.

    Science.gov (United States)

    Van Campenhout, Jelle; Vanreusel, Ann; Van Belleghem, Steven; Derycke, Sofie

    2015-12-03

    Bathyal cold seeps are isolated extreme deep-sea environments characterized by low species diversity while biomass can be high. The Håkon Mosby mud volcano (Barents Sea, 1,280 m) is a rather stable chemosynthetic driven habitat characterized by prominent surface bacterial mats with high sulfide concentrations and low oxygen levels. Here, the nematode Halomonhystera hermesi thrives in high abundances (11,000 individuals 10 cm(-2)). Halomonhystera hermesi is a member of the intertidal Halomonhystera disjuncta species complex that includes five cryptic species (GD1-5). GD1-5's common habitat is characterized by strong environmental fluctuations. Here, we compared the transcriptomes of H. hermesi and GD1, H. hermesi's closest relative. Genes encoding proteins involved in oxidative phosphorylation are more strongly expressed in H. hermesi than in GD1, and many genes were only observed in H. hermesi while being completely absent in GD1. Both observations could in part be attributed to high sulfide concentrations and low oxygen levels. Additionally, fatty acid elongation was also prominent in H. hermesi confirming the importance of highly unsaturated fatty acids in this species. Significant higher amounts of transcription factors and genes involved in signaling receptor activity were observed in GD1 (many of which were completely absent in H. hermesi), allowing fast signaling and transcriptional reprogramming which can mediate survival in dynamic intertidal environments. GC content was approximately 8% higher in H. hermesi coding unigenes resulting in differential codon usage between both species and a higher proportion of amino acids with GC-rich codons in H. hermesi. In general our results showed that most pathways were active in both environments and that only three genes are under natural selection. This indicates that also plasticity should be taken in consideration in the evolutionary history of Halomonhystera species. Such plasticity, as well as possible

  14. Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kyotani, Yoji, E-mail: cd147@naramed-u.ac.jp [Department of Pharmacology, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Department of Pharmacy, Nara Medical University Hospital, Kashihara 634-8522 (Japan); Ota, Hiroyo [Second Department of Internal Medicine, Nara Medical University School of Medicine, Kashihara 634-8522 (Japan); Department of Biochemistry, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Itaya-Hironaka, Asako; Yamauchi, Akiyo; Sakuramoto-Tsuchida, Sumiyo [Department of Biochemistry, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Zhao, Jing; Ozawa, Kentaro; Nagayama, Kosuke; Ito, Satoyasu [Department of Pharmacology, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Takasawa, Shin [Department of Biochemistry, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Kimura, Hiroshi [Second Department of Internal Medicine, Nara Medical University School of Medicine, Kashihara 634-8522 (Japan); Uno, Masayuki [Department of Pharmacy, Nara Medical University Hospital, Kashihara 634-8522 (Japan); Yoshizumi, Masanori [Department of Pharmacology, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan)

    2013-11-15

    Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned medium significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation. - Highlights: ●In vitro system for intermittent hypoxia (IH) and sustained hypoxia (SH). ●IH, but not SH, induces the proliferation of rat vascular smooth muscle cell. ●Epiregulin m

  15. Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

    International Nuclear Information System (INIS)

    Kyotani, Yoji; Ota, Hiroyo; Itaya-Hironaka, Asako; Yamauchi, Akiyo; Sakuramoto-Tsuchida, Sumiyo; Zhao, Jing; Ozawa, Kentaro; Nagayama, Kosuke; Ito, Satoyasu; Takasawa, Shin; Kimura, Hiroshi; Uno, Masayuki; Yoshizumi, Masanori

    2013-01-01

    Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned medium significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation. - Highlights: ●In vitro system for intermittent hypoxia (IH) and sustained hypoxia (SH). ●IH, but not SH, induces the proliferation of rat vascular smooth muscle cell. ●Epiregulin m

  16. Identification of a novel antagonist of the ErbB1 receptor capable of inhibiting migration of human glioblastoma cells

    DEFF Research Database (Denmark)

    Staberg, Mikkel; Riemer, Christian; Xu, Ruodan

    2013-01-01

    B1 targeting peptide, termed Herfin-1, was designed based on a model of the tertiary structure of the EGF-EGFR ternary complex. The binding kinetics of this peptide were determined employing surface plasmon resonance analyses. ErbB1-4 expression and phosphorylation in human glioblastoma cell lines U...... processing. RESULTS: The present study shows that Herfin-1 functions as an ErbB1 antagonist. It binds to the extracellular domain of ErbB1 with a KD value of 361 nM. In U87 and U118 cells, both expressing high levels of ErbB1, Herfin-1 inhibits EGF-induced ErbB1 phosphorylation and cell migration....... Additionally, Herfin-1 was found to increase neurite outgrowth in cerebellar granule neurons, likely through the inhibition of a sustained weak ErbB1 activation. CONCLUSIONS: Targeting the ErbB1 receptor dimerization interface is a promising strategy to inhibit receptor activation in ErbB1-expressing glioma...

  17. Polymer carriers for anticancer drugs targeted to EGF receptor

    Czech Academy of Sciences Publication Activity Database

    Studenovský, Martin; Pola, Robert; Pechar, Michal; Etrych, Tomáš; Ulbrich, Karel; Kovář, Lubomír; Kabešová, Martina; Říhová, Blanka

    2012-01-01

    Roč. 12, č. 12 (2012), s. 1714-1720 ISSN 1616-5187 R&D Projects: GA AV ČR IAAX00500803 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z50200510 Institutional support: RVO:61389013 ; RVO:61388971 Keywords : biocompatibility * EGFR * peptides Subject RIV: CD - Macromolecular Chemistry; EC - Immunology (MBU-M) Impact factor: 3.742, year: 2012

  18. EGF-R is Expressed and AP-1 and NF-κ:B Are Activated in Stromal Myofibroblasts Surrounding Colon Adenocarcinomas Paralleling Expression of COX-2 and VEGF

    Directory of Open Access Journals (Sweden)

    Panagiotis A. Konstantinopoulos

    2007-01-01

    Full Text Available Background: COX-2 and VEGF are important triggers of colon cancer growth, metastasis and angiogenesis. Cox-2 promoter contains transcriptional regulatory elements for AP-1 and NF-κ:B transcription factors whilst vegf is a known AP-1 downstream target gene. We investigated whether stromal myofibroblasts surrounding colon adenocarcinomas express COX-2 and VEGF and whether activation of AP-1 and NF-κ:B, as well as expression of EGF-R parallel expression of COX-2 and VEGF in these cells. Methods: Immunohistochemical methodology was performed on archival sections from 40 patients with colon adenocarcinomas. We evaluated c-FOS, p-c-JUN (phosphorylated c-JUN, p-Iκ:B-α (phosphorylated Iκ:B-α, EGF-R, COX-2, NF-κ:B and VEGF expression in stromal myofibroblasts surrounding colon adenocarcinomas. Double immunostaining with a-smooth muscle actin and each antibody was done to verify the expression of these molecules in stromal myofibroblasts. Results: VEGF, p-Iκ:B-α, NF-κ:B, c-FOS, p-c-JUN, EGF-R and COX-2 were expressed in stromal myofibroblasts surrounding colon adenocarcinomas in the majority of cases. EGF-R, p-Iκ:B-α, NF-κ:B, c-FOS and p-c-JUN correlated positively with COX-2 and VEGF expression. Conclusion: Stromal myofibroblasts surrounding colon adenocarcinomas are an important source of VEGF and COX-2 production, while AP-1 and NF-κ:B transcription factors are activated and EGF-R is expressed in these cells and associated with COX-2 and VEGF production.

  19. Epidermal growth factor (EGF) A61G polymorphism and EGF gene expression in normal colon tissue from patients with colorectal cancer

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise G; Nielsen, Jens N; Ornskov, Dorthe

    2007-01-01

    Introduction. EGF/EGFR interactions are important mechanisms behind colorectal tumour development and growth. Recently a single nucleotide polymorphism in the EGF gene has been identified (EGF A61G). It may be a potential predictor for survival of patients receiving EGFR-inhibitor cetuximab...

  20. Heparin-Binding EGF-like Growth Factor (HB-EGF) Therapy for Intestinal Injury: Application and Future Prospects

    Science.gov (United States)

    Yang, Jixin; Su, Yanwei; Zhou, Yu; Besner, Gail E.

    2014-01-01

    Throughout the past 20 years, we have been investigating the potential therapeutic roles of heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor family, in various models of intestinal injury including necrotizing enterocolitis (NEC), intestinal ischemia/reperfusion (I/R) injury, and hemorrhagic shock and resuscitation (HS/R). Our studies have demonstrated that HB-EGF acts as an effective mitogen, a restitution-inducing reagent, a cellular trophic factor, an anti-apoptotic protein and a vasodilator, via its effects on various cell types in the intestine. In the current paper, we have reviewed the application and therapeutic effects of HB-EGF in three classic animal models of intestinal injury, with particular emphasis on its protection of the intestines from NEC. Additionally, we have summarized the protective functions of HB-EGF on various target cells in the intestine. Lastly, we have provided a brief discussion focusing on the future development of HB-EGF clinical applications for the treatment of various forms of intestinal injury including NEC. PMID:24345808

  1. Tc-{sup 99m} direct radiolabeling of monoclonal antibody ior egf/r3: quality control and image studies in mice

    Energy Technology Data Exchange (ETDEWEB)

    Dias, Carla Roberta; Marczewski, Barbara; Moraes, Vanessa; Barboza, Marycel Figols de; Osso Junior, Joao Alberto [Instituto de Pesquisas Energeticas e Nucleares (IPEN-CNEN/SP), SP (Brazil). Centro de Radiofarmacia]. E-mail: crdias@ipen.br

    2005-10-15

    Monoclonal antibodies (Mabs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in the practice of nuclear medicine. The ior egf/r3 (Centis, Cuba) is a murine monoclonal antibody against epidermal growth factor receptor (EGF-R) and has been widely used in the radioimmunodiagnosis of tumors of epithelial origin. Labeled with 99m Tc, its main application in Nuclear Medicine is the follow up, detection and evaluation of tumor recurrences. The objective of this work is to describe the preparation of a lyophilized formulation (kit) for radiolabeling the Mab ior egf/r3 with 99m Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, image studies and stability of the formulation are reported. The study demonstrated that the kit formulation can be labeled with 99m Tc at high yields and can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies.(author)

  2. Near Infrared Optical Visualization of Epidermal Growth Factor Receptors Levels in COLO205 Colorectal Cell Line, Orthotopic Tumor in Mice and Human Biopsies

    Directory of Open Access Journals (Sweden)

    Philip Lazarovici

    2013-07-01

    Full Text Available In this study, we present the applicability of imaging epidermal growth factor (EGF receptor levels in preclinical models of COLO205 carcinoma cells in vitro, mice with orthotopic tumors and ex vivo colorectal tumor biopsies, using EGF-labeled with IRDye800CW (EGF-NIR. The near infrared (NIR bio-imaging of COLO205 cultures indicated specific and selective binding, reflecting EGF receptors levels. In vivo imaging of tumors in mice showed that the highest signal/background ratio between tumor and adjacent tissue was achieved 48 hours post-injection. Dissected colorectal cancer tissues from different patients demonstrated ex vivo specific imaging using the NIR bio-imaging platform of the heterogeneous distributed EGF receptors. Moreover, in the adjacent gastrointestinal tissue of the same patients, which by Western blotting was demonstrated as EGF receptor negative, no labeling with EGF-NIR probe was detected. Present results support the concept of tumor imaging by measuring EGF receptor levels using EGF-NIR probe. This platform is advantageous for EGF receptor bio-imaging of the NCI-60 recommended panel of tumor cell lines including 6–9 colorectal cell lines, since it avoids radioactive probes and is appropriate for use in the clinical setting using NIR technologies in a real-time manner.

  3. EGF suppresses hydrogen peroxide induced Ca2+ influx by inhibiting L-type channel activity in cultured human corneal endothelial cells.

    Science.gov (United States)

    Mergler, Stefan; Pleyer, Uwe; Reinach, Peter; Bednarz, Jürgen; Dannowski, Haike; Engelmann, Katrin; Hartmann, Christian; Yousif, Tarik

    2005-02-01

    Endogenous generated hydrogen peroxide during eye bank storage limits viability. We determined in cultured human corneal endothelial cells (HCEC) whether: (1) this oxidant induces elevations in intracellular calcium concentration [Ca2+]i; (2) epidermal growth factor (EGF) medium supplementation has a protective effect against peroxide mediated rises in [Ca2+]i. Whereas pathophysiological concentrations of H2O2 (10 mM) induced irreversible large increases in [Ca2+]i, lower concentrations (up to 1 mM) had smaller effects, which were further reduced by exposure to either 5 microM nifedipine or EGF (10 ng ml(-1)). EGF had a larger protective effect against H2O2-induced rises in [Ca2+]i than nifedipine. In addition, icilin, the agonist for the temperature sensitive transient receptor potential protein, TRPM8, had complex dose-dependent effects (i.e. 10 and 50 microM) on [Ca2+]i. At 10 microM, it reversibly elevated [Ca2+]i whereas at 50 microM an opposite effect occurred suggesting complex effects of temperature on endothelial viability. Taken together, H2O2 induces rises in [Ca2+]i that occur through increases in Ca2+ permeation along plasma membrane pathways that include L-type Ca2+ channels as well as other EGF-sensitive pathways. As EGF overcomes H2O2-induced rises in [Ca2+]i, its presence during eye bank storage could improve the outcome of corneal transplant surgery.

  4. Insulin treatment promotes tyrosine phosphorylation of PKR and inhibits polyIC induced PKR threonine phosphorylation.

    Science.gov (United States)

    Swetha, Medchalmi; Ramaiah, Kolluru V A

    2015-11-01

    Tyrosine phosphorylation of insulin receptor beta (IRβ) in insulin treated HepG2 cells is inversely correlated to ser(51) phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) that regulates protein synthesis. Insulin stimulates interaction between IRβ and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IRβ and anti-phosphotyrosine antibodies, recombinant IRβ and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IRβ and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IRβ, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2α phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IRβ, reduces PKR association with the receptor, IRβ in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Functional characterization of autophosphorylation sites of the activated insulin receptor-tyrosine kinase

    International Nuclear Information System (INIS)

    Flores-Riveros, J.R.; Lane, M.D.

    1987-01-01

    Insulin receptor, solubilized from 3T3-L1 cellular membranes and then purified, was autophosphorylated with [γ- 32 P]ATP in the absence or presence of insulin. Specific phosphopeptides generated by trypsin digestion of the 32 P-labeled β-subunit were identified and separated by reverse phase HPLC. In the absence of insulin, radioactivity of the phosphopeptides is evenly distributed among four major peaks designated as sites I, II, III and IV, according to their order of elution. This pattern is maintained for at least the first 30 min of autophosphorylation. When the reaction is carried out in the presence of insulin, > 50% of the total 32 P radioactivity is found in site I and the rate of 32 P incorporation into this site is markedly higher than into sites II, III and IV. Maximal activation of tyrosine kinase activity, as estimated by substrate phosphorylation, is coincident with the nearly complete phosphorylation of site I. Delayed activation of previously autophosphorylated receptor by insulin, but not by EGF or IGF-I, produced a similar pattern where phosphorylated site I predominates. These observations indicate that one major insulin-regulated autophosphorylation site in the β-subunit is responsible for activation of the insulin receptor tyrosine kinase. The isolation of this phosphopeptide on a preparative scale and its characterization are now in progress

  6. The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

    OpenAIRE

    Hoskins, J.T.; Zhou, Z.; Harding, P.A.

    2008-01-01

    A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser108/121, HB-EGF-Cys/Ser116/132, and HB-EGF-Cys/Ser134/143) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with Mr of 6.5, 21 and 24kDa were observed from lys...

  7. Insulin-Mimetic Action of Rhoifolin and Cosmosiin Isolated from Citrus grandis (L. Osbeck Leaves: Enhanced Adiponectin Secretion and Insulin Receptor Phosphorylation in 3T3-L1 Cells

    Directory of Open Access Journals (Sweden)

    Yerra Koteswara Rao

    2011-01-01

    Full Text Available Citrus grandis (L. Osbeck (red wendun leaves have been used in traditional Chinese medicine to treat several illnesses including diabetes. However, there is no scientific evidence supporting these actions and its active compounds. Two flavone glycosides, rhoifolin and cosmosiin were isolated for the first time from red wendun leaves and, identified these leaves are rich source for rhoifolin (1.1%, w/w. In differentiated 3T3-L1 adipocytes, rhoifolin and cosmosiin showed dose-dependent response in concentration range of o.oo1–5 μM and 1–20 μM, respectively, in biological studies beneficial to diabetes. Particularly, rhoifolin and cosmosiin at 0.5 and 20 μM, respectively showed nearly similar response to that 10 nM of insulin, on adiponectin secretion level. Furthermore, 5 μM of rhoifolin and 20 μM of cosmosiin showed equal potential with 10 nM of insulin to increase the phosphorylation of insulin receptor-β, in addition to their positive effect on GLUT4 translocation. These findings indicate that rhoifolin and cosmosiin from red wendun leaves may be beneficial for diabetic complications through their enhanced adiponectin secretion, tyrosine phosphorylation of insulin receptor-β and GLUT4 translocation.

  8. Dorsal hippocampal NMDA receptor blockade impairs extinction of naloxone-precipitated conditioned place aversion in acute morphine-treated rats by suppressing ERK and CREB phosphorylation in the basolateral amygdala.

