Loss of downregulation: a mechanism for oncogenic activation of receptor -derived oncoproteins.

Hayley Hoi Lam Mak

A thesis submitted to the Faculty of Graduate and Post-doctoral studies in the partial Fulfillment of the requirements for the degree of Master of Science

© Hayley Hoi Lam Mak, August 2006

Department of Biochemistry McGill University Montreal, Quebec, Canada Library and Bibliothèque et 1+1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'édition

395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada

Your file Votre référence ISBN: 978-0-494-32842-2 Our file Notre référence ISBN: 978-0-494-32842-2

NOTICE: AVIS: The author has granted a non­ L'auteur a accordé une licence non exclusive exclusive license allowing Library permettant à la Bibliothèque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l'Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans loan, distribute and sell th es es le monde, à des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, électronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats.

The author retains copyright L'auteur conserve la propriété du droit d'auteur ownership and moral rights in et des droits moraux qui protège cette thèse. this thesis. Neither the thesis Ni la thèse ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent être imprimés ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission.

ln compliance with the Canadian Conformément à la loi canadienne Privacy Act some supporting sur la protection de la vie privée, forms may have been removed quelques formulaires secondaires from this thesis. ont été enlevés de cette thèse.

While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. ••• Canada 11

Abstract Many human have been associated with the deregulation of receptor tyrosine kinases. This occurs through various molecular mechanisms, including amplification, translocation and point mutations sorne of which involve loss of negative regulatory signaIs. Negative regulation of RTKs involves their internalisation and subsequent degradation in the lysosome. RTK oncoproteins activated following chromosomal translocation are no longer transmembrane proteins and would be predicted to escape lysosomal degradation. In this study, we addressed wh ether the Tpr-Met oncogene, produced via chromosomal translocation, escapes downregulation thereby leading to its oncogenic activity. Unlike the Met receptor, Tpr-Met is localized to the cytoplasm and lacks the for Cbl, E3 ubiquitin . Ubiquitination of RTKs, including the Met receptor, target them for efficient degradation in the lysosome. To address whether the subcellular localization of Tpr-Met, and/or loss of its Cbl binding site, is important for its oncogenic activity, Tpr-Met variants, targeted to the plasma membrane with and without Cbl recruitment were examined. The presence of a Cbl binding site and ubiquitination of cytosolic Tpr-Met oncoproteins does not alter Tpr-Met transforming activity, or protein stability, and these proteins fail to enter the endocytic pathway. However membrane targeting allows Tpr-Met to enter the endocytic pathway and transformation and protein stability are decreased in a Cbl dependent manner. These data demonstrate that the oncogenic activity ofTpr-Met is in part dependent on its ability to escape normal down-regulatory mechanisms and provides a paradigm for many RTK­ derived oncoproteins activated following chromos omal translocation. III

Resumé Plusieurs cancers humains sont associés avec la dérégulation des récepteurs à activité tyrosine kinase (RTK). Ce phénomène peut se produire via plusieurs mécanismes moléculaires telles que l'amplification de gènes, la translocation chromosomique ainsi que des mutations ponctuelles qui sont impliquées dans la perte des signaux de régulation négative. L'internalisation des RTK suivie par leur dégradation dans les lysosomes fait partie de cette régulation négative. Les oncoprotéines dérivées de la translocation chromosomique des RTK (tel que Tpr-Met) ne sont plus des protéines transmembranaires et présentent la faculté d'échapper à la dégradation lysosomale. Dans cette étude, nous nous proposons de déterminer si l'activité oncogénique de Tpr-Met est due à son échappement aux mécanismes d'insensibilisation. A l'inverse du récepteur Met, Tpr-Met est localisé dans le cytoplasme et ne présente pas de site de liaison à Cbl, une d'ubiquitination de type E3. L'ubiquitination permet aux RTK, dont le récepteur Met, d'être dégradés efficacement dans les lysosomes. Afin d'évaluer si la localisation subcellulaire de Tpr-Met et/ou la perte de sites de liaison de Cbl sont importants pour son activité oncogénique, des mutants de Tpr-Met ciblés à la membrane plasmique avec ou sans un site de recrutement de Cbl ont été étudiés. La présence d'un site de liaison de Cbl et l'ubiquitination des oncoprotéines Tpr-Met cytoplasmiques ne modifient pas leur pouvoir transformant ou leur stabilité comparativement à Tpr-Met. En outre, ces protéines ne sont pas capables d'entrer dans la voie endocytaire. Par contre, le ciblage à la membrane permet à Tpr-Met d'entrer dans la voie endocytaire et son pouvoir transformant ainsi que sa stabilité sont diminués de façon dépendamment de la présence de CbI. Ces résultats démontrent que le pouvoir oncogénique de Tpr-Met est en partie dû à capacité d'échapper aux mécanismes normaux d'insensibilisation. Cette étude fournit donc un éventuel paradigme pour plusieurs autres oncoprotéines dérivés des RTK qui sont activés suite à une translocation chromosomique. IV

Acknowledgements

1 would like to thank Dr. Morag Park for her guidance and support throughout my studies. 1 am very grateful for having the opportunity to work in her lab and to take on this interesting project.

A special thankyou goes to Dr. Pascal Peschard and Dr. Lina Musallam for giving comments and suggestions. l've leamed a lot from you both. Thank you Lina and David Germain for translating my abstract into French and reading over my thesis.

1 am also grateful to Kelly Fathers and Stephanie Petkiewicz for their friendships over the past two years. Thank you for reading my thesis and giving me helpful suggestions. 1 also appreciate your help for the mice work.

A big thank you to Monica Naujokas, Dongmei Zuo, Tina Lin and Anie Monast for their technical assistance. 1 will miss tuming to you guys for a chat.

Thank you to other members of the Park lab for making my two years a little easier. You have made me feel very welcome in the lab and 1 will cherish the friendships that l've made. Thanks for aU the encouragement and making me laugh whenever 1 am down.

1 would also like aIl my family and friends for always being there whenever 1 need help and need advice.

Financial support during these studies were provided by MURC Research Institute and CIHR Consortium. v

Preface

This thesis is a manuscript-based thesis. It contains one manuscript provisionally accepted, Oncogene. The thesis is organized into three chapters:

1) a general introduction and literature review

2) manuscript, which inc1udes the abstract, introduction, materials and methods, results, discussion and references.

3) a general discussion. VI

Publication arising from the work of the thesis /----

1. Mak, H., Peschard, P., Naujokas, M.A., Lin, T., and Park, M. (2006). Oncogenic activation ofthe Met fusion protein, Tpr-Met, involves exclusion from the endocytic degradative pathway. Provisionally accepted, Oncogene.

