Novel Regulation of Mtor Complex 1 Signaling by Site-Specific Mtor Phosphorylation

Total Page:16

File Type:pdf, Size:1020Kb

Novel Regulation of Mtor Complex 1 Signaling by Site-Specific Mtor Phosphorylation Novel Regulation of mTOR Complex 1 Signaling by Site-Specific mTOR Phosphorylation by Bilgen Ekim Üstünel A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cell and Developmental Biology) in The University of Michigan 2012 Doctoral Committee: Assistant Professor Diane C. Fingar, Chair Associate Professor Billy Tsai Associate Professor Anne B. Vojtek Assistant Professor Patrick J. Hu Assistant Professor Ken Inoki “Our true mentor in life is science.” (“Hayatta en hakiki mürşit ilimdir.”) Mustafa Kemal Atatürk, the founder of Turkish Republic © Bilgen Ekim Üstünel 2012 Acknowledgements This thesis would not have been possible without the enormous support and encouragement of my Ph.D. advisor Diane C. Fingar. I am sincerely thankful for her research insight and guidance during my Ph.D. training. I would like to express my great appreciation to Billy Tsai, Anne B. Vojtek, Ken Inoki, and Patrick J. Hu for serving on my thesis committee, whose advice and help have been valuable. I would like to thank all members of the Fingar, Tsai, and Verhey labs for the discussion in our group meetings. I also would like to thank the CDB administrative staff, especillay Kristen Hug, for their help. I thank Ed Feener for performing the liquid chromatography tandem mass spectrometry analysis to identify novel phosphorylation sites on mTOR and Steve Riddle for performing the in vitro kinome screen to identify candidate kinases for mTOR S2159 phosphorylation site. I thank Brian Magnuson, Hugo A. Acosta-Jaquez, and Jennifer A. Keller for contributing to my first-author paper published in Molecular and Cellular Biology Journal in 2011. I also thank Diane C. Fingar for generating data presented in Figure 2.2 Part D. I cannot finish without saying how grateful I am with my family; my father Halil İbrahim Ekim, my mother Meral Ekim, my sister Çiğdem Ekim, and most importantly my husband Eser Üstünel for their unconditional love and support. ii Table of Contents Acknowledgements .............................................................................................. ii List of Figures .......................................................................................................iv List of Abbreviations ............................................................................................. v Abstract ................................................................................................................ x Chapter .................................................................................................................. 1. Introduction ................................................................................................... 1 2. mTOR Kinase Domain Phosphorylation Promotes mTORC1 Signaling, Cell Growth, and Cell Cycle Progression .................................................... 38 3. Novel Regulation of mTORC1 by TBK1 and IKKε ...................................... 76 4. Conclusion ................................................................................................ 107 References ....................................................................................................... 113 iii List of Figures Figure 1.1 mTOR domain structure and phosphorylation sites 35 1.2 Intra- and extra-cellular signals regulate mTORC1 signaling 36 1.3 S6K1 isoforms, domain structure, and phosphorylation sites 37 1.4 4E-BP1 domain structure and phosphorylation sites 37 2.1 Identification of S2159 and T2164 as novel sites of mTOR phosphorylation 62 2.2 mTOR S2159/T2164 phosphorylation promotes mTORC1 signaling to S6K1 64 2.3 mTOR S2159/T2164 phosphorylation increases the sensitivity and duration of insulin-stimulated mTORC1 signaling 66 2.4 Phosphorylation of mTOR S2159/T2164 promotes mTORC1 