DOCSLIB.ORG

IL13 Receptor A2 Signaling Requires a Scaffold Protein, FAM120A, to Activate the FAK and PI3K Pathways in Colon Cancer Metastasis Ruben A

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
IL13 Receptor A2 Signaling Requires a Scaffold Protein, FAM120A, to Activate the FAK and PI3K Pathways in Colon Cancer Metastasis Ruben� A Published OnlineFirst April 20, 2015; DOI: 10.1158/0008-5472.CAN-14-3650 Cancer Microenvironment and Immunology Research IL13 Receptor a2 Signaling Requires a Scaffold Protein, FAM120A, to Activate the FAK and PI3K Pathways in Colon Cancer Metastasis Ruben A. Bartolome1, Irene García-Palmero1, Sofía Torres1, María Lopez-Lucendo 2, Irina V. Balyasnikova3, and J. Ignacio Casal1 Abstract IL13 signaling through its receptor IL13Ra2 plays a critical involved in cargo movement along microtubules. IL13Ra2-trig- role in colon cancer invasion and liver metastasis, but the gered activation of the FAK and PI3K/AKT/mTOR pathways was mechanistic features of this process are obscure. In this study, mediated by FAM120A, which also recruited PI3K and func- we identified a scaffold protein, FAM120A (C9ORF10), as a tioned as a scaffold protein to enable phosphorylation and signaling partner in this process. FAM120A was overexpressed activation of PI3K by Src family kinases. FAM120A silencing in human colon cancer cell lines and 55% of human colon abolished IL13-induced cell migration, invasion, and survival. cancer specimens. IL13Ra2-FAM120A coimmunoprecipitation Finally, antibody blockade of IL13Ra2 or FAM120A silencing experiments revealed further signaling network associations precluded liver colonization in nude mice or metastasis. In that could regulate the activity of IL13Ra2, including FAK, SRC, conclusion, we identified FAM120A in the IL13/IL13Ra2signal- PI3K, G-protein–coupled receptors, and TRAIL receptors. In ing pathway as a key mediator of invasion and liver metastasis addition, FAM120A associated with kinesins and motor proteins in colon cancer. Cancer Res; 75(12); 2434–44. Ó2015 AACR. Introduction IL13 binding to IL13Ra2 induced a significant increase in cell adhesion, migration, and invasion capacity of colorectal cancer Interleukin 13 (IL13) has been associated to different pathologic cells (2). Cell signaling was independent of type II IL4 receptor conditions (asthma, autoimmune diseases, ulcerative colitis, and and IL13Ra1 receptor as KM12 cells did not express these others; ref. 1). More recently, we demonstrated that IL13 signaling alternative IL13 receptors on the cell surface (2). Moreover, through IL13 receptor subunit a-2 (IL13Ra2) plays a critical role in silencing of IL13Ra2 increased mice survival and provoked a colon cancer invasion and liver metastasis (2). We described the clear reduction in liver colonization in mouse xenograft models overexpression of IL13Ra2 in 66% of tumor samples from patients (2). Although initially was considered a decoy receptor (11), with colon cancer, which was associated tolatestages ofprogression there are multiple evidences that IL13Ra2 is functional and (metastasis in lymph nodes or liver) and poor outcome of patients induces the activation of several pathways and the transcription with colorectal cancer (2). IL13Ra2 is overexpressed in a variety of factor AP-1, inducing the expression of TGFb (2, 12, 13). human tumor types, such as colon, glioblastoma, renal cell carci- IL13Ra2 cytoplasmic domain is very short, consisting of only noma, pancreatic, melanoma, head and neck, mesothelioma, and 14 amino acids residues, which does not contain any recog- ovarian, where it has been proposed as biomarker and potential nizable motif and makes difficult its interaction with other therapeutic target (2–10). In glioblastoma multiforme, IL13Ra2 proteins. Nevertheless, this receptor participates in signal trans- expression occurs in 75% of tumors and is associated with poor duction, triggering the activation of several signaling proteins, prognosis (3). A similar situation occurs for head and neck squa- such as PI3K and Src kinases (2, 12, 13). Little was known about mous cell carcinoma (8) and ovarian cancer (9). the signaling mechanisms of IL13 through IL13Ra2inmetas- tasis and cancer progression. fi a 1Department of Cellular and Molecular Medicine, Centro de Investiga- Here, we have identi ed the molecular partners of IL13R 2 ciones Biologicas (CIB-CSIC), Madrid, Spain. 