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Characterization of Nedd4 Function and Its Interaction with Angiomotin

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Characterization of Nedd4 Function and Its Interaction with Angiomotin Characterization of Nedd4 Function and its Interaction with Angiomotin by Madhvi Nath A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Biochemistry University of Toronto © Copyright by Madhvi Nath 2014 Characterization of Nedd4 Function and its Interaction with Angiomotin Madhvi Nath Master of Science Department of Biochemistry University of Toronto 2014 Abstract The HECT E3 ubiquitin ligase Nedd4-1 was previously shown to regulate diverse processes such as cell and animal growth, insulin signaling, and lysosomal trafficking. To further elucidate the cellular functions of Nedd4-1, Nedd4-1 knockout mouse embryonic fibroblasts were characterized relative to their wild type counterparts. Immunofluorescence experiments revealed an altered lysosomal distribution in the knockout cells, although their lysosomal proteolytic function appeared normal. Transmission Electron Microscopy revealed striking morphological differences, especially regarding the lysosome and endoplasmic reticulum of the knockout cells. Another aspect of my studies examined the interaction between Nedd4-1 and Angiomotin (p130-AMOT), which involves the same motifs required to sequester transcriptional co-activators YAP and TAZ in the cytoplasm. To test either a competitive or non-competitive mode of binding, co-immunoprecipitation experiments involving p130-AMOT, the Nedd4 proteins, and YAP or TAZ were performed, with results not supporting a competitive mode of interaction. Overall, my results demonstrate new Nedd4-1 cellular functions. ii Acknowledgements Firstly and foremost, I would like to thank my supervisor Dr. Daniela Rotin for her guidance and encouragement throughout the past few years. Your knowledge and enthusiasm for Science know no bounds. I would also like to thank my committee members, Dr. Allen Volchuk and Dr. Vuk Stambolic, for their suggestions and guidance throughout my studies. I would also like to thank the past and present members of the Rotin Lab for all of their support and really making the lab a welcoming place to come in everyday. Thanks to Dr. Philipp Alberts and Dr. Avinash Persaud for introducing me to all of the Cell Biology techniques I used in my studies. To Chong, Ruth, and Wioletta for always pointing me in the right direction. I will continue to covet the friendship and support I have received here. iii Table of Contents Abstract ii Acknowledgements iii Table of Contents iv List of Tables vii List of Figures viii Abbreviations x Chapter 1: Introduction 1 Par t A: Characterization of Nedd4-1 knockout (KO) mouse embryonic fibroblasts 2 (MEFs) 1. Ubiquitin- Proteasome System 2 2. The Nedd4 Family of HECT E3 Ligases 5 3. Functions of Nedd4 Family Members 5 4. Studies on Nedd4-1 Knockout (KO) MEFs 8 A) Observations in the Nedd4-1 KO MEFs 8 B) Grb10, insulin signaling and Nedd4-1 9 C) Grb10 and mTORC1 10 5. Other Functions of Nedd4-1 14 A) Examples of other, known binding partners of Nedd4-1 14 B) Nedd4-1 Interaction with Angiomotin 15 Part B: Nedd4-1 interaction with Angiomotin 15 