Contribution to Authors

1. 1 performed - Coimmunoprecipitation of Cbl TKB with Tpr-Met variants - Colocalization studies with EEAI - Focus forming assays - Generated stable celllines ofTpr Met variants - Pulse Chase analysis - Tumorigenic assay

P. Peschard generated the cytoplasmic Tpr Met variants. M.A.Naujokas performed the soft agar assay. T. Lin performed the ubiquitination assay. VIl

Table of Contents

Abstract ...... ii Resume ...... iii Acknow ledgments ...... i V Preface ...... V Publications arising from work of the thesis ...... vi Table of contents ...... vii List of figures ...... ix

Chapter 1- Literature review

1. Introduction ...... l 2. Receptor tyrosine kinase ...... 1 3. RTK family and activation ...... 1 4. The Met receptor ...... 4 5. Met signaling ...... 6 6. Normal regulation ofRTKs ...... 7 7. Ubiquitination ...... 8 8. E3 ubiquitin ligases ...... 10 9. Clathrin mediated endocytosis ...... 12 10.Endocytic pathway ...... 13 Il.RTK receptor endocytosis...... 14 12.0ther mechanisms involved in degradation of Met...... 15 13.Mechanisms of deregulation ...... 15 14.RTK-derived oncoproteins ...... 18 Abbreviations ...... 20 References...... 22

Chapter 2- Mak et al. to be submitted

1. Abstract ...... 2 2. Introduction ...... , ...... 3 3. Results ...... 6 4. Discussion ...... Il 5. Materials and Methods ...... 15 6. Acknowledgements ...... 19 7. References ...... 20 8. Figures and Figure legends ...... 22 V111

8. Supplemental Data ...... 36

Chapter 3- General discussion

1. Insertion of the juxtamembrane domain of the Met receptor into Tpr-Met is required for itsubiquitination...... 2 2. Membrane targeting ofTpr-Met is sufficient for intemalization...... 3 3. Tac Tpr-Met Juxta YI003 has a shorter halflife ...... 4 4. Membrane targeting ofTpr-Met and ubiquitination is required to reduce the transforrning ability ofTpr-Met...... 4 5. Proposed Mechanism...... 6 6. Summary and Perspectives...... 7 7. References...... 9 IX

List of Figures

Chapter 1: Literature Review

Figure 1. Ruman receptor tyrosine kinases ...... 3 Figure 2. The Met receptor and the human oncogene, Tpr-Met ...... 4 Figure 3. Met signaling ...... 6 Figure 4. The ubiquitination cascade ...... 7 Figure 5. Ubiquitin modifications ...... 9 Figure 6. Different ubiquitin protein ligases ...... 11 Figure 7. Structural organization ofhuman CbI ubiquitin-protein Iigases ...... 12 Figure 8. RTKs targeted for degradation through the endocytic pathway ...... 14 Figure 9. Mechanisms ofRTK deregulation ...... 15 Figure 10. RTK oncoproteins that have lost their ability to recruit Cbl in a TKB- dependent manner ...... 18

Chapter 2: Mak et al. Submitted.

Figure 1. Schematic diagram of the Met receptor, Tpr-Met and Tpr-Met variants ...... 11 Figure 2. Expression and association ofTpr-Met variants with Cbl TKB ...... 13 Figure 3. Ubiquitination ofTpr-Met variants ...... 15 Figure 4. Localization ofTpr-Met variants in EEA1 positive endosomes ...... 17 Figure 5. Stability ofTpr-Met variants ...... 19 Figure 6. Anchorage independent growth ofTpr-Met variants ...... 23 Figure 7. Tumorigenic capacity of membrane localized Tpr-Met variants ...... 25 Supplemental Figure 1. Localization ofTpr-Met variants in EEA1 positive endosomes.36

Chapter 3: General Discussion.

Figure 1. An illustration showing the proposed mechanism ...... 7 Literature review 1-1

Chapter 1

Literature Review Literature review 1-2

1. Introduction The development of multicellular organisms requires proper cell growth and cell division. Cell signaling is part of a complex system of communication that govems basic cellular activities and coordinates cell actions. The ability of cells to perceive and correctly respond to their microenvironment is the basis of development, tissue repair, and immunity as well as normal tissue homeostasis. Deregulation of cell signaIs may lead to uncontrolled growth, a characteristic of cancer development and many other hum an diseases. The cellular changes associated with cells allow them to multiply uncontrollably, invading other tissues either by direct growth into adjacent tissue or by implantation into distant sites by metastasis. Receptor tyrosine kinases (RTKs) [1] are cell surface receptors and are key players in maintaining normal physiological processes through their role in transmitting extracellular signaIs to within the cell.

2. Receptor Tyrosine Kinase

Many intracellular signalling networks have been shown to be activated by receptor tyrosine kinases. Receptor tyrosine kinases are central components of cell signaling networks and play crucial roles in normal physiological processes, such as embryogenesis, cell proliferation and cell death (apoptosis). They are single pass transmembrane proteins that are comprised of an extracellular ligand binding domain and an intracellular domain. The cytoplasmic domain contains the catalytic kinase domain as well as a juxtamembrane and carboxy terminal domains which contain tyrosine residues which recently have been shown to be phosphorylated in several RTKs that play a role in the activation and normal signaling of the receptors [7].

3. RTK family and activation

RTKs are classified into 20 subfamilies based on their structure and biological function [1] (Figure 1). AU RTKs share structural similarity as they aU have an large extracellular domain, a single transmembrane domain, and a cytoplasmic region which contains a weIl conserved catalytic kinase domain [2, 3]. The kinase domain contains a conserved Gly-x-Gly-x-x-Gly motifwithin the ATP-binding domain which is critical for 2 the association of Mg ++ -ATP molecules with the catalytic pocket of the receptor [5-7]. A Literature review 1-3

conserved lysine residue is present to aid the transfer of a phosphate from the ATP to tyrosine residues residing within the activation loop of the receptor, leading to its activation [4-6]. Most RTKs are monomers when they are inactive except for the insu lin receptor and the insulin-like growth factor (IGF-1) receptor which exists as disulfide dimers [7]. Receptor dimerization following ligand binding induces a conformational change which activates the intrinsic catalytic kinase activity. This will subsequently allow phosphorylation of key tyrosine residues in the kinase domain, juxtamembrane and c­ terminal tai!. Once the receptor is phosphorylated, the tyrosine residues located outside the kinase domain recruit adaptor proteins involved in the transmission of the biological signalofRTKs.

1 L o cysteine-rich Il fibronectin 1.11 type III

::> 'g

liliiii EGF • leucine·rler. @ cadherin

qh discoidin @!;f • krlngle

tyrosine kinase EGFR InsR PDGFRa Flli FGFRI TrkA Ror1 MuSK Met AxI 11e EpI;A j Ret Ryk DDR 1 Ros ErbB2 IGFIR PDGFR~ KDR FGFRZ TrkB Ror2 Ron Eyk Tek DDR2 EroS:> IRR CSFtR Flt4 FGFR3 TrtC Sea Tyro3 EiJ!?SI 1 ErbEl4 Kil FGFR4 N'IX F!k2 • SAM

Figure 1. Human receptor tyrosine kinases. Domain organization of a variety of receptor tyrosine kinases. Adapted from Hubbard and Till. 2000. Annu. Rev. Biochem. 69: 373- 398.

Tyrosine phosphorylation is a rare event in normal cells as it makes up about 0.05% of total cellular phosphorylation [8]. Phosphorylation of tyrosine residues recruits proteins with src homology 2 (SH2) and phosphotyrosine binding (PTB) domain containing proteins. SH2 and PTB containing proteins either possess enzymatic activity such as Literature review 1-4 phospholipase C-y (PLC- y) and phosphatidylinositol 3-kinase (PI3K) or lack enzymatic activitiy such as Grb2 and Shc. These proteins which are normally localized in the cytoplasm translocates to the plasma membrane following stimulation where they get phosphorylated on specific residues or change conformation. This activates their catalytic activity and/or generates binding sites for signaling proteins thus allowing these proteins to interact with new partners. Proteins that can be recruited include adaptor and docking proteins, phospholipases, kinases, phosphatases, transcription factors, guanine nucleotide exchange factors (GEFs), and GTPases activating proteins (GAPs) [1]. Formation of signaling complexes after RTK activation requires additional protein-protein interactions and protein-lipid interactions that occur through a range of protein domains such as Src Homology 3 (SH3), WW, PDZ, pleckstrin homology (PH), and FYVE [9]. The generation of the activated receptor complex plays a role in signal transduction to downstream pathways to modulate the cytoskeleton, cell-matrix and cell adhesion complexes [10].