2Proteomics Facility, and the mechanisms of signal transduction by using immuno- Centro de Investigaciones Biologicas (CIB-CSIC), Madrid, Spain. 3The precipitation experiments combined with a proteomic Brain Tumor Center, University of Chicago, Chicago, Illinois. approach. We identified the adaptor FAM120A as the scaffold Note: Supplementary data for this article are available at Cancer Research protein necessary for IL13Ra2-mediated signaling. FAM120A, Online (http://cancerres.aacrjournals.org/). also known as c9orf10 or OSSA (oxidative stress-associated Src Corresponding Author: J. Ignacio Casal, Functional Proteomics Laboratory, activator), was required for the activation and recruitment of Centro de Investigaciones Biologicas (CIB-CSIC), Ramiro de Maeztu, 9, 28040 FAK, Src, PI3K, and most of the proteins involved in IL13Ra2 Madrid. Spain. Phone: 34-91-8373112; Fax: 34-91-5360432; E-mail: signaling, providing an overall picture of IL13 signaling in [email protected] colorectal cancer cells and its relevance in liver metastasis. doi: 10.1158/0008-5472.CAN-14-3650 FAM120A and/or IL13Ra2 targeting abolished liver coloniza- Ó2015 American Association for Cancer Research. tion in a mouse model. 2434 Cancer Res; 75(12) June 15, 2015 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst April 20, 2015; DOI: 10.1158/0008-5472.CAN-14-3650 IL13Ra2 Signaling in Colorectal Cancer Metastasis Materials and Methods ions were submitted to collision-induced dissociation in the LTQ using normalized collision energy of 35% and a target value of 104 Cell culture and reagents ions. Dynamic exclusion was enabled with a repeat count of one KM12C and KM12SM human colon cancer cells were obtained and exclusion duration of 30 seconds. Mass spectra were searched from I. Fidler's laboratory (MD Anderson Cancer Center. Hous- using SEQUEST search engine with Proteome Discoverer (Thermo ton, TX). These cell lines were authenticated by short tandem Scientific) against the Uniprot Database. Search parameters repeat analysis. Other cell lines were obtained directly from the included precursor and fragment mass tolerances of 10 p.p.m. ATCC. These cell lines were passaged fewer than 6 months after and 0.8 Da, respectively, a maximum of two missed cleavages purchase for all the experiments. All cell lines were cultured in allowed, a fixed modification of carbamidomethyl cysteine and a DMEM (Gibco-Life Technologies) containing 10% FCS (Sigma- variable modification of methionine oxidation. Identified pep- Aldrich) and antibiotics at 37Cina5%CO-humidified 2 tides were validated using Percolator algorithm with a q-value atmosphere. threshold 0.01. IL13 was used at 10 ng/mL and purchased from PeproTech. PP2 (used at 3 mmol/L), PP3 (3 mmol/L), and UO126 (5 mmol/L) IHC analysis inhibitors were from Calbiochem. LY294002 (25 mmol/L) was Paraffin samples were obtained from 119 patients diagnosed from Sigma-Aldrich and FAK inhibitor 14 (10 mmol/L) from Santa and treated of colorectal adenocarcinoma between 2001 and Cruz Biotechnology. All antibodies used in this article are listed in 2014 in Fundacion Jimenez Díaz (Madrid, Spain). Clinicopatho- Supplementary Table S1. logical data are shown in Supplementary Table S2. Each sample was deparaffinated for antigen retrieval using citrate sodium siRNAs transfections buffer at pH 6.0 for 20 minutes and subsequent incubation with For transient transfections, siRNAs targeting specifically the primary antibody against FAM120A. The reaction was FAM120A (SASI-Hs01-00149752, Sigma-Aldrich) or control revealed using DAB as chromogen and hematoxylin for counter- siRNAs were transfected with JetPrime (Polyplus Transfection) staining. In all cases, sections from normal colonic mucosa distant according to the manufacturer's instructions. from the tumor site were used as negative controls. Western blot analysis and immunoprecipitation assays Wound healing Cells were detached, washed, and lysed with protease and Cells were cultured to confluence in Matrigel-coated plates phosphatase inhibitors in lysis buffer (1% Igepal, 100 mmol/L (0.4 mg/mm2). A 1-mm wide wound was done across the mono- NaCl, 2 mmol/L MgCl , 10% Glycerol in 50 mmol/L Tris-HCl). 