signaling to 4E-BP1 68 2.5 mTOR S2159/T2164 phosphorylation weakens the mTOR-raptor and raptor-PRAS40 interactions and augments mTORC1 intrinsic kinase activity 69 2.6 mTOR S2159/T2164 phosphorylation promotes mTORC1-mediated cell growth and G1-phase cell cycle progression 71 2.7 mTOR-S2159D/T2164E (DE) but not S2159A/T2164A (AA) promotes cell growth to an increased cell size during a cell cycle block 73 2.8 Model 75 3.1 In vitro kinome screen to identify potential mTOR S2159 kinases 98 3.2 Recombinant TBK1 and IKKε phosphorylate mTOR S2159 in vitro 100 3.3 TBK1/IKKε and mTOR coimmunoprecipitate 102 3.4 TBK1-/- and IKKε-/- MEFs show reduced mTOR S2159 phosphorylation 103 3.5 Overexpression of TBK1 and IKKε increases mTOR S2159 phosphorylation and S2481 autophosphorylation in intact cells 104 3.6 Activation of TLR3 and TLR4 promotes mTOR signaling in RAW264.7 murine macrophages 106 iv List of Abbreviations 4E-BP1 Eukaryotic translation initiation factor 4E binding protein 5’TOP 5’track of polypyrimidines m7GTP 7-Methylguanosine 5’-triphosphate AGC Protein kinase A, Protein kinase G and Protein kinase C AMP Adenosine monophosphate AMPK Adenosine monophosphate activated kinase AP-1 Activator protein 1 ATG Autophagy-related protein ATM Ataxia telangiectasia mutated ATR Ataxia telangiectasia and Rad3-related protein CBP CREB binding protein CD Cluster of differentiation Cdc Cell division control CNS Central nervous system Deptor DEP domain-containing mTOR-interacting protein DNA-PK DNA dependent protein kinase dsRNA Double stranded RNA eEF2 Eukaryotic elongation factor 2 eEF2K Eukaryotic elongation factor 2 kinase EGF Epidermal growth factor EGFR Epidermal growth factor receptor eIF Eukaryotic translation initiation factor EMT Epithelial mesenchymal transition ERK Extracellular signal-regulated kinase v FAK Focal adhesion kinase FDA Food and Drug Administration FIP200 FAK family-interacting protein of 200 kDa FKBP12 FK506-binding protein 12-kDa FRAP The FKBP-12-rapamycin associated protein FRB FKBP12-rapamcyin binding GAP GTPase activating protein GEF GTP exchange factor Glut4 Glucose transporter type 4 GSK3β Glycogen synthase kinase 3 beta GTP Guanosine 5’ triphosphate HEAT Huntingtin, elongation factor 3, protein phosphatase 2A (PP2A), and the yeast PI3-kinase TOR1 HIF-1α Hypoxia-inducible factor 1-alpha HM Hydrophobic motif hVPS34 Mammalian vacuolar protein sorting 34 homologue IB Immunoblot IGF Insulin-like growth factor IκBα I-kappa-B-alpha IKK Inhibitor of nuclear factor-kappa B- kinase ILK Integrin-linked kinase IP Immunoprecipitate IRS Insulin receptor substrate IVK In vitro kinase JAK Janus kinase KOG1 Kontroller of growth protein 1 LKB1 Liver kinase B1 LPS Lipopolysaccharide LRL Leucine rich repeat MAP4K3 MAP kinase kinase kinase kinase 3 MAPK Mitogen-activated protein (MAP) kinase vi MAPKAPK-2 MAP kinase-activated protein kinase 2 MBH Mediobasal hypothalamus MEF Mouse embryonic fibroblast MEK MAPK/ERK kinase mLST8 mTOR associated lethal with Sec 13 8/G protein β subunit-like MP1 MEK-binding partner 1 mRNA Messenger RNA mSin1 Mammalian stress-activated protein kinase interacting protein mTOR Mammalian target of rapamycin mTORC mTOR complex MyD88 Myeloid differentiation primary response protein NDRG1 N-Myc downregulated gene 1 NEMO NF-kappa-B essential modulator NF-1 Neurofibromatosis-related protein NF-kB Nuclear factor-kappa B NLS Nuclear localization signal PA Phosphatidic acid PAK1 p21-activated kinase 1 PAMP Pathogen associated molecular patterns pDC Plasmacytoid dendritic cell PDCD4 Programmed cell death protein 4 PDCD4 PDGF Platelet derived growth factor PDK1 Phosphoinositide-dependent kinase-1 PGC1α Peroxisome-proliferator-activated receptor gamma coactivator-1 alpha PI3K Phosphatidylinositol 3 kinase PIKK Phosphoinositide kinase-related kinase PIP3 Phosphatidylinositol-3,4,5-trisphosphate PKCα Protein kinase c alpha isoform PLD Phospholipase D vii Pol DNA polymerase Poly(I:C) Polyinosinic:polycytidylic