Investigation of the Role of Different Regions of the Cbl Protein in Its Function

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Nurettin Ilter Sever, M.Sc.

Graduate Program in Molecular, Cellular and Developmental Biology

The Ohio State University

2013

Dissertation Committee

Amanda Simcox, Advisor

Nancy L. Lill

David Bisaro

Mamuka Kvaratskhelia

Nurettin Ilter Sever

2013

ABSTRACT

The duration and amplitude of any signal transduction pathway should be tightly regulated for the maintenance of homeostasis in any organism. Cbl protein is one of the crucial modulators of epidermal growth factor receptor (EGFR) signaling. Cbl is a RING-type, E3 ubiquitin ligase which marks its substrates with ubiquitin and destines them for degradation. Upon stimulation by a ligand, Cbl is associated with and phosphorylated by

EGFR, ubiquitinates it, helps to internalize it and directs it to the lysosomal degradation pathway. Reanalysis of the crystal structure depicted in 2000 by Zhang et al. suggested that Cbl may be present as dimers when complexed with ZAP-70 and its E2 ubiquitin- conjugating partner UbcH7. The V431 and F434 residues of the Cbl RING finger tail (RF tail), found at one interface of this putative dimer, were shown to play different roles in

EGFR trafficking. For this study, the other residues in the same putative dimer interface were analyzed in terms of their necessity for Cbl to enhance EGFR degradation and ubiquitination. All mutations were presented in the context of the full length Cbl protein.

Our degradation assay showed that all the tested mutants degraded EGFR as efficiently as wild type 90 minutes after EGF stimulation. All mutants could associate with EGFR and ubiquitinate it. Interestingly, even mutations that were predicted to induce major structural disruption at the putative dimer interface failed to affect the degradation of EGFR. Based on these data, I concluded that the published 2000 crystal structure from Zhang et al. did not largely reflect the active form of Cbl dimers. My finding is discussed here in the context of a new Cbl structure reported in 2012 by Dou et al. ii

In further studies, I extended earlier research by our lab that led us to hypothesize the presence of an inhibitory activity within Cbl, which mapped C-terminal to amino acid 357.

This novel activity is normally masked by the ubiquitin ligase activity of Cbl, but becomes evident when E3 activity is inhibited and ubiquitin is present in Cbl-EGFR complexes via recombinant fusion to Cbl. Using ubiquitin-fused truncated Cbl-Y371F constructs, which lack E3 activity but can bind to ubiquitin interacting motifs of the endocytosis machinery, I mapped more definitively the location of the inhibitory element. I then tested whether this inhibitory activity involved binding of the signaling adapter Grb2 to EGFR in complex with

Cbl. Our findings suggest that Grb2 is not involved in this inhibitory activity.

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Dedication

Dedicated to my wife, my family and my beloved friend Dr. Yaman Kocak

ACKNOWLEDGEMENTS

First of all, I would like to thank Dr. Nancy L.Lill, for her endless support throughout my doctoral studies. No words can actually describe how grateful I am to her. She had given me a hand at one of my darkest moments of my career. Working with her was a great pleasure and a great journey for me. I would not have completed my studies if she had not given me the confidence, enthusiasm and encouragement. She always believed in me at times even when I did not believe in myself. She will be missed so much.

My other appreciations go to my colleagues Dr. Zahida Qamri, Jennifer Zhang, Kevin Henschel, Aimee Schmenk, Elise Blankenship, Mark Riley and Rajeev Sachdeva for their enormous help and friendship throughout my studies. Especially, I would like to thank Kevin and Rajeev for the materials they constructed which were used in my study.

Also, I am very thankful to my current official advisor, Dr. Amanda Simcox, who has given me a space in her lab to finish my graduate studies and her support this year. It was so meaningful to me that I was able to conduct my last experiments this year when we had to move our lab. Also, I would like to thank her graduate student, Sathiya Manivannan for his kind help for the last months.

I would like to thank Dr. David Bisaro, MCDB Program Director and my thesis committee member, who always helped me at my tough times throughout my Ph.D tenure. His advises helped me to solve the problems I faced. Also, I would like to thank Jan Zinaich and Wayne Dellinger for their help about the administrative issues.

My other thanks go to Dr. Mamuka Kvaratskhelia, my thesis committee member, for his advises in conducting our research. And also, I would like to thank the laboratories of Dr. Susan Cole and Dr. Harold Fisk for sharing their lab resources with me.

Personally, I would like to thank my wife, Nimet Kardes Sever very very much. Again, no words can describe my feelings for her endless love, support, encouragement, tolerance she had given me for a decade. She has always been with me. I have struggled so much in my last decade and she always helped me to overcome those obstacles. In other words, I owe her a lot so that I could manage to finish my studies. I would like to apologize to her since I gave her so many rough times.

My next great appreciations go to my family. You are being missed so much here. You have always believed in me and given me so much support and love in my tough times. Sometimes, I felt your absence here so much that life had become unbearable to me. I know that you always prayed for me so that I would finish my Ph.D studies and I am really grateful for that. It was very difficult for me to be far away from you.

Finally, I would like to thank my friends; Gulsen Colakoglu-Onur Hamsici, Yelda Serinagaoglu, Serdar Vural, Seda-Ozan Koyluoglu, Deniz-Doruk Bozdag, Aysegul-Serdar Kizilgul, Merve-Olcay Sertel, Berna-Murat Inalpolat, Elif-Caner Sakin, Nil Apaydin, Ozan Bilgin, Marion Nicolet-Utku Solpuker, Sahika-Gokhan Korkmaz, Duygu Ucar, Ozge Ozcakir and Adam Groseclose for the enormous friendship they had given me in my last decade and made my Ph.D life bearable.

VITA

October 2, 1979……………..Born, Denizli, Turkey

2001…………………………..B.Sc. Molecular Biology and Genetics, Middle East Technical University, Ankara, Turkey

2003………………………….M.Sc. Molecular Biology and Genetics, Bilkent University, Ankara, Turkey

2003-present……………….Graduate Research Associate, Molecular, Cellular and Developmental Biology, The Ohio State University, Columbus, Ohio, USA

PUBLICATIONS

Nancy L.Lill and Nurettin Ilter Sever (2012). Where EGF receptors transmit their signals. Science Signaling. 5(243):pe41.

FIELDS OF STUDY

Major Field: Molecular, Cellular and Developmental Biology

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TABLE OF CONTENTS

ABSTRACT………………………………………………………………………………………ii

DEDICATION………………………………………………………………...... iv

ACKNOWLEDGEMENTS………………………………………………………………...... v

VITA………………………………………………………………………………………...... vii

TABLE OF CONTENTS……………………………………………………………………….viii

LIST OF FIGURES………………………………………………………………………...... xi

LIST OF TABLES………………………………………………………………………………xiii

ABBREVIATIONS………………………………………………………………………...... xiv

CHAPTER 1: INTRODUCTION………………………………………………………………..1

1.1 ErbB Signaling…………………………………………………………………………..1 1.1.1 ErbB Receptors…………………………………………………………………1 1.1.2 ErbB Ligands……………………………………………………………………2 1.1.3 Signaling Pathways Activated by ErbB Receptors…………………...... 3 1.1.4 ErbB Ligands and Receptors in Mammalian Development…………………6 1.1.5 ErbB Ligands and Receptors in Cancer………………………………………8

1.2 Cbl Protein……………………………………………………………………………...10 1.2.1 Discovery, Elucidation of Structure and Function………………………….10 1.2.2 Cbl Structural Effects on Regulation of EGFR……………………………..14

1.3 EGFR Endocytosis and Regulation of Signaling……………………………………17 1.3.1 Mechanism of EGFR Endocytosis…………………………………………..19 1.3.2 Hrs Protein and Its Role in EGFR Endocytosis…………………………….20 1.3.3 EGFR Signaling Potency Along Its Endocytosis……………………………22

1.4 c-Cbl Crystal Structure and Its Relevance to Our Study……………………………23 viii

CHAPTER 2: MATERIALS AND METHODS……………………………………………….33 2.1 Mutagenesis of cbl-wt plasmid………………………………………………………33 2.1.1 Mutagenesis Protocol……………………………………………………….35

2.2 Construction of Truncated Ubiquitin-Fusion Cbl Plasmids……………………….36 2.2.1 Mutagenesis of Cbl-Y371F-1-520-Ub Plasmid…………………………..38

2.3 Cell Lines, Transfections, EGF Stimulation and EGFR Degradation Assay……39 2.3.1 Propagation of HEK 293 Cell Line.…………………...... 39 2.3.2 Transfection of HEK 293 Cell Line…………………………………………39 2.3.3 EGF Stimulation and Harvesting of Cells…………………………………40 2.3.4 Antibodies……………………………………………………………………40 2.3.5 Immunoprecipitation and Immunoblotting…………………………………41

CHAPTER 3: TESTS TO DETERMINE WHETHER CBL DIMER INTERFACE MUTANTS ARE COMPROMISED IN THEIR ABILITY TO ENHANCE EGFR DEGRADATION………………………………………………………………………………..43

3.1 Mutagenesis of c-Cbl Putative Dimer Interface Residues……………………….43

3.2 Degradation Assay of Putative c-Cbl Dimer Interface Mutants……………….....45

3.2.1 Statistical Analysis for Set 1………………………………………………50

3.3 Association of Putative c-Cbl Interface Mutants with EGFR upon EGF Stimulation……………………………………………………………………...... 52

3.4 Impact of Putative c-Cbl Interface Mutants on EGFR Ubiquitination upon EGF Stimulation……………………………………………………………………...... 52

3.5 Summary of Results…………………………………………………………………55

CHAPTER 4: INVESTIGATION OF THE ROLE OF GRB2 IN A NOVEL INHIBITORY ACTIVITY THAT MAPS BETWEEN CBL AMINO ACIDS 358-480……………………...56

4.1 Degradation Assay of Grb-2 Binding Mutant of c-Cbl……………………………..56

CHAPTER 5: DISCUSSION…………………………………………………………………..59

5.1 The Role of Putative Cbl Dimer Interface Mutants in Cbl Function………………59

5.2 Investigation of Role of Grb2 in Regulation of Putative Cbl C-terminal Inhibitory

Activity………………………………………………………………………………….61

REFERENCES………………………………………………………………………………….64

LIST OF FIGURES

Figure 1.1 Generic structure of ErbB proteins…………………………………………………2

Figure 1.2 Regulatory phosphorylation sites on ErbB1, the EGFR…………………………4

Figure 1.3 Signaling pathways that are activated upon stimulation of EGFR by ligand……5

Figure 1.4 The structure of Cbl orthologues………………………………………………....12

Figure 1.5 Steps of EGFR endocytosis……………………………………………………….18

Figure 1.6 Where EGFR signals………………………………………………………………24

Figure 1.7 Putative Cbl dimer contained within the 2000 structure reported by Zhang....25

Figure 1.8 Dimerization analysis of Cbl RF tail mutants…………………………………….30

Figure 1.9 The residues located at the putative dimer interface of Cbl crystal structure….31

Figure 2.1 The strategy in construction of truncated GFP-Cbl-Y371F-Ub plasmids……...37

Figure 3.1 Impact of Cbl putative dimer interface mutants on EGFR degradation………..47

Figure 3.2 Box and whiskers plot of degradation assays of Set 1 mutants…………...... 51

Figure 3.3 Immmunoprecipitation analysis of the putative Cbl dimer interface mutants…53

Figure 3.4 EGFR ubiquitination analysis of the putative Cbl dimer interface mutants……54

Figure 4.1 Impact of mutation of Grb2 binding site on EGFR degradation………………...58

Figure 5.1 The position of V431 and F434 amino acid residues in crystal structures of

Y371-phosphorylated Cbl…………………………………………………………63

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LIST OF TABLES

Table 1.1 Manual sequence analysis of RF tail regions of different RING/E3 proteins……………………………………………………………………………27

Table 3.1 Mann-Whitney U test of each Set 1 mutant……………………….51

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ABBREVIATIONS

ADAM A disintegrin and a metalloprotease AP-2 Adaptor protein complex 2 APS Ammonium persulfate AR Amphiregulin ATP Adenosine-3-phosphate BARD1 BRCA1-associated RING domain protein 1. BRCA1 Breast cancer susceptibility gene 1. BSA Bovine serum albumin BTC Betacellulin C- Carbon- CAPS N-cyclohexyl-3-aminopropanesulfonic Cbl Casitas-B-lymphoma c-Cbl Cellular Cbl protein cDNA Complementary DNA CHO Chinese hamster ovary CO2 Carbon dioxide COX2 Cyclooxygenase 2 CSF-1 Colony stimulating factor-1 Cys Cysteine D-Cbl Drosophila Cbl protein DMEM Dulbecco’s medium DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate EEA1 Early endosomal marker 1 EGF Epidermal growth factor EGFR Epidermal growth factor receptor ELK1 ETS domain-containing protein EPG Epigen EPR Epiregulin ERK Extracellular signal-regulated kinase ESCRT Endosomal sorting complex required for transport FAK Focal adhesion kinase FBS Fetal bovine serum FW Forward

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GBM Glioblastoma multiforme GFP Green fluorescent protein Grb2 Growth factor receptor-bound 2 GST Gluthathione-S-transferase GTPase Guanosine-3-phosphatase HB-EGF Heparan-binding epidermal growth factor HEK 293 Human embryonic kidney 293 HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid HRP Houseradish peroxidase Hrs Hepatocyte-growth factor regulated tyrosine kinase substrate I.B. Immunoblotting I.P. Immunoprecipitation LB Luria broth Lys Lysate LZ Leucine zipper mAb Monoclonal antibody MAPK Mitogen-activated protein kinase MEK Mitogen-activated protein kinase kinase MVB Multivesicular body Mdm2 Mouse double minute 2. N- Amino- Na3VO4 Sodium orthovanadate NaCl Sodium chloride NaF Sodium fluoride NRG Neuregulin NSCLC Non-small cell lung cancer NT Non-transfected PAGE Polyacrylamide gel electrophoresis PBS Phosphate-buffered saline PCR Polymerase chain reaction PDGF Platelet-derived growth factor PDGFR Platelet-derived growth factor receptor PI3-K Phosphadityl inositol 3’-kinase PLCγ Phospholipase C-gamma PMSF Phenylmethanesulfonyl fluoride PRO Proline-rich domain PTB Phosphotyrosine binding PVDF Polyvinylidene difluoride RING Really interesting RF RING Finger RV Reverse xv

RTK Receptor tyrosine kinase S.D. Standard deviation SDS Sodium dodecyl sulfate SH2 Src-homology 2 SH3 Src-homology 3 Sos Son-of-sevenless STAM Signal-transducing adaptor molecule STAT3 Signal-transducer and activator of transcription 3 TBS Tris-buffered saline TBS-T Tris-buffered saline with Tween-20 TEMED Tetramethylethylenediamine TGF-α Transforming growth factor-alpha TKB Tyrosine kinase binding TKI Tyrosine kinase inhibitor TM Transmembrane TSG101 Tumor suppressor gene 101 Tyr Tyrosine Ub Ubiquitin Ubc Ubiquitin-conjugating enzyme UIM Ubiquitin-interacting motif v-Cbl Viral Cbl protein VPS34A Vacuolar protein sorting 34A Vul Vulvaless Wt Wild-type ZAP-70 Zeta-chain-associated protein kinase 70

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CHAPTER 1 INTRODUCTION

1.1 ErbB Signaling

Cellular responses to environmental stimuli (growth factors, cytokines, etc.) are mediated by various signal transduction pathways. Receptor tyrosine kinases (RTKs) play a very important role in converting external stimuli to internal effects for a cell, mostly by exerting their kinase activity. ErbB receptors constitute a very extensively studied family of RTKs which is involved in physiological processes such as development and pathological events such as cancer.