4. The Met receptor

The Met receptor was first identified as the human oncogene, Tpr-Met, which is the product of chromosomal translocation induced by a chemical carcinogen N' -methyl­ N' -nitro-N' -nitrosoguanadine in the human osteogenic cell line [l1]. The resulting protein is a fusion of a leucine zipper dimerization motif known as the translocated promoter region (tpr) , and the intracellular domain of the Met receptor. This 65 kDa forms a dimer and gives rise to a constituitively activated protein kinase [12].

F ! p ./ Brcnk~)olnt E ! 0/ Q! Met

Y1356

Figure 2. The Met receptor and the human oncogene, Tpr Met Literature review 1-5

The gene encoding the dimerization domain of Tpr-Met is located on and when expressed normally, it is a large coiled-coil protein that forms intranuclear filaments attached to the inner surface of nuclear pore complexes (NPCs). Tpr directly interacts with several components of the NPC. It is required for the nuclear export of mRNAs and sorne proteins. The normal cellular counterpart, Met, is a transmembrane receptor and was mapped to human chromosome 7q31 [13] (Figure 2). The mammalian Met receptor is the receptor for hepatocyte growth factor (HGF) and de fines a subfamily of RTKs which include the macrophage stimulating protein (MSPlRon) receptor and the avian c-Sea receptor. Members of this subfamily have been implicated in the activation of proliferation, cell motility, morphogenesis as weIl as differentiation.

The Met receptor is comprised of a and ~ subunits, where the ~ chain spans the plasma membrane, a cytoplasmic region containing the kinase domain as well as tyrosines residues which serve as docking sites [14, 15]. Met is synthesized as a single chain precursor that undergoes proteolytic cleavage within the extracellular domain, thereby allowing disulfide bond formation of the a and ~ chains which facilitates ligand binding to the receptor [16]. The receptor tyrosine kinase, Met, is expressed in ectodermal derivatives and endothelial cells such as neuronal cells, hematopoetic cens, melanocytes, and various types of cancer cells [17]. Not long after its identification, the ligand for Met was identified as the hepatocyte growth factor (HGF). Hepatocyte growth factor (HGF) was originally identified as the mitogen for rat hepatocytes and epithelial cells [18]. Another group identified that the scatter factor (SF) as the potent motility factor secreted by fibroblasts and it induces dissocation, motility and invasiveness of epithelial cells [19]. Through sequence analysis, it was later identified that the hepatocyte growth factor (HGF) was identical to scatter factor (SF), since they were both encoded by the same gene; thus, HGF is a multifunctional factor [20, 21]. Unlike the receptor, HGF is produced by cells which have a mesenchymal origin inc1uding platelets, macrophages, monocytes, endothelial cells, nonparenchymalliver cells, leukocytes, bone marrow cens and placental cens [22]. HGF plays a role in development, angiogenesis, tumorigenesis as well as organ regeneration in vivo [23, 24]. It has been demonstrated that Met and its growth factor, HGF, has a potential role in epithelial-mesenchymal transition as it can promote Literature review 1-6 branching epithelial morphogenesis, wound healing, cell scattering and invasion in 3D matrices [17, 25] (Figure 3).

/ Prolifelation / , / / ,-,-'~~~~~-~

,// / / "1" HGr

Epithell'll Celh

Invasion

+HCF

The Met receptor Branch€

Figure 3. Met signaling. When stimulated with hepatocyte growth factor (HGF) in epithelial cells, the Met receptor recruits a wide range of adaptor proteins leading to signaIs that promote proliferation, cell migration, invasion and branching morphogenesis.

5. Met signaling Following Met activation, two unique bidentate docking sites are created [26, 27]. Tyrosine phosphorylation of c-terminal tyrosines Y1349 and Y1356 allows the recruitment of adaptor proteins such as Grb2, Shc, Gab1 and PI3K [26, 28, 29]. A versatile adaptor protein, Gab1, is a key modulator of downstream signaling of RTKs. Gab 1 binds to Met directly through a 13 amino acid sequence known as the Met binding motif (MBM) in Gab 1 and also indirectly through the Grb2 adaptor protein [30-34]. This 13 amino acid sequence is not present in other Gab family members [31, 35]. Upon stimulation of RTKs, Gab1 becomes tyrosine phosphorylated thereby creating multiple docking sites which mediates recruitment of SH2-domain containing proteins such as SH2 domain containing phosphatase (SHP2) p85 subunit of PI3K. Recruitment of SHP2 Literature review 1-7 increases its phosphatase activity which in turn leads to activation of the Ras-MAPK cascade known to mediate cell proliferation, differentiation and survival [34, 36]. Association of PI3K with Gab 1 mediates PI3K1 Akt signaling pathway which induces cell transformation, survival, proliferation, cell growth, cell motility and glycogen metabolism [37, 38]. PI3K facilitates the conversion of phosphatidylinositol -4,5-bisphosphate (PtdIns-4,5-P2) to phosphatidylinositol -3,4,5- trisphosphate. The phosphorylated product subsequently recruits Akt family members to the inner leaflet of the plasma membrane and stimulates their protein kinase activity. In addition, Gab 1 contains multiple YXXP motifs which allow recruitment ofPLCy or Crk family pro teins [39,40]. There has been increasing interest in the interaction between Crk and Gab 1 which subsequently leads to anchorage independent growth in response to Met activation [39].

6. Normal Regulation of RTKs

Activated receptors must be tightly regulated in normal cells. Prolonged activation leads to sustained downstream signaling which promo tes the development of many hurnan diseases, such as cancer. Fifty eight have been identified to encode RTKs; of which 30 have been implicated in human tumors [1]. Deregulation of RTKs occurs through gene amplification, point mutation and chromosomal translocations [8]. This results in either increased dimerization or oligomerization of receptor molecules in the absence ofligand or enhanced catalytic activity leading to altered signaling patterns [1, 6]. Rence, downregulation of these receptors is crucial to control downstream signaling pathways thus maintaining a normal, physiological state. The quantity of a prote in within a cell is determined not only by rates of synthesis, but also by rates of degradation. Protein turnover is an essential cellular process that is required to maintain cell homeostasis, structure and function by regulating the levels of components of cell cycle, signal transduction, apoptosis and transcription [41]. The half­ lives of proteins within cells vary widely, from minutes to several days, and rates of protein degradation are an important aspect of cell regulation. Many rapidly degraded proteins function as regulatory molecules, such as transcription factors. The rapid turnover of these proteins is necessary to allow their levels to change quickly in response to external and internaI stimuli. In addition, faulty or damaged proteins are recognized Literature review 1-8 and rapidly degraded within ceUs, thereby eliminating the consequences of mistakes made during protein synthesis. In eukaryotic ceUs, two major pathways-the ubiquitin­ proteasome pathway and lysosomal proteolysis-mediate protein degradation.

7. Ubiquitination Ubiquitin is a protein of 76 amino acid residues, found in aU eukaryotic ceUs and whose sequence is extremely weU conserved from protozoan to vertebrates. Ubiquitin acts through its post-translational covalent attachment (ubiquitination) to other proteins, where these modifications alter the function, location or trafficking of the protein, or targets it for irreversible destruction by the 26S proteasome [42].

ATP AMI' + pp, V

..

Figure 4. The Ubiquitination cascade. Adapted From www.mdc- berlin.deldittmar/Research. htm 1.

Ubiquitination of the pro teins in the ceU is a multistep process (Figure 4). First, the carboxy terminal glycine of the ubiquitin molecule forms a thiol ester bond with the El ubiquitin activating in an ATP dependent manner. The activated ubiquitin is then transferred to the ubiquitin conjugating enzyme, E2. Ubiquitin conjugation to a specifie substrate is provided by an E3 ubiquitin ligase which brings the E2 conjugating enzyme containing ubiquitin in close proximity to the targeted substrate [43]. The conjugation of ubiquitin to substrates is a complex process. That is, within a single ubiquitin moiety, there are seven intemallysines (Lys6, Lys 11 , Lys27, Lys29, Lys33, Lys48 Literature review 1-9 and Lys63) and all can be conjugated to ubiquitin [44]. Depending on how the ubiquitin is linked, the marked proteins are destined for distinct processes (Figure 5).