2 layer. Cells were incubated in serum-free medium containing Protein extracts were separated in SDS-PAGE, transferred to nitro- IL13, inhibitors, and antibodies. Pictures were taken either imme- cellulose membranes, which were incubated with primary anti- diately (0 hour) or after 48 hours in culture at 37 C after the injury. bodies (Supplementary Table S1) followed by incubation with Migration was quantified as a percentage of wound closure. either HRP-anti-mouse IgG (Thermo Scientific) or HRP-anti-rab- bit IgG (Sigma-Aldrich). Specific reactive proteins were visualized with SuperSignal West Pico Chemiluminescent Substrate Invasion assays  4 (Thermo Scientific). For Matrigel invasion assays, 6 10 KM12C or KM12SM cells For immunoprecipitation, cells were lysed and 500 mg of cell were resuspended in invasion medium (serum-free DMEM con- m fi lysate were incubated with the indicated antibodies. The immu- taining 0.4% BSA) and loaded onto 8 m pore-size lters coated m nocomplex was captured by adding 100 mL of Protein G-sephar- with 35 L of Matrigel (1:3 dilution; BD Biosciences) in Transwell ose beads (Sigma-Aldrich). After washing, samples were resus- plates (Costar) in presence of inhibitors or antibodies. The lower  compartment
Load more

Recommended publications
  • The Wnt Pathway Scaffold Protein Axin Promotes Signaling Specificity by Suppressing Competing Kinase Reactions
    bioRxiv preprint doi: https://doi.org/10.1101/768242; this version posted September 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. The Wnt pathway scaffold protein Axin promotes signaling specificity by suppressing competing kinase reactions Maire Gavagan1,2, Erin Fagnan1,2, Elizabeth B. Speltz1, and Jesse G. Zalatan1,* 1Department of Chemistry, University of Washington, Seattle, WA 98195, USA 2These authors contributed equally to this work *Correspondence: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/768242; this version posted September 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract GSK3β is a multifunctional kinase that phosphorylates β-catenin in the Wnt signaling network and also acts on other protein targets in response to distinct cellular signals. To test the long-standing hypothesis that the scaffold protein Axin specifically accelerates β-catenin phosphorylation, we measured GSK3β reaction rates with multiple substrates in a minimal, biochemically-reconstituted system. We observed an unexpectedly small, ~2-fold Axin-mediated rate increase for the β-catenin reaction. The much larger effects reported previously may have arisen because Axin can rescue GSK3β from an inactive state that occurs only under highly specific conditions.
    [Show full text]
  • CK1 Is Required for a Mitotic Checkpoint That Delays Cytokinesis
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Current Biology 23, 1920–1926, October 7, 2013 ª2013 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2013.07.077 Report CK1 Is Required for a Mitotic Checkpoint that Delays Cytokinesis Alyssa E. Johnson,1 Jun-Song Chen,1 isoforms were detected, which collapsed into a discrete ladder and Kathleen L. Gould1,* upon phosphatase treatment (Figure 1A, lanes 1 and 2). These 1Department of Cell and Developmental Biology, Vanderbilt bands are ubiquitinated isoforms because they collapse into a University School of Medicine, Nashville, TN 37232, USA single band in the absence of dma1+ (Figure 1A, lane 4) and Dma1 is required for Sid4 ubiquitination [6]. In dma1D cells, a single slower-migrating form of Sid4 was detected, which Summary was collapsed by phosphatase treatment, indicating that Sid4 is phosphorylated in vivo (Figure 1A, lanes 3 and 4). In vivo Failure to accurately partition genetic material during cell radiolabeling experiments validated Sid4 as a phosphoprotein division causes aneuploidy and drives tumorigenesis [1]. and revealed that Sid4 is phosphorylated on serines and thre- Cell-cycle checkpoints safeguard cells from such catastro- onines (see Figures S1A–S1C available online). The constitu- phes by impeding cell-cycle progression when mistakes tive presence of an unmodified Sid4 isoform indicates that arise. FHA-RING E3 ligases, including human RNF8 [2] and only a subpopulation of Sid4 is modified (Figure 1A). Collec- CHFR [3] and fission yeast Dma1 [4], relay checkpoint signals tively, these data indicate that Sid4 is ubiquitinated and phos- by binding phosphorylated proteins via their FHA domains phorylated in vivo.