    Science.gov (United States)

    Wang, Wei-Sheng; Chen, Zhong-Guo; Liu, Wen-Tao; Chi, Zhi-Qiang; He, Ling; Liu, Jing-Gen

    2015-01-01

    Substantial evidence shows that negative reinforcement resulting from the aversive affective consequences of opiate withdrawal may play a crucial role in drug relapse. Understanding the mechanisms underlying the loss (extinction) of conditioned aversion of drug withdrawal could facilitate the treatment of drug addiction. Naloxone-induced conditioned place aversion (CPA) of Sprague-Dawley rats was used to measure conditioned aversion. An NMDA receptor antagonist and MAPK kinase inhibitor were applied through intracranial injections. The phosphorylation of ERK and cAMP response element-binding protein (CREB) was detected using Western blot. The extinction of CPA behaviour increased the phosphorylation of ERK and CREB in the dorsal hippocampus (DH) and basolateral amygdala (BLA), but not in the central amygdala (CeA). Intra-DH injection of AP5 or intra-BLA injection of AP-5 or U0126 before extinction training significantly attenuated ERK and CREB phosphorylation in the BLA and impaired the extinction of CPA behaviour. Although intra-DH injections of AP-5 attenuated extinction training-induced activation of the ERK-CREB pathway in the BLA, intra-BLA injection of AP5 had no effect on extinction training-induced activation of the ERK-CREB pathway in the DH. These results suggest that activation of ERK and CREB in the BLA and DH is involved in the extinction of CPA behaviour and that the DH, via a direct or indirect pathway, modulates the activity of ERK and CREB in the BLA through activation of NMDA receptors after extinction training. Understanding the mechanisms underlying the extinction of conditioned aversion could facilitate the treatment of drug addiction. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The British Pharmacological Society.

  9. Gallic acid-capped gold nanoparticles inhibit EGF-induced MMP-9 expression through suppression of p300 stabilization and NFκB/c-Jun activation in breast cancer MDA-MB-231 cells.

    Science.gov (United States)

    Chen, Ying-Jung; Lee, Yuan-Chin; Huang, Chia-Hui; Chang, Long-Sen

    2016-11-01

    Triple-negative breast cancers (TNBCs) are highly invasive and have a higher rate of distant metastasis. Matrix metalloproteinase-9 (MMP-9) plays a crucial role in EGF/EGFR-mediated malignant progression and metastasis of TNBCs. Various studies have revealed that treatment with gallic acid down-regulates MMP-9 expression in cancer cells, and that conjugation of phytochemical compounds with gold nanoparticles (AuNPs) increases the anti-tumor activity of the phytochemical compounds. Thus, the effect of gallic acid-capped AuNPs (GA-AuNPs) on MMP-9 expression in EGF-treated TNBC MDA-MB-231 cells was analyzed in the present study. The so-called green synthesis of AuNPs by means of gallic acid was performed at pH10, and the resulting GA-AuNPs had spherical shape with an average diameter of approximately 50nm. GA-AuNPs notably suppressed migration and invasion of EGF-treated cells, and inhibited EGF-induced MMP-9 up-regulation. GA-AuNPs abrogated EGF-induced Akt/p65 and ERK/c-Jun phosphorylation, leading to down-regulation of MMP-9 mRNA and protein expression in EGF-treated cells. Meanwhile, EGF-induced p300 stabilization was found to be involved in MMP-9 expression, whereas GA-AuNPs inhibited the EGF-promoted stability of the p300 protein. Although GA-AuNPs and gallic acid suppressed EGF-induced MMP-9 up-regulation via the same signaling pathway, the effective concentration of gallic acid was approximately 100-fold higher than that of GA-AuNPs for inhibition of MMP-9 expression in EGF-treated cells to a similar extent. Collectively, our data indicate that, in comparison with gallic acid, GA-AuNPs have a superior ability to inhibit EGF/EGFR-mediated MMP-9 expression in TNBC MDA-MB-231 cells. Our findings also point to a way to improve the anti-tumor activity of gallic acid. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. ABA renewal involves enhancements in both GluA2-lacking AMPA receptor activity and GluA1 phosphorylation in the lateral amygdala.

    Directory of Open Access Journals (Sweden)

    Kyungjoon Park

    Full Text Available Fear renewal, the context-specific relapse of fear following fear extinction, is a leading animal model of post-traumatic stress disorders (PTSD and fear-related disorders. Although fear extinction can diminish fear responses, this effect is restricted to the context where the extinction is carried out, and the extinguished fear strongly relapses when assessed in the original acquisition context (ABA renewal or in a context distinct from the conditioning and extinction contexts (ABC renewal. We have previously identified Ser831 phosphorylation of GluA1 subunit in the lateral amygdala (LA as a key molecular mechanism for ABC renewal. However, molecular mechanisms underlying ABA renewal remain to be elucidated. Here, we found that both the excitatory synaptic efficacy and GluA2-lacking AMPAR activity at thalamic input synapses onto the LA (T-LA synapses were enhanced upon ABA renewal. GluA2-lacking AMPAR activity was also increased during low-threshold potentiation, a potential cellular substrate of renewal, at T-LA synapses. The microinjection of 1-naphtylacetyl-spermine (NASPM, a selective blocker of GluA2-lacking AMPARs, into the LA attenuated ABA renewal, suggesting a critical role of GluA2-lacking AMPARs in ABA renewal. We also found that Ser831 phosphorylation of GluA1 in the LA was increased upon ABA renewal. We developed a short peptide mimicking the Ser831-containing C-tail region of GluA1, which can be phosphorylated upon renewal (GluA1S; thus, the phosphorylated GluA1S may compete with Ser831-phosphorylated GluA1. This GluA1S peptide blocked the low-threshold potentiation when dialyzed into a recorded neuron. The microinjection of a cell-permeable form of GluA1S peptide into the LA attenuated ABA renewal. In support of the GluA1S experiments, a GluA1D peptide (in which the serine at 831 is replaced with a phosphomimetic amino acid, aspartate attenuated ABA renewal when microinjected into the LA. These findings suggest that enhancements

  11. ABA renewal involves enhancements in both GluA2-lacking AMPA receptor activity and GluA1 phosphorylation in the lateral amygdala.

    Science.gov (United States)

    Park, Kyungjoon; Song, Beomjong; Kim, Jeongyeon; Hong, Ingie; Song, Sangho; Lee, Junuk; Park, Sungmo; Kim, Jihye; An, Bobae; Lee, Hyun Woo; Lee, Seungbok; Kim, Hyun; Lee, Justin C; Lee, Sukwon; Choi, Sukwoo

    2014-01-01

    Fear renewal, the context-specific relapse of fear following fear extinction, is a leading animal model of post-traumatic stress disorders (PTSD) and fear-related disorders. Although fear extinction can diminish fear responses, this effect is restricted to the context where the extinction is carried out, and the extinguished fear strongly relapses when assessed in the original acquisition context (ABA renewal) or in a context distinct from the conditioning and extinction contexts (ABC renewal). We have previously identified Ser831 phosphorylation of GluA1 subunit in the lateral amygdala (LA) as a key molecular mechanism for ABC renewal. However, molecular mechanisms underlying ABA renewal remain to be elucidated. Here, we found that both the excitatory synaptic efficacy and GluA2-lacking AMPAR activity at thalamic input synapses onto the LA (T-LA synapses) were enhanced upon ABA renewal. GluA2-lacking AMPAR activity was also increased during low-threshold potentiation, a potential cellular substrate of renewal, at T-LA synapses. The microinjection of 1-naphtylacetyl-spermine (NASPM), a selective blocker of GluA2-lacking AMPARs, into the LA attenuated ABA renewal, suggesting a critical role of GluA2-lacking AMPARs in ABA renewal. We also found that Ser831 phosphorylation of GluA1 in the LA was increased upon ABA renewal. We developed a short peptide mimicking the Ser831-containing C-tail region of GluA1, which can be phosphorylated upon renewal (GluA1S); thus, the phosphorylated GluA1S may compete with Ser831-phosphorylated GluA1. This GluA1S peptide blocked the low-threshold potentiation when dialyzed into a recorded neuron. The microinjection of a cell-permeable form of GluA1S peptide into the LA attenuated ABA renewal. In support of the GluA1S experiments, a GluA1D peptide (in which the serine at 831 is replaced with a phosphomimetic amino acid, aspartate) attenuated ABA renewal when microinjected into the LA. These findings suggest that enhancements in both the

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  13. SNP analyses of growth factor genes EGF, TGF{beta}-1, and HGF reveal haplotypic association of EGF with autism

    Energy Technology Data Exchange (ETDEWEB)

    Toyoda, Takao; Thanseem, Ismail; Kawai, Masayoshi; Sekine, Yoshimoto [Department of Psychiatry and Neurology, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); Nakamura, Kazuhiko; Anitha, Ayyappan; Suda, Shiro [Department of Psychiatry and Neurology, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); Yamada, Kazuo [Laboratory of Molecular Psychiatry, RIKEN Brain Science Institute, Saitama (Japan); Tsujii, Masatsugu [Faculty of Sociology, Chukyo University, Toyota, Aichi (Japan); [The Osaka-Hamamatsu Joint Research Center for Child Mental Development, Hamamatsu University School of Medicine, Hamamatsu (Japan); Iwayama, Yoshimi; Hattori, Eiji; Toyota, Tomoko; Yoshikawa, Takeo [Laboratory of Molecular Psychiatry, RIKEN Brain Science Institute, Saitama (Japan); Miyachi, Taishi; Tsuchiya, Kenji; Sugihara, Gen-ichi; Matsuzaki, Hideo [The Osaka-Hamamatsu Joint Research Center for Child Mental Development, Hamamatsu University School of Medicine, Hamamatsu (Japan); Iwata, Yasuhide; Suzuki, Katsuaki [Department of Psychiatry and Neurology, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); Mori, Norio [Department of Psychiatry and Neurology, Hamamatsu University School of Medicine, Hamamatsu 431-3192 (Japan); [The Osaka-Hamamatsu Joint Research Center for Child Mental Development, Graduate School of Medicine, Osaka University (Japan); Ouchi, Yasuomi [The Osaka-Hamamatsu Joint Research Center for Child Mental Development, Hamamatsu University School of Medicine, Hamamatsu (Japan); [The Positron Medical Center, Hamamatsu Medical Center, Hamamatsu (Japan); Sugiyama, Toshiro [Aichi Children' s Health and Medical Center, Obu, Aichi (Japan); Takei, Nori [The Osaka-Hamamatsu Joint Research Center for Child Mental Development, Hamamatsu University School of Medicine, Hamamatsu (Japan)

    2007-09-07

    Autism is a pervasive neurodevelopmental disorder diagnosed in early childhood. Growth factors have been found to play a key role in the cellular differentiation and proliferation of the central and peripheral nervous systems. Epidermal growth factor (EGF) is detected in several regions of the developing and adult brain, where, it enhances the differentiation, maturation, and survival of a variety of neurons. Transforming growth factor-{beta} (TGF{beta}) isoforms play an important role in neuronal survival, and the hepatocyte growth factor (HGF) has been shown to exhibit neurotrophic activity. We examined the association of EGF, TGF{beta}1, and HGF genes with autism, in a trio association study, using DNA samples from families recruited to the Autism Genetic Resource Exchange; 252 trios with a male offspring scored for autism were selected for the study. Transmission disequilibrium test revealed significant haplotypic association of EGF with autism. No significant SNP or haplotypic associations were observed for TGF{beta}1 or HGF. Given the role of EGF in brain and neuronal development, we suggest a possible role of EGF in the pathogenesis of autism.

  14. SNP analyses of growth factor genes EGF, TGFβ-1, and HGF reveal haplotypic association of EGF with autism

    International Nuclear Information System (INIS)

    Toyoda, Takao; Nakamura, Kazuhiko; Yamada, Kazuo; Thanseem, Ismail; Anitha, Ayyappan; Suda, Shiro; Tsujii, Masatsugu; Iwayama, Yoshimi; Hattori, Eiji; Toyota, Tomoko; Miyachi, Taishi; Iwata, Yasuhide; Suzuki, Katsuaki; Matsuzaki, Hideo; Kawai, Masayoshi; Sekine, Yoshimoto; Tsuchiya, Kenji; Sugihara, Gen-ichi; Ouchi, Yasuomi; Sugiyama, Toshiro; Takei, Nori; Yoshikawa, Takeo; Mori, Norio

    2007-01-01

    Autism is a pervasive neurodevelopmental disorder diagnosed in early childhood. Growth factors have been found to play a key role in the cellular differentiation and proliferation of the central and peripheral nervous systems. Epidermal growth factor (EGF) is detected in several regions of the developing and adult brain, where, it enhances the differentiation, maturation, and survival of a variety of neurons. Transforming growth factor-β (TGFβ) isoforms play an important role in neuronal survival, and the hepatocyte growth factor (HGF) has been shown to exhibit neurotrophic activity. We examined the association of EGF, TGFβ1, and HGF genes with autism, in a trio association study, using DNA samples from families recruited to the Autism Genetic Resource Exchange; 252 trios with a male offspring scored for autism were selected for the study. Transmission disequilibrium test revealed significant haplotypic association of EGF with autism. No significant SNP or haplotypic associations were observed for TGFβ1 or HGF. Given the role of EGF in brain and neuronal development, we suggest a possible role of EGF in the pathogenesis of autism

  15. Mechanisms of Inhibition of the Epidermal Growth Factor Receptor: Implications for Novel Anti-Cancer Therapies

    National Research Council Canada - National Science Library

    Klein, Daryl E

    2005-01-01

    .... No secreted or extracellular ErbB receptor inhibitors have been reported in mammals. However, two natural inhibitors of the highly homologous Drosophila EGF receptor are found in Drosophila melanogaster...

  16. Odor preference learning and memory modify GluA1 phosphorylation and GluA1 distribution in the neonate rat olfactory bulb: testing the AMPA receptor hypothesis in an appetitive learning model.

    Science.gov (United States)

    Cui, Wen; Darby-King, Andrea; Grimes, Matthew T; Howland, John G; Wang, Yu Tian; McLean, John H; Harley, Carolyn W

    2011-01-01

    An increase in synaptic AMPA receptors is hypothesized to mediate learning and memory. AMPA receptor increases have been reported in aversive learning models, although it is not clear if they are seen with memory maintenance. Here we examine AMPA receptor changes in a cAMP/PKA/CREB-dependent appetitive learning model: odor preference learning in the neonate rat. Rat pups were given a single pairing of peppermint and 2 mg/kg isoproterenol, which produces a 24-h, but not a 48-h, peppermint preference in the 7-d-old rat pup. GluA1 PKA-dependent phosphorylation peaked 10 min after the 10-min training trial and returned to baseline within 90 min. At 24 h, GluA1 subunits did not change overall but were significantly increased in synaptoneurosomes, consistent with increased membrane insertion. Immunohistochemistry revealed a significant increase in GluA1 subunits in olfactory bulb glomeruli, the targets of olfactory nerve axons. Glomerular increases were seen at 3 and 24 h after odor exposure in trained pups, but not in control pups. GluA1 increases were not seen as early as 10 min after training and were no longer observed 48 h after training when odor preference is no longer expressed behaviorally. Thus, the pattern of increased GluA1 membrane expression closely follows the memory timeline. Further, blocking GluA1 insertion using an interference peptide derived from the carboxyl tail of the GluA1 subunit inhibited 24 h odor preference memory providing causative support for our hypothesis. PKA-mediated GluA1 phosphorylation and later GluA1 insertion could, conjointly, provide increased AMPA function to support both short-term and long-term appetitive memory.

  17. Antibody-induced activation of the epidermal growth factor receptor tyrosine kinase requires the presence of detergent

    NARCIS (Netherlands)

    Spaargaren, M.; Defize, L. H.; de Laat, S. W.; Boonstra, J.

    1990-01-01

    Activation of the epidermal growth factor receptor (EGF-R) tyrosine kinase was investigated in membrane preparations as well as intact A431 cells, using anti-EGF-R antibodies directed against extra- and intracellular receptor domains. In vitro assay conditions were mimicked on whole cells by a mild

  18. Arsenic interferes with the signaling transduction pathway of T cell receptor activation by increasing basal and induced phosphorylation of Lck and Fyn in spleen cells

    International Nuclear Information System (INIS)

    Soto-Pena, Gerson A.; Vega, Libia

    2008-01-01

    Arsenic is known to produce inhibition as well as induction of immune cells proliferative responses depending on the doses as one of its mechanisms of immunotoxicity. Here we evaluate the effect of arsenic exposure on the activation of splenic mononuclear cells (SMC) in male CD57BL6N mice. Intra-gastric exposure to arsenic (as sodium arsenite) for 30 days (1, 0.1, or 0.01 mg/kg/day), reduced the proportion of CD4+ cells and the CD4+/CD8+ ratio in the spleen, increasing the proportion of CD11b+ cells. Arsenic exposure did not modify the proportion of B cells. SMC showed an increased level of phosphorylation of lck and fyn kinases (first kinases associated to TCR complex when activated). Although normal levels of apoptosis were observed on freshly isolated SMC, an increase in apoptotic cells related with the increase in phosphorylation of lck and fyn was observed when SMC were activated with Concanavalin-A (Con-A). Arsenic exposure reduced the proliferative response of SMC to Con-A, and also reduced secretion of IL-2, IL-6, IL-12 and IFNγ. No effect was observed on IL-4, and IL-10 secretion. The same effects were observed when SMC of exposed animals were activated with anti-CD3/CD28 antibodies for 24 h, but these effects were transitory since a recovery, up to control levels or even higher, were observed after 72 h of stimulation. This study demonstrates that repeated and prolonged exposure to arsenic alters cell populations and produces functional changes depending on the specific activation pathway, and could be related with the phosphorylation status of lck and fyn kinases

  19. N-terminal truncation of the dopamine transporter abolishes phorbol ester- and substance P receptor-stimulated phosphorylation without impairing transporter internalization

    DEFF Research Database (Denmark)

    Granas, Charlotta; Ferrer, Jasmine; Loland, Claus Juul

    2003-01-01

    (q)-coupled human substance P receptor (hNK-1) co-expressed with hDAT in HEK293 cells and in N2A neuroblastoma cells. In both cell lines, activation of the hNK-1 receptor by substance P reduced the V(max) for [(3)H]dopamine uptake to the same degree as did PMA ( approximately 50 and approximately 20% in HEK293...

  20. Single molecule force spectroscopy for in-situ probing oridonin inhibited ROS-mediated EGF-EGFR interactions in living KYSE-150 cells.