Lys4~ •• Lysl1· Of Lys29. MOnrHJbiquitin llnked poIy-ubi::!unin chalns

, ,, 1/'/ i'; l ,:' Recrunmoolof Modulation of protEi'in (lownstream afftC!Of mnctlon ttlrough p;otetns (eg, endocyllc conformatlonsl changes, p;oteinsJ through proteln-proteil'l inleracUons ul:i;lqmllo.l)lndlng Of changes in subœUular domalns. localisation.

Figure 5. Ubiquitin modifications. Consequence of ubiquitination is dependent upon the type of ubiquitin modification. Adapted from Passmore and Barford. 2004. Biochem J. 379:513-525.

The ubiquitin proteasome system (UPS) is the major pathway in which intracellular proteins are destroyed. Pro teins targeted for the proteasome are marked with a minimium of four ubiquitin moieties. Several studies have proposed that the tetraubiquitin chain is required to allow association of the targeted protein with ubiquitin binding proteins which shuttle the polyubiquitinated protein to the proteosome, an organelle where proteins are digested into short peptides [45, 46]. Several groups have identified an E4, polyubiquitin-chain elongation factor that aids efficient polyubiquitination of the targeted substrate [47]. Polyubiquitin chains linked through Lys48 are a primary signal for proteasomal degradation [48]. Multimonoubiquitin Lys63 chains serves as a signal for DNA repair, signal transduction and lysosomal degradation. Recently, it has also been shown that Lys63 can be polyubiquitinated as well [49]. However, the function of polyubiquitinated Lys63 remains unknown. Since polyubiquitination has been widely accepted to be the signal to target proteins to the proteasome, monoubiquitination oftransmembrane proteins seems to serve as a sorting signal for lysosomal degradation. Initial studies were performed in yeast Literature review 1-10 where a single ubiquitin moiety was able to induce Ste2p transmembrane protein intemalization [50-52]. Overexpression of free mutant ubiquitin did not inhibit intemalization of Ste2p suggesting that polyubiquitination is not required for intemalization of transmembrane proteins [50, 51]. Monoubiquitin and polyubiquitin chains cannot form within the same lysine residue of the substrate because when a single ubiquitin is linked through Lys63 it masks the Lys48 site which allows attachment of polyubiquitin chains. Transmembrane proteins in mammalian cells such as EGFR, PDGFR, FGFR and Met receptor are thought to be monoubiquitinated following ligand binding [53, 54]. Several years ago, it was believed that the smeared appearance of the ubiquitin signal associated with these transmembrane pro teins was polyubiquitinated and had somehow escaped proteasomal degradation. Rowever a series of experiments involving the use of anti-ubiquitin antibodies which differentiated poly and. monoubiquitination, revealed that these transmembrane proteins were multimonoubiquitinated thus giving a smeared ubiquitin signal appearance [55]. Interestingly, when a single moiety of ubiquitin was fused to the C-terminus of EGFR, intemalization was induced in a ligand independent manner which provides support that ubiquitin can act as an endocytic signal in yeast and mammalian cells [52]. Unlike EGFR, Met does not seem to require a ubiquitin moiety to aid intemalization. As it has been demonstrated that the Met YI003F mutant receptor which is ubiquitination deficient had similar intemalization rates as the Met wt receptor [56].

8. E3 ubiquitin ligases The role of E3 ubiquitin ligases is to regulate substrate ubiquitination and promote precise targeting of specific substrates leading to its degradation. Over one hundred E3 ubiquitin ligases have been identified in humans and they can be grouped into subfamilies by the presence of several defining motifs such as RECT (homologous to E6-associated protein), RING (really interesting new gene) and U-box (a modified RING motifwithout the full complement of Zn- 2+-binding ligands) domains. RECT E3s directly catalyze ubiquitination of the substrate whereas RING and U-box E3's bring the E2 ubquitin conjugating enzyme and the substrate in close proximity to promote its ubiquitination Literature review 1-11

(Figure 6). Mutation, absence or malfuntional E3 ligases have been implicated in neurodegenerative disorders and cancers [57, 58].

b

Figure 6. Different ubiquitin protein ligases. Ubiquitin protein ligase coordinates transfer of ubiquitin to a lysine residue of the target protein. A. Rect E3 ligase and B. Ring E3 ligase. Adapted from Sullivan, Shirasu and Deng. 2003. Nature reviews genetics. 4: 948- 958.

The first E3 identified was the E6-associated protein 1 (E6-APl), which has a 350 aa RECT catalytic domain. E6-API belongs to the RECT domain E3s and they function like El and E2s. RECT E3s contain a cysteine residue in the RECT domain where the sul fur forms a thioester bond with the carboxy group of the C-terminal glycine residue facilitating the transfer of ubiquitin to the substrate [59]. Although the substrates for this E3 ligase have yet to be identified, loss ofE6-APl has been associated with an inherited disorder known as the Angelman syndrome, characterized by mental retardation and seizures [60]. The disease is caused by maternaI epigenetic imprinting at chromosome l5qll-q13, a region that encodes for E6-APl. The RING family of proteins constitutes the largest family of E3s [61, 62]. As mentioned earlier, the RING E3's function differently from RECT E3's as they act as scaffolding proteins that brings the E2s and the targeted substrates in close proximity. AlI Cbl family members possess an SR2 domain, a RING finger domain, a proline rich region as well as a leucine zipper. RING finger domains are cyteine/histidine-rich, zinc­ chelating do main that promotes both protein-protein and protein-DNA interactions. Two zinc atoms are complexed by cysteine/histidine residues to provide correct folding and activity of the RING domain. c-Cbl is a RING finger E3 which is involved in endocytosis and degradation of RTKs by promoting their ubiquitination, a modification required for endocytic sorting [53, 63]. c-Cbl was originally identified as an oncogene, v-CbI, encoded by the Cas NS-l retrovirus which was induced by pre-B cell lymphoma [64]. Like c-Cbl, two family Literature review 1-12 members Cbl-b and Cbl-3, also possess a RING finger, SH2 domain (Figure 7). However, Cbl-3 lacks parts of the proline rich region and the carboxy leucine zipper [65, 66]. c-Cbl and Cbl-b are ubiquitiously expressed and are localized in the cytoplasm [67]. Mice deficient in c-Cbl or Cbl-b have no developmental abnormalities [68-71]. However, later in life, c-Cbl knockout mice develop lymphoid hyperplasia and Cbl-b null mice are highly susceptible to auto-immune diseases [54-57]. c-Cbl and Cbl-b double knockout mice are embryonically lethal suggesting that they may play similar roles. It has been shown that Cbl can act as an adaptor protein which recruits other adaptor proteins, e.g. CIN85- at the level of RTK internalization. CIN85 is constitutively associated with endophilin, an enzyme that catalyzes membrane curvature [72]. Tyrosine phospho- TKB RING Proline-nch regto,n rylation sites UBA/LZ

474

Figure 7. Structural organization of human Cbl ubiquitin-protein ligases. Adapted from Swaminathan and Tsygankov·2006. J Cell Physiol. 209: 21-43.