1.1.1 ErbB Receptors:

The ErbB receptor family is composed of four members: ErbB-1 (EGFR), ErbB-2 (HER- 2), ErbB-3 (HER-3) and ErbB-4 (HER-4). The common properties of each member include an extracellular domain to which ligands can bind, a single transmembrane domain and a cytoplasmic domain which typically has kinase activity and phosphorylation sites (Figure 1.1). Defective ligand binding by ErbB-2 and lack of kinase activity of ErbB-3 are the unique properties of these members. ErbB receptors function as homo- or heterodimers upon activation, as described below (Yarden and Sliwkowski, 2001; Normanno et al., 2005; Linggi and Carpenter, 2006).

In order to exert their function, ErbB receptors other than ErbB2 first bind to different extracellular ligands. When a ligand binds, ErbB receptors are activated via dimerization. In the unliganded state, ErbB-1, ErbB-3 and ErbB-4 have a closed conformation keeping a beta hairpin arm from subdomain II (called the dimerization loop) in a hidden state. Upon ligand binding, the dimerization loop is exposed at the surface, mediating the interaction

Figure 1.1 Generic structure of ErbB proteins. N-terminal cysteine-rich (Cys) sequences serve as the ligand binding site.

Cys rich repeats A single transmembrane (TM) domain bridges the extracellular environment and intracellular space. Kinase domain TM region phosphorylates tyrosine residues within Kinase domain ErbB partners and other associated proteins. The C-terminal tyrosine phosphorylation C-terminal region with sites can undergo inducible modification Tyr phosphorylation sites when ErbB protein or its partner is activated by ligand binding

between receptor monomers (Ferguson et al., 2003; Cho et al., 2002; Bouyain et al., 2006). ErbB-2 was shown to have the dimerization loop exposed in its unliganded state, enabling its active conformation without the need for activation by a ligand (Garrett et al., 2003).

Receptor dimerization enables the receptor’s kinase activity. Asymmetric interaction between the C-lobe of a kinase domain of a receptor with the N-lobe of its dimerization partner facilitates the kinase activities of both dimerized receptors (Zhang et al., 2006).

1.1.2 ErbB Ligands:

ErbB ligands are classified into three groups. The first group consists of epidermal growth factor (EGF), amphiregulin (AR) and transforming growth factor-α (TGF-α), which bind to epidermal growth factor receptor (EGFR) exclusively. Betacellulin (BTC), heparin binding EGF (HB-EGF), epigen (EPG) and epiregulin (EPR) form the second group, as they bind both ErbB1 and ErbB4. Neuregulins (NRG) constitute the third group with two subgroups:

NRG-1 and NRG-2 bind both ErbB3 and ErbB4 whereas NRG-3 and NRG-4 bind only ErbB4 (reviewed in Holbro et al., 2003).

ErbB ligands are synthesized as transmembrane precursor proteins. They commonly contain one EGF module in their extracellular domains, except for EGF, which has nine of them. The EGF module is the functional unit of ErbB ligands and is responsible for ligand binding to ErbB receptors. The EGF module is a 40 amino acids-long sequence characterized by six cysteine residues having three disulfide bonds among them (Schneider and Wolf, 2008; Harris et al., 2003). To elicit endocrine (distant cell), paracrine (neighbor cell) or autocrine (original cell) signaling, the precursor ligands are processed via proteolytic cleavage by ADAM (A Disintegrin And a Metalloprotease) proteins following physiological responses. The cleavage releases the EGF modules of ligands into the extracellular milieu, converting the ligands into their soluble forms. For EGF, only the membrane-closest EGF module is processed (Sahin et al., 2004). Also, in some cases, unprocessed membrane-bound EGF ligands bind a contacting cell’s ErbB receptor, mediating a juxtacrine form of signaling (Singh and Harris, 2005).

1.1.3 Signaling Pathways Activated by ErbB Receptors:

Receptor dimerization mediates auto- or transphosphorylation of several tyrosine residues in the cytoplasmic region of ErbB receptors. The signaling pathways to be activated by ErbB receptors are determined by the identity of the phosphorylated residues. The phosphorylated residues recruit different proteins whose identities are determined by the phosphorylation sites. As a typical example, proteins recruited to ErbB-1 phosphorylation sites are illustrated in Figure 1.2.

ErbB receptor activity yields activation of various signaling pathways in a cell, resulting in different cellular processes like proliferation, survival, migration and angiogenesis. The pathways activated are illustrated in Figure 1.3 and briefly explained below.

Figure 1.2 Regulatory phosphorylation sites on ErbB1, the EGFR. A subset of known phosphorylation sites is presented. Tyrosines 974, 1045 and 1068 recruit proteins that are direct or indirect effectors of endocytosis. Tyrosines 1068, 1086 and 1173 bind to signaling effectors.

Grb2 (Growth factor-receptor-bound) is the adaptor protein that links ErbB receptor to Ras/MAPK pathway. Grb2 both directly and indirectly (via Shc) interacts with EGFR (Batzer et al, 1994; Sasaoka et al., 1994a). Grb2’s two SH2 (Src-homology 2) and one SH3 domains enable it to bind directly to phosphorylated EGFR. Indirect interaction involves Shc, of which SH2 and PTB domains mediate interaction with phosphorylated EGFR residues (Sakaguchi et al., 1998). Grb2 complexes with Sos (son-of-sevenless) and the Grb2/Sos complex mediates signal transduction from the ErbB receptor to Ras protein, leading to activation of the Ras/MAPK pathway (Sasaoka et al., 1994b).

Figure 1.3 Signaling pathways that are activated upon stimulation of EGFR by ligand. Lipid-dependent signaling is effected by PLC and PI3-K, two proteins which share a lipid substrate. Because the substrate localizes to the inside of the plasma membrane, signaling by these effectors occurs largely at that site. The EGFR binding proteins Shc and Grb2 act through SOS and Ras to relay their signals by phosphorylating messengers in the MAPK cascade. The outcome of this is cell proliferation. (The figure is modified from Singh and Harris, 2005).

Phosphatidylinositol 3’-kinase (PI3K) is activated by ErbB receptors both directly and indirectly, leading to activation of Akt-mediated pathways, which play critical roles in different cellular processes like survival and proliferation. The regulatory subunit of PI3K, p85, interacts with ErbB3 and ErbB4 directly via the former’s SH2 domains. On the other

5 hand, PI3K interaction with ErbB1 and ErbB2 requires Cbl, as an adaptor protein (Soltoff et al., 1994 and Soltoff et al., 1996).

Phospholipase C-γ (PLC-γ) directly interacts with ErbB1 through its SH2 domain. It is phosphorylated by ErbB-1, leading to its activation (Chattopadhyay, 1999; Wahl et al, 1990). Activation of PLC increases cellular calcium levels, leading to activation of calcium- dependent protein kinases.

Besides the above-mentioned signaling mechanisms, ErbB receptors utilize other mechanisms to relay signals. Src is a kinase which has strong association with EGFR through its SH2 domain. This interaction involves EGFR phosphorylation by Src, rather than EGFR autophosphorylation (Stover et al., 1995). Upon binding to EGFR, Src phosphorylates substrates important for cytoskeletal organization such as FAK and Eps8 (Schlaepfer et al., 1999; Provenzano et al., 1998).

ErbB receptors themselves can function as direct transcriptional activators. ErbB4 translocates to the nucleus, after a two-step proteolytic cleavage by ADAM-17 and γ- secretase, to activate transcription (Ni et al., 2001). Intact ErbB1 and ErbB2 have also been reported to translocate to the nucleus; the former interacts with STAT3 whereas the latter binds the COX2 promoter directly (Lo et al., 2005; Wang et al., 2004).

1.1.4. ErbB Ligands and Receptors in Mammalian Development:

Various studies investigated the roles of ErbB ligands and receptors in mammalian development. Knockout mice models have highlighted the importance of ErbB ligands and receptors especially in development of cardiovascular and nervous systems and mammary gland. ErbB1, ErbB2-, ErbB3-, ErbB4- and NRG1-knockout mice are embryonic lethal at different stages of embryonic development (Gassmann et al., 1995; Lee et al., 1995; Meyer et al., 1995; Erickson et al., 1997). ErbB3-knockout mice die at E13.5 (Embryonic day 13.5) whereas the other knockout mice die at E10.5 due to impaired cardiac development. ErbB3-knockout mice fail to develop cardiac cushion, the cardiac tissue that develops valves of the heart (Erickson et al., 1997). ErbB2 and ErbB4-knockout mice die due to lack of formation of ventricular trabeculae, where cardiac myocytes proliferate to thicken the cardiac muscular wall (Gassmann et al., 1995; Lee et al., 1995).

In NRG1-knockout mice, both cardiac cushion and ventricular trabeculae do not develop (Meyer et al., 1995). In conditional knockout mice of ErbB2 and ErbB4 where the genes are knocked-out postnatally, severe dilated cardiomyopathy develops, pointing out the importance of ErbB2 and ErbB4 in adult heart function (Crone et al. 2002; Garcia-Rivello et al., 2005).

Targeted mutations of ErbB2, ErbB3 and NRG-1 led to malformation of the sympathetic nervous system, due to the blocked migration of neural crest cells to the mesenchyme lateral of the dorsal aorta (Britsch et al., 1998). ErbB3-knockout mice have no Schwann cells or Schwann cell precursors, leading to lack of myelination, pointing out the importance of ErbB3 receptor for Schwann cell development (Riethmacher et al., 1997). Also, conditional ErbB2-mutant mice displayed peripheral nerve hypomyelination (Garratt et al., 2000).

The mammary gland is unique in the sense that most of the development of the tissue takes place postnatally. During mammary development at the postnatal stage, temporal expression of ligands is observed. A constant expression of AR, TGF-α, BTC, EPR and HB-EGF was observed from prepuberty until late pregnancy. Their expression is decreased in late pregnancy, lactation and involution. Among them, TGF-α is the only ligand whose expression is increased in involution. EGF expression is observed in late pregnancy and lactation (Schroeder and Lee, 1998). NRG expression was observed highly in mammary stroma in pregnancy during lobuloalveloar development. Its expression diminishes through lactation and involution (Yang et al,, 1995). TGF-α, EGF and AR single knockout mice were viable, pointing out the redundancy between the ligands, However, AR-knockout mice have impaired ductal growth during puberty. The pups of triple knockout mice of these ligands die due to lack of nursing. It shows that these ligands work redundantly for lactation (Luetteke et al., 1999).

ErbB1 and ErbB2 expression is observed throughout all of mammary development whereas ErbB3 expression starts in mid-pregnancy and ErbB4 expression starts in late pregnancy (Schroeder and Lee, 1998). ErbB1 was shown to maintain ductal growth; ErbB1 knockout mice fail to develop a mature mammary gland (Sebastian et al., 1998; Wiesen et al., 1999). On the other hand, ErbB2 and ErbB4 play roles in lobuloalveolar development and lactation (Sebastian et al., 1998; Jones et al., 1999; Long et al., 2003).

1.1.5. ErbB Ligands and Receptors in Cancer:

Aberrations of the expression and function of ErbB ligands and receptors is a common phenomenon in human cancer. Overexpression of ErbB1 was observed in various types of cancer such as head and neck, breast, glioma and non-small-cell lung cancer (reviewed in Yarden and Sliwkowski, 2001 and Normanno et al., 2006). ErbB1 overexpression may be linked with shorter disease-free and overall survival. ErbB1 overexpression is correlated with higher grade and reduced survival in gliomas where overexpression is observed in 40% of the cases (Wong et al., 1992). Overexpression is usually due to ErbB1 gene amplification.

Mutations are found in various cancer cases for ErbB1 which contribute to the malignant potential of the receptor. Deletions in different exons of EGFR gene and some point mutations in the kinase domain are among them (reviewed in Normanno et al., 2006). The most frequent mutant form is EGFRvIII, in which the exons 2-7 are deleted. This deletion yields an extracellular domain-truncated EGFR protein, which is constitutively active due to ligand independence (Kuan et al., 2001). This mutant is observed in 50% of the glioblastoma multiforme (GBM) cases where mutant EGFR protein is present (Kuan et al., 2001). This mutant form has also been observed in breast, ovarian and lung cancer (Pedersen et al., 2001). Point mutations in the kinase domain are observed in some non- small-cell lung cancer (NSCLC) cases (Lynch et al., 2004 and Paez et al., 2004).

ErbB2 overexpression is observed also in many types of cancer such as breast, lung and ovary (reviewed in Yarden and Sliwkowski, 2001 and Normanno et al., 2006). Correlation of ErbB2 overexpression is found with tumor size, high grade and spread of the tumor to lymph nodes in breast cancer (Ross and Fletcher, 1998). Mutation in the ERBB2 gene is not as common as in the EGFR gene; in-frame insertions and missense substitutions are reported in some NSCLC cases (Stephens et al., 2004). Alternatively splice variants of ErbB2 are also observed in some breast cancer cases where ErbB2 is constitutively active (Siegel and Muller, 1996).