    Science.gov (United States)

    Pi, Jiang; Jin, Hua; Jiang, Jinhuan; Yang, Fen; Cai, Huaihong; Yang, Peihui; Cai, Jiye; Chen, Zheng W

    2017-05-01

    As the active anticancer component of Rabdosia Rubescens, oridonin has been proved to show strong anticancer activity in cancer cells, which is also found to be closely related to its specific inhibition effects on the EGFR tyrosine kinase activity. In this study, atomic force microscopy based single molecule force spectroscopy (AFM-SMFS) was used for real-time and in-situ detection of EGF-EGFR interactions in living esophageal cancer KYSE-150 cells to evaluate the anticancer activity of oridonin for the first time. Oridonin was found to induce apoptosis and also reduce EGFR expression in KYSE-150 cells. AFM-SMFS results demonstrated that oridonin could inhibit the binding between EGF and EGFR in KYSE-150 cells by decreasing the unbinding force and binding probability for EGF-EGFR complexes, which was further proved to be closely associated with the intracellular ROS level. More precise mechanism studies based on AFM-SMFS demonstrated that oridonin treatment could decrease the energy barrier width, increase the dissociation off rate constant and decrease the activation energy of EGF-EGFR complexes in ROS dependent way, suggesting oridonin as a strong anticancer agent targeting EGF-EGFR interactions in cancer cells through ROS dependent mechanism. Our results not only suggested oridonin as a strong anticancer agent targeting EGF-EGFR interactions in ROS dependent mechanism, but also highlighted AFM-SMFS as a powerful technique for pharmacodynamic studies by detecting ligand-receptor interactions, which was also expected to be developed into a promising tool for the screening and mechanism studies of drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Oxidative phosphorylation revisited

    DEFF Research Database (Denmark)

    Nath, Sunil; Villadsen, John

    2015-01-01

    The fundamentals of oxidative phosphorylation and photophosphorylation are revisited. New experimental data on the involvement of succinate and malate anions respectively in oxidative phosphorylation and photophosphorylation are presented. These new data offer a novel molecular mechanistic...

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

    Science.gov (United States)

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

    2013-01-01

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

  3. Genetic variations in EGF and EGFR and glioblastoma outcome

    DEFF Research Database (Denmark)

    Sjöström, Sara; Andersson, Ulrika; Liu, Yanhong

    2010-01-01

    associated EGF polymorphisms in haplotype block 4 were validated in a set from MDACC; however, none of the associations were clearly replicated. rs379644 had a hazard ratio (HR) of 1.19 (0.94-1.51) in the whole population with 18.6 months survival in the risk genotype compared with 24.5 in the reference...

  4. Identification of a novel EGF-sensitive cell cycle checkpoint

    International Nuclear Information System (INIS)

    Walker, Francesca; Zhang Huihua; Burgess, Antony W.

    2007-01-01

    The site of action of growth factors on mammalian cell cycle has been assigned to the boundary between the G1 and S phases. We show here that Epidermal Growth Factor (EGF) is also required for mitosis. BaF/3 cells expressing the EGFR (BaF/wtEGFR) synthesize DNA in response to EGF, but arrest in S-phase. We have generated a cell line (BaF/ERX) with defective downregulation of the EGFR and sustained activation of EGFR signalling pathways: these cells undergo mitosis in an EGF-dependent manner. The transit of BaF/ERX cells through G2/M strictly requires activation of EGFR and is abolished by AG1478. This phenotype is mimicked by co-expression of ErbB2 in BaF/wtEGFR cells, and abolished by inhibition of the EGFR kinase, suggesting that sustained signalling of the EGFR, through impaired downregulation of the EGFR or heterodimerization, is required for completion of the cycle. We have confirmed the role of EGFR signalling in the G2/M phase of the cell cycle using a human tumor cell line which overexpresses the EGFR and is dependent on EGFR signalling for growth. These findings unmask an EGF-sensitive checkpoint, helping to understand the link between sustained EGFR signalling, proliferation and the acquisition of a radioresistant phenotype in cancer cells

  5. dlk acts as a negative regulator of Notch1 activation through interactions with specific EGF-like repeats

    International Nuclear Information System (INIS)

    Baladron, Victoriano; Ruiz-Hidalgo, Maria Jose; Nueda, Maria Luisa; Diaz-Guerra, Maria Jose M.; Garcia-Ramirez, Jose Javier; Bonvini, Ezio; Gubina, Elena; Laborda, Jorge

    2005-01-01

    The protein dlk, encoded by the Dlk1 gene, belongs to the Notch epidermal growth factor (EGF)-like family of receptors and ligands, which participate in cell fate decisions during development. The molecular mechanisms by which dlk regulates cell differentiation remain unknown. By using the yeast two-hybrid system, we found that dlk interacts with Notch1 in a specific manner. Moreover, by using luciferase as a reporter gene under the control of a CSL/RBP-Jk/CBF-1-dependent promoter in the dlk-negative, Notch1-positive Balb/c 14 cell line, we found that addition of synthetic dlk EGF-like peptides to the culture medium or forced expression of dlk decreases endogenous Notch activity. Furthermore, the expression of the gene Hes-1, a target for Notch1 activation, diminishes in confluent Balb/c14 cells transfected with an expression construct encoding for the extracellular EGF-like region of dlk. The expression of Dlk1 and Notch1 increases in 3T3-L1 cells maintained in a confluent state for several days, which is associated with a concomitant decrease in Hes-1 expression. On the other hand, the decrease of Dlk1 expression in 3T3-L1 cells by antisense cDNA transfection is associated with an increase in Hes-1 expression. These results suggest that dlk functionally interacts in vivo with Notch1, which may lead to the regulation of differentiation processes modulated by Notch1 activation and signaling, including adipogenesis

  6. Binding of sFRP-3 to EGF in the extra-cellular space affects proliferation, differentiation and morphogenetic events regulated by the two molecules.

    Directory of Open Access Journals (Sweden)

    Raffaella Scardigli

    Full Text Available BACKGROUND: sFRP-3 is a soluble antagonist of Wnts, widely expressed in developing embryos. The Wnt gene family comprises cysteine-rich secreted ligands that regulate cell proliferation, differentiation, organogenesis and oncogenesis of different organisms ranging from worms to mammals. In the canonical signal transduction pathway Wnt proteins bind to the extracellular domain of Frizzled receptors and consequently recruit Dishevelled (Dsh to the cell membrane. In addition to Wnt membrane receptors belonging to the Frizzled family, several other molecules have been described which share homology in the CRD domain and lack the putative trans-membrane domain, such as sFRP molecules (soluble Frizzled Related Protein. Among them, sFRP-3 was originally isolated from bovine articular cartilage and also as a component of the Spemann organizer. sFRP-3 blocks Wnt-8 induced axis duplication in Xenopus embryos and binds to the surface of cells expressing a membrane-anchored form of Wnt-1. Injection of sFRP-3 mRNA blocks expression of XMyoD mRNA and leads to embryos with enlarged heads and shortened trunks. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that sFRP-3 specifically blocks EGF-induced fibroblast proliferation and foci formation. Over-expression of sFRP-3 reverts EGF-mediated inhibition of hair follicle development in the mouse ectoderm while its ablation in Xenopus maintains EGF-mediated inhibition of ectoderm differentiation. Conversely, over-expression of EGF reverts the inhibition of somitic myogenesis and axis truncation in Xenopus and mouse embryos caused by sFRP-3. In vitro experiments demonstrated a direct binding of EGF to sFRP-3 both on heparin and on the surface of CHO cells where the molecule had been membrane anchored. CONCLUSIONS/SIGNIFICANCE: sFRP-3 and EGF reciprocally inhibit their effects on cell proliferation, differentiation and morphogenesis and indeed are expressed in contiguous domains of the embryo, suggesting that in

  7. Stimulation of casein kinase II by epidermal growth factor: Relationship between the physiological activity of the kinase and the phosphorylation state of its beta subunit

    International Nuclear Information System (INIS)

    Ackerman, P.; Osheroff, N.; Glover, C.V.C.

    1990-01-01

    To determine relationships between the hormonal activation of casein kinase II and its phosphorylation state, epidermal growth factor (EGF)-treated and EGF-naive human A-431 carcinoma cells were cultured in the presence of [ 32 P]orthophosphate. Immunoprecipitation experiments indicated that casein kinase II in the cytosol of EGF-treated cells contained approximately 3-fold more incorporated [ 32 P]phosphate than did its counterpart in untreated cells. Levels of kinase phosphorylation paralleled levels of kinase activity over a wide range of EGF concentrations as well as over a time course of hormone action. Approximately 97% of the incorporated [ 32 P]phosphate was found in the β subunit of casein kinase II. Both activated and hormone-naive kinase contained radioactive phosphoserine and phosphothreonine but no phosphotyronsine. On the basis of proteolytic mapping experiments, EGF treatment of A-431 cells led to an increase in the average [ 32 P]phosphate content (i.e., hyperphosphorylation) of casein kinase II β subunit peptides which were modified prior to hormone treatment. Finally, the effect of alkaline phosphatase on the reaction kinetics of activated casein kinase II indicated that hormonal stimulation of the kinase resulted from the increase in its phosphorylation state

  8. Brief study about the distribution of recombinant human Epidermic Growth Factor (rh-EGF)

    International Nuclear Information System (INIS)

    Rodriguez Garcia, J.C.; De Dios D Espaux, R.; Bello Garciga, J.L.

    1997-01-01

    This report describes results of the study about biodistribution of I-125 recombinant human Epidermic Growth Factor (rhEGF). The radiolabelled product was administrated to Sprague Dawley rats in three different ways: intramuscular, subcutaneous and epidermic; the highest concentration of EGF in blood was found 4 hours after rhEGF administration, with a greater distribution in the plasma with regard to cellular pellet. The slowest plasma clearance corresponded to the intramuscular administration. The highest concentration of radiolabelled rhEGF was found in liver, kidney and intestine. It was found that radiolabelled EGF is excreted mainly throughout urine and faeces although other excretion pathways could exist

  9. Phosphorylation of Rac1 T108 by Extracellular Signal-Regulated Kinase in Response to Epidermal Growth Factor: a Novel Mechanism To Regulate Rac1 Function

    Science.gov (United States)

    Tong, Junfeng; Li, Laiji; Ballermann, Barbara

    2013-01-01

    Accumulating evidence has implicated Rho GTPases, including Rac1, in many aspects of cancer development. Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that Rac1 T108 within the 106PNTP109 motif is likely an extracellular signal-regulated kinase (ERK) phosphorylation site and that Rac1 also has an ERK docking site, 183KKRKRKCLLL192 (D site), at the C terminus. Indeed, we show here that both transfected and endogenous Rac1 interacts with ERK and that this interaction is mediated by its D site. Green fluorescent protein (GFP)-Rac1 is threonine (T) phosphorylated in response to epidermal growth factor (EGF), and EGF-induced Rac1 threonine phosphorylation is dependent on the activation of ERK. Moreover, mutant Rac1 with the mutation of T108 to alanine (A) is not threonine phosphorylated in response to EGF. In vitro ERK kinase assay further shows that pure active ERK phosphorylates purified Rac1 but not mutant Rac1 T108A. We also show that Rac1 T108 phosphorylation decreases Rac1 activity, partially due to inhibiting its interaction with phospholipase C-γ1 (PLC-γ1). T108 phosphorylation targets Rac1 to the nucleus, which isolates Rac1 from other guanine nucleotide exchange factors (GEFs) and hinders Rac1's role in cell migration. We conclude that Rac1 T108 is phosphorylated by ERK in response to EGF, which plays an important role in regulating Rac1. PMID:24043306

  10. Immunohistochemical and quantitative changes in salivary EGF, amylase and haptocorrin following radiotherapy for oral cancer

    DEFF Research Database (Denmark)

    Christensen, M E; Hansen, H S; Poulsen, Steen Seier

    1996-01-01

    Epidermal growth factor (EGF), amylase and haptocorrin are molecules produced in the salivary glands. The aim of the present study was to determine immunohistochemical and quantitative alterations in EGF as compared with haptocorrin and amylase following radiotherapy for oral cancer. Changes in t...... a reduction in the mitogenic peptide EGF both immunohistochemically and quantitatively following irradiation for oral cancer, results which may contribute to the understanding of the clinical signs of mucositis.......Epidermal growth factor (EGF), amylase and haptocorrin are molecules produced in the salivary glands. The aim of the present study was to determine immunohistochemical and quantitative alterations in EGF as compared with haptocorrin and amylase following radiotherapy for oral cancer. Changes....... The concentration of EGF in saliva before treatment was significantly higher in patients than in the control group (p oral tumors contribute with EGF to the saliva. In conclusion we have demonstrated...

  11. The Epstein-Barr virus BILF1 gene encodes a G protein-coupled receptor that inhibits phosphorylation of RNA-dependent protein kinase

    NARCIS (Netherlands)

    Beisser, P.S.; Verzijl, D.; Gruijthuijsen, Y.K.; Beuken, E.V.; Smit, M.J.; Leurs, R.; Bruggeman, C.A.; Vink, C.

    2005-01-01

    Epstein-Barr vires (EBV) infection is associated with many lymphoproliferative diseases, such as infectious mononucleosis and Burkitt's lymphoma. Consequently, EBV is one of the most extensively studied herpesvirases. Surprisingly, a putative G protein-coupled receptor (GPCR) gene of EBV, BILF1, has

  12. The Cu-Zn superoxide dismutase (SOD1) inhibits ERK phosphorylation by muscarinic receptor modulation in rat pituitary GH3 cells

    International Nuclear Information System (INIS)

    Secondo, Agnese; De Mizio, Mariarosaria; Zirpoli, Laura; Santillo, Mariarosaria; Mondola, Paolo

    2008-01-01

    The Cu-Zn superoxide dismutase (SOD1) belongs to a family of isoenzymes that are able to dismutate the oxygen superoxide in hydrogen peroxide and molecular oxygen. This enzyme is secreted by many cellular lines and it is also released trough a calcium-dependent depolarization mechanism involving SNARE protein SNAP 25. Using rat pituitary GH3 cells that express muscarinic receptors we found that SOD1 inhibits P-ERK1/2 pathway trough an interaction with muscarinic M1 receptor. This effect is strengthened by oxotremorine, a muscarinic M agonist and partially reverted by pyrenzepine, an antagonist of M1 receptor; moreover this effect is independent from increased intracellular calcium concentration induced by SOD1. Finally, P-ERK1/2 inhibition was accompanied by the reduction of GH3 cell proliferation. These data indicate that SOD1 beside the well studied antioxidant properties can be considered as a neuromodulator able to affect mitogen-activated protein kinase in rat pituitary cells trough a M1 muscarinic receptor

  13. TGF beta-1 dependent fast stimulation of ATM and p53 phosphorylation following exposure to ionizing radiation does not involve TGF beta-receptor I signalling

    NARCIS (Netherlands)

    Wiegman, Erwin M.; Blaese, Marcet A.; Loeffler, Heidi; Coppes, Rob P.; Rodemann, H. Peter

    Background and purpose: It has been proposed that radiation induced stimulation of ATM and downstream components involves activation of TGF beta-1 and that this may be due to TGF beta-1-receptor I-Smad signalling. Therefore, the aim of this study was to clarify the distinct role of TGF

  14. Tyrosine phosphorylation of Grb14 by Tie2

    Directory of Open Access Journals (Sweden)

    Dumont Daniel J

    2010-10-01

    Full Text Available Abstract Background Growth factor receptor bound (Grb proteins 7, 10 and 14 are a family of structurally related multi-domain adaptor proteins involved in a variety of biological processes. Grb7, 10 and 14 are known to become serine and/or threonine phosphorylated in response to growth factor (GF stimulation. Grb7 and 10 have also been shown to become tyrosine phosphorylated under certain conditions. Under experimental conditions Grb7 is tyrosine phosphorylated by the Tie2/Tie-2/Tek angiogenic receptor tyrosine kinase (RTK. Furthermore, Grb14 has also been shown to interact with Tie2, however tyrosine phosphorylation of this Grb family member has yet to be reported. Results Here we report for the first time tyrosine phosphorylation of Grb14. This phosphorylation requires a kinase competent Tie2 as well as intact tyrosines 1100 and 1106 (Y1100 and Y1106 on the receptor. Furthermore, a complete SH2 domain on Grb14 is required for Grb14 tyrosine phosphorylation by Tie2. Grb14 was also able to become tyrosine phosphorylated in primary endothelial cells when treated with a soluble and potent variant of the Tie2 ligand, cartilage oligomeric matrix protein (COMP Ang1. Conclusion Our results show that Grb14, like its family members Grb7 and Grb10, is able to be tyrosine phosphorylated. Furthermore, our data indicate a role for Grb14 in endothelial signaling downstream of the Tie2 receptor.

  15. Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M

    1992-01-01

    of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell......Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data...... demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression...

  16. Brk activates rac1 and promotes cell migration and invasion by phosphorylating paxillin.

    Science.gov (United States)

    Chen, Hsin-Yi; Shen, Che-Hung; Tsai, Yuh-Tyng; Lin, Feng-Chi; Huang, Yuan-Ping; Chen, Ruey-Hwa

    2004-12-01

    Brk (for breast tumor kinase) is a nonreceptor tyrosine kinase containing SH3, SH2, and tyrosine kinase catalytic domains. Brk was originally identified from a human metastatic breast tumor, and its overexpression is frequently observed in breast cancer and several other cancer types. However, the molecular mechanism by which this kinase participates in tumorigenesis remains poorly characterized. In the present study, we not only identified paxillin as the binding partner and substrate of Brk but also discovered a novel signaling pathway by which Brk mediates epidermal growth factor (EGF)-induced paxillin phosphorylation. We show that EGF stimulation activates the catalytic activity of Brk, which in turn phosphorylates paxillin at Y31 and Y118. These phosphorylation events promote the activation of small GTPase Rac1 via the function of CrkII. Through this pathway, Brk is capable of promoting cell motility and invasion and functions as a mediator of EGF-induced migration and invasion. In accordance with these functional roles, Brk translocates to membrane ruffles, where it colocalizes with paxillin during cell migration. Together, our findings identify novel signaling and biological roles of Brk and indicate the first potential link between Brk and metastatic malignancy.

