Regulation of the 3Bp2 Adaptor Protein by the Nedd4 Family of Hect E3 Ubiquitin Ligases

REGULATION OF THE 3BP2 ADAPTOR PROTEIN BY THE NEDD4 FAMILY OF HECT E3 UBIQUITIN LIGASES

Lisa Gabrielli

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Medical Biophysics University of Toronto

ABSTRACT

Regulation of the 3BP2 Adaptor Protein by the Nedd4 Family of HECT Domain Containing E3 Ubiquitin Ligases

Lisa Gabrielli Master of Science, 2009 Department of Medical Biophysics, University of Toronto

3BP2 has been previously described as the protein mutated in the osteoporotic disorder, Cherubism. The gain of function mutation that characterizes Cherubism is the result of an uncoupling of its interaction with Tankyrase 2, which has been reported to stimulate 3BP2 ubiquitination. Here we describe an attempt at identifying the E3 ligase responsible for mediating this ubiquitination using four candidate members from the Nedd4 family. Based on their respective abilities to bind and ubiquitinate 3BP2, as well as their sensitivity to the presence of Tankyrase 2 and to 3BP2 mutations (including Cherubism mutations and mutations within the 3BP2 PPxY motif thought to confer binding to the Nedd4 proteins), we have determined that Smurf1 best fits our model. Further supporting these findings, we have seen an elevation in 3BP2 protein levels in macrophages derived from Smurf1-/-/Smurf2+/- mice. This work supports a role for the Nedd4 family member, Smurf1, in mediating 3BP2 ubiquitination.

ACKNOWLEDGEMENTS

Upon completion of this decidedly brief career as a graduate student, I find myself indebted to several people who have helped me in so many different ways.

To my supervisor, Dr. Robert Rottapel: Thank you for providing me with an abundance of experimental advice and suggestions; thank you for always challenging me to think critically, and finally, thank you for supporting my decisions and helping me to reach my goals.

To my committee members, Dr. Anne-Claude Gingras and Dr. Jane McGlade: Thank you for providing me with so many helpful comments and suggestions during our meetings and for always being approachable if I needed any additional advice.

To my friends and family: Thank you for providing me with an outlet to vent frustration and for being so supportive along the way. I would like to especially thank my father for showing such an interest in my work and for trying so hard to understand exactly what it is I do.

Finally, to the members of the Rottapel lab:

To our technicians Jose LaRose and Yael Levaot: Thank you for ensuring that the lab always ran smoothly. I would like to especially thank Jose, for answering so many of my questions and for giving me so much technical advice, but most importantly, for genuinely caring about both me and my work.

To the post-docs and associates, past and present, Liliana Clemenza, Yiannis Dimitriou, Noam Levaot, Dedi Meiri, Andy Scotter, Dan Simoncic and Oleksander Voytyuk,: Thank you for all of your advice and assistance and, more importantly, for being such wonderful coworkers.

Finally to the students, Andrea Brunet, Grace Chen, Jane Cullis, Noah Fine, Fiona Guerra, Bryan Kim and Mauricio Medrano: Thank you for being such great friends and for making every day in the lab as fun as humanly possible. I would like to especially thank Andrea, for so quickly becoming one

iii of my dearest friends, I'm not sure if I would have lasted a day without you, nor am I sure if I'll be able to handle our imminent separation. You will all be missed!

TABLE OF CONTENTS

Page ABSTRACT...... ii ACKNOWLEDGEMENTS...... iii TABLE OF CONTENTS...... v LIST OF FIGURES...... viii LIST OF TABLES...... x LIST OF SYMBOLS AND ABBREVIATIONS...... xi

CHAPTER 1: INTRODUCTION…………………………………………………………...………1 1.1 The Ubiquitin Reaction……………………………………………………………..………….1 1.1.1 E1 Ubiquitin Activating Enzyme……………………………………………..………..2 1.1.2 E2 Ubiquitin Conjugating Enzyme…………………………………...………..………2 1.1.3 E3 Ubiquitin Ligases…………………………………………………………..…...... 4 1.2 The Nedd4 Family of HECT E3 Ligases………………………………………….…...………6 1.2.1 Domain Organization……………………………………………………….….………8 1.2.2 Molecular Targets and Related Diseases……………………………………..……..…9 1.2.3 Regulation of Nedd4 Family of HECT E3s………………………………………..…12 1.3 The c-Abl Src Homology 3 Domain-Binding Protein 2 (3BP2)………………………..…….13 1.3.1 Domain Organization.…………………………………………………………..…….14 1.3.2 The 3BP2 Signaling Complex………………….…………………...... …….…….16 1.3.3 3BP2 and Cherubism..…………………………………………………….………….18 1.4 3BP2 Regulation…...……………………………..……………………………….……...…19 1.4.1 Regulation by Phosphorylation……………………………………………...... …...…19 1.4.2 3BP2 Regulation by Poly-(Adenosine Diphosphate) Ribosylation……….………….20 1.4.3 3BP2 Regulation by Ubiquitination………………………………………….....…….23 1.5 Thesis Rationale: Identification of a 3BP2-Associated E3 Ubiquitin Ligase…...... 24 CHAPTER 2: MATERIALS AND METHODS..………………………………….……….…….27 2.1 Mice…………………………………………………………………...…….……….…...…27 2.2 Reagents and Antibodies………..………………………………………….…………...…..27 2.3 Plasmids…..………..……………………………………………….…………….………...27 v

2.4 Cell Culture and Transfection………….………..………………….………………………28 2.5 Co-Immunoprecipitation………………..………..…………………………………………29 2.6 Re-precipitation Experiments…………..………..…………………………………….……29 2.7 Western Blotting………..…………………………………………………………………..30 2.8 Isolation of Bone Marrow Derived Macrophages……………………………………….…30 2.9 Software………..…………………………………………………………………………...31 CHAPTER 3: RESULTS..………..……………………………………………………...... ……32 3.1 3BP2 Interacts with Nedd4 Family HECT E3 Ligases………………………………….…32 3.2 3BP2 Ubiquitination is Stimulated in the Presence of Nedd4 Family E3 Ligases….....…..34 3.3 Ability of 3BP2_Y202A Mutant to Interact with Nedd4 Family E3 Ligases…….………..34 3.4 Ability of Y202A 3BP2 to become Ubiquitinated by Nedd4 E3 Ligases……………….…36 3.5 Ability of Tankyrase 2 to Stimulate 3BP2 Ubiquitination in the Presence of Nedd4 E3 Ligases…………………………………………………………………………………………....38 3.6 Ability of Cherubism Mutant 3BP2 (R413Q) to Bind Nedd4 Family E3 Ligases…….…...40 3.7 Ability of Cherubism Mutant 3BP2 (R413Q) to be Ubiquitinated in the Presence of Nedd4 Family Members………………………………………………………………………..…42 3.8 hSmurf1 Best Fits Proposed Model.………………………………………....………...... …45 3.9 Analysis of Endogenous 3BP2 Protein Levels in Smurf1-/-/Smurf2+/- Macrophages…….45 CHAPTER 4: DISCUSSION………..……………………………………………………………..48 4.1 3BP2 Interacts with Nedd4 Family HECT E3 Ligases………..……………………..……..48 4.2 3BP2 Ubiquitination is Stimulated in the Presence of Nedd4 Family E3 Ligases……..…..48 4.3 Ability of 3BP2_Y202A Mutant to Interact with Nedd4 Family E3 Ligases………….…..49 4.4 Ability of 3BP2_Y202A to become Ubiquitinated by Nedd4 E3 Ligases……………….....51 4.5 Ability of Tankyrase 2 to Stimulate 3BP2 Ubiquitination in the Presence of Nedd4 E3 Ligases…………………………………………………………………………………………....52 4.6 Ability of Cherubism Mutant 3BP2 (R413Q) to Bind to Nedd4 Family E3 Ligases……...54 4.7 Ability of Cherubism Mutant 3BP2 (R413Q) to be Ubiquitinated in the Presence of Nedd4 Family Members…………………………………………………………………………..55 4.8 Analysis of Endogenous 3BP2 Protein Levels in Smurf1-/-/Smurf2+/- Macrophages…….56 CHAPTER 5: FUTURE DIRECTIONS………………………………………………..………...58 5.1 A Role for the Nedd4 Family E3 Ligase AIP4/Itch in 3BP2 Ubiquitination………………58 5.2 Assessment of Endogenous/Semi-Endogenous Complex Formation between 3BP2 and

Nedd4 Family Proteins……………………………………………………………………...... 59 5.3 In Vitro Ubiquitination Reaction……….………..…………………………………………61 5.4 Knockdown Experiments………………………..………………………………………….61 5.5 Analysis of Smurf1-/-/Smurf2+/- and Smurf1+/-/Smurf2-/- Mice…………………...... 62 5.6 Analysis of "Itchy" Mice……………….………..………………………………………….63 CHAPTER 6: REFERENCES………………..………..…………………………………………..65 APPENDIX 1: REPLICATE EXPERIMENTS…………………………………………...... 76

vii

LIST OF FIGURES

Figure 1-1 The Ubiquitin Reaction……………...... 3 Figure 1-2 Nedd4 Family of HECT Domain E3 Ligases………...... 7 Figure 1-3 Domain Architecture of Src Homology 3 Binding Protein 2…...... 15 Figure 1-4 The 3BP2 Signaling Complex...... 17 Figure 1-5 Schematic Representation of Tankyrase 2...... 22 Figure 1-6 Model for 3BP2 Ubiquitination...... 26 Figure 3-1 3BP2 Interacts with Nedd4 Family E3 Ligases……...... 33 Figure 3-2 Nedd4 Family E3 Ligases Stimulate 3BP2 Ubiquitination...... 35 Figure 3-3 Ability of 3BP2_Y202A Mutant to Interact with Nedd4 Family E3 Ligases...... 37 Figure 3-4 Ability of 3BP2_Y202A to become Ubiquitinated by Nedd4 E3 Ligases...... 39 Figure 3-5 Ability of Tankyrase 2 to Stimulate 3BP2 Ubiquitination in the Presence of Nedd4 E3 Ligases...... 41 Figure 3-6 Ability of Cherubism Mutant 3BP2 (R413Q) to Bind to Nedd4 Family E3 Ligases…….43 Figure 3-7 Ability of Cherubism Mutant 3BP2 (R413Q) to be Ubiquitinated in the Presence of Nedd4 Family Members...... 44 Figure 3-8 Analysis of Endogenous 3BP2 Protein Levels in Smurf1-/-/Smurf2+/- Macrophages...... 46 Figure 5-1 Itch Interacts with 3BP2...... 60 Figure 5-2 Tankyrase 2 Augments the Ability of Itch to Mediate 3BP2 Ubiquitination...... 60 Figure A1-1(i) 3BP2 Interacts with Nedd4 Family E3 Ligases...... 76 Figure A1-1 (ii) 3BP2 Interacts with Nedd4 Family E3 Ligases...... 77 Figure A1-2 (i) Nedd4 Family E3 Ligases Stimulate 3BP2 Ubiquitination...... 78 Figure A1-2 (ii) Nedd4 Family E3 Ligases Stimulate 3BP2 Ubiquitination...... 78 Figure A1-3 (i) Ability of 3BP2_Y202A Mutant to Interact with Nedd4 Family E3 Ligases……….79 Figure A1-3 (ii) Ability of 3BP2_Y202A Mutant to Interact with Nedd4 Family E3 Ligases...... 80 Figure A1-4 (i) Ability of 3BP2_Y202A to become Ubiquitinated by Nedd4 E3 Ligases...... 81 Figure A1-4 (ii) Ability of 3BP2_Y202A to become Ubiquitinated by Nedd4 E3 Ligases ...... 82 Figure A1-5 (i) Ability of Tankyrase 2 to Stimulate 3BP2 Ubiquitination in the Presence of Nedd4 E3 Ligases ...... 83 Figure A1-5 (ii) Ability of Tankyrase 2 to Stimulate 3BP2 Ubiquitination in the Presence of Nedd4 E3 Ligases...... 84 Figure A1-6 (i) Ability of Cherubism Mutant 3BP2 (R413Q) to Bind to Nedd4 Family E3 Ligases ...... 85 Figure A1-6 (ii) Ability of Cherubism Mutant 3BP2 (R413Q) to Bind to Nedd4 Family E3 Ligases ...... 86 Figure A1-7 (i) Ability of Cherubism Mutant 3BP2 (R413Q) to be Ubiquitinated in the Presence of Nedd4 Family Members...... 87 Figure A1-7 (ii) Ability of Cherubism Mutant 3BP2 (R413Q) to be Ubiquitinated in the Presence of Nedd4 Family Members ...... 88 Figure A1-8 (i) Itch Interacts with 3BP2...... 89 Figure A1-8 (ii) Itch Interacts with 3BP2...... 89 Figure A1-9 (i) Tankyrase 2 Augments the Ability of Itch to Mediate 3BP2 Ubiquitination...... 90 viii

Figure A1-9 (ii) Tankyrase 2 Augments the Ability of Itch to Mediate 3BP2 Ubiquitination...... 90

LIST OF TABLES

Table 1 Summary of Binding and Ubiquitination Data……………………………………..…….46

LIST OF SYMBOLS AND ABBREVIATIONS

Amino Acids Ala/A/Alanine; Arg/R/Arginine; Asn/N/Asparagine; Asp/D/Aspartic Acid; Cys/C/Cysteine; Glu/E/Glutamic Acid; Gln/Q/Glutamine; Gly/G/Glycine; His/H/Histidine; Ile/I/Isoleucine; Leu/L/Leucine; Lys/K/Lysine; Met/M/Methionine; Phe/F/Phenylalanine; Pro/P/Proline; Ser/S/Serine; Thr/T/Threonine; Trp/W/Tryptophan; Tyr/Y/Tyrosine; Val/V/Valine

α alpha β beta γ gamma θ theta m milli, 1x10 -3 micro, 1x10 -6 n nano, 1x10 -9 U units M molar L litre g gram °C Degrees Celcius

19S 19 Svedberg 26S 26 Svedberg 3BP1 SH3 binding protein 1 3BP2 SH3 binding protein 2 αMEM alpha modified eagles medium ACK isotonic ammonium chloride buffer ADP adenosine diphosphate ANA anti-nuclear antibody AIP4 atrophin interacting protein 4 (also Itch) ATP adenosine triphospate BMP bone morphogenic protein Ca calcium Cbl protooncogene product of murine Cas NS-1 B cell lymphoma Cdc42 cell division cycle 42 cDNA complementary DNA CO 2 carbon dioxide C-terminal carboxy terminal CSF-1 colony stimulating factor 1 DMEN Dulbecco’s modified eagles medium DNA deoxyribonucleic acid E1 ubiquitin-activating enzyme E2 ubiquitin-conjugating enzyme E3 ubiquitin ligase EBNA1 Epstein-Barr virus nuclear antigen 1 ECL enhanced chemiluminescence xi

EDTA ethylenediaminetetraacetic acid EGTA ethylene glycol tetraacetic acid ENaC epithelial sodium channel ERK extracellular related kinase Flt3 FMS-like tyrosine kinase 3 (also fetal liver kinase 2, Flk-2) G-CSFR granulocyte colony stimulating factor receptor GEF guanine nucleotide exchange factor GTP guanosine triphosphate HA hemagglutinin HECT homology to E6 associated protein carboxy terminus HEK human embryonic kidney HPV human papilloma virus HRP horseradish peroxidase IB immunoblot IP immunoprecipitated IRAP insulin-responsive aminopeptidase JNK Jun N-terminal kinase kDa kilodalton Lck lymphocyte specific protein tyrosine kinase MAPK mitogen activated protein kinase MEF murine embryonic fibroblast MEKK1/2 MAP-ERK kinase kinase 1/2 Mg magnesium mRNA messenger ribonucleic acid NAD nicotinamide adenine dinucleotide Nedd4 neuronal precursor cells expressed developmentally downregulated 4 NEDL1/2 Nedd-like 1/2 (also HecW1/2) NFAT nuclear factor of activated T-cells N-terminal amino-terminal NuMA nuclear/mitotic apparatus protein PARP poly-ADP ribose polymerase PBS phosphate buffered saline PBS-T phosphate buffered saline tween PH pleckstrin homology PHD plant homeodomain PKC θ protein kinase C theta PLC γ phospholipase C gamma PMSF phenylmethanesulfonyl fluoride PR proline rich pS phosphoserine pT phosphothreonine PTKs protein tyrosine kinases PVDF polyvinylidene fluoride pY phosphotyrosine Rac ras-related C3 botulinum toxin substrate RING really interesting new gene RTKs receptor tyrosine kinases SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis xii

SGK1 serum and glucocorticoid-inducible kinase 1 SH2 Src homology 2 SH3 Src homology 3 shRNA short hairpin ribonucleic acid Smurf Smad Ubiquitin Regulatory Factor Syk spleen tyrosine kinase TAB182 182-kDa tankyrase-binding protein TGF-β Transforming Growth Factor-β Tiul1 TGIF-interacting ubiquitin ligase 1 (also WWP1) TNF tumor necrosis factor TRF1 telomere-repeat binding factor-1 Ub ubiquitin U-box UFD2-homology WCL whole cell lysate WT wild type WWP2 WW domain containing E3 ubiquitin protein ligase 2 ZAP70 zeta chain associated protein kinase 70

xiii

CHAPTER 1: INTRODUCTION

1.1 The Ubiquitin Reaction Ubiquitination is an elegant process by which protein levels (and by extension, protein functions) are controlled within the cell (reviewed by (Fang and Weissman, 2004)). The aptly named “ubiquitin” is a highly conserved, 76 amino acid polypeptide that is expressed throughout all eukaryotes. Covalent attachment of ubiquitin monomers to protein substrates occurs through a highly regulated chain of events that can drastically alter the fate and functions of these molecules, and whose disruption can lead to a multitude of pathological effects, which include (among countless others) cancer, neurodegenerative disorders and diabetes (reviewed by

(Petroski, 2008)).

Modification by ubiquitination has been reported to have multiple and diverse effects on protein signaling including protein kinase activation, transcription factor activity, DNA repair mechanisms and protein trafficking (Deng et al. , 2000; Hoege et al. , 2002; Pickart, 2002; Wang et al. , 2001). Two such modifications are monoubiquitination (the transfer of one ubiquitin moiety to a lysine sidechain) or multiubiquitination (the addition of one ubiquitin moiety to multiple lysine sidechains). Mono or multiubiquitination are important mechanisms by which membrane proteins are internalized and sorted by the endocytic compartment. Once internalized, the protein is either recycled (i.e.: returned to the plasma membrane) or sent to the multivesicular body and then to the lysosome for degradation (Bonifacino and Traub, 2003).

Newly synthesized proteins may also be monoubiquitinated, which targets them to the trans-

Golgi network for sorting, either destined for the plasma membrane or the lysosome (Bonifacino and Traub, 2003).

2 Perhaps the best characterized downstream effect of this covalent modification, however, is the targeting of ubiquitinated proteins to the 26S proteasome for degradation. This occurs through the attachment of polyubiquitin chains (i.e.: four or more monomers) linked by lysine

48 (K48) of ubiquitin, which results in their recognition by the 19S cap of the 26S proteasome

(Thrower et al. , 2000). The highly coordinated activities of the E1, E2 and E3 enzymes mediate this process, and serve as important points of regulation for this crucial cellular event.

1.1.1 E1 Ubiquitin Activating Enzyme The E1 (or ubiquitin activating) enzyme executes the first step of the ubiquitination pathway. Upon first binding to MgATP, the E1 forms a ubiquitin adenylate intermediate

(Pickart, 2001). This adenylate then serves as a donor to create a thiol-ester bond between the

E1 reactive site cysteine and the carboxy-terminal glycine of ubiquitin (Haas and Rose, 1982;

Hershko et al. , 1983). Each “fully loaded” E1 enzyme carries two ubiquitin monomers, one as an adenylate and one as a thiol-ester. The ubiquitin moiety is then said to be “activated” and can subsequently be transferred to the E2 (discussed below). The E1 enzyme shows the least diversity among the three classes of enzymes in the pathway and it is generally accepted that there is only one E1 governing all ubiquitin-associated processes (McGrath et al. , 1991), though there appear to be two isoforms in mammals (E1a and E1b) that are the result of two translation start sites (Handley-Gearhart et al. , 1994).

1.1.2 E2 Ubiquitin Conjugating Enzyme The E1, carrying the activated ubiquitin moiety, can then transfer it directly to the E2 (or ubiquitin conjugating) enzyme. Common to all E2s is an approximately 150 amino acid stretch known as the UBC domain. This domain contains an invariant cysteine residue that is

ATP Ub Ub + E1 E1 E2

E3 E2 SUBSTRATE Ub E2 Ub Ub Ub Ub SUBSTRATE E3 Ub SUBSTRATE

PROTEASOMAL SUBSTRATE E3 + DEGRADATION

Figure 1-1: The Ubiquitin Reaction. E1 Ubiquitin Activating Enzyme activates ubiquitin in an ATP-dependent step. Ubiquitin is then transferred to the E2 Ubiquitin Conjugating enzyme. The E2, charged with active Ubiquitin, interacts with the E3 Ubiquitin Ligase which carries substrate. Ubiquitin is transferred to the substrate and the cycle is repeated a minimum of three times. A polyubiquitin chain of four or more monomers gets recognized by the 26S proteasome and targets the complex for proteasomal degradation.

4 responsible for accepting the activated ubiquitin molecule from the E1. There are approximately 30 E2 enzymes reported throughout the mammalian genomes (von Arnim, 2001), and the vast majority of these proteins are between 14 and 36kDa in size. Most E2s can interact with multiple E3 ubiquitin ligases, which are generally believed to confer substrate specificity

(Hershko and Ciechanover, 1998; Hochstrasser, 1996).

1.1.3 E3 Ubiquitin Ligases Though there is only one E1 and approximately 30 E2s in mammals, the number of E3 ubiquitin ligases is comparatively vast in higher organisms. Though it is believed that there are still many E3 ligases that are yet unidentified, the estimate appears to be anywhere between several hundred to well over a thousand (reviewed by (Hershko and Ciechanover, 1998)), thus, it is the E3 that confers the most specificity toward protein targets. E3 enzymes are classified into families; of these the most common are the RING domain E3s and the HECT domain E3s.

The first identified family was the Homology to E6-Associated Protein Carboxy

Terminus (HECT) domain-containing proteins (Huibregtse et al. , 1995), whose prototypic member (i.e.: E6-AP) is encoded by the human papilloma virus (HPV), and is responsible for conferring the proteasomal degradation of p53, which is essential to the development of HPV- associated cervical cancer (Scheffner et al. , 1994). The HECT domain is a highly conserved structure of approximately 350 amino acids and contains an essential reactive cysteine residue approximately 35 amino acids upstream of its C-terminus (Scheffner et al. , 1995). This cysteine residue accepts the ubiquitin moiety from the E2 and directly transfers it to the substrate

(Weissman, 2001). Substrate specificity and subcellular localization are conferred by domains within the amino-termini of the molecules (Weissman, 2001).

5 The second group of E3 ligases for which there has been extensive study are the RING

(Really Interesting New Gene) finger domain containing family (Aravind and Koonin, 2000;

Jackson et al. , 2000; Joazeiro and Weissman, 2000). RING fingers are approximately 40 to 100 amino acids in length and are characterized by a stretch of histidine and cysteine residues that are capable of coordinating zinc ions (Borden and Freemont, 1996). The main functional distinction between HECT and RING E3s is that while HECT E3s directly ubiquitinate their substrates (via the catalytic cysteine residue present within the HECT domain), RING domain

E3s merely serve as scaffolds to bring the E2 and substrate into proximity such that the E2 can transfer ubiquitin onto the substrate (Borden, 2000).