ErbB3 and ErbB4 expression is present in breast and prostate cancer and heterodimerization with ErbB2 in childhood medulloblastoma and oral squamous cell

8 cancer may be important for prognosis of these cancer types (Xia et al., 1999; Lyne et al., 1997; Kew et al., 2000; Gilbertson et al., 1997).

Overexpression of TGF-α is observed in many cancer types like breast, lung, colon, ovary and head and neck (Salomon et al., 1995). In some tumor types such as breast, a less differentiated phenotype is associated with TGF-α expression (Salomon et al., 1990). TGF-α expression is also found in in situ carcinomas and colon adenomas, it may be important in early steps of tumorigenesis (Salomon et al., 1995; Normanno et al., 2001).

In contrast, AR expression is found to be correlated with a more differentiated phenotype in breast and colon cancers (Normanno et al., 1995; Saeki et al., 1992). AR expression is an indicative of low proliferative activity and low grade in ovary cancer (D’Antonio at al., 2002). HB-EGF expression is found in different cancers such as breast, ovary and colon. NRG expression is found in breast and ovary cancer as well (Normanno et al., 2005).

Overexpression and hyperactivation of ErbB receptors in cancer make them important cancer therapeutic targets. The pharmacological agents targeting ErbB receptors can be divided into two groups: Monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs). Two examples of mAbs are trastuzumab and cetuximab. Trastuzumab (Herceptin, Genentech) is a humanized mAb developed against ErbB2 that now has a widespread usage in breast cancer. Ongoing trials are present for endometrial, ovary and bladder cancer. Cetuximab (Erbitux, Bristol Myers Squibb) is a chimaeric mAb developed against ErbB1 that is now approved for colorectal and head and neck carcinoma. Ongoing trials using it exist for breast, pancreas and cervix cancer (reviewed in Bublil and Yarden, 2007; Citri and Yarden, 2006). MAbs both interfere with signaling machinery of ErbB receptors and bring cytotoxic lymphocytes to the tumor site (Clynes et al., 2000; Nagata et al., 2004).

TKIs are small molecules that bind the kinase domain of ErbB receptors and block kinase function. Gefitinib (Iressa, AstraZeneca) and erlotinib (Tarceva, Genentech) inhibit ErbB1 and are used against tumors that have hyperactive ErbB1 mutants (reviewed in Bublil and Yarden, 2007; Citri and Yarden, 2006). Both have widespread usage for NSCLC. Ongoing trials exist for gastrointestinal, head and neck, breast (for gefitinib), colorectal cancer and glioma (for erlotinib). Dual specific TKIs that target both ErbB1 and ErbB2 such as

Lapatinib (GlaxoSmithKline), CI-1033 (Pfizer) and EKB-569 (Wyeth-Ayerst Research) are used in ongoing trials (reviewed in Bublil and Yarden, 2007; Citri and Yarden, 2006).

1.2. Cbl Protein

1.2.1. Discovery, Elucidation of Structure and Function:

Cbl was first identified as the etiological agent in pre-B-cell lymphomas of mice by Langdon et al. (1989). The investigators had determined that a retrovirus, Cas-Br-M, was found in cells of a cancer-bearing mouse from the Lake Casitas region of California. Tumor extracts from the cells induced new tumors in NFS/N mice, and when the extracts were applied to NIH3T3 and NRK rodent fibroblasts, the cells became transformed.

To understand the molecular basis for these transformations, Langdon and coworkers generated bacteriophage libraries of genomic DNA from the transformed NRK cells. The libraries were screened for the presence of retrovirus genome sequences. They discovered that the entire transforming unit was contained within a single clone, which was sufficient to transform NIH3T3 fibroblasts. Sequencing of the clone revealed the presence of a Cbl (Casitas B-lymphoma)-coding fragment fused to the viral gag and pol genes. The clone was designated as v-cbl. Southern blotting experiments, using v-cbl as the probe, showed that cellular counterparts to the viral sequences were present in rat, mouse and human cells.

Subsequent studies performed by Blake et al. (1991) included screen of cDNA libraries from the human T-cell leukemia cell lines CCRF-CEM and HUT-78 and mouse pre-B cell line 70Z/3. They conducted Southern blotting experiments using v-cbl as the probe. Hybridizations revealed the human and murine c-cbl genes. The cDNA clones were cloned into bacteriophage vectors and then sequenced. The human and murine c-Cbl gene sequences demonstrated that v-cbl gene is a truncated version of its cellular homologue and encodes only 355 amino acids, whereas human c-cbl has 906 amino acids. The detailed analysis of the c-cbl sequences revealed that there is a proline-rich domain and

10 a leucine zipper domain in the C-terminal region absent from v-cbl. The sequence analysis of the transforming c-cbl gene from 70Z/3 cell line revealed a deletion between amino acids 365-383 (Δ366-382), therefore this mutant gene was designated as 70Z/3 c-Cbl. This mutant protein’s transforming capacity on NIH3T3 fibroblasts and its tumorigenic potential in nude mice were high and were demonstrated by Andoniou et al. (1994).

The worm orthologue of c-cbl protein was identified in a genetic screen of C. elegans, which rescued the activity of a hypomorphic allele of the worm EGF receptor LET-23. Vulval development was used as a readout in their experiments, because vulva development requires proper LET-23 function. A hypomorphic mutation in LET-23 results in a vulvaless (Vul) phenotype due to a reduction of LET-23 signaling. The investigators injected mutant X chromosome DNA fragments into Vul mutant animals and then determined that one fragment, a mutant known as sli-1 (sy143), could restore the full-vulva phenotype. Analysis of the sequence of the clone revealed homology with c-cbl. Restoration of wild type sli-1 to the double-mutant animals restored the vulvaless phenotype. Therefore, wild type sli-1 normally functions to impair signaling by LET- 23/EGF receptor, and the mutant sy143 was able to restore vulva formation due to its loss of suppression of EGFR signaling. This study was groundbreaking because it revealed the authentic function of Cbl as suppressing EGF receptor signaling [Yoon et al. (1995)].

Meisner and coworkers (1997) first discovered the fly orthologue of c-Cbl. Drosophila Cbl (D-Cbl) was first cloned after screening of a Drosophila eye cDNA library using of a probe consisting of a DNA fragment amplified from Drosophila DNA using degenerate primers corresponding to c-cbl sequences. When they compared the amino acids of D-Cbl with human c-Cbl and C.elegans Sli-1, it was discovered that D-Cbl is shorter than its counterparts and lacks proline rich regions. It was reported that D-Cbl-overexpressing flies failed to develop R7 photoreceptors using eye development as readout. It was also shown that D-Cbl, unlike human Cbl, fails to bind Grb2. These investigators also showed that D- Cbl is both phosphorylated and binds EGFR upon activation by EGF in COS-1 cells and this binding and the phosphorylation is not present when a Cbl mutant G305E is overexpressed in COS-1 cells. Immunofluorescence experiments revealed the colocalization of Cbl and EGFR in COS-1 cells upon stimulation with EGF.

Based on the aforementioned studies, the Cbl protein domain structure in different organisms is shown in Figure 1.4.

Figure 1.4 The structure of Cbl orthologues. All three orthologues have tyrosine kinase binding (TKB) and RING finger domains. D-Cbl, the D.melanogaster orthologue, lacks C- terminal proline-rich regions and a leucine zipper domain (LZ) possessed by the other orthologues.

Numerous studies published in 1995 (Galisteo et al., Meisner at al., Tanaka et al., Bowtell et al., Odai et al., Fukazawa et al.) revealed the importance of c-Cbl protein in signal transduction networks. These studies showed that c-Cbl protein is tyrosine phosphorylated in different cellular systems like cell lines NIH3T3 (or EGFR overexpressing variants like HER-14 or E10) and HEK 293 or macrophages upon stimulation by different growth stimuli such as EGF, platelet-derived growth factor (PDGF) or colony stimulating factor (CSF-1). Association of Cbl with EGFR in HEK 293 cells upon stimulation with EGF, with different non-receptor cellular kinases such as Src, Shc, Fyn

12 and Lyn and with different adaptor proteins such as Grb2 and Nck was demonstrated in these studies.

A study by Miyake et al. (1998) was the first to demonstrate the ability of c-cbl to bind to and enhance the ubiquitination and degradation of a RTK. In this case, Cbl expression downregulated PDGFR from the cell surface and enhanced its degradation in PDGF- stimulated NIH3T3 cells that stably overexpress PDGFR. However, this report did not show how Cbl exerts its effect.

A subsequent breakthrough study that further elucidated the function of Cbl protein was conducted by Levkowitz et al. (1998). When Chinese hamster ovary (CHO) cells were transfected with c-Cbl or v-Cbl and were stimulated with EGF, c-Cbl but not v-Cbl mediated both enhanced downregulation of EGFR from the cell surface and its degradation. Downregulation refers to the extent of removal of EGFR receptors from the cell surface. As a specificity control, neuregulin stimulation of CHO cells expressing ErbB3 resulted in no downregulation and degradation of that receptor when Cbl is overexpressed. Double immunofluorescence analysis revealed that ErbB1 but not ErbB3 colocalized with c-Cbl after the receptor-expressing cells were stimulated with the corresponding growth factor.

It was determined that v-Cbl mediated EGFR recycling, on the other hand, c-Cbl provoked EGFR internalization without recycling. The ability of c-Cbl to downregulate and degrade EGFR levels upon EGF stimulation was partially blocked by chloroquine and MG132, inhibitors of lysosomal and proteosomal degradation, respectively. This showed that c- Cbl-mediated EGFR degradation involves both lysosomal and proteosomal proteolysis processes. Importantly, it did not show whether the proteasomes and lysosomes were both degrading the EGFR directly. Levkowitz et al. demonstrated that c-Cbl overexpression enhances EGFR ubiquitination in the same way that it enhanced PDGFR ubiquitination (Miyake et al., 1998). Finally, v-Cbl and 70Z/3 Cbl mutants failed to enhance EGFR ubiquitination upon EGF stimulation.

The exact function of c-Cbl protein was first demonstrated by Joazeiro et al. (1999) and Yokouchi et al (1999). Given the above findings, these researchers investigated whether c-Cbl itself could be a ubiquitin ligase enzyme. In the studies by Joazeiro et al., substrate- independent in vitro ubiquitination reactions were performed using GST-fused wt Cbl

RING domain (aa 380-425) or mutant Cbl RING domains, E1 Ub-activating enzyme, Histidine (His)-tagged E2 Ubc4 and ATP. Iodine-labeled or unlabeled ubiquitin was also included in separate experiments. The isolated Cbl-wt RING domain enhanced ubiquitination of the target proteins (Ubc4 or GST-RING); on the other hand, Cbl 70Z/3 RING domain failed to ubiquitinate itself or Ubc4 as determined by the above approach. GST-bound Cbl mutants ∆Y368 and C372A could ubiquitinate themselves and Ubc4 as efficiently as wild-type Cbl, whereas C381A and W408A mutants could not. This pointed out the importance of the RING domain (C381 and W408 reside in RING domain). GST pull down assays revealed that GST Cbl wt and the Cbl ∆Y368 and C372A mutants could bind Ubc4, whereas Cbl C381A and W408A Cbl mutants failed to bind Ubc4. Thus, Ubc4 binding correlated with ubiquitin ligase activity.

Yokouchi et al. (1999) revealed that UbcH7 is the E2 enzyme that recruits ubiquitin to c- Cbl for its ligation to the substrate, i.e. EGFR. UbcH7 was identified by a yeast two-hybrid screen. GST-bound Cbl RING domain was used as bait where it was conjugated to a GAL4 binding domain. The screen revealed cDNA of UbcH7 when β-galactisodase assay was used as a readout. When UbcH7 or wt Cbl is overexpressed in HEK 293 cells by transient transfection and HEK293 is stimulated by EGF, the ubiquitination of EGFR is enhanced. On the other hand, Cbl mutant 70Z/3 failed to ubiquitinate EGFR in HEK 293 cells. When cell extracts from stably wt Cbl-expressing HEK293 cells were harvested after EGF stimulation, EGFR was found to be ubiquitinated in vitro when UbcH7 is included in the ubiquitination reaction. On the contrary, cell extracts from 70Z.3 Cbl failed to show the in vitro ubiquitination of EGFR.

An opposing study was recently published by Umebayashi et al. (2012), stating that Ubc 4/5 rather than UbcH7 is the E2 enzyme for c-Cbl. In this study, FLAG-tagged Ubc 4/5 but not UbcH7 colocalized with c-Cbl and compromised EGFR degradation after EGF stimulation. This supported the conclusion that the E2 partner for c-Cbl is Ubc4/5.

1.2.2 Cbl Structural Effects on Regulation of EGFR:

Andoniou et al. (1994) investigated the tumorigenic potential of wild-type and differentially truncated c-Cbl proteins on nude mice. It was discovered that both wt Cbl and Cbl proteins

14 truncated back to residue 388 had no or minimal transforming activity when retrovirally expressed in nude mice. On the other hand, cells transduced with retrovirally expressed v-Cbl (c-Cbl 1-357) formed tumors in nude mice. In the same study, tumors formed when cells were retrovirally infected with 70Z/3 mutant Cbl and also mutant Cbls with deletions of tyrosine residue 368 or 371. Interestingly, Cbl mutants Y368F and Y371F did not form tumors, implying that Y368 and Y371 phosphorylation is not crucial for inducing the oncogenic potential of c-Cbl protein. This study also revealed that the oncogenic forms of c-Cbl were constitutively tyrosine phosphorylated, unlike the wild type c-Cbl.

After the discovery of c-Cbl association and tyrosine phosphorylation upon cell stimulation with EGF as described above, Thien and Langdon (1997a) investigated which regions of c-Cbl protein play a crucial role in those activities. In this study, wild-type Cbl and different Cbl mutants were stably expressed in NIH3T3 fibroblasts. The cells were then serum starved and stimulated by EGF. Immunoprecipitations followed by immunoblotting revealed that Cbl proline region mutants (Δ472-540, Δ543-548, Δ543-645) were phosphorylated as much as wt Cbl were, but did not associate with EGFR as much, pointing out that Cbl binding to EGFR is enhanced by SH3 containing adaptor proteins. However, this enhanced association was not so important for Cbl phosphorylation. N- terminal deletions of c-Cbl (Δ262-300 and Δ302-350), on the other hand, were phosphorylated much less than wild-type c-Cbl, revealing the importance of residues within the deleted regions for tyrosine phosphorylation. This does not necessarily mean that the critical residues in these regions are tyrosines. The association with EGFR was affected very slightly for these N-terminal mutants. It was also discovered that a G306E mutation in the c-Cbl protein, which is analogous to the loss-of-function mutation found in sli-1, decreased c-Cbl association with, and phosphorylation of, EGFR.