acid PPARγ Peroxisome proliferator-activated receptor gamma PRAS40 Proline rich Akt substrate of 40 KDa P-Rex1 Phosphatidylinositol-3,4,5-trisphosphate -dependent Rac exchange factor PRR5/Protor Proline-rich protein 5 PTEN Phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase Rac 1 Ras-related C3 botulinum toxin substrate 1 RAFT1 Rapamycin and FKBP12 target RAPT Rapamycin target Raptor Regulatory associated protein of mTOR Ras Rat sarcoma RCC Renal cell carcinoma rDNA Ribosomal DNA Redd1 Regulated in development and DNA damage response Rheb Ras homolog enriched in brain Rictor Rapamycin-insensitive companion of mTOR RNAi RNA interference ROS Reactive oxygen species RR Rapamycin resistant rRNA Ribosomal RNA S6K S6 kinase SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEP Sirolimus effector protein SGK1 Serum/Glucocorticoid Regulated Kinase 1 SH3 SRC Homology 3 Domain SKAR S6K1 Aly/REF-like target SLC7A5/SLC3A2 Solute carrier family 7, member 5/solute carrier family 3, member 2 viii SREBP Sterol response element binding protein STAT3 Signal transducer and activator of transcription 3 Tssk3 Testis-specific serine/threonine-protein kinase 3 TBK1 TANK-binding kinase 1 TIF Transcription initiation factor TIR Toll/interleukin-1 receptor domain TM Turn motif TNFα Tumor necrosis factor alpha TOS TOR signaling TRAM TRIF-related adaptor molecule tRNA Transfer RNA TRIF Toll/IL-1 Receptor Domain-Containing Adaptor Inducing IFN-β TSC Tuberous sclerosis complex TTRAP Trafficking protein particle UBF Upstream-binding factor ULK Unc-51-like kinase UPR Unfolded protein response VEGF Vascular endothelial growth factor YY1 Yin-yang 1 ix Abstract Aberrant regulation of the kinase mTOR (mammalian target of rapamycin)
Recommended publications
  • Supplemental Information to Mammadova-Bach Et Al., “Laminin Α1 Orchestrates VEGFA Functions in the Ecosystem of Colorectal Carcinogenesis”
    Supplemental information to Mammadova-Bach et al., “Laminin α1 orchestrates VEGFA functions in the ecosystem of colorectal carcinogenesis” Supplemental material and methods Cloning of the villin-LMα1 vector The plasmid pBS-villin-promoter containing the 3.5 Kb of the murine villin promoter, the first non coding exon, 5.5 kb of the first intron and 15 nucleotides of the second villin exon, was generated by S. Robine (Institut Curie, Paris, France). The EcoRI site in the multi cloning site was destroyed by fill in ligation with T4 polymerase according to the manufacturer`s instructions (New England Biolabs, Ozyme, Saint Quentin en Yvelines, France). Site directed mutagenesis (GeneEditor in vitro Site-Directed Mutagenesis system, Promega, Charbonnières-les-Bains, France) was then used to introduce a BsiWI site before the start codon of the villin coding sequence using the 5’ phosphorylated primer: 5’CCTTCTCCTCTAGGCTCGCGTACGATGACGTCGGACTTGCGG3’. A double strand annealed oligonucleotide, 5’GGCCGGACGCGTGAATTCGTCGACGC3’ and 5’GGCCGCGTCGACGAATTCACGC GTCC3’ containing restriction site for MluI, EcoRI and SalI were inserted in the NotI site (present in the multi cloning site), generating the plasmid pBS-villin-promoter-MES. The SV40 polyA region of the pEGFP plasmid (Clontech, Ozyme, Saint Quentin Yvelines, France) was amplified by PCR using primers 5’GGCGCCTCTAGATCATAATCAGCCATA3’ and 5’GGCGCCCTTAAGATACATTGATGAGTT3’ before subcloning into the pGEMTeasy vector (Promega, Charbonnières-les-Bains, France). After EcoRI digestion, the SV40 polyA fragment was purified with the NucleoSpin Extract II kit (Machery-Nagel, Hoerdt, France) and then subcloned into the EcoRI site of the plasmid pBS-villin-promoter-MES. Site directed mutagenesis was used to introduce a BsiWI site (5’ phosphorylated AGCGCAGGGAGCGGCGGCCGTACGATGCGCGGCAGCGGCACG3’) before the initiation codon and a MluI site (5’ phosphorylated 1 CCCGGGCCTGAGCCCTAAACGCGTGCCAGCCTCTGCCCTTGG3’) after the stop codon in the full length cDNA coding for the mouse LMα1 in the pCIS vector (kindly provided by P.
