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Defining the Role of Folliculin and Its Interacting Partners

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Defining the Role of Folliculin and Its Interacting Partners DEFINING THE ROLE OF FOLLICULIN AND ITS INTERACTING PARTNERS Sara Seifan 2016 Thesis submitted to Cardiff University in fulfilment of the requirements for the degree of Doctor of Philosophy This work is dedicated to Rienk Doetjes It is through your presence in our hearts that your absence is felt every day CONTENTS Table of contents …………………………………………………………….….…………..I Acknowledgments……………………………...………………………………..………...VII Summary………………...……………………………………………………….…….….VIII Declaration…………………..……………………..………………………………….…....IX Abbreviations……………………………………………………………….….…………...XI 1 CHAPTER 1: GENERAL INTRODUCTION ........................................................ 1 1.1 BIRT-HOGG-DUBÉ SYNDROME ............................................................................ 1 1.2 CLINICAL MANIFESTATION ................................................................................... 2 1.2.1 Skin .......................................................................................................... 2 1.2.2 Lung ......................................................................................................... 5 1.2.3 Kidney ...................................................................................................... 7 1.2.4 Other clinical manifestations associated with BHD ................................. 10 1.2.4.1 Colorectal polyps and colorectal cancer .......................................... 10 1.2.4.2 Thyroid nodules and cancer ............................................................ 11 1.2.4.3 Parotid tumours ............................................................................... 12 1.3 FOLLICULIN ...................................................................................................... 12 1.3.1 Structure and post-translational modifications ........................................ 15 1.3.2 Mutations ................................................................................................ 17 1.3.3 Interacting partners ................................................................................ 19 1.3.3.1 FLCN interacting protein 1 (FNIP1) ................................................. 19 1.3.3.2 FLCN interacting protein 2 (FNIP2) ................................................. 20 1.3.3.3 Plakophilin-4 .................................................................................... 21 1.3.3.4 Rpt4 ................................................................................................. 21 1.3.3.5 Rag proteins .................................................................................... 21 I 1.3.4 Signalling pathways ................................................................................ 22 1.3.4.1 Mammalian target of rapamycin (mTOR) signalling ........................ 22 1.3.4.2 AMP-activated protein kinase (AMPK) signalling ............................ 24 1.3.4.3 Hypoxia Inducible Factor (HIF) signalling ........................................ 26 1.3.4.4 The Janus kinase/signal transducers and activators of transcription (JAK/STAT) and TGF-β signalling ................................................................. 27 1.3.4.5 Ciliogenesis ..................................................................................... 29 1.3.4.6 The Ras-Raf-MAPK signalling ......................................................... 29 1.3.4.7 Wnt/Cadherin signalling pathway .................................................... 30 1.4 AUTOPHAGY .................................................................................................... 32 1.4.1 Autophagy-related proteins (Atgs) .......................................................... 36 1.4.1.1 Atg8 family of proteins ..................................................................... 36 1.4.1.1.1 LIR motif ...................................................................................... 37 1.4.2 p62 (Sequestosome 1, SQSTM1) .......................................................... 38 1.4.3 unc-51 like autophagy activating kinase 1 (ULK1) .................................. 39 1.4.4 AMP-activated protein kinase (AMPK) ................................................... 41 1.4.5 Mammalian/mechanistic target of rapamycin (mTOR) ........................... 43 1.4.6 Rabs ....................................................................................................... 45 1.5 AIMS AND OBJECTIVE OF THIS PROJECT .............................................................. 46 2 CHAPTER 2: MATERIALS AND METHODS ................................................... 47 2.1 REAGENTS AND CHEMICALS .............................................................................. 47 2.2 BUFFERS AND SOLUTIONS ................................................................................. 49 2.3 ANTIBODIES ..................................................................................................... 52 2.4 PLASMIDS ........................................................................................................ 53 2.5 METHODOLOGY ............................................................................................... 54 II 2.5.1 Cell culture ............................................................................................. 54 2.5.2 Transfection ............................................................................................ 55 2.5.3 Bradford protein assay ........................................................................... 55 2.5.4 In vivo radiolabelling ............................................................................... 56 2.5.5 Immunoprecipitation (IP) ........................................................................ 56 2.5.6 GST purification ...................................................................................... 56 2.5.7 Western blot analysis ............................................................................. 56 2.5.8 Site-Directed Mutagenesis ..................................................................... 57 2.5.9 Plasmid purification (Miniprep/ Maxiprep)............................................... 58 2.5.10 Sequencing ............................................................................................ 58 2.5.11 Cyquant cell proliferation assay .............................................................. 58 2.5.12 RNA extraction ....................................................................................... 59 2.5.13 Reverse transcription ............................................................................. 59 2.5.14 Quantitative-PCR ................................................................................... 60 2.5.15 Modelling of FLCN structure ................................................................... 60 2.5.16 Densitometry .......................................................................................... 60 2.5.17 Statistical analysis .................................................................................. 61 3 CHAPTER 3: INVOLVEMENT OF FLCN IN AUTOPHAGY ............................. 62 3.1 INTRODUCTION ................................................................................................ 62 3.2 METHODS ........................................................................................................ 65 3.3 RESULTS ......................................................................................................... 67 3.3.1 FLCN could possibly be involved in autophagy ...................................... 67 3.3.2 Autophagy is down-regulated in FLCN-deficient cells ............................ 69 3.3.3 FLCN interacts with ULK1 ...................................................................... 71 3.3.4 FLCN is an ULK1 substrate .................................................................... 73 III 3.3.5 ULK1 phosphorylates FLCN within a conserved C-terminus .................. 75 3.3.6 ULK1 phosphorylation sites within the C-terminus are shown to be on the surface and are available for phosphorylation ................................................... 76 3.3.7 WT-FLCN rescues basal autophagy in FLCN deficient cells but not the 3A mutant .......................................................................................................... 78 3.3.8 GABARAP interacts with FLCN .............................................................. 81 3.3.9 FLCN/GABARAP binding is impaired in BHD patient-derived mutants .. 83 3.3.10 FLCN interacts with Rab GTPases associated with autophagy .............. 85 3.3.11 FLCN is not a typical GEF ...................................................................... 87 3.4 DISCUSSION .................................................................................................... 89 4 CHAPTER 4: FAMILIAL MULTIPLE DISCOID FIBROMA (FMDF) ................. 93 4.1 INTRODUCTION ................................................................................................ 93 4.2 METHODS ........................................................................................................ 95 4.3 RESULTS ......................................................................................................... 98 4.3.1 Families with FMDF have common ancestors and affected members have FNIP1 c.1989delT mutation ...................................................................... 98 4.3.2 The c.1989delT mutation predicts the frameshift p.Val663fsX which is a conserved residue in vertebrates .................................................................... 100 4.3.3
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