A concomitant study by the same authors [Thien and Langdon (1997b)] showed that 70Z/Cbl (Δ365-382) mutant protein enhanced both EGFR binding and phosphorylation in both serum-starved and EGF-stimulated NIH3T3 fibroblasts. This result revealed that 70/Z Cbl induces constitutive activation of EGFRs.

Studies conducted by Waterman et al. (1999) and Levkowitz et al. (1999) further uncovered the mechanism by which Cbl ubiquitinates EGFR, leading to its degradation. Waterman et al. (1999) identified the importance of the RING finger domain in EGFR

15 ubiquitination, downregulation and degradation. Both 70Z/Cbl and C381A mutant proteins, which had no intact RING finger domains, failed to ubiquitinate, downregulate and degrade EGFR when they were overexpressed in CHO cells upon EGF stimulation. The studies carried out by Levkowitz et al. (1999) revealed the importance of Y1045 residue of EGFR protein and Y371 residue of Cbl protein in ubiquitination, downregulation and degradation of EGFR, because the phenylalanine mutations of aforementioned residues of both proteins failed to regulate EGFR like wild-type Cbl. Y1045 was found to serve as the docking site for Cbl association. The truncation mutants which lacked an intact TKB domain showed that direct EGFR binding by this region is a prerequisite for EGFR ubiquitination.

Lill et al. (2000) reported which contiguous minimal domains of c-Cbl are sufficient to exert its function on EGFR. The effects of wild-type c-Cbl, c-Cbl G306E, c-Cbl 1-357 (v-cbl), c- Cbl 1-357 (G306E) and Cbl 1-440 were tested. When the proteins were overexpressed in HEK293 cells, only Cbl wild-type and Cbl 1-440 downregulated EGFR from the cell surface. The other mutant c-Cbl proteins were unable to downregulate, ubiquitinate and degrade EGFR upon EGF stimulation. This study revealed that a Cbl fragment containing the TKB domain (1-357) and RING domain (380-425), as well as additional sequences to residue 440, together were sufficient for c-Cbl to exert its function on EGFR. One important conclusion from this finding is that the non-conserved proline-rich and leucine-rich domains of the Cbl C-terminus are not essential for Cbl-mediated regulation of the EGFR.

Thien et al. (2001) investigated the correlation between inability of different domain-mutant c-Cbl proteins for EGFR downregulation/ubiquitination and transformation of cultured cells. In this study, it was revealed that RING finger mutant Cbl proteins could not transform cells despite their inability to ubiquitinate and downregulate EGFR in NIH3T3 fibroblasts. Rather, mutants such as 70Z/Cbl, ΔY368 and ΔY371 (linker region deletion mutants), which also were unable to ubiquitinate and downregulate EGFR, did transform NIH 3T3 fibroblasts. It pointed out that the linker region between TKB domain and RING finger domain is crucial for oncogenic potential for c-Cbl protein.

Waterman et al. (2002) discovered that c-Cbl function (EGFR downregulation, ubiquitination and degradation) was partially restored in CHO cells with EGFR Y1045F mutation by Grb2 overexpression, implying that indirect binding of c-Cbl to EGFR via Grb2

16 through proline-rich regions found on C-terminal part of c-Cbl may serve as an auxillary mechanism for c-Cbl function.

Duan et al. (2002) used two different approaches to study the effect of the endogenous Cbl on EGFR. They used Cbl-knockout cells and also a temperature-dependent ubiquitination system in CHO cells. Internalization of EGFR in these two types of cells was not different from cells expressing endogenous Cbl. On the other hand, EGFR ubiquitination, EGFR localization on late endosomes/lysosomes and EGFR degradation upon EGF stimulation were impaired when endogenous Cbl function was blocked. These findings revealed that ubiquitination is critical for EGFR endocytosis not at the internalization step, but in downstream steps. The data of Huang et al. (2007) further supported that EGFR internalization upon EGF stimulation does not require EGFR ubiquitination, as EGFRs with lysine mutations (ubiquitination-defective) were internalized as efficiently as wild-type. On the other hand, degradation of EGFR was impaired for these mutants.

1.3 EGFR Endocytosis and Regulation of Signaling:

In order to maintain the desired response for a signal, the duration and amplitude of the signal must be tightly regulated. Endocytosis is one of the mechanisms in which signaling through RTKs is controlled upon ligand binding. Related to endocytosis, “downregulation” is the term referring to describe the decrease in the RTK levels in the cell membrane. Among ErbB family members, EGFR (ErbB1) is the only member where extensive downregulation of the receptor occurs upon ligand binding (Wiley et al., 1991). On the other hand, ErbB3 and ErbB4 are downregulated in a very little extent when activated (Baulida et al., 1996; Baulida et al., 1997). ErbB2 downregulation depends on its expression level. ErbB2 is downregulated in parallel to EGFR when its expression is low, whereas ErbB2 is retained in the cell membrane upon activation when its expression is high such as in breast cancer cell lines (Worthylake et al., 1997; Worthylake et al, 1999; Haslekas et al, 2005). A contradictory study by Muthuswamy et al. (1999) suggests that

17 only ErBB1 homodimers, rather than ErbB2 homodimers or ErbB1/ErbB2 heterodimers are associated with and can phosphorylate Cbl.

Like the other RTKs, ErbBs have two different fates upon endocytosis: They either go through the complete endocytic pathway and are degraded in lysosomes or recycle back to the cell membrane at different points of endocytosis (reviewed in Wiley 2003; Sorkin and Goh, 2008; Grandhal and Madshus, 2008). EGFR endocytosis activated by EGF is the most extensively studied example of RTK endocytosis. The steps of endocytosis of EGFR include internalization of the receptor, formation of early endosome, fusion with other endosomes leading to maturation into multivesicular bodies (MVB), followed by degradation of the receptor in the lysosome. These steps and also recycling are illustrated in Figure 1.5.

Figure 1.5 Steps of EGFR endocytosis. Ligand-activated EGF-R is: ubiquitinated at the cell surface; internalized (* outside the cell) on vesicles that fuse with early endosomes; and either recycled or sorted onto lumenal membranes that are internalized from endosomal limiting membranes. Lumenal vesicle formation (* within the cell), initiated at

18 early endosomes, is complete at late endosomes (multivesicular morphology). Lysosomal delivery allows for destruction of internal membranes and their cargo.

1.3.1 Mechanism of EGFR Endocytosis:

EGFR endocytosis starts with the internalization of the receptor from the cell membrane. It has been widely accepted that internalization is mediated generally by clathrin-coated pits (Kazazic et al., 2006; Huang et al., 2004). However, a second route for EGFR internalization has been clearly defined by Polo and colleagues: This pathway brings activated EGFRs inside the cell through a clathrin-independent mechanism utilizing caveolae (Sigismund et al., 2005).

Grb2 is recruited to active EGFR via phosphorylated Y1068 and Y1086 residues. The observation that strong blockade of EGFR internalization occurs when these residues are mutated shows that Grb2 plays a very important role for EGFR internalization (Jiang et al., 2003). In the same study, it was also shown that depletion of Grb2 by siRNA decreased EGFR internalization. It points out that besides the signal relaying role by activating RAS/MAPK pathway that was mentioned in section 1.1.3 of this introduction, Grb2 may serve as an adaptor/scaffold molecule that recruits other proteins involved in EGFR downregulation to the receptor. Some investigators have discovered in their model systems that Cbl ubiquitinates activated (phosphorylated) EGFR via direct or Grb-2 mediated indirect binding to EGFR (reviewed in Thien and Langdon, 2001; Thien and Langdon, 2005; Schmidt and Dikic, 2005). However, only the direct binding of Cbl to EGFR via G306 of Cbl and Y1045 of EGFR is universally acknowledged to lead to receptor ubiquitination in all tested model systems. For this reason, research in Lill laboratory has focused on events that arise from the direct association of EGFR with Cbl, which is the only evolutionarily conserved mechanism resulting in receptor ubiquitination.

Once clathrin-coated vesicles (pits) are budded totally from the plasma membrane, they become ready to fuse with endosomes (early), compartmentalized structures of vesicular membranes, by uncoating their clathrins (Hopkins et al., 1985; Miller at al., 1986). The fusion events continue as the early endosomes mature into late endosomes and then

MVBs. The fusion events are mediated by small GTPase proteins called Rabs. Rab proteins are also involved in recycling of vesicles (reviewed in Stenmark, 2009).

MVB formation is mediated by the actions of ESCRT (endosomal sorting complex required for transport) family of proteins. The ubiquitinated EGFRs in early endosomes are first handed to ESCRT-0 via interaction through UIM found in ESCRT-0 components, and then consecutively transferred to ESCRT-I, ESCRT-II and ESCRT-III complexes. Hrs, STAM and Vps27 are components of ESCRT-0, TSG-101 is a component of ESCRT-I. EGFR is deubiquitinated when bound to ESCRT-III. ESCRT-III complex mediates invagination of the endosomal membrane, leading to formation of multiple lumenal vesicles. Once EGFR is transferred to the internal membranes of MVB, it is destined for lysosomal degradation, that is, it cannot be recycled to the cell membrane (Henne et al., 2011; Sorkin and Goh, 2008; Grandal and Madshus, 2008).

EGFR degradation requires the persistence of ligand binding to the receptor and receptor phosphorylation. Low affinity binding of TGF-α to EGFR leads to dissociation of the complex as early endosomes begin to acidify. Under the same conditions, EGF-activated receptors remain bound to their ligand. The outcome of these differential events is that TGF-α stimulated receptors recycle to the cell surface while their EGF-stimulated counterparts move along the degradative pathway to lysosomes (French at al., 1995).

1.3.2 Hrs Protein and Its Role in EGFR Endocytosis;

Hrs (Hepatocyte-growth factor regulated tyrosine kinase substrate) is a protein which plays an important role in endosomal protein sorting. Hrs complexes with STAM (signal- transducing adaptor molecule) and the HRS-STAM complex is localized on the early endosomal membrane. The complex constitutes ESCRT-0 of endocytic sorting machinery. Hrs has FYVE domain which mediates its interaction with phosphadityl- inositol-3-phosphate (PtdIns(3)P), helping its localization to endosomal membrane (Komada et al., 1997; Mizuno et al., 2004; Bache et al., 2003). Hrs binds clathrin via its clathrin binding domain and forms bilayered clathrin-rich microdomains around the endosomal membrane (Sachse et al., 2002). Hrs has a UIM (ubiquitin interacting motif) which mediates Hrs interaction with ubiquitinated cargo (i.e. EGFR). Depletion of Hrs by

20 siRNA compromises EGFR degradation (Malerod et al., 2007) indicating its critical role in this process. Hrs interacts with TSG-101 (a component of ESCRT-I) which also can interact with ubiquitinated cargo, so it is proposed that Hrs/STAM complex’s role in degradative trafficking is to recruit ESCRT-I and relay ubiquitinated cargo to TSG101 of ESCRT-I (Komada and Kitamura, 2005).

Hrs is both phosphorylated and ubiquitinated upon growth-factor stimulation (Komoda and Kitamura, 1995; Polo et al., 2002). Ubiquitination enhances Hrs/STAM complex interaction with TSG-101, therefore contributing the recruitment of ESCRT-I complex. It is also proposed that ubiquitination makes UIM of Hrs inaccessible to ubiquitinated cargo, which may be another form of facilitation to relay ubiquitinated cargo to ESCRT-I (Stern et al., 2007). Hrs is phosphorylated via its Y334 and Y329 residues upon EGFR activation, the former is the predominant phosphorylation site (Steen et al, 2002; Urbe et al., 2003). Hrs is proposed to translocate from endosomal membrane to cytosol upon phosphorylation, which causes its attenuation of function (Urbe et al., 2000; Stern et al., 2007).

It is demonstrated that when Cbl is overexpressed in HEK 293 cells, Hrs is rapidly ubiquitinated and phosphorylated, then translocated to cytosol, dephosphorylated and degraded in the cytosol. This shows that Cbl has another role rather than EGFR ubiquitination on the cell membrane. Hrs phosphorylation and degradation displays a significant correlation with EGFR degradation, pointing out that it is an important checkpoint for degradative trafficking of EGFR following its activation (Stern et al., 2007).

The Lill laboratory showed that when Cbl-F434A is overexpressed in HEK 293 cells, Hrs dephosphorylation is delayed and EGFR degradation is compromised after ligand activation (Visser Smit et al., 2009). This was an important finding for the field, as it was the first demonstration that the site of Cbl action after receptor internalization was the ESCRT-0-positive endosomal compartment. Moreover, the group reported that clusters of EGFR-loaded endosomes docked together without fusing when Cbl mutant F434A was expressed. In wild-type Cbl expressing cells, endosomes docked and then quickly fused (Visser Smit et al., 2009). Biochemical studies revealed that the defect in endosome fusion, induced by F434A-Cbl, correlated with the inability of the mutant to effect the dephosphorylation and the degradation of Hrs on those endosomes. It had been reported by the laboratory of Andrew Bean that Hrs is a critical regulator of endosome fusion (Sun

21 et al., 2003). Therefore, it was concluded that the Cbl-dependent regulation of Hrs activity on early endosomes likely was controlling the maturation of trafficking endosomes via fusion and that this checkpoint was the principle molecular mechanism through which Cbl controls EGFR degradation.

1.3.3 EGFR Signaling Potency Along Its Endocytosis:

Recent reports by Sousa et al. (2012) and Brankatschk et al. (2012) studied EGFR signaling potency throughout endocytosis. In either study, they interfered with separate steps of EGFR endocytosis and concluded that signaling elicited by EGFR activation in a cell mainly stems from the receptors at the plasma membrane and then receptors at the early endosomes. The receptors after endosome fusion had little or minimum effect on signaling elicited by EGFR activation.