    [Show full text]
  • ROS Production Induced by BRAF Inhibitor Treatment Rewires
    Cesi et al. Molecular Cancer (2017) 16:102 DOI 10.1186/s12943-017-0667-y RESEARCH Open Access ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells Giulia Cesi, Geoffroy Walbrecq, Andreas Zimmer, Stephanie Kreis*† and Claude Haan† Abstract Background: Most melanoma patients with BRAFV600E positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant BRAFV600E has been shown to affect the metabolism. Methods: Time course experiments and a series of western blots were performed in a panel of BRAFV600E and BRAFWT/ NRASmut human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested. Results: We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in BRAFV600E and in BRAFWT/NRASmut harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation.
    [Show full text]
  • PDK1 Acquires PDK2 Activity in the Presence of a Synthetic Peptide
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Research Paper 393 PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2 Anudharan Balendran*†, Antonio Casamayor*†, Maria Deak†, Andrew Paterson†, Piers Gaffney‡, Richard Currie§, C. Peter Downes§ and Dario R. Alessi† Background: Protein kinase B (PKB) is activated by phosphorylation of Thr308 Addresses: *MRC Protein Phosphorylation Unit, and of Ser473. Thr308 is phosphorylated by the 3-phosphoinositide-dependent Department of Biochemistry, University of Dundee, ‡ protein kinase-1 (PDK1) but the identity of the kinase that phosphorylates Dundee DD1 5EH, UK. Ludwig Institute of Cancer Research, London W1P 8BY, UK. §Department of Ser473 (provisionally termed PDK2) is unknown. Biochemistry, University of Dundee, Dundee DD1 5EH, UK. Results: The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF). PIF is Correspondence: Dario R. Alessi E-mail: [email protected] situated carboxy-terminal to the kinase domain of PRK2, and contains a consensus motif for phosphorylation by PDK2 similar to that found in PKBα, †A.B. and A.C. contributed equally to this work. except that the residue equivalent to Ser473 is aspartic acid. Mutation of any of the conserved residues in the PDK2 motif of PIF prevented interaction of PIF Received: 12 January 1999 Revised: 18 February 1999 with PDK1. Remarkably, interaction of PDK1 with PIF, or with a synthetic Accepted: 3 March 1999 peptide encompassing the PDK2 consensus sequence of PIF, converted PDK1 from an enzyme that could phosphorylate only Thr308 of PKBα to one that Published: 8 April 1999 phosphorylates both Thr308 and Ser473 of PKBα in a manner dependent on Current Biology 1999, 9:393–404 phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P3).
    [Show full text]
  • AGC Kinases in Mtor Signaling, in Mike Hall and Fuyuhiko Tamanoi: the Enzymes, Vol
    Provided for non-commercial research and educational use only. Not for reproduction, distribution or commercial use. This chapter was originally published in the book, The Enzymes, Vol .27, published by Elsevier, and the attached copy is provided by Elsevier for the author's benefit and for the benefit of the author's institution, for non-commercial research and educational use including without limitation use in instruction at your institution, sending it to specific colleagues who know you, and providing a copy to your institution’s administrator. All other uses, reproduction and distribution, including without limitation commercial reprints, selling or licensing copies or access, or posting on open internet sites, your personal or institution’s website or repository, are prohibited. For exceptions, permission may be sought for such use through Elsevier's permissions site at: http://www.elsevier.com/locate/permissionusematerial From: ESTELA JACINTO, AGC Kinases in mTOR Signaling, In Mike Hall and Fuyuhiko Tamanoi: The Enzymes, Vol. 27, Burlington: Academic Press, 2010, pp.101-128. ISBN: 978-0-12-381539-2, © Copyright 2010 Elsevier Inc, Academic Press. Author's personal copy 7 AGC Kinases in mTOR Signaling ESTELA JACINTO Department of Physiology and Biophysics UMDNJ-Robert Wood Johnson Medical School, Piscataway New Jersey, USA I. Abstract The mammalian target of rapamycin (mTOR), a protein kinase with homology to lipid kinases, orchestrates cellular responses to growth and stress signals. Various extracellular and intracellular inputs to mTOR are known. mTOR processes these inputs as part of two mTOR protein com- plexes, mTORC1 or mTORC2. Surprisingly, despite the many cellular functions that are linked to mTOR, there are very few direct mTOR substrates identified to date.