9. Clathrin mediated endocytosis Cells take up nutrients and modulate the concentration of cell surface proteins and control their responses by c1athrin mediated endocytosis. This process involves the formation of vesic1e budding from the plasma membrane, generation of specific "coats" as well as many protein-protein/lipid interactions. After growth factor receptors are stimulated by their ligand, the receptor-ligand complexes become highly c1ustered into specifie locations enriched with c1athrin coats of the plasma membrane, known as c1athrin coated vesic1es (CCV). Clathrin is a triskeleton protein composed of proximal and distal segments which are available for binding heavy chains of other triskelia with a defined geometry, thus allowing its polymerization to form regular lattices characteristic of coated vesic1es [73]. It is believed that c1athrin Literature review 1-13 polymerization aids invagination and budding of the vesicle from the plasma membrane to facilitate receptor sorting into clathrin coated pits [74]. Concentration of transmembrane pro teins into CCV's may require specific amino acid sequences in their cytoplasmic domain. These specific sequences are also known as "sorting motifs" are believed to promote intemalization of transmembrane proteins through sequence recognition by the AP complexes [75]. Two types of sorting motifs have been identified through yeast two-hybrid screening and other biochemical assays,

YXX0 tyrosine based motif and di leucine motifs [76].

Once the transmembrane pro teins are concentrated in the budded vesicle, it is excisioned from the plasma membrane by a GTPase, dynamin [77]. This process is facilitated by amphiphysin, which also binds to clathrin and AP-2, and promo tes liposome-fragmenting activity of dynamin [78]. The function of dynamin is the promotion of vesicle fission. Purified recombinant dynamin has been shown to bind acidic lipid vesicles to form he lie al tubes and constrict them upon the addition of GTP [79]. This process is thought to be mediated by an effector, endophilin 1, which exhibit lyosphosphatidic acid acyl activity and change membrane curvature by working with dynamin facilitating fission from the plasma membrane [80).

10. Endocytic pathway Endocytosis is an efficient and complex process where tight regulation is required to aid extensive membrane exchange events. The endocytic pathway consists of distinct membrane-bound compartments and degradative compartments. After CCV s lose their coats, they are known as early endosomes. It is at this compartment where the intemalized cargo can either recycle back up to the membrane or be directed to the later compartments of the endocytic pathway[7 4]. Once the intemalized cargo reaches the sorting endosome, it can undergo inward vesiculation provided they have the proper signais, where the Iimiting membrane can pinches off and forms multivesicular bodies (MVBs). The purpose of inward vesiculation is to prevent intemalized cargo from recycling back to the plasma membrane, thereby terminating signaling cascade and efficiently directing it to the lysosome [81]. Literature review 1-14

11. RTK receptor endocytosis Ubiquitination of RTKs such as EGFR mediated by c-Cbl E3 ligase correlates to its rapid degradation [82-90]. Like the EGFR receptor, Met is ubiquitinated by c-Cbl E3 ubiquitin ligase and is also targeted for lysosomal degradation. It has been shown that recruitment of c-Cbl to the Met receptor occurs in two ways. Cbl interacts indirectly through association with the SH2 domain of the adaptor protein, Grb2 and directly through associating with a phosphotyrosine YI003 residue. The YI003 site provides a binding site for the tyrosine kinase binding (TKB) domain of c-Cbl and has been shown to be required for proper ubiquitination of the Met receptor [91]. Autophosphorylation as weIl as intemalization is induced once the Met receptor is stimulated with its ligand, HGF. The Met receptor reaches the sorting endosome, where the inward vesiculation process is regulated by a class of proteins that have ubiquitin interacting motifs (UlM) [56]. A UIM containing protein, hepatocyte growth factor regulated substrate (Hrs), has been proposed to act as an adaptor which links ubiquitinated receptors to the endosomal sorting complex required for transport (ESCRT) machinery. The ESCRT complexes are known to promote luminal budding and the generation of luminal vesicles of multivesicular bodies (MVB' s) [92]. These vesicles then deliver its cargo to the proteolytic interior of the lysosome (Figure 8).

Recycling n Endosome~

Early ~ Endosome 1 -

Lysosome Mullivesicular Body

Figure 8. RTKs targeted for degradation through the endocytic pathway. (provided by Dr. Pascal Peschard). Literature review 1-15

12. Other mechanisms involved in the degradation of Met Sorne reports suggested that degradation of Met could occur via the prote as omal pathway since Met levels were sensitive to proteasomal inhibitors such as lactacystin and MG132- a property which other RTKs do not share [93]. Jeffers et al. showed that Met degradation is blocked by inhibitors of the proteasome [80]. Proton pump inhibitors (where ATP is generated and is required for proteasomal activity) which have been able to show inhibition Met degradation [94]. This suggests that there may be sorne overlap between proteasomal and lysosomal degradation of Met. Hammond et al. have demonstrated that expression of the dominant-negative mutant dynamin K44A in Hela celIs did not completely block Met degradation. Therefore, a portion of the Met receptor can be degraded through the proteasomal pathway [95].

13. Mechanisms of deregulation Alterations in RTK signaling and structure are frequently associated with human cancers. Molecular mechanisms that are involved in RTK deregulation are the formation of an autocrine loop, overexpression, point mutations and chromosomal translocations (Figure 9). Most deregulated RTKs were identified as oncogenic retroviruses. Deregulation of RTKs would lead to amplification of downstream signaling preventing celIs from maintaining their normal physiological state.

Cîlromosomsl Point mutation Atltocnne loop Imnslor.:FIIII)f1

1 1 1 1

Figure 9. Mechanisms ofRTK deregulation. (provided by Dr. Pascal Peschard) Literature review 1-16

In cancer cells, it has been observed that RTKs and ligands may be both expressed leading to continous receptor activation, known as the formation of an autocrine loop. In vitro studies by Vande Woude's group have shown that fibroblasts expressing both Met and HGF induce tumor formation in nude mice [24]. Other RTKs such platelet derived (PDGFR) and epidermal growth factor recptor (EGFR) are implicated in many cancers such as gliomas, astrocytomas, pancreatic and breast carcinomas also uses the formation of an autocrine loop mechanism to induce tumor formation [96-98]. Overexpression ofRTKs is another mechanism by which downstream signaling is increased. Expression of an increased amount of R TKs concentrates the plasma membrane with RTKs increasing the chance for each monomer to encounter its counterpart to promote downstream signaIs in a ligand independent manner. This mechanism may result from gene amplification which led to increased transcription and translation. Amplification of RTKs, such as ErbB2 has been implicated in 10-30% of breast cancers [99, 100], gastric cancers [101], osephageal cancers [102], ovarian cancers [95]. EGFR is amplified in 40-50% of glioblastomas [103], breast cancers [104] and squamous cell carcinomas [105]. PDGFRa has been shown to be amp1ified in gliob1astomas [106]. R TK overexpression in human tumors has been associated with poor prognoSlS. Another mechanism by which RTKs are activated in a ligand independent manner is through point mutations. The ErbB2/Neu receptor is activated through a point mutation in the transmembrane domain inducing dimerization and activation of downstream signaling [107, 108]. Use of transgenic mouse models showed that overexpression of a ErbB2 protooncogene in the mammary gland gave rise to an altered ErbB2 receptor. The altered ErbB2 receptors are activated by removal of a cysteine residue in the extracellular domain yielding an uneven number of cysteines which promo tes disulfide bond formation between the receptor mono mers in the absence of ligand. This mechanism has been implicated in the multiple endocrine neoplasia (MEN) type 2A syndrome where the mutated Ret receptor is expressed [109-111]. A gain of a cysteine residue in the extracellular or transmembrane domain in the FGFR3 has been found in multiple myeloma, bladder and cervical carcinomas [112, 113]. Apart from point mutations in the Literature review 1-17 extracellular and transmembrane domains, R TKs mutated in the kinase domains have also been found. Such mutations alter the substrate specificity of Ret from that of a RTK to that of a non-RTK which is expressed in the MEN 2B syndrome [114]. In addition, point mutations of c- were found in mast cell tumors [115, 116]. Point mutations within Met kinase domain are found in renal and hepatocellular carcinomas [117]. Mutations in sorne residues of the kinase domain are believed to change contacts between residues which relieve the inhibitory conformation of the activation loop. Additionally, a study has suggested that mutations at the hinge regions facilitates movement between subdomains of the activated kinase [118]. Mutations within the juxtamembrane domain of several RTKs have been found in tumor tissue including c-Kit [119, 120], FIt3 [121-123] and the Met receptor [124, 125]. Several studies have suggested the juxtamembrane domain of certain receptors such as VEGFR-1 [126], Eph [127, 128], Flt-3 [129] and c-Kit [130] autoinhibits their activity by adopting a conformation that prevents proper orientation of key residues in the kinase domain therefore preventing activation of the receptor [131, 132]. Once the receptor becomes activated, phosphorylation of the juxtamembrane tyrosine residue renders the do main incapable of distorting the small lobe in the kinase domain thus, allowing full receptor activation. Chromosomal translocation occurs frequently in human neoplasms and constitutes an important mechanism for oncogene activation. Over 25 RTK-derived oncoproteins have been identified and are associated with human tumors. This event occurs by fusion of a protein dimerization domain with the cytoplasmic kinase domain of RTKs resulting in the constitutive activation of the protein kinase [27]. As mentioned previously, Tpr­ Met is a protein kinase that was identified when the hum an osteogenic sarcoma (HOS) cell line was treated with MNNG, a carcinogenic agent. The result of chromosomal translocation fused the Tpr dimerization domain upstream of the Met kinase domain generating an activated protein kinase [12]. Other RTKs that are also activated by chromosomal translocation include FGFRl, FGFR3, PDGFR~, Ret, Ros, TrkA, TrkC[8]. The tpr dimerization domain has also been found upstream of the TrkA receptor, a neutrophin receptor [133]. Another dimerization domain ETS, derived from transcription factors, has been involved in the activation ofRTKs such as the PDGFR~ and TrkC [134]. Literature review 1-18