  17. Freeze-dried formulation for direct {sup 99m}Tc-labeling ior-egf/r3 MAb: additives, biodistribution, and stability

    Energy Technology Data Exchange (ETDEWEB)

    Morales, Alejo A. Morales; Nunez-Gandolff, Gilda; Perez, Niuvis Perez; Veliz, Belkis Chico; Caballero-Torres, Idania; Duconge, Jorge; Fernandez, Eduardo; Crespo, Francisco Zayas; Veloso, Ana; Iznaga-Escobar, Normando E-mail: normando@ict.sld.cu

    1999-08-01

    Monoclonal antibodies (MAbs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in nuclear medicine practice. The MAb ior egf/r3 developed at the Center of Molecular Immunology (Havana, Cuba) is a murine antibody that recognizes the human epidermal growth factor receptor (EGF-R) and has been used widely in the radioimmunodiagnosis of tumors of epithelial origin. Based on the direct Schwarz method, the present report describes the preparation of a freeze-dried formulation for radiolabeling the MAb ior egf/r3 with {sup 99m}Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, biodistribution, pharmacokinetic, and stability of the formulation are reported. The study demonstrated that the freeze-dried formulation can be labeled with {sup 99m}Tc at high yield. The resulting {sup 99m}Tc-labeled ior egf/r3 MAb can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies. The kit does not need any other addition or purification at the time of tagging other than the requisite amount of pertechnetate (40-50 mCi). Because the contents of the kit are lyophilized, no special storage or transportation is required.

  18. Freeze-dried formulation for direct 99mTc-labeling ior-egf/r3 MAb: additives, biodistribution, and stability

    International Nuclear Information System (INIS)

    Morales, Alejo A. Morales; Nunez-Gandolff, Gilda; Perez, Niuvis Perez; Veliz, Belkis Chico; Caballero-Torres, Idania; Duconge, Jorge; Fernandez, Eduardo; Crespo, Francisco Zayas; Veloso, Ana; Iznaga-Escobar, Normando

    1999-01-01

    Monoclonal antibodies (MAbs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in nuclear medicine practice. The MAb ior egf/r3 developed at the Center of Molecular Immunology (Havana, Cuba) is a murine antibody that recognizes the human epidermal growth factor receptor (EGF-R) and has been used widely in the radioimmunodiagnosis of tumors of epithelial origin. Based on the direct Schwarz method, the present report describes the preparation of a freeze-dried formulation for radiolabeling the MAb ior egf/r3 with 99m Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, biodistribution, pharmacokinetic, and stability of the formulation are reported. The study demonstrated that the freeze-dried formulation can be labeled with 99m Tc at high yield. The resulting 99m Tc-labeled ior egf/r3 MAb can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies. The kit does not need any other addition or purification at the time of tagging other than the requisite amount of pertechnetate (40-50 mCi). Because the contents of the kit are lyophilized, no special storage or transportation is required

  19. Fc gamma receptor activation induces the tyrosine phosphorylation of both phospholipase C (PLC)-gamma 1 and PLC-gamma 2 in natural killer cells

    OpenAIRE

    1992-01-01

    Crosslinking of the low affinity immunoglobulin G (IgG) Fc receptor (Fc gamma R type III) on natural killer (NK) cells initiates antibody- dependent cellular cytotoxicity. During this process, Fc gamma R stimulation results in the rapid activation of phospholipase C (PLC), which hydrolyzes membrane phosphoinositides, generating inositol-1,4,5- trisphosphate and sn-1,2-diacylglycerol as second messengers. We have recently reported that PLC activation after Fc gamma R stimulation can be inhibit...

  20. Decline in Proliferation and Immature Neuron Markers in the Human Subependymal Zone during Aging: Relationship to EGF- and FGF-related Transcripts

    Directory of Open Access Journals (Sweden)

    Christin Weissleder

    2016-11-01

    Full Text Available Neuroblasts exist within the human subependymal zone (SEZ; however, it is debated to what extent neurogenesis changes during normal aging. It is also unknown how precursor proliferation may correlate with the generation of neuronal and glial cells or how expression of growth factors and receptors may change throughout the adult lifespan. We provided evidence of dividing cells in the human SEZ in conjunction with a dramatic age-related decline (n=50; 21-103 years of mRNAs indicative of proliferating cells (Ki67 and immature neurons (doublecortin. Microglia mRNA (ionized calcium-binding adapter molecule 1 increased during aging, whereas transcript levels of stem/precursor cells (glial fibrillary acidic protein delta and achaete-scute homolog 1, astrocytes (vimentin and glial fibrillary acidic protein and oligodendrocytes (oligodendrocyte lineage transcription factor 2 remained stable. Epidermal growth factor receptor (EGFR and fibroblast growth factor 2 (FGF2 mRNAs increased throughout adulthood, while transforming growth factor alpha (TGFα, EGF, Erb-B2 receptor tyrosine kinase 4 (ErbB4 and FGF receptor 1 (FGFR1 mRNAs were unchanged across adulthood. Cell proliferation mRNA positively correlated with FGFR1 transcripts. Immature neuron and oligodendrocyte expression positively correlated with TGFα and ErbB4 mRNAs, whilst astrocyte transcripts positively correlated with EGF, FGF2 and FGFR1 mRNAs. Microglia mRNA positively correlated with EGF and FGF2 expression. Our findings indicate that neurogenesis in the human SEZ continues well into adulthood, although proliferation and neuronal differentiation may decline across adulthood. We suggest that mRNA expression of EGF- and FGF-related family members do not become limited during aging and may modulate neuronal and glial fate determination in the SEZ throughout human life.

  1. Phosphorylation of mouse serine racemase regulates D-serine synthesis

    DEFF Research Database (Denmark)

    Foltyn, Veronika N; Zehl, Martin; Dikopoltsev, Elena

    2010-01-01

    Serine racemase (SR) catalyses the synthesis of the transmitter/neuromodulator D-serine, which plays a major role in synaptic plasticity and N-methyl D-aspartate receptor neurotoxicity. We now report that SR is phosphorylated at Thr71 and Thr227 as revealed by mass spectrometric analysis and in v...... with a phosphorylation-deficient SR mutant indicate that Thr71 phosphorylation increases SR activity, suggesting a novel mechanism for regulating D-serine production....

  2. Dopamine D1 receptors and phosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein-32 in the medial preoptic area are involved in experience-induced enhancement of male sexual behavior in rats.

    Science.gov (United States)

    McHenry, Jenna A; Bell, Genevieve A; Parrish, Bradley P; Hull, Elaine M

    2012-08-01

    The medial preoptic area (MPOA) is an integral site for male sexual behavior. Dopamine is released in the MPOA before and during copulation and facilitates male rat sexual behavior. Repeated sexual experience and noncopulatory exposures to an estrous female facilitate subsequent copulation. However, the neurobiological mechanisms that mediate such enhancement remain unclear. Here, we examined the role of dopamine D₁ receptors in the MPOA in experience-induced enhancement of male sexual behavior in rats. In experiment 1, microinjections of the D₁ antagonist SCH-23390 into the MPOA before each of seven daily 30-min noncopulatory exposures to a receptive female impaired copulation on a drug-free test on Day 8, compared to vehicle-treated female-exposed animals. Copulatory performance in drug-treated animals was similar to that of vehicle-treated males that had not been preexposed to females. This effect was site specific. There were no group differences in locomotor activity in an open field on the copulation test day. In experiment 2, a separate cohort of animals was used to examine phosphorylation of dopamine- and cAMP-regulated phosphoprotein (DARPP-32) in the MPOA of animals with acute and/or chronic sexual experience. DARPP-32 is a downstream marker of D₁ receptor signaling and substrate of cAMP-dependent protein kinase (PKA). Western immunoblot analysis revealed that p-DARPP-32 expression was greatest in the MPOA of males that received both acute and chronic sexual experience, compared to all other mated conditions and naïve controls. These data suggest that D₁ receptors in the MPOA contribute to experience-induced enhancement of male sexual behavior, perhaps through a PKA regulated mechanism.

  3. The effects of chronic administration of epidermal growth factor (EGF) to rats on the levels of endogenous EGF in the submandibular glands and kidneys

    DEFF Research Database (Denmark)

    Vinter-Jensen, Lars; Jøgensen, P E; Poulsen, Steen Seier

    1996-01-01

    Epidermal growth factor (EGF) is mainly produced in the submandibular glands (SMG) and in the kidneys. It has recently been reported that EGF-related ligands may induce their own biosynthesis (autoinduction) in vitro. In the present paper, we investigated whether chronic systemic treatment with E...

  4. Involvement of Angiopoietin-2 and Tie2 Receptor Phosphorylation in STEC-HUS Mediated by Escherichia coli O104:H4

    Directory of Open Access Journals (Sweden)

    Alexander Lukasz

    2015-01-01

    Full Text Available Escherichia coli O104:H4-associated hemolytic uremic syndrome (HUS is characterized by Shiga toxin-induced vascular damage. As indicated by recent studies, dysregulation of the angiopoietin (Angpt/Tie2 ligand receptor system may be crucial for endothelial dysfunction in HUS. Early Angpt-2 levels quantified in 48 adult HUS patients were predictive for a complicated clinical course, in particular for need of hemodialysis and mechanical ventilation as well as occurrence of seizures. In vitro challenge of human umbilical vein endothelial cells with patients’ sera indicated an injurious mediator role of Angpt-2 opening future perspectives for mitigating endothelial activation in HUS.

  5. Role of protein kinase C and epidermal growth factor receptor signalling in growth stimulation by neurotensin in colon carcinoma cells

    Directory of Open Access Journals (Sweden)

    Dajani Olav

    2011-10-01

    Full Text Available Abstract Background Neurotensin has been found to promote colon carcinogenesis in rats and mice, and proliferation of human colon carcinoma cell lines, but the mechanisms involved are not clear. We have examined signalling pathways activated by neurotensin in colorectal and pancreatic carcinoma cells. Methods Colon carcinoma cell lines HCT116 and HT29 and pancreatic adenocarcinoma cell line Panc-1 were cultured and stimulated with neurotensin or epidermal growth factor (EGF. DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA. Levels and phosphorylation of proteins in signalling pathways were assessed by Western blotting. Results Neurotensin stimulated the phosphorylation of both extracellular signal-regulated kinase (ERK and Akt in all three cell lines, but apparently did so through different pathways. In Panc-1 cells, neurotensin-induced phosphorylation of ERK, but not Akt, was dependent on protein kinase C (PKC, whereas an inhibitor of the β-isoform of phosphoinositide 3-kinase (PI3K, TGX221, abolished neurotensin-induced Akt phosphorylation in these cells, and there was no evidence of EGF receptor (EGFR transactivation. In HT29 cells, in contrast, the EGFR tyrosine kinase inhibitor gefitinib blocked neurotensin-stimulated phosphorylation of both ERK and Akt, indicating transactivation of EGFR, independently of PKC. In HCT116 cells, neurotensin induced both a PKC-dependent phosphorylation of ERK and a metalloproteinase-mediated transactivation of EGFR that was associated with a gefitinib-sensitive phosphorylation of the downstream adaptor protein Shc. The activation of Akt was also inhibited by gefitinib, but only partly, suggesting a mechanism in addition to EGFR transactivation. Inhibition of PKC blocked neurotensin-induced DNA synthesis in HCT116 cells. Conclusions While acting predominantly through PKC in Panc-1 cells and via EGFR transactivation in HT29 cells, neurotensin used both these pathways in HCT116

  6. Role of protein kinase C and epidermal growth factor receptor signalling in growth stimulation by neurotensin in colon carcinoma cells

    International Nuclear Information System (INIS)

    Müller, Kristin M; Tveteraas, Ingun H; Aasrum, Monica; Ødegård, John; Dawood, Mona; Dajani, Olav; Christoffersen, Thoralf; Sandnes, Dagny L

    2011-01-01

    Neurotensin has been found to promote colon carcinogenesis in rats and mice, and proliferation of human colon carcinoma cell lines, but the mechanisms involved are not clear. We have examined signalling pathways activated by neurotensin in colorectal and pancreatic carcinoma cells. Colon carcinoma cell lines HCT116 and HT29 and pancreatic adenocarcinoma cell line Panc-1 were cultured and stimulated with neurotensin or epidermal growth factor (EGF). DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA. Levels and phosphorylation of proteins in signalling pathways were assessed by Western blotting. Neurotensin stimulated the phosphorylation of both extracellular signal-regulated kinase (ERK) and Akt in all three cell lines, but apparently did so through different pathways. In Panc-1 cells, neurotensin-induced phosphorylation of ERK, but not Akt, was dependent on protein kinase C (PKC), whereas an inhibitor of the β-isoform of phosphoinositide 3-kinase (PI3K), TGX221, abolished neurotensin-induced Akt phosphorylation in these cells, and there was no evidence of EGF receptor (EGFR) transactivation. In HT29 cells, in contrast, the EGFR tyrosine kinase inhibitor gefitinib blocked neurotensin-stimulated phosphorylation of both ERK and Akt, indicating transactivation of EGFR, independently of PKC. In HCT116 cells, neurotensin induced both a PKC-dependent phosphorylation of ERK and a metalloproteinase-mediated transactivation of EGFR that was associated with a gefitinib-sensitive phosphorylation of the downstream adaptor protein Shc. The activation of Akt was also inhibited by gefitinib, but only partly, suggesting a mechanism in addition to EGFR transactivation. Inhibition of PKC blocked neurotensin-induced DNA synthesis in HCT116 cells. While acting predominantly through PKC in Panc-1 cells and via EGFR transactivation in HT29 cells, neurotensin used both these pathways in HCT116 cells. In these cells, neurotensin-induced activation of ERK

  7. THE UNCOVERING OF A NOVEL REGULATORY MECHANISM FOR PLD2: FORMATION OF A TERNARY COMPLEX WITH PROTEIN TYROSINE PHOSPHATASE PTP1B AND GROWTH FACTOR RECEPTOR-BOUND PROTEIN GRB2

    Science.gov (United States)

    Horn, Jeff; Lopez, Isabel; Miller, Mill; Gomez-Cambronero, Julian

    2011-01-01

    The regulation of PLD2 activation is poorly understood at present. Transient transfection of COS-7 with a mycPLD2 construct results in elevated levels of PLD2 enzymatic activity and tyrosyl phosphorylation. To investigate whether this phosphorylation affects PLD2 enzymatic activity, anti-myc immunoprecipitates were treated with recombinant protein tyrosine phosphatase PTP1B. Surprisingly, lipase activity and PY levels both increased over a range of PTP1B concentrations. These increases occurred in parallel to a measurable PTP1B-associated phosphatase activity. Inhibitor studies demonstrated that an EGF-receptor type kinase is involved in phosphorylation. In a COS-7 cell line created in the laboratory that stably expressed myc-PLD2, PTP1B induced a robust (>6-fold) augmentation of myc-PLD2 phosphotyrosine content. The addition of growth factor receptor-bound protein 2 (Grb2) to cell extracts also elevated PY levels of myc-PLD (>10-fold). Systematic co-immunoprecipitation-immunoblotting experiments pointed at a physical association between PLD2, Grb2 and PTP1B in both physiological conditions and in overexpressed cells. This is the first report of a demonstration of the mammalian isoform PLD2 existing in a ternary complex with a protein tyrosine phosphatase, PTP1b, and the docking protein Grb2 which greatly enhances tyrosyl phosphorylation of the lipase. PMID:15896299

  8. TRPP2 and TRPV4 form an EGF-activated calcium permeable channel at the apical membrane of renal collecting duct cells.

    Directory of Open Access Journals (Sweden)

    Zhi-Ren Zhang

    Full Text Available Regulation of apical calcium entry is important for the function of principal cells of the collecting duct. However, the molecular identity and the regulators of the transporter/channel, which is responsible for apical calcium entry and what factors regulate the calcium conduction remain unclear.We report that endogenous TRPP2 and TRPV4 assemble to form a 23-pS divalent cation-permeable non-selective ion channel at the apical membrane of renal principal cells of the collecting duct. TRPP2\\TRPV4 channel complex was identified by patch-clamp, immunofluorescence and co-immunprecipitation studies in both principal cells that either possess normal cilia (cilia (+ or in which cilia are absent (cilia (-. This channel has distinct biophysical and pharmacological and regulatory profiles compared to either TRPP2 or TRPV4 channels. The rate of occurrence detected by patch clamp was higher in cilia (- compared to cilia (+ cells. In addition, shRNA knockdown of TRPP2 increased the prevalence of TRPV4 channel activity while knockdown of TRPV4 resulted in TRPP2 activity and knockdown of both proteins vastly decreased the 23-pS channel activity. Epidermal growth factor (EGF stimulated TRPP2\\TRPV4 channel through the EGF receptor (EGFR tyrosine kinase-dependent signaling. With loss of cilia, apical EGF treatment resulted in 64-fold increase in channel activity in cilia (- but not cilia (+ cells. In addition EGF increased cell proliferation in cilia (- cell that was dependent upon TRPP2\\TRPV4 channel mediated increase in intracellular calcium.We conclude that in the absence of cilia, an EGF activated TRPP2\\TRPV4 channel may play an important role in increased cell proliferation and cystogenesis.

  9. Tyrosine phosphorylation in signal transduction

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  10. Epidermal growth factor (EGF) inhibits stimulated thyroid hormone secretion in the mouse

    International Nuclear Information System (INIS)

    Ahren, B.

    1987-01-01

    It is known that epidermal growth factor (EGF) inhibits iodide uptake in the thyroid follicular cells and lowers plasma levels of thyroid hormones upon infusion into sheep and ewes. In this study, the effects of EGF on basal and stimulated thyroid hormone secretion were investigated in the mouse. Mice were pretreated with 125 I and thyroxine; the subsequent release of 125 I is an estimation of thyroid hormone secretion. It was found that basal radioiodine secretion was not altered by intravenous injection of EGF (5 micrograms/animal). However, the radioiodine secretion stimulated by both TSH (120 microU/animal) and vasoactive intestinal peptide (VIP; 5 micrograms/animal) were inhibited by EGF (5 micrograms/animal). At a lower dose level (0.5 microgram/animal), EGF had no influence on stimulated radioiodine secretion. In conclusion, EGF inhibits stimulated thyroid hormone secretion in the mouse

  11. Combined immunotherapy of breast cancer with EGF and VEGF vaccines from DNA shuffling in a mouse model.

    Science.gov (United States)

    Jin, Dong; Yu, Xin; Chen, Bing; Li, Zhitao; Ding, Jia; Zhao, Xiuyun; Qi, Gaofu

    2017-06-01

    Development of EGF and VEGF vaccines with high antigenicity for combined immunotherapy of EGF-EGFR signaling-dependent epithelial tumors such as breast cancer. EGF genes from mouse, human and chicken were randomly assembled to chimeric genes by DNA shuffling, then a chimeric EGF was selected out by PCR, SDS-PAGE and immunization for combined immunotherapy of breast cancer with a previously constructed chimeric VEGF vaccine from shuffling. Combined vaccination with chimeric EGF and VEGF from shuffling could induce high titer of antibodies against EGF and VEGF to inhibit tumor growth and angiogenesis, and improve the survival rate of mice with breast cancer. Combined vaccination with EGF and VEGF from shuffling showed better immunotherapy on EGF-EGFR signaling-dependent epithelial tumors such as breast cancer than the single-agent EGF vaccination.