    [Show full text]
  • Plant Mitogen-Activated Protein Kinase Signaling Cascades Guillaume Tena*, Tsuneaki Asai†, Wan-Ling Chiu‡ and Jen Sheen§
    392 Plant mitogen-activated protein kinase signaling cascades Guillaume Tena*, Tsuneaki Asai†, Wan-Ling Chiu‡ and Jen Sheen§ Mitogen-activated protein kinase (MAPK) cascades have components that link sensors/receptors to target genes emerged as a universal signal transduction mechanism that and other cellular responses. connects diverse receptors/sensors to cellular and nuclear responses in eukaryotes. Recent studies in plants indicate that In the past few years, it has become apparent that mitogen- MAPK cascades are vital to fundamental physiological functions activated protein kinase (MAPK) cascades play some of the involved in hormonal responses, cell cycle regulation, abiotic most essential roles in plant signal transduction pathways stress signaling, and defense mechanisms. New findings have from cell division to cell death (Figure 1). MAPK cascades revealed the complexity and redundancy of the signaling are evolutionarily conserved signaling modules with essen- components, the antagonistic nature of distinct pathways, and tial regulatory functions in eukaryotes, including yeasts, the use of both positive and negative regulatory mechanisms. worms, flies, frogs, mammals and plants. The recent enthu- siasm for plant MAPK cascades is backed by numerous Addresses studies showing that plant MAPKs are activated by hor- Department of Molecular Biology, Massachusetts General Hospital, mones, abiotic stresses, pathogens and pathogen-derived Department of Genetics, Harvard Medical School, Wellman 11, elicitors, and are also activated at specific stages during the 50 Blossom Street, Boston, Massachusetts 02114, USA cell cycle [2]. Until recently, studies of MAPK cascades in *e-mail: [email protected] †e-mail: [email protected] plants were focused on cDNA cloning [3,4] and used a ‡e-mail: [email protected] MAPK in-gel assay, MAPK and tyrosine-phosphate anti- §e-mail: [email protected] bodies, and kinase inhibitors to connect signals to MAPKs Current Opinion in Plant Biology 2001, 4:392–400 [2].
    [Show full text]
  • A Calcium- and Calmodulin-Dependent Kinase I␣/ Microtubule Affinity Regulating Kinase 2 Signaling Cascade Mediates Calcium-Dependent Neurite Outgrowth
    The Journal of Neuroscience, April 18, 2007 • 27(16):4413–4423 • 4413 Cellular/Molecular A Calcium- and Calmodulin-Dependent Kinase I␣/ Microtubule Affinity Regulating Kinase 2 Signaling Cascade Mediates Calcium-Dependent Neurite Outgrowth Nataliya V. Uboha,1 Marc Flajolet,2 Angus C. Nairn,1,2 and Marina R. Picciotto1 1Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut 06508, and 2Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021 Calcium is a critical regulator of neuronal differentiation and neurite outgrowth during development, as well as synaptic plasticity in adulthood. Calcium- and calmodulin-dependent kinase I (CaMKI) can regulate neurite outgrowth; however, the signal transduction cascades that lead to its physiological effects have not yet been elucidated. CaMKI␣ was therefore used as bait in a yeast two-hybrid assay and microtubule affinity regulating kinase 2 (MARK2)/Par-1b was identified as an interacting partner of CaMKI in three independent screens. The interaction between CaMKI and MARK2 was confirmed in vitro and in vivo by coimmunoprecipitation. CaMKI binds MARK2 within its kinase domain, but only if it is activated by calcium and calmodulin. Expression of CaMKI and MARK2 in Neuro-2A (N2a) cells and in primary hippocampal neurons promotes neurite outgrowth, an effect dependent on the catalytic activities of these enzymes. In addition, decreasing MARK2 activity blocks the ability of the calcium ionophore ionomycin to promote neurite outgrowth. Finally, CaMKI phosphorylates MARK2 on novel sites within its kinase domain. Mutation of these phosphorylation sites decreases both MARK2 kinase activity and its ability to promote neurite outgrowth.