Sousa et al. (2012) used dynamin-knockout mouse fibroblasts in their study. As expected, dynamin depletion blocked EGFR endocytosis, and therefore EGFR degradation upon EGF stimulation. Also, it caused enhanced EGFR activation and ubiquitination. When the ERK and Akt phosphorylation patterns, as proximal signaling events, were compared even upon different amounts of EGF activation, no difference was found between wild-type and dynamin-knockout cells. Therefore, they concluded that EGFR signaling primarily occurs at the plasma membrane.

Brankatschk et al. (2012) used a more systemic approach to determine which step along EGFR endocytosis is crucial for the transcriptional response elicited by EGFR activation. Using siRNA knockdown system, they blocked expression of different molecules which are involved in EGFR endocytosis. The molecules they silenced were clathrin, dynamin, c-Cbl, Cbl-b, Hrs (ESCRT-0), TSG101 (ESCRT-I), VPS4A and Alix (an MVB formation suppressor).

ELK1 luciferase assay and EGR1 and FOS transcriptions were used as readouts for transcriptional response elicited by EGFR activation when ESCRT components were depleted. No change in luciferase assay or reporter transcription was evident between the mock-treated and ESCRT-component-silenced cells. Also, they used NanoString technology to observe the global transcriptome in both control and experimental cells.

Little difference was observed between mock-depleted and Hrs- and TSG-101 depleted cells. Even no difference in global transcriptome was observed between mock-depleted and VPS4A and ALIX-depleted cells. On the other hand, proximal signaling events such as MEK1/2 and ERK1/2 phosphorylation events were enhanced when the aforementioned components were silenced. This pointed out that the outcomes of early signaling events may not be good reflectors of the late transcriptional responses, at least for the short term.

On the contrary, when clathrin, dynamin and Cbls were depleted in HeLa cells, the investigators found out increased luciferase activity in ELK1 luciferase assay and also a significant increase in global transcriptome of the cells. Also, EGFR phosphorylation and proximal signaling events such as MEK1/2 and ERK1/2 phosphorylation were also enhanced.

Immunofluorescence studies revealed that 10 min after EGF stimulation, little colocalization with EEA1 (early endosome marker) was observed in dynamin and clathrin- depleted cells, more colocalization was observed when Cbls were depleted, a significant amount of colocalization was observed when Hrs and TSG101 were depleted.

The data revealed in these studies suggest that therapeutic approaches should be directed toward the events occurring at or in close proximity to the plasma membrane. Since the site of action of c-Cbl is at or in close proximity to the cell membrane, these findings point out the importance of our studies described in this dissertation.

The findings depicted in these studies are summarized in Figure 1.6.

1.4 c-Cbl Crystal Structure and Its Relevance to Our Study:

In 2000, Zhang et al. published the first-ever molecular structure of Cbl. The protein used for this study actually was a truncated version of c-Cbl (amino acids 47-447, encompassing the TKB, linker and RING finger domains, as well as sequences through the RING finger tail). Cocrystallized in this study were UbcH7 and an 11 residue-peptide of ZAP-70 which contains a c-Cbl binding site. Importantly, the Cbl protein was purified from bacteria that do not effect post-translational modifications of the recombinant protein. The investigators found that when c-Cbl complexes with UbcH7, this interaction is

23 mediated both via RING domain and also the linker region, via van der Waals interactions and hydrogen bonds. The computer model revealed a surface channel which connects UbcH7 and ZAP-70 peptide and which might play an important role in transfer of ubiquitin. It was concluded that c-Cbl, as a member of the RING family of E3 ubiquitin ligases, possibly functions via recruiting an E2 enzyme close to the substrate thereby serving as a scaffold. Importantly, no functional data were provided to support the biological relevance of the reported crystal.

Figure 1.6 Where EGFR signals. Proteins that regulate EGFR trafficking and the receptor- induced transcriptional response. A subset of the regulatory proteins that increase (+) or decrease (–) each process is listed above the site of action. The question mark next to Cbl indicates the arguable function of the protein in promoting EGFR internalization. The times shown in minutes (min) are the approximate periods required to move activated receptors from the plasma membrane to the indicated compartments. The reports by

(Figure 1.6 continued) Brankatschk et al. and Sousa et al. have mapped EGF-induced signaling events to distinct trafficking checkpoints. Reproduced from Lill and Sever, 2012.

Cbl #1 Cbl

UbcH7

UbcH7 #2

Figure 1.7 Putative Cbl dimer contained within the 2000 structure reported by Zhang et al. The figure resulted from a reanalysis of the published coordinates by S.Ramaswamy and R.Friemann.

The c-Cbl RF tail residues (421-436) lay within the solved crystal, suggesting that this region might play an important role for c-Cbl function. The Lill lab had investigated the function of the RF tail previously. The first study used truncated Cbl proteins to test their role in EGFR regulation. The truncation analysis demonstrated that amino acid residue F434, found in the RF tail region, is the C-terminal limit of functional c-Cbl (Smit GD and Lill NL, 2005). A later study reported that alanine scanning mutagenesis of residues 428- 436 produced a panel of full-length Cbl mutants with different abilities to effect EGFR

25 downregulation, ubiquitination and degradation. The V431A and F434A mutants were found to be compromised for EGFR downregulation, ubiquitination and degradation. An unexpected discovery was that these two substitutions, physically close on the Cbl molecule, led to endocytosis defects at different subcellular sites. hSprouty degradation at the plasma membrane was impaired in Cbl-V431A-overexpressing cells, whereas timely dephosphorylation and degradation of Hrs on early endosomes was compromised in Cbl-F434A-overexpressing cells. Immunofluorescence experiments showed that early endosome fusion was blocked in F434A-overexpressing cells, as reflected by the accumulation of docked clusters of endosomes carrying EGFR on their limiting membranes [Visser-Smit et al. (2009)].

Since the crystal structure of Zhang et al. contained only a portion of the c-Cbl protein and a fragment of a tyrosine kinase, and also because the c-Cbl used in the study was unphosphorylated, the biological relevance of the structure was uncertain. Therefore, the role of the RF tail within the structure remained to be tested. In collaboration with Drs. S. Ramaswamy and R. Friemann at the University of Iowa, we reanalyzed the published coordinates to determine the position of the RF tail within the more extensive crystal. When interactions between Cbl units were considered, no regular repeating structure was observed. The Cbl-Cbl interactions could be due to crystal packing; alternatively, some of them might represent genuine Cbl-Cbl associations. The best interface observed (Figure 1.7) was selected for testing the impact of a putative Cbl dimer on EGFR degradation. There was no certainty that the best-fit dimer structure is biologically relevant. However, the Lill laboratory proposed to test the hypothesis that formation of the dimer interface is critical for Cbl-mediated regulation of EGFR. It is essential to note that the theory that dimerization controls the activity of the Cbl/E3 ubiquitin ligase was based on published observations that RF tail sequences of other E3 proteins serve as alpha-helical structures at the interface of dimers formed by those RING/E3 proteins.

Two studies particularly supported the idea that a dimer analysis of Cbl’s RF tail region was warranted. First, a study by Brzovic et al. (2001) showed that heterodimerization of BRCA1, another RING-type E3 ligase, with its partner BARD1 requires regions in the C- terminal flank of their RING domains. This interaction is important for the E3 function of BRCA1 protein. A more recent study by Poyurovsky et al (2007) demonstrated that

26 oligomerization of Mdm2, a RING-type E3 ligase, is mediated by residues C-terminal to the RING domain. Mutational analysis of this region by Uldrijan et al. (2007) further supported the hypothesis. A manual sequence analysis of this region revealed striking similarity to the RF tail region of c-Cbl and other E3 proteins (Table 1.1) (Vander Kooi CW et al., 2006 and Linke K et al., 2008).

Table 1.1 Manual sequence analysis of RF tail regions of different RING/E3 proteins.

Basing a dissertation only on these data would present a high risk. However, unpublished data from the Lill laboratory revealed that the Cbl RF tail appears to play a role in Cbl dimer formation and maintenance. In these studies, GFP- and HA-tagged Cbl proteins were co-expressed in HEK 293 cells. If the proteins could form dimers in the presence or absence of EGF, immunoprecipitated GFP-Cbl would co-precipitate HA-tagged Cbl. The converse must also be true. In our laboratory, Drs. Nancy Lill and Kathryn Stern (Clausen) undertook the experiment shown in Figure 1.8. The figure reveals that the formation and kinetics of Cbl-V431A and Cbl-F434A mutant dimers differ from those of wild-type Cbl.

Wild-type Cbl dimers were present in low amounts in unstimulated cells, then increased progressively over the 25 min stimulation period. On the other hand, V431A and F434A dimers were observed after EGF stimulation with different kinetics, suggesting that V431A and F434A substitutions affect Cbl dimer formation as well as Cbl function. Specifically, V431A showed an increase in Cbl dimerization at 10 min of EGF stimulation, but the dimer amount decreased by 25 min post-stimulation. In the case of F434A, no increase in dimer levels was observed after 10 min of cell stimulation with EGF, but abundant dimerization was apparent at 25 min post-stimulation.

Based on the differences in the kinetics of Cbl dimer formation, we concluded that Cbl dimers are induced upon cell activation by EGF, but those dimers form less efficiently or with less sustained interaction when the Cbl RF tail residues V431 and F434 are mutated. Thus, the functional defects of V431A and F434A mutants correlate with abnormal kinetics of Cbl-Cbl dimer formation.

The Figure 1.7 image of Ramaswamy and Friemann seemed to present a reasonable starting point for subsequent structure-function studies of Cbl dimers. At the least, introducing mutations into the free bearing RF tail would provide certain information about the importance of that region. By testing residues at the interface that were contributed by the other Cbl molecule, we would directly test whether that particular structure might be functionally relevant.

For my Ph.D dissertation study, I chose to test whether targeted disruption of amino acid residues on either side of the putative dimer interface of Cbl led to a loss of Cbl-mediated EGFR degradation (Figure 1.9). Target residues were separately mutated to alanine or to a residue of altered charge to maximize the disruption of the putative dimer interface. The functional study of choice for analyzing these mutants was the EGFR degradation assay previously reported by Lill laboratory. The results of these experiments constitute Chapter III of the dissertation.

In further studies (Chapter IV of the dissertation), I extended earlier research by our lab that led us to hypothesize the presence of an activity within Cbl, which inhibited degradation trafficking of EGFR and which mapped C-terminal to amino acid 357. This novel activity is normally masked by the ubiquitin ligase activity of Cbl, but becomes

28 evident when E3 activity is inhibited and ubiquitin is present in Cbl-EGFR complexes via recombinant fusion to Cbl. Using ubiquitin-fused truncated Cbl-Y371F constructs, which lack E3 activity but can bind to ubiquitin interacting motifs of the endocytosis machinery, I mapped more definitively the location of the inhibitory element. I then tested whether this inhibitory activity involved binding of Cbl-interacting proteins to consensus Grb2 binding sites within Cbl. Our findings suggest that these sites are not involved in this inhibitory activity.

Figure 1.8 Dimerization analysis of Cbl RF tail mutants. HEK 293 cells were co- transfected with GFP-tagged and HA-tagged constructs. 48 hours after transfection, cells were stimulated with EGF, and harvested at 10 and 25 min timepoints. 100 µg of whole cell lysate was directly run on the gel and GFP immunoblotting was performed. 1 mg of lysate was immunoprecipitated with anti-GFP antibody followed by immunoblotting with anti-HA and anti-GFP anti

A K58

H286 Figure 1.8 Q409 D52

E410

D348

R333

Figure 1.9 The residues located at the putative dimer interface of Cbl crystal structure. (A-C) Possible amino acid interactions at the interface of adjacent Cbl molecules in the solved crystal of unmodified Cbl (Zhang et al., 2000). Residues within the same Cbl molecule are highlighted in red rectangle; amino acids on the other side of the Cbl/Cbl interface (second Cbl protein) are highlighted in blue rectangle. (continued on next page)

Figure 1.9

E410

D348 R333

K424

C E394

Q345

N346

T426

CHAPTER 2 MATERIALS AND METHODS

2.1 Mutagenesis of Cbl-Wt Plasmid:

In order to express mutant Cbl proteins in the HEK 293 cell line, wild-type Cbl encoding DNA was mutagenized to change the desired bases. Primers were designed to introduce mutations. 33-mer sense primers harboring mutations on their midpoint codons for desired sites, along with complementary antisense primers were synthesized by Integrated DNA Technologies (IDT). The primers corresponding to the desired mutations are indicated below. The bold codons represent the mutated sequences whereas the codons above the lines indicate the corresponding wild-type codon.

