Regulation of EGF receptor trafficking by the lysine deacetylase HDAC6 [Elektronische Ressource] / von Yonathan Lissanu Deribe

Yonathan Ebguy

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Regulation of EGF receptor trafficking by the
lysine deacetylase HDAC6




Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften



vorgelegt beim Fachbereich 14
Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main





von
Yonathan Lissanu Deribe
aus Addis Ababa, Äthiopien



Frankfurt am Main 2009
D30





















vom Fachbereich Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität als Dissertation angenommen


Dekan: Prof. Dr. Dieter Steinhilber

Gutachter: Prof. Dr. Volker Dötsch
Prof. Dr. Ivan Dikic


Datum der Disputation: 5. Juli 2010



Table of contents

ABSTRACT ..............................................................................................................................2
ZUSAMMENFASSUNG.........................................4
1. INTRODUCTION..............................................................................11
1.1 EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR)...............................................11
1.1.1 Growth factors and their cognate receptors ................................................11
1.1.2 ErbB family receptors .................................................................................12
1.1.3 Mechanism of activation of EGFR .............................................................12
1.1.4 Signaling through EGFR.............................................................................13
1.1.4.1 Principles of signal transduction downstream of RTKs..................................13
1.1.4.2 Signaling pathways activated by EGFR..........................................................15
1.1.5 Physiological functions of EGFR signaling................................................16
1.1.6 Pathological perturbations of EGFR and its signaling pathway .................17
1.1.7 Mechanisms of EGFR signal attenuation .................................................18
1.2 EGFR TRAFFICKING ...........................................................................................18
1.2.1 Endocytosis .................................................................................................18
1.2.1.1 Clathrin-mediated endocytosis (CME) ...........................................................19
1.2.1.1 Clathrin independent endocytosis (CIE).........................22
1.2.1.1.1 Caveolin mediated endocytosis....................................................................23
1.2.1.1.2 Dynamin-independent cdc42 regulated endocytosis...23
1.2.1.1.3 Internalization of large volumes of membrane – macropinocytosis and
phagocytosis................................................................................................................24
1.2.1.1.4 Pathogens and the endocytic pathway..........................25
1.2.3 EGFR endocytosis ......................................................................................26
1.3 HDAC6 AND PROTEIN ACETYLATION.................................................................29
1.3.1 Lysine acetylation as a post-translational modification..............................29
1.3.2 Deacetylases................................................................................................30
1.3.3 Histone deacetylase 6 (HDAC6)31
1.4 MICROTUBULE DEPENDENT INTRACELLULAR TRAFFICKING ...............................34
1.5 MEMBRANE BASED YEAST TWO-HYBRID SCREENING (MYTH)...........................35
1.6 RELATED ORIGINAL WORK- A CBL-CIN85 COMPLEX IN EGFR ENDOCYTOSIS ...38
1.7 AIMS OF THE CURRENT STUDY ...........................................................................40
2. MATERIALS AND METHODS.......................................................................................41
2.1 MATERIALS.....................................................................................................41
2.1.1 Chemical compounds..................................................................................41


Table of contents

2.1.2 Recombinant proteins and speciality reagents............................................41
2.1.3 Antibodies ...................................................................................................43
2.1.4 Buffers, solutions and media for routine use ..............................................44
2.1.5 Plasmids ......................................................................................................45
2.1.6 Oligonucleotides .........................................................................................46
2.1.7 Cell lines, bacterial and yeast strains ..........................................................46
2.2 METHODS ........................................................................................................47
2.2.1 Basic molecular biology techniques ...........................................................47
2.2.1.1 Plasmid DNA transformation into E.coli........................................................47
2.2.1.2 Plasmid DNA extraction from bacteria...........................47
2.2.1.3 Plasmid DNA transformation into S.cerevisae using the lithium acetate
method.........................................................................................................................47
2.2.1.4 Plasmid DNA extraction from yeast- the lyticase approach ...........................48
2.2.1.5 Site-directed mutagenesis................................................................................48
2.2.1.6 PCR amplification and restriction digestion of DNA.....48
2.2.1.7 Preparation of MFα-EGFR-C-T construct......................49
2.2.2 Membrane-based yeast two-hybrid screen..................................................49
2.2.3 Cell culture methods ...................................................................................50
2.2.3.1 Cultivation of mammalian cells ......................................................................50
2.2.3.2 Transfection of mammalian cells....50
2.2.3.3 Lentivirus transduction and stable shRNA expression cell line generation....51
2.2.3.4 Real time cell analysis (RTCA) ......................................................................51
2.2.4 Biochemical assays .....................................................................................52
2.2.4.1 SDS-PAGE and Western blot.........52
2.2.4.2 Immunoprecipitation .......................................................................................52
2.2.4.3 GST-GATE16 purification and GST pull down assay ...................................53
2.2.4.4 In vitro kinase assay........................54
2.2.4.5 Deacetylase assay............................................................................................54
2.2.4.6 Ligand induced EGFR degradation.................................54
1252.2.4.7 EGFR internalization assay using I-labelled EGF......55
2.2.4.8 Mass spectrometry sample preparation...........................................................55
2.2.5 Cell imaging studies....................................................................................56
2.2.5.1 Immunofluorescence microscopy ...................................................................56
2.2.5.2 Live cell imaging.............................................................57
2.2.6 Bioinformatic analyses................................................................................57

Table of contents

3. RESULTS............................................................................................................................59
3.1 MYTH BASED SCREENING OF LIGAND-UNOCCUPIED EGFR...............................59
3.1.1 Generation of bait EGFR-Cub-TF construct...............................................59
3.1.2 MYTH screening using MFα-EGFR-C-T...................................................61
3.1.3 Biochemical validation of putative interactions .........................................65
3.2 INTERACTION BETWEEN THE CYTOPLASMIC DEACETYLASE HDAC6 AND EGFR 67
3.2.1 HDAC6 binds to EGFR ..............................................................................68
3.2.2 Mapping of EGFR and HDAC6 binding regions .......................................70
3.3 HDAC6 REGULATES LIGAND-INDUCED DEGRADATION OF EGFR .......................73
3.3.1 HDAC6 overexpression slows ligand-induced degradation of EGFR .......73
3.3.2 HDAC6 knockdown accelerated the degradation of EGFR .......................75
3.4 HDAC6 MODULATES THE KINETICS OF EGFR INTRACELLULAR TRAFFICKING ...75
3.4.1 HDAC6 has no effect on EGFR internalization .........................................77
3.4.2 Knockdown of HDAC6 accelerates the delivery of EGF late endosomes .77
3.5 ACETYLATION AND RECEPTOR TRAFFICKING.......................................................80
3.5.2 Acetylation of α-tubulin is increased upon EGF stimulation......................82
3.5.3 Acetylation of α-tubulin Lys40 is important for efficient motility of
endosomes............................................................................................................83
3.6 PHOSPHORYLATION OF HDAC6 M