There is an emerging body of evidence implicating two RING finger related domains in the ubiquitination reaction, namely, the PHD (plant homeodomain) and U-box (UFD2- homology) domains. The PHD finger is a RING variant that has a cysteine in place of a histidine in the forth predicted zinc coordinating position and a tryptophan residue directly upstream of the seventh zinc binding residue (Capili et al. , 2001). An important PHD finger containing protein is the mammalian MEKK1 which has not only been shown to be implicated in the activation of Mitogen Activating Protein Kinase (MAPK) but has also been shown to confer its ubiquitination (Lu et al. , 2002).

U-box containing proteins, conversely, are unable to coordinate zinc ions and are characterized by stretches of charged and polar residues which structural predictions have shown to resemble the three-dimensional structure of a RING domain (Aravind and Koonin,

2000). In addition, numerous studies have shown them to mediate ubiquitination in a manner which is quite similar to that of the classic RING finger proteins (Hatakeyama et al. , 2001).

6 Still further classification of E3s depends on their existence as either single or multi- subunit proteins, and additionally, whether or not the ligase acts as a monomer or whether it has the capacity to homo- or heterodimerize in order to further diversify or fine tune its function.

1.2 The Nedd4 Family of HECT E3 Ligases Of the members of the HECT domain containing E3 ubiquitin ligases, members of the neural precursor cells-expressed developmentally down-regulated 4 (Nedd4) family have been the most extensively studied. Since Nedd4’s original discovery in 1992 (Kumar et al. , 1992), eight related proteins in mouse and human have been identified (namely, Nedd4-2/Nedd4L,

WWP1/Tiul1, WWP2, AIP4/Itch, Smurf1, Smurf2, HecW1/NEDL1 and HecW2/NEDL2). To add further complexity to this family of molecules, many of these proteins have also been shown to contain multiple splice variants (Chen et al. , 2001; Dunn et al. , 2002; Flasza et al. , 2002; Itani et al. , 2003).

In Saccharomyces cerevisiae the family consists of only one member (Rsp5p) but is known to regulate several cellular processes including internalization of cell surface receptors

(reviewed by (Rotin et al. , 2000)), gene transcription (Huibregtse et al. , 1997) and mitochondrial inheritance (Fisk and Yaffe, 1999). Additionally, it has further been shown that disruption of the rsp5 gene is lethal (Hein et al. , 1995).

In Schizosaccharomyces pombe , Caenorhabditis elegans and Drosophila melanogaster the family has expanded to have 3 members. Genetic disruption of 2 of the 3 members in S. pombe (namely Pubp1 and Pubp3 ) results in embryonic lethality (Tamai and Shimoda, 2002) whereas disruption in only one gene in C. elegans ( ceWWP1 ) or in D. melanogaster ( dSmurf ) causes defects in the developing embryo (Huang et al. , 2000; Podos et al. , 2001). Though the human and murine family members do share limited overlapping functional similarities,

C2 WW HECT H. sapiens

hNEDD4-1

hNEDD4-2

AIP4/Itch hWWP-1 hWWP-2

hSmurf2 hSmurf1 hNEDL hNEDL2

Figure 1-2: Nedd4 Family of HECT Domain E3 Ligases. Schematic representation of H. sapiens Nedd4 family of HECT domain containing E3 ligases. C2 domain represented by green box, WW domain depicted by lilac circle and HECT domain displayed by blue bar. Relative sizes and domain architecture not to scale.

8 specificity has been demonstrated for a number of these proteins, as will be described in detail below.

1.2.1 Domain Organization As previously discussed, the Nedd4 family of E3 ligases all contain a C-terminal HECT domain, which confers the ligase activity of the molecule. Upstream of the HECT are a C2 domain and between 2 and 4 WW domains in the human and murine family members (reviewed by (Ingham et al. , 2004)). The C2 domain is responsible for intracellular localization and was first identified as a Ca 2+ dependent phospholipid binding domain (reviewed by (Rizo and

Sudhof, 1998)). Consisting of approximately 120 amino acids, the C2 domain has multiple conserved aspartate residues that are responsible for Ca 2+ binding (Essen et al. , 1996; Shao et al. , 1996; Sutton et al. , 1995) and has been shown to interact with several proteins and phospholipids (Nalefski and Falke, 1996).

The WW domains are approximately 35 amino acids in length and are characterized by two tryptophan residues spaced 20-22 amino acids apart (reviewed by (Macias et al. , 2002;

Zarrinpar and Lim, 2000)). WW domains are generally involved in modulating protein-protein interactions and have been reported to bind proline-rich motifs. These motifs include those of sequence PPxY (single letter amino acid code, where x is any amino acid) (Chen and Sudol,

1995), PPLP (Bedford et al. , 1997) and phospho-serine and threonine residues directly upstream of a proline (Lu et al. , 1999; Yaffe et al. , 1997). While the PPxY motif is the most common interacting partner of a Nedd4 family WW domain, interactions with phospho-serine and threonine residues have also been reported (Lu et al. , 1999).

9 1.2.2 Molecular Targets and Related Diseases While there does seem to be a degree of functional overlap between Nedd4 family members, there is a definite preference and selectivity among targets. Nedd4 family members

Nedd4-1 (Staub et al. , 1996) WWP2 (McDonald et al. , 2002) and Nedd4-2 (Debonneville et al. ,

2001; Harvey et al. , 2001) are essential to stimulating the ubiquitination, internalization and subsequent lysosomal degradation of the epithelial sodium channel (ENaC) (Staub et al. , 1997).

This structure is crucial to the maintenance of fluid and electrolyte concentrations in the kidney, lung and colon (Garty and Palmer, 1997; Rossier et al. , 2002). Specificity for the ENaC is accomplished through preferential interaction with a specific WW domain over others within the

E3 ligase (reviewed by (Ingham et al. , 2004)).

Disruption of this interaction is thought to be linked to the hypertensive disorder,

Liddle’s Syndrome (Botero-Velez et al. , 1994). Indeed, mutations that cause Liddle’s syndrome are localized to the carboxy-terminal tail of the β or γ subunit of the ENaC (Botero-Velez et al. ,

1994) and cause disruption or complete abolishment of the PPxY motif found within this structure, leading to an increase in its stability and a subsequent increase in the uptake of sodium ions (Abriel et al. , 1999; Goulet et al. , 1998; Schild et al. , 1996; Snyder et al. , 1995). The

Nedd4:ENaC interaction has also been reported to be augmented by phosphorylation of threonine residues in both the β and γ subunits by the extracellular regulated kinases (ERK) 1 and 2 (Shi et al. , 2002). Nedd4-1 overexpression has also been detected in prostate and bladder cancer (reviewed by (Chen and Matesic, 2007)).

Nedd4 family members Smurf (Sm ad Ubiquitin Regulatory Factor) 1 and 2 are important regulators of the Transforming Growth Factor-β (TGF-β) superfamily which regulate critical signaling processes including growth, differentiation and apoptosis (reviewed by (Sporn and Roberts, 1990)). The effectors of TGF-β signaling are Smad proteins; there are eight Smads in total, Smads 1, 2, 3, 5 and 8 are the receptor-regulated (R-Smads) (Massague, 1998) which,

10 when phosphorylated, bind to Smad 4 (also known as the common Smad) and are then capable of translocating to the nucleus to affect gene transcription (Akiyoshi et al. , 1999; Luo et al. ,

1999; Stroschein et al. , 1999; Sun et al. , 1999; Xu et al. , 2000). Smad proteins 6 and 7 are known as inhibitory or I-Smads that interact with the activated receptor to block the ability of R-

Smads to become phosphorylated (Hayashi et al. , 1997; Imamura et al. , 1997; Nakao et al. ,

1997). Though Smurf1 and Smurf2 (sharing high sequence homology) both regulate this pathway, their molecular targets are quite specific.

Smurf1’s activity is restricted to R-Smads 1 and 5 which are effectors specifically related to Bone Morphogenic Protein (BMP) signaling (Zhu et al. , 1999). Through interaction with PPxY motifs present on both Smad 1 and Smad 5, Smurf1 is capable of binding to them in order to stimulate their degradation. I-Smad 7 also contains a PPxY motif, and while Smurf1 can stimulate its ubiquitination and subsequent degradation, it is also capable of forming a complex with this molecule in order to mediate ubiquitination of the TGF-β type I receptor

(Ebisawa et al. , 2001). This occurs as a result of Smurf1’s translocation to the nucleus where it interacts with Smad 7, the protein complex is then transported to the cytoplasm, where Smad7 acts as an adaptor protein in order to mediate an interaction between Smurf1 and the TGF-β type

I receptor. This, in turn, results in the receptor’s ubiquitination and subsequent degradation via the proteasome (Ebisawa et al. , 2001).

Smurf1 has also been shown to stimulate degradation of the osteoblast specific transcription factor, Runx2 (Ying et al. , 2003; Zhao et al. , 2004; Zhao et al. , 2003; Zhu et al. ,

1999) in a Tumor Necrosis Factor (TNF) dependent manner (Kaneki et al. , 2006). Smurf1 has further been reported to stimulate MEKK2 degradation, thus regulating osteoblast activity and bone homeostasis (Yamashita et al. , 2005). Smurf-1 -/- mice, though they survive to adulthood

(presumably due to compensation by the closely related homolog Smurf2), have an age-

11 dependent increase in bone mass (Yamashita et al. , 2005). Overexpression of Smurf1 has also been detected in pancreatic cancer (reviewed by (Chen and Matesic, 2007)).

Smurf2 has similarly been reported to interact with R-Smads 1 and 5, I-Smad 7 (Kavsak et al. , 2000) and can also stimulate Runx2 degradation stimulated by TNF overexpression

(Kaneki et al. , 2006). In conjunction with Smurf1, Smurf2 has also been reported to regulate planar cell polarity through non canonical Wnt signaling (Narimatsu et al. , 2009). Distinct from

Smurf1, however, Smurf2 is localized to the nucleus where it can interact with R-Smads 2 and 3

(Lin et al. , 2000; Zhang et al. , 2001). Smurf2 is found to be overexpressed in particularly metastatic and invasive forms of esophageal squamous cell carcinoma (Fukuchi et al. , 2002).

Alternative splice forms of Smurf2 have also been shown to be present in both prostate and breast cancer cell lines (Chen and Matesic, 2007).

Itch/Atrophin Interacting Protein 4 (AIP4) is yet another member of the Nedd4 family about which an extensive body of literature has been produced. Most Itch targets are signaling proteins that are crucial to immune system function. Itch is known to bind both JunB and c-Jun via their PPxY motifs and stimulates their downregulation in T-cells, thus regulating T-cell differentiation (Fang et al. , 2002; Fang and Kerppola, 2004; Oberst et al. , 2007). Indeed, mice homozygous for an Itch loss of function mutation display T-cell hyperproliferation and abnormal allergic responses (Fang et al. , 2002).

Other Itch targets important for T-cell function include PKC θ and PLC γ-1. Itch has been shown to stimulate monoubiquitination of these proteins, thus targeting them for lysosomal degradation (Heissmeyer et al. , 2004). Itch deficient mice display impaired T-cell anergy (i.e.: self-tolerance) as a direct result of an increase in PKC θ and PLC γ-1 protein levels (Heissmeyer et al. , 2004).

12 A third and similarly crucial Itch target is the Notch protein (Qiu et al. , 2000). Though

Notch1 does not contain a PPxY motif, binding to Itch is instead accomplished through interactions with Notch ankyrin repeats. Notch is said to be one of the most important Itch targets contributing to the autoimmune phenotype, as an increase in Notch1 signaling results in an autoimmune-like disease which phenocopies the autoimmune and inflammatory symptoms that are displayed by Itch deficient mice (Matesic et al. , 2006). Finally, Itch has been shown to be an important regulator of p53 family members p73 and p63 which are regulators of cell cycle arrest and apoptosis (Rossi et al. , 2006; Rossi et al. , 2005).

1.2.3 Regulation of Nedd4 Family of HECT E3s Due to the overwhelming number of proteins regulated by the Nedd4 family of HECT domain containing E3 ligases, regulation of the ligases themselves is critically important.

Though the Nedd4 family E3s are generally ubiquitously expressed, there does seem to be preferential expression of certain members over others in a tissue-specific manner. For example, Nedd4-1 mRNA expression in mouse is highest in kidney, liver, muscle, brain and heart (Kumar et al. , 1997) whereas Nedd4-2 mRNA levels have been shown to be most elevated in rat testis, kidney, liver, lung, brain and heart (Umemura et al. , 2006). Human Smurf1 mRNA shows highest expression in placenta, pancreas and testis (Komuro et al. , 2004) while Itch expression in humans is concentrated in the heart, brain, placenta, muscle and pancreas, though it is also expressed quite highly in spleen, lung, testis and liver (Wood et al. , 1998).

Additionally, mRNA expression of some Nedd4 family members has been shown to be responsive to growth factor signals, for example, the transcription of Smurf1 and Smurf2 mRNA is induced by both BMP and TGF β (Ohashi et al. , 2005) and (along with Nedd4-1) also induced by TNF α (Judge et al. , 2007; Kaneki et al. , 2006).

13 Regulation is also accomplished, in part, by phosphorylation of the E3 ligases. Itch phosphorylation by JNK increases its ability to ubiquitinate both JunB and c-Jun (Gao et al. ,

2004). Conversely, however, Fyn phosphorylation of Itch on tyrosine 371 decreases its capacity to stimulate JunB ubiquitination (Yang et al. , 2006). Nedd4-2 is also sensitive to regulation by phosphorylation; its serine phosphorylation by serum and glucocorticoid-inducible kinase

(SGK1) promotes 14-3-3 binding and significantly decreases its ability to stimulate ENaC degradation (Snyder et al. , 2004).

Not surprisingly, subcellular localization also plays an important part in regulation of the

Nedd4 family E3s. Itch, for example, has been shown to localize to the endosomal compartment, mediated by its C2 domain (Angers et al. , 2004; Marchese et al. , 2003). Nedd4-1 and Smurf1 have been reported to contain nuclear export signals (Hamilton et al. , 2001; Tajima et al. , 2003) whereas Smurf2 is reported to localize to the nucleus but also has the capacity to travel to the cytoplasm to stimulate TGF-β type I receptor degradation (Ebisawa et al. , 2001).

1.3 The c-Abl Src Homology 3 Domain-Binding Protein 2 (3BP2) The c-Abl Src Homology 3 Domain-Binding Protein 2 (3BP2) was first identified (along with 3BP1) as an interacting partner for the c-Abl Src Homology 3 (SH3) domain (Ren et al. ,

1993). The gene is located on the human chromosome 4p16.3 which is frequently deleted in bladder cancer and Wolf-Hirschorn syndrome (Bell et al. , 1997; Zollino et al. , 2000). The 13 exon gene encodes a 561 amino acid gene product in humans (and 559 in mouse), yielding a protein of an approximate molecular weight of 63 kDa (Bell et al. , 1997; Deckert et al. , 1998).

3BP2 expression is thought to be ubiquitous in humans, though highest expression is reported in cells of hematopoetic and lymphoid origin (Deckert et al. , 1998; Foucault et al. , 2005;

Jevremovic et al. , 2001; Sada et al. , 2002). In mouse, 3BP2 expression is highest in B

14 lymphocytes, osteoclasts, osteoblasts, macrophages and oocytes (BioGPS Website and

Database, Genomics Institute of the Novartis Research Foundation, 2009).

1.3.1 Domain Organization As mentioned, the 3BP2 adaptor protein is approximately 63 kDa in size. It consists of an amino-terminal Pleckstrin Homology (PH) domain, a carboxy-terminal Src Homology 2

(SH2) domain and an intervening Proline Rich (PR) region. The presence of a PH domain suggests that 3BP2 may bind phospholipids, perhaps indicating that it is localized to the plasma membrane. Indeed, work by our lab has shown that 3BP2 does interact with several membrane phospholipids (unpublished data). Experiments in both COS and Jurkat T cells show that while

3BP2 is largely localized to the cytoplasm there is basal association with the plasma membrane

(Deckert et al. , 1998). Additionally upon anti-CD3 stimulation in COS cells or T cell receptor stimulation in Jurkats, 3BP2 was rapidly distributed to the membrane, suggesting that its membrane localization is largely inducible (Deckert et al. , 1998).

The proline rich region that stretches between the PH and SH2 domains confers the SH3 binding ability of 3BP2. Work by our lab and others have shown that this region can bind not only to the c-Abl SH3 (Ren et al. , 1993) but also that of guanine nucleotide exchange factor Vav

(Foucault et al. , 2005), and non-receptor tyrosine kinases Fyn (Deckert et al. , 1998), Lyn

(Maeno et al. , 2003) and Src (Levaot et al ., manuscript in preparation). Work in our lab also suggests that the SH3-domain mediated binding of 3BP2 to Src and Abl leads to their activation

(Levaot et al., manuscript in preparation). Finally, the proline rich region contains multiple putative binding sites for both WW and EVH1 domains (Kay et al. , 2000), as both structures are known to bind sequences with high proline content.

PH SH2

Figure 1-3: Domain Architecture of Src Homology 3 Binding Protein 2. The 3BP2 adaptor protein consists of an amino-terminal pleckstrin homology (PH) domain, a carboxy-terminal Src Homology 2 (SH2) domain and an intervening proline rich (PR) region. The full length polypeptide is approximately 63 kDa in size.

The most C-terminal domain is the SH2, responsible for binding phospho-tyrosine residues, with the optimal binding sequence reported as YEN (single letter amino acid code)

(Deckert and Rottapel, 2006). Through its SH2, 3BP2 has been shown to interact with receptor tyrosine kinases Granulocyte-Colony Stimulating Factor Receptor (G-CSFR) (Kendrick et al.,

2004) and Flt3 (our unpublished data) and non-receptor tyrosine kinases Syk and ZAP70

(Deckert et al., 1998; Foucault et al., 2005). Other SH2 interacting partners include the tyrosine phosphorylated versions of Vav2 (Foucault et al., 2005), PLC γ and Cbl (Deckert et al., 1998;

Foucault et al., 2005; Jevremovic et al., 2001; Sada et al., 2002).

1.3.2 The 3BP2 Signaling Complex As an adaptor protein (having no enzymatic activity itself), 3BP2 can act as a scaffold to bring its interacting partners into close proximity in order to mediate and regulate their respective functions. In osteoclasts 3BP2 has been shown to activate Src, downstream of the

-/- αvβ3 integrin (Levaot et al ., manuscript in preparation). 3BP2 osteoclasts fail to properly activate not only Src but also the non-receptor tyrosine kinase Syk, the guanine nucleotide exchange factor Vav and the Rac1 GTPase, thus suggesting that 3BP2 is required to organize these proteins into a productive signaling complex in order to achieve proper osteoclast formation and function (Levaot et al., manuscript in preparation). 3BP2 deficient neutrophils similarly show defects in the activation of Src, Vav1, and the GTPases Rac2 and cdc42 (Chen et al., manuscript in preparation). Finally, 3BP2 null B lymphocytes show impaired B cell receptor signaling, including a defect in Syk activation (Chen et al. , 2007). The recurrence of common signaling defects relating to the Src and Syk family of non-receptor tyrosine kinases, the Vav family of guanine nucleotide exchange factors and the Rac and Cdc families of

OPN αv β3

3BP2 Src

Vav

Actin Remodeling

Figure 1-4: The 3BP2 Signaling Complex. Upon ligand binding, integrin activation results in recruitment of 3BP2 which then serves to activate signaling molecules Src and Vav, leading to downstream effects such as Actin remodeling and cytoskeletal rearrangement in osteoclasts.

18 GTPases in 3BP2 null cells (of various lineages) strongly implicates 3BP2 as an important modulator of their functions in vivo .

1.3.3 3BP2 and Cherubism 3BP2 mutation is the cause of the autosomal dominant, osteoporotic disorder Cherubism, which is characterized by an increase in osteoclast function and giant cell granuloma formation

(Ueki et al. , 2001). Patients affected are mainly males, and present with excessive bone resorption in the mandible and maxilla, resulting in what has been described as a “heavenly gaze” (Quan et al. , 1995). A mouse model for Cherubism has recently been established; it harbours the most common mutation occurring in Cherubism patients (P416R) and exhibits trabecular bone loss, cortical bone erosion and TNFα dependent systemic inflammation (Ueki et al. , 2007). While survival of heterozygotes is comparable to wild type mice, homozygous mutants display a significant decrease in viability, with only 50% survival at 25 weeks due to widespread macrophage infiltration into the skin, synovial lining, and visceral organs including the liver, lung, and stomach and also possess excessive amounts of serum TNF α (Ueki et al. ,

2007).

Mutations in 3BP2 that cause Cherubism are restricted to a defined hexapeptide region of sequence RSP PDG (mutated residues highlighted) which resides 31-36 amino acids upstream of the SH2 domain. The gain of function phenotype that characterizes Cherubism is the result of an increase in 3BP2 steady state protein levels (Voytyuk et al ., manuscript submitted).

Additionally, consistent with our data on 3BP2 -/- mice, osteoclasts derived from Cherubism knock-in mice show hyperphosphorylation of Src, Syk and Vav with a correlated increase in

Rac1GTP loading (Voytyuk et al ., manuscript submitted). .

19 1.4 3BP2 Regulation As it is implicated in the activation of many downstream signaling effectors, regulation of the adaptor protein itself becomes critically important. Acting downstream of αvβ3 integrin,

3BP2 deregulation (as seen in Cherubism) results in hyperactivation of both Src and Vav

(Voytyuk et al ., manuscript submitted). We have shown that, in osteoclasts, this results in an increase in osteoclastogenesis and activity, but in a different cellular background, (for example, in epithelial cells), the same deregulation may result in malignancy. Indeed, tumours with increased osteopontin expression (an αvβ3 ligand) are associated with increased tumor invasion, progression and/or metastasis in cancers of the breast, stomach, lung, liver, prostate and colon

(Rittling and Chambers, 2004; Wai and Kuo, 2004). It is therefore attractive to speculate that this may be due to an increase in Src and/or Vav activity (Katzav, 2007; Yeatman, 2004) and thus an increase in 3BP2 protein expression may result in a similar phenotype, though to date this has not been formally tested.

1.4.1 Regulation by Phosphorylation Important to the regulation of many proteins is phosphorylation, both on tyrosine and serine/threonine residues. 3BP2, containing multiple sites of phosphorylation has been shown to be regulated in this manner. Phosphorylation of serine residues S225 and S277 (in human) result in binding of the 14-3-3 chaperone protein (Foucault et al. , 2003). This interaction negatively regulates 3BP2 function as NFAT activation in T lymphocytes (which is a known

3BP2 target) is increased when serine to alanine mutations are introduced (as these residues are no longer capable of being phosphorylated, thus preventing 14-3-3 binding) (Deckert et al. ,

1998; Foucault et al. , 2003).

3BP2 also contains several tyrosine residues that have been shown to confer sites of phosphorylation on the polypeptide. Tyrosine phosphorylation by the Syk family of protein

20 tyrosine kinases has been reported at residues Y174, Y183 and Y448 (in human) in response to both T and B cell receptor activation, as well as activation of Fc receptors (Maeno et al. , 2003;

Qu et al. , 2005). Upon phosphorylation, Y183 mediates an interaction with the Vav SH2 domain and with PLC γ (Jevremovic et al. , 2001) while Y448 phosphorylation leads to binding of both the Lyn and Lck SH2 domains (Maeno et al. , 2003; Qu et al. , 2005). These phosphotyrosine dependent interactions have all been reported to stimulate activation of the binding partner and lead to the associated activation of downstream effector signaling (Jevremovic et al. ,

2001; Maeno et al. , 2003; Qu et al. , 2005).

1.4.2 3BP2 Regulation by Poly-(Adenosine Diphosphate) Ribosylation Poly-(Adenosine Diphosphate) ribosylation (poly-(ADP) ribosylation) is a post- translational modification that has been reported to affect a myriad of cellular processes including modulating protein-protein interactions and protein-nucleic acid interactions as well as affecting chromatin structure, DNA repair, replication, transcription and telomere homeostasis

(reviewed by (Hsiao and Smith, 2008)). Roles for poly-ADP ribosylation have also been postulated for controlling cell cycle progression, cell division, cell proliferation and even for the control of programmed cell death (reviewed by (Hakme et al. , 2008)).

