JUHA GRÖNHOLM Evolutionary Conserved Regulatory Mechanisms of the JAK/STAT Pathway ACADEMIC DISSERTATION To be presented, with the permission of the board of Institute of Biomedical Technology of the University of Tampere, for public discussion in the Jarmo Visakorpi Auditorium, of the Arvo Building, Lääkärinkatu 1, Tampere, on November 2nd, 2012, at 12 o’clock. UNIVERSITY OF TAMPERE ACADEMIC DISSERTATION University of Tampere, Institute of Biomedical Technology and BioMediTech, Tampere University Hospital, Department of Pediatrics, Finland Supervised by Reviewed by Professor Olli Silvennoinen Professor Jean-Luc Imler University of Tampere University of Strasbourg Finland France Professor Mika Rämet Docent Sampsa Matikainen University of Tampere University of Helsinki Finland Finland Copyright ©2012 Tampere University Press and the author Distribution Tel. +358 40 190 9800 Bookshop TAJU [email protected] P.O. Box 617 www.uta.fi/taju 33014 University of Tampere http://granum.uta.fi Finland Cover design by Mikko Reinikka Acta Universitatis Tamperensis 1775 Acta Electronica Universitatis Tamperensis 1249 ISBN 978-951-44-8948-8 (print) ISBN 978-951-44-8949-5 (pdf) ISSN-L 1455-1616 ISSN 1456-954X ISSN 1455-1616 http://acta.uta.fi Tampereen Yliopistopaino Oy – Juvenes Print Tampere 2012 Contents Contents .............................................................................................................................. 3 List of original communications ......................................................................................... 6 Abbreviations...................................................................................................................... 7 Abstract............................................................................................................................. 10 Tiivistelmä ........................................................................................................................ 12 1. Introduction................................................................................................................... 14 2. Review of the literature................................................................................................. 16 2.1 Cytokines and the immune system ......................................................................... 16 2.2 The JAK/STAT pathway ........................................................................................ 18 2.2.1 Biological roles of the JAK/STAT pathway.................................................... 19 2.2.2 Domain structure of JAKs ............................................................................... 20 2.2.3 Domain structure of STATs............................................................................. 21 2.3 Regulation of the JAK/STAT pathway................................................................... 23 2.3.1 Protein tyrosine phosphatases.......................................................................... 23 2.3.2 Suppressors of cytokine signaling, SOCS ....................................................... 25 2.3.3 Protein inhibitors of activated STAT, PIAS .................................................... 26 2.3.4 Post-translational modifications in the regulation of the JAK/STAT pathway........................................................................................................... 29 2.3.4.1 Tyrosine and serine phosphorylation, methylation, acetylation, O-glycosylation and ISGylation ............................................................... 29 2.3.4.2 Ubiquitination ........................................................................................... 31 2.4 Sumoylation ............................................................................................................ 32 2.4.1 Physiological consequences of protein sumoylation ....................................... 36 2.4.2 Sumoylation of STATs .................................................................................... 38 2.5 Drosophila as a model organism in biomedical research ....................................... 40 2.6 Overview of the Drosophila immune system ......................................................... 41 2.6.1 Cellular response.............................................................................................. 41 2.6.2 Humoral response ............................................................................................ 44 3 2.7 The Drosophila JAK/STAT pathway..................................................................... 46 2.7.1 Roles of the JAK/STAT pathway in Drosophila development and the immune response ............................................................................................ 48 2.7.2 Regulation of the Drosophila JAK/STAT pathway ........................................ 51 3. Aims of the study.......................................................................................................... 54 4. Materials and methods .................................................................................................. 55 4.1. Plasmid constructs and cloning.............................................................................. 55 4.2 Antibodies............................................................................................................... 57 4.3 Cell culture.............................................................................................................. 57 4.4 Generation of stable overexpression S2 cell lines .................................................. 58 4.5 Transfections........................................................................................................... 58 4.6 Immunoprecipitation, Co-immunoprecipitation and Western blotting................... 59 4.6.1 Immunoprecipitation........................................................................................ 60 4.6.2 Co-immunoprecipitation.................................................................................. 60 4.6.3 Western blotting............................................................................................... 60 4.7 dsRNA synthesis and RNA interference................................................................. 61 4.8 Luciferase reporter gene assay................................................................................ 62 4.9 RNAi screen to identify novel JAK/STAT pathway regulators ............................. 62 4.10 Electrophoretic mobility shift assay (EMSA)....................................................... 64 5. Results........................................................................................................................... 65 5.1 Sumoylation of Stat92E (Article I)......................................................................... 65 5.1.1 Lysine 187 is a putative sumoylation site in Stat92E ...................................... 65 5.1.2 Stat92E is sumoylated in mammalian cells ..................................................... 66 5.1.3 Stat92E is sumoylated in Drosophila S2 cells and lysine 187 is the acceptor site for SUMO conjugation .............................................................. 67 5.1.4 Sumoylation negatively regulates Stat92E driven gene expression ................ 67 5.2 CG14225/ET and CG31716/Not4 as regulators of the Drosophila JAK/STAT pathway (Articles II and III) ................................................................................. 68 5.2.1 CG14225 and CG31716 are novel Drosophila JAK/STAT pathway regulators found in a genome-wide RNAi screen in Drosophila S2 cells....................... 68 4 5.2.2 CG14225 encodes a gp130 related transmembrane protein called Eye transformer (ET) (Article II)........................................................................... 71 5.2.3 ET negatively regulates Upd-induced Stat92E phosphorylation (Article II)... 72 5.2.4 CG31716 overexpression enhances JAK/STAT pathway mediated gene responses in S2 cells (Article III).................................................................... 73 5.2.5 Drosophila CG31716 gene encodes Not4, a protein related to mammalian CNOT4 (Article III)........................................................................................ 74 5.2.6 Human CNOT4 regulates STAT1- and STAT6-mediated signal transduction (Article III)...................................................................................................... 74 5.2.7 Drosophila Not4 is a Stat92E interacting protein (Article III)........................ 75 5.2.8 Not4/CNOT4 does not affect STAT tyrosine phosphorylation (Article III) ... 75 5.2.9 Not4 is required for efficient Stat92E DNA binding (Article III) ................... 77 6. Discussion..................................................................................................................... 78 6.1 Drosophila melanogaster as a model for studying the JAK/STAT signaling cascade .................................................................................................................. 78 6.2 Sumoylation in the regulation of transcription factor activity................................ 80 6.3 SUMO as a regulator of STAT-mediated gene expression .................................... 81 6.4 Genome-wide screens to identify genes that regulate the activity of the JAK/STAT pathway.............................................................................................. 83 7. Conclusions..................................................................................................................