    [Show full text]
  • Impact of Digestive Inflammatory Environment and Genipin
    International Journal of Molecular Sciences Article Impact of Digestive Inflammatory Environment and Genipin Crosslinking on Immunomodulatory Capacity of Injectable Musculoskeletal Tissue Scaffold Colin Shortridge 1, Ehsan Akbari Fakhrabadi 2 , Leah M. Wuescher 3 , Randall G. Worth 3, Matthew W. Liberatore 2 and Eda Yildirim-Ayan 1,4,* 1 Department of Bioengineering, College of Engineering, University of Toledo, Toledo, OH 43606, USA; [email protected] 2 Department of Chemical Engineering, College of Engineering, University of Toledo, Toledo, OH 43606, USA; [email protected] (E.A.F.); [email protected] (M.W.L.) 3 Department of Medical Microbiology and Immunology, University of Toledo, Toledo, OH 43614, USA; [email protected] (L.M.W.); [email protected] (R.G.W.) 4 Department of Orthopaedic Surgery, University of Toledo Medical Center, Toledo, OH 43614, USA * Correspondence: [email protected]; Tel.: +1-419-530-8257; Fax: +1-419-530-8030 Abstract: The paracrine and autocrine processes of the host response play an integral role in the success of scaffold-based tissue regeneration. Recently, the immunomodulatory scaffolds have received huge attention for modulating inflammation around the host tissue through releasing anti- inflammatory cytokine. However, controlling the inflammation and providing a sustained release of anti-inflammatory cytokine from the scaffold in the digestive inflammatory environment are predicated upon a comprehensive understanding of three fundamental questions. (1) How does the Citation: Shortridge, C.; Akbari release rate of cytokine from the scaffold change in the digestive inflammatory environment? (2) Fakhrabadi, E.; Wuescher, L.M.; Can we prevent the premature scaffold degradation and burst release of the loaded cytokine in the Worth, R.G.; Liberatore, M.W.; digestive inflammatory environment? (3) How does the scaffold degradation prevention technique Yildirim-Ayan, E.
    [Show full text]
  • Crosstalk Between Casein Kinase II and Ste20-Related Kinase Nak1
    University of Massachusetts Medical School eScholarship@UMMS GSBS Student Publications Graduate School of Biomedical Sciences 2013-03-05 Crosstalk between casein kinase II and Ste20-related kinase Nak1 Lubos Cipak University of Vienna Et al. Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_sp Part of the Biochemistry Commons, Cell Biology Commons, Cellular and Molecular Physiology Commons, and the Molecular Biology Commons Repository Citation Cipak L, Gupta S, Rajovic I, Jin Q, Anrather D, Ammerer G, McCollum D, Gregan J. (2013). Crosstalk between casein kinase II and Ste20-related kinase Nak1. GSBS Student Publications. https://doi.org/ 10.4161/cc.24095. Retrieved from https://escholarship.umassmed.edu/gsbs_sp/1850 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Student Publications by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. EXTRA VIEW Cell Cycle 12:6, 884–888; March 15, 2013; © 2013 Landes Bioscience Crosstalk between casein kinase II and Ste20-related kinase Nak1 Lubos Cipak,1,2,† Sneha Gupta,3,† Iva Rajovic,1 Quan-Wen Jin,3,4 Dorothea Anrather,1 Gustav Ammerer,1 Dannel McCollum3,* and Juraj Gregan1,5,* 1Max F. Perutz Laboratories; Department of Chromosome Biology; University of Vienna; Vienna, Austria; 2Cancer Research Institute; Slovak Academy of Sciences; Bratislava, Slovak Republic; 3Department of Microbiology and Physiological Systems and Program in Cell Dynamics; University of Massachusetts Medical School; Worcester, MA USA; 4Department of Biological Medicine; School of Life Sciences; Xiamen University; Xiamen, China; 5Department of Genetics; Comenius University; Bratislava, Slovak Republic †These authors contributed equally to this work.