Poly-ADP ribosylation is accomplished through the action of poly-ADP ribose polymerases (PARPs). Poly-ADP ribosylation occurs through the addition of ADP ribose moieties onto acidic residues by PARP containing proteins, using nicotinamide adenine dinucleotide (NAD +) as a substrate in an ATP-dependent manner.

Of the 17 PARP family members in humans, Tankyrase 1 and 2 (or PARP5a and 5b) are of particular interest to the study of 3BP2 modification as they bind to proteins containing consensus RxxPDG (reviewed by (Hsiao and Smith, 2008)) which precisely describes the 3BP2

21 hexapeptide found to be mutated in Cherubism. Tankyrase 1, consists of an amino terminal

HPS region (that is, homopolymeric tracts of His Pro and Ser), followed by an ankyrin repeat region responsible for binding to protein targets and a sterile alpha module or SAM domain which is responsible for homo- or hetero-multimerization of Tankyrase molecules (De Rycker and Price, 2004; De Rycker et al. , 2003; Sbodio et al. , 2002). The PARP domain is located at the carboxy terminus, and, as previously discussed, confers the enzymatic activity of the molecule. The domain structure of Tankyrase 2 is quite similar to Tankyrase 1, though it lacks an HPS region.

Tankyrase 1 and 2 interact with several proteins, including TRF1, IRAP, Tab182, NuMA and EBNA1, most of which contain the RXXPDG consensus (or are only slightly divergent)

(reviewed by (Hsiao and Smith, 2008)). Poly-ADP ribosylation by Tankyrase molecules has been shown to be essential to many cellular processes including maintenance of telomere length, sister telomere association, and trafficking of glut4-containing vesicles (Chang et al. , 2005;

Cook et al. , 2002; Dynek and Smith, 2004; Smith and de Lange, 2000; Yeh et al. , 2007).

Having 85% amino acid sequence identity (Kuimov et al. , 2001; Smith et al. , 1998), Tankyrase

1 and 2 have been shown to display partial functional redundancy as Tankyase 1 seems to compensate for a lack of Tankyrase 2 in the cell (Chiang et al. , 2006; Hsiao et al. , 2006). The

Tankyrase1 -/-/Tankyrase 2 -/- mouse, however, displays embryonic lethality at day E10.5 (Chiang et al. , 2008). Thus, though the two proteins show some functional redundancy, Tankyrase is indeed essential to survival of the developing embryo.

Using 3BP2 as bait, Tankyrase 2 was identified as a putative binding partner via a yeast two-hybrid screen using a hematopoetic cell library and this interaction was then further validated by co-immunoprecipitation experiments (Voytyuk et al ., manuscript submitted).

3BP2 was also found to be a bona fide substrate of Tankyrase 2 as Tankyrase 2 can stimulate

Ankyrin Repeat Region SAM PARP

Figure 1-5 Schematic Representation of Tankyrase 2. The 120 kDa Tankyrase 2 consists of an N-terminal ankyrin repeat region responsible for mediating protein-protein interactions, an internal sterile alpha module (SAM) responsible for modulating homo- and hetero-multimerization of Tankyrase molecules and a C-terminal poly-ADP ribose polymerase (PARP) domain which confers the protein’s enzymatic activity.

23 3BP2 ribosylation in vitro (Voytyuk et al ., manuscript submitted). Complex formation and subsequent ribosylation of 3BP2 by Tankyrase 2 is abolished if a Cherubism mutation is introduced into the wild type 3BP2 protein sequence (Voytyuk et al ., manuscript submitted).

The poly-ADP ribosylation of 3BP2 by Tankyrase 2 seems to have a negative effect on its steady state protein levels. Indeed, in the presence of Tankyrase 2, 3BP2 protein levels are decreased as assayed by Western blot (Voytyuk et al ., manuscript submitted). In addition, if one compares wild type murine embryonic fibroblasts (MEFs) to MEFs derived from mice with a PARP domain deleted version of Tankyrase 2, a dramatic increase in 3BP2 steady state protein levels is observed (Voytyuk et al ., manuscript submitted). Thus, it appears that the increase in

3BP2 protein levels observed in Cherubism is a direct result of the inability of this mutated form of 3BP2 to bind to and be ribosylated by Tankyrase 2.

1.4.3 3BP2 Regulation by Ubiquitination As discussed in section 1.1, ubiquitination is an important modification which is crucial to a plethora of cellular processes, and most commonly results in protein degradation. Work in our lab has shown that 3BP2 is indeed sensitive to proteasomal degradation, as assayed by its sensitivity to the proteasome inhibitor, MG132. Though 3BP2 protein levels decrease in the presence of Tankyrase 2, this effect can be rescued with MG132 treatment (Voytyuk et al ., manuscript submitted).

Also of interest is the ability of Tankyrase 2 to stimulate 3BP2 ubiquitination. In the absence of Tankyrase 2, 3BP2 ubiquitination is virtually undetectable, however, with overexpression of Tankyrase 2 in the system, 3BP2 ubiquitination is dramatically increased

(Voytyuk et al ., manuscript submitted). While this data is encouraging, the discovery of an E3 ubiquitin ligase responsible for mediating 3BP2 ubiquitination will be essential to a better

24 understanding of 3BP2 regulation at the protein level and may prove to be critical for further elucidation of the methods by which the 3BP2 signaling pathway is controlled.

1.5 Thesis Rationale: Identification of a 3BP2-Associated E3 Ubiquitin Ligase 3BP2, which has been shown to undergo ubiquitination in a Tankyrase 2 dependent manner and is also sensitive to proteasomal degradation, contains, within its proline rich region, a canonical PPxY motif (single letter amino acid code, where x is any amino acid) of sequence

PPAY. PPxY motifs are known to interact with WW domains (Chen and Sudol, 1995), which are structures present throughout the Nedd4 family of HECT domain containing E3 ubiquitin ligases. Based on the hypothetical interaction between the 3BP2 PPxY motif and the Nedd4 family WW domains, they appear to be excellent candidate E3 ligases for 3BP2. We favour a model by which 3BP2, upon poly-ADP ribosylation by Tankyrase 2, becomes more sensitive to ubiquitination mediated by a member (or members) of the Nedd4 family of HECT E3 ligases. It is attractive to speculate that this enhanced sensitivity could be a direct result of an increase in binding affinity for a poly-ADP ribosylated molecule, thus stabilizing the interaction with a

Nedd4 family E3 ubiquitin ligase and subsequently enhancing its ability to stimulate 3BP2 ubiquitination. In this manner, the binding of 3BP2 to a Nedd4 family E3 would be bidentate, binding to a WW domain at the site of its PPxY motif, and, via a poly-ADP ribose modification, binding to another (yet currently unidentified) site on the Nedd4 family protein. While we do favour this model, it is also a distinct possibility that, upon poly-ADP ribosylation, 3BP2 may undergo a change in conformation that would make it more susceptible to ubiquitination, or that this modification may somehow activate the ligase upon binding. Though 3BP2 has been reported to interact with the Cbl E3 ubiquitin ligase via its SH2 domain as a result of Cbl tyrosine phosphorylation (Deckert et al. , 1998), there have been no reports to date to suggest

25 that this results in 3BP2 ubiquitination or that it stimulates its degradation, though this possibility requires formal experimental testing.

The goal of this thesis work was to identify a potential E3 ubiquitin ligase responsible for mediating 3BP2 ubiquitination and to determine whether this ligase showed sensitivity to the presence of Tankyrase 2. The experimental work reported in Chapter 3, which was completed by the author, establishes Nedd4 family members Nedd4-1, Nedd4-2, Smurf1 and Smurf2 as potential candidates for mediating 3BP2 ubiquitination in vivo . Based on the criteria of ability to bind and ubiquitinate 3BP2, sensitivity to PPxY motif mutation on said binding and ubiquitination and the sensitivity of ubiquitination to both the presence of Tankyrase 2 and/or a

3BP2 Cherubism mutation, Smurf1 and Smurf2 appear to be the best candidates of the four family members tested. Chapter 4 summarizes and interprets these results, while Chapter 5 discusses the directions of future work, including the possible role for a fifth Nedd4 family member, Itch, as an important candidate E3 ligase for mediating 3BP2 regulation.

3BP2 + Tank2 3BP2 Tank2 PPAY PPAY

3BP2 Nedd4 + Tank2 E3 Ligase PPAY

3BP2 Tank2 PPAY Nedd4 E3 Ligase 3BP2 Tank2 26S PPAY

Nedd4

E3 Ligase

Figure 1-6: Model for 3BP2 Ubiquitination: 3BP2 interacts with Tankyrase 2, stimulating its poly-ADP ribosylation. Through a yet unidentified mechanism, this stimulates interaction with a Nedd4 family HECT domain containing E3 ligase via the PPAY motif present on 3BP2. This would consequently result in 3BP2 ubiquitination, therefore targeting it to the 26S proteasome for degradation.

CHAPTER 2: MATERIALS AND METHODS

2.1 Mice 129(ICR) Smurf1 -/-/Smurf2 +/- mice were a kind gift from Dr. Jeffrey Wrana (Samuel

Lunenfeld Research Institute, Toronto, Ontario, Canada).

2.2 Reagents and Antibodies Unless otherwise stated, all chemicals were from Sigma-Aldrich (Oakville, Ontario,

Canada). Antibodies were obtained from the following sources: anti-Actin, anti-Flag (Sigma), anti-Myc (Santa Cruz Biotechnologies, Santa Cruz, California, USA), anti-V5 (Invitrogen

Canada, Burlington, Ontario, Canada) and anti-HA (Hybridoma Core Facility, Sunnybrook

Research Institute, Toronto, Ontario, Canada). 3BP2 antibodies have been previously described

(Deckert et al ., 1998). Protein-A Sepharose beads were from GE Healthcare (Uppsala,

Sweden), Protein-G sepharose beads were from Sigma. Complete protease inhibitor cocktail tablets were obtained from Roche Diagnostics (Manheim, Germany). Carbobenzoxy-L-leucyl-

L-leucyl-L-leucinal (MG132) and Calpain Inhibitor I (ALLN) were purchased from Calbiochem

(EMD Chemicals Inc., Gibbstown, New Jersey, USA).

2.3 Plasmids pCMV-3X-FLAG vector was purchased from Addgene (Cambridge, Massachusetts,

USA). pCDNA3.1/Myc-His(-) B vector was purchased from Invitrogen Canada (Burlington,

Ontario, Canada). The construction of full-length murine 3BP2 expression vector was described previously (Chen et al ., 2007). Mutation of full-length 3BP2 was accomplished using the

28 Quickchange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, California, USA) according to the manufacturer’s instructions. Full length Tankyrase2 cDNA (a kind gift from Dr. Nai-wen

Chi, University of California, San Diego, California, USA) was cloned into the pEF6-Myc/His vector (Invitrogen) using Bam HI and Xba I sites. Full-length pCDNA6.2-V5-tagged human

Nedd4-1, Nedd4-2, Smurf1 and Smurf2 were a kind gift from Dr. Daniela Rotin (Sickkids

Research Institute, Toronto, Ontario, Canada). Full-length pCDNA3.1-Myc/His-Itch cDNA was a kind gift from Dr. Robert Ingham (University of Alberta, Edmonton, Alberta, Canada). pCMV-HA-Ubiquitin was a kind gift from Dr. Jeffrey Wrana (Samuel Lunenfeld Research

Institute, Toronto, Ontario, Canada).

2.4 Cell Culture and Transfection Human Embryonic Kidney (HEK) 293T cells (ATCC, Manassas, Virginia, USA) were transfected with 2-3g of the indicated plasmids using the Polyfect transfection reagent

(QIAGEN Inc., Mississauga, Ontario, Canada) according to manufacturer’s recommendations for a period of 24-48hours. 293T cells were maintained in Dulbecco’s Modified Eagles Medium

(Gibco, Invitrogen Canada) containing 10% Cosmic Calf Serum (Hyclone, Thermo Fisher

Scientific, Waltham Massachusetts, USA), 50U/L penicillin and 50 g/L streptomycin.

Bone marrow derived macrophages were maintained in α-Modified Eagle’s Medium

(Gibco, Invitrogen Canada) containing 10% Fetal Bovine Serum (Hyclone, Thermo Fisher

Scientific), 50U/L penicillin, 50 g/L streptomycin, and 10ng/mL CSF-1.

All cells were cultured at 37 oC in an incubator with a humidified atmosphere containing

5% CO 2.

29 2.5 Co-Immunoprecipitation For co-immunoprecipitation experiments, 293T cells were lysed in Kinase Lysis Buffer

(KLB) (20mM Tris, pH 7.5, 150mM NaCl, 1mM EDTA, 1% NP-40, 0.5% Sodium

Deoxycholate) supplemented with 10mM Sodium Fluoride (NaF), 2mM Sodium Orthovanadate

(Na 3VO 4), 1mM phenylmethanesulphonylfluoride (PMSF) and a complete protease inhibitor tablet (Roche). Lysates were cleared by centrifugation for 10 minutes at 14 000 rpm and 4°C, and incubated with Protein-G sepharose beads and 1 L Benzonase nuclease (Novagen, EMD

Chemicals Inc.) for 30 minutes to reduce non-specific binding. Pre-cleared lysates were incubated with 1 L anti-Flag antibody for 2 hours, and then incubated with Protein-A sepharose beads for 1 hour. Samples were washed three times with KLB and boiled for 5 minutes in sodium dodecyl sulfate (SDS) sample buffer and resolved by SDS-PAGE as described in section

2.7.

2.6 Re-precipitation Experiments For re-precipitation experiments (used to detect 3BP2 ubiquitination), cells were lysed in

TNTE buffer (50mM Tris, pH 7.4, 150mM NaCl, 0.5% Triton X-100, 1mM EDTA) supplemented with 10mM NaF, 2mM Na 3VO 4, 1mM PMSF, 10 M MG-132, 25 M ALLN and a complete protease inhibitor tablet (Roche). Lysates were cleared by centrifugation for 10 minutes at 14 000 rpm and 4°C, and incubated with Protein-G sepharose beads for 30 minutes to reduce non-specific binding. Precleared lysates were subject to anti-FLAG immunoprecipitation, performed for 1 hour at 4°C with 1 L anti-FLAG antibody and then incubated with Protein-A sepharose beads for 1 hour. Samples were then washed three times with TNTE buffer containing 0.1% SDS. Samples were then washed twice with RIPA buffer

(50mM Tris, pH 7.4, 150mM NaCl, 1mM EDTA, 0.5% NP-40, 1% sodium deoxycholate) containing 0.1% SDS and then once again with TNTE + 0.1% SDS. Samples were boiled for 5

30 minutes in 100 L RIPA buffer containing 1% SDS and then added to 1mL RIPA containing no

SDS. A second anti-Flag immunoprecipitation was performed for 1 hour at 4°C with 1 L anti-

FLAG antibody and then incubated with Protein-A sepharose beads for 1 hour. Samples were washed three times with TNTE + 0.1% SDS and then boiled for 5 minutes in SDS sample buffer and resolved by SDS-PAGE as described in section 2.7.

2.7 Western Blotting After boiling in SDS, samples were resolved by SDS-polyacrylamide gel electrophoresis

(PAGE). Gels were then transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon,

Millipore). Membranes were incubated with 5% non fat dried milk (Carnation, Markham,

Ontario, Canada) in phosphate buffered saline containing 0.05% Tween 20 (PBS-T) for one hour. Membranes were then probed with the indicated primary antibodies diluted in blocking buffer at 4°C overnight. Membranes were then washed three times with PBS-T, incubated with horse radish peroxidase (HRP)-conjugated secondary antibodies for 30 minutes at room temperature, washed an additional three times with PBS-T and developed using an enhanced chemiluminescence kit (ECL) (Amersham GE Healthcare, Uppsala, Sweden) according to the manufacturer’s instructions.

2.8 Isolation of Bone Marrow Derived Macrophages For macrophage isolation, mice were sacrificed at 8 weeks of age and bone marrow cells were isolated from mice femurs and tibias. The bone marrow cells were then treated with ACK buffer (0.15mM NH 4Cl, 10mM KHCO 3, 0.1mM EDTA, pH 7.3) to remove red blood cells.

Cells were plated at 10 6 cells/mL in α-Modified Eagle’s Medium (Gibco, Invitrogen Canada) containing 10% Fetal Bovine Serum (Hyclone, Thermo Fisher Scientific), 50U/L penicillin,

31 50 g/L streptomycin and 10ng/mL CSF-1 for one day to remove adherent cells. The resulting, non-adherent bone marrow monocytes were then resuspended in the same media and plated at 1 x 10 6 cells per 10cm plate. Media was changed after 4 days of culture and cells were harvested

th on the 5 day in HNMETG buffer (50mM Hepes (pH 7.5), 150mM NaCl, 1.5mM MgCl 2,

1.0mM EGTA, 1.0% Triton X-100, 10% glycerol), supplemented with 10mM NaF, 2mM

Na 3VO 4, 1mM PMSF and a complete protease inhibitor tablet (Roche). Samples were normalized for protein content using Bradford reagent according to the manufacturer instructions (Bio-Rad, Hercules California, USA) and 3BP2 levels were analyzed via Western blot (see section 2.7)

2.9 Software Western blots were quantified with Image J software (Macbiophotonics, McMaster

University, Hamilton, Ontario, Canada).

32 CHAPTER 3: RESULTS

3.1 3BP2 Interacts with Nedd4 Family HECT E3 Ligases To assess whether 3BP2 could interact with the Nedd4 family E3 ligases I utilized an overexpression system. 3BP2 was shown to form a stable protein complex with the four tested members of the Nedd4 family of HECT domain containing E3 ligases. Co-expression of pCMV3X-FLAG-3BP2 with pCDNA6.2-V5-tagged versions of hNedd4-1 (Fig. 3-1A, lane 3), hNedd4-2 (Fig. 3-1A, lane 7), hSmurf1 (Fig. 3-1B, lane 3) and hSmurf2 (Fig 3-1B, lane 7) yielded productive complexes as assayed by anti-FLAG immunoprecipitation followed by anti-

V5 Western blot.

Co-expression of pCMV3X-FLAG vector with pCDNA6.2-V5-tagged versions of hNedd4-1 (Fig. 3-1A, lane 1), hNedd4-2 (Fig. 3-1A, lane 5), hSmurf1 (Fig. 3-1B, lane 1) and hSmurf2 (Fig. 3-1B, lane 5) served as negative controls as assayed by the lack of signal detected in these lanes after anti-FLAG immunoprecipitation followed by anti-V5 Western Blot. Mock immunoprecipitation experiments were also performed (i.e.: bead controls) in order to serve as further negative controls for non-specific interactions with sepharose beads alone (Figs. 3-1A and 3-2B, lanes 2, 4, 6 and 8)

Based on the results of this experiment, one can conclude that each of the four Nedd4 family members tested do form a complex with 3BP2 when overexpressed. However, hSmurf1 seems to be the strongest interacting partner as compared to hNedd4-1, hNedd4-2 and hSmurf2 as it yielded a stronger signal in the co-immunoprecipitation experiment relative to the individual expression levels of the ligases themselves present in the whole cell lysate (Fig. 3-

1C). This trend is consistent in repeated experiments (see Appendix 1, Fig. A1-1).

A V5-hNedd4-1 V5-hNedd4-2 B V5-hSmurf1 V5-hSmurf2 pCMV3X-Flag + + - - + + - - pCMV3X-Flag + + - - + + - - pCMV3X-Flag-3BP2 - - + + - - + + pCMV3X-Flag-3BP2 - - + + - - + + IP: Flag + - + - + - + - IP: Flag + - + - + - + -

180kDa 180kDa 115kDa 115kDa 82kDa 82kDa IB: αV5 IB: αV5 64kDa 64kDa

IB: αFlag 82kDa IB: αFlag 82kDa

Nedd41 Nedd42 Smurf1 Smurf2 C pCMV3X-Flag + - + - + - + - pCMV3X-Flag-3BP2 - + - + - + - +

180kDa 115kDa 82kDa IB: αV5

49kDa IB: αActin 37kDa

82kDa IB: αFlag

Figure 3-1 3BP2 Interacts with Nedd4 Family E3 Ligases. (A) pCMV3X-FLAG-3BP2 was co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 3 and 4) or pCDNA6.2-V5-hNedd4-2 (lanes 7 and 8). Control experiments include pCMV3X-FLAG vector co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2) and with pCDNA6.2-V5-hNedd4-2 (lanes 5 and 6). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti-V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (B) pCMV3X-FLAG-3BP2 was co- expressed with pCDNA6.2-V5-hSmurf1 (lanes 3 and 4) or pCDNA6.2-V5-hSmurf2 (lanes 7 and 8). Control experiments include pCMV3X- FLAG vector co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) and with pCDNA6.2-V5-Smurf2 (lanes 5 and 6). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti-V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (C) Whole cell lysate samples. pCMV3X-FLAG vector is expressed in lanes 1, 3, 5 and 7. pCMV3X-FLAG-3BP2 is expressed in lanes 2, 4, 6 and 8. pCNDA6.2-V5-hNedd4-1 was co-expressed in lanes 1 and 2, pCDNA6.2-V5-hNedd4-2 was co-expressed in lanes 3 and 4, pCDNA6.2-V5- hSmurf1 was co-expressed in lanes 5 and 6 and pCDNA6.2-V5-hSmurf2 was co-expressed in lanes 7 and 8. Membrane in top panel was probed with anti-V5 (top) and anti-Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

34 3.2 3BP2 Ubiquitination is Stimulated in the Presence of Nedd4 Family E3 Ligases The ability of each Nedd4 family E3 ligase to ubiquitinate 3BP2 was then tested by a re- precipitation assay. Briefly, the immunoprecipitation step was followed by a denaturing step in order to remove contamination from binding partners and then the protein of interest was re- immunoprecipitated before Western blot analysis. pCMV3X-FLAG-3BP2 was co-expressed with pCMV-HA-Ubiquitin alone or with pCMV-HA-Ubiquitin and each of the abovementioned

Nedd4 family members (see section 3.1) While expression of pCMV3X-FLAG-3BP2 and pCMV-HA-Ubiquitin alone yielded minimal ubiquitination (Fig. 3-2A, lane 1), co-expression of pCDNA6.2-V5-tagged versions of hNedd4-1 (Fig. 3-2 A, lane 2), hNedd4-2 (Fig. 3-2A, lane 3), hSmurf1 (Fig. 3-2A, lane 4) and hSmurf2 (Fig. 3-2 A, lane 5) dramatically increased the incorporation of ubiquitin onto 3BP2.

Consistent with the immunoprecipitation data, hSmurf1 and hSmurf2 seem to be quite efficient at incorporating ubiquitin onto 3BP2, though hNedd4-1 is also quite a strong inducer of

3BP2 ubiquitination. Again, as relative expression levels are constant (Fig 3-2 B), ubiquitination can be directly compared. Replicate experiments show similar results (see

Appendix 1, Fig. A1-2).

