SUBSTRATE SPECIFICITY AND REGULATION OF NEDD4 PROTEINS by Mary Christine Bruce A thesis submitted in conformity with the requirements for the Degree of Doctor of Philosophy Graduate Department of Biochemistry University of Toronto © Copyright by Mary Christine Bruce 2009 Substrate Specificity and Regulation of Nedd4 proteins Doctor of Philosophy, 2009 Mary Christine Bruce, Department of Biochemistry, University of Toronto Abstract Nedd4-1 and Nedd4-2 are closely related E3 ubiquitin protein ligases that contain a C2 domain, 3-4 WW domains, and a catalytic ubiquitin ligase HECT domain. The WW domains of Nedd4 proteins recognize substrates for ubiquitination by binding the sequence L/PPxY (the PY-motif) found in target proteins. Nedd4-2 functions as a suppressor of the epithelial Na+ channel (ENaC), which interacts with Nedd4-2 WW domains via PY-motifs located at its C-terminus. The importance of Nedd4-2 mediated ENaC regulation is highlighted by the fact that mutations affecting the ENaC PY-motifs cause Liddle syndrome, a hereditary hypertension. Since all Nedd4 family members recognize the same core sequence in their target proteins, the question was raised of how substrate specificity for Nedd4 family members is achieved. Using intrinsic tryptophan florescence to measure the binding affinity of Nedd4-1/-2 WW domains for their substrate PY-motifs, we demonstrate the importance of both PY-motif and WW domain residues, outside the core binding residues, in determining the specificity of WW domain-ligand interactions. Little was known about regulation of catalytic activity for this family of E3 ligases, and hence was the second focus of my work. Notably, Nedd4-2 contains a PY-motif within its HECT domain, raising the possibility that its catalytic activity is regulated by an interaction between its WW domains and HECT domain. Here I present evidence supporting a model in which a low-affinity interaction between the Nedd4-2 WW domains and its HECT domain ii regulate Nedd4-2 stability by preventing self-ubiquitination and subsequent degradation. Furthermore, evidence is presented suggesting that interaction between Nedd4-2 and the RING- E3 ligase Rnf11, a Nedd4-2 substrate, may also serve to regulate Nedd4-2 stability, as this interaction leads to decreased Nedd4-2 self-ubiquitination. Collectively, the studies presented here further our understanding of the substrate specificity and regulation of Nedd4-1 and Nedd4-2. iii Acknowledgments First of all, I would like to thank my supervisor Daniela Rotin for her support and guidance over the past 7 years. Daniela’s passion for science first inspired me while I was a summer student in her lab and is ultimately what led to my decision to pursue graduate studies. Thank you Daniela for allowing me to pursue my interests, for providing me the opportunity to work on multiple collaborations, and most of all for always believing in me. Thank you to all the members of the Rotin Lab, past and present, without whom this ride wouldn’t have been as enjoyable. Special thanks to Chris Fladd and Pauline Henry for teaching me the basics when I was ‘green’. Thanks to Voula Kanelis for helping me appreciate (and understand) structural biology, but more importantly for her inspirational passion for science and life. Thanks to Chong Jiang for doing an awesome job at running the lab and for fixing everything (or at least trying!) I would especially like to thank Wioletta Glowacka, my bench mate and my best friend, for her endless support, both scientific and personal, over the last three years. I will miss working beside you! I would also like to thank my graduate committee (Dr. Christine Bear and Dr. Sean Egan) for their guidance over the years and for always asking the tough questions. Special thanks to Carrie Harber in the Department of Biochemistry for being amazing at her job and for making life as a graduate student much easier. Finally I would like to thank my family. Thanks to my sisters Jenny, Lauren and Catherine for laughter, love and friendship. Thanks to my parents Linda and Bill for always believing in me. Your love, patience and encouragement made this PhD possible. Thanks to my second family the Bruces, especially Bob and Susan, for your continuous love and support. Above all, thanks to my husband Neil, for always being there, for your encouragement and confidence in me and for your endless love. iv Table of Contents Abstract ii Acknowledgments iv Table of Contents v List of Figures ix List of Tables xi Abbreviations xii Chapter 1: Introduction 1 I) Thesis Overview 2 II) The Ubiquitination Pathway 4 A) Components of the Pathway 5 i) Ubiquitin and Ubiquitin-like proteins 5 ii) Ubiquitin-activating enzymes (E1s) 11 iii) Ubiquitin-conjugating enzymes (E2s) 12 iv) Ubiquitin protein