14. RTK derived oncoproteins It has been shown that several RTK-derived oncoproteins have lost their ability to associate with the ubiquitin ligase c-Cbl through its TKB domain (Figure 10). Examples of these are the Met, CSF-IR, c-Kit, and EGFR [135]. The normal counterparts of these pro teins can be ubiquitinated by Cbl E3 ligase leading to their degradation.

TKE3"I~'dJated TfanshHrnin~.. Chi blodil19 ablU1y

yas

no

no +

v,Se43 no

yes

no

no

EGFR yes

no ND

no +

yas

no

Figure 10. RTK oncoproteins that have lost their ability to recruit Cbl III a TKB dependent manner. Adapted from Peschard. 2003. Cancer cell. 3: 1-5.

Colony stimulating factor CSF -1 R was first identified as the viral oncogene v­ FMS, derived from the feline sarcoma virus. The last 50 amino acids containing the Cbl TKB domain have been replaced with 14 unrelated amino acids [136]. Moreover, mutations of the Cbl TKB binding site have been observed in hum an myelodysplasia and acute myeloblastic leukemia [137]. Indeed, it has been shown that mutation of the TKB binding site residue tyrosine 969 to phenylalanine, increases the transforming ability of CSF-IR [138]. This suggests that loss of Cbl TKB binding site may contribute to its oncogenic activity.

The c-Kit and PDGFR~ recruits Cbl through an adaptor protein, adaptor containing PH and SH2 domains (APS). This association has been implicated in PDGFR c-Kit downregulation [139]. Mutation of the sites required for APS recruitment has been shown to enhance the transforming ability of c-Kit [140]. The v-ErbB protein encoded by the avian erythroblastosis virus is a isoform of the EGF receptor that has gained an internaI deletion of 21 amino acids that inc1udes the Cbl Literature review 1-19

TKB binding site [141]. Like Met and CSF-1R, EGF receptor lacking the Cbl TKB binding site seems to induce stronger mitogenic signaIs than EGFR. The retroviral product v-Sea, which belongs to the Met family has also lost its ability to recruit CbI though TKB domain [135]. Tpr Met is an oncogenically active protein kinase which lacks 47 aa iofits juxtamembrane inc1uding Y1003, the binding site of cbl TKB domaine as a result of chromosomal translocation. We predict its inability to recruit Cbl might explain sorne ofits oncogenic acitivity [91].

Rationale Recent studies have revealed that RTK-derived oncoproteins may have escaped downregulation after chromosomal rearrangement thereby leading to its oncogenic activity. Many RTK-derived oncoproteins seem to have lost the c-Cbl TKB binding site and are localized in the cytoplasm following chromosomal translocation. In this thesis work, l will address whether these two factors contribute the oncogenic activity of Tpr­ Met (Chapter 2). Understanding the molecular mechanisms by which RTKs are deregulated aids theraupeutic approaches to target these R TKs in cancer. Literature review 1-20

Abbreviations

Cbl Casitas B-lineage Lymphoma CCV Clathrin Coated Vesicle CIN85 85K Cbl-Interacting Protein Crk CT10 Regulator of Kinase CUE Coupling of Ubiquitin conjugation to ER degradation El Ubiquitin-activating Enzyme E2 Ubiquitin-conjugation Enzyme E3 Ubiquitin-Protein Ligase E4 Polyubquitin Protein Ligase E6AP E6 associated Protein EGF(R) Epidermal Growth Factor (Receptor) Epsl5 Epidermal Growth Factor Receptor Substrate 15 Epsin Eps 15 Interacator ESCRT Endosomal Sorting Complex Required for Transport FGF(R) Fibroblast Growth Factor (Receptor) Fms Feline McDonough Strain FYVE Fab-1, YGL023, Vps27, and EEAI Gabl Grb2 Associated Binding Protein GAP GTPase activating Protein GEF Guanine Nucleotide Exchange Factor GH(R) Growth Hormone (Receptor) GPCR G-Protein Coupled Receptor Grb2 Growth Factor Receptor-Bound Protein 2 HECT Homologous to E6AP Carboxy Terminus HGF Hepatocyte Growth Factor HOS Human Osteogenic Sarcoma Hrs Hepatocyte Growth Factor Regulated Substrate IR MAPK Mitogen Activating Protein Kinase MDCK Madin Darby Canine Kidney MEN Multiple Endocrine Leukemia Met Cloned in N' -methyl-N' -nitronitrosoguanine treated cells MNNG N' -methyl-N' -nitronitrosoguanine treated cells MSP Macrophage Stimuating Protein MVB Multivesicular Bodies p53 Tumour Suppressor Protein 53 p85 Regulatory Subunit ofPI3'K PDGF(R) Platelet Derived Growth Factor (Receptor) PH Pleckstrin Homology PI3'K Phosphoinositide-3-Kinase PTB Phospho Tyrosine Binding RING Really Interesting New Gene RTK Receptor Tyrosine Kinase ~ Sea Sarcoma, Erythroblastosis and anemia SF Scattor Factor Literature review 1-21

SH2 Src Homology 2 SH3 Src Homology 3 Shc Src homology 2 domain containing SHP-2 SH2 Domain-containing Protein-Tyrosine Phosphatase Sos Sons of Sevenless STAM Signal Transducing Adaptor Molecule STAT3 Signal Transducer and Activator of Transcription 3 TKB Tyrosine Kinase Binding Tpr Translocated Promoter Region UBA Ubiquitin-associated domain UBD Ubiquitin Binding Domain UCH Ubiquitin Carboxy-Terminal UlM Ubiquitin Interacting motif VEGF(R) Vascular Endothelial Growth Factor (Receptor) Vps23 Vacuolar Protein Sorting 23 WW Domain that contains two highly conserved tryptophan residues Literature review 1-22