3.3 Ability of 3BP2_Y202A to Interact with Nedd4 E3 Ligases We have proposed that 3BP2 interacts with the WW domain-containing Nedd4 E3s via a

PPxY motif (where x is any amino acid), found within the proline rich region of the polypeptide

(residues 199-202 in the murine amino acid sequence). Tyrosine 202 in the full-length mouse pCMV3X-FLAG-3BP2 construct (which describes the fourth position of the PPxY motif) was mutated to an alanine residue (henceforth Y202A). It should be noted that this mutation does not seem to alter the stability of the protein, as its steady state levels are not different as compared to wild type. Though this could suggest that this mutation is not involved in 3BP2

A pCMV3X-FLAG-3BP2

_ hSmurf-1 hSmurf-2 hNedd4-2 hNedd4-1

180kDa _

115kDa Nedd4-1 Nedd4-2 Smurf-1 Smurf-2 180kDa 82kDa IP: αFlag 115kDa IB: α HA-Ub IB: V5 64kDa 82kDa

82kDa IB: Flag

IP: α Flag 49kDa 82kDa IB: Actin IB: α Flag 37kDa

Figure 3-2 Nedd4 Family E3 Ligases Stimulate 3BP2 Ubiquitination. (A) pCMV-HA-Ubiquitin and pCMV3X-FLAG-3BP2 were expressed alone (lane 1) or co-expressed with pCDNA6.2-V5-hNedd4-1 (lane 2), pCDNA6.2-V5-hNedd4-2 (lane 3), pCDNA6.2-V5-hSmurf1 (lane 4) or pCDNA6.2-V5-hSmurf2 (lane 5). Re-precipitation experiment was performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (B) Whole cell lysate samples. pCMV-HA-Ubiquitin and pCMV3X-FLAG-3BP2 were expressed alone (lane 1) or co-expressed with pCDNA6.2-V5-hNedd4-1 (lane 2), pCDNA6.2-V5-hNedd4-2 (lane 3), pCDNA6.2-V5-hSmurf1 (lane 4) or pCDNA6.2-V5-hSmurf2 (lane 5). Membrane in top panel was probed with anti-V5, membrane in middle panel probed with anti-FLAG and membrane in bottom panel probed with anti-Actin.

36 regulation, the overexpression system may mask the effect, as perhaps subtle differences cannot be distinguished.

pCMV3X-FLAG-3BP2 or the pCMV-3X-FLAG-3BP2_Y202A mutant were co- transfected with each of the above-mentioned pCDNA6.2-V5-tagged Nedd4 E3s and subject to a co-immunoprecipitation experiment in order to assess the effect of this mutation on the ability of 3BP2 to bind to the Nedd4 E3s. While the ability of hNedd4-1 to bind 3BP2_Y202A was only slightly reduced as compared to wild type (Fig. 3-3A, lanes 1 and 3), the interaction between hNedd4-2 and the Y202A mutant was more drastically affected (Fig. 3-3A, lanes 5 and

7) as assayed by anti-FLAG immunoprecipitation followed by anti-V5 Western blot. The identical experiment was carried out with wild type and Y202A mutant 3BP2 with hSmurf1

(Fig. 3-3B, lanes 1 and 3) and with hSmurf2 (Fig. 3-3B, lanes 5 and 7). These experiments showed an appreciable decrease in the ability of 3BP2_Y202A to bind to hSmurf1 (as compared to wild type 3BP2), while binding between wild type and Y202A 3BP2 to hSmurf2 was virtually unchanged.

Analysis of loading controls as assayed by total cell lysate anti-FLAG, anti-V5 and anti-

Actin Western blot (Fig. 3-3 C) show equal amounts of wild type and 3BP2_Y202A protein levels, as well as similar amounts of Nedd4 family members and total protein. Similar co- immunoprecipitation results were seen in triplicate (see Appendix 1, Figure A1-3).

3.4 Ability of Y202A 3BP2 to become Ubiquitinated by Nedd4 E3 Ligases Based on the reduced affinity of the Nedd4 family E3 ligases for 3BP2_Y202A as compared to wild type, we then tested the ability of 3BP2_Y202A to become ubiquitinated by the Nedd4 family HECT E3s. pCMV3X-FLAG-3BP2 or PCMV3X-FLAG-3BP2_Y202A was

V5 -hNedd4 -1 V5-hNedd4-2 pCMV3X-Flag-3BP2 + + - - + + - - pCMV3X-Flag-3BP2_Y202A - - + + - - + + A IP: Flag + - + - + - + -

180kD hNedd4-2 hNedd4-1

82kD IB: αV5 64kD

82kD IB: αFlag

V5-hSmurf1 V5-hSmurf2 B pCMV3X-Flag-3BP2 + + - - + + - - pCMV3X-Flag-3BP2_Y202A - - + + - - + + IP: Flag + - + - + - + - 180kD

115kD hSmurf hSmurf

64kD IB: αV5

IB: αFlag 82kD

Nedd41 Nedd42 Smurf1 Smurf2 C pCMV3X-Flag-3BP2 + - + - + - + - pCMV3X-Flag-3BP2_Y202A - + - + - + - + 180kDa 115kDa 82kDa IB: αV5

49kDa 37kDa IB: αActin

IB: αFlag 82kDa

Figure 3-3 Ability of 3BP2_Y202A to Interact with Nedd4 E3 Ligases. (A) pCMV3X-FLAG-3BP2 was co-expressed with pCDNA6.2-V5- hNedd4-1 (lanes 1 and 2) or pCDNA6.2-V5-hNedd4-2 (lanes 5 and 6). pCMV3X-FLAG-3BP2_Y202A was co-expressed with pCDNA6.2-V5- hNedd4-1 (lanes 3 and 4) and with pCDNA6.2-V5-hNedd4-2 (lanes 7 and 8). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti- V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (B) pCMV3X-FLAG-3BP2 was co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) or pCDNA6.2-V5-hSmurf2 (lanes 5 and 6). pCMV3X-FLAG-3BP2_Y202A was co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 3 and 4) and with pCDNA6.2-V5-Smurf2 (lanes 7 and 8). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti-V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (C) Whole cell lysate samples. pCMV3X- FLAG-3BP2 is expressed in lanes 1, 3, 5 and 7. pCMV3X-FLAG-3BP2_Y202A is expressed in lanes 2, 4, 6 and 8. pCNDA6.2-V5-hNedd4-1 was co-expressed in lanes 1 and 2, pCDNA6.2-V5-hNedd4-2 was co-expressed in lanes 3 and 4, pCDNA6.2-V5-hSmurf1 was co-expressed in lanes 5 and 6 and pCDNA6.2-V5-hSmurf2 was co-expressed in lanes 7 and 8. Membrane in top panel was probed with anti-V5 (top) and anti- Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

co-transfected with pCMV-HA-Ubiquitin and each of the abovementioned Nedd4 family members in question.

Co-transfection of hNedd4-1 with wild type and Y202A 3BP2 resulted in equivalent ubiquitin incorporation between the two 3BP2 constructs as assayed by anti-FLAG re- precipitation experiments similar to those carried out in section 3.2 (Fig. 3-4A, lanes 1 and 2).

Co-transfection of hNedd4-2 with wild type and Y202A mutant 3BP2, however, showed a significant decrease in the ability of the Y202A mutant to become ubiquitinated (Fig 3-4A, lanes

3 and 4). This data is consistent with the relative abilities of hNedd4-1 and hNedd4-2 to bind to the Y202A mutant 3BP2 as compared to their abilities to bind to the wild type polypeptide.

Again, consistent with the co-immunoprecipitation data reported in section 3.3, the ability of hSmurf1 to stimulate 3BP2 ubiquitination was greatly reduced after the introduction of the Y202A mutation (Fig. 3-4B, lanes 1 and 2), while the ability of hSmurf2 to ubiquitinate

Y202A mutant 3BP2 was unchanged as compared to wild type (Fig. 3-4B, lanes 3 and 4).

Controls indicate equivalent protein loading between experiments containing both wild type and

Y202A mutant 3BP2 (Fig. 3-4C). Consistent trends for triplicate experiments are depicted in

Appendix 1, Fig. A1-4.

3.5 Ability of Tankyrase 2 to Stimulate 3BP2 Ubiquitination in the Presence of Nedd4 E3 Ligases Co-expression of Tankyrase 2 has been shown to augment the ubiquitination of endogenous 3BP2 (Voytyuk et al ., manuscript submitted) To test the ability of Tankyrase 2 to augment the ubiquitination of 3BP2 in the presence of each Nedd4 E3 ligase, a re-precipitation experiment was performed using wild type pCMV3X-FLAG-3BP2, pCDNA6.2-V5-tagged

Nedd4 E3s, pCMV-HA-Ubiquitin and either empty myc vector or pEF6-Myc/His-Tankyrase 2.

A V5-hNedd4-1 V5-hNedd4-2 pCMV3X-Flag-3BP2 + - + - pCMV3X-Flag-3BP2_Y202A - + - +

180kDa

115kDa IP: αFlag 82kDa IB: α HA-Ub C pCMV3X -Flag pCMV3X -Flag 3BP2 3BP2_Y202A 64kDa

IP: α Flag 82kDa IB: α Flag Nedd4-1 Nedd4-1 Nedd4-2 Smurf-1 Smurf-2 Nedd4-2 Smurf-1 Smurf-2 180kDa 115kDa IB: αV5 B 82kDa 49kDa V5-hSmurf-1 V5-hSmurf-2 IB: αActin pCMV3X-Flag-3BP2 + - + - 37kDa pCMV3X-Flag-3BP2_Y202A - + - + 82kDa 180kDa IB: αFlag

115kDa IP: αFlag 82kDa IB: α HA-Ub 64kDa

IP: α Flag 82kDa IB: α Flag

Figure 3-4 Ability of 3BP2_Y202A Shows Reduced Ubiquitination in the Presence of Nedd4 E3 Ligases. (A) pCMV-HA-Ubiquitin and pCMV3X-FLAG-3BP2 (lanes 1 and 3) or pCMV3X-FLAG-3BP2_Y202A (lanes 2 and 4) were co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2) or pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (B) pCMV-HA-Ubiquitin and pCMV3X-FLAG-3BP2 (lanes 1 and 3) or pCMV3X-FLAG-3BP2_Y202A (lanes 2 and 4) were co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) or pCDNA6.2-V5-hSmurf2 (lanes 3 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (C) Whole cell lysate samples. pCMV-HA-Ubiquitin and pCMV3X-FLAG-3BP2 (lanes 1, 3, 5 and 7) or pCMV3X-FLAG-3BP2_Y202A (lanes 2, 4, 6 and 8) were co- expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2), pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4), pCDNA6.2-V5-hSmurf1 (lanes 5 and 6) or pCDNA6.2-V5-hSmurf2 (lanes 7 and 8). Membrane in top panel was probed with anti-V5 (top) and anti-Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

40 The ability of hNedd4-1 to ubiquitinate 3BP2 was diminished by the presence of

Tankyrase 2 (Fig. 3-5A, lanes 1 and 2), whereas ubiquitination of 3BP2 by hNedd4-2 was slightly increased in the presence of Tankyrase 2 (Fig. 3-5A, lanes 3 and 4).

3BP2 ubiquitination by hSmurf1 (Fig. 3-5B lanes 1 and 2) and hSmurf2 (Fig. 3-5B, lanes 3 and 4) were, however, quite dramatically increased upon co-expression of Tankyrase 2.

Western blots of total cell lysates from each experiment show equivalent 3BP2 protein levels in both the presence and absence of Tankyrase 2 (Fig. 3-5C). Equivalent results were seen in triplicate, with repeat experiments depicted in Appendix 1, Fig. A1-5.

3.6 Ability of Cherubism Mutant 3BP2 (R413Q) to Bind Nedd4 Family E3 Ligases The introduction of a Cherubism mutation into the wild type 3BP2 amino acid sequence yields higher steady state protein levels as a result of the protein’s inability to interact with

Tankyrase 2 (Voytyuk et al ., manuscript submitted). Cherubism mutation also decreases the ability of 3BP2 to become ubiquitinated in the presence of Tankyrase 2 (Voytyuk et al ., manuscript submitted). Based on the ability of Tankyrase 2 to augment 3BP2 ubiquitination in the presence of some Nedd4 family E3 ligases, we then tested their ability to interact with a

Cherubism mutated version of 3BP2 (henceforth, R413Q). pCMV3X-FLAG-3BP2 or pCMV3X-FLAG-3BP2_R413Q was co-transfected with each of the pCDNA6.2-V5-tagged human Nedd4 family members and a co-immunoprecipitation experiment was performed

(similar to those described in section 3.1).

Cherubism mutated 3BP2 showed a slightly reduced affinity for hNedd4-1 when co-expressed as compared to wild type 3BP2 as assayed by anti-FLAG immunoprecipitation followed by anti-

V5 Western blot (Fig. 3-6A, lanes 1 and 3). R413Q mutant 3BP2 showed a more dramatic decrease in affinity for hNedd4-2, when this E3 ligase was co-expressed with either the wild

pCMV3X-Flag-3BP2 A V5-hNedd4-1 V5-hNedd4-2 pCNDA3.1 + - + - pCDNA3.1-Myc/His-Tankyrase 2 - + - +

180kDa

115kDa IP: αFlag 82kDa IB: α HA-Ub pCMV3X-Flag-3BP2 64kDa

IP: α Flag C Nedd4-1 Nedd4-2 Nedd4-2 82kDa IB: α Flag Smurf-1 Smurf-2 Nedd4-1 Smurf-1 Smurf-2 180kDa IB: αV5 pCMV3X-Flag-3BP2 115kDa 82kDa B V5-hSmurf1 V5-hSmurf2 IB: αActin pCNDA3.1 + - + - 49kDa pCDNA3.1-Myc/His-Tankyrase 2 - + - + 37kDa

82kDa IB: αFlag 180kDa 115kDa IP: αFlag 82kDa IB: α HA-Ub 64kDa

IP: α Flag 82kDa IB: α Flag

Figure 3-5 Ability of Tanykrase 2 to Augment Ability of Nedd4 Family E3 Ligases to Ubiquitinate 3BP2. (A) pCMV3X-FLAG-3BP2 and pCMV-HA-Ubiquitin were co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2) or pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4) and either empty pCDNA3.1-Myc/His vector (lanes 1 and 3) or pCDNA3.1-Myc/His-Tankyrase 2 (lanes 2 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (B) pCMV3X-FLAG-3BP2 and pCMV-HA-Ubiquitin were co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) or pCDNA6.2- V5-hSmurf2 (lanes 3 and 4) and either empty pCDNA3.1-Myc/His vector (lanes 1 and 3) or pCDNA3.1-Myc/His-Tankyrase 2 (lanes 2 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (C) Whole cell lysates. pCMV3X-FLAG-3BP2 and pCMV-HA-Ubiquitin were co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2), pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4), pCDNA6.2-V5-hSmurf1 (5 and 6) or pCDNA6.2-V5- hSmurf2 (lanes 7 and 8) and either empty pCDNA3.1-Myc/His vector (lanes 1, 2, 3 and 4) or pCDNA3.1-Myc/His-Tankyrase 2 (lanes 5, 6, 7 and 8). Membrane in top panel was probed with anti-V5 (top) and anti-Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

42 type or mutant forms of the adaptor (Fig. 3-6A, lanes 2 and 4). Both hSmurf1 (Fig. 3-6B, lanes

1 and 3) and hSmurf2 (Fig. 3-6B, lanes 2 and 4) show reduced affinity for the Cherubism mutant (R413Q) version of 3BP2 as compared to their affinity for the wild type polypeptide.

This result was repeated in triplicate, with alternate experiments depicted in Appendix 1,

Fig. A1-6. While there is some variation in the degree to which this mutation affects Nedd4 family member binding, the trends noted above are consistent.

3.7 Ability of Cherubism Mutant 3BP2 (R413Q) to be Ubiquitinated in the Presence of Nedd4 Family Members Based on the differential affinity of some Nedd4 family members for wild type versus

Cherubism mutated 3BP2 (R413Q) we then tested the ability of Cherubic 3BP2 to be ubiquitinated by each Nedd4 family member as compared to wild type. pCMV3X-FLAG-3BP2 wild type or pCMV3X-FLAG-3BP2_R413Q were co-expressed with pCMV-HA-Ubiquitin and each pCDNA6.2-V5-tagged Nedd4 ligase mentioned above. Samples were subject to an anti-

FLAG re-precipitation experiment to detect 3BP2 ubiquitination.

Co-expression of wild type or R413Q 3BP2 with hNedd4-1 showed equivalent ubiquitin incorporation onto 3BP2 (Fig. 3-7A, lanes 1 and 2) as tested by re-precipitation with anti-FLAG antibody. Consistent with the proposed model, ubiquitination of R413Q 3BP2 was reduced as compared to wild type with co-expression of hNedd4-2 (Fig. 3-7A, lanes 3 and 4). While an increase in 3BP2 ubiquitination was observed with Cherubism mutant R413Q 3BP2 as compared to wild type 3BP2 in the presence of Smurf1 (Fig. 3-7B, lanes 1 and 2), co-expression of hSmurf-2 with Cherubic 3BP2 showed reduced ubiquitination as compared to its ability to ubiquitinate the wild type protein (Fig. 3-7B, lanes 3 and 4).

Loading controls show equivalent 3BP2 protein expression in either its wild type or

R413Q mutated forms as assayed by anti-FLAG Western blot on whole cell lysates (Fig. 3-7C).

A V5-hNedd4-1 V5-hNedd4-2 pCMV3X-Flag-3BP2 + + - - + + - - pCMV3X-Flag-3BP2_R413Q - - + + - - + + IP: Flag + - + - + - + -

hNedd4-1 115kD hNedd4-2 82kD IB: αV5 64kD

82kD IB: αFlag

B V5-hSmurf1 V5-hSmurf2 pCMV3X-Flag-3BP2 + + - - + + - - pCMV3X-Flag-3BP2_R413Q - - + + - - + + IP: Flag + - + - + - + - 180kD 115kD hSmurf1 82kD hSmurf2

64kD IB: αV5

82kD IB: αFlag

Nedd41 Nedd42 Smurf1 Smurf2 pCMV3X-Flag-3BP2 + - + - + - + - pCMV3X -Flag -3BP2_R413Q - + - + - + - + 180kDa 115kDa 82kDa IB: αV5

49kDa 37kDa IB: αActin

82kDa IB: αFlag

Figure 3-6 Ability of 3BP2_R413Q to Bind to Nedd4 Family E3 Ligases. (A) pCMV3X-FLAG-3BP2 was co-expressed with pCDNA6.2- V5-hNedd4-1 (lanes 1 and 2) or pCDNA6.2-V5-hNedd4-2 (lanes 5 and 6). pCMV3X-FLAG-3BP2_R413Q was co-expressed with pCDNA6.2- V5-hNedd4-1 (lanes 3 and 4) and with pCDNA6.2-V5-hNedd4-2 (lanes 7 and 8). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti- V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (B) pCMV3X-FLAG-3BP2 was co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) or pCDNA6.2-V5-hSmurf2 (lanes 5 and 6). pCMV3X-FLAG-3BP2_R413Q was co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 3 and 4) and with pCDNA6.2-V5-Smurf2 (lanes 7 and 8). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti-V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (C) Whole cell lysate samples. pCMV3X- FLAG-3BP2 is expressed in lanes 1, 3, 5 and 7. pCMV3X-FLAG-3BP2_R413Q is expressed in lanes 2, 4, 6 and 8. pCNDA6.2-V5-hNedd4-1 was co-expressed in lanes 1 and 2, pCDNA6.2-V5-hNedd4-2 was co-expressed in lanes 3 and 4, pCDNA6.2-V5-hSmurf1 was co-expressed in lanes 5 and 6 and pCDNA6.2-V5-hSmurf2 was co-expressed in lanes 7 and 8. Membrane in top panel was probed with anti-V5 (top) and anti- Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

A V5 -hNedd4 -1 V5 -hNedd4 -2 pCMV3X-Flag-3BP2 + - + - pCMV3X-Flag-3BP2-R413Q - + - +

180kD 115kD 82kD IP: αFlag IB: α HA- 64kD Ub

IP: α Flag 82kD IB: α Flag

B V5 -hSmurf1 V5 -hSmurf2 pCMV3X-Flag-3BP2 + - + - pCMV3X-Flag-3BP2-R413Q - + - + 180kD 115kD 82kD IP: αFlag IB: α HA- Ub 64kD

IP: α Flag 82kD IB: α Flag

Nedd41 Nedd42 Smurf1 Smurf2 pCMV3X-Flag-3BP2 + - + - + - + - pCMV3X-Flag-3BP2_R413Q - + - + - + - + 180kD 115kD α 82kD IB: V5

49kD IB: αActin 37kDa

82kD IB: αFlag

Figure 3-7 Ability of 3BP2_R413Q Shows to be Ubiquitinated in the Presence of Nedd4 Family E3 Ligases. (A) HA-Ubiquitin and pCMV3X-FLAG-3BP2 (lanes 1 and 3) or pCMV3X-FLAG-3BP2_R413Q (lanes 2 and 4) were co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2) or pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (B) HA-Ubiquitin and pCMV3X- FLAG-3BP2 (lanes 1 and 3) or pCMV3X-FLAG-3BP2_R413Q (lanes 2 and 4) were co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) or pCDNA6.2-V5-hSmurf2 (lanes 3 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (C) Whole cell lysate samples. HA-Ubiquitin and pCMV3X-FLAG-3BP2 (lanes 1, 3, 5 and 7) or pCMV3X-FLAG-3BP2_R413Q (lanes 2, 4, 6 and 8) were co-expressed with pCDNA6.2-V5- hNedd4-1 (lanes 1 and 2), pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4), pCDNA6.2-V5-hSmurf1 (lanes 5 and 6) or pCDNA6.2-V5-hSmurf2 (lanes 7 and 8). Membrane in top panel was probed with anti-V5 (top) and anti-Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

45 This experiment was completed in triplicate (see Appendix 1, Fig. A1-7) and while trends are consistent, the degree to which the Cherubism mutation affects ubiquitination is occasionally variable.

3.8 hSmurf1 Best Fits Proposed Model. Based on each of the criteria enumerated in this chapter, the Nedd4 Family HECT E3 that most closely adheres to the proposed model is hSmurf1. It interacts most tightly with the wild type 3BP2 and is a rather strong inducer of 3BP2 ubiquitination. It is most sensitive to

PPxY mutation in both its reduced ability to bind and to ubiquitinate the Y202A mutant. It is also sensitive to the presence of Tankyrase 2, which augments its ability to ubiquitinate the adaptor. Finally, though it is sensitive to Cherubism mutation with respect to its ability to bind

3BP2, Cherubism has the reverse effect on the capacity of Smurf1 to stimulate 3BP2 ubiquitination. Though Smurf2 is rather insensitive to Y202A mutation, it does show sensitivity to Cherubism mutation, as well as to the presence of Tankyrase 2, thus it will be included in the future directions of this project. These results are summarized in tabular format (Table 1) and are discussed in detail in Chapter 4.

3.9 Analysis of Endogenous 3BP2 Protein Levels in Smurf1 -/-/Smurf2 +/- Macrophages Based on the experiments described in 3.1-3.7 (the results of which are summarized in

Table 1), Smurf1 and Smurf2 seem to be the best possible candidate E3 ligases for 3BP2, as they not only interact most strongly with the adaptor and lead to its efficient ubiquitination, but they also seem to be sensitive to the presence of Tankyrase 2 and to the introduction of a 3BP2

Cherubism mutation. 3BP2 plays an important role in macrophage function (our unpublished data), thus, we attempted to study the affects that the Smurf proteins have on 3BP2 protein levels in this cell type.

46 Criteria Nedd4-1 Nedd4-2 Smurf1 Smurf2

Bind + + +++ +

Ubiquitinate +++ + ++ ++

Sensitivity to Y202A Mutation + ++ +++ - (Bind) Sensitivity to Y202A Mutation - ++ +++ - (Ubiquitinate) Sensitivity to Tankyrase 2 - + ++ +++ (Ubiquitinate) Sensitivity to R413Q Mutation - +++ +++ +++ (Bind) Sensitivity to R413Q Mutation - + - +++ (Ubiquitinate

Table 1. Summary of Binding and Ubiquitination Data. Table summarizes results of experiments described in 3.1-3.7. Rankings are arbitrarily assigned, and reflect relative sensitivities binding efficiencies, ubiquitination capabilities and sensitivity to 3BP2 mutation of each Nedd4 family member tested.