    [Show full text]
  • Diverse Physiological Functions for Dual-Specificity MAP Kinase
    Commentary 4607 Diverse physiological functions for dual-specificity MAP kinase phosphatases Robin J. Dickinson and Stephen M. Keyse* Cancer Research UK Stress Response Laboratory, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK *Author for correspondence (e-mail: [email protected]) Accepted 19 September 2006 Journal of Cell Science 119, 4607-4615 Published by The Company of Biologists 2006 doi:10.1242/jcs.03266 Summary A structurally distinct subfamily of ten dual-specificity functions in mammalian cells and tissues. However, recent (Thr/Tyr) protein phosphatases is responsible for the studies employing a range of model systems have begun to regulated dephosphorylation and inactivation of mitogen- reveal essential non-redundant roles for the MKPs in activated protein kinase (MAPK) family members in determining the outcome of MAPK signalling in a variety mammals. These MAPK phosphatases (MKPs) interact of physiological contexts. These include development, specifically with their substrates through a modular kinase- immune system function, metabolic homeostasis and the interaction motif (KIM) located within the N-terminal non- regulation of cellular stress responses. Interestingly, these catalytic domain of the protein. In addition, MAPK binding functions may reflect both restricted subcellular MKP is often accompanied by enzymatic activation of the C- activity and changes in the levels of signalling through terminal catalytic domain, thus ensuring specificity of multiple MAPK pathways. action. Despite our knowledge of the biochemical and structural basis for the catalytic mechanism of the MKPs, we know much less about their regulation and physiological Key words: MAPK, MKP, Signal transduction, Phosphorylation Introduction the activation motif is required for MAPK activity, Mitogen-activated protein kinases (MAPKs) constitute a dephosphorylation of either residue inactivates these enzymes.
    [Show full text]
  • Glycogen Synthase Kinase-3 Is Activated in Neuronal Cells by G 12
    The Journal of Neuroscience, August 15, 2002, 22(16):6863–6875 Glycogen Synthase Kinase-3 Is Activated in Neuronal Cells by G␣ ␣ 12 and G 13 by Rho-Independent and Rho-Dependent Mechanisms C. Laura Sayas, Jesu´ s Avila, and Francisco Wandosell Centro de Biologı´a Molecular “Severo Ochoa”, Consejo Superior de Investigaciones Cientı´ficas, Universidad Auto´ noma de Madrid, Cantoblanco, Madrid 28049, Spain ␣ ␣ ␣ ␣ Glycogen synthase kinase-3 (GSK-3) was generally considered tively active G 12 (G 12QL) and G 13 (G 13QL) in Neuro2a cells a constitutively active enzyme, only regulated by inhibition. induces upregulation of GSK-3 activity. Furthermore, overex- Here we describe that GSK-3 is activated by lysophosphatidic pression of constitutively active RhoA (RhoAV14) also activates ␣ acid (LPA) during neurite retraction in rat cerebellar granule GSK-3 However, the activation of GSK-3 by G 13 is blocked by neurons. GSK-3 activation correlates with an increase in GSK-3 coexpression with C3 transferase, whereas C3 does not block ␣ tyrosine phosphorylation. In addition, LPA induces a GSK-3- GSK-3 activation by G 12. Thus, we demonstrate that GSK-3 is ␣ ␣ mediated hyperphosphorylation of the microtubule-associated activated by both G 12 and G 13 in neuronal cells. However, ␣ protein tau. Inhibition of GSK-3 by lithium partially blocks neu- GSK-3 activation by G 13 is Rho-mediated, whereas GSK-3 ␣ rite retraction, indicating that GSK-3 activation is important but activation by G 12 is Rho-independent. The results presented not essential for the neurite retraction progress. GSK-3 activa- here imply the existence of a previously unknown mechanism of ␣ tion by LPA in cerebellar granule neurons is neither downstream GSK-3 activation by G 12/13 subunits.