References: 1. Blume-Jensen, P. and T. Hunter, Oncogenic kinase signalling. Nature, 2001. 411(6835): p. 355-65. 2. Fantl, W.J., D.E. Johnson, and L.T. Williams, Signalling by receptor tyrosine kinases. Annu Rev Biochem, 1993.62: p. 453-81. 3. Heldin, C.H., Protein tyrosine kinase receptors. Cancer SUry, 1996.27: p. 7-24. 4. Schlessinger, J., Signal transduction by allosteric receptor oligomerization. Trends Biochem Sci, 1988. 13(11): p. 443-7. 5. Yarden, Y. and A. Ullrich, Growth factor receptor tyrosine kinases. Annu Rev Biochem, 1988.57: p. 443-78. 6. Hanks, S.K., A.M. Quinn, and T. Hunter, The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science, 1988. 241(4861): p. 42-52. 7. Heldin, C.H., Dimerization of cell surface receptors in signal transduction. Cell, 1995. 80(2): p. 213-23. 8. Lamorte, L. and M. Park, The receptor tyrosine kinases: role in cancer progression. Surg Oncol Clin N Am, 2001. 10(2): p. 271-88, viii. 9. Pawson, T., M. Raina, and P. Nash, Interaction domains: from simple binding events to complex cellular behavior. FEBS Lett, 2002. 513(1): p. 2-10. 10. Pawson, T., Protein modules and signalling networks. Nature, 1995.373(6515): p. 573-80. Il. Cooper, C.S., et al., Molecular cloning of a new transforming gene from a chemically transformed human ceilline. Nature, 1984.311(5981): p. 29-33. 12. Rodrigues, G.A. and M. Park, Dimerization mediated through a leucine zipper activates the oncogenic potential of the met receptor tyrosine kinase. Mol Cell Biol, 1993. 13(11): p. 6711-22. 13. Park, M., et al., Two rearranged MET alleles in MNNG-HOS cells reveal the orientation ofMET on chromosome 7 to other markers tightly linked to the cystic fibrosis locus. Proc Natl Acad Sci USA, 1988.85(8): p. 2667-71. Literature review 1-23

14. Gonzatti-Haces, M., et al., Characterization of the TPR-MET oncogene p65 and the MET protooncogene p140 protein-tyrosine kinases. Proc Nat! Acad Sci USA, 1988.85(1): p. 21-5. 15. Giordano, S., et al., Tyrosine kinase receptor indistinguishable from the c-met protein. Nature, 1989. 339(6220): p. 155-6. 16. Komada, M., et al., Proteolytic processing of the hepatocyte growthfactor/scatter factor receptor by furin. FEBS Lett, 1993.328(1-2): p. 25-9. 17. Brinkmann, V., et al., Hepatocyte growthfactor/scatter factor induces a variety of tissue-specific morphogenic programs in epithelial ceUs. J Cell Biol, 1995. 131(6 Pt 1): p. 1573-86. 18. Nakamura, T., et al., Molecular cloning and expression of human hepatocyte growthfactor. Nature, 1989.342(6248): p. 440-3. 19. Stoker, M., et al., Scatter factor is a fibroblast-derived modulator of epithelial ceU mobility. Nature, 1987.327(6119): p. 239-42. 20. Bottaro, D.P., et al., Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science, 1991. 251 (4995): p. 802-4. 21. Weidner, K.M., et al., Evidence for the identity of human scatter factor and hum an hepatocyte growth factor. Proc Nat! Acad Sci USA, 1991. 88(16): p. 7001-5. 22. Zamegar, Rand G.K. Michalopoulos, The many faces of hepatocyte growth factor: from hepatopoiesis to hematopoiesis. J Cell Biol, 1995. 129(5): p. 1177-80. 23. Birchmeier, W., et al., Role of HGF/SF and c-Met in morphogenesis and metastasis of epithelial ceUs. Ciba Found Symp, 1997.212: p. 230-40; discussion 240-6. 24. Jeffers, M., S. Rong, and G.F. Woude, Hepatocyte growth factor/scatter factor­ Met signaling in tumorigenicity and invasion/metastasis. J Mol Med, 1996. 74(9): p.505-13. 25. Montesano, R, et al., Identification of a fibroblast-derived epithelial morphogen as hepatocyte growthfactor. Cell, 1991. 67(5): p. 901-8. Literature review 1-24

26. Ponzetto, C., et al., A multifunctional docking site mediates signaling and transformation by the hepatocyte growth factor/scatter factor receptor family. Cell, 1994.77(2): p. 261-71. 27. Rodrigues, G.A. and M. Park, Oncogenic activation oftyrosine kinases. CUIT Opin Genet Dev, 1994.4(1): p. 15-24. 28. Fixman, E.D., et al., Efficient cellular transformation by the Met oncoprotein requires a functional Grb2 binding site and correlates with phosphorylation of the Grb2-associated proteins, Cbl and Cab1. J Biol Chem, 1997. 272(32): p. 20167- 72. 29. Fixman, E.D., et al., Efficient cel! transformation by the Tpr-Met oncoprotein is dependent upon tyrosine 489 in the carboxy-terminus. Oncogene, 1995. 10(2): p. 237-49. 30. Nguyen, L., et al., Association ofthe multisubstrate docking protein Gab1 with the hepatocyte growth factor receptor requires a functional Grb2 binding site involving tyrosine 1356. J Biol Chem, 1997.272(33): p. 20811-9. 31. Lock, L.S., et al., Identification of an atypical Grb2 carboxyl-terminal SH3 domain binding site in Gab docking proteins reveals Grb2-dependent and - independent recruitment of Gab1 to receptor tyrosine kinases. J Biol Chem, 2000. 275(40): p. 31536-45. 32. Weidner, K.M., et al., Interaction between Gab1 and the c-Met receptor tyrosine kinase is responsible for epithelial morphogenesis. Nature, 1996. 384(6605): p. 173-6. 33. Schaeper, u., et al., Coupling of Gab1 to c-Met, Grb2, and Shp2 mediates biological responses. J Cell Biol, 2000. 149(7): p. 1419-32. 34. Rosario, M. and W. Birchmeier, How to make tubes: signaling by the Met receptor tyrosine kinase. Trends Cell Biol, 2003. 13(6): p. 328-35. 35. Maroun, C.R., et al., The Gab1 PH domain is requiredfor localization ofGab1 at sites of cell-cel! contact and epithelial morphogenesis downstream from the met receptor tyrosine kinase. Mol Cell Biol, 1999. 19(3): p. 1784-99. 36. Gu, H. and B.G. Neel, The "Gab" in signal transduction. Trends Cell Biol, 2003. 13(3): p. 122-30. Literature review 1-25