  12. Network analysis of epidermal growth factor signaling using integrated genomic, proteomic and phosphorylation data.

    Directory of Open Access Journals (Sweden)

    Katrina M Waters

    Full Text Available To understand how integration of multiple data types can help decipher cellular responses at the systems level, we analyzed the mitogenic response of human mammary epithelial cells to epidermal growth factor (EGF using whole genome microarrays, mass spectrometry-based proteomics and large-scale western blots with over 1000 antibodies. A time course analysis revealed significant differences in the expression of 3172 genes and 596 proteins, including protein phosphorylation changes measured by western blot. Integration of these disparate data types showed that each contributed qualitatively different components to the observed cell response to EGF and that varying degrees of concordance in gene expression and protein abundance measurements could be linked to specific biological processes. Networks inferred from individual data types were relatively limited, whereas networks derived from the integrated data recapitulated the known major cellular responses to EGF and exhibited more highly connected signaling nodes than networks derived from any individual dataset. While cell cycle regulatory pathways were altered as anticipated, we found the most robust response to mitogenic concentrations of EGF was induction of matrix metalloprotease cascades, highlighting the importance of the EGFR system as a regulator of the extracellular environment. These results demonstrate the value of integrating multiple levels of biological information to more accurately reconstruct networks of cellular response.

  13. Network Analysis of Epidermal Growth Factor Signaling using Integrated Genomic, Proteomic and Phosphorylation Data

    Energy Technology Data Exchange (ETDEWEB)

    Waters, Katrina M.; Liu, Tao; Quesenberry, Ryan D.; Willse, Alan R.; Bandyopadhyay, Somnath; Kathmann, Loel E.; Weber, Thomas J.; Smith, Richard D.; Wiley, H. S.; Thrall, Brian D.

    2012-03-29

    To understand how integration of multiple data types can help decipher cellular responses at the systems level, we analyzed the mitogenic response of human mammary epithelial cells to epidermal growth factor (EGF) using whole genome microarrays, mass spectrometry-based proteomics and large-scale western blots with over 1000 antibodies. A time course analysis revealed significant differences in the expression of 3172 genes and 596 proteins, including protein phosphorylation changes measured by western blot. Integration of these disparate data types showed that each contributed qualitatively different components to the observed cell response to EGF and that varying degrees of concordance in gene expression and protein abundance measurements could be linked to specific biological processes. Networks inferred from individual data types were relatively limited, whereas networks derived from the integrated data recapitulated the known major cellular responses to EGF and exhibited more highly connected signaling nodes than networks derived from any individual dataset. While cell cycle regulatory pathways were altered as anticipated, we found the most robust response to mitogenic concentrations of EGF was induction of matrix metalloprotease cascades, highlighting the importance of the EGFR system as a regulator of the extracellular environment. These results demonstrate the value of integrating multiple levels of biological information to more accurately reconstruct networks of cellular response.

  14. Elevated EGF Levels in the Blood Serum of Dogs with Periodontal Diseases and Oral Tumours.

    Science.gov (United States)

    Sobczyńska-Rak, Aleksandra; Żylińska, Beata; Polkowska, Izabela; Szponder, Tomasz

    2018-01-01

    Paradontopathy and neoplasms of the oral cavity represent one of the greatest challenges in human and animal dentistry. EGF plays a key role in maintaining the integrity and proper rate of cell proliferation in normal oral epithelium. The aim of the present study was to study serum levels of EGF in dogs diagnosed with periodontal diseases and oral cavity tumours. The samples comprised of cancerous tissue sections and serum obtained from dogs of various breeds, aged between 5-13 years. Serum EGF concentrations were measured by an immunoenzymatic method. The median for EGF concentration in serum of dogs suffered from severe periodontal diseases was greater when compared to the control group. EGF concentration in dogs with malignant tumours was significantly higher than in those with non-malignant growths. A positive correlation between EGF concentration and tumour size was also observed. EGF level in dogs diagnosed with benign tumours was comparable to the control group. The blood serum level of EGF increases significantly in patients with malignant oral tumours and advanced periodontal disease. In malignant tumours, the high level of EGF correlates with the size and invasiveness of the neoplasm. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  15. HB-EGF expression as a potential biomarker of acquired middle ear cholesteatoma.

    Science.gov (United States)

    Xie, Shumin; Wang, Xiaoli; Ren, Hongmiao; Liu, Xiaoyu; Ren, Jihao; Liu, Wei

    2017-08-01

    The heparin-binding epidermal growth factor-like growth factor (HB-EGF) plays an essential role in the development and invasiveness of cholesteatoma. This study may help to realize the molecular mechanisms underlying the pathogenesis of cholesteatoma and make HB-EGF a promising target for drug intervention of cholesteatoma. To detect HB-EGF expression in human surgical specimens of acquired middle ear cholesteatoma and analyze its functional role as a regulator of epithelial keratinocytes hyperproliferation. A total of 34 patients who underwent surgical treatment for middle ear cholesteatoma were recruited in the study. The mRNA and protein expression of HB-EGF in middle ear cholesteatoma tissues and normal postauricular skin tissues was investigated by real-time quantitative reverse-transcription-polymerase chain reaction (RT-qPCR), immunohistochemical staining, and western blot. The correlation between bone resorption degree and HB-EGF expression was also analyzed. On average, compared with normal postauricular skin, expression of HB-EGF mRNA in the cholesteatoma epithelium was significantly elevated 2.41-fold by RT-qPCR, and HB-EGF protein significantly upregulated 2.32-fold by western blot. Positive HB-EGF immunostaining observed in the basal and suprabasal layers of cholesteatoma epithelium was significantly stronger than in normal postauricular skin. Meanwhile, an obviously positive correlation between HB-EGF protein expression and bone resorption degree was discovered.

  16. Src kinase regulation by phosphorylation and dephosphorylation

    International Nuclear Information System (INIS)

    Roskoski, Robert

    2005-01-01

    Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPα, PTPε, and PTPλ. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined

  17. 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.

  18. Tyrosine phosphorylation switching of a G protein.

    Science.gov (United States)

    Li, Bo; Tunc-Ozdemir, Meral; Urano, Daisuke; Jia, Haiyan; Werth, Emily G; Mowrey, David D; Hicks, Leslie M; Dokholyan, Nikolay V; Torres, Matthew P; Jones, Alan M

    2018-03-30

    Heterotrimeric G protein complexes are molecular switches relaying extracellular signals sensed by G protein-coupled receptors (GPCRs) to downstream targets in the cytoplasm, which effect cellular responses. In the plant heterotrimeric GTPase cycle, GTP hydrolysis, rather than nucleotide exchange, is the rate-limiting reaction and is accelerated by a receptor-like regulator of G signaling (RGS) protein. We hypothesized that posttranslational modification of the Gα subunit in the G protein complex regulates the RGS-dependent GTPase cycle. Our structural analyses identified an invariant phosphorylated tyrosine residue (Tyr 166 in the Arabidopsis Gα subunit AtGPA1) located in the intramolecular domain interface where nucleotide binding and hydrolysis occur. We also identified a receptor-like kinase that phosphorylates AtGPA1 in a Tyr 166 -dependent manner. Discrete molecular dynamics simulations predicted that phosphorylated Tyr 166 forms a salt bridge in this interface and potentially affects the RGS protein-accelerated GTPase cycle. Using a Tyr 166 phosphomimetic substitution, we found that the cognate RGS protein binds more tightly to the GDP-bound Gα substrate, consequently reducing its ability to accelerate GTPase activity. In conclusion, we propose that phosphorylation of Tyr 166 in AtGPA1 changes the binding pattern with AtRGS1 and thereby attenuates the steady-state rate of the GTPase cycle. We coin this newly identified mechanism "substrate phosphoswitching." © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes.

    Science.gov (United States)

    Panayotou, G; Bax, B; Gout, I; Federwisch, M; Wroblowski, B; Dhand, R; Fry, M J; Blundell, T L; Wollmer, A; Waterfield, M D

    1992-01-01

    Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3-kinase, a closely related p85 beta protein, and a recombinant SH2 domain-containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase-insert region of the platelet-derived growth factor beta-receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain-containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain-containing proteins is discussed. Images PMID:1330535

  20. Differentiation of human keratinocytes: changes in lipid synthesis, plasma membrane lipid composition, and 125I-EGF binding upon administration of 25-hydroxycholesterol and mevinolin

    International Nuclear Information System (INIS)

    Ponec, M.; Kempenaar, J.; Weerheim, A.; Boonstra, J.

    1987-01-01

    We have studied the relationship between differentiation capacity, plasma membrane composition, and epidermal growth factor (EGF) receptor expression of normal keratinocytes in vitro. The plasma membrane composition of the cells was modulated experimentally by cholesterol depletion, using specific inhibitors of cholesterol synthesis, such as 25-hydroxycholesterol and mevinolin. Exposure of the cells towards these inhibitors resulted in a drastic decrease of cholesterol biosynthesis, as determined from 14 C-acetate incorporation into the various lipid fractions. This effect on cholesterol biosynthesis was reflected by changes in plasma membrane composition, as determined by lipid analysis of isolated plasma membrane fractions, these resulting in a decreased cholesterol-phospholipid ratio. The experimental modulation of plasma membrane composition by 25-hydroxycholesterol or mevinolin were accompanied by a decreased cornified envelope formation and by high expression of EGF binding sites. These phenomena were more pronounced in cells induced to differentiate by exposure of cells grown under low Ca2+ to normal Ca2+ concentrations, as compared to cells grown persistently under low Ca2+ concentrations. These results suggest a close correlation between plasma membrane composition, differentiation capacity, and EGF receptor expression

  1. Administration of Menadione, Vitamin K3, Ameliorates Off-Target Effects on Corneal Epithelial Wound Healing Due to Receptor Tyrosine Kinase Inhibition.

    Science.gov (United States)

    Rush, Jamie S; Bingaman, David P; Chaney, Paul G; Wax, Martin B; Ceresa, Brian P

    2016-11-01

    The antiangiogenic receptor tyrosine kinase inhibitor (RTKi), 3-[(4-bromo-2,6-difluorophenyl)methoxy]-5-[[[[4-(1-pyrrolidinyl) butyl] amino] carbonyl]amino]-4-isothiazolecarboxamide hydrochloride, targets VEGFR2 (half maximal inhibitory concentration [IC50] = 11 nM); however, off-target inhibition of epidermal growth factor receptor (EGFR) occurs at higher concentrations. (IC50 = 5.8 μM). This study was designed to determine the effect of topical RTKi treatment on EGF-mediated corneal epithelial wound healing and to develop new strategies to minimize off-target EGFR inhibition. In vitro corneal epithelial wound healing was measured in response to EGF using a transformed human cell line (hTCEpi cells). In vivo corneal wound healing was assessed using a murine model. In these complementary assays, wound healing was measured in the presence of varying RTKi concentrations. Immunoblot analysis was used to examine EGFR and VEGFR2 phosphorylation and the kinetics of EGFR degradation. An Alamar Blue assay measured VEGFR2-mediated cell biology. Receptor tyrosine kinase inhibitor exposure caused dose-dependent inhibition of EGFR-mediated corneal epithelial wound healing in vitro and in vivo. Nanomolar concentrations of menadione, a vitamin K3 analog, when coadministered with the RTKi, slowed EGFR degradation and ameliorated the inhibitory effects on epithelial wound healing both in vitro and in vivo. Menadione did not alter the RTKi's IC50 against VEGFR2 phosphorylation or its inhibition of VEGF-induced retinal endothelial cell proliferation. An antiangiogenic RTKi exhibited off-target effects on the corneal epithelium that can be minimized by menadione without deleteriously affecting its on-target VEGFR2 blockade. These data indicate that menadione has potential as a topical supplement for individuals suffering from perturbations in corneal epithelial homeostasis, especially as an untoward side effect of kinase inhibitors.

  2. Preparation of epidermal growth factor (EGF) conjugated iron oxide nanoparticles and their internalization into colon cancer cells

    International Nuclear Information System (INIS)

    Creixell, Mar; Herrera, Adriana P.; Ayala, Vanessa; Latorre-Esteves, Magda; Perez-Torres, Marianela; Torres-Lugo, Madeline; Rinaldi, Carlos

    2010-01-01

    Epidermal growth factor (EGF) was conjugated with carboxymethyldextran (CMDx) coated iron oxide magnetic nanoparticles using carbodiimide chemistry to obtain magnetic nanoparticles that target the epidermal growth factor receptor (EGFR). Epidermal growth factor modified magnetic nanoparticles were colloidally stable when suspended in biological buffers such as PBS and cell culture media. Both targeted and non-targeted nanoparticles were incubated with CaCo-2 cancer cells, known to overexpress EGFR. Nanoparticle localization within the cell was visualized by confocal laser scanning microscopy and light microscopy using Prussian blue stain. Results showed that targeted magnetic nanoparticles were rapidly accumulated in both flask-shaped small vesicles and large circular endocytic structures. Internalization patterns suggest that both clathrin-dependent and clathrin-independent receptors mediated endocytosis mechanisms are responsible for nanoparticle internalization.

  3. Recurrent exposure to nicotine differentiates human bronchial epithelial cells via epidermal growth factor receptor activation

    International Nuclear Information System (INIS)

    Martinez-Garcia, Eva; Irigoyen, Marta; Anso, Elena; Martinez-Irujo, Juan Jose; Rouzaut, Ana

    2008-01-01

    Cigarette smoking is the major preventable cause of lung cancer in developed countries. Nicotine (3-(1-methyl-2-pyrrolidinyl)-pyridine) is one of the major alkaloids present in tobacco. Besides its addictive properties, its effects have been described in panoply of cell types. In fact, recent studies have shown that nicotine behaves as a tumor promoter in transformed epithelial cells. This research focuses on the effects of acute repetitive nicotine exposure on normal human bronchial epithelial cells (NHBE cells). Here we show that treatment of NHBE cells with recurrent doses of nicotine up to 500 μM triggered cell differentiation towards a neuronal-like phenotype: cells emitted filopodia and expressed neuronal markers such as neuronal cell adhesion molecule, neurofilament-M and the transcription factors neuronal N and Pax-3. We also demonstrate that nicotine treatment induced NF-kB translocation to the nucleus, phosphorylation of the epidermal growth factor receptor (EGFR), and accumulation of heparin binding-EGF in the extracellular medium. Moreover, addition of AG1478, an inhibitor of EGFR tyrosine phosphorylation, or cetuximab, a monoclonal antibody that precludes ligand binding to the same receptor, prevented cell differentiation by nicotine. Lastly, we show that differentiated cells increased their adhesion to the extracellular matrix and their protease activity. Given that several lung pathologies are strongly related to tobacco consumption, these results may help to better understand the damaging consequences of nicotine exposure

  4. A multiscale computational approach to dissect early events in the Erb family receptor mediated activation, differential signaling, and relevance to oncogenic transformations.

    Science.gov (United States)

    Liu, Yingting; Purvis, Jeremy; Shih, Andrew; Weinstein, Joshua; Agrawal, Neeraj; Radhakrishnan, Ravi

    2007-06-01

    We describe a hierarchical multiscale computational approach based on molecular dynamics simulations, free energy-based molecular docking simulations, deterministic network-based kinetic modeling, and hybrid discrete/continuum stochastic dynamics protocols to study the dimer-mediated receptor activation characteristics of the Erb family receptors, specifically the epidermal growth factor receptor (EGFR). Through these modeling approaches, we are able to extend the prior modeling of EGF-mediated signal transduction by considering specific EGFR tyrosine kinase (EGFRTK) docking interactions mediated by differential binding and phosphorylation of different C-terminal peptide tyrosines on the RTK tail. By modeling signal flows through branching pathways of the EGFRTK resolved on a molecular basis, we are able to transcribe the effects of molecular alterations in the receptor (e.g., mutant forms of the receptor) to differing kinetic behavior and downstream signaling response. Our molecular dynamics simulations show that the drug sensitizing mutation (L834R) of EGFR stabilizes the active conformation to make the system constitutively active. Docking simulations show preferential characteristics (for wildtype vs. mutant receptors) in inhibitor binding as well as preferential enhancement of phosphorylation of particular substrate tyrosines over others. We find that in comparison to the wildtype system, the L834R mutant RTK preferentially binds the inhibitor erlotinib, as well as preferentially phosphorylates the substrate tyrosine Y1068 but not Y1173. We predict that these molecular level changes result in preferential activation of the Akt signaling pathway in comparison to the Erk signaling pathway for cells with normal EGFR expression. For cells with EGFR over expression, the mutant over activates both Erk and Akt pathways, in comparison to wildtype. These results are consistent with qualitative experimental measurements reported in the literature. We discuss these

  5. Shoc2 is targeted to late endosomes and required for Erk1/2 activation in EGF-stimulated cells.

    Directory of Open Access Journals (Sweden)

    Emilia Galperin

    Full Text Available Shoc2 is the putative scaffold protein that interacts with RAS and RAF, and positively regulates signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2. To elucidate the mechanism by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor (EGF receptor (EGFR, we studied subcellular localization of Shoc2. Upon EGFR activation, endogenous Shoc2 and red fluorescent protein tagged Shoc2 were translocated from the cytosol to a subset of late endosomes containing Rab7. The endosomal recruitment of Shoc2 was blocked by overexpression of a GDP-bound H-RAS (N17S mutant and RNAi knockdown of clathrin, suggesting the requirement of RAS activity and clathrin-dependent endocytosis. RNAi depletion of Shoc2 strongly inhibited activation of ERK1/2 by low, physiological EGF concentrations, which was rescued by expression of wild-type recombinant Shoc2. In contrast, the Shoc2 (S2G mutant, that is myristoylated and found in patients with the Noonan-like syndrome, did not rescue ERK1/2 activation in Shoc2-depleted cells. Shoc2 (S2G was not located in late endosomes but was present on the plasma membrane and early endosomes. These data suggest that targeting of Shoc2 to late endosomes may facilitate EGFR-induced ERK activation under physiological conditions of cell stimulation by EGF, and therefore, may be involved in the spatiotemporal regulation of signaling through the RAS-RAF module.