    [Show full text]
  • G Protein Regulation of MAPK Networks
    Oncogene (2007) 26, 3122–3142 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW G Protein regulation of MAPK networks ZG Goldsmith and DN Dhanasekaran Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA, USA G proteins provide signal-coupling mechanisms to hepta- the a-subunits has been used as a basis for the helical cell surface receptors and are criticallyinvolved classification of G proteins into Gs,Gi,Gq and G12 in the regulation of different mitogen-activated protein families in which the a-subunits that show more than kinase (MAPK) networks. The four classes of G proteins, 50% homology are grouped together (Simon et al., defined bythe G s,Gi,Gq and G12 families, regulate 1991). In G-protein-coupled receptor (GPCR)-mediated ERK1/2, JNK, p38MAPK, ERK5 and ERK6 modules by signaling pathways, ligand-activated receptors catalyse different mechanisms. The a- as well as bc-subunits are the exchange of the bound GDP to GTP in the a-subunit involved in the regulation of these MAPK modules in a following which the GTP-bound a-subunit disassociate context-specific manner. While the a- and bc-subunits from the receptor as well as the bg-subunit. The GTP- primarilyregulate the MAPK pathwaysvia their respec- bound a-subunit and the bg-subunit stimulate distinct tive effector-mediated signaling pathways, recent studies downstream effectors including enzymes, ion channels have unraveled several novel signaling intermediates and small GTPase, thus regulating multiple signaling including receptor tyrosine kinases and small GTPases pathways including those involved in the activation of through which these G-protein subunits positivelyas well mitogen-activated protein kinase (MAPK) modules as negativelyregulate specific MAPK modules.
    [Show full text]
  • Table S1. List of Oligonucleotide Primers Used
    Table S1. List of oligonucleotide primers used. Cla4 LF-5' GTAGGATCCGCTCTGTCAAGCCTCCGACC M629Arev CCTCCCTCCATGTACTCcgcGATGACCCAgAGCTCGTTG M629Afwd CAACGAGCTcTGGGTCATCgcgGAGTACATGGAGGGAGG LF-3' GTAGGCCATCTAGGCCGCAATCTCGTCAAGTAAAGTCG RF-5' GTAGGCCTGAGTGGCCCGAGATTGCAACGTGTAACC RF-3' GTAGGATCCCGTACGCTGCGATCGCTTGC Ukc1 LF-5' GCAATATTATGTCTACTTTGAGCG M398Arev CCGCCGGGCAAgAAtTCcgcGAGAAGGTACAGATACGc M398Afwd gCGTATCTGTACCTTCTCgcgGAaTTcTTGCCCGGCGG LF-3' GAGGCCATCTAGGCCATTTACGATGGCAGACAAAGG RF-5' GTGGCCTGAGTGGCCATTGGTTTGGGCGAATGGC RF-3' GCAATATTCGTACGTCAACAGCGCG Nrc2 LF-5' GCAATATTTCGAAAAGGGTCGTTCC M454Grev GCCACCCATGCAGTAcTCgccGCAGAGGTAGAGGTAATC M454Gfwd GATTACCTCTACCTCTGCggcGAgTACTGCATGGGTGGC LF-3' GAGGCCATCTAGGCCGACGAGTGAAGCTTTCGAGCG RF-5' GAGGCCTGAGTGGCCTAAGCATCTTGGCTTCTGC RF-3' GCAATATTCGGTCAACGCTTTTCAGATACC Ipl1 LF-5' GTCAATATTCTACTTTGTGAAGACGCTGC M629Arev GCTCCCCACGACCAGCgAATTCGATagcGAGGAAGACTCGGCCCTCATC M629Afwd GATGAGGGCCGAGTCTTCCTCgctATCGAATTcGCTGGTCGTGGGGAGC LF-3' TGAGGCCATCTAGGCCGGTGCCTTAGATTCCGTATAGC RF-5' CATGGCCTGAGTGGCCGATTCTTCTTCTGTCATCGAC RF-3' GACAATATTGCTGACCTTGTCTACTTGG Ire1 LF-5' GCAATATTAAAGCACAACTCAACGC D1014Arev CCGTAGCCAAGCACCTCGgCCGAtATcGTGAGCGAAG D1014Afwd CTTCGCTCACgATaTCGGcCGAGGTGCTTGGCTACGG LF-3' GAGGCCATCTAGGCCAACTGGGCAAAGGAGATGGA RF-5' GAGGCCTGAGTGGCCGTGCGCCTGTGTATCTCTTTG RF-3' GCAATATTGGCCATCTGAGGGCTGAC Kin28 LF-5' GACAATATTCATCTTTCACCCTTCCAAAG L94Arev TGATGAGTGCTTCTAGATTGGTGTCggcGAAcTCgAGCACCAGGTTG L94Afwd CAACCTGGTGCTcGAgTTCgccGACACCAATCTAGAAGCACTCATCA LF-3' TGAGGCCATCTAGGCCCACAGAGATCCGCTTTAATGC RF-5' CATGGCCTGAGTGGCCAGGGCTAGTACGACCTCG
    [Show full text]
