PhD degree in Molecular Medicine (curriculum in Molecular Oncology) European School of Molecular Medicine (SEMM), University of Milan and University of Naples “Federico II” Settore disciplinare: Bio/10 Characterization of the role of USP25 in EGFR endocytosis Nadine Caroline Woessner IFOM, Milan Matricola n. R08892 Supervisor: Dr. Simona Polo IFOM, Milan Anno accademico 2013-2014 TABLE OF CONTENTS LIST OF ABBREVIATIONS................................................................................................ 8 FIGURE AND TABLE INDEX .......................................................................................... 11 ABSTRACT ........................................................................................................................ 13 INTRODUCTION ............................................................................................................... 15 1 Endocytosis .................................................................................................................. 15 1.1 Endocytic entry routes .......................................................................................... 15 1.1.1 Clathrin-mediated endocytosis (CME) .......................................................... 16 1.1.2 Non-clathrin endocytosis (NCE) ................................................................... 16 1.2 Endocytic sorting .................................................................................................. 19 1.3 Transferrin as a model substrate for CME ............................................................ 20 1.4 Endocytosis and signalling.................................................................................... 21 1.4.1 Endocytosis regulates signalling.................................................................... 21 1.4.2 Signalling regulates endocytosis.................................................................... 23 1.4.3 Signalling endosomes .................................................................................... 25 2 The ubiquitin system .................................................................................................... 26 2.1 Ubiquitin in endocytosis ....................................................................................... 29 2.1.1 Ubiquitination of cargoes .............................................................................. 30 2.1.2 Ubiquitin and endocytic adaptors .................................................................. 31 2.1.3 Ubiquitin in endosomal sorting ..................................................................... 33 2.2 Deubiquitinating enzymes (DUBs) in endocytosis ............................................... 35 2.2.1 Mechanism of catalysis .................................................................................. 36 2 2.2.2 DUB families .................................................................................................37 2.2.3 DUB activity in early steps of endocytosis ....................................................39 2.2.4 DUB activity in later steps of endocytosis .....................................................40 2.2.5 DUB activity in the secretory pathway ..........................................................43 3 The EGFR system ........................................................................................................44 3.1 Ligand-induced EGFR signal transduction ...........................................................45 3.2 EGFR endocytosis and trafficking ........................................................................46 3.3 Role of ubiquitin in EGFR endocytosis ................................................................47 3.4 Role of DUBs in EGFR endocytosis .....................................................................51 4 USP25 ..........................................................................................................................52 5 Cullin 3 .........................................................................................................................55 5.1 Structure and regulation of Cullins........................................................................55 5.2 Cullin 3 in endocytosis ..........................................................................................56 MATERIAL AND METHODS ...........................................................................................58 1 Solutions .......................................................................................................................58 1.1 Phosphate-buffered saline .....................................................................................58 1.2 Tris-HCl (1 M) ......................................................................................................58 1.3 Tris-buffered saline (TBS) ....................................................................................58 1.4 10X SDS-PAGE running buffer ............................................................................59 1.5 10X transfer buffer ................................................................................................59 1.6 50X TAE (Tris-Acetate-EDTA) ............................................................................59 2 Protein buffers ..............................................................................................................59 3 2.1 1X JS buffer .......................................................................................................... 59 2.2 1X RIPA buffer ..................................................................................................... 60 2.3 1X Laemmli buffer................................................................................................ 60 3 Reagents ....................................................................................................................... 61 3.1 Antibodies ............................................................................................................. 61 3.2 RNAi oligos .......................................................................................................... 62 3.2.1 Negative control siRNA ................................................................................ 62 3.2.2 Specific RNAi oligos ..................................................................................... 62 3.3 TaqMAN assays for qRT-PCR (Applied Biosystems) ......................................... 62 4 Cloning techniques ...................................................................................................... 63 4.1 Agarose gel electrophoresis .................................................................................. 63 4.2 Minipreps .............................................................................................................. 63 4.3 Diagnostic DNA restriction .................................................................................. 63 4.4 Large scale plasmid preparation ........................................................................... 63 4.5 Transformation of competent cells ....................................................................... 64 5 Constructs and plasmids .............................................................................................. 64 6 Cell culture ................................................................................................................... 65 6.1 Cell culture media ................................................................................................. 65 6.2 Transfections ......................................................................................................... 65 6.2.1 RNAi transfections ........................................................................................ 65 6.2.2 DNA transfections ......................................................................................... 66 6.3 Retroviral and lentiviral infection ......................................................................... 66 4 7 Protein procedures ........................................................................................................67 7.1 Cell lysis ................................................................................................................67 7.2 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) .......................................67 7.3 Immunoblot (IB) ....................................................................................................68 7.4 Anti-ubiquitin immunoblot ....................................................................................69 7.5 Immmunoprecipitation ..........................................................................................69 8 DUB library screening .................................................................................................70 9 Protein production and purification .............................................................................70 9.1 GST-fusion protein production .............................................................................70 9.2 GST pull-down ......................................................................................................72 10 Assays with 125I-EGF and 125I-Tf .................................................................................72 10.1 Receptor internalization assays with 125I-EGF and 125I-Tf ................................72 10.2 Measurement of the number of EGF/Tf receptors at the cell surface by saturation binding