  6. Brain Injury Expands the Numbers of Neural Stem Cells and Progenitors in the SVZ by Enhancing Their Responsiveness to EGF

    Directory of Open Access Journals (Sweden)

    Dhivyaa Alagappan

    2009-04-01

    Full Text Available There is an increase in the numbers of neural precursors in the SVZ (subventricular zone after moderate ischaemic injuries, but the extent of stem cell expansion and the resultant cell regeneration is modest. Therefore our studies have focused on understanding the signals that regulate these processes towards achieving a more robust amplification of the stem/progenitor cell pool. The goal of the present study was to evaluate the role of the EGFR [EGF (epidermal growth factor receptor] in the regenerative response of the neonatal SVZ to hypoxic/ischaemic injury. We show that injury recruits quiescent cells in the SVZ to proliferate, that they divide more rapidly and that there is increased EGFR expression on both putative stem cells and progenitors. With the amplification of the precursors in the SVZ after injury there is enhanced sensitivity to EGF, but not to FGF (fibroblast growth factor-2. EGF-dependent SVZ precursor expansion, as measured using the neurosphere assay, is lost when the EGFR is pharmacologically inhibited, and forced expression of a constitutively active EGFR is sufficient to recapitulate the exaggerated proliferation of the neural stem/progenitors that is induced by hypoxic/ischaemic brain injury. Cumulatively, our results reveal that increased EGFR signalling precedes that increase in the abundance of the putative neural stem cells and our studies implicate the EGFR as a key regulator of the expansion of SVZ precursors in response to brain injury. Thus modulating EGFR signalling represents a potential target for therapies to enhance brain repair from endogenous neural precursors following hypoxic/ischaemic and other brain injuries.

  7. Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

    Directory of Open Access Journals (Sweden)

    Béatrice Marquèze-Pouey

    Full Text Available Signaling mediated by the epidermal growth factor (EGF is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer. In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

  8. Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

    Science.gov (United States)

    Marquèze-Pouey, Béatrice; Mailfert, Sébastien; Rouger, Vincent; Goaillard, Jean-Marc; Marguet, Didier

    2014-01-01

    Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

  9. Discoidin domain receptor 1 is activated independently of beta(1) integrin

    DEFF Research Database (Denmark)

    Vogel, W; Brakebusch, C; Fässler, R

    2000-01-01

    independent of the epidermal growth factor (EGF) receptor. In cells that endogenously express both DDR1 and the EGF receptor, stimulation with EGF does not induce DDR activation. Third, we detected full DDR1 activation after collagen stimulation in cells that have been treated with blocking antibodies...... for alpha(2)beta(1) integrin or in cells with a targeted deletion of the beta(1) integrin gene. Finally, we show that overexpression of dominant negative DDR1 in the myoblast cell line C2C12 blocks cellular differentiation and the formation of myofibers....

  10. Differential effects of EGFR ligands on endocytic sorting of the receptor

    DEFF Research Database (Denmark)

    Roepstorff, Kirstine; Grandal, Michael Vibo; Henriksen, Lasse

    2009-01-01

    signalling and is a more potent mitogen than EGF. In addition to EGF and TGF-alpha, five EGFR ligands have been identified. Although many of these ligands are upregulated in cancers, very little is known about their effect on EGFR trafficking. We have compared the effect of six different ligands on endocytic...... trafficking of EGFR. We find that, whereas they all stimulate receptor internalization, they have very diverse effects on endocytic sorting. Heparin-binding EGF-like growth factor and Betacellulin target all EGFRs for lysosomal degradation. In contrast, TGF-alpha and epiregulin lead to complete receptor...

  11. Time-dependent changes of levels of endogenous epidermal growth factor in submandibular glands, in kidneys, and in urine in rats during systemic treatment with EGF

    DEFF Research Database (Denmark)

    Vinter-Jensen, Lars; Jørgensen, P E; Thulesen, J

    1998-01-01

    Exogenous EGF influences the levels of endogenous EGF differently in the submandibular glands (SMG) and the kidneys. The aim of the present study was to examine the time-dependent changes in levels of endogenous EGF during 1-4 weeks of EGF treatment.......Exogenous EGF influences the levels of endogenous EGF differently in the submandibular glands (SMG) and the kidneys. The aim of the present study was to examine the time-dependent changes in levels of endogenous EGF during 1-4 weeks of EGF treatment....

  12. The role of sialoadenectomy and epıdermal growth factor (EGF) in ...

    African Journals Online (AJOL)

    USER

    2010-05-17

    May 17, 2010 ... result, epidermal growth factor was concluded to have an important role in skin development. Key words: Epidermal growth factor, ... sialoadenectomy on epiderm and the role of EGF and antiserum EGF in prevention of .... remarkable finding in skin healing of sialoadenectomy and normal rats. According to ...

  13. Expression of polyhedrin-hEGF fusion protein in cultured cells and ...

    African Journals Online (AJOL)

    For mass production of human epidermal growth factor (hEGF), silkworm baculovirus expression vector system (BEVS) was adopted in this study. hEGF gene was in-frame fused with polyhedrin (Ph) gene under the control of Ph promoter and was used to co-transfect BmN cell with the modified. Bombyx mori baculovirus ...

  14. δ-Tocopherol inhibits receptor tyrosine kinase-induced AKT activation in prostate cancer cells.

    Science.gov (United States)

    Wang, Hong; Hong, Jungil; Yang, Chung S

    2016-11-01

    The cancer preventive activity of vitamin E is suggested by epidemiological studies and supported by animal studies with vitamin E forms, γ-tocopherol and δ-tocopherol (δ-T). Several recent large-scale cancer prevention trials with high dose of α-tocopherol, however, yielded disappointing results. Whether vitamin E prevents or promotes cancer is a serious concern. A better understanding of the molecular mechanisms of action of the different forms of tocopherols would enhance our understanding of this topic. In this study, we demonstrated that δ-T was the most effective tocopherol form in inhibiting prostate cancer cell growth, by inducing cell cycle arrest and apoptosis. By profiling the effects of δ-T on the cell signaling using the phospho-kinase array, we found that the most inhibited target was the phosphorylation of AKT on T308. Further study on the activation of AKT by EGFR and IGFR revealed that δ-T attenuated the EGF/IGF-induced activation of AKT (via the phosphorylation of AKT on T308 induced by the activation of PIK3). Expression of dominant active PIK3 and AKT in prostate cancer cell line DU145 in which PIK3, AKT, and PTEN are wild type caused the cells to be reflectory to the inhibition of δ-T, supporting that δ-T inhibits the PIK3-mediated activation of AKT. Our data also suggest that δ-T interferes with the EGF-induced EGFR internalization, which leads to the inhibition of the receptor tyrosine kinase-dependent activation of AKT. In summary, our results revealed a novel mechanism of δ-T in inhibiting prostate cancer cell growth, supporting the cancer preventive activity δ-T. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  15. Endogenous EGF as a potential renotrophic factor in ischemia-induced acute renal failure.

    Science.gov (United States)

    Schaudies, R P; Nonclercq, D; Nelson, L; Toubeau, G; Zanen, J; Heuson-Stiennon, J A; Laurent, G

    1993-09-01

    The time course for the increases in soluble renal epidermal growth factor (EGF) after ischemia has been established. These elevated levels of EGF have been compared with the degree of tissue injury as well as the extent of cell proliferation in the recovering tissue. Levels of soluble immunoreactive EGF (irEGF) in control animals were 9.74 +/- 1.1 ng/g wet wt (n = 4-8 for all values) and rose to 83.9 +/- 30 ng/g within 12 h after injury. Soluble irEGF content peaked at 88.8 +/- 15 ng/g at 24 h postinjury and returned to control values by 72 h. We previously reported that trypsin digestion of crude renal membranes (CRM) generates rat EGF that is indistinguishable from that isolated from the submandibular gland. Initial levels of trypsin-releasable membrane-associated irEGF were 439 +/- 26 ng/g. These levels fell to 46.6 +/- 9.6 ng/g at 48 h after injury. The total renal EGF demonstrated an 80% decline 48 h after injury but returned to 50% of the initial values after 72 h representing significant new synthesis of EGF-containing proteins between 48 and 72 h postinjury. Immunohistochemical staining of kidney paraffin sections for EGF immunoreactivity demonstrated staining intensities that paralleled the amount of irEGF in the trypsin-digested CRM fraction, suggesting that the membrane-associated irEGF is the predominant form detected by this technique. Regenerative hyperplasia subsequent to tubular insult was monitored by immunostaining nuclei of S phase cells after pulse labeling with the thymidine analogue 5-bromo-2'-deoxyuridine. Cell proliferation was particularly prominent in the outer stripe of outer medulla of kidneys exposed to ischemia and reached a maximum (19-fold higher than the baseline value) 48 h after reperfusion. Renal cell turnover returned to control values by day 7. The observation that the peak in soluble EGF levels (24 h) precedes the peak in tubular regeneration (48 h) by 24 h is consistent with the hypothesis that EGF is one of the mitogenic

  16. DNA methylation dynamics in the rat EGF gene promoter after partial hepatectomy

    Directory of Open Access Journals (Sweden)

    Deming Li

    2014-06-01

    Full Text Available Epidermal growth factor (EGF, a multifunctional growth factor, is a regulator in a wide variety of physiological processes. EGF plays an important role in the regulation of liver regeneration. This study was aimed at investigating the methylation level of EGF gene throughout liver regeneration. DNA of liver tissue from control rats and partial hepatectomy (PH rats at 10 time points was extracted and a 354 bp fragment including 10 CpG sites from the transcription start was amplified after DNA was modified by sodium bisulfate. The result of sequencing suggested that methylation ratio of four CpG sites was found to be significantly changed when PH group was compared to control group, in particular two of them were extremely striking. mRNA expression of EGF was down-regulated in total during liver regeneration. We think that the rat EGF promoter region is regulated by variation in DNA methylation during liver regeneration.

  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

    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....... Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization...... 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. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands.

    Directory of Open Access Journals (Sweden)

    Lasse Henriksen

    Full Text Available The epidermal growth factor receptor (EGFR regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF and transforming growth factor-α (TGF-α. For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF or betacellulin (BTC was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.

  19. Internalization Mechanisms of the Epidermal Growth Factor Receptor after Activation with Different Ligands

    Science.gov (United States)

    Henriksen, Lasse; Grandal, Michael Vibo; Knudsen, Stine Louise Jeppe; van Deurs, Bo; Grøvdal, Lene Melsæther

    2013-01-01

    The epidermal growth factor receptor (EGFR) regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown. PMID:23472148

  20. Novel Role of Src in Priming Pyk2 Phosphorylation.

    Directory of Open Access Journals (Sweden)

    Ming Zhao

    Full Text Available Proline-rich tyrosine kinase 2 (Pyk2 is a member of the focal adhesion kinase (FAK family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.

  1. Inhibition of the epidermal growth factor receptor in bladder cancer cells treated with the DNA-damaging drug etoposide markedly increases apoptosis

    DEFF Research Database (Denmark)

    Munk, Mathias; Memon, Ashfaque Ahmed; Nexo, Ebba

    2007-01-01

    : The bladder cancer cell lines RT4 and T24, representing low- and high-malignancy grades respectively, were treated with VP16 (10 or 50 microM) and the level of apoptosis determined using a commercial kit. EGFR receptor activity was determined by western blotting using antibodies against phosphorylated EGFR....... The EGFR was either activated by heparin-binding (HB)-EGF (1 nM) or inhibited with the specific EGFR inhibitor gefitinib (1 or 5 microM). The pan-caspase inhibitor Z-VAD (30 microM) was used to test the involvement of caspase activity. RESULTS: Treatment of T24 bladder cancer cells with VP16 (50 micro...... suggest that activation of the EGFR induced a cell-survival function when bladder cancer cells were treated with the DNA-damaging drug VP16, and that combined treatment with VP16 and the EGFR inhibitor gefitinib might improve the efficacy of treatment. Udgivelsesdato: 2007-Jan...

  2. Resveratrol modulates MED28 (Magicin/EG-1) expression and inhibits epidermal growth factor (EGF)-induced migration in MDA-MB-231 human breast cancer cells.

    Science.gov (United States)

    Lee, Ming-Fen; Pan, Min-Hsiung; Chiou, Yi-Siou; Cheng, An-Chin; Huang, Han

    2011-11-09

    Resveratrol and pterostilbene exhibit diverse biological activities. MED28, a subunit of the mammalian Mediator complex for transcription, was also identified as magicin, an actin cytoskeleton Grb2-associated protein, and as endothelial-derived gene (EG-1). Several tumors exhibit aberrant MED28 expression, whereas the underlying mechanism is unclear. Triple-negative breast cancers, often expressing epidermal growth factor (EGF) receptor (EGFR), are associated with metastasis and poor survival. The objective of this study is to compare the effect of resveratrol and pterostilbene and to investigate the role of MED28 in EGFR-overexpressing MDA-MB-231 breast cancer cells. Pretreatment of resveratrol, but not pterostlbene, suppressed EGF-mediated migration and expression of MED28 and matrix metalloproteinase (MMP)-9 in MDA-MB-231 cells. Moreover, overexpression of MED28 increased migration, and the addition of EGF further enhanced migration. Our data indicate that resveratrol modulates the effect of MED28 on cellular migration, presumably through the EGFR/phosphatidylinositol 3-kinase (PI3K) signaling pathway, in breast cancer cells.

  3. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands

    DEFF Research Database (Denmark)

    Henriksen, Lasse; Grandal, Michael Vibo; Knudsen, Stine Louise Jeppe

    2013-01-01

    after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist....... Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown...... fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand...

  4. Pretreatment with oleic acid accelerates the entrance into the mitotic cycle of EGF-stimulated fibroblasts.

    Science.gov (United States)

    Zugaza, J L; Casabiell, X A; Bokser, L; Eiras, A; Beiras, A; Casanueva, F F

    1995-07-01

    We have previously demonstrated that pretreatment of several cell lines with cis-unsaturated fatty acids, like oleic acid, blocks epidermal growth factor (EGF)-induced early ionic signals, and in particular the [Ca2+]i rise. In the present work we show that this blockade does not alter EGF-stimulated cellular proliferation evaluated by direct cell counting, but induces a powerful enhancement in the pulsed thymidine incorporation assay. The lack of effect of oleic acid on EGF-stimulated cellular proliferation was confirmed by repeated cell counts, cumulative thymidine incorporation, and protein synthesis, but a clear synergistic effect between oleic acid and EGF was again obtained by means of time course experiments with pulsed thymidine. Combined flow cytometry analysis and cell counts at earlier times in EGF-stimulated cells showed that oleic acids accelerates the entrance of cells into the replicative cycle leading to an earlier cell division. Afterward, these oleic acid-pretreated cells became delayed by an unknown compensatory mechanism in such a way that at 48 h post-EGF, the cell count in control and oleic acid-pretreated cells was equal. In conclusion (a) oleic acid accelerates or enhances the EGF mitogenic action and (b) in the long term cells compensate the initial perturbation with respect to untreated cells. As a side observation, the widely employed pulsed thymidine incorporation method as a measure of cell division could be extremely misleading unless experimental conditions are well controlled.

  5. The preventive effect of recombinant human growth factor (rhEGF) on the recurrence of radiodermatitis

    International Nuclear Information System (INIS)

    Ryu, Seung-Hee; Kim, Yeun-Hwa; Lee, Sang-Wook; Hong, Joon-Pio

    2010-01-01

    The effects of topical application of recombinant human epidermal growth factor (rhEGF) on wound healing and the recurrence of radiodermatitis were assessed in the irradiated skin of BALB/c Nu/Nu mice. Mice irradiated with 45 Gy of radiation were divided into 5 groups and treated with 10, 50, and 100 μg/g rhEGF ointment, vehicle alone, or no treatment (control) for 6 months. Wounds were observed initially in all groups and complete healing time (HT 100 ) for initial wound repair did not differ significantly among groups. However, the rate of recurrence over 6 months was significantly lower in the EGF-treated groups than in the control group (p<0.05). Histological examination showed that treatment with the optimum dose of EGF (50 μg/g) accelerated normal wound healing when compared with the higher dose of EGF (100 μg/g), vehicle alone, or no treatment, with the latter group showing irregular epidermal thickness, poor definition of epidermis and dermis, and unstable dermal structure. Collagen distribution was also significantly increased in mice treated with 50 μg/g rhEGF (p<0.05) compared with the control or vehicle-treated group. Taken together, these results indicate that treatment with exogenous EGF (50 μg/g dose) can enhance radiation-induced wound repair while preserving structural tissue stability and preventing the recurrence of radiodermatitis. (author)

  6. Arf6 regulates EGF-induced internalization of E-cadherin in breast cancer cells.

    Science.gov (United States)

    Xu, Rui; Zhang, Yujie; Gu, Luo; Zheng, Jianchao; Cui, Jie; Dong, Jing; Du, Jun

    2015-01-01

    E-cadherin internalization facilitates dissolution of adherens junctions and promotes tumor cell epithelial-mesenchymal transition (EMT) and migration. Our previous results have shown that Arf6 exerts pro-migratory action in breast cancer cells after EGF stimulation. Despite the fact that EGF signaling stimulates EMT of breast cancer cells, the effect of Arf6 on internalization of E-cadherin of breast cancer cells under EGF treatment remains to be determined. Here, we showed that EGF dose-dependently stimulated E-cadherin internalization by MCF-7 cells with the maximal effect at 50 ng/ml. Meanwhile, EGF treatment markedly increased Arf6 activation. Arf6 was involved in complexes of E-cadherin, and more E-cadherin was pulled down with Arf6 when the activity of the latter was increased. Immunoblotting and immunofluorescence assays showed that transfection breast cancer cells with Arf6-T27N or Arf6 siRNA suppressed EGF-induced E-cadherin internalization. Taken together, our study demonstrated that Arf6 activation plays a potential role in EGF-induced E-cadherin internalization, providing new mechanism underlying the effect of Arf6 on promoting breast cancer cell metastasis.