  • Protein Kinases Phosphorylation/Dephosphorylation Protein Phosphorylation Is One of the Most Important Mechanisms of Cellular Re
    Protein Kinases Phosphorylation/dephosphorylation Protein phosphorylation is one of the most important mechanisms of cellular responses to growth, stress metabolic and hormonal environmental changes. Most mammalian protein kinases have highly a homologous 30 to 32 kDa catalytic domain. • Most common method of reversible modification - activation and localization • Up to 1/3 of cellular proteins can be phosphorylated • Leads to a very fast response to cellular stress, hormonal changes, learning processes, transcription regulation .... • Different than allosteric or Michealis Menten regulation Protein Kinome To date – 518 human kinases known • 50 kinase families between yeast, invertebrate and mammaliane kinomes • 518 human PKs, most (478) belong to single super family whose catalytic domain are homologous. • Kinase dendrogram displays relative similarities based on catalytic domains. • AGC (PKA, PKG, PKC) • CAMK (Casein kinase 1) • CMGC (CDC, MAPK, GSK3, CLK) • STE (Sterile 7, 11 & 20 kinases) • TK (Tryosine kinases memb and cyto) • TKL (Tyrosine kinase-like) • Phosphorylation stabilized thermodynamically - only half available energy used in adding phosphoryl to protein - change in free energy forces phosphorylation reaction in one direction • Phosphatases reverse direction • The rate of reaction of most phosphatases are 1000 times faster • Phosphorylation occurs on Ser/The or Tyr • What differences occur due to the addition of a phosphoryl group? • Regulation of protein phosphorylation varies depending on protein - some turned on or off
    [Show full text]
  • Targeting the Hippo Pathway in Prostate Cancer: What's New?
    cancers Review Targeting the Hippo Pathway in Prostate Cancer: What’s New? Kelly Coffey Solid Tumour Target Discovery Laboratory, Translational and Clinical Research Institute, Newcastle University Centre for Cancer, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK; [email protected] Simple Summary: Prostate cancer is the most commonly diagnosed cancer in men in the UK, accounting for the deaths of over 11,000 men per year. A major problem in this disease are tumours which no longer respond to available treatments. Understanding how this occurs will reveal new ways to treat these patients. In this review, the latest findings regarding a particular group of cellular factors which make up a signalling network called the Hippo pathway will be described. Accumulating evidence suggests that this network contributes to prostate cancer progression and resistance to current treatments. Identifying how this pathway can be targeted with drugs is a promising area of research to improve the treatment of prostate cancer. Abstract: Identifying novel therapeutic targets for the treatment of prostate cancer (PC) remains a key area of research. With the emergence of resistance to androgen receptor (AR)-targeting therapies, other signalling pathways which crosstalk with AR signalling are important. Over recent years, evidence has accumulated for targeting the Hippo signalling pathway. Discovered in Drosophila melanogasta, the Hippo pathway plays a role in the regulation of organ size, proliferation, migration and invasion. In response to a variety of stimuli, including cell–cell contact, nutrients and stress, a kinase cascade is activated, which includes STK4/3 and LATS1/2 to inhibit the effector proteins YAP and its paralogue TAZ.
    [Show full text]