1. Discovery and Structure of Angiomotin (AMOT) 15 2. Functions of AMOT 18 A) AMOT in Junctional Integrity 18 B) Angiomotin in Development 18 C) Regulation of Ras/MAPK signaling by Angiomotin 19 3. Differences between the p130 and p80 isoforms of Angiotmotin 19 4. The Hippo Signalling Pathway 20 5. Angiomotin in Hippo Signalling 23 A) p130-AMOT Interacts with YAP and TAZ 23 B) YAP and TAZ Function 23 6. p130 AMOT interaction with Nedd4-1 and Nedd4-2 26 7. Nedd4 family of E3 ligases in Hippo Signalling 26 Rationale and Hypotheses 28 iv Chapter 2: Methods and Materials 29 Par t A: Characterization of Nedd4-1 knockout (KO) mouse embryonic 30 fibroblasts (MEFs) 1. Cell Culture 30 2. Immunofluorescence 30 A) Fixation 30 B) Immunostaining 30 C) Imaging 31 3. Immunoblot Analysis 32 4. DQ BSA Proteolysis Assay - Live Immunofluorescence Imagining 33 5. Tranmission Electron Microscopy of MEFs 34 Part B: Nedd4-1 interaction with Angiomotin 34 1. Cell Culture 34 2. Mutagenesis 34 3. Cloning and Plasmids 35 4. Transfection 35 5. Immunoprecipitation 35 Chapter 3: Results 38 Par t A: Characterization of Nedd4-1 knockout (KO) mouse embryonic 39 fibroblasts (MEFs) 1. Grb10 is increased in the Nedd4-1 KO MEFs 39 2. Lysosomes have altered distribution in the Nedd4-1 KO MEFS 41 3. mTORC1 signalling is not overactive in Nedd4-1 KO MEFs (in 43 response to amino acid restimulation) 4. Lysosomes from the Nedd4-1 KO MEFs have normal proteolytic 47 activity 5. Morphological Differences between the Nedd4-1 WT and KO MEFs 50 analyzed by EM 6. Comparing Levels of ER Stress Markers in Nedd4-1 KO MEFs 53 relative to WT MEFs 7. There are Lower Levels of Rac1 protein in the Nedd4-1 KO MEFs 55 Part B: Nedd4-1 interaction with Angiomotin 57 1. AMOT and Nedd4-1 interact in HEK293T cells 57 2. Nedd4-1 knockdown in HEK293T results in an increase of 59 endogenous AMOT levels v 3. Interaction with Nedd4-1 is abrogated when Angiomotin's three PY 61 motifs are mutated 4. AMOT interacts with Nedd4-1 and Nedd4-1 in HeLa cells 63 5. Increased ubiquitination of Angiomotin in the presence of Nedd4-1 65 and Nedd4-2 in HeLa cells. 6. Nedd4-1 and Nedd4-2 do not disrupt the interactions of Angiomotin 68 with TAZ in HeLa cells 7. The introduction of Nedd4s does not disrupt AMOT and YAP1 69 interaction Chapter 4: Discussion 75 Part A: Characterization of the Nedd4-1 KO MEFs 76 Part B: Angiomotin (AMOT) and Nedd4-1 interactions 83 Future Directions 87 Conclusions 90 References 91 vi List of Tables Chapter 1: Introduction Table 1: Interactors of Nedd4-1 14 Chapter 2: Methods and Materials Table 2: Mutagenesis Primers 34 vii List of Figures Chapter 1: Introduction Figure 1: The Ubiquitination Cascade Involving HECT or RING E3 3 Ligases Figure 2: Nedd4 family of HECT E3 ligases 7 Figure 3: mTOR signalling pathway 12 Figure 4: mTORC1 Regulation of Grb10 stability 13 Figure 5: Domain Architecture of the Motin family 17 Figure 6: The Mammalian Hippo Signalling Cascade 22 Figure 7: Angiomotin's role in Hippo Signalling 25 Chapter 3: Results Figure 8: Grb10 levels are elevated in the Nedd4-1 KO MEFs 40 Figure 9: Lysosomal Distribution is Altered in the Nedd4-1 KO MEFs 42 Figure 10: mTORC1 Signaling is Not Overactive in the Nedd4-1 KO 45 MEFs Figure 11: DQ BSA Assay in the WT and Nedd4-1 