  7. Ticagrelor Improves Endothelial Function by Decreasing Circulating Epidermal Growth Factor (EGF

    Directory of Open Access Journals (Sweden)

    Francesco Vieceli Dalla Sega

    2018-04-01

    Full Text Available Ticagrelor is one of the most powerful P2Y12 inhibitor. We have recently reported that, in patients with concomitant Stable Coronary Artery Disease (SCAD and Chronic Obstructive Pulmonary Disease (COPD undergoing percutaneous coronary intervention (PCI, treatment with ticagrelor, as compared to clopidogrel, is associated with an improvement of the endothelial function (Clinical Trial NCT02519608. In the present study, we showed that, in the same population, after 1 month treatment with ticagrelor, but not with clopidogrel, there is a decrease of the circulating levels of epidermal growth factor (EGF and that these changes in circulating levels of EGF correlate with on-treatment platelet reactivity. Furthermore, in human umbilical vein endothelial cells (HUVEC incubated with sera of the patients treated with ticagrelor, but not with clopidogrel there is an increase of p-eNOS levels. Finally, analyzing the changes in EGF and p-eNOS levels after treatment, we observed an inverse correlation between p-eNOS and EGF changes only in the ticagrelor group. Causality between EGF and eNOS activation was assessed in vitro in HUVEC where we showed that EGF decreases eNOS activity in a dose dependent manner. Taken together our data indicate that ticagrelor improves endothelial function by lowering circulating EGF that results in the activation of eNOS in the vascular endothelium.

  8. Growth of intestinal epithelium in organ culture is dependent on EGF signalling

    International Nuclear Information System (INIS)

    Abud, Helen E.; Watson, Nadine; Heath, Joan K.

    2005-01-01

    Differentiation of endoderm into intestinal epithelium is initiated at E13.5 of mouse development when there are significant changes in morphology resulting in the conversion of undifferentiated stratified epithelium into a mature epithelial monolayer. Here we demonstrate that monolayer formation is associated with the selective apoptosis of superficial cells lining the lumen while cell proliferation is progressively restricted to cells adjacent to the basement membrane. We describe an innovative embryonic gut culture system that maintains the three-dimensional architecture of gut and in which these processes are recapitulated in vitro. Explants taken from specific regions of the gut and placed into organ culture develop and express molecular markers (Cdx1, Cdx2 and A33 antigen) in the same spatial and temporal pattern observed in vivo indicating that regional specification is maintained. Inhibition of the epidermal growth factor receptor (EGFR) tyrosine kinase using the specific inhibitor AG1478 significantly reduced the proliferation and survival of cells within the epithelial cell layer of cultured gut explants. This demonstrates an essential role for the EGF signalling pathway during the early stages of intestinal development

  9. Does phosphorylation of cofilin affect the progression of human bladder cancer?

    International Nuclear Information System (INIS)

    Chung, Hong; Kim, Hong Sup; Kim, Bokyung; Jung, Seung-Hyo; Won, Kyung-Jong; Jiang, Xiaowen; Lee, Chang-Kwon; Lim, So Dug; Yang, Sang-Kuk; Song, Ki Hak

    2013-01-01

    We determined the differently expressed protein profiles and their functions in bladder cancer tissues with the aim of identifying possible target proteins and underlying molecular mechanisms for taking part in their progression. We examined the expression of proteins by proteomic analysis and western blot in normal urothelium, non-muscle-invasive bladder cancers (NMIBCs), and muscle-invasive bladder cancers (MIBCs). The function of cofilin was analyzed using T24 human bladder cancer cells. The expression levels of 12 proteins were altered between bladder cancers and normal bladder tissues. Of these proteins, 14-3-3σ was upregulated in both NMIBCs and MIBCs compared with controls. On the other hand, myosin regulatory light chain 2, galectin-1, lipid-binding AI, annexin V, transthyretin, CARD-inhibitor of NF-κB-activating ligand, and actin prepeptide were downregulated in cancer samples. Cofilin, an actin-depolymerizing factor, was prominent in both NMIBCs and MIBCs compared with normal bladder tissues. Furthermore, we confirmed that cofilin phosphorylation was more prominent in MIBCs than in NMIBCs using immunoblotting and immunohistochemcal analyses. Epidermal growth factor (EGF) increased the phosphorylation of cofilin and elevated the migration in T24 cells. Knockdown of cofilin expression with small interfering RNA attenuated the T24 cell migration in response to EGF. These results demonstrate that the increased expression and phosphorylation of cofilin might play a role in the occurrence and invasiveness of bladder cancer. We suspected that changes in cofilin expression may participate in the progression of the bladder cancer

  10. Both sides of the same coin: Rac1 splicing regulating by EGF signaling.

    Science.gov (United States)

    Fu, Xiang-Dong

    2017-04-01

    EGF, a well-studied mitogen for cancer cells, is revealed to induce an E3 ubiquitin ligase adaptor SPSB1, which recruits the Elongin B/C-Collin complex to trigger ubiquitylation of the negative splicing regulator hnRNP A1. This event is synergized with EGF-activated SR proteins to alter alternative splicing of a key small GTPase Rac1 to enhance cell migration, highlighting converging EGF signals on both negative and positive splicing regulators to jointly promote a key cancer pathway.

  11. Immunoautoradiographic analysis of epidermal growth factor receptors: a sensitive method for the in situ identification of receptor proteins and for studying receptor specificity

    International Nuclear Information System (INIS)

    Fernandez-Pol, J.A.

    1982-01-01

    The use of an immunoautoradiographic system for the detection and analysis of epidermal growth factor (EGF) receptors in human epidermoid carcinoma A-431 cells is reported. By utilizing this technique, the interaction between EGF and its membrane receptor in A-431 cells can be rapidly visualized. The procedure is simple, rapid, and very sensitive, and it provides conclusive evidence that the 150K dalton protein is the receptor fo EGF in A-431 cells. In summary, the immunoautoradiographic procedure brings to the analysis of hormone rceptor proteins the power that the radioimmunoassay technique has brought to the analysis of hormones. Thus, this assay system is potentially applicable in a wide spectrum in many fields of nuclear medicine and biology

  12. Gene expression response of A253 human salivary cell line to radiation, Cis-Pt, and EGF

    International Nuclear Information System (INIS)

    Woloschak, G.; Paunesku, T.; Mittal, B.; Dyck, P.; Pauloski, B.; Rademaker, A.; Logemann, J.; Quigg, R.

    2003-01-01

    We are interested in long and short term effects of head and neck cancers treatment, and prior to the studies of patient samples, experiments were designed to observe treatment effects in cultured cells, and examine gene expression profiles from A253 human salivary cells (derived from a head and neck tumor) following exposure to gamma-rays, cisplatin (cis-Pt), and a combination of either with epidermal growth factor (EGF) treatment. A253 cells were treated by: 2 Gy or 10 Gy of γ-rays (Cs137 source, 77 cGy/min), Cis-Pt at 50 μ/mL, and EGF at 40 ng/mL. RNAs were processed and hybridized with Affymetrix Hu95A arrays according to the manufacturer's instructions. Data were scanned and analyzed and we found significant differences in the expression patterns of numerous genes were observed. Some of the more interesting genes are: Requeim [a protein required for apoptosis]; Cyclin D1 (prad1/bcl1) [a cyclin that can function as an oncogene]; FK506 Binding Protein [which may be competing with TGF-beta type I receptor for binding with FK506 thus acting against this powerful immunosuppressant]; Thioredoxin (TXN) [an oxidoreductase with multiple in vitro substrates, including ribonuclease, choriogonadotropins, coagulation factors, glucocorticoid receptor, and insulin]; Glutathione Peroxidase (GPX) [whose role in protection against oxidative stress was long ago well documented]; Aquaporin 3 (AQP3) [protein with a water-channel function that was confirmed by functional expression in Xenopus oocytes]; Eukaryotic Initiation Factor 1A (EIF1A) [a translation factor, proposed as a candidate gene for Turner syndrome]; and finally Insulin-like Growth Factor-Binding Protein 6 (IGFBP6) [an autocrine growth inhibitor shown to inhibit growth of HaCat cells and other keratinocyte cell lines

  13. Ketamine inhibits tumor necrosis factor-α and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation

    International Nuclear Information System (INIS)

    Wu, G.-J.; Chen, T.-L.; Ueng, Y.-F.; Chen, R.-M.

    2008-01-01

    Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 μM ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 μM of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-α and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-α and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 μM) significantly inhibited LPS-induced TNF-α and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-α and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-α and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms occur through suppression of TLR4-mediated

  14. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    Directory of Open Access Journals (Sweden)

    Yueh-Hsiung Kuo

    2016-06-01

    Full Text Available This study investigated the potential effects of dehydroeburicoic acid (TT, a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom on membrane glucose transporter 4 (GLUT4 and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4 and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels, fenofibrate (Feno (at 0.25 g/kg body weight, metformin (Metf (at 0.3 g/kg body weight or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%. TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase, an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator

  15. ErbB1-4-dependent EGF/neuregulin signals and their cross talk in the central nervous system: pathological implications in schizophrenia and Parkinson’s disease

    Directory of Open Access Journals (Sweden)

    Yuriko eIwakura

    2013-02-01

    Full Text Available Ligands for ErbB1-4 receptor tyrosine kinases, such as epidermal growth factor (EGF and neuregulins, regulate brain development and function. Thus, abnormalities in their signaling are implicated in the etiology or pathology of schizophrenia and Parkinson’s disease. Among the ErbB receptors, ErbB1 and ErbB4 are expressed in dopamine and GABA neurons, while ErbB1, 2, and/or 3 are mainly present in oligodendrocytes, astrocytes and their precursors. Thus, deficits in ErbB signaling might contribute to schizophrenia neuropathology stemming from these cell types. By incorporating the latest cancer molecular biology as well as our recent progress, we discuss signal cross talk between the ErbB1-4 subunits and their neurobiological functions in each cell type. The potential contribution of virus-derived cytokines (virokines that mimic EGF and neuregulin-1 in brain diseases are also discussed.

  16. Resveratrol prevents angiotensin II-induced hypertrophy of vascular smooth muscle cells through the transactivation of growth factor receptors.

    Science.gov (United States)

    Hossain, Ekhtear; Anand-Srivastava, Madhu B

    2017-08-01

    We previously showed that augmented levels of endogenous angiotensin II (AngII) contribute to vascular smooth muscle cell (VSMC) hypertrophy through the transactivation of growth factor receptors in spontaneously hypertensive rats. Resveratrol (RV), a polyphenolic component of red wine, has also been shown to attenuate AngII-evoked VSMC hypertrophy; however, the molecular mechanism mediating this response is obscure. The present study was therefore undertaken to examine whether RV could prevent AngII-induced VSMC hypertrophy through the transactivation of growth factor receptor and associated signaling pathways. AngII treatment of VSMC enhanced the protein synthesis that was attenuated towards control levels by RV pretreatment as well as by the inhibitors of NADPH oxidase, c-Src, and growth factor receptors. Furthermore, RV pretreatment also inhibited enhanced levels of superoxide anion, NADPH oxidase activity, increased expression of NADPH oxidase subunits, and phosphorylation of c-Src, EGF-R, PDGE-R, ERK1/2, and AKT1/2. In conclusion, these results indicate that RV attenuates AngII-induced VSMC hypertrophy through the inhibition of enhanced oxidative stress and activation of c-Src, growth factor receptors, and MAPK/AKT signaling. We suggest that RV could be used as a therapeutic agent in the treatment of vascular complications associated with hypertension and hypertrophy.

  17. LPA, HGF, and EGF utilize distinct combinations of signaling pathways to promote migration and invasion of MDA-MB-231 breast carcinoma cells

    International Nuclear Information System (INIS)

    Harrison, Susan MW; Knifley, Teresa; Chen, Min; O’Connor, Kathleen L

    2013-01-01

    Various pathways impinge on the actin-myosin pathway to facilitate cell migration and invasion including members of the Rho family of small GTPases and MAPK. However, the signaling components that are considered important for these processes vary substantially within the literature with certain pathways being favored. These distinctions in signaling pathways utilized are often attributed to differences in cell type or physiological conditions; however, these attributes have not been systematically assessed. To address this question, we analyzed the migration and invasion of MDA-MB-231 breast carcinoma cell line in response to various stimuli including lysophosphatidic acid (LPA), hepatocyte growth factor (HGF) and epidermal growth factor (EGF) and determined the involvement of select signaling pathways that impact myosin light chain phosphorylation. LPA, a potent stimulator of the Rho-ROCK pathway, surprisingly did not require the Rho-ROCK pathway to stimulate migration but instead utilized Rac and MAPK. In contrast, LPA-stimulated invasion required Rho, Rac, and MAPK. Of these three major pathways, EGF-stimulated MDA-MB-231 migration and invasion required Rho; however, Rac was essential only for invasion and MAPK was dispensable for migration. HGF signaling, interestingly, utilized the same pathways for migration and invasion, requiring Rho but not Rac signaling. Notably, the dependency of HGF-stimulated migration and invasion as well as EGF-stimulated invasion on MAPK was subject to the inhibitors used. As expected, myosin light chain kinase (MLCK), a convergence point for MAPK and Rho family GTPase signaling, was required for all six conditions. These observations suggest that, while multiple signaling pathways contribute to cancer cell motility, not all pathways operate under all conditions. Thus, our study highlights the plasticity of cancer cells to adapt to multiple migratory cues

  18. Protein tyrosine phosphatase-1B (PTP1B) helps regulate EGF-induced stimulation of S-phase entry in human corneal endothelial cells

    Science.gov (United States)

    Ishino, Yutaka; Zhu, Cheng; Harris, Deshea L.

    2008-01-01

    Purpose Human corneal endothelial cells (HCEC), particularly from older donors, only proliferate weakly in response to EGF. The protein tyrosine phosphatase, PTP1B, is known to negatively regulate EGF-induced signaling in several cell types by dephosphorylating the epidermal growth factor receptor (EGFR). The current studies were conducted to determine whether PTP1B plays a role in regulating cell cycle entry in HCEC in response to EGF stimulation. Methods Donor corneas were obtained from the National Disease Research Interchange and accepted for study based on established exclusion criteria. PTP1B was localized in the endothelium of ex vivo corneas and in cultured cells by immunocytochemistry. Western blot analysis verified PTP1B protein expression in HCEC and then compared the relative expression of EGFR and PTP1B in HCEC from young (60 years old). The effect of inhibiting the activity of PTP1B on S-phase entry was tested by comparing time-dependent BrdU incorporation in subconfluent HCEC incubated in the presence or absence of the PTP1B inhibitor, CinnGEL 2Me, before EGF stimulation. Results PTP1B was localized in a punctate pattern mainly within the cytoplasm of HCEC in ex vivo corneas and cultured cells. Western blots revealed the presence of three PTP1B-positive bands in HCEC and the control. Further western blot analysis showed no significant age-related difference in expression of EGFR (p=0.444>0.05); however, PTP1B expression was significantly higher in HCEC from older donors (p=0.024<0.05). Pre-incubation of HCEC with the PTP1B inhibitor significantly increased (p=0.019<0.05) the number of BrdU positive cells by 48 h after EGF stimulation. Conclusions Both immunolocalization and western blot studies confirmed that PTP1B is expressed in HCEC. Staining patterns strongly suggest that at least a subset of PTP1B is localized to the cytoplasm and most likely to the endoplasmic reticulum, the known site of EGFR/PTP1B interaction following EGF stimulation. PTP1B

  19. Linking γ-aminobutyric acid A receptor to epidermal growth factor receptor pathways activation in human prostate cancer.

    Science.gov (United States)

    Wu, Weijuan; Yang, Qing; Fung, Kar-Ming; Humphreys, Mitchell R; Brame, Lacy S; Cao, Amy; Fang, Yu-Ting; Shih, Pin-Tsen; Kropp, Bradley P; Lin, Hsueh-Kung

    2014-03-05

    Neuroendocrine (NE) differentiation has been attributed to the progression of castration-resistant prostate cancer (CRPC). Growth factor pathways including the epidermal growth factor receptor (EGFR) signaling have been implicated in the development of NE features and progression to a castration-resistant phenotype. However, upstream molecules that regulate the growth factor pathway remain largely unknown. Using androgen-insensitive bone metastasis PC-3 cells and androgen-sensitive lymph node metastasis LNCaP cells derived from human prostate cancer (PCa) patients, we demonstrated that γ-aminobutyric acid A receptor (GABA(A)R) ligand (GABA) and agonist (isoguvacine) stimulate cell proliferation, enhance EGF family members expression, and activate EGFR and a downstream signaling molecule, Src, in both PC-3 and LNCaP cells. Inclusion of a GABA(A)R antagonist, picrotoxin, or an EGFR tyrosine kinase inhibitor, Gefitinib (ZD1839 or Iressa), blocked isoguvacine and GABA-stimulated cell growth, trans-phospohorylation of EGFR, and tyrosyl phosphorylation of Src in both PCa cell lines. Spatial distributions of GABAAR α₁ and phosphorylated Src (Tyr416) were studied in human prostate tissues by immunohistochemistry. In contrast to extremely low or absence of GABA(A)R α₁-positive immunoreactivity in normal prostate epithelium, elevated GABA(A)R α₁ immunoreactivity was detected in prostate carcinomatous glands. Similarly, immunoreactivity of phospho-Src (Tyr416) was specifically localized and limited to the nucleoli of all invasive prostate carcinoma cells, but negative in normal tissues. Strong GABAAR α₁ immunoreactivity was spatially adjacent to the neoplastic glands where strong phospho-Src (Tyr416)-positive immunoreactivity was demonstrated, but not in adjacent to normal glands. These results suggest that the GABA signaling is linked to the EGFR pathway and may work through autocrine or paracine mechanism to promote CRPC progression. Copyright © 2013 Elsevier

  20. About phosphorylation of lappaconitine

    International Nuclear Information System (INIS)

    Burdelnaya, E.V.; Turmukhambetov, A.Zh.