ligases (E3s) 13 1) RING E3s 14 2) HECT E3s 18 v) E4 enzymes 22 vi) Deubiquitinating enzymes (DUBs) 23 vii) Ubiquitin-binding domains (UBDs) 24 B) Fates of Ubiquitinated Proteins 28 i) Protein Degradation: The Ubiquitin-Proteasome Pathway 28 ii) Role of Ubiquitination in trafficking, endocytosis and protein sorting 30 1) Endocytosis of plasma membrane proteins 30 2) Protein sorting at the MVB/Late Endosome 32 3) Protein sorting at the Trans-Golgi Network (TGN) 34 4) Viral Budding 35 III) Nedd4 family of E3 ligases 37 A) Function of the different domains of Nedd4 proteins 37 v i) C2 domain 37 ii) WW domains 40 iii) HECT domain 41 B) Members of the Nedd4 family 41 C) Cellular targets of Nedd4-1 and Nedd4-2 42 i) Nedd4-2 43 ii) Nedd4-1 44 IV) Project Goals and Rationale 46 Chapter 2: Affinity and specificity of Nedd4-1 and -2 WW domains 47 for substrate PY motifs I) Summary 48 II) Introduction 49 III) Experimental Procedures 55 IV) Results 58 A) Contribution of WW3* domain to the Nedd4-ENaC interaction 58 i) Comparison of binding affinity of WW3* and WW4 toward the PY 58 motif of βENaC ii) Suppression of ENaC activity by the WW3*-containing dNedd4-1 62 iii) Homology modeling and comparison of WW3* and WW4 complexes 65 iv) Mutation analysis to test the role of WW3* Ala-504/Pro-505 in 66 conferring high affinity binding to PY motifs B) Identification of residues involved in high affinity interaction between 69 Drosophila Nedd4 WW3* and the Commissureless PY-motif i) Contributions of LPSY peptide residues to binding affinity 74 ii) Contributions of dNedd4 WW3* domain residues to binding affinity 75 V) Discussion 78 A) Importance of the WW3* domain in Nedd4-ENaC interactions 78 B) Role of the WW3* residues in high affinity binding with substrate PY motifs 79 C) Biological significance of Nedd4 WW domain specificity 80 vi Chapter 3: Regulation of Nedd4-2 self-ubiquitination and stability by a 83 PY-motif located within its HECT-domain I) Summary 84 II) Introduction 85 III) Experimental Procedures 86 IV) Results 88 A) The Nedd4-2 WW domains bind to the HECT-PY motif 88 and regulate catalytic activity of the HECT domain B) Nedd4-2 stability is regulated by self-ubiquitination 95 and subsequent degradation C) The HECT-PY motif regulates self-, but not substrate, 100 ubiquitination and stability V) Discussion 108 Chapter 4: Characterization of the Nedd4-2 interacting protein Rnf11 115 I) Summary 116 II) Introduction 117 III) Experimental Procedures 124 IV) Results 127 A) Expression and localization of Rnf11 127 B) Rnf11 binds both Nedd4-1 and Nedd4-2 132 C) Rnf11 exhibits in vitro ubiquitin ligase activity 135 D) Role of the Nedd4-2/Rnf11 interaction 144 i) Ubiquitination of Rnf11 by Nedd4-2 144 ii) Effect of Rnf11 on Nedd4-2 ubiquitination 148 V) Discussion 151 Chapter 5: Thesis Summary and Future Directions 158 I) Molecular determinants of WW domain specificity of Nedd4 family members 159 II) Regulation of Nedd4 E3 catalytic activity 161 vii III) Nedd4-2 binding protein Rnf11 163 References 167 Appendix I: Interactions between the three CIN85 SH3 Domains and Ubiquitin: 182 Implications for CIN85 Ubiquitination Appendix II: Transport of LAPTM5 to lysosomes requires association with 184 the ubiquitin ligase Nedd4, but not LAPTM5 ubiquitination Appendix III: Molecular determinants of voltage-gated sodium channel 186 regulation by the Nedd4/Nedd4-like proteins viii List of Figures Chapter 1 Figure 1-1 The ubiquitination cascade 7 Figure 1-2 Schematic view of opposite faces of ubiquitin 10 Figure 1-3 RING-finger E3s 17 Figure 1-4 The HECT domain 21 Figure 1-5 The Nedd4 family 39 Chapter 2 Figure 2-1 Alignment of Nedd4-1 and Nedd4-2 proteins 54 Figure 2-2 Representative curves of fluorescence emission from rNedd4-1 WW4, 60 x/m/hNedd4-2 WW3*, or hNedd4-1 WW3* binding to αPY, βPY, or γPY peptides of ENaC Figure 2-3 Suppression of ENaC by dNedd4-1 64 Figure 2-4 Homology model of Nedd4-2 WW3*·βENaC PY motif complex 68 LPSY Peptide Complex 71-כFigure 2-5 Solution Structure of the dNedd4 WW3 Domain-LPSY Peptide and rNedd4 73 כFigure 2-6 Comparison of the dNedd4 WW3 WW4 Domain-βENaC Complexes Chapter 3 Figure 3-1 A PY motif is present within the HECT domain 90 Figure 3-2 The Nedd4-2 HECT PY motif binds its WW domains and regulates 94 catalytic activity Figure 3-3 Both Nedd4-2(WT) and the HECT-PY mutant, Nedd4-2(YA), display 97 ubiquitination activity in an in vitro assay Figure 3-4 Mutation of the HECT PY motif affects Nedd4-2 stability 99 ix Figure 3-5 Nedd4-2(YA) ubiquitinates and regulates substrate as effectively as 102 Nedd4-2 (WT) Figure 3-6 Substrate ubiquitination promotes Nedd4-2 self-ubiquitination 105 Figure 3-7 Homology model of a Nedd4-2 WW3 domain–HECT PY motif complex 107 Figure 3-8 Nedd4-2(FL) (phenylalanine to leucine