DISSERTATION submitted to the Combined Faculty of Natural Sciences and Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Lucie Magdalena Wolf, M.Sc. born in Nuremberg, Germany Date of oral examination: 2nd February 2021 Ubiquitin-dependent regulation of the WNT cargo protein EVI/WLS Referees: Prof. Dr. Michael Boutros apl. Prof. Dr. Viktor Umansky If you don’t think you might, you won’t. Terry Pratchett This work was accomplished from August 2015 to November 2020 under the supervision of Prof. Dr. Michael Boutros in the Division of Signalling and Functional Genomics at the German Cancer Research Center (DKFZ), Heidelberg, Germany. Contents Contents ......................................................................................................................... ix 1 Abstract ....................................................................................................................xiii 1 Zusammenfassung .................................................................................................... xv 2 Introduction ................................................................................................................ 1 2.1 The WNT signalling pathways and cancer ........................................................................ 1 2.1.1 Intercellular communication ........................................................................................ 1 2.1.2 WNT ligands are conserved morphogens .................................................................. 2 2.1.3 WNT ligand maturation and secretion ........................................................................ 3 2.1.3.1 WNT ligand secretion ............................................................................................ 4 2.1.3.2 The WNT cargo protein EVI/WLS......................................................................... 5 2.1.4 β-catenin-dependent/canonical WNT signalling ........................................................ 8 2.1.5 Non-canonical WNT signalling by WNT5A and WNT11 .......................................... 10 2.1.6 Deregulated WNT signalling and melanoma ............................................................ 12 2.1.6.1 Cutaneous melanoma ......................................................................................... 12 2.1.6.2 WNT signalling and phenotype switching in melanoma cells .......................... 14 2.2 Ubiquitin and Ubiquitination ............................................................................................. 15 2.2.1 Ub and Ub-binding domains ...................................................................................... 16 2.2.2 The ubiquitination cascade........................................................................................ 16 2.2.3 Linkage types and non-lysine ubiquitination ............................................................ 18 2.3 Ub-mediated protein degradation .................................................................................... 19 2.3.1 The Ub-proteasome system (UPS) ........................................................................... 20 2.3.2 ER-associated degradation (ERAD) .......................................................................... 20 2.3.2.1 Protein folding and recognition of ERAD clients ............................................... 23 2.3.2.2 ERAD-associated E3 Ub ligase complexes and their E2 partners ................... 23 2.3.2.3 VCP/Cdc48 provides energy for retrotranslocation.......................................... 25 2.3.2.4 Retrotranslocation – shuttling ERAD substrates back to the cytoplasm ......... 25 2.3.2.5 Targeting to the proteasome and degradation of ERAD substrates ................ 27 2.3.3 Protein quantity control by regulatory ERAD ........................................................... 27 2.3.4 Autophagy and lysosomal degradation .................................................................... 30 2.3.5 Endocytosis and ubiquitination.................................................................................. 30 2.4 Aim of this thesis ............................................................................................................... 31 ix Contents 3 Material & Methods ................................................................................................... 33 3.1 Material ............................................................................................................................. 33 3.1.1 Antibodies ................................................................................................................... 33 3.1.2 Buffers and solutions ................................................................................................. 35 3.1.3 Cell lines and culture media ...................................................................................... 36 3.1.4 Consumables .............................................................................................................. 37 3.1.5 Enzymes, reagents, chemicals, and drugs ............................................................... 39 3.1.6 Kits….. ......................................................................................................................... 41 3.1.7 Oligonucleotides and antisense oligonucleotides.................................................... 41 Primers for reverse-transcription quantitative PCR (RT-qPCR) ....................... 41 Small interfering ribonucleic acids (siRNAs) ..................................................... 42 3.1.8 Plasmids ...................................................................................................................... 49 3.1.9 Software ...................................................................................................................... 51 3.1.10 Technical equipment ............................................................................................... 52 3.2 Methods ............................................................................................................................. 54 3.2.1 Cell biological methods.............................................................................................. 54 Cell lines, culture media, and cell handling ....................................................... 54 Plasmid transfection ............................................................................................ 54 siRNA transfection ............................................................................................... 55 Inhibitor treatments ............................................................................................. 56 Gelatin degradation assay .................................................................................. 56 3.2.2 Molecular biological methods.................................................................................... 57 Molecular cloning and sequencing .................................................................... 57 Gateway cloning to generate FLAG-tagged constructs ............................ 57 Site-directed mutagenesis ........................................................................... 58 Plasmid DNA amplification and sequencing ............................................... 58 Isolation of total RNA and synthesis of complementary DNA (cDNA) ............. 59 RT-qPCR .............................................................................................................. 59 3.2.3 Protein biochemical methods .................................................................................... 60 Cell lysis and determination of protein concentration ...................................... 60 Isolation of secreted WNTs from supernatant ................................................... 61 andem Ub Binding Entity (TUBE) Assays ........................................................ 62 Immunoprecipitation (IP)..................................................................................... 64 SDS-PAGE and Western blotting ....................................................................... 65 3.2.4 Microscopy ................................................................................................................. 66 Immunofluorescence staining, imaging, and image analysis ........................... 66 x Contents Migration and proliferation live cell imaging assays ......................................... 67 4 Results ...................................................................................................................... 69 4.1 RNAi screen identified novel candidates involved in the ERAD of EVI/WLS ................ 69 4.2 ERLIN2, FAF2, and UBXN4 regulate EVI/WLS ............................................................... 72 4.2.1 EVI/WLS and VCP interact with ERLIN2-FLAG, FAF2-FLAG, and UBXN4-FLAG..72 4.2.2 FAF2 knock-down impedes EVI/WLS turn-over ...................................................... 75 4.3 EVI/WLS is modified with Ub by multiple E2 enzymes ................................................... 76 4.3.1 EVI/WLS is ubiquitinated at several positions .......................................................... 77 4.3.2 K63-linked Ub chains regulate WNT secretion ........................................................ 79 4.4 EVI/WLS is ubiquitinated and degraded in cells with endogenous WNT ligands......... 81 4.5 EVI/WLS is modified with K11-, K48-, and K63-linked Ub.............................................