A Smurf1 -/- B Wild Type Smurf2 +/- R elative 3B P2 Protein L evels 180kD Wild Type vs . S murf1-/- S murf2+/- 115kDa a82kD 1.2 IB: 1 a α 3BP2 0.8 0.6 0.4 49kD

IB: (Arbitrary 0.2Units) 37kDa Relative Intensity αActin 0 a WT KO

Figure 3-8. Analysis of Endogenous 3BP2 Protein Levels in Smurf1 -/-/Smurf2 +/- Macrophages (A) Whole cell lysates from macrophages derived from wild type (lanes 1-3) or Smurf1 -/- / Smurf2 +/- mice. Membrane is probed with anti-3BP2 (top) and anti-Actin (bottom) antibodies. (B) Intensity of 3BP2 bands were quantified using Image J software and then normalized to the intensity of Actin bands. Triplicate experiments were averaged (bars) and the standard deviations plotted as error bars. The result is not significant (p=0.31).

47 Whole cell lysates from bone marrow derived macrophages were obtained from three, age- matched wild type and Smurf1 -/-/Smurf2 +/- mice. These lysates were normalized for protein content, subject to SDS-PAGE and then probed with an anti-3BP2 specific antibody to assess whether the lack of Smurf protein resulted in a change in 3BP2 protein levels. The result was an increase in 3BP2 protein levels in Smurf1 -/-/Smurf2 +/- mice as compared to wild type controls.

This was assessed by measuring the 3BP2 signal intensity relative to the intensity of an irrelevant protein ( β-Actin). This difference, though visible by eye, was not statistically significant (as determined using a Student’s t-test, with p=0.31) (Fig. 3.8).

48 CHAPTER 4: DISCUSSION

4.1 3BP2 Interacts with Nedd4 Family HECT E3 Ligases Based on our co-immunoprecipitation data, we can conclude that 3BP2 interacts with each of the tested Nedd4 Family members (i.e.: Nedd4-1, Nedd4-2, Smurf1 and Smurf2) when both binding partners are overexpressed in 293Ts. This binding, though not extremely tight (as observed by comparing the relative signals from the amount of immunoprecipitated 3BP2 and the amount of each Nedd4 family member associating with it via Western blot) is detectable over both bead and pCMV3X-FLAG vector controls.

Comparing the relative binding affinities for 3BP2 among the E3 ligases tested, Smurf1 seems to be the strongest 3BP2 interacting partner. As the purpose of this thesis work was to identify a bona fide 3BP2 E3 ligase, the fact that Smurf1 binds 3BP2 most tightly may indicate that this interaction is recapitulated in vivo , though this supposition would require validation, perhaps via endogenous complex formation (see Chapter 5: Future Directions).

4.2 3BP2 Ubiquitination is Stimulated in the Presence of Nedd4 Family E3 Ligases Though the Nedd4 family of HECT E3 ligases tested in this thesis work have been shown to interact with 3BP2, this does not necessarily indicate that they are in fact bona fide E3 ligases for the adaptor. Binding alone, though quite suggestive, does not prove a functional relationship, especially in an overexpression system. Additionally, though the binding may affect 3BP2 stability or downstream signaling, this may not be due to ubiquitination of 3BP2 itself, but perhaps a third binding partner that requires the adaptor function of 3BP2 to mediate its interaction with a Nedd4 HECT E3 (similar to the adaptor function that I-Smad7 plays in mediating Smurf1 dependent ubiquitination of the TGF β type I receptor (Ebisawa et al. , 2001)).

To formally test whether 3BP2 is indeed ubiquitinated by these Nedd4 family proteins,

49 we performed a re-precipitation experiment in the absence and presence of each of the E3 ligases in our study. This experiment, by design, prevents signal contamination by molecules associated with 3BP2 and/or other Nedd4 family binding partners, as the denaturing step followed by a second anti-FLAG immunoprecipitation would prevent other interacting partners from being pulled down with the complex.

In the absence of co-expression of an E3 ligase with 3BP2, its ubiquitination is virtually undetectable. In the presence of any Nedd4 family member tested, however, the incorporation of ubiquitin onto 3BP2 is dramatically increased (as assayed by anti-HA-Ubiquitin Western blot). This lends further confidence to our hypothesis that these HECT domain containing proteins are responsible for ubiquitinating 3BP2 in vivo . Again, the nature of this experiment does not exclude the possibility that mere overexpression of an E3 ligase that binds to 3BP2 would stimulate its ubiquitination, even if this phenomenon was not replicated in vivo . Further confidence in this observation may be obtained via an in vitro ubiquitination assay (see Chaper

5: Future Directions).

Also noteworthy is the relative abilities of each Nedd4 family member to stimulate 3BP2 ubiquitination. Based on these experiments, it appears that Nedd4-1 is the strongest stimulator of 3BP2 ubiquitination, though Smurf1 and Smurf2 are also quite efficient at incorporating ubiquitin onto 3BP2. As all of these players seem to be capable of binding and ubiquitinating

3BP2 (albeit to varying degrees) more experiments are required to further validate the model.

4.3 Ability of 3BP2_Y202A Mutant to Interact with Nedd4 Family E3 Ligases Based on the assumption that 3BP2 interacts with the WW domains present within the

Nedd4 Family HECT E3 domain structure, we then decided to introduce a mutation in 3BP2 that would disrupt its PPxY motif (i.e.: the presumed site of interaction with said WW domains).

50 To this end we utilized site directed mutagenesis to change the 3BP2 amino acid code, replacing tyrosine 202 (i.e.: the fourth position in the 3BP2 PPxY motif) for an alanine residue

(henceforth 3BP2_Y202A). We then carried out co-immunoprecipitation experiments similar to those completed in section 3.1 to compare the respective abilities of wild type and

3BP2_Y202A to bind to the Nedd4 family HECT E3s.

While Nedd4-1, Nedd4-2 and Smurf1 all showed reduced binding to 3BP2_Y202A,

Smurf2 showed no such effect. Furthermore, of the three family members showing sensitivity to the Y202A mutation, Smurf1 tended to be the most sensitive (with the exception of one experiment, see Appendix 1), followed by Nedd4-2 and finally Nedd4-1. Sensitivity to the

PPxY mutation gives further confidence to our model, as this may suggest a genetic interaction between 3BP2 and these HECT domain containing E3s, similar to what we have previously described with respect to the 3BP2:Tankyrase 2 interaction and its dependence on an in tact

Cherubism hexapeptide (Voytyuk et al., manuscript submitted). The fact that Smurf1 shows the most sensitivity to this mutation and in conjunction with its strong capacity to bind and ubiquitinate 3BP2, these experiments may indicate that Smurf1 is indeed a bona fide E3 ligase for 3BP2.

That there is residual binding of 3BP2_Y202A to each of the Nedd4 family members in question is not surprising. 3BP2 contains a large hydrophobic region, promoting potential non- specific interactions with these Nedd4 family members (which would be intensified due to the limitations of our overexpression system). It is, however, also formally possible that the PPxY motif is not the only site of interaction between 3BP2 and these Nedd4 E3 ligases. Indeed, it has been reported in the literature that phosphorylated serine and threonine residues directly upstream of a proline also confer binding to WW domain containing molecules (Lu et al. , 1999;

Yaffe et al. , 1997). Indeed there are many serine and threonine residues within the 3BP2 amino

51 acid sequence that are directly upstream of prolines and are predicted sites of phosphorylation by several serine/threonine kinases (Scansite, (Obenauer et al. , 2003). Mutation of these residues both separately and in conjunction with the Y202A mutation may lend further information on the mechanism by which 3BP2 is able to associate with the Nedd4 family of

HECT E3 ligases.

4.4 Ability of 3BP2_Y202A to become Ubiquitinated by Nedd4 E3 Ligases In order to further assess the dependence on an in tact PPxY motif on the ability of 3BP2 to form a productive interaction with the Nedd4 family HECT E3s, we then subject the Y202A mutant to re-precipitation experiments similar to those completed in section 3.2 and compared the respective abilities of wild type and mutant 3BP2 to become ubiquitinated by the Nedd4 family proteins. Wild type and Y202A mutant 3BP2 were equally ubiquitinated by Nedd4-1 and Smurf2 while there was a dramatic decrease in the ability of 3BP2_Y202A to be ubiquitinated in the presence of Nedd4-2 and Smurf1.

The lack of sensitivity to the PPxY mutation displayed by Nedd41 and Smurf2 may simply be due to the fact that their interaction with and subsequent ubiquitination of 3BP2 is non-specific and merely an artifact of the overexpression system. It may also be possible that the two proteins, being so effective at incorporating ubiquitin onto 3BP2 (see Fig. 3-2) that the slight decrease in binding affinity for the mutant is not enough to completely abrogate ubiquitination. This reasoning may also be extended to the drastic decrease in the ability of

Nedd4-2 to ubiquitinate 3BP2_Y202A, as both its binding and ability to ubiquitinate 3BP2 are relatively weak, such that even a slight reduction in the stability of this interaction (as would be the case with the introduction of a PPxY mutation) may completely obliterate the ability of this molecule to stimulate ubiquitination of the adaptor.

52 Of note once again is Nedd4 family member Smurf1, who not only binds 3BP2 most strongly, and ubiquitinates it quite efficiently, but is also quite sensitive to Y202A mutation, both in its capacity to bind 3BP2 and in its ability to ubiquitinate the adaptor. This again brings

Smurf1 to the forefront of our small scale screen for a potential E3 ligase responsible for mediating 3BP2 ubiquitination in an in vivo system. Again, recapitulation of this experiment in an in vitro setting will reduce the possibility of confounding factors present in the 293T cell lysate and would add confidence to the possibility that Smurf1 is the E3 ligase responsible for mediating 3BP2 ubiquitination.

4.5 Ability of Tankyrase 2 to Stimulate 3BP2 Ubiquitination in the Presence of Nedd4 E3 Ligases As discussed in Chapter 1, 3BP2 is negatively regulated by the poly-ADP ribose polymerase, Tankyrase 2, which is the result of its capacity to stimulate a decrease in 3BP2 steady state protein levels (Voytyuk et al., manuscript submitted). Further investigation into the method by which Tankyrase 2 stimulates the downregulation of 3BP2 resulted in the discovery that Tankyrase 2 actually stimulates ubiquitination of the adaptor (Voytyuk et al., manuscript submitted). In our search for an E3 ubiquitin ligase responsible for mediating this ubiquitination, we then tested whether the Nedd4 family members showed sensitivity to the presence or absence of Tankyrase 2 with respect to their abilities to mediate 3BP2 ubiquitination. As there is a known genetic interaction between 3BP2 and Tankyrase 2

(Voytyuk et al., manuscript submitted), sensitivity to Tankyrase 2 would lend a great degree of confidence to our model.

In all cases but Nedd4-1, Tankyrase 2 has been shown to augment 3BP2 ubiquitination in the presence of the tested HECT E3 ligases. Nedd4-1, stimulating the most impressive ubiquitination of 3BP2 may be insensitive to Tankyrase 2, as its capacity to stimulate 3BP2

53 ubiquitination may have already reached saturation. Though Nedd4-2 does show slight sensitivity to the presence of Tankyrase 2, the increase in 3BP2 ubiquitination is modest, and does not compare to the drastic effect that Tankyrase 2 has on endogenous 3BP2 ubiquitination

(Voytyuk et al., manuscript submitted).

The activities of Smurf1 and Smurf2 however show significant sensitivity to the presence of Tankyrase 2 as their respective abilities to mediate 3BP2 ubiquitination are dramatically increased with addition of the poly-ADP ribose polymerase. Though Smurf1 seems to best fit our model, based on the above-mentioned observations, due to the overlapping functions of the two proteins and the fact that loss of one Smurf protein results in compensation by the other (Yamashita et al. , 2005), they may have similar regulation, and may both be sensitive to poly-ADP ribosylation of their substrates.

What is still unclear, however, is how poly-ADP ribosylation stimulates ubiquitination, and how this may relate to the activities of Smurf 1 and 2, or perhaps other Nedd4 E3 ligases.

Further investigation into the other Nedd4 family members brought about the discovery that

Itch/AIP4 contains a putative poly-ADP ribose (PAR) binding motif (Gagne et al. , 2008), consisting of a stretch of hydrophobic and basic residues, as described by Pleschke et al .

((Pleschke et al. , 2000). This would suggest that poly-ADP ribosylation of 3BP2 serves as a docking site for this Nedd4 family HECT E3 ligase, thus allowing it to stimulate 3BP2 ubiquitination. This PAR binding motif is located within the Itch HECT domain, thus it is tempting to speculate that this motif is required to position 3BP2 in such a way that is accessible to the HECT domain, or that perhaps this binding stimulates the ligase activity of the molecule.

Further discussion and preliminary data suggesting Itch as a potential E3 ubiquitin ligase for

3BP2 will be covered in detail in Chapter 5.

54 4.6 Ability of Cherubism Mutant 3BP2 (R413Q) to Bind to Nedd4 Family E3 Ligases Mutation at any of the identified Cherubism sites (namely R413, P416 and G418 in the murine polypeptide) (Ueki et al. , 2007; Ueki et al. , 2001) results in an uncoupling of the interaction between 3BP2 and Tankyrase 2, resulting in an inability of the adaptor to become poly-ADP ribosylated and leading to an increase in 3BP2 steady state protein levels (Voytyuk et al., manuscript submitted). Work in our lab has also shown that the ability of Tankyrase 2 to stimulate 3BP2 ubiquitination is dramatically decreased when wild type 3BP2 is exchanged for its “Cherubic” form (Voytyuk et al., manuscript submitted). Based on these results we then attempted to determine whether Cherubism mutated 3BP2 (henceforth 3BP2_R413Q) showed any decrease in its ability to bind the Nedd4 family of HECT E3 ligases, as this version of 3BP2 cannot become poly-ADP ribosylated and, as mentioned in section 4.5, this modification may have a positive effect on the ability of 3BP2 to interact with the Nedd4 family of proteins.

Of the four HECT E3s tested, while Nedd4-1 showed a slight reduction in binding to

3BP2_R413Q, the results obtained from Nedd4-2, Smurf1 and Smurf2 showed more dramatic effects as a result of a 3BP2 Cherubism mutation. Consistent with our findings concerning the ability of Tankyrase 2 to augment 3BP2 ubiquitination most drastically in the presence of

Smurf1 and Smurf2 (and to a lesser extend, Nedd4-2), this result suggests poly-ADP ribosylation by Tankyrase 2 encourages interaction between 3BP2 and the Smurf (and Nedd4-2) proteins. Again, showing the greatest sensitivity to the Cherubism mutation is Smurf1, who has consistently fit our model and lends itself to being the best candidate E3 ligase for 3BP2, based on the four Nedd4 family members tested.

It should also be noted that the ability of 3BP2_R413Q to bind the Nedd4 family members is reduced, but not obliterated. As mentioned previously, the binding is believed to be a result of an interaction between the Nedd4 WW domains and the 3BP2 PPxY motif. Though the presence of poly-ADP ribosylation may augment the ability of the Nedd4 family members to

55 bind 3BP2, a residual interaction is not unexpected (and may also partially explain why

3BP2_Y202A shows residual binding to the Nedd4 family members, as this molecule would still be able to undergo ribosylation). It would therefore be interesting to combine the

Cherubism and PPxY mutations in order to determine if this combination completely obliterates the interaction.

4.7 Ability of Cherubism Mutant 3BP2 (R413Q) to be Ubiquitinated in the Presence of Nedd4 Family Members As a final experiment to determine the importance of 3BP2 poly-ADP riboslyation on its ability to be ubiquitinated by the Nedd4 family of E3 ligases, we repeated the re-precipitation experiments, this time comparing wild type 3BP2 to the Cherubism mutated R413Q version of the polypeptide. Again, one would expect that since Tankyrase 2 augments the ability of 3BP2 to become ubiquitinated by the Nedd4 family members, a Cherubism mutated form of the protein would show a reduced ability to incorporate ubiquitin.

The results of this experiment are somewhat inconsistent with the previous data reported.

Though Nedd4-2 and Smurf2 did show a reduction in the their respective abilities to incorporate ubiquitin onto Cherubism mutated 3BP2, this was not as drastic as the stimulatory effect that

Tankyrase 2 had on 3BP2 ubiquitination. Additionally, while Nedd4-1 was insensitive to

Tankyrase 2 (and similarly to a Cherubism mutation), Smurf1’s ability to ubiquitinate Cherubic

3BP2 was increased as compared to wild type, however its activity was also augmented in the presence of Tankyrase 2. Smurf1’s ability to bind 3BP2 was also significantly decreased upon introduction of a Cherubism mutation, though, again, this seems to have the reverse effect on its ability to ubiquitinate the molecule.

These results, though puzzling, may be an artifact of the overexpression system. Indeed, much of our work on Cherubism and Tankyrase 2 was accomplished in primary cells, thus it is a

56 formal possibility that the results may be confounded by factors present in the overexpression system that are not of issue in a primary cell culture. It would be interesting to see the effects of the abovementioned Nedd4 family members on endogenous “Cherubic” 3BP2, by perhaps transfecting these proteins into, for example, primary macrophages derived from the Cherubism knock-in mouse model and assessing the degree to which 3BP2 is able to incorporate ubiquitin.

4.8 Analysis of Endogenous 3BP2 Protein Levels in Smurf1 -/-/Smurf2 +/- Macrophages A problem common to the entirety of the abovementioned experiments has been the potential confounding factors that may be present in a 293T overexpression system. Based on the results of the experiments completed within this system, however, we identified Smurf1 as the best candidate E3 ligase for 3BP2, due to its consistent adherence to our working model.

Additionally, due to its sensitivity to Cherubism mutation and high sequence homology, Smurf2 cannot be excluded from our future experiments. To this end, we have begun an attempt at validating these results using primary macrophages derived from Smurf1 -/-/Smurf2 +/- mice (a kind gift from Dr. Jeffrey Wrana).

Cell lysates from 3 independent, age matched pairs of wild type and Smurf1 -/-/Smurf2 +/- mice were used to assess the effect of a decrease in Smurf protein on endogenous 3BP2 levels.

As previously noted, in the absence of one another, the Smurf proteins, with such high sequence similarity, may compensate for one another thus masking the effect of the knockout (Yamashita et al. , 2005), thus the use of a Smurf1 -/-/Smurf2 -/- mouse would have been ideal. However though this mouse has been generated, these mutations are embryonic lethal as a result of either gastrulation or neural tube defects (Narimatsu et al. , 2009).

Though there is a slight, yet observable increase in 3BP2 protein levels in the Smurf1 -/-

/Smurf2 +/- macrophages, this result is not significant upon quantification of triplicate

57 experiments normalized to an irrelevant protein ( β-Actin). While this issue may be resolved with more replicates, it is also known that in the absence of Smurf1, Smurf2 levels increase

(presumably as a compensatory mechanism). Thus, though the result is quite suggestive, it may be masked by the compensatory action of an increase in Smurf2 protein levels. The abundance of Smurf2 (a protein we’ve shown can also bind and ubiquitinate 3BP2 in an overexpression system) would then take over some of the burden resulting from a lack of Smurf1 protein.

58 CHAPTER 5: FUTURE DIRECTIONS

The work presented in Chapter 3 describes a small scale screen looking for a potential

E3 ubiquitin ligase responsible for mediating 3BP2 ubiquitination and subsequent degradation.

The limitation of this work was that it was largely completed via overexpression in 293Ts. This type of experimental system lends itself to the possibility of nonspecific interactions or confounding based on the overabundance of protein expressed in the cell as they are not representative of real, in vivo systems. Thus, the future directions for this project will focus less on overexpression systems and instead will be based on the utilization of primary cell culture, knockdown approaches, in vitro reactions and mouse models.

As discussed in Chapter 4, the Nedd4 Family member Smurf1 seems to be the best candidate 3BP2 E3 ubiquitin ligase, and will be the focus of much of the future directions described below. Additionally, due to its high sequence similarity and potential overlapping function, Smurf2 will also be included in future experiments. Finally, as mentioned in Chapter

4, a new candidate Nedd4 E3 ligase, AIP4/Itch will also be a large focus for the future of this project.

5.1 A Role for the Nedd4 Family E3 Ligase AIP4/Itch in 3BP2 Ubiquitination As discussed in Chapter 4, the Nedd4 family HECT E3 ligase Atrophin Interacting

Protein 4 (AIP4)/Itch (henceforth Itch) is an attractive candidate protein which could be responsible for mediating 3BP2 ubiquitination in vivo . Itch, containing a putative poly-ADP ribose binding motif (Gagne et al. , 2008) may explain the dependence on the poly-ADP ribose polymerase, Tankyrase 2, in the regulation of 3BP2 ubiquitination.

To this end we have begun a series of preliminary experiments in order to address the possibility of Itch as a bona fide E3 ubiquitin ligase for 3BP2. The first of these experiments

59 was a co-immunoprecipitation wherein pCDNA3.1-Myc/His-Itch was co-transfected with either pCMV3X-FLAG vector or pCMV3X-FLAG-3BP2. As depicted in Figure 5-1, after an anti-

FLAG immunoprecipitation and an anti-Myc Western blot, Itch forms a complex with 3BP2 over FLAG vector controls in this overexpression system. In addition, mock immunoprecipitation (i.e.: bead control) yields no detectable signal. Similar results were seen in triplicate experiments (see Appendix 1, Fig. A1-8).

An interaction between Itch and 3BP2 is an important result as it suggests a possible functional relationship between the two molecules. The basis of our interest in Itch, however, is its putative poly-ADP ribose binding motif. Thus, we then tested first whether Itch was capable of stimulating 3BP2 ubiquitination and second (and perhaps more importantly) if this ubiquitination was sensitive to the presence of Tankyrase 2. As depicted in Figure 5-2, not only is Itch capable of stimulating 3BP2 ubiquitination, but this ubiquitination is enhanced upon co- transfection of Tankyrase 2 (based on an anti-FLAG re-precipitation experiment as described in

Chapter 2). This experiment was repeated in triplicate, and yielded similar observations (see

Appendix 1, Fig. A1-9).

Based on these preliminary results, our future directions will include experiments similar to those completed in Chapter 3, namely, we will test the sensitivity of Itch binding and ubiquitination on both 3BP2 PPxY mutation (i.e.: Y202A) and 3BP2 Cherubism mutation (i.e.:

R413Q). Upon completion of these experiments, we will then go on to complete the Itch-related directions described in the remainder of this chapter.

5.2 Assessment of Endogenous/Semi-Endogenous Complex Formation between 3BP2 and Nedd4 Family Proteins As mentioned previously, much of our work has been carried out using an overexpression system, which may lend itself to non-specific interactions. We have recently

A B

pCDNA3.1 - Myc/His- pCMV3X-Flag + Itch + - - Itc pCMV3X-Flag-3BP2 - - + + pCMV3X-Flag + - IP: Flag + - + - pCMV3X-Flag-3BP2 - + 180kD 115kD 115kD Itch α 82kD 82kD IB: Myc IB: αMyc 64kD 49kD IB: αActin 37kD 82kD IB: αFlag α 82kD IB: Flag

Figure 5-1. Itch Interacts with 3BP2. (A) pCDNA3.1-Myc/His-Itch was co-expressed with pCMV3X-FLAG vector (lanes 1 and 2) or pCMV3X-FLAG-3BP2 (lanes 3 and 4). Each sample was divided equally and subject to anti-FLAG immunoprecipitation (lanes 1 and 3) or a bead control experiment (lanes 2 and 4). Membrane was probed with anti-Myc antibody (top panel) then stripped and re-probed with anti- FLAG antibody (bottom panel). (B) Whole cell lysates. pCDNA3.1-Myc/His-Itch was co-expressed with pCMV3X-FLAG vector (lane 1) or pCMV3X-FLAG-3BP2 (lanes 2). Membrane in top panel was probed with anti-Myc (top) and anti-Actin (bottom) antibodies, membrane in bottom panel was probed with anti-FLAG antibody.