D52A GAC FW: 5’-CCG-CCG-GGG-ACG-GTG-GCC-AAG-AAG-ATG-GTG-GAG-3

RV: 5’-CTC-CAC-CAT-CTT-CTT-GGC-CAC-CGT-CCC-CGG-CGG-3’

D52N GAC FW: 5’-CCG-CCG-GGG-ACG-GTG-AAC-AAG-AAG-ATG-GTG-GAG-3’

RV: 5’-CTC-CAC-CAT-CTT-CTT-GTT-CAC-CGT-CCC-CGG-CGG-3’

K58A AAG

FW: 5’-AAG-AAG-ATG-GTG-GAG-GCG-TCC-TCC-AAG-CTC-ATG-3’

RV: 5’-CAT-GAG-CTT-CCA-GCA-CGC-CTC-CAC-CAT-CTT-CTT-3’

K58E AAG

FW: 5’-AAG-AAG-ATG-GTG-GAG-GAG-TCC-TCC-AAG-CTC-ATG-3’

RV: 5’-CAT-GAG-CTT-CCA-GCA-CTC-CTC-CAC-CAT-CTT-CTT-3’\

H286A C AC

FW: 5’-CTC GGC TCC AGA AAT TCA TTG CCA AAC CTG GCA GTT ATA TC-3

RV: 5’-GAT-ATA-ACT-GCC-AGG-TTT-GGC-AAT-GAA-TTT-CTG-GAG-CCG-AG-3’

R333A A GG

FW: 5’-CAC-TGA-TTG-ATG-GCT-TCG-CGG-AAG-GCT-TCT-ATT-TG-3’

RV: 5’-CAA-ATA-GAA-GCC-TTC-CGC-GAA-GCC-ATC-AAT-CAG-TG-3’

Q345A CA G FW: 5’-GTT-TCC-TGA-TGG-ACG-AAA-TGC-GAA-TCC-TGA-TCT-GAC-TGG-3’

RV: 5’-CCA-GTC-AGA-TCA-GGA-TTC-GCA-TTT-CGT-CCA-TCA-GGA-AAC-3’

N346A AAT

FW: 5’-GAT-GGA-CGA-AAT-CAG-GCT-CCT-GAT-CTG-ACT-G-3’

RV: 5’-CAG-TCA-GAT-CAG-GAG-CCT-GAT-TTC-GTC-CAT-C-3’

D348A G AT

FW: 5’-GAA-ATC-AGA-ATC-CTG-CTC-TGA-CTG-GCT-TAT-G-3’

RV; 5’-CAT-AAG-CCA-GTC-AGA-GCA-GGA-TTC-TGA-TTT-C-3’

E394A GAG FW: 5’-AAG-GAT-GTA-AAG-ATT-GCG-CCC-TGT-GGA-CAC-CTC-3’

RV: 5’-GAG-GTG-TCC-ACA-GGG-CGC-AAT-CTT-TAC-ATC-CTT-3’

Q409A CAG

FW: 5’-TGT-CTT-ACA-TCC-TGG-GCA-GAA-TCA-GAA-GGT-CAG-3’

RV: 5’-CTG-ACC-TTC-TGA-TTC-TGC-CCA-GGA-TGT-AAG-ACA-3’

Q409E CAG

FW: 5’-TGT-CTT-ACA-TCC-TGG-GAG-GAA-TCA-GAA-GGT-CAG-3

RV: 5’-CTG-ACC-TTC-TGA-TTC-CTC-CCA-GGA-TGT-AAG-ACA-3’

E410A GAA

FW: 5’-CTT-ACA-TCC-TGG-CAG-GCA-TCA-GAA-GGT-CAG-GGC-3’

RV: 5’-GCC-CTG-ACC-TTC-TGA-TGC-CTG-CCA-GGA-TGT-AAG-3’

E410Q GAA FW: 5’-CTT-ACA-TCC-TGG-CAG-CAA-TCA-GAA-GGT-CAG-GGC-3’

RV: 5’-GCC-CTG-ACC-TTC-TGA-TGC-CTG-CCA-GGA-TGT-AAG-3’

K424A AAA

FW: 5’-TGC-CGA-TGT-GAA-ATT-GCA-GGT-ACT-GAA-CCC-ATC-3’

RV: 5’-GAT-GGG-TTC-AGT-ACC-TGC-AAT-TTC-ACA-TCG-GCA-3’

2.1.1 Mutagenesis Protocol:

QuikChange Lightning Site Directed Mutagenesis Kit was used to introduce mutations into the parental plasmid containing wild-type Cbl cDNA (Agilent Technologies). The mutations were introduced by PCR reaction using sense and antisense primers that were specified above and Cbl-wt pcDNA3 plasmid as the template. PCR reactions and PCR conditions were as specified by the manufacturer’s instructions. After the amplification reaction, 2 µl DpnI was added to PCR products to digest methylated template DNA at 37°C for 5

35 minutes. The bacterial transformation reaction followed this digestion. First, 3 µl β- mercaptoethanol was added to 67.5 µl XL-10 Gold Bacteria (Agilent Technologies) and the mixture was incubated on ice for 5 minutes. Then, 5 µl DpnI-treated DNA was added to bacteria and the mixture was incubated on ice for 30 minutes. The mixture was heat- shocked at 42°C for 50 seconds and incubated on ice for 5 minutes. 500 µl pre-warmed LB media was added to the mixture and incubated at 37°C for 1 hour with constant shaking. Then 250 µl mixture was plated on LB plate supplemented with ampicillin and incubated overnight at 37°C. The next day, grown colonies were picked up individually and placed in 5 ml miniculture of 5 ml of LB supplemented with 5 µl ampicillin (net concentration equals 100 ng/ml) and grown overnight at 37°C with constant shaking. DNA isolation from the minicultures was performed using Qiagen Miniprep DNA Isolation Kit according to manufacturer’s instructions. The isolated DNAs were quantified and sent to the Nucleic Acid Shared Resources facility of The Ohio State University for sequencing.

The presence of the mutations of desired bases was verified using BLAST software (PubMed). The isolated DNAs harboring the desired mutations were used to transform competent JM109 cells. One colony was picked up and grown for mini- and maxi-culture (500 ml LB+500 µl Ampicillin). DNAs were isolated using Qiagen Plasmid Maxi Kit. The plasmids were qualitatively checked by BamHI digestion, quantified and also sent to the sequencing facility as specified above for the verification of the desired mutation.

2.2 Construction of Truncated Ubiquitin-Fusion Cbl Plasmids:

The constructs GFP-Cbl-wt-Ub, GFP-Cbl-Y371F-Ub and GFP-Cbl-N-Ub had previously been produced by Kathryn Stern, Ph.D. The construction of plasmids encoding the truncated ubiquitin-fusion Cbl proteins was performed by Kevin Henschel. The truncations of Cbl protein at residues 560, 520 and 480 were generated using GFP-Cbl-Y371F-Ub as the template plasmid. The procedure is summarized on Figure 2.1.

Y371F FW2 RV2

GFP Cbl Ub

FW1 RV1 EcoRI XbaI

Figure 2.1 The strategy in construction of truncated GFP-Cbl-Y371F-Ub plasmids

The procedure involved three PCR reactions [Ex Taq DNA Polymerase supplemented with Ex Taq DNA Buffer and dNTPs (RR001A, Takara)] in which the third reaction used the products of the first two PCR reactions as the templates. As depicted in the figure, the first PCR reaction involved amplification of the Cbl fragment starting from EcoRI restriction site until the desired Cbl truncation residue, plus the codons for several ubiquitin amino acids beginning with residue 2. A single sense primer served as the forward primer for all reaction 1 amplifications. It incorporated an EcoRI site into the 5’-end of the PCR product (FW1). Distinct antisense primers were necessary for annealing at the 3’-end, as each incorporated sequences complementary to the initial codons of ubiquitin plus the C- terminal limit of the truncated Cbl protein (RV1). The designed primers are displayed below.

FW1: 5’-GCG GAA TTC TCA GCC TAG GCG-3’

RV1-560: 5’-TAA CGT CTT GAC GAA GAT CTG AGG TCG GGA TTC TGC TCC-3’

RV1-520: 5’-TAA CGT CTT GAC GAA GAT CTG AGA AGC AGT TCC AAG AGC-3’

RV1-480: 5’-TAA CGT CTT GAC GAA GAT CTG CCG TTC CAC CTT GGC ACC-3’

The second PCR reaction for each construct involved a sense primer incorporating sequences from the C-terminal limit of the truncated Cbl protein plus the initial codons for ubiquitin (FW2). A single antisense primer was used for all second PCR reactions. It incorporated coding sequences for an SpeI site, two stop codons and the 3’ codons for ubiquitin DNA (RV2). The designed primers are displayed below.

FW2-560: 5’-GGA GCA GAA TCC CGA CCT CAG ATC TTC GTC AAG ACG-3’

FW2-520: 5’-CTG CTC TTG GAA CTG CTT CTC AGA TCT TCG TCA AGA CG-3’

FW2-480: 5’-CTG GTG CCA AGG TGG AAC GGC AGA TCT TCG TCA AGA CG-3’

RV2: 5’-GCG ACT AGT TTA CTA ACC ACC TCT TAG TCT TAA GAC-3’

The purified products of each pair of first and second PCR reactions described above had overlapping sequences constituting the junction region of the 3’ Cbl truncation limit and the initial codons for ubiquitin. Therefore, disruption of DNA duplexes by high temperature heating in a third PCR reaction generated single-stranded DNAs that could anneal to one another and act as primers for further extension.

The products of reactions I and II were run on a 2% agarose gel and purified from the gel by using GeneClean Kit II (MP Biomedicals). The appropriate pairs of purified PCR products were combined for the third PCR reaction. The products of the third reaction were run on a 2% agarose gel and purified using GeneClean Kit II.

For cloning, the parental GFP-Cbl-Y371F-Ub vector and each purified PCR product from the third reaction were digested with EcoRI and SpeI, run on a 2% agarose gel, purified from the gel using GeneClean Kit II and ligated using T4 DNA Ligase (Invitrogen). The ligation mixtures were used for the bacterial transformation. Isolated colonies were cultured and then extracted for purification of the recombinant plasmids. Each DNA was characterized by restriction endonuclease mapping and subsequent DNA sequencing.

2.2.1 Mutagenesis of Cbl-Y371F-1-520-Ub Plasmid

P494 and P495 residues in Cbl-Y371F-1-520-Ub plasmid were to be mutated to alanine. To introduce these two mutations into a plasmid, primers were obtained from IDT. The sequences of the primers are indicated below. Codons above the line represent the wild- type codons.

P494A, P495A: CCC-CCG FW: 5’-CCA-CAA-GCT-TCC-CTT-GCC-GCG-GTG-CCA-CCA-CGA-CTT-3’

RV: 5’-AAG-TCG-TGG-TGG-CAC-CGC-GGC-AAG-GGA-AGC-TTG-TGG-3’

Cbl-Y371F-1-520-Ub plasmid was used as the template plasmid in the mutagenesis reaction. Please refer to the “Mutagenesis Protocol” section depicted above for the production of the double-alanine harboring plasmid.

2.3 Cell Lines, Transfections, EGF Stimulation and EGFR Degradation Assay:

2.3.1 Propagation of HEK 293 Cell Line:

HEK 293 cell line was maintained in DMEM media supplemented with 10% FBS, 20 mM HEPES, 1mM sodium pyruvate and 0.1 mM non-essential amino acids. Cell cultures were grown in an incubator at 37°C with 5% CO2. Starvation media used in the experiments included 0.5% FBS with all the other non-serum constituents specified above.

2.3.2 Transfection of HEK 293 Cell Line

Cells were seeded onto respective plates 24 hours before transfection. On transfection day, 7 ml fresh complete DMEM media were placed 2-4 hours prior to transfection. Lipofectamine 2000 (Life Technologies) was used for HEK 293 cell line transfection. 0.5 µg EGFR plasmid per 10-cm plate was added to 1.5 ml OPTI-MEM (Life Technologies) transfection media in 15-ml polypropylene tubes. 3 µg GFP-only or 4 µg Cbl-wild type or 4 µg Cbl-mutant protein expressing plasmid DNAs were added to the respective polypropylene tubes and incubated for 5 minutes at ambient temperature. On the other hand, 45 ml Lipofectamine 2000 reagent per 10 cm plate was added to 1.5 ml OPTI-MEM transfection media and incubated for 5 minutes at ambient temperature. 1.5 Lipofectamine

2000 mixture was added to the each polypropylene tube of DNA mixtures. The Lipofectamine-DNA mixtures were incubated for 20 minutes at ambient temperature. Then, the mixtures were added to the respective 10-cm plates.

Each transfected plate of cells was split into 3 10-cm plates on the day following transfection, in order to generate matched cultures for time course of EGF stimulation experiments.

2.3.3 EGF Stimulation and Harvesting of Cells

2 days post transfection, cells were serum starved for 4 hours with 4 ml starvation media. Then, the cells were stimulated with EGF at final concentration of 100 ng/ml at 37°C. This dose of EGF is biologically relevant and sufficient to saturate all receptors at the surface of serum-starved cells. For each set, one plate was left unstimulated (0 minute timepoint). Other plates were stimulated for 10 minutes or 90 minutes with EGF. For harvest, all plates were placed on ice to arrest protein trafficking and to minimize changes in receptor levels and modification. Then, the media of cells were aspirated. The cells were washed with 4 ml PBS, which was then removed. To harvest cellular proteins , each plate received 350 µl Triton X-100 Lysis Buffer (composed of 0.5% Triton X-100, 150 mM NaCl and 50 mM

Tris pH 7.5), supplemented with 1 mM PMSF, 10 mM NaF, 1 mM Na3VO4, 1% aprotinin and 1% protease inhibitor cocktail. The plate contents were collected in 1.5-ml microcentrifuge tubes, shaken at 4°C for 30 minutes for complete lysis, and then centrifuged at 4°C for 10 minutes to precipitate cell debris. The supernatants were then transferred into fresh 1.5-ml microcentrifuge tubes. Proteins were quantified using BioRad Protein Assay Dye Reagent Concentrate (BioRad Laboratories). BSA protein was used as the standard in protein quantification.

2.3.4 Antibodies

The primary antibodies used in immunoblotting experiments included mouse monoclonal anti-EGFR Ab (H9B4, Neomarker), rabbit polyclonal anti-GFP Ab (ab-290, Abcam), mouse monoclonal anti-gamma-tubulin Ab and mouse monoclonal anti-Ub Ab (VU-101,

LifeSensor). The secondary reagents used in immunoblotting experiments included HRP- conjugated protein A and HRP-conjugated rabbit polyclonal anti-mouse IgG. The antibodies used in immunoprecipitation experiments were mouse monoclonal anti-EGFR (4 µg/ml, sc-120, Santa Cruz Biotechnology) and rabbit polyclonal anti-GFP Ab (ab-290, Abcam).

2.3.5 Immunoprecipitation and Immunoblotting:

100 µg of whole cell lysate protein was used for immunoblotting. The proteins were aliquotted into 70 µl of 2X sample buffer and stored at -20°C until electrophoresis through acrylamide gels.

For immunoprecipitations, 1 mg lysate protein was incubated with mouse monoclonal EGFR antibody (sc-120) at a final amount of 4 µg and was shaken for overnight at 4°C. On the following day, BSA-blocked protein A Sepharose beads (GE Healthcare) were rinsed, resuspended in complete lysis buffer at 1:1 (v/v) ratio, and added to the immunoprecipitation tubes. 60 µl of bead suspension was used for each reaction. The immunoprecipitates with beads were shaken for at least 4 hours at 4°C. Then, the samples were washed and centrifuged briefly at 13000 rpm, 4 times, with 1 ml supplemented Triton- X 100 lysis buffer each time. The final washed bead pellet was transferred to a fresh 1.5- ml microcentrifuge tube. After this, 70 µl 2X sample buffer was added to each pellet and the samples were stored at -20°C until electrophoresis through acrylamide gels.