    [Show full text]
  • Electrical Synaptic Transmission Requires a Postsynaptic Scaffolding Protein
    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.03.410696; this version posted December 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. TITLE: Electrical synaptic transmission requires a postsynaptic scaffolding protein Abagael M. Lasseigne1*, Fabio A. Echeverry2*, Sundas Ijaz2*, Jennifer Carlisle Michel1*, E. Anne Martin1, Audrey J. Marsh1, Elisa Trujillo1, Kurt C. Marsden3, Alberto E. Pereda2#, Adam C. Miller1#@ * denotes co-first author # denotes co-corresponding author @ denotes lead contact Affiliations: 1 Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA 2 Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA 3 Department of Biological Sciences, NC State University, Raleigh, NC 27695, USA Correspondence: [email protected] [email protected] Keywords: gap junction; connexin; ZO1 ZO-1; synaptic development; electrical coupling 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.03.410696; this version posted December 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. SUMMARY Electrical synaptic transmission relies on neuronal gap junctions containing channels constructed by Connexins. While at chemical synapses neurotransmitter-gated ion channels are critically supported by scaffolding proteins, it is unknown if channels at electrical synapses require similar scaffold support.
    [Show full text]
  • Head Formation Requires Dishevelled Degradation That Is Mediated By
    © 2018. Published by The Company of Biologists Ltd | Development (2018) 145, dev143107. doi:10.1242/dev.143107 RESEARCH ARTICLE Head formation requires Dishevelled degradation that is mediated by March2 in concert with Dapper1 Hyeyoon Lee*,**, Seong-Moon Cheong‡,**, Wonhee Han, Youngmu Koo, Saet-Byeol Jo, Gun-Sik Cho§, Jae-Seong Yang¶, Sanguk Kim and Jin-Kwan Han‡‡ ABSTRACT phosphorylate LRP6 (Bilic et al., 2007). Inhibition of GSK3β by Dishevelled (Dvl/Dsh) is a key scaffold protein that propagates Wnt the phospho-LRP6-containing activated receptor complex protects β signaling essential for embryogenesis and homeostasis. However, -catenin from proteasomal degradation (Logan and Nusse, 2004; β whether the antagonism of Wnt signaling that is necessary for MacDonald et al., 2009). Consequently, -catenin accumulates and vertebrate head formation can be achieved through regulation of Dsh interacts with T-cell factor/lymphoid-enhancer factor (TCF/LEF) in protein stability is unclear. Here, we show that membrane-associated the nucleus, thereby initiating transcription. RING-CH2 (March2), a RING-type E3 ubiquitin ligase, antagonizes During vertebrate development, the canonical Wnt pathway plays Wnt signaling by regulating the turnover of Dsh protein via ubiquitin- a crucial role in head formation (Glinka et al., 1997; Kiecker and mediated lysosomal degradation in the prospective head region of Niehrs, 2001; Yamaguchi, 2001). Proper head formation requires Xenopus. We further found that March2 acquires regional and inhibition of the canonical
    [Show full text]
  • INVESTIGATION of the ROLE of the SCAFFOLD PROTEIN SHC in ERK SIGNALING Kin Man Suen
    The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 5-2015 INVESTIGATION OF THE ROLE OF THE SCAFFOLD PROTEIN SHC IN ERK SIGNALING kin man suen Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Biochemistry, Biophysics, and Structural Biology Commons, and the Medicine and Health Sciences Commons Recommended Citation suen, kin man, "INVESTIGATION OF THE ROLE OF THE SCAFFOLD PROTEIN SHC IN ERK SIGNALING" (2015). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 549. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/549 This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. INVESTIGATION OF THE ROLE OF THE SCAFFOLD PROTEIN SHC IN ERK SIGNALING by Kin Man Suen, B.Sc. APPROVED: ______________________________ Dr. John E. Ladbury
    [Show full text]
  • The Regulatory Role of Key Metabolites in the Control of Cell Signaling
    biomolecules Review The Regulatory Role of Key Metabolites in the Control of Cell Signaling Riccardo Milanesi, Paola Coccetti * and Farida Tripodi Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126 Milan, Italy; [email protected] (R.M.); [email protected] (F.T.) * Correspondence: [email protected]; Tel.: +39-02-6448-3521 Received: 8 May 2020; Accepted: 3 June 2020; Published: 5 June 2020 Abstract: Robust biological systems are able to adapt to internal and environmental perturbations. This is ensured by a thick crosstalk between metabolism and signal transduction pathways, through which cell cycle progression, cell metabolism and growth are coordinated. Although several reports describe the control of cell signaling on metabolism (mainly through transcriptional regulation and post-translational modifications), much fewer information is available on the role of metabolism in the regulation of signal transduction. Protein-metabolite interactions (PMIs) result in the modification of the protein activity due to a conformational change associated with the binding of a small molecule. An increasing amount of evidences highlight the role of metabolites of the central metabolism in the control of the activity of key signaling proteins in different eukaryotic systems. Here we review the known PMIs between primary metabolites and proteins, through which metabolism affects signal transduction pathways controlled by the conserved kinases Snf1/AMPK, Ras/PKA and TORC1. Interestingly, PMIs influence also the mitochondrial retrograde response (RTG) and calcium signaling, clearly demonstrating that the range of this phenomenon is not limited to signaling pathways related to metabolism. Keywords: Snf1/AMPK/SnRK1; Ras/PKA; TORC1; RTG; calcium; glucose; glycolysis; TCA; amino acids; protein-metabolite interaction 1.