A B

Myc -Itch Myc -Itch pCMV3X-Flag-3BP2 + + pCMV3X-Flag-3BP2 + + pCNDA3.1 + - pCDNA3.1 + - pCDNA3.1-Myc/His-Tank2 - + pCDNA3.1-Myc/His-Tank2 - +

180kD 115kD 115kD IB: αMyc 82kD a82kD IP: αFlag IB: α HA- Ub 64kD a 49kD IB: αActin

82kD IP: α Flag IB: α Flag IB: αFlag a 82kD

Figure 5-2 Tankyrase 2 Augments the Ability of Itch to Mediate 3BP2 Ubiquitination. (A) pCMV-HA-Ubiquitin, pCMV3X-FLAG-3BP2 and pCDNA3.1-Myc/His-Itch were co-expressed with either empty pCDNA3.1 vector (lane 1) or pCDNA3.1-Myc/His-Tankyrase2 (lane 2) and subject to anti-FLAG re-precipitation experiment. Blot was probed with anti-HA antibody (top panel) then stripped and re-probed with anti- FLAG antibody. (B) Whole cell lysates. pCMV-HA-Ubiquitin, pCMV3X-FLAG-3BP2 and pCDNA3.1-Myc/His-Itch were co-expressed with either empty pCDNA3.1 vector (lane 1) or pCDNA3.1-Myc/His-Tankyrase2 (lane 2). Membrane in top panel was probed with anti-Myc antibody (top) and anti-Actin antibody (bottom), membrane in bottom panel was probed with anti-FLAG antibody.

61 come into possession of antibodies that are able to detect endogenous Smurf1, Smurf2 and Itch proteins. We are also in possession of antibodies capable of immunoprecipitating and detecting endogenous 3BP2. 3BP2 protein is abundantly expressed in bone marrow derived macrophages and osteoclasts, thus these cell types may serve as an effective starting point. Alternatively, if we are unable to detect an endogenous complex, perhaps a semi-endogenous complex may result from the overexpression of one binding partner (i.e.: 3BP2 or Smurf1/Smurf2/Itch) and detection of the other via protein-specific antibody Western blot. The results of this experiment will not only help us to determine which of these proteins is a real in vivo binding partner of

3BP2 (if any), but will also add confidence to our previous findings in 293T overexpression studies.

5.3 In Vitro Ubiquitination Reaction In collaboration with Dr. Jeffrey Wrana, we are attempting to assess the dependence of

Smurf 1 and 2 mediated 3BP2 ubiquitination on the presence of Tankyrase 2. To this end we have begun attempts at reconstituting our model system using recombinantly expressed 3BP2,

Smurf1, Smurf2 and the PARP domain of Tankyrase 2. Together with an E1 and E2 enzyme, ubiquitin, NAD + (the substrate for a poly-ADP ribose reaction) and ATP we will be able to determine the effects of poly-ADP ribosylation on Smurf mediated 3BP2 ubiquitination in the absence of potential confounding factors present in a 293T cell lysate.

5.4 Knockdown Experiments We have also recently acquired shRNA constructs against Itch (a kind gift from Dr.

Robert Ingham), Smurf1 and Smurf2 (a kind gift from Dr. Jeffrey Wrana). Using these constructs we would like to attempt to knock down these E3 ligases (alone or in concert) to

62 determine their effects on 3BP2 protein levels, as an effect would again give confidence to our findings in the overexpression system. This experiment does lend itself to inherent problems however as the ubiquitously expressed Nedd4 family members have been reported to compensate for one another. Since our overexpression data does suggest that most (if not all) family members are capable of stimulating 3BP2 ubiquitination, loss of just one may not have any effect at all.

If however we are successful in altering 3BP2 protein levels it would be quite interesting to knockdown the relevant Nedd4 family member in cells where 3BP2 is known to play an important role. We have been successful at viral infection of osteoclasts in past experiments, which are a cell type that require 3BP2 for proper function (Levaot et al ., manuscript in preparation). If we could manipulate this system and knockdown a Nedd4 family member of interest, we may potentially see an increase in osteoclastogenesis as would be a direct result of an increase in 3BP2 protein levels (similar to what we have observed in “Cherubic” cells)

(Voytyuk et al ., manuscript submitted).

5.5 Analysis of Smurf1 -/-/Smurf2 +/- and Smurf1 +/-/Smurf2 -/- Mice As described in Chapter 3, analysis of Smurf1 -/-/Smurf2 +/- bone marrow derived macrophages yielded a slight, yet insignificant increase in 3BP2 protein levels as compared to wild type controls. As the sample size was quite small (n=3) it would be interesting to see whether this result will approach significance with increased experiments. Smurf1 +/-/Smurf2 -/- mice have also been generated and analysis of 3BP2 protein levels in macrophages derived from these animals may lend clues as to which Smurf protein is more important to 3BP2 regulation in vivo . Also as previously mentioned, as the Smurf proteins tend to compensate for one another,

63 thus, knockdown of the remaining Smurf allele in either mouse model may yield a more drastic increase in 3BP2 protein levels.

Finally, if dependence on 3BP2 protein levels by Smurf 1 or 2 is established, it will be interesting to further investigate whether cell types where 3BP2 is known to have an important function are affected. Again, as we have seen with Cherubism mice, it is expected that if a lack of Smurf protein leads to an increase in 3BP2 protein levels, we should see a hyperactive osteoclast phenotype.

5.6 Analysis of “Itchy” Mice The Itch null or “Itchy” mouse has a phenotype characterized by massive, widespread inflammation and a severe autoimmune and allergic disorder ((Fang et al. , 2002). We have recently received this mouse as a kind gift from Dr. Pamela Ohashi. In addition to experiments studying the effects on 3BP2 protein levels in the Itch null cells and perhaps their ability to form osteoclasts in in vitro culture, it may also be interesting to cross this mouse with a 3BP2 -/- mouse whose derivation has been previously described (Chen et al. , 2007).

In addition to the osteoporotic phenotype characteristic of the Cherubism mouse, the animal displays massive, widespread TNF α dependent systemic inflammation, presumably as a result of an increase in 3BP2 protein levels (Ueki et al. , 2007). As the Itch null mouse also displays widespread inflammation, and since it is our presumption that Itch results in 3BP2 downregulation, we hypothesize that perhaps crossing the Itch and 3BP2 null mice may, at least to some degree, curtail this phenotype.

To add further interest to the possible in vivo association between Itch and 3BP2, T cells derived from the Cherubism mouse model have recently been shown by our lab to possess a significant increase in the amount of anti-nuclear antibody (ANA) staining as compared to T

64 cells derived from wild type mice. This preliminary data would be suggestive of a potential auto-immune phenotype as a result of an increase in 3BP2 protein levels. As the Itch null mouse also displays a dramatic autoimmune disorder, this may further implicate a role for Itch in mediating 3BP2 regulation in an in vivo system.

In conclusion, though our work to date suggests a potential role for the Nedd4 family of

HECT E3 ligases in the regulation of 3BP2 ubiquitination, the limitations of our experimental system make any definitive statements about the in vivo relationship between these proteins impossible to state with any confidence. Using the wealth of reagents that have recently become available to us, not only will we be able to add genetic validation to our findings, but we may also be able to put our work into a larger, biological context.

65 CHAPTER 6: REFERENCES

Abriel H, Loffing J, Rebhun JF, Pratt JH, Schild L, Horisberger JD et al (1999). Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle's syndrome. J Clin Invest 103: 667- 73.

Akiyoshi S, Inoue H, Hanai J, Kusanagi K, Nemoto N, Miyazono K et al (1999). c-Ski acts as a transcriptional co-repressor in transforming growth factor-beta signaling through interaction with smads. J Biol Chem 274: 35269-77.

Angers A, Ramjaun AR, McPherson PS (2004). The HECT domain ligase itch ubiquitinates endophilin and localizes to the trans-Golgi network and endosomal system. J Biol Chem 279: 11471-9.

Aravind L, Koonin EV (2000). The U box is a modified RING finger - a common domain in ubiquitination. Curr Biol 10: R132-4.

Bedford MT, Chan DC, Leder P (1997). FBP WW domains and the Abl SH3 domain bind to a specific class of proline-rich ligands. Embo J 16: 2376-83.

Bell SM, Shaw M, Jou YS, Myers RM, Knowles MA (1997). Identification and characterization of the human homologue of SH3BP2, an SH3 binding domain protein within a common region of deletion at 4p16.3 involved in bladder cancer. Genomics 44: 163-70.

Bonifacino JS, Traub LM (2003). Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu Rev Biochem 72: 395-447.

Borden KL (2000). RING domains: master builders of molecular scaffolds? J Mol Biol 295: 1103-12.

Borden KL, Freemont PS (1996). The RING finger domain: a recent example of a sequence- structure family. Curr Opin Struct Biol 6: 395-401.

Botero-Velez M, Curtis JJ, Warnock DG (1994). Brief report: Liddle's syndrome revisited--a disorder of sodium reabsorption in the distal tubule. N Engl J Med 330: 178-81.

Capili AD, Schultz DC, Rauscher IF, Borden KL (2001). Solution structure of the PHD domain from the KAP-1 corepressor: structural determinants for PHD, RING and LIM zinc-binding domains. Embo J 20: 165-77.

Chang P, Coughlin M, Mitchison TJ (2005). Tankyrase-1 polymerization of poly(ADP-ribose) is required for spindle structure and function. Nat Cell Biol 7: 1133-9.

Chen C, Matesic LE (2007). The Nedd4-like family of E3 ubiquitin ligases and cancer. Cancer Metastasis Rev 26: 587-604.

66 Chen G, Dimitriou ID, La Rose J, Ilangumaran S, Yeh WC, Doody G et al (2007). The 3BP2 adapter protein is required for optimal B-cell activation and thymus-independent type 2 humoral response. Mol Cell Biol 27: 3109-22.

Chen H, Ross CA, Wang N, Huo Y, MacKinnon DF, Potash JB et al (2001). NEDD4L on human chromosome 18q21 has multiple forms of transcripts and is a homologue of the mouse Nedd4-2 gene. Eur J Hum Genet 9: 922-30.

Chen HI, Sudol M (1995). The WW domain of Yes-associated protein binds a proline-rich ligand that differs from the consensus established for Src homology 3-binding modules. Proc Natl Acad Sci U S A 92: 7819-23.

Chiang YJ, Hsiao SJ, Yver D, Cushman SW, Tessarollo L, Smith S et al (2008). Tankyrase 1 and tankyrase 2 are essential but redundant for mouse embryonic development. PLoS ONE 3: e2639.

Chiang YJ, Nguyen ML, Gurunathan S, Kaminker P, Tessarollo L, Campisi J et al (2006). Generation and characterization of telomere length maintenance in tankyrase 2-deficient mice. Mol Cell Biol 26: 2037-43.

Cook BD, Dynek JN, Chang W, Shostak G, Smith S (2002). Role for the related poly(ADP- Ribose) polymerases tankyrase 1 and 2 at human telomeres. Mol Cell Biol 22: 332-42.

De Rycker M, Price CM (2004). Tankyrase polymerization is controlled by its sterile alpha motif and poly(ADP-ribose) polymerase domains. Mol Cell Biol 24: 9802-12.

De Rycker M, Venkatesan RN, Wei C, Price CM (2003). Vertebrate tankyrase domain structure and sterile alpha motif (SAM)-mediated multimerization. Biochem J 372: 87-96.

Debonneville C, Flores SY, Kamynina E, Plant PJ, Tauxe C, Thomas MA et al (2001). Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+) channel cell surface expression. Embo J 20: 7052-9.

Deckert M, Rottapel R (2006). The adapter 3BP2: how it plugs into leukocyte signaling. Adv Exp Med Biol 584: 107-14.

Deckert M, Tartare-Deckert S, Hernandez J, Rottapel R, Altman A (1998). Adaptor function for the Syk kinases-interacting protein 3BP2 in IL-2 gene activation. Immunity 9: 595-605.

Deng L, Wang C, Spencer E, Yang L, Braun A, You J et al (2000). Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103: 351-61.

Dunn DM, Ishigami T, Pankow J, von Niederhausern A, Alder J, Hunt SC et al (2002). Common variant of human NEDD4L activates a cryptic splice site to form a frameshifted transcript. J Hum Genet 47: 665-76.

67 Dynek JN, Smith S (2004). Resolution of sister telomere association is required for progression through mitosis. Science 304: 97-100.

Ebisawa T, Fukuchi M, Murakami G, Chiba T, Tanaka K, Imamura T et al (2001). Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation. J Biol Chem 276: 12477-80.

Essen LO, Perisic O, Cheung R, Katan M, Williams RL (1996). Crystal structure of a mammalian phosphoinositide-specific phospholipase C delta. Nature 380: 595-602.

Fang D, Elly C, Gao B, Fang N, Altman Y, Joazeiro C et al (2002). Dysregulation of T lymphocyte function in itchy mice: a role for Itch in TH2 differentiation. Nat Immunol 3: 281-7.

Fang D, Kerppola TK (2004). Ubiquitin-mediated fluorescence complementation reveals that Jun ubiquitinated by Itch/AIP4 is localized to lysosomes. Proc Natl Acad Sci U S A 101: 14782- 7.

Fang S, Weissman AM (2004). A field guide to ubiquitylation. Cell Mol Life Sci 61: 1546-61.

Fisk HA, Yaffe MP (1999). A role for ubiquitination in mitochondrial inheritance in Saccharomyces cerevisiae. J Cell Biol 145: 1199-208.

Flasza M, Gorman P, Roylance R, Canfield AE, Baron M (2002). Alternative splicing determines the domain structure of WWP1, a Nedd4 family protein. Biochem Biophys Res Commun 290: 431-7.

Foucault I, Le Bras S, Charvet C, Moon C, Altman A, Deckert M (2005). The adaptor protein 3BP2 associates with VAV guanine nucleotide exchange factors to regulate NFAT activation by the B-cell antigen receptor. Blood 105: 1106-13.

Foucault I, Liu YC, Bernard A, Deckert M (2003). The chaperone protein 14-3-3 interacts with 3BP2/SH3BP2 and regulates its adapter function. J Biol Chem 278: 7146-53.

Fukuchi M, Fukai Y, Masuda N, Miyazaki T, Nakajima M, Sohda M et al (2002). High-level expression of the Smad ubiquitin ligase Smurf2 correlates with poor prognosis in patients with esophageal squamous cell carcinoma. Cancer Res 62: 7162-5.

Gagne JP, Isabelle M, Lo KS, Bourassa S, Hendzel MJ, Dawson VL et al (2008). Proteome- wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes. Nucleic Acids Res 36: 6959-76.

Gao M, Labuda T, Xia Y, Gallagher E, Fang D, Liu YC et al (2004). Jun turnover is controlled through JNK-dependent phosphorylation of the E3 ligase Itch. Science 306: 271-5.

Garty H, Palmer LG (1997). Epithelial sodium channels: function, structure, and regulation. Physiol Rev 77: 359-96.

68 Goulet CC, Volk KA, Adams CM, Prince LS, Stokes JB, Snyder PM (1998). Inhibition of the epithelial Na+ channel by interaction of Nedd4 with a PY motif deleted in Liddle's syndrome. J Biol Chem 273: 30012-7.

Haas AL, Rose IA (1982). The mechanism of ubiquitin activating enzyme. A kinetic and equilibrium analysis. J Biol Chem 257: 10329-37.

Hakme A, Wong HK, Dantzer F, Schreiber V (2008). The expanding field of poly(ADP- ribosyl)ation reactions. 'Protein Modifications: Beyond the Usual Suspects' Review Series. EMBO Rep 9: 1094-100.

Hamilton MH, Tcherepanova I, Huibregtse JM, McDonnell DP (2001). Nuclear import/export of hRPF1/Nedd4 regulates the ubiquitin-dependent degradation of its nuclear substrates. J Biol Chem 276: 26324-31.

Handley-Gearhart PM, Stephen AG, Trausch-Azar JS, Ciechanover A, Schwartz AL (1994). Human ubiquitin-activating enzyme, E1. Indication of potential nuclear and cytoplasmic subpopulations using epitope-tagged cDNA constructs. J Biol Chem 269: 33171-8.

Harvey KF, Dinudom A, Cook DI, Kumar S (2001). The Nedd4-like protein KIAA0439 is a potential regulator of the epithelial sodium channel. J Biol Chem 276: 8597-601.

Hatakeyama S, Yada M, Matsumoto M, Ishida N, Nakayama KI (2001). U box proteins as a new family of ubiquitin-protein ligases. J Biol Chem 276: 33111-20.

Hayashi H, Abdollah S, Qiu Y, Cai J, Xu YY, Grinnell BW et al (1997). The MAD-related protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGFbeta signaling. Cell 89: 1165-73.

Hein C, Springael JY, Volland C, Haguenauer-Tsapis R, Andre B (1995). NPl1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin-protein ligase. Mol Microbiol 18: 77-87.

Heissmeyer V, Macian F, Im SH, Varma R, Feske S, Venuprasad K et al (2004). Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins. Nat Immunol 5: 255-65.

Hershko A, Ciechanover A (1998). The ubiquitin system. Annu Rev Biochem 67: 425-79.

Hershko A, Heller H, Elias S, Ciechanover A (1983). Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J Biol Chem 258: 8206- 14.

Hochstrasser M (1996). Ubiquitin-dependent protein degradation. Annu Rev Genet 30: 405-39.

Hoege C, Pfander B, Moldovan GL, Pyrowolakis G, Jentsch S (2002). RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419: 135-41.

69 Hsiao SJ, Poitras MF, Cook BD, Liu Y, Smith S (2006). Tankyrase 2 poly(ADP-ribose) polymerase domain-deleted mice exhibit growth defects but have normal telomere length and capping. Mol Cell Biol 26: 2044-54.

Hsiao SJ, Smith S (2008). Tankyrase function at telomeres, spindle poles, and beyond. Biochimie 90: 83-92.

Huang K, Johnson KD, Petcherski AG, Vandergon T, Mosser EA, Copeland NG et al (2000). A HECT domain ubiquitin ligase closely related to the mammalian protein WWP1 is essential for Caenorhabditis elegans embryogenesis. Gene 252: 137-45.

Huibregtse JM, Scheffner M, Beaudenon S, Howley PM (1995). A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc Natl Acad Sci U S A 92: 5249.

Huibregtse JM, Yang JC, Beaudenon SL (1997). The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase. Proc Natl Acad Sci U S A 94: 3656-61.

Imamura T, Takase M, Nishihara A, Oeda E, Hanai J, Kawabata M et al (1997). Smad6 inhibits signalling by the TGF-beta superfamily. Nature 389: 622-6.

Ingham RJ, Gish G, Pawson T (2004). The Nedd4 family of E3 ubiquitin ligases: functional diversity within a common modular architecture. Oncogene 23: 1972-84.

Itani OA, Campbell JR, Herrero J, Snyder PM, Thomas CP (2003). Alternate promoters and variable splicing lead to hNedd4-2 isoforms with a C2 domain and varying number of WW domains. Am J Physiol Renal Physiol 285: F916-29.

Jackson PK, Eldridge AG, Freed E, Furstenthal L, Hsu JY, Kaiser BK et al (2000). The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases. Trends Cell Biol 10: 429-39.

Jevremovic D, Billadeau DD, Schoon RA, Dick CJ, Leibson PJ (2001). Regulation of NK cell- mediated cytotoxicity by the adaptor protein 3BP2. J Immunol 166: 7219-28.

Joazeiro CA, Weissman AM (2000). RING finger proteins: mediators of ubiquitin ligase activity. Cell 102: 549-52.

Judge AR, Koncarevic A, Hunter RB, Liou HC, Jackman RW, Kandarian SC (2007). Role for IkappaBalpha, but not c-Rel, in skeletal muscle atrophy. Am J Physiol Cell Physiol 292: C372- 82.

Kaneki H, Guo R, Chen D, Yao Z, Schwarz EM, Zhang YE et al (2006). Tumor necrosis factor promotes Runx2 degradation through up-regulation of Smurf1 and Smurf2 in osteoblasts. J Biol Chem 281: 4326-33.

Katzav S (2007). Flesh and blood: the story of Vav1, a gene that signals in hematopoietic cells but can be transforming in human malignancies. Cancer Lett 255: 241-54.

70 Kavsak P, Rasmussen RK, Causing CG, Bonni S, Zhu H, Thomsen GH et al (2000). Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation. Mol Cell 6: 1365-75.

Kay BK, Williamson MP, Sudol M (2000). The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains. Faseb J 14: 231-41.

Kendrick TS, Lipscombe RJ, Rausch O, Nicholson SE, Layton JE, Goldie-Cregan LC et al (2004). Contribution of the membrane-distal tyrosine in intracellular signaling by the granulocyte colony-stimulating factor receptor. J Biol Chem 279: 326-40.

Komuro A, Imamura T, Saitoh M, Yoshida Y, Yamori T, Miyazono K et al (2004). Negative regulation of transforming growth factor-beta (TGF-beta) signaling by WW domain-containing protein 1 (WWP1). Oncogene 23: 6914-23.

Kuimov AN, Kuprash DV, Petrov VN, Vdovichenko KK, Scanlan MJ, Jongeneel CV et al (2001). Cloning and characterization of TNKL, a member of tankyrase gene family. Genes Immun 2: 52-5.

Kumar S, Kao WH, Howley PM (1997). Physical interaction between specific E2 and Hect E3 enzymes determines functional cooperativity. J Biol Chem 272: 13548-54.

Kumar S, Tomooka Y, Noda M (1992). Identification of a set of genes with developmentally down-regulated expression in the mouse brain. Biochem Biophys Res Commun 185: 1155-61.

Lin X, Liang M, Feng XH (2000). Smurf2 is a ubiquitin E3 ligase mediating proteasome- dependent degradation of Smad2 in transforming growth factor-beta signaling. J Biol Chem 275: 36818-22.

Lu PJ, Zhou XZ, Shen M, Lu KP (1999). Function of WW domains as phosphoserine- or phosphothreonine-binding modules. Science 283: 1325-8.

Lu Z, Xu S, Joazeiro C, Cobb MH, Hunter T (2002). The PHD domain of MEKK1 acts as an E3 ubiquitin ligase and mediates ubiquitination and degradation of ERK1/2. Mol Cell 9: 945-56.

Luo K, Stroschein SL, Wang W, Chen D, Martens E, Zhou S et al (1999). The Ski oncoprotein interacts with the Smad proteins to repress TGFbeta signaling. Genes Dev 13: 2196-206.

Macias MJ, Wiesner S, Sudol M (2002). WW and SH3 domains, two different scaffolds to recognize proline-rich ligands. FEBS Lett 513: 30-7.

Maeno K, Sada K, Kyo S, Miah SM, Kawauchi-Kamata K, Qu X et al (2003). Adaptor protein 3BP2 is a potential ligand of Src homology 2 and 3 domains of Lyn protein-tyrosine kinase. J Biol Chem 278: 24912-20.

Marchese A, Raiborg C, Santini F, Keen JH, Stenmark H, Benovic JL (2003). The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G protein-coupled receptor CXCR4. Dev Cell 5: 709-22.

Massague J (1998). TGF-beta signal transduction. Annu Rev Biochem 67: 753-91.

Matesic LE, Haines DC, Copeland NG, Jenkins NA (2006). Itch genetically interacts with Notch1 in a mouse autoimmune disease model. Hum Mol Genet 15: 3485-97.

McDonald FJ, Western AH, McNeil JD, Thomas BC, Olson DR, Snyder PM (2002). Ubiquitin- protein ligase WWP2 binds to and downregulates the epithelial Na(+) channel. Am J Physiol Renal Physiol 283: F431-6.

McGrath JP, Jentsch S, Varshavsky A (1991). UBA 1: an essential yeast gene encoding ubiquitin-activating enzyme. Embo J 10: 227-36.

Nakao A, Afrakhte M, Moren A, Nakayama T, Christian JL, Heuchel R et al (1997). Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling. Nature 389: 631-5.

