THESIS ON NATURALAND EXACT SCIENCES B99 Vertebrate Homologues of Drosophila Fused Kinase and Their Roles in Sonic Hedgehog Signalling Pathway ALLA MALOVERJAN PRESS TALLINN UNIVERSITY OF TECHNOLOGY Faculty of Science Department of Gene Technology Dissertation was accepted for the defence of the degree of Doctor of Philosophy in Natural and Exact Sciences on November 3, 2010 Supervisors: Senior Researcher Priit Kogerman, PhD, Department of Gene Technology, Tallinn University of Technology, Tallinn, Estonia Senior Researcher Torben Østerlund, PhD, Department of Gene Technology, Tallinn University of Technology, Tallinn, Estonia Docent Marko Piirsoo,, PhD Department of Gene Technology, Tallinn University of Technology, Tallinn, Estonia Opponents: Prof. Mart Ustav,, PhD Institute of Technology, University of Tartu, Tartu, Estonia ResearchAssociate Viljar Jaks, PhD, Department of Biosciences and Nutrition, Center for Biosciences, Karolinska Institutet, Huddinge, Sweden Defence of the thesis: December 17, 2010, Tallinn University of Technology Declaration: Hereby I declare that this doctoral thesis, my original investigation and achievement, submitted for the doctoral degree at Tallinn University of Technology has not been submitted for any academic degree. /Alla Maloverjan/ Copyright: Alla Maloverjan, 2010 ISSN 1406-4723 ISBN 978-9949-23-043-3 LOODUS- JA TÄPPISTEADUSED B99 Selgroogsete fu kinaasi homoloogide roll Sonic Hedgehogi signaali ülekanderajas ALLA MALOVERJAN CONTENTS INTRODUCTION ..................................................................................................... 7 OUTLINE AND AIMS OF THIS THESIS ............................................................. 9 ORIGINAL PUBLICATIONS ............................................................................... 10 ABBREVIATIONS ................................................................................................. 11 1. REVIEW OF THE LITERATURE ................................................................... 12 1.1. Discovery and biological significance of Hh proteins ................................... 12 1.2. Expression and biological roles of Hh proteins ............................................. 13 1.2.1. Hh controls segmentation and wings development in Drosophila .............. 13 1.2.2. Shh controls development of CNS and limbs in vertebrates ....................... 15 1.3. Maturation and secretion of Hh proteins ........................................................ 17 1.4. Reception of Hh at the plasma membrane ..................................................... 18 1.4.1. Reception of hh in Drosophila cells ............................................................ 19 1.4.2. Reception of Shh in vertebrate cells ............................................................ 21 1.5. Transcription factors activities in Hh signalling ............................................ 23 1.5.1. Ci/Gli proteins are transcriptional activators and repressors ....................... 23 1.5.2. Regulation of ci protein ............................................................................... 25 1.5.3. Regulation of Gli2/3 proteins ...................................................................... 26 1.5.4. Protein kinases participating in regulation of Gli proteins .......................... 27 1.6. Intracellular events induced by Hh proteins ................................................... 27 1.6.1. Hedgehog Signalling Complex comprises interactions between fu, cos2, sufu and ciFL in Drosophila ................................................................................... 27 1.6.2. HSC regulates the ci transcription factor in Drosophila ............................. 30 1.6.3. Sufu and Kif7 are main regulators of Gli transcription factors in vertebrates ............................................................................................................. 31 1.7. Serine/threonine kinase Fu in Drosophila and mammalian Hh pathways ..... 33 1.7.1. The fu gene encodes a protein kinase maternally required for Drosophila development .......................................................................................................... 33 1.7.2. fu plays kinase activity dependent and independent roles .......................... 35 1.7.3. fu is required for the regulation of hh signalling pathway .......................... 36 1.7.4. fu is required for post-translational modifications of transcription factor ci ............................................................................................ 37 1.7.5. Indications of fu-independent hh pathway .................................................. 38 1.7.6. Mammalian serine/threonine kinase Stk36 is a putative Drosophila fu homologue ............................................................................................................. 38 1.7.7. Unc51-like kinases (Ulk) are the closest relatives of Stk36 ........................ 40 2. AIMS OF THE STUDY ...................................................................................... 42 3. MATERIALS AND METHODS ........................................................................ 43 4. RESULTS AND DISCUSSION .......................................................................... 44 4.1 Stk36 and ULK3 are serine/threonine kinases sharing similarity with fu (Publications I and II) ............................................................................................ 44 4.2. Expression pattern of Stk36 and ULK3 (Publications I and II) ...................... 45 4.3. Stk36 and ULK3 enhance transcriptional activity of GLI1 and GLI2 proteins (Publications I and II) ............................................................................................ 45 5 4.4. Stk36 and ULK3 regulate transcriptional activity of GLI proteins in a different way: ULK3 does and Stk36 does not require kinase activity (Publications I and II) ............................................................................................ 46 4.5. KD and CTD of Stk36 and ULK3 are involved in GLI regulation (Publications I and III) .......................................................................................... 47 4.6. ULK3 is an autophosphorylated kinase that possesses self-regulation properties and is able to phosphorylate GLI proteins in vitro (Publications II and III) ......................................................................................... 48 4.7. Ulk3 and Stk36 are required for Shh signal transduction (Publications I and III) .......................................................................................... 49 4.8. ULK3 directly interacts with SUFU through its KD that leads to loss of ULK3 autophosphorylation activity (Publication III) ........................................... 50 4.9. The ULK3-SUFU complex promotes generation of GLI2Rep form (Publication III) ..................................................................................................... 51 4.10. An emerging Shh signalling model involving Ulk3 (Publication III and here) ...................................................................................... 52 CONCLUSIONS ...................................................................................................... 54 REFERENCES ........................................................................................................ 55 ACKNOWLEDGEMENTS .................................................................................... 74 SUMMARY .............................................................................................................. 75 KOKKUVÕTE ........................................................................................................ 77 CURRICULUM VITAE ........................................................................................... 78 ELULOOKIRJELDUS ........................................................................................... 80 PUBLICATION I .................................................................................................... 83 PUBLICATION II ................................................................................................... 95 PUBLICATION III ............................................................................................... 109 6 INTRODUCTION Hedgehog signalling molecules trigger one of the fundamental developmental pathways required for proper formation of body plan. Hh was first discovered as a gene responsible for the segment polarity phenotype in fruit fly Drosophila melanogaster. At present, it is evident that the Hh signalling pathway is conserved in many aspects from flies to humans. However, severe variations in the Hh signalling cascade generated during evolution by mutations or gene duplication events illustrate divergences of Hh pathway between different species. Sonic Hedgehog (Shh) is the most widely expressed and best-studied mammalian homologue of Hh proteins and is vitally required for numerous developmental and postnatal processes. However, inappropriate activation and/or deregulation of the Shh pathway are linked to several developmental abnormalities and tumorigenesis in humans. Several molecules implicated in the Shh pathway are proto-oncoproteins or tumour suppressors. Although mechanisms of Shh signal transduction have been investigated for almost two decades, many aspects of intracellular events triggered by Shh are still unclear. Therefore unravelling the molecular mechanisms of Shh signal transduction is a matter of particular