KO MEFs 48 Figure 12: Transmission electron micrographs of both the Nedd4-1 WT 51 and KO MEFs Figure 13: ER Stress Marker Comparison between WT and Nedd4-1 KO 54 MEFs Figure 14: Decreased levels of Rac1 in the Nedd4-1 KO MEFs, relative 56 to WT Figure 15: Validation of the Nedd4-1 and Angiomotin interaction 58 Figure 16: Nedd4-1 knock down leads to an increase in endogenous 60 Angiomotin levels viii Figure 17: Mutation of AMOT's three PY motifs abrogates interaction 62 with Nedd4-1 and Nedd4-2 Figure 18: Nedd4-1 and AMOT interaction is Preserved in HeLa cells 64 Figure 19: In HeLa cells, Wild type Angiomotin ubiquitination increases 66 in the presence of Nedd4-1 and Nedd4-2 Figure 20: The introduction of V5-tagged Nedd4s does not disrupt the 70 interaction between FLAG-TAZ and HA-Angiomotin wild type Figure 21: The introduction of Nedd4 variants does not disrupt binding 72 between Angiomotin and YAP1 Chapter 4: Discussion Figure 22: Comparison of Nedd4-1 KO MEFs to Galactosialidosis Patient 80 Fibroblasts Figure 23: Proposed models for AMOT interaction with Nedd4 and 84 YAP/TAZ ix List of Abbreviations 4-MU-NANA 2-(4-Methylumbelliferyl)- α-D-N- acetylneuraminic acid 4eBP-1 eIF4E Binding-Protein 1 AIP4 Atrophin Interacting Protein 4 (Itch) AMOT Angiomotin AMOTL1 Angiomotin-like 1 AMOTL2 Angiomotin like-2 BPS (domain) Between Pleckstrin Homology and SH3 CF Cystic Fibrosis CFTR Cystic Fibrosis Transmembrane Receptor Comm. Commissureless DAPI 4',6-diamidino-2-phenylindole DMEM Dulbecco's Modified Essential Medium DNedd4 Drosophila Nedd4 DQ BSA De-quenched Bovine Serum Albumin DUBs De-ubiquitinating enzymes Dvl1 Dishevelled 1 eIF2 α eukaryotic Initiation Factor 2 α eIF4E eukaryotic Initiation Factor 4E eIF4F eukaryotic Intiation Factor 4F ENaC Epithelial Sodium Channel ERAD Endoplasmic Reticulum-Associated Degradation x FBS Fetal Bovine Serum FGF Fibroblast Growth Factor FGFR1 FIbroblast Growth Factor Receptor 1 Grb10 Growth Factor Receptor Bound 10 HECT Homologous to E6-AP Carboxy-Terminus HEK293T cells Human Embryonic Kidney 293T cells HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid IGFR-1 Insulin-like growth factor receptor 1 IR Insulin Receptor IRS1 Insulin Receptor Substrate 1 KD Knock down KO Knock out LAMP1 Lysosomal-Associated Membrane Protein 1 LAPTM4 Lysosomal Protein Transmembrane 4 LAPTM5 Lysosomal Protein Transmembrane 5 LATS1/2 Large Tumour Suppressor Kinase 1/2 LSD Lysosomal Storage Disorder MAE cells Mouse Aortic Endothelial Cells MAPK Mitogen Activated Protein Kinase MEFs Mouse Embryonic Fibroblasts Merlin Moesin-Ezrin-Radixin-Like Protein, Neurofibromin 2 mTOR mammalian Target of Rapamycin xi mTORC1 mammalian Target of Rapamycin Complex 1 NCC Na +/Cl - Co-transporter Nedd4-1 Neural precursor cell expressed, developmentally down-regulated 4-1 p130-AMOT 130 kDa isoform of Angiomotin p80-AMOT 80 kDa isoform of Angiomotin pAKT phospho-Akt Pals1 Protein Associted with Lin Seven 1 PBS Phosphate Buffered Saline PFA Paraformaldehyde PH (domain) Pleckstrin Homology PI-3-K Phosphatidylinositide 3-kinase PMSF phenylmethylsulfonyl fluoride PPCA Protective Protein/Cathepsin A PSF Penicillin/Streptomycin/Fungizone Rac1 Ras-related C3 botulinum toxin substrate
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