    [Show full text]
  • Parkin Represses 6-Hydroxydopamine-Induced Apoptosis Via Stabilizing Scaffold Protein P62 in PC12 Cells
    npg Acta Pharmacologica Sinica (2015) 36: 1300-1307 © 2015 CPS and SIMM All rights reserved 1671-4083/15 www.nature.com/aps Original Article Parkin represses 6-hydroxydopamine-induced apoptosis via stabilizing scaffold protein p62 in PC12 cells Xiao-ou HOU1, Jian-min SI2, Hai-gang REN1, Dong CHEN1, Hong-feng WANG1, Zheng YING1, Qing-song HU1, Feng GAO1, *, Guang-hui WANG1, * 1Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China; 2Laboratory of Molecular Neuropathology, Key Laboratory of Brain Function and Diseases and School of Life Sciences, University of Science & Technology of China, Chinese Academy of Sciences, Hefei 230027, China Aim: Parkin has been shown to exert protective effects against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in different models of Parkinson disease. In the present study we investigated the molecular mechanisms underlying the neuroprotective action of parkin in vitro. Methods: HEK293, HeLa and PC12 cells were transfected with parkin, parkin mutants, p62 or si-p62. Protein expression and ubiquitination were assessed using immunoblot analysis. Immunoprecipitation assay was performed to identify the interaction between parkin and scaffold protein p62. PC12 and SH-SY5Y cells were treated with 6-OHDA (200 μmol/L), and cell apoptosis was detected using PI and Hoechst staining. Results: In HEK293 cells co-transfected with parkin and p62, parkin was co-immunoprecipitated with p62, and parkin overexpression increased p62 protein levels. In parkin-deficient HeLa cells, transfection with wild-type pakin, but not with ligase activity-deficient pakin mutants, significantly increased p62 levels, suggesting that parkin stabilized p62 through its E3 ligase activity.
    [Show full text]
  • The Scaffold Protein IB1/JIP-1 Is a Critical Mediator of Cytokine-Induced Apoptosis in Pancreatic Β Cells
    Research Article 1463 The scaffold protein IB1/JIP-1 is a critical mediator of cytokine-induced apoptosis in pancreatic β cells Jacques-Antoine Haefliger1,*,‡, Thomas Tawadros1,*, Laure Meylan1, Sabine Le Gurun1, Marc-Estienne Roehrich1, David Martin1, Bernard Thorens2 and Gérard Waeber1 1Department of Internal Medicine, CHUV-1011 Lausanne, Switzerland 2Institute of Pharmacology and Toxicology, University Hospital, CHUV-1011 Lausanne, Switzerland *These authors contributed equally to this work ‡Author for correspondence (e-mail: jhaefl[email protected]) Accepted 7 January 2003 Journal of Cell Science 116, 1463-1469 © 2003 The Company of Biologists Ltd doi:10.1242/jcs.00356 Summary In insulin-secreting cells, cytokines activate the c-Jun N- cells by overproducing IB1/JIP-1 and this effect was terminal kinase (JNK), which contributes to a cell signaling associated with inhibition of caspase-3 cleavage. towards apoptosis. The JNK activation requires the Conversely, reducing IB1/JIP-1 content in INS-1 cells and presence of the murine scaffold protein JNK-interacting isolated pancreatic islets induced a robust increase in basal protein 1 (JIP-1) or human Islet-brain 1(IB1), which and cytokine-stimulated apoptosis. In heterozygous mice organizes MLK3, MKK7 and JNK for proper signaling carrying a selective disruption of the IB1/JIP-1 gene, the specificity. Here, we used adenovirus-mediated gene reduction in IB1/JIP-1 content in happloinsufficient transfer to modulate IB1/JIP-1 cellular content in order to isolated pancreatic islets was associated with an increased investigate the contribution of IB1/JIP-1 to β-cell survival. JNK activity and basal apoptosis. These data demonstrate Exposure of the insulin-producing cell line INS-1 or that modulation of the IB1-JIP-1 content in β cells is a isolated rat pancreatic islets to cytokines (interferon-γ, crucial regulator of JNK signaling pathway and of tumor necrosis factor-α and interleukin-1β) induced a cytokine-induced apoptosis.
