Novel Functions of the Neurofibromatosis 2 Tumour Suppressor Protein Merlin Mikaela Grönholm Neuroscience Program and Department of Pathology Biomedicum Helsinki and Haartman Institute Faculty of Medicine University of Helsinki Finland Academic dissertation To be publicly discussed with the permission of the Faculty of Medicine of the University of Helsinki, on April 29th, 2005, at 12 noon in lecture hall Arppeanum, Snellmaninkatu 3, Helsinki HELSINKI 2005 Thesis supervisor Professor Olli Carpén, M.D., Ph.D. Department of Pathology Neuroscience Program Biomedicum Helsinki University of Helsinki Helsinki, Finland Thesis reviewers Professor John E. Eriksson, Ph.D. Department of Biology University of Turku Turku, Finland and Professor Dan Lindholm, M.D., Ph.D. Minerva Foundation Institute for Medical Research Biomedicum Helsinki Helsinki, Finland Thesis opponent Dr. Marco Giovannini, M.D., Ph.D. Director Inserm U674 "Génomique Fonctionnelle des Tumeurs Solides" Foundation Jean Dausset-CEPH Paris, France ISBN 952-91-8477-8 (paperback) ISBN 952-10-2395-3 (PDF) http://ethesis.helsinki.fi Yliopistopaino Helsinki 2005 - 2 - To my grandparents and parents who taught me to value curiosity, knowledge and understanding ”Jag söker den fråga på vilken människolivet är ett svar” ”I search for the question to which life is the answer" (Willy Kyrklund, Mästaren Ma, 1952) - 3 - CONTENTS CONTENTS .............................................................................................................. 4 ABBREVIATIONS ..................................................................................................... 6 ORIGINAL PUBLICATIONS...................................................................................... 8 ABSTRACT............................................................................................................... 9 REVIEW OF THE LITERATURE............................................................................. 11 1. THE CELL DIVISION CYCLE......................................................................................................11 2. CANCER GENES.............................................................................................................................12 2.1 Oncogenes ....................................................................................................................................12 2.2 Tumour suppressor genes .............................................................................................................13 3. THE ACTIN CYTOSKELETON....................................................................................................13 3.1 The actin cytoskeleton in disease..................................................................................................14 4. THE BAND 4.1 PROTEIN SUPERFAMILY ................................................................................14 4.1 The ERM protein family...............................................................................................................15 4.1.1 The ERM proteins..................................................................................................................16 4.1.1.1 Structure of the ERM proteins........................................................................................17 4.1.2 The NF2 protein, merlin ........................................................................................................17 4.1.3 Ezrin and merlin in neoplasia ...............................................................................................19 4.1.3.1 Neurofibromatosis 2, NF2 ..............................................................................................19 5. MOLECULAR INTERACTIONS AND FUNCTIONS OF MERLIN AND EZRIN..................20 5.1 Association with the cytoskeleton.................................................................................................20 5.2 Association with the plasma membrane........................................................................................22 5.3 Cell adhesion and cell-cell communication ..................................................................................23 5.4 Cell proliferation ..........................................................................................................................24 6. REGULATION OF MERLIN AND EZRIN ..................................................................................26 6.1 Phosphorylation............................................................................................................................26 6.2 RHO GTPases ..............................................................................................................................29 6.3 Homotypic and heterotypic binding..............................................................................................30 6.4 Other mechanisms of regulation ...................................................................................................30 AIMS OF THE STUDY ............................................................................................ 32 METHODS .............................................................................................................. 33 RESULTS AND DISCUSSION ................................................................................ 34 1. HETEROTYPIC AND HOMOTYPIC INTERACTIONS OF MERLIN AND EZRIN (I) .......34 1.1 Heterotypic interactions................................................................................................................34 1.2 Homotypic interactions.................................................................................................................35 1.3 Merlin N-ERMAD and C-ERMAD..............................................................................................36 1.4 Regulation of the dormant and active states .................................................................................37 - 4 - 2. NUCLEO-CYTOPLASMIC SHUTTLING OF MERLIN (II).....................................................39 2.1 Nucleo-cytoplasmic shuttling is dependent on the CRM1/exportin pathway ...............................40 2.2 Nucleo-cytoplasmic shuttling is cell cycle-dependent ..................................................................40 2.3 Nucleo-cytoplasmic shuttling is dependent on cell adhesion........................................................41 2.4 Regulation of the nucleo-cytoplasmic shuttling............................................................................42 3. INTERACTION WITH HEI-10 CONNECTS MERLIN TO CELL CYCLE REGULATION (III).........................................................................................................................................................43 3.1 Merlin and HEI10 interact through coiled-coil domains ..............................................................44 3.2 Merlin and HEI10 interaction requires regulation of both proteins..............................................44 3.3 The colocalisation of merlin and HEI10 is dependent on cell adhesion, cell cycle stage and merlin expression levels.................................................................................................................................45 3.4 Merlin and HEI10 affect cell cycle progression ...........................................................................47 4. MERLIN IN THE CNS (IV)............................................................................................................49 4.1 Analysis of merlin and ezrin in the developing and adult brain....................................................49 4.2 Subcellular distribution of merlin and ezrin in brain cells............................................................50 5. RIβ, A POTENTIAL INTERACTION PARTNER FOR MERLIN IN THE SYNAPTIC JUNCTIONS (IV, V) ............................................................................................................................51 5.1 The merlin-RIβ interaction...........................................................................................................51 5.2 Regulation of the merlin-RIβ interaction......................................................................................51 5.3 Mapping of the AKAP interaction site .........................................................................................52 5.4 Potential functions for the merlin-PKA association......................................................................52 CONCLUSIONS AND FUTURE DIRECTIONS ....................................................... 55 ACKNOWLEDGEMENTS ....................................................................................... 57 REFERENCES........................................................................................................ 59 - 5 - ABBREVIATIONS ABD Actin binding domain AKAP A-kinase anchoring protein APC Adenomatosis polyposis coli BRCA Breast cancer gene cAMP Cyclic adenosine 5'-monophosphate Cdk Cyclic-dependent kinase CNC Carney complex CNS Central nervous system CC Coiled-coiled CCD Cytochalasin D DAL 1 Differentially expressed in adenocarcinoma of the lung E3KARP NHE3 Kinase A Regulatory Protein EBP50 Ezrin binding protein 50 ECM Extracellular matrix E.Coli Escherichia coli EGF Epidermal growth factor EGFR Epidermal growth factor receptor ERK Extracellular signal-regulated kinase ERM Ezrin, Radixin, Moesin ERMAD ERM association domain EVH1 Enabled/VASP-1 domain F-actin Filamentous actin FAK Focal adhesion kinase FBD FERM