Nalefski EA, Falke JJ (1996). The C2 domain calcium-binding motif: structural and functional diversity. Protein Sci 5: 2375-90.

Narimatsu M, Bose R, Pye M, Zhang L, Miller B, Ching P et al (2009). Regulation of planar cell polarity by Smurf ubiquitin ligases. Cell 137: 295-307.

Obenauer JC, Cantley LC, Yaffe MB (2003). Scansite 2.0: Proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res 31: 3635-41.

Oberst A, Malatesta M, Aqeilan RI, Rossi M, Salomoni P, Murillas R et al (2007). The Nedd4- binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch. Proc Natl Acad Sci U S A 104: 11280-5.

Ohashi N, Yamamoto T, Uchida C, Togawa A, Fukasawa H, Fujigaki Y et al (2005). Transcriptional induction of Smurf2 ubiquitin ligase by TGF-beta. FEBS Lett 579: 2557-63.

Petroski MD (2008). The ubiquitin system, disease, and drug discovery. BMC Biochem 9 Suppl 1: S7.

Pickart CM (2001). Mechanisms underlying ubiquitination. Annu Rev Biochem 70: 503-33.

Pickart CM (2002). DNA repair: right on target with ubiquitin. Nature 419: 120-1.

Pleschke JM, Kleczkowska HE, Strohm M, Althaus FR (2000). Poly(ADP-ribose) binds to specific domains in DNA damage checkpoint proteins. J Biol Chem 275: 40974-80.

Podos SD, Hanson KK, Wang YC, Ferguson EL (2001). The DSmurf ubiquitin-protein ligase restricts BMP signaling spatially and temporally during Drosophila embryogenesis. Dev Cell 1: 567-78.

72 Qiu L, Joazeiro C, Fang N, Wang HY, Elly C, Altman Y et al (2000). Recognition and ubiquitination of Notch by Itch, a hect-type E3 ubiquitin ligase. J Biol Chem 275: 35734-7.

Qu X, Kawauchi-Kamata K, Miah SM, Hatani T, Yamamura H, Sada K (2005). Tyrosine phosphorylation of adaptor protein 3BP2 induces T cell receptor-mediated activation of transcription factor. Biochemistry 44: 3891-8.

Quan F, Grompe M, Jakobs P, Popovich BW (1995). Spontaneous deletion in the FMR1 gene in a patient with fragile X syndrome and cherubism. Hum Mol Genet 4: 1681-4.

Ren R, Mayer BJ, Cicchetti P, Baltimore D (1993). Identification of a ten-amino acid proline- rich SH3 binding site. Science 259: 1157-61.

Rittling SR, Chambers AF (2004). Role of osteopontin in tumour progression. Br J Cancer 90: 1877-81.

Rizo J, Sudhof TC (1998). C2-domains, structure and function of a universal Ca2+-binding domain. J Biol Chem 273: 15879-82.

Rossi M, Aqeilan RI, Neale M, Candi E, Salomoni P, Knight RA et al (2006). The E3 ubiquitin ligase Itch controls the protein stability of p63. Proc Natl Acad Sci U S A 103: 12753-8.

Rossi M, De Laurenzi V, Munarriz E, Green DR, Liu YC, Vousden KH et al (2005). The ubiquitin-protein ligase Itch regulates p73 stability. Embo J 24: 836-48.

Rossier BC, Pradervand S, Schild L, Hummler E (2002). Epithelial sodium channel and the control of sodium balance: interaction between genetic and environmental factors. Annu Rev Physiol 64: 877-97.

Rotin D, Staub O, Haguenauer-Tsapis R (2000). Ubiquitination and endocytosis of plasma membrane proteins: role of Nedd4/Rsp5p family of ubiquitin-protein ligases. J Membr Biol 176: 1-17.

Sada K, Miah SM, Maeno K, Kyo S, Qu X, Yamamura H (2002). Regulation of FcepsilonRI- mediated degranulation by an adaptor protein 3BP2 in rat basophilic leukemia RBL-2H3 cells. Blood 100: 2138-44.

Sbodio JI, Lodish HF, Chi NW (2002). Tankyrase-2 oligomerizes with tankyrase-1 and binds to both TRF1 (telomere-repeat-binding factor 1) and IRAP (insulin-responsive aminopeptidase). Biochem J 361: 451-9.

Scheffner M, Huibregtse JM, Howley PM (1994). Identification of a human ubiquitin- conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53. Proc Natl Acad Sci U S A 91: 8797-801.

Scheffner M, Nuber U, Huibregtse JM (1995). Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature 373: 81-3.

73 Schild L, Lu Y, Gautschi I, Schneeberger E, Lifton RP, Rossier BC (1996). Identification of a PY motif in the epithelial Na channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome. Embo J 15: 2381-7.

Shao X, Davletov BA, Sutton RB, Sudhof TC, Rizo J (1996). Bipartite Ca2+-binding motif in C2 domains of synaptotagmin and protein kinase C. Science 273: 248-51.

Shi H, Asher C, Chigaev A, Yung Y, Reuveny E, Seger R et al (2002). Interactions of beta and gamma ENaC with Nedd4 can be facilitated by an ERK-mediated phosphorylation. J Biol Chem 277: 13539-47.

Smith S, de Lange T (2000). Tankyrase promotes telomere elongation in human cells. Curr Biol 10: 1299-302.

Smith S, Giriat I, Schmitt A, de Lange T (1998). Tankyrase, a poly(ADP-ribose) polymerase at human telomeres. Science 282: 1484-7.

Snyder PM, Olson DR, Kabra R, Zhou R, Steines JC (2004). cAMP and serum and glucocorticoid-inducible kinase (SGK) regulate the epithelial Na(+) channel through convergent phosphorylation of Nedd4-2. J Biol Chem 279: 45753-8.

Snyder PM, Price MP, McDonald FJ, Adams CM, Volk KA, Zeiher BG et al (1995). Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na+ channel. Cell 83: 969-78.

Sporn MB, Roberts AB (1990). TGF-beta: problems and prospects. Cell Regul 1: 875-82.

Staub O, Dho S, Henry P, Correa J, Ishikawa T, McGlade J et al (1996). WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome. Embo J 15: 2371-80.

Staub O, Gautschi I, Ishikawa T, Breitschopf K, Ciechanover A, Schild L et al (1997). Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination. Embo J 16: 6325-36.

Stroschein SL, Wang W, Zhou S, Zhou Q, Luo K (1999). Negative feedback regulation of TGF- beta signaling by the SnoN oncoprotein. Science 286: 771-4.

Sun Y, Liu X, Eaton EN, Lane WS, Lodish HF, Weinberg RA (1999). Interaction of the Ski oncoprotein with Smad3 regulates TGF-beta signaling. Mol Cell 4: 499-509.

Sutton RB, Davletov BA, Berghuis AM, Sudhof TC, Sprang SR (1995). Structure of the first C2 domain of synaptotagmin I: a novel Ca2+/phospholipid-binding fold. Cell 80: 929-38.

Tajima Y, Goto K, Yoshida M, Shinomiya K, Sekimoto T, Yoneda Y et al (2003). Chromosomal region maintenance 1 (CRM1)-dependent nuclear export of Smad ubiquitin regulatory factor 1 (Smurf1) is essential for negative regulation of transforming growth factor- beta signaling by Smad7. J Biol Chem 278: 10716-21.

Tamai KK, Shimoda C (2002). The novel HECT-type ubiquitin-protein ligase Pub2p shares partially overlapping function with Pub1p in Schizosaccharomyces pombe. J Cell Sci 115: 1847-57.

Thrower JS, Hoffman L, Rechsteiner M, Pickart CM (2000). Recognition of the polyubiquitin proteolytic signal. Embo J 19: 94-102.

Ueki Y, Lin CY, Senoo M, Ebihara T, Agata N, Onji M et al (2007). Increased myeloid cell responses to M-CSF and RANKL cause bone loss and inflammation in SH3BP2 "cherubism" mice. Cell 128: 71-83.

Ueki Y, Tiziani V, Santanna C, Fukai N, Maulik C, Garfinkle J et al (2001). Mutations in the gene encoding c-Abl-binding protein SH3BP2 cause cherubism. Nat Genet 28: 125-6.

Umemura M, Ishigami T, Tamura K, Sakai M, Miyagi Y, Nagahama K et al (2006). Transcriptional diversity and expression of NEDD4L gene in distal nephron. Biochem Biophys Res Commun 339: 1129-37. von Arnim AG (2001). A hitchhiker's guide to the proteasome. Sci STKE 2001: PE2.

Wai PY, Kuo PC (2004). The role of Osteopontin in tumor metastasis. J Surg Res 121: 228-41.

Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ (2001). TAK1 is a ubiquitin- dependent kinase of MKK and IKK. Nature 412: 346-51.

Weissman AM (2001). Themes and variations on ubiquitylation. Nat Rev Mol Cell Biol 2: 169- 78.

Wood JD, Yuan J, Margolis RL, Colomer V, Duan K, Kushi J et al (1998). Atrophin-1, the DRPLA gene product, interacts with two families of WW domain-containing proteins. Mol Cell Neurosci 11: 149-60.

Xu W, Angelis K, Danielpour D, Haddad MM, Bischof O, Campisi J et al (2000). Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type beta transforming growth factor. Proc Natl Acad Sci U S A 97: 5924-9.

Yaffe MB, Schutkowski M, Shen M, Zhou XZ, Stukenberg PT, Rahfeld JU et al (1997). Sequence-specific and phosphorylation-dependent proline isomerization: a potential mitotic regulatory mechanism. Science 278: 1957-60.

Yamashita M, Ying SX, Zhang GM, Li C, Cheng SY, Deng CX et al (2005). Ubiquitin ligase Smurf1 controls osteoblast activity and bone homeostasis by targeting MEKK2 for degradation. Cell 121: 101-13.

Yang C, Zhou W, Jeon MS, Demydenko D, Harada Y, Zhou H et al (2006). Negative regulation of the E3 ubiquitin ligase itch via Fyn-mediated tyrosine phosphorylation. Mol Cell 21: 135-41.

75 Yeatman TJ (2004). A renaissance for SRC. Nat Rev Cancer 4: 470-80.

Yeh TY, Sbodio JI, Tsun ZY, Luo B, Chi NW (2007). Insulin-stimulated exocytosis of GLUT4 is enhanced by IRAP and its partner tankyrase. Biochem J 402: 279-90.

Ying SX, Hussain ZJ, Zhang YE (2003). Smurf1 facilitates myogenic differentiation and antagonizes the bone morphogenetic protein-2-induced osteoblast conversion by targeting Smad5 for degradation. J Biol Chem 278: 39029-36.

Zarrinpar A, Lim WA (2000). Converging on proline: the mechanism of WW domain peptide recognition. Nat Struct Biol 7: 611-3.

Zhang Y, Chang C, Gehling DJ, Hemmati-Brivanlou A, Derynck R (2001). Regulation of Smad degradation and activity by Smurf2, an E3 ubiquitin ligase. Proc Natl Acad Sci U S A 98: 974-9.

Zhao M, Qiao M, Harris SE, Oyajobi BO, Mundy GR, Chen D (2004). Smurf1 inhibits osteoblast differentiation and bone formation in vitro and in vivo. J Biol Chem 279: 12854-9.

Zhao M, Qiao M, Oyajobi BO, Mundy GR, Chen D (2003). E3 ubiquitin ligase Smurf1 mediates core-binding factor alpha1/Runx2 degradation and plays a specific role in osteoblast differentiation. J Biol Chem 278: 27939-44.

Zhu H, Kavsak P, Abdollah S, Wrana JL, Thomsen GH (1999). A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation. Nature 400: 687-93.

Zollino M, Di Stefano C, Zampino G, Mastroiacovo P, Wright TJ, Sorge G et al (2000). Genotype-phenotype correlations and clinical diagnostic criteria in Wolf-Hirschhorn syndrome. Am J Med Genet 94: 254-61.

76 APPENDIX 1: REPLICATE EXPERIMENTS

180kDa 180kDa 115kDa 115kDa 82kDa 82kDa IB: αV5 IB: αV5

64kDa 64kDa

82kDa IB: αFlag 82kDa IB: αFlag

Nedd41 Nedd42 Smurf1 Smurf2 C pCMV3X-Flag + - + - + - + - pCMV3X-Flag-3BP2 - + - + - + - +

180kDa 115kDa 82kDa IB: αV5

49kDa 37kDa IB: αActin

82kDa IB: αFlag

Figure A1-1 (i) 3BP2 Interacts with Nedd4 Family E3 Ligases. (A) pCMV3X-FLAG-3BP2 was co-expressed with pCDNA6.2-V5-hNedd4- 1 (lanes 3 and 4) or pCDNA6.2-V5-hNedd4-2 (lanes 7 and 8). Control experiments include pCMV3X-FLAG vector co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2) and with pCDNA6.2-V5-hNedd4-2 (lanes 5 and 6). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti-V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (B) pCMV3X-FLAG-3BP2 was co- expressed with pCDNA6.2-V5-hSmurf1 (lanes 3 and 4) or pCDNA6.2-V5-hSmurf2 (lanes 7 and 8). Control experiments include pCMV3X- FLAG vector co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) and with pCDNA6.2-V5-Smurf2 (lanes 5 and 6). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti-V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (C) Whole cell lysate samples. pCMV3X-FLAG vector is expressed in lanes 1, 3, 5 and 7. pCMV3X-FLAG-3BP2 is expressed in lanes 2, 4, 6 and 8. pCNDA6.2-V5-hNedd4-1 was co-expressed in lanes 1 and 2, pCDNA6.2-V5-hNedd4-2 was co-expressed in lanes 3 and 4, pCDNA6.2-V5- hSmurf1 was co-expressed in lanes 5 and 6 and pCDNA6.2-V5-hSmurf2 was co-expressed in lanes 7 and 8. Membrane in top panel was probed with anti-V5 (top) and anti-Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

180kDa 180kDa 115kDa 115kDa 82kDa IB: αV5 82kDa IB: αV5

64kDa 64kDa

82kDa IB: αFlag 82kDa IB: αFlag

Nedd41 Nedd42 Smurf1 Smurf2 C pCMV3X-Flag + - + - + - + - pCMV3X-Flag-3BP2 - + - + - + - +

180kDa 115kDa 82kDa IB: αV5

49kDa IB: αActin 37kDa

82kDa IB: αFlag

Figure A1-1 (ii) 3BP2 Interacts with Nedd4 Family E3 Ligases. (A) pCMV3X-FLAG-3BP2 was co-expressed with pCDNA6.2-V5-hNedd4- 1 (lanes 3 and 4) or pCDNA6.2-V5-hNedd4-2 (lanes 7 and 8). Control experiments include pCMV3X-FLAG vector co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2) and with pCDNA6.2-V5-hNedd4-2 (lanes 5 and 6). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti-V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (B) pCMV3X-FLAG-3BP2 was co- expressed with pCDNA6.2-V5-hSmurf1 (lanes 3 and 4) or pCDNA6.2-V5-hSmurf2 (lanes 7 and 8). Control experiments include pCMV3X- FLAG vector co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) and with pCDNA6.2-V5-Smurf2 (lanes 5 and 6). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti-V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (C) Whole cell lysate samples. pCMV3X-FLAG vector is expressed in lanes 1, 3, 5 and 7. pCMV3X-FLAG-3BP2 is expressed in lanes 2, 4, 6 and 8. pCNDA6.2-V5-hNedd4-1 was co-expressed in lanes 1 and 2, pCDNA6.2-V5-hNedd4-2 was co-expressed in lanes 3 and 4, pCDNA6.2-V5- hSmurf1 was co-expressed in lanes 5 and 6 and pCDNA6.2-V5-hSmurf2 was co-expressed in lanes 7 and 8. Membrane in top panel was probed with anti-V5 (top) and anti-Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

A pCMV3X -FLAG - B 3BP2

_ hNedd4-1 hSmurf-1 hSmurf-2 hNedd4-2

180kDa _ Nedd4-1 Nedd4-2 Smurf-1 Smurf-2 115kDa 180kDa 82kDa IP: αFlag 115kDa IB: α HA-Ub IB: V5 64kDa 82kDa

82kDa IB: Flag

IP: α Flag 49kDa 82kDa IB: Actin IB: α Flag 37kDa

Figure A1-2 (i) Nedd4 Family E3 Ligases Stimulate 3BP2 Ubiquitination. (A) pCMV-HA-Ubiquitin and pCMV3X-FLAG-3BP2 were expressed alone (lane 1) or co-expressed with pCDNA6.2-V5-hNedd4-1 (lane 2), pCDNA6.2-V5-hNedd4-2 (lane 3), pCDNA6.2-V5-hSmurf1 (lane 4) or pCDNA6.2-V5-hSmurf2 (lane 5). Re-precipitation experiment was performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (B) Whole cell lysate samples. pCMV-HA-Ubiquitin and pCMV3X-FLAG-3BP2 were expressed alone (lane 1) or co-expressed with pCDNA6.2-V5-hNedd4-1 (lane 2), pCDNA6.2-V5-hNedd4-2 (lane 3), pCDNA6.2-V5-hSmurf1 (lane 4) or pCDNA6.2-V5-hSmurf2 (lane 5). Membrane in top panel was probed with anti-V5, membrane in middle panel probed with anti-FLAG and membrane in bottom panel probed with anti-Actin.

A pCMV3X -FLAG - B 3BP2

_ hNedd4-1 hSmurf-1 hSmurf-2 hNedd4-2

180kDa _ Smurf-1 Nedd4-1 Nedd4-2 Smurf-2 115kDa 180kDa 82kDa IP: αFlag 115kDa IB: V5 IB: α HA-Ub 82kDa

82kDa IB: Flag

IP: α Flag 82kDa IB: α Flag 49kDa IB: Actin 37kDa

Figure A1-2 (ii) Nedd4 Family E3 Ligases Stimulate 3BP2 Ubiquitination. (A) pCMV-HA-Ubiquitin and pCMV3X-FLAG-3BP2 were expressed alone (lane 1) or co-expressed with pCDNA6.2-V5-hNedd4-1 (lane 2), pCDNA6.2-V5-hNedd4-2 (lane 3), pCDNA6.2-V5-hSmurf1 (lane 4) or pCDNA6.2-V5-hSmurf2 (lane 5). Re-precipitation experiment was performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (B) Whole cell lysate samples. pCMV-HA-Ubiquitin and pCMV3X-FLAG-3BP2 were expressed alone (lane 1) or co-expressed with pCDNA6.2-V5-hNedd4-1 (lane 2), pCDNA6.2-V5-hNedd4-2 (lane 3), pCDNA6.2-V5-hSmurf1 (lane 4) or pCDNA6.2-V5-hSmurf2 (lane 5). Membrane in top panel was probed with anti-V5, membrane in middle panel probed with anti-FLAG and membrane in bottom panel probed with anti-Actin.

V5 -hNedd4 -1 V5-hNedd4-2 pCMV3X-Flag-3BP2 + + - - + + - - pCMV3X-Flag-3BP2_Y202A - - + + - - + + A IP: Flag + - + - + - + -

180kD a hNedd4-1 hNedd4-2 115kD a 82kD a IB: αV5 64kD a

82kD IB: αFlag a

V5-hSmurf1 V5-hSmurf2 B pCMV3X-Flag-3BP2 + + - - + + - - pCMV3X-Flag-3BP2_Y202A - - + + - - + + IP: Flag + - + - + - + -

180kD a 115kD a hSmurf1 hSmurf2 82kD a 64kD a

82kD a

Nedd41 Nedd42 Smurf1 Smurf2 C pCMV3X-Flag-3BP2 + - + - + - + - pCMV3X-Flag-3BP2_Y202A - + - + - + - + 180kD 115kDa a82kD IB: αV5 a

49kD α 37kDa IB: Actin a

82kD IB: αFlag a

Figure A1-3 (i) Ability of 3BP2_Y202A to Bind to Nedd4 Family E3s. (A) pCMV3X-FLAG-3BP2 was co-expressed with pCDNA6.2-V5- hNedd4-1 (lanes 1 and 2) or pCDNA6.2-V5-hNedd4-2 (lanes 5 and 6). pCMV3X-FLAG-3BP2_Y202A was co-expressed with pCDNA6.2-V5- hNedd4-1 (lanes 3 and 4) and with pCDNA6.2-V5-hNedd4-2 (lanes 7 and 8). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti- V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (B) pCMV3X-FLAG-3BP2 was co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) or pCDNA6.2-V5-hSmurf2 (lanes 5 and 6). pCMV3X-FLAG-3BP2_Y202A was co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 3 and 4) and with pCDNA6.2-V5-Smurf2 (lanes 7 and 8). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti-V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (C) Whole cell lysate samples. pCMV3X- FLAG-3BP2 is expressed in lanes 1, 3, 5 and 7. pCMV3X-FLAG-3BP2_Y202A is expressed in lanes 2, 4, 6 and 8. pCNDA6.2-V5-hNedd4-1 was co-expressed in lanes 1 and 2, pCDNA6.2-V5-hNedd4-2 was co-expressed in lanes 3 and 4, pCDNA6.2-V5-hSmurf1 was co-expressed in lanes 5 and 6 and pCDNA6.2-V5-hSmurf2 was co-expressed in lanes 7 and 8. Membrane in top panel was probed with anti-V5 (top) and anti- Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

V5-hNedd4-1 V5-hNedd4-2 pCMV3X-Flag-3BP2 + + - - + + - - pCMV3X-Flag-3BP2_Y202A - - + + - - + + A IP: Flag + - + - + - + -

180kDa hNedd4-1 115kDa hNedd4-2

82kDa IB: αV5 64kDa

82kDa IB: αFlag

V5-hSmurf1 V5-hSmurf2 B pCMV3X-Flag-3BP2 + + - - + + - - pCMV3X-Flag-3BP2_Y202A - - + + - - + + IP: Flag + - + - + - + - 180kDa 115kDa

hSmurf1 hSmurf2 82kDa

64kDa IB: αV5

82kDa IB: αFlag

Nedd41 Nedd42 Smurf1 Smurf2 C pCMV3X-Flag-3BP2 + - + - + - + - pCMV3X-Flag-3BP2_Y202A - + - + - + - + 180kD 115kDa a α 82kD IB: V5 a

49kD IB: αActin 37kDa a 82kD IB: αFlag

a Figure A1-3 (ii) Ability of 3BP2_Y202A to Bind to Nedd4 Family E3s. (A) pCMV3X-FLAG-3BP2 was co-expressed with pCDNA6.2-V5- hNedd4-1 (lanes 1 and 2) or pCDNA6.2-V5-hNedd4-2 (lanes 5 and 6). pCMV3X-FLAG-3BP2_Y202A was co-expressed with pCDNA6.2-V5- hNedd4-1 (lanes 3 and 4) and with pCDNA6.2-V5-hNedd4-2 (lanes 7 and 8). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti- V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (B) pCMV3X-FLAG-3BP2 was co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) or pCDNA6.2-V5-hSmurf2 (lanes 5 and 6). pCMV3X-FLAG-3BP2_Y202A was co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 3 and 4) and with pCDNA6.2-V5-Smurf2 (lanes 7 and 8). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti-V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (C) Whole cell lysate samples. pCMV3X- FLAG-3BP2 is expressed in lanes 1, 3, 5 and 7. pCMV3X-FLAG-3BP2_Y202A is expressed in lanes 2, 4, 6 and 8. pCNDA6.2-V5-hNedd4-1 was co-expressed in lanes 1 and 2, pCDNA6.2-V5-hNedd4-2 was co-expressed in lanes 3 and 4, pCDNA6.2-V5-hSmurf1 was co-expressed in lanes 5 and 6 and pCDNA6.2-V5-hSmurf2 was co-expressed in lanes 7 and 8. Membrane in top panel was probed with anti-V5 (top) and anti- Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