    2005-01-01

    In the article chemical modifications of alkaloid lappaconitine are investigated. It was shown that synthesis of the phosphorylated derivatives are the ways to create new biologically active compounds. Interaction of lappaconitine with phosphorus pentachloride was used to obtain new phosphoric derivatives of alkaloid. The composition and structure of the new phosphorus-containing compounds were confirmed by elemental analysis: IR, UV and 13 C, 1 H, 31 P NMR -spectroscopy

  1. INTERACTION OF RECOMBINANT DIPHTHERIA TOXOIDS WITH CELLULAR RECEPTORS in vitro

    Directory of Open Access Journals (Sweden)

    K. Yu. Manoilov

    2016-06-01

    Full Text Available The aim of the research was to compare in vitro characteristics of reception of the natural diphtheria toxin — DT and its nontoxic recombinant analogs — toxoids. For assessing ligand-receptor interaction the method of immunoenzyme analysis and ELISA was used, where the bonding layer recombinant analogues of diphtheria toxin cell receptor HB-EGF from sensitive and resistant to the toxin of the organisms were served. According to the results of ELISA the natural diphtheria toxin, in contrast to recombinant toxoids — CRM197, and B subunit, interacted with mouse HB-EGF with a very low affinity. While human HB-EGF with an equally high affinity connected as toxoids as native diphtheria toxin. Therefore, the analyzed recombinant analogs of toxin obtained in E. coli cells did not reproduce in full measure the receptor specificity of the natural toxin, which should be considered in the case of using these proteins as biotech products.

  2. EGF increases expression and activity of PAs in preimplantation rat embryos and their implantation rate

    Directory of Open Access Journals (Sweden)

    Har-Vardi Iris

    2007-01-01

    Full Text Available Abstract Background Embryo implantation plays a major role in embryogenesis and the outcome of pregnancy. Plasminogen activators (PAs have been implicated in mammalian fertilization, early stages of development and embryo implantation. As in-vitro developing embryos resulted in lower implantation rate than those developed in-vivo we assume that a reduced PAs activity may be involved. In the present work we studied the effect of EGF on PAs activity, quantity and embryo implantation. Methods Zygotes were flushed from rat oviducts on day one of pregnancy and grown in-vitro in R1ECM supplemented with EGF (10 ng/ml and were grown up to the blastocyst stage. The control groups were grown in the same medium without EGF. The distribution and quantity of the PAs were examined using fluorescence immunohistochemistry followed by measurement of PAs activity using the chromogenic assay. Implantation rate was studied using the embryo donation model. Results PAs distribution in the embryos was the same in EGF treated and untreated embryos. Both PAs were localized in the blastocysts' trophectoderm, supporting the assumption that PAs play a role in the implantation process in rats. EGF increased the quantity of uPA at all stages studied but the 8-cell stage as compared with controls. The tissue type PA (tPA content was unaffected except the 8-cell stage, which was increased. The activity of uPA increased gradually towards the blastocyst stage and more so due to the presence of EGF. The activity of tPA did not vary with the advancing developmental stages although it was also increased by EGF. The presence of EGF during the preimplantation development doubled the rate of implantation of the treated group as compared with controls.

  3. A functional EGF+61 polymorphism is associated with severity of obstructive sleep apnea.

    Science.gov (United States)

    Ding, Qunli; Cao, Chao; Chen, Zhongbo; Tabusi, Mahebali; Chen, Li; Deng, Zaichun

    2015-05-01

    Involvement of epidermal growth factor (EGF) is reported in diseases caused by hypoxia. Its functional polymorphism may alter its transcription, affecting EGF expression, contributing to obstructive sleep apnea (OSA). The aim of this study was to investigate associations of EGF+61 polymorphism and risk of OSA. Two hundred two participants were enrolled in this case-control study. DNA was extracted from peripheral blood, and EGF 61A/G polymorphism was determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. No significant association between EGF 61 A/G polymorphism and risk of OSA was observed in any of the gene models tested (AA vs. GG: OR = 0.97, 95% CI = 0.37-2.55; P = 0.95). However, compared with GG genotype, AG genotype associated with decreased risk of severe OSA (AG vs. GG: OR = 0.32, 95% CI = 0.11-0.94). Our study showed that AG genotype has a protective effect on OSA patients against severe disease, although EGF 61A/G polymorphisms have no role on the risk of the disease. Additional large studies should further validate our findings.

  4. Restoration of CpG Methylation in The Egf Promoter Region during Rat Liver Regeneration

    Science.gov (United States)

    Deming, Li; Ziwei, Li; Xueqiang, Guo; Cunshuan, Xu

    2015-01-01

    Epidermal growth factor (EGF) is an important factor for healing after tissue damage in diverse experimental models. It plays an important role in liver regeneration (LR). The objective of this experiment is to investigate the methylation variation of 10 CpG sites in the Egf promoter region and their relevance to Egf expression during rat liver regenera- tion. As a follow up of our previous study, rat liver tissue was collected after rat 2/3 partial hepatectomy (PH) during the re-organization phase (from days 14 to days 28). Liver DNA was extracted and modified by sodium bisulfate. The methylation status of 10 CpG sites in Egf promoter region was determined using bisulfite sequencing polymerase chain reaction (PCR), as BSP method. The results showed that 3 (sites 3, 4 and 9) out of 10 CpG sites have strikingly methylation changes during the re-organization phase compared to the regeneration phase (from 2 hours to 168 hours, P=0.002, 0.048 and 0.018, respectively). Our results showed that methylation modification of CpGs in the Egf promoter region could be restored to the status before PH operation and changes of methylation didn’t affect Egf mRNA expression during the re-organization phase. PMID:26464832

  5. HB-EGF is necessary and sufficient for Müller glia dedifferentiation and retina regeneration

    Science.gov (United States)

    Wan, Jin; Ramachandran, Rajesh; Goldman, Daniel

    2011-01-01

    Summary Müller glia (MG) dedifferentiation into a cycling population of multipotent progenitors is crucial to zebrafish retina regeneration. The mechanisms underlying MG dedifferentiation are unknown. Here we report that heparin-binding epidermal-like growth factor (HB-EGF) is rapidly induced in MG residing at the injury site and that proHB-EGF ectodomain shedding is necessary for retina regeneration. Remarkably, HB-EGF stimulates the formation of multipotent MG-derived progenitors in the uninjured retina. We show that HB-EGF mediates its effects via an EGFR/MAPK signal transduction cascade that regulates the expression of regeneration-associated genes, like ascl1a and pax6b. We also uncover an HB-EGF/Ascl1a/Notch/hb-egfa signaling loop that helps define the zone of injury-responsive MG. Finally, we show that HB-EGF acts upstream of the Wnt/β-catenin signaling cascade that controls progenitor proliferation. These data provide a link between extracellular signaling and regeneration-associated gene expression in the injured retina and suggest strategies for stimulating retina regeneration in mammals. PMID:22340497

  6. Engineering of PDMS surfaces for use in microsystems for capture and isolation of complex and biomedically important proteins: epidermal growth factor receptor as a model system.

    Science.gov (United States)

    Lowe, Aaron M; Ozer, Byram H; Wiepz, Gregory J; Bertics, Paul J; Abbott, Nicholas L

    2008-08-01

    Elastomers based on poly(dimethylsiloxane) (PDMS) are promising materials for fabrication of a wide range of microanalytical systems due to their mechanical and optical properties and ease of processing. To date, however, quantitative studies that demonstrate reliable and reproducible methods for attachment of binding groups that capture complex receptor proteins of relevance to biomedical applications of PDMS microsystems have not been reported. Herein we describe methods that lead to the reproducible capture of a transmembrane protein, the human epidermal growth factor (EGF) receptor, onto PDMS surfaces presenting covalently immobilized antibodies for EGF receptor, and subsequent isolation of the captured receptor by mechanical transfer of the receptor onto a chemically functionalized surface of a gold film for detection. This result is particularly significant because the physical properties of transmembrane proteins make this class of proteins a difficult one to analyze. We benchmark the performance of antibodies to the human EGF receptor covalently immobilized on PDMS against the performance of the same antibodies physisorbed to conventional surfaces utilized in ELISA assays through the use of EGF receptor that was (32)P-radiolabeled in its autophosphorylation domain. These results reveal that two pan-reactive antibodies for the EGF receptor (clones H11 and 111.6) and one phosphospecific EGF receptor antibody (clone pY1068) capture the receptor on both PDMS and ELISA plates. When using H11 antibody to capture EGF receptor and subsequent treatment with a stripping buffer (NaOH and sodium dodecylsulfate) to isolate the receptor, the signal-to-background obtained using the PDMS surface was 82 : 1, exceeding the signal-to-background measured on the ELISA plate (<48 : 1). We also characterized the isolation of captured EGF receptor by mechanical contact of the PDMS surface with a chemically functionalized gold film. The efficiency of mechanical transfer of the

  7. 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”.

  8. Odor Preference Learning and Memory Modify GluA1 Phosphorylation and GluA1 Distribution in the Neonate Rat Olfactory Bulb: Testing the AMPA Receptor Hypothesis in an Appetitive Learning Model

    Science.gov (United States)

    Cui, Wen; Darby-King, Andrea; Grimes, Matthew T.; Howland, John G.; Wang, Yu Tian; McLean, John H.; Harley, Carolyn W.

    2011-01-01

    An increase in synaptic AMPA receptors is hypothesized to mediate learning and memory. AMPA receptor increases have been reported in aversive learning models, although it is not clear if they are seen with memory maintenance. Here we examine AMPA receptor changes in a cAMP/PKA/CREB-dependent appetitive learning model: odor preference learning in…

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

    Science.gov (United States)

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

    2012-01-01

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

  10. The heparin-binding domain of HB-EGF as an efficient cell-penetrating peptide for drug delivery.

    Science.gov (United States)

    Luo, Zhao; Cao, Xue-Wei; Li, Chen; Wu, Miao-Dan; Yang, Xu-Zhong; Zhao, Jian; Wang, Fu-Jun

    2016-11-01

    Cell-penetrating peptides (CPPs) have been shown to be potential drug carriers for cancer therapy. The inherently low immunogenicity and cytotoxicity of human-derived CPPs make them more suitable for intracellular drug delivery compared to other delivery vehicles. In this work, the protein transduction ability of a novel CPP (termed HBP) derived from the heparin-binding domain of HB-EGF was evaluated. Our data shows, for the first time, that HBP possesses similar properties to typical CPPs and is a potent drug delivery vector for improving the antitumor activity of impermeable MAP30. The intrinsic bioactivities of recombinant MAP30-HBP were well preserved compared to those of free MAP30. Furthermore, HBP conjugated to the C-terminus of MAP30 promoted the cellular uptake of recombinant MAP30-HBP. Moreover, the fusion of HBP to MAP30 gave rise to significantly enhanced cytotoxic effects in all of the tumor cell lines tested. In HeLa cells, this cytotoxicity was mainly caused by the induction of cell apoptosis. Further investigation revealed that HBP enhanced MAP30-induced apoptosis through the activation of the mitochondrial- and death receptor-mediated signaling pathways. In addition, the MAP30-HBP fusion protein caused more HeLa cells to become arrested in S phase compared to MAP30 alone. These results highlight the MAP30-HBP fusion protein as a promising drug candidate for cancer therapy and demonstrate HBP, a novel CPP derived from human HB-EGF, as a new potential vector for antitumor drug delivery. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  11. Thyroid hormone regulation of epidermal growth factor receptor levels in mouse mammary glands

    International Nuclear Information System (INIS)

    Vonderhaar, B.K.; Tang, E.; Lyster, R.R.; Nascimento, M.C.

    1986-01-01

    The specific binding of iodinated epidermal growth factor ([ 125 I]iodo-EGF) to membranes prepared from the mammary glands and spontaneous breast tumors of euthyroid and hypothyroid mice was measured in order to determine whether thyroid hormones regulate the EGF receptor levels in vivo. Membranes from hypothyroid mammary glands of mice at various developmental ages bound 50-65% less EGF than those of age-matched euthyroid controls. Treatment of hypothyroid mice with L-T4 before killing restored binding to the euthyroid control level. Spontaneous breast tumors arising in hypothyroid mice also bound 30-40% less EGF than tumors from euthyroid animals even after in vitro desaturation of the membranes of endogenous growth factors with 3 M MgCl2 treatment. The decrease in binding in hypothyroid membranes was due to a decrease in the number of binding sites, not to a change in affinity of the growth factor for its receptor, as determined by Scatchard analysis of the binding data. Both euthyroid and hypothyroid membranes bound EGF primarily to a single class of high affinity sites [dissociation constant (Kd) = 0.7-1.8 nM]. Euthyroid membranes bound 28.4 +/- (SE) 0.6 fmol/mg protein, whereas hypothyroid membranes bound 15.5 +/- 1.0 fmol/mg protein. These data indicate that EGF receptor levels in normal mammary glands and spontaneous breast tumors in mice are subject to regulation by thyroid status

  12. Tyrosine Phosphorylation of Jak2 in the JH2 Domain Inhibits Cytokine Signaling

    OpenAIRE

    Feener, Edward P.; Rosario, Felicia; Dunn, Sarah L.; Stancheva, Zlatina; Myers, Martin G.

    2004-01-01

    Jak family tyrosine kinases mediate signaling by cytokine receptors to regulate diverse biological processes. Although Jak2 and other Jak kinase family members are phosphorylated on numerous sites during cytokine signaling, the identity and function of most of these sites remains unknown. Using tandem mass spectroscopic analysis of activated Jak2 protein from intact cells, we identified Tyr221 and Tyr570 as novel sites of Jak2 phosphorylation. Phosphorylation of both sites was stimulated by c...

  13. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr

    2015-01-01

    by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were...... also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent....

  14. The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor.

    Science.gov (United States)

    Klippel, A; Escobedo, J A; Fantl, W J; Williams, L T

    1992-01-01

    Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor. Images PMID:1312663

  15. Novel nanofibrous dressings containing rhEGF and Aloe vera for wound healing applications.

    Science.gov (United States)

    Garcia-Orue, Itxaso; Gainza, Garazi; Gutierrez, Franciso Borja; Aguirre, Jose Javier; Evora, Carmen; Pedraz, Jose Luis; Hernandez, Rosa Maria; Delgado, Araceli; Igartua, Manoli

    2017-05-25

    Nanofibrous membranes produced by electrospinning possess a large surface area-to-volume ratio, which mimics the three-dimensional structure of the extracellular matrix. Thus, nanofibrous dressings are a promising alternative for chronic wound healing, since they can replace the natural ECM until it is repaired. Therefore, in this study we have developed a PLGA nanofibrous membrane that contains recombinant human Epidermal Growth Factor (rhEGF) and Aloe vera (AV) extract. Both of them promote wound healing, as EGF is a wound healing mediator and AV stimulates the proliferation and activity of fibroblast. The obtained membranes were composed of uniform and randomly oriented fibers with an average diameter of 356.03±112.05nm, they presented a porosity of 87.92±11.96% and the amount of rhEGF was 9.76±1.75μg/mg. The in vitro viability assay demonstrated that the membranes containing rhEGF and AV improved fibroblast proliferation, revealing the beneficial effect of the combination. Furthermore, these membranes accelerated significantly wound closure and reepithelisation in an in vivo full thickness wound healing assay carried out in db/db mice. Overall, these findings demonstrated the potential of PLGA nanofibers containing rhEGF and AV for the treatment of chronic wounds. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Excess HB-EGF, which promotes VEGF signaling, leads to hydrocephalus

    Science.gov (United States)

    Shim, Joon W.; Sandlund, Johanna; Hameed, Mustafa Q.; Blazer-Yost, Bonnie; Zhou, Feng C.; Klagsbrun, Michael; Madsen, Joseph R.

    2016-01-01

    Heparin binding epidermal growth factor-like growth factor (HB-EGF) is an angiogenic factor mediating radial migration of the developing forebrain, while vascular endothelial growth factor (VEGF) is known to influence rostral migratory stream in rodents. Cell migratory defects have been identified in animal models of hydrocephalus; however, the relationship between HB-EGF and hydrocephalus is unclear. We show that mice overexpressing human HB-EGF with β-galactosidase reporter exhibit an elevated VEGF, localization of β-galactosidase outside the subventricular zone (SVZ), subarachnoid hemorrhage, and ventriculomegaly. In Wistar polycystic kidney rats with hydrocephalus, alteration of migratory trajectory is detected. Furthermore, VEGF infusions into the rats result in ventriculomegaly with an increase of SVZ neuroblast in rostral migratory stream, whereas VEGF ligand inhibition prevents it. Our results support the idea that excess HB-EGF leads to a significant elevation of VEGF and ventricular dilatation. These data suggest a potential pathophysiological mechanism that elevated HB-EGF can elicit VEGF induction and hydrocephalus. PMID:27243144

  17. Tangeretin and its metabolite 4'-hydroxytetramethoxyflavone attenuate EGF-stimulated cell cycle progression in hepatocytes; role of inhibition at the level of mTOR/p70S6K.

    Science.gov (United States)

    Cheng, Z; Surichan, S; Ruparelia, K; Arroo, R; Boarder, M R

    2011-04-01

    The mechanisms by which the dietary compound tangeretin has anticancer effects may include acting as a prodrug, forming an antiproliferative product in cancer cells. Here we show that tangeretin also inhibits cell cycle progression in hepatocytes and investigate the role of its primary metabolite 4'-hydroxy-5,6,7,8-tetramethoxyflavone (4'-OH-TMF) in this effect. We used epidermal growth factor (EGF)-stimulated rat hepatocytes, with [(3)H]-thymidine incorporation into DNA as an index of progression to S-phase of the cell cycle, and Western blots for phospho-proteins involved in the cell signalling cascade. Incubation of tangeretin with microsomes expressing CYP1A, or with hepatocytes, generated a primary product we identified as 4'-OH-TMF. Low micromolar concentrations of tangeretin or 4'-OH-TMF gave a concentration-dependent inhibition of EGF-stimulated progression to S-phase while having little effect on cell viability. To determine whether time for conversion of tangeretin to an active metabolite would enhance the inhibitory effect we used long pre-incubations; this reduced the inhibitory effect, in parallel with a reduction in the concentration of tangeretin. The EGF-stimulation of hepatocyte cell cycle progression requires signalling through Akt/mTOR/p70S6K kinase cascades. The tangeretin metabolite 4'-OH-TMF selectively inhibited S6K phosphorylation in the absence of significant inhi