    [Show full text]
  • The Protein Kinase CK2 Facilitates Repair of Chromosomal DNA Single-Strand Breaks
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Cell, Vol. 117, 17–28, April 2, 2004, Copyright 2004 by Cell Press The Protein Kinase CK2 Facilitates Repair of Chromosomal DNA Single-Strand Breaks Joanna I. Loizou,1 Sherif F. El-Khamisy,1 sion is itself deregulated in most tumors, and expression Anastasia Zlatanou,1 David J. Moore,5 of a CK2␣ promotes cancer in transgenic mouse models Douglas W. Chan,2 Jun Qin,2 (Kelliher et al., 1996; Litchfield, 2003; Seldin and Leder, Stefania Sarno,3,4 Flavio Meggio,3 1995). CK2 is also implicated in the response of mamma- Lorenzo A. Pinna,3,4 and Keith W. Caldecott1,* lian cells to a variety of stresses, including UV, heat 1Genome Damage and Stability Centre shock, inflammation, and damage to the mitotic spindle University of Sussex (Ahmed et al., 2002; Keller et al., 2001; Miyata et al., Science Park Road 1997). Some of the roles of CK2 in stress response ap- Falmer, Brighton BN1 9RQ pear to involve promoting the transcriptional activity of United Kingdom p53 by phosphorylation of Ser 392 (Hupp et al., 1992; 2 Baylor College of Medicine Keller et al., 2001; Keller and Lu, 2002; Meek et al., 1990). Houston, Texas 77030 In addition, CK2 can inhibit apoptosis by promoting the 3 Universita` di Padova antiapoptotic activity of the apoptosis repressor with Colombo 3 caspase recruitment domain (ARC) and by inhibiting 35121 Padova caspase-mediated cleavage of key apoptotic regulators 4 Venetian Institute for Molecular Medicine such as Bid (Desagher et al., 2001; Li et al., 2002b).
    [Show full text]
  • Integrin Signaling in Cell Adhesion and Mechanotransduction: Regulation of PI3K, AKT, And
    To my parents Und für Gerda, Margarete, Ida und Maria Everything has been said already, but not yet by everyone. Karl Valentin List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Zeller1, K.S., Idevall-Hagren1, O., Stefansson1, A., Velling, T., Jackson, S.P., Downward, J., Tengholm, A., Johansson, S. (2010) PI3-kinase p110α mediates β1 integrin-induced AKT activation and membrane protrusion during cell attachment and initial spreading. Cellular Signalling 22:1838–1848 1Equal contribution II Riaz, A., Zeller, K.S., Johansson, S. (2012) Receptor-specific mechanisms regulate phosphorylation of AKT at Ser473: Role of RICTOR in β1 integrin-mediated cell survival. PLoS ONE 7(2): e32081 III Zeller, K.S., Riaz, A., Sarve, H., Johansson, S. (2012) The role of mechanical force and ROS in integrin-dependent signals. Manuscript Reprints were made with permission from the respective publishers. The cover picture shows fibroblast-like BJ hTERT cells. Contents Introduction ...................................................................................................11 Background ...................................................................................................13 Integrin structure and activation...............................................................13 Integrin activating proteins.......................................................................15 Integrin signaling......................................................................................17
    [Show full text]