For electrophoresis, whole cell lysates and EGFR-immunoprecipitates were pre-heated at 95°C for 5 minutes and then briefly centrifuged at 13000 rpm to pellet the beads from which the immunocomplexes had been eluted. The supernatants were loaded onto 8% SDS-polyacrylamide gels for protein resolution. The proteins on the gels were then transferred onto PVDF membrane (Immobilon, Millipore) with transfer cassettes immersed in CAPS buffer (10 mM CAPS, pH 11 [Sigma], 10% methanol, 0.01% SDS [BioRad Laboratories]) at 250 V for overnight.

On the following day, the membranes were removed from transfer cassettes, washed with distilled water 3 times for 10 minutes and 1 time with TBS-T (150 mM NaCl, 10 mM Tris, pH 8.0, 0.05% Tween-20 [Bio-Rad Laboratories]) for 10 minutes. Then, the membranes

41 were blocked with 2% gelatin (BioRad Laboratories) with 0.025% Na azide in TBS-T for 1 hour and then washed with TBS-T for 10 minutes. Afterwards, the membranes were incubated with the primary antibody for 4 hours. Membranes were then washed with TBS- T 3 times for 10 minutes. Membranes were incubated with respective secondary reagents for 1 hour followed by 3 washes with TBS-T for 10 minutes. The membranes were finally treated with Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer Life Sciences) for 2 minutes and placed into cassettes. The proteins were detected using film exposures (GE Amersham). For reprobing, membranes were stripped with stripping solution for 10 minutes at 56°C, followed by extensive washes with distilled water and TBS-T. Then, they were reprobed with another primary antibody.

Ubiquitin detection required some pre-treatments before anti-Ub antibody incubation. Membranes were first washed with distilled water and then treated with 0.5% glutaraldehyde solution for 20 minutes at ambient temperature. Membranes were washed with PBS 3 times for 10 minutes and then were blocked with 5% milk for 30 minutes at ambient temperature. They were extensively washed with distilled water to remove residual blocking solution and then incubated with anti-Ub antibody for overnight at 4°C. The same procedure for chemiluminescent detection described above was used for these experiments.

CHAPTER 3 TESTS TO DETERMINE WHETHER CBL DIMER INTERFACE MUTANTS ARE COMPROMISED IN THEIR ABILITY TO ENHANCE EGFR DEGRADATION

Previous studies in the Lill laboratory had demonstrated that the Cbl RING finger tail residues V431 and F434 are critical for Cbl-mediated EGFR degradation. We subsequently hypothesized that the residues were important because they mapped to a putative interface between interacting Cbl molecules. To test this hypothesis, I analyzed a newly generated panel of full-length Cbl mutants carrying single amino acid substitutions of residues that lay at the interface of the putative Cbl dimer. Residues mapping to either side of the interface were studied for their impact on EGFR degradation. Statistical analysis was performed on data from three independent experiments to determine whether the behavior of any mutant differed significantly from that of the wild-type Cbl protein. The results are shown here.

3.1. Mutagenesis of c-Cbl Putative Dimer Interface Residues:

The functional importance of c-Cbl residues localized to the asymmetric c-Cbl dimer interface was analyzed in degradation assays as described in Materials and Methods. Because of the number of the mutants to be analyzed, they were grouped into three sets to make the experiments manageable. The mutants studied are listed below.

Set 1: Set 2: Set 3:

1) D52A 1) Q409A 1) H286A

2) D52N 2) Q409E 2) R333A

3) K58A 3) E410A 3) Q345A

4) K58E 4) E410Q 4) N346A

5) E394A 5) K424A 5) D348A

The residues on c-Cbl putative dimer interface were mutated to either alanine or a residue of a changed charge/polarity using QuikChange Site Directed Mutagenesis Kit (Stratagene) as described above in subsection 2.1.1. The amino acid changes and their putative structural impact within the dimer follow.

As seen in Figure 1.9, H286 residue putatively interacts with K58 residue via its oxygen atom in its backbone. It interacts with D52 residue via its nitrogen atom in its side chain. K58 putatively interacts with H286A via its nitrogen atom in its side chain. D52 interacts with H286 via its oxygen atom in its side chain. Q409 interacts with R333 via its oxygen atom; with D348 via its nitrogen atom. Both interactions are mediated by side chain of Q409. The interaction between R333 and E410 are putatively mediated by their side chains. It requires the nitrogen atom of R333 and the oxygen atom of E410. The interaction between D348 and K424 are putatively mediated by backbone regions of the residues; nitrogen atom of D348 and oxygen atom of K424. So, any mutations of these residues are not expected to have an impact. There is a hydrophobic interaction between Q345 and E394. N346 and T424 putatively interact via their backbones; each oxygen and nitrogen atom in one backbone interacts with the oxygen and nitrogen atom of the other residue.

D52 (set 1) and H286 (set 3) mutations disrupted hydrogen bonds between the side chains of these residues. K58 (set 1) mutations disrupted its hydrogen bond with the backbone of H286 residue. Q409 (set 2) mutations disrupted its hydrogen bonds with the backbones of R333 (set 3) and D348 (set 3) and vice versa. R333 (Set 3) mutation also disrupted its

44 backbone interaction of E410 (set 2) and vice versa [Figure 1.9 (A)]. D348 (set 3) and K424 (set 2) mutations did not change the hydrogen bonds between the residues since the hydrogen bonds are between the backbones of these residues [Figure 1.9 (B)]. N346 (set 3) mutation did not change its interaction with T426 since the hydrogen bonds are between the backbones of these residues. Q345 (set 3) and E394 (set 1) mutations disrupted the hydrophobic interactions between these residues [Figure 1.9 (C)].

3.2. Degradation Assay of Putative c-Cbl Dimer Interface Mutants:

The procedure followed for this experiment was summarized above in “Materials and Methods” under the section 2.3 “Cell Lines, Transfections, EGF Stimulation and EGFR Degradation Assay”. Below in Figure 3.1, degradation assay results are displayed for each set of mutants.

Each degradation assay includes a positive control and a negative control. Wild-type GFP- Cbl protein serve as the positive control, on the other hand, GFP serve as the negative control to see the effect of endogenous Cbl in our experiments. Since HEK 293 cells has almost no EGFR protein, these cells were transfected with EGFR-expressing along with each of the GFP-Cbl harboring plasmid (GFP only for the negative control). This assay is performed by overexpressing the Cbl protein. This is a necessary approach because endogenous Cbl activity must be overcome by the ectopic expression of wild-type or mutant Cbl proteins. The EGFR levels 90 min post-stimulation are compared with EGFR levels of unstimulated cells (0 min). In negative control cells, EGFR degradation level is in a low extent, on the other hand, positive control cells degrade EGFR in a high extent. GFP blot verifies the equal expression of ectopic constructs. Tubulin blot serve as the loading control of proteins.

Two bands of EGFR were observed 90 min after EGF stimulation. We suspect that the fast migrating bands represent aither a degradation product or non-activated EGFRs. Therefore, slow migrating EGFRs (upper band) were taken into account for the analysis and the quantification.

Figure 3.1 (a, c, e) reveal that the all the analyzed mutants (Set 1, Set 2 and Set 3) degraded EGFR as efficiently as wild-type Cbl. The experiments were repeated at least three times and the percentage of EGFR remaining for each of the constructs was quantified using ImageJ and ImageQuant software. The quantification of the immunoblotting results of three different experiments revealed that there is no significant difference in terms of EGFR degradation for the analyzed mutants [Figure 3.1(b, d)]. GFP expression analysis confirmed the similar level expression of the constructs. Tubulin blot was used as the loading control.

Immunoprecipitation of the lysates with anti-EGFR antibody followed by anti-EGFR immunoblotting further supported the conclusions drawn by the analysis of whole cell lysates. The immunoprecipitation results are displayed on Figure 3.2 (a, b, c).

Figure 3.1. Impact of Cbl putative dimer interface mutants on EGFR degradation. (a, c, e) HEK 293 cells were transfected with pAlterMAX-EGF-R (0.05 µg per 10 cm dish) and either GFP or the indicated GFP-Cbl expression constructs (4 µg per 10 cm dish). The cells were stimulated with EGF over the time course shown. 100 μg of lysate protein from each sample were resolved by SDS-PAGE and transferred to PVDF membranes for sequential immunoblotting (I.B.) (n=3 for Set 1 and Set 2; n=1 for Set 3). (b, d) Data obtained in three independent experimental repeats of the Figure 3.1 degradation assay were used for statistical analysis. In each assay, the amount of degradation induced was expressed as the percentage of EGFR remaining after 90 minutes. Error bars represent standard deviations (S.D.) of the means. The activities of all mutant proteins were statistically different from the GFP control at a confidence interval of 95% (+/- 2 S.D.). Figure 3.1 (a-e) are showed on pages 48-50.

Figure 3.1

a NT GFP WT D52A D52N K58A K58E E394A T P EE 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’

EGFR R

GFP P

Tubulin

b 90

%EGFR %EGFR Remaining 30

0 GFP-2X WT D52A D52N K58A K58E E394A

Figure 3.1 continued

Figure 3.1

c NT GFP WT Q409A Q409E E410A E410Q K424A T Q 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’

EGFR

GFP P

Tubulin

d 80

30 %EGFR Remaining 20

0 GFP-2X WT Q409A Q409E E410A E410Q K424A

Figure 3.1 continued

Figure 3.1

e NT GFP WT H286A R333A Q345A N346A D348A T P A 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’

EGFR R

GFP P

Tubulin

Figure 3.1 (b) reveals that there is a variation between the values of each mutant when compared to GFP and Cbl-wt. Therefore, a detailed statistical analysis was needed, which will be explained in detail below in section 3.1.1. Figure 3.1 (d) shows that the set 2 mutants functioned as wild-type protein.

3.2.1 Statistical Analysis for Set 1:

Due to the highly variable results in different experiments in which Set 1 mutant Cbl plasmids were used, a detailed statistical analysis was performed with the raw data using SPSS statistics software (IBM). Figure 3.2 illustrates a box and whiskers plot with the raw data from three independent experiments of Set 1 mutants. Then, an asymptotic Mann- Whitney U test was performed to determine whether the tested mutants statistically were different from either GFP or wild-type constructs with a confidence interval of 95%. Each mutant value was compared with GFP or wild-type value separately. Table 3.1 shows the p-values calculated when the Mann-Whitney test was performed.

Figure 3.2 Box and whiskers plot of degradation assays of Set 1 mutants. Percentage of EGFR remaining after 90 minutes of EGF stimulation is shown for each of the three experiments. The end of each whisker represent the minimum and maximum value. The median value is represented in the boxes as the horizontal line.

Tested Mutants p values vs GFP p values vs WT

D52A .127 .827

D52N .050 .050

K58A .050 .827

K58E .050 .127

E394A .050 .513

Table 3.1 Mann-Whitney U test of each Set 1 mutant. The raw data from three independent experiments were compared pairwise to either GFP or WT plasmids. p values above .05 were regarded as no statistical difference between the samples.

As shown above in Table 3.1, the EGFR degradation levels of Cbl mutants K58A, K58E and E394A are not statistically different from WT protein, so it can be concluded that these mutants functioned normally. EGFR degradation level of D52A mutant is not significantly different from both GFP and WT, so any conclusion cannot be drawn for this mutant. D52N mutant EGFR degradation level is statistically significant from both GFP and WT, so this mutant functioned as a compromised protein, the values are intermediate between GFP and WT protein.

3.3 Association of Putative c-Cbl Interface Mutants with EGFR upon EGF Stimulation:

Immunoprecipitation of the lysates with anti-EGFR antibody followed by anti-GFP immunoblotting showed that all the analyzed mutants associated with EGFR after 10 minutes of EGF stimulation as efficiently as did wild-type c-Cbl. The association decreased by time, as observed after 90 minutes of EGF stimulation, as expected, for all the mutants [Figure 3.3 (a, b, c)] (page 46).

3.4 Impact of Putative c-Cbl Interface Mutants on EGFR Ubiquitination upon EGF Stimulation:

Immunoprecipitation of the lysates with anti-EGFR antibody followed by anti-ubiquitin antibody revealed that the ubiquitination patterns did not differ among the analyzed mutants from the wild-type Cbl. As expected, 10 minutes after EGF stimulation, EGFRs were ubiquitinated by the mutant Cbls as efficiently as wild-type Cbl. The ubiquitination levels were decreased by time, as observed 90 minutes after EGF stimulation [Figure 3.4 (a, b)] (page 47). The results are from independent experiments. The analyses of sets 2 and 3 are shown. Anti-EGFR blotting of EGFR immunoprecipitates and anti-GFP blotting of whole cell lysates are also showed to reveal the quality of the experiment.

a GFP WT D52A D52N K58A K58E E394A EE 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ GFPP 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ EGFR

GFP P

b GFP WT Q409A Q409E E410A E410Q K424A

0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’

EGFR

GFP P

c GF P WT H286A R333A Q345A N346A D348A

0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’

EGFR R

GFP P

IP: EGFR

Figure 3.3 Immunooprecipitation analysis of the putative Cbl dimer interface mutants. (a, b, c) 1 mg of lysate protein was first immunoprecipitated by anti-EGFR antibody and then resolved by SDS-PAGE and transferred to PVDF membranes for sequential immunoblotting (I.B.).

a GFP WT Q409A Q409E E410A E410Q K424A 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ IP: EGFR GFP EGFR

Lys: GFP

b GFP WT H286A R333A Q345A N346A D348A

0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ 0’ 10’ 90’ IP: EGFR

EGFR

Lys: GFP

Figure 3.4 EGFR ubiquitination analysis of the putative Cbl dimer interface mutants (a, b) 1 mg of lysate protein was first immunoprecipitated by anti-EGFR antibody and then resolved by SDS-PAGE and transferred to PVDF membranes for sequential immunoblotting (I.B.). GFP blot is of 100 µg whole cell lysate.

3.5. Summary of Results:

All tested mutants could associate with EGFR and ubiquitinate it similar to Cbl wild-type protein. Interestingly, even mutations that were predicted to induce major structural disruption at the putative dimer interface failed to affect the degradation of EGFR. Detailed statistical analysis revealed that any statistical conclusion cannot be drawn for the Cbl- D52A mutant.Cbl-D52N mutant functioned differently from both GFP and wild-type Cbl, as an intermediate. However, since H286, D52-interacting residue, functioned as wild-type, pointing out the fact that the intermediate behavior of D52N mutant is not biologically significant. All the other tested mutants degraded EGFR as efficiently as wild-tyoe Cbl.