A B V5-hNedd4-1 V5 -hNedd4 -2 V5-hSmurf-1 V5-hSmurf-2 pCMV3X-Flag-3BP2 + - + - pCMV3X-Flag-3BP2 + - + - pCMV3X-Flag-3BP2_Y202A - + - pCMV3X-Flag-3BP2_Y202A - + - + + 180kD 180kD a a 115kD IP: αFlag 115kD IP: αFlag a82kD IB: α HA- a82kD IB: α HA- a Ub a Ub 64kD 64kD a a IP: α Flag IP: α Flag 82kD 82kD IB: α Flag IB: α Flag a

Nedd41 Nedd42 Smurf1 Smurf2 C pCMV3X-Flag-3BP2 + - + - + - + - pCMV3X-Flag-3BP2_Y202A - + - + - + - + 180kD 115kDa a82kD a IB: αV5

49kD IB: αActin a37kD a 82kD IB: αFlag

a Figure A1-4 (i) Ability of 3BP2_Y202A to be Ubiquitinated in the Presence of Nedd4 Family E3 Ligases. (A) pCMV-HA-Ubiquitin and pCMV3X-FLAG-3BP2 (lanes 1 and 3) or pCMV3X-FLAG-3BP2_Y202A (lanes 2 and 4) were co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2) or pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (B) pCMV-HA-Ubiquitin and pCMV3X-FLAG-3BP2 (lanes 1 and 3) or pCMV3X-FLAG-3BP2_Y202A (lanes 2 and 4) were co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) or pCDNA6.2-V5-hSmurf2 (lanes 3 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (C) Whole cell lysate samples. pCMV-HA-Ubiquitin and pCMV3X-FLAG-3BP2 (lanes 1, 3, 5 and 7) or pCMV3X-FLAG-3BP2_Y202A (lanes 2, 4, 6 and 8) were co- expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2), pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4), pCDNA6.2-V5-hSmurf1 (lanes 5 and 6) or pCDNA6.2-V5-hSmurf2 (lanes 7 and 8). Membrane in top panel was probed with anti-V5 (top) and anti-Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

A B V5-hNedd4-1 V5-hNedd4-2 V5-hSmurf-1 V5-hSmurf-2 pCMV3X-Flag-3BP2 + - + - pCMV3X-Flag-3BP2 + - + - pCMV3X-Flag-3BP2_Y202A - + - + pCMV3X-Flag-3BP2_Y202A - + - +

180kD 180kDa a 115kD IP: αFlag 115kDa IP: αFlag a82kD IB: α HA-Ub 82kD IB: α HA-Ub a a 64kD 64kD a a IP: α Flag IP: α Flag 82kD 82kD IB: α Flag IB: α Flag a a

Nedd4 Nedd4 Smurf1 Smurf2 C pCMV3X-Flag-3BP2 1+ - 2+ - + - + - pCMV3X-Flag-3BP2_Y202A - + - + - + - +

180kDa 115kDa 82kD IB: αV5 a

49kD IB: αActin 37kDa a 82kD IB: αFlag a Figure A1-4 (ii) Ability of 3BP2_Y202A to be Ubiquitinated in the Presence of Nedd4 Family E3 Ligases. (A) pCMV-HA-Ubiquitin and pCMV3X-FLAG-3BP2 (lanes 1 and 3) or pCMV3X-FLAG-3BP2_Y202A (lanes 2 and 4) were co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2) or pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (B) pCMV-HA-Ubiquitin and pCMV3X-FLAG-3BP2 (lanes 1 and 3) or pCMV3X-FLAG-3BP2_Y202A (lanes 2 and 4) were co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) or pCDNA6.2-V5-hSmurf2 (lanes 3 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (C) Whole cell lysate samples. pCMV-HA-Ubiquitin and pCMV3X-FLAG-3BP2 (lanes 1, 3, 5 and 7) or pCMV3X-FLAG-3BP2_Y202A (lanes 2, 4, 6 and 8) were co- expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2), pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4), pCDNA6.2-V5-hSmurf1 (lanes 5 and 6) or pCDNA6.2-V5-hSmurf2 (lanes 7 and 8). Membrane in top panel was probed with anti-V5 (top) and anti-Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

pCMV3X-Flag-3BP2 pCMV3X-Flag-3BP2 B A V5-hNedd4-1 V5-hNedd4-2 V5-hSmurf1 V5-hSmurf2 pCNDA3.1 + - + - pCNDA3.1 + - + - pCDNA3.1-Myc/His-Tank2 - + - + pCDNA3.1-Myc/His-Tank2 - + - +

180kDa 180kDa 115kDa 115kDa IP: αFlag 82kDa IP: αFlag 82kDa IB: α HA-Ub IB: α HA-Ub 64kDa 64kDa

IP: α Flag 82kDa IP: α Flag 82kDa IB: α Flag IB: α Flag

Nedd41 Nedd42 Smurf1 Smurf2 C pCDNA3.1 + - + - + - + - pCDNA3.1-Myc/His-Tank2 - + - + - + - +

180kDa 115kDa 82kDa IB: αV5

49kDa IB: αActin 37kDa

82kDa IB: αFlag

Figure A1-5 (i) Ability of Tanykrase 2 to Augment 3BP2 Ubiquitination in the Presence of Nedd4 E3 Ligases. (A) pCMV3X-FLAG-3BP2 and pCMV-HA-Ubiquitin were co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2) or pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4) and either empty pCDNA3.1-Myc/His vector (lanes 1 and 3) or pCDNA3.1-Myc/His-Tankyrase 2 (lanes 2 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (B) pCMV3X-FLAG-3BP2 and pCMV-HA-Ubiquitin were co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) or pCDNA6.2-V5-hSmurf2 (lanes 3 and 4) and either empty pCDNA3.1-Myc/His vector (lanes 1 and 3) or pCDNA3.1-Myc/His-Tankyrase 2 (lanes 2 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (C) Whole cell lysates. pCMV3X-FLAG-3BP2 and pCMV-HA-Ubiquitin were co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2), pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4), pCDNA6.2-V5-hSmurf1 (5 and 6) or pCDNA6.2-V5-hSmurf2 (lanes 7 and 8) and either empty pCDNA3.1-Myc/His vector (lanes 1, 2, 3 and 4) or pCDNA3.1-Myc/His-Tankyrase 2 (lanes 5, 6, 7 and 8). Membrane in top panel was probed with anti-V5 (top) and anti-Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

pCMV3X-Flag-3BP2 pCMV3X-Flag-3BP2 B A V5-hNedd4-1 V5-hNedd4-2 V5-hSmurf1 V5-hSmurf2 pCNDA3.1 + - + - pCNDA3.1 + - + - pCDNA3.1-Myc/His-Tank2 - + - + pCDNA3.1-Myc/His-Tank2 - + - +

180kDa 180kDa 115kDa 115kDa IP: αFlag 82kDa IP: αFlag 82kDa IB: α HA-Ub IB: α HA-Ub 64kDa 64kDa

IP: α Flag 82kDa IP: α Flag 82kDa IB: α Flag IB: α Flag

Nedd41 Nedd42 Smurf1 Smurf2 C pCDNA3.1 + - + - + - + - pCDNA3.1-Myc/His-Tank2 - + - + - + - +

180kDa 115kDa 82kDa IB: αV5

49kDa IB: αActin 37kDa 82kDa IB: αFlag

Figure A1-5 (ii) Ability of Tanykrase 2 to Augment 3BP2 Ubiquitination in the Presence of Nedd4 E3 Ligases. (A) pCMV3X-FLAG- 3BP2 and pCMV-HA-Ubiquitin were co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2) or pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4) and either empty pCDNA3.1-Myc/His vector (lanes 1 and 3) or pCDNA3.1-Myc/His-Tankyrase 2 (lanes 2 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (B) pCMV3X-FLAG-3BP2 and pCMV-HA-Ubiquitin were co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) or pCDNA6.2-V5-hSmurf2 (lanes 3 and 4) and either empty pCDNA3.1-Myc/His vector (lanes 1 and 3) or pCDNA3.1-Myc/His-Tankyrase 2 (lanes 2 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (C) Whole cell lysates. pCMV3X-FLAG-3BP2 and pCMV-HA-Ubiquitin were co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2), pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4), pCDNA6.2-V5-hSmurf1 (5 and 6) or pCDNA6.2-V5-hSmurf2 (lanes 7 and 8) and either empty pCDNA3.1-Myc/His vector (lanes 1, 2, 3 and 4) or pCDNA3.1-Myc/His-Tankyrase 2 (lanes 5, 6, 7 and 8). Membrane in top panel was probed with anti-V5 (top) and anti-Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

A V5-hNedd4-1 V5-hNedd4-2 pCMV3X-Flag-3BP2 + + - - + + - - pCMV3X-Flag-3BP2_R413Q - - + + - - + + IP: Flag + - + - + - + -

180kDa hNedd4-2 hNedd4-1 115kDa 82kDa IB: αV5 64kDa

82kDa IB: αFlag B V5-hSmurf1 V5-hSmurf2 pCMV3X-Flag-3BP2 + + - - + + - - pCMV3X-Flag-3BP2_R413Q - - + + - - + + IP: Flag + - + - + - + - 180kDa 115kDa hSmurf1 hSmurf2 82kDa

64kDa IB: αV5

82kDa IB: αFlag

Nedd4 Nedd4 Smurf Smurf pCMV3X-Flag-3BP2 + - + - + - + - pCMV3X -Flag -3BP2_R413Q - + - + - + - +

180kD 115kD α 82kD IB: V5

49kD IB: αActin 37kD 82kD IB: αFlag

Figure A1-6 (i) Ability of 3BP2_R413Q to Bind to Nedd4 Family E3 Ligases. (A) pCMV3X-FLAG-3BP2 was co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2) or pCDNA6.2-V5-hNedd4-2 (lanes 5 and 6). pCMV3X-FLAG-3BP2_R413Q was co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 3 and 4) and with pCDNA6.2-V5-hNedd4-2 (lanes 7 and 8). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti-V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (B) pCMV3X-FLAG-3BP2 was co- expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) or pCDNA6.2-V5-hSmurf2 (lanes 5 and 6). pCMV3X-FLAG-3BP2_R413Q was co- expressed with pCDNA6.2-V5-hSmurf1 (lanes 3 and 4) and with pCDNA6.2-V5-Smurf2 (lanes 7 and 8). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti-V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (C) Whole cell lysate samples. pCMV3X-FLAG-3BP2 is expressed in lanes 1, 3, 5 and 7. pCMV3X-FLAG-3BP2_R413Q is expressed in lanes 2, 4, 6 and 8. pCNDA6.2-V5-hNedd4-1 was co-expressed in lanes 1 and 2, pCDNA6.2-V5-hNedd4-2 was co-expressed in lanes 3 and 4, pCDNA6.2-V5- hSmurf1 was co-expressed in lanes 5 and 6 and pCDNA6.2-V5-hSmurf2 was co-expressed in lanes 7 and 8. Membrane in top panel was probed with anti-V5 (top) and anti-Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

A V5-hNedd4-1 V5-hNedd4-2 pCMV3X-Flag-3BP2 + + - - + + - - pCMV3X-Flag-3BP2_R413Q - - + + - - + + IP: Flag + - + - + - + - 180kD hNedd4-1a hNedd4-2 115kD 82kDa IB: αV5 64kD a

82kD IB: αFlag a B V5-hSmurf1 V5-hSmurf2 pCMV3X-Flag-3BP2 + + - - + + - - pCMV3X-Flag-3BP2_R413Q - - + + - - + + IP: Flag + - + - + - + - 180kD 115kD hSmurf1a 82kD hSmurf2 a 64kD a IB: αV5

82kD IB: αFlag a

Nedd41 Nedd42 Smurf1 Smurf2

pCMV3X-Flag-3BP2 + - + - + - + - pCMV3X-Flag-3BP2_R413Q - + - + - + - + 180kD 115kDa a IB: αV5 82kD a

49kD 37kDa IB: αActin a 82kD IB: αFlag

a Figure A1-6 (ii) Ability of 3BP2_R413Q to Bind to Nedd4 Family E3 Ligases. (A) pCMV3X-FLAG-3BP2 was co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2) or pCDNA6.2-V5-hNedd4-2 (lanes 5 and 6). pCMV3X-FLAG-3BP2_R413Q was co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 3 and 4) and with pCDNA6.2-V5-hNedd4-2 (lanes 7 and 8). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti-V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (B) pCMV3X-FLAG-3BP2 was co- expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) or pCDNA6.2-V5-hSmurf2 (lanes 5 and 6). pCMV3X-FLAG-3BP2_R413Q was co- expressed with pCDNA6.2-V5-hSmurf1 (lanes 3 and 4) and with pCDNA6.2-V5-Smurf2 (lanes 7 and 8). Each sample was divided equally, samples in lanes 1, 3, 5 and 7 are anti-FLAG immunoprecipitation experiments while lanes 2, 4, 6 and 8 are bead control experiments. The membrane was probed with anti-V5 antibody (upper panel), stripped and then re-probed with anti-FLAG (lower panel). (C) Whole cell lysate samples. pCMV3X-FLAG-3BP2 is expressed in lanes 1, 3, 5 and 7. pCMV3X-FLAG-3BP2_R413Q is expressed in lanes 2, 4, 6 and 8. pCNDA6.2-V5-hNedd4-1 was co-expressed in lanes 1 and 2, pCDNA6.2-V5-hNedd4-2 was co-expressed in lanes 3 and 4, pCDNA6.2-V5- hSmurf1 was co-expressed in lanes 5 and 6 and pCDNA6.2-V5-hSmurf2 was co-expressed in lanes 7 and 8. Membrane in top panel was probed with anti-V5 (top) and anti-Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

A V5-hNedd4-1 V5-hNedd4-2 pCMV3X-Flag-3BP2 + - + - pCMV3X-Flag-3BP2-R413Q - + - +

180kDa 115kDa 82kDa IP: αFlag IB: α HA-Ub 64kDa

IP: α Flag 82kDa IB: α Flag

B V5-hSmurf1 V5-hSmurf2 pCMV3X-Flag-3BP2 + - + - pCMV3X-Flag-3BP2-R413Q - + - +

180kDa 115kDa 82kDa IP: αFlag IB: α HA-Ub 64kDa

IP: α Flag 82kDa IB: α Flag

Nedd41 Nedd42 Smurf1 Smurf2 pCMV3X-Flag-3BP2 + - + - + - + - pCMV3X-Flag-3BP2_R413Q - + - + - + - + 180kD 115kDa a82kD IB: αV5 a

49kD IB: αActin 37kDa a82kD IB: αFlag a

Figure A1-7 (i) Ability of 3BP2_R413Q to be Ubiquitinated in the Presence of Nedd4 Family E3 Ligases. (A) HA-Ubiquitin and pCMV3X-FLAG-3BP2 (lanes 1 and 3) or pCMV3X-FLAG-3BP2_R413Q (lanes 2 and 4) were co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2) or pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (B) HA-Ubiquitin and pCMV3X- FLAG-3BP2 (lanes 1 and 3) or pCMV3X-FLAG-3BP2_R413Q (lanes 2 and 4) were co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) or pCDNA6.2-V5-hSmurf2 (lanes 3 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (C) Whole cell lysate samples. HA-Ubiquitin and pCMV3X-FLAG-3BP2 (lanes 1, 3, 5 and 7) or pCMV3X-FLAG-3BP2_R413Q (lanes 2, 4, 6 and 8) were co-expressed with pCDNA6.2-V5- hNedd4-1 (lanes 1 and 2), pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4), pCDNA6.2-V5-hSmurf1 (lanes 5 and 6) or pCDNA6.2-V5-hSmurf2 (lanes 7 and 8). Membrane in top panel was probed with anti-V5 (top) and anti-Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

A V5-hNedd4-1 V5-hNedd4-2 pCMV3X-Flag-3BP2 + - + - pCMV3X-Flag-3BP2-R413Q - + - +

180kDa 115kDa 82kDa IP: αFlag IB: α HA-Ub 64kDa

82kDa IP: α Flag IB: α Flag

B V5-hSmurf1 V5-hSmurf2 pCMV3X-Flag-3BP2 + - + - pCMV3X-Flag-3BP2-R413Q - + - +

180kDa

115kDa IP: αFla g 82kDa IB: α HA-Ub

64kDa

IP: α Flag 82kDa IB: α Flag

Nedd41 Nedd42 Smurf1 Smurf2 pCMV3X-Flag-3BP2 + - + - + - + - pCMV3X-Flag-3BP2_R413Q - + - + - + - + 180kD 115kD IB: αV5 82kD

49kD IB: αActin 37kD

82kD IB: αFlag

Figure A1-7 (ii) Ability of 3BP2_R413Q to be Ubiquitination in the Presence of Nedd4 Family E3 Ligases. (A) HA-Ubiquitin and pCMV3X-FLAG-3BP2 (lanes 1 and 3) or pCMV3X-FLAG-3BP2_R413Q (lanes 2 and 4) were co-expressed with pCDNA6.2-V5-hNedd4-1 (lanes 1 and 2) or pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (B) HA-Ubiquitin and pCMV3X- FLAG-3BP2 (lanes 1 and 3) or pCMV3X-FLAG-3BP2_R413Q (lanes 2 and 4) were co-expressed with pCDNA6.2-V5-hSmurf1 (lanes 1 and 2) or pCDNA6.2-V5-hSmurf2 (lanes 3 and 4). Re-precipitation experiments were performed with anti-FLAG antibody and membrane was probed with anti-HA (top panel), then stripped and re-probed with anti-FLAG (bottom panel). (C) Whole cell lysate samples. HA-Ubiquitin and pCMV3X-FLAG-3BP2 (lanes 1, 3, 5 and 7) or pCMV3X-FLAG-3BP2_R413Q (lanes 2, 4, 6 and 8) were co-expressed with pCDNA6.2-V5- hNedd4-1 (lanes 1 and 2), pCDNA6.2-V5-hNedd4-2 (lanes 3 and 4), pCDNA6.2-V5-hSmurf1 (lanes 5 and 6) or pCDNA6.2-V5-hSmurf2 (lanes 7 and 8). Membrane in top panel was probed with anti-V5 (top) and anti-Actin (bottom) antibodies. Membrane in bottom panel was probed with anti-FLAG antibody.

A B

pC DNA3.1 - Myc/His-Itch pCMV3X-Flag + + - - Itch pCMV3X-Flag-3BP2 - - + + pCMV3X-Flag + - IP: Flag + - + - pCMV3X-Flag-3BP2 - + 180kDa 180kDa 115kDa 115kDa α 82kDa Itch 82kDa IB: Myc

IB: αMyc

64kDa 49kDa IB: αActin 37kDa 82kDa IB: αFlag 82kDa IB: αFlag

Figure A1-8 (i). Itch Interacts with 3BP2. (A) pCDNA3.1-Myc/His-Itch was co-expressed with pCMV3X-FLAG vector (lanes 1 and 2) or pCMV3X-FLAG-3BP2 (lanes 3 and 4). Each sample was divided equally and subject to anti-FLAG immunoprecipitation (lanes 1 and 3) or a bead control experiment (lanes 2 and 4). Membrane was probed with anti-Myc antibody (top panel) then stripped and re-probed with anti- FLAG antibody (bottom panel). (B) Whole cell lysates. pCDNA3.1-Myc/His-Itch was co-expressed with pCMV3X-FLAG vector (lane 1) or pCMV3X-FLAG-3BP2 (lanes 2). Membrane in top panel was probed with anti-Myc (top) and anti-Actin (bottom) antibodies, membrane in bottom panel was probed with anti-FLAG antibody.

A B

pCDNA3.1 - Myc/His-Itch pCMV3X-Flag + + - - Itch pCMV3X-Flag-3BP2 - - + + IP: Flag + - + - pCMV3X-Flag + - pCMV3X-Flag-3BP2 - + 115kDa 180kDa 82kDa 115kDa IB: αMyc Itch 82kDa

IB: αMyc 64kDa 49kDa α 37kDa IB: Actin α 82kDa IB: 82kDa IB: Flag αFlag

Figure A1-8 (ii). Itch Interacts with 3BP2. (A) pCDNA3.1-Myc/His-Itch was co-expressed with pCMV3X-FLAG vector (lanes 1 and 2) or pCMV3X-FLAG-3BP2 (lanes 3 and 4). Each sample was divided equally and subject to anti-FLAG immunoprecipitation (lanes 1 and 3) or a bead control experiment (lanes 2 and 4). Membrane was probed with anti-Myc antibody (top panel) then stripped and re-probed with anti- FLAG antibody (bottom panel). (B) Whole cell lysates. pCDNA3.1-Myc/His-Itch was co-expressed with pCMV3X-FLAG vector (lane 1) or pCMV3X-FLAG-3BP2 (lanes 2). Membrane in top panel was probed with anti-Myc (top) and anti-Actin (bottom) antibodies, membrane in bottom panel was probed with anti-FLAG antibody.

A B

Myc-Itch Myc-Itch pCMV3X-Flag-3BP2 + + pCNDA3.1 + - pCMV3X-Flag-3BP2 + + pCDNA3.1-Myc/His-Tank2 - + pCDNA3.1 + - pCDNA3.1-Myc/His-Tank2 - + 180kDa 180kDa 115kDa 115kDa 82kDa 82kDa IB: αMyc IP: αFlag IB: α HA-Ub 64kDa

49kDa α 82kDa IP: α Flag IB: Actin IB: α Flag 37kDa

82kDa IB: αFlag

Figure A1-9 (i) Tankyrase 2 Augments the Ability of Itch to Mediate 3BP2 Ubiquitination. (A) pCMV-HA-Ubiquitin, pCMV3X-FLAG- 3BP2 and pCDNA3.1-Myc/His-Itch were co-expressed with either empty pCDNA3.1 vector (lane 1) or pCDNA3.1-Myc/His-Tankyrase2 (lane 2) and subject to anti-FLAG re-precipitation experiment. Blot was probed with anti-HA antibody (top panel) then stripped and re-probed with anti-FLAG antibody. (B) Whole cell lysates. pCMV-HA-Ubiquitin, pCMV3X-FLAG-3BP2 and pCDNA3.1-Myc/His-Itch were co-expressed with either empty pCDNA3.1 vector (lane 1) or pCDNA3.1-Myc/His-Tankyrase2 (lane 2). Membrane in top panel was probed with anti-Myc antibody (top) and anti-Actin antibody (bottom), membrane in bottom panel was probed with anti-FLAG antibody.

A B

Myc-Itch Myc-Itch pCMV3X-Flag-3BP2 + + pCNDA3.1 + - pCMV3X-Flag-3BP2 + + pCDNA3.1-Myc/His-Tank2 - + pCDNA3.1 + - pCDNA3.1-Myc/His-Tank2 - + 180kD 180kD 115kD 115kD IB: αMyc 82kD 82kD IP: αFlag a IB: α HA- 64kD Ub

49kD α 82kD IP: α IB: Actin Flag 37kD IB: α 82kD IB: αFlag

a Figure A1-9 (ii) Tankyrase 2 Augments the Ability of Itch to Mediate 3BP2 Ubiquitination. (A) pCMV-HA-Ubiquitin, pCMV3X-FLAG- 3BP2 and pCDNA3.1-Myc/His-Itch were co-expressed with either empty pCDNA3.1 vector (lane 1) or pCDNA3.1-Myc/His-Tankyrase2 (lane 2) and subject to anti-FLAG re-precipitation experiment. Blot was probed with anti-HA antibody (top panel) then stripped and re-probed with anti-FLAG antibody. (B) Whole cell lysates. pCMV-HA-Ubiquitin, pCMV3X-FLAG-3BP2 and pCDNA3.1-Myc/His-Itch were co-expressed with either empty pCDNA3.1 vector (lane 1) or pCDNA3.1-Myc/His-Tankyrase2 (lane 2). Membrane in top panel was probed with anti-Myc antibody (top) and anti-Actin antibody (bottom), membrane in bottom panel was probed with anti-FLAG antibody.