Based on these data, I concluded that the published 2000 crystal structure from Zhang et al. did not largely reflect the active form of Cbl dimers. In the discussion of this dissertation, I explain how my results support data presented in a 2012 publication by Dou et al., which demonstrate that the 2000 crystal failed to reflect the active conformation of Cbl and why RING finger tail residues V431 and F434 are critical for its function.

CHAPTER 4 INVESTIGATION OF THE ROLE OF GRB2 IN A NOVEL INHIBITORY ACTIVITY THAT MAPS BETWEEN CBL AMINO ACIDS 358-480

4.1. Degradation Assay of Grb-2 Binding Mutant of c-Cbl:

Lill laboratory investigated the role of ubiquitin (found in EGFR-GFP complexes but not directly associated with EGFR) in EGFR degradation. Ubiquitin-fused GFP-Cbl constructs were generated by Kathryn Clausen and Kevin Henschel in the laboratory as described in section 2.2.

Previous experiments conducted in our laboratory showed that a ubiquitin-fused truncation mutant of Cbl-N (1-357-Ub) enhanced EGFR degradation after 120 min of EGF stimulation. This result was unexpected, as Cbl-N without ubiquitin acts to suppress EGFR degradation. An additional analysis of other Cbl-Ub fusions revealed that EGFR degradation is compromised when either Cbl-Y371F-Ub or Cbl-Y371F is overexpressed (Clausen et al., submitted). This result was expected, as neither protein possesses ubiquitin ligase activity. The combined results raised a new question: why could Cbl-N- Ub enhance EGFR degradation while Y371F-Ub could not, despite the fact that both Cbl proteins lack Ub ligase activity?

In the aforementioned experiments, Cbl-wt-Ub construct served as the positive control. Expectedly, EGFRs were efficiently degraded when this protein is overexpressed.

One potential explanation is that sequences present in the full-length Y371F Cbl protein but absent from Cbl-N have a previously undetected activity, which becomes apparent only when Cbl’s E3 activity is lost. What must be the nature of this activity? It must be the suppression of EGFR degradation, as the presence of additional amino acids in the

Y371F fusion protein abrogates the unexpected EGFR degradation ability of the smaller Cbl-N-Ub fusion protein. The inability of Cbl-Y371F-Ub to enhance EGFR degradation as does wt-Cbl-Ub suggests that Cbl’s ubiquitin ligase activity normally masks the inhibitory function demonstrated by Y371F-Ub.

To better define the precise region which is responsible for the unmasked inhibitory activity, plasmids encoding truncation mutants which contain the Y371F mutation plus fused ubiquitin were constructed in our laboratory (Kevin Henschel and Nancy L. Lill). Initial experiments suggested that the inhibitory activity was retained by Cbl 1-520-Ub but lost from Cbl 1-480-Ub (not shown). The protein sequences between Cbl amino acids 480 and 520 include some proline-rich regions containing P-X-X-P motifs. Such motifs were reported by others to constitute consensus recruitment sites for the signaling and trafficking regulatory protein Grb2. Therefore, we hypothesized that Grb2, which could potentially bind this region of Cbl via its SH3 domain, may be involved in the novel inhibitory c-Cbl function.

To investigate Grb2’s role in the putative inhibitory activity, amino acids 494 and 495 were mutated jointly to alanine in the Cbl 1-520-Y371F-Ub construct. This construct were designated as 3X-MUT, reflecting its triple mutations: Truncation (1-520), Y371F mutation, and P(494,495)A mutations. The mutation of both prolines jointly disrupts critical residues in adjacent P-X-X-P motifs that individually are consensus Grb2-binding motifs.

The degradation assay was performed for the 3X-MUT along with the ubiquitin-fused Y371F-harboring truncation constructs. The assay result is displayed below in Figure 4.1. Both whole cell lysate and EGFR immunoprecipitation analysis revealed that 3X-MUT had impaired EGFR degradation, just like its non-proline mutated counterpart (Cbl-Y371F 1- 520-Ub). This result showed that binding of Grb2 or any other Cbl-interacting protein to the adjacent P-X-X-P consensus binding motifs, which incorporated prolines 494 and 495, is unlikely to play a primary role in the inhibitory activity.

In our assays, in correlation with the early experiments, Cbl-wt-Ub protein functioned like wild-type Cbl protein. Cbl-N-Ub protein degraded EGFR efficiently, in conjunction with the previous data. On the other hand, both full length Y371F-Ub and the truncated proteins 1-

560, 1-520 and 1-480 Y371F-Ub proteins have compromised EGFR degradation. Besides the last construct, our data is consistent with the previous observations.

Y371F Y371F Y371F WT WT-Ub Y371F-Ub 1-560-Ub 1-520-Ub 3X-MUT 1-480-Ub 1-357-Ub

0’ 10’ 120’ 0’ 10’ 120’ 0’ 10’ 120’ 0’ 10’120’ 0’ 10’ 120’ 0’ 10’ 120’ 0’ 10’ 120’ 0’ 10’ 120’

EGFR R

GFP P

IP:EGFR

IB:EGFR

Figure 4.1 Impact of mutation of Grb2 binding site on EGFR degradation. HEK 293 cells were transfected with pAlterMAX-EGF-R (0.05 µg per 10 cm dish) and each of the indicated truncated Cbl-Y371F constructs (4 µg per 10 cm dish). The cells were stimulated with EGF over the time course shown. 100 μg of lysate protein from each sample were resolved by SDS-PAGE and transferred to PVDF membranes for sequential immunoblotting (I.B.). 1 mg of lysate protein was immunoprecipitated by anti-EGFR antibody, resolved by SDS-PAGE and transferred to PVDF membranes for sequential immunoblotting (I.B.).

CHAPTER 5 DISCUSSION

5.1 The Role of Putative Cbl Dimer Interface Mutants in Cbl Function:

Signal transduction mechanisms must be tightly regulated for the homeostasis of an organism. ErbB receptors play a very important role in relaying extracellular signals to the interior of a cell, thereby inducing desired cellular responses like differentiation and proliferation. Cbl protein is a crucial regulator of the prototype member of the ErbB receptor family, the epidermal growth factor receptor (EGFR). The results of my dissertation research have extended our understanding of Cbl, beyond the fact that it is a RING finger- type E3 ubiquitin ligase which associates with EGFR, ubiquitinates it, and destines the receptor for degradation by acting on EGFR-bearing early endosomes to enhance their fusion.

In Chapter III, I have summarized the results of my studies testing whether a specific putative Cbl dimer plays a role in the protein’s ability to target EGFR for degradation. My cumulative data indicate that this structure does not explain Cbl’s activity. Rather, my results support a different model reported by Dou et al. (2012). In their study, those authors solved the structure of a Cbl protein that was expressed in bacteria but was post- translationally modified by tyrosine phosphorylation. The resulting structure revealed that a major conformational change within Cbl occurs when the protein is phosphorylated on Tyr 371. The RING domain of the modified Cbl swings into a new position, rendering the RING finger capable of binding to its partner E2. Importantly, maintenance of the active conformation requires the integrity of the RING finger tail amino acids V431 and F434. Dou and colleagues used the published results from the Lill laboratory (Visser Smit et al., 2009) to predict the impact of an F434A mutation on the active conformation of Cbl. They reported that mutation of this residue would destabilize intramolecular interactions crucial

59 for the formation of the active structure (Figure 5.1-Dou et al., 2012, reproduced with the author’s permission).

Like the results of Dou et al., my data testing the Cbl dimer interface of unmodified Cbl (based on the 2000 structure of Zhang and coworkers) indicate that the published structure of a non-phosphorylated Cbl protein is not useful to explain the molecular basis for RING finger tail-dependent Cbl activities. Specifically, interfering with interactions between residues on either side of the putative Cbl dimerization interface failed to change Cbl’s activity in enhancing EGFR degradation (Chapter III).

The statistical analysis of EGFR degradation assay reveals that besides D52 mutants, all the tested mutants functioned like wild-type protein, in terms of their impact on EGFR degradation. D52A mutant cannot be statistically interpreted. D52N differs from both GFP and wild-type Cbl. But, due to lack of abnormality of its putative interacting partner, H286A, in terms of EGFR degradation, this unique function of D52N is not regarded as biologically important. All the tested mutants were associated with and ubiquitinated EGFR as efficiently as wild-type 10 minutes after EGF stimulation.

To determine whether these tested residues play an important role in Cbl dimerization through a different mechanism, dimerization studies can be performed in which the mutant constructs are differentially tagged (such as with HA and GFP), as in Figure 1.8. Immunoprecipitation/coprecipitation studies using HA-specific versus GFP-specific antibodies will allow the detection by immunoblotting of the alternatively tagged molecule that coprecipitates with the tagged Cbl directly recognized by the precipitating antibody.

Downregulation assays could be performed to determine if these residues are crucial for the internalization of EGFR upon EGF stimulation. Costaining in immunofluorescence studies, to detect both EGFR and GFP-tagged mutant Cbl proteins would reveal any differences in EGFR trafficking.

5.2. Investigation of Role of Grb2 in Regulation of Putative Cbl C-terminal Inhibitory Activity;

To test whether ubiquitin present in EGFR-Cbl complexes is a sufficient signal for EGFR degradation, plasmids encoding Cbl fused to ubiquitin at their C-termini were constructed in our laboratory. EGFR-Ub constructs that cannot bind to Cbl were not used because their half-lives are too long to reflect the impact of fused ubiquitin on the receptor. Furthermore, the use of Cbl-ubiquitin fusions allowed us to look at ligand-induced events, which could not be evaluated within EGFR-Ub proteins.

Previous studies in Lill laboratory showed that a Cbl-N-Ub (1-357-Ub) fusion protein could induce EGFR degradation as efficiently as could Cbl-wt-Ub (unpublished). This result presented some challenges for interpretation, in light of the published report that Cbl appears to enhance EGFR degradation by ubiquitinating Hrs on EGFR-positive early endosomes (Visser Smit et al., 2009). Specifically, Cbl-N-Ub can bind to EGFR and move with it to early endosomes; however, the recombinant Cbl protein lacks E3 activity and therefore could not possibly effect Hrs ubiquitination. Does this prove that Hrs ubiqutination actually is dispensable for Cbl-mediated degradative targeting of EGFRs in cells expressing wild-type receptor and Cbl? I believe that it is not. One reason for this is that ubiquitin fused to the recombinant Y371F-Cbl proteins can bind to the UIMs of ESCRT machinery other than the UIM of Hrs. Bypassing the Hrs checkpoint by binding directly to the UIM of TSG101, downstream in the trafficking pathway, would eliminate the need for Y371F-Cbl-ubiquitin fusion proteins to ubiquitinate Hrs.

If the bypass were sufficient for any Cbl-Ub/EGFR complex to move on unimpeded to lysosomes for degradation, all of the Y371F-Cbl-Ub fusions would have enhanced EGFR degradation. However, they did not. Only the fusion protein containing Cbl residues truncated beyond amino acid 480 could target EGFR for degradation. Based on this result, I propose a model in which endogenous wild-type Cbl binds EGFR, is internalized onto early endosomes with it, then experiences the Hrs checkpoint. If Cbl retains E3 activity at this site, it can ubiquitinate Hrs and regulate its phosphorylation/dephosphorylation. This permits endosome fusion and passes the ubiquitinated EGFR/Cbl complexes to the next UIM motif in the ESCRT pathway: that of TSG-101. At this stage, the E3 function of Cbl neutralizes the novel inhibitory activity of Cbl which requires amino acids mapping to

61 residues 358-479 (Chapter IV). In my model, the inhibitory activity must lie downstream of the Hrs checkpoint because my full panel of Y371F-Cbl-Ub fusion proteins should bind to the UIMs of both Hrs and TSG101, thereby allowing all recombinant complexes to localize at the later trafficking checkpoint; however, only the Cbl-N-Ub-associated receptors were able to proceed from the TSG-101 checkpoint to the lysosome in my experiments (Chapter IV).

Preliminary experiments in the Lill laboratory had suggested that the inhibitory motif required Cbl amino acid residues between 480 and 520. My results disproved this. Instead, I have shown that Cbl amino acids between residues 358 and 479 are essential for the inhibitory activity. Further experiments will be required for finer mapping of the function. Those experiments are beyond the scope of this dissertation. However, one possibility is that the inhibitory activity is caused by the RF tail-mediated Cbl homodimers. In our studies, Cbl oligomerization correlates with its E3 activity. At late stages of the endocytic pathway, it is desirable to disrupt Ub-UIM interactions so Cbl-EGFR complexes can move beyond UIM-controlled steps. Either dephosphorylation of EGFRs, with associated loss of Cbl phosphorylation and dimerization, or deubiquitination of the trafficking complexes could achieve this end. Interestingly, the loss of EGFR phosphorylation has been shown to correlate with loss of signaling and with degradation. It will be exciting to see whether the mutations that alter Cbl dimerizaltion kinetics also impair EGFR trafficking at these late stages of endocytosis.

The question of whether Grb2 binding to Cbl contributes to the inhibitory activity also is not fully addressed here. It is important to note that even if there is no difference in the amount of associated with EGFR in complex with the various Cbl-Ub fusion proteins, this does not mean that Grb2 in those complexes is functionally equal. Post-translational modifications and other protein-protein interactions (such as with Sos) may be qualitatively different. This also is a subject for investigations beyond the scope of this dissertation.

In summary, the results presented here show that the 2000 crystal structure reported by Zhang et al. does not adequately explain the formation of Cbl dimers that are unmodified. Rather, the 2012 crystal structure reported by Dou et al. reflects the importance of RING finger tail residues in regulating EGFR ubiquitination and degradation. Additional results shown here reveal the existence of a novel inhibitory activity within Cbl that may regulate

62 the degradative trafficking of Cbl/EGFR complexes at sites downstream of Hrs. My data have revealed novel concepts about the mechanism through which Cbl regulates EGFR trafficking and signaling. This opens the door for new investigations by other researchers in the field.

Figure 5.1 The position of V431 and F434 amino acid residues in crystal structure of Y371-phosphorylated Cbl. The positions are based on the crystal structure depicted in the paper. Reproduced from Dou et al., 2012 with the permission of the author.

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