37. Ingham, RJ., et al., The Gabl docking protein links the b cell antigen receptor to the phosphatidylinositol 3-kinase/Akt signaling pathway and to the SHP2 tyrosine phosphatase. J Biol Chem, 2001. 276(15): p. 12257-65. 38. Ho1gado-Madruga, M., et al., Grb2-associated binder-l mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growthfactor. Proc Nat! Acad Sci USA, 1997.94(23): p. 12419-24. 39. Lamorte, L., D.M. Kamikura, and M. Park, A switch from p130Cas/Crk to Gabl/Crk signaling correlates with anchorage independent growth and JNK activation in cells transformed by the Met receptor oncoprotein. Oncogene, 2000. 19(52): p. 5973-8l. 40. Garcia-Guzman, M., et al., Met-induced JNK activation is mediated by the adapter protein Crk and correlates with the Gabl - Crk signaling complex formation. Oncogene, 1999. 18(54): p. 7775-86. 41. Ciechanover, A., A. Orian, and A.L. Schwartz, Ubiquitin-mediated proteolysis: biological regulation via destruction. Bioessays, 2000.22(5): p. 442-5l. 42. Burger, A.M. and A.K. Seth, The ubiquitin-mediated protein degradation pathway in cancer: therapeutic implications. Eur J Cancer, 2004. 40(15): p. 2217-29. 43. Pickart, C.M., Back to the future with ubiquitin. Cell, 2004. 116(2): p. 181-90. 44. Peng, J., et al., A proteomics approach to understanding protein ubiquitination. Nat Biotechno1, 2003.21(8): p. 921-6. 45. Thrower, J.S., et al., Recognition of the polyubiquitin proteolytic signal. Embo J, 2000. 19(1):p. 94-102. 46. Rich1y, H., et al., A series of ubiquitin binding factors connects CDC48/p97 to substrate multiubiquitylation and proteasomal targeting. Cell, 2005. 120(1): p. 73-84. 47. Koeg1, M., et al., A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly. Cell, 1999.96(5): p. 635-44. 48. Deveraux, Q., et al., A 26 S protease subunit that binds ubiquitin conjugates. J Biol Chem, 1994.269(10): p. 7059-61. Literature reyiew 1-26

49. Huang, F., et al., Differentiai regulation of EGF receptor internalization and degradation by multiubiquitination within the kinase domain. Mol Cell, 2006. 21(6): p. 737-48. 50. Hicke, L. and R. Dunn, Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins. Annu Rev Cell Dey Biol, 2003. 19: p. 141-72. 51. Dupre, S., D. Urban-Grimal, and R. Haguenauer-Tsapis, Ubiquitin and endocytic internalization in yeast and animal cells. Biochim Biophys Acta, 2004. 1695(1-3): p.89-111. 52. Mosesson, Y., et al., Endocytosis of receptor tyrosine kinases is driven by monoubiquitylation, not polyubiquitylation. J Biol Chem, 2003. 278(24): p. 21323-6. 53. Marmor, M.D. and Y. Yarden, Role of protein ubiquitylation in regulating endocytosis ofreceptor tyrosine kinases. Oncogene, 2004.23(11): p. 2057-70. 54. Carter, S., S. Urbe, and M.J. Clague, The met receptor degradation pathway: requirement for Lys48-linked polyubiquitin independent ofproteasome activity. J Biol Chem, 2004. 279(51): p. 52835-9. 55. Haglund, K., et al., Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation. Nat Cell Biol, 2003.5(5): p. 461-6. 56. Abella, J.V., et al., Met/Hepatocyte growth factor receptor ubiquitination suppresses transformation and is required for Hrs phosphorylation. Mol Cell Biol, 2005.25(21): p. 9632-45. 57. Ardley, H.C., C.c. Hung, and P.A. Robinson, The aggravating role of the ubiquitin-proteasome system in neurodegeneration. FEBS Lett, 2005. 579(3): p. 571-6. 58. Michael, D. and M. Oren, The p53 and Mdm2 families in cancer. CUIT Opin Genet Dey, 2002. 12(1): p. 53-9. 59. Beaudenon, S., A. Dastur, and J.M. Huibregtse, Expression and assay of HEeT domain ligases. Methods Enzymol, 2005.398: p. 112-25. 60. Kishino, T., M. Lalande, and 1. Wagstaff, UBE3A/E6-AP mutations cause Angelman syndrome. Nat Genet, 1997. 15(1): p. 70-3. Literature review 1-27

61. Borden, K.L. and P.S. Freemont, The RING finger domain: a recent example of a sequence-structurefami/y. Curr Opin Struct Biol, 1996.6(3): p. 395-401. 62. Jackson, P.K., et al., The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases. Trends CeU Biol, 2000. 10(10): p. 429-39. 63. Dikic, L, Mechanisms controlling EGF receptor endocytosis and degradation. Biochem Soc Trans, 2003. 31(Pt 6): p. 1178-81. 64. Langdon, W.Y., et al., v-cbl, an oncogene from a dual-recombinant murine retro virus that induces early B-lineage lymphomas. Proc Natl Acad Sci USA, 1989.86(4): p. 1168-72. 65. Sawasdikosol, S., et al., Adapting to multiple personalities: Cbl is also a RING finger ubiquitin ligase. Biochim Biophys Acta, 2000. 1471(1): p. M1-M12. 66. Miyake, S., et al., The Cbl protooncogene product: from an enigmatic oncogene to center stage ofsignal transduction. Crit Rev Oncog, 1997.8(2-3): p. 189-218. 67. Andoniou, C.E., C.B. Thien, and W.Y. Langdon, Tumour induction byactivated involves tyrosine phosphorylation of the product of the cbl oncogene. Embo J, 1994. 13(19): p. 4515-23. 68. Naramura, M., et al., Altered thymic positive selection and intracellular signais in Cbl-deficient mice. Proc Natl Acad Sci USA, 1998. 95(26): p. 15547-52. 69. Murphy, M.A., et al., Tissue hyperplasia and enhanced T-cell signalling via ZAP- 70 in c-Cbl-deficient mice. Mol CeU Biol, 1998. 18(8): p. 4872-82. 70. Bachmaier, K., et al., Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b. Nature, 2000. 403(6766): p. 211-6. 71. Chiang, Y.J., et al., Cbl-b regulates the CD28 dependence of T-cell activation. Nature, 2000. 403(6766): p. 216-20. 72. Soubeyran, P., et aL, Cbl-CIN85-endophilin complex mediates ligand-induced downregulation ofEGF receptors. Nature, 2002. 416(6877): p. 183-7. 73. Heuser, J. and T. Kirchhausen, Deep-etch views of clathrin assemblies. J Ultrastruct Res, 1985.92(1-2): p. 1-27. 74. Harris, T.W., et aL, Mutations in synaptojanin disrupt synaptic vesicle recycling. J CeU Biol, 2000. 150(3): p. 589-600. Literature review 1-28

75. Ohno, H., et al., Interaction of tyrosine-based sorting signais with clathrin­ associated proteins. Science, 1995.269(5232): p. 1872-5. 76. Letourneur, F. and R.D. Klausner, A novel di-leucine motif and a tyrosine-based motif independently mediate Iysosomal targeting and endocytosis of CD3 chains. Cell, 1992.69(7): p. 1143-57. 77. Cao, H., F. Garcia, and M.A. McNiven, Differentiai distribution of dynamin isoforms in mammalian cells. Mol Biol Cell, 1998.9(9): p. 2595-609. 78. Takei, K., et al., Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis. Nat Cell Biol, 1999. 1(1): p. 33-9. 79. Sweitzer, S.M. and lE. Hinshaw, Dynamin undergoes a GTP-dependent conformational change causing vesiculation. Cell, 1998.93(6): p. 1021-9. 80. Schmidt, A., et al., Endophilin 1 mediates synaptic vesicle formation by transfer of arachidonate to Iysophosphatidic acid. Nature, 1999.401(6749): p. 133-41. 81. Luzio, J.P., et al., Lysosome-endosome fusion and lysosome biogenesis. J Cell Sei, 2000. 113 (Pt 9): p. 1515-24. 82. Thien, C.B., F. Walker, and W.Y. Langdon, RING finger mutations that abolish c­ Cbl-directed polyubiquitination and downregulation of the EGF receptor are insufficientfor cell transformation. Mol Cell, 2001. 7(2): p. 355-65. 83. Dikie, 1., 1. Szymkiewiez, and P. Soubeyran, Cbl signaling networks in the regulation ofcell function. Cell Mol Life Sei, 2003. 60(9): p. 1805-27. 84. Levkowitz, G., et al., c-Cbl/Sli-l regulates endocytic sorting and ubiquitination of the epidermal growthfactor receptor. Genes Dev, 1998. 12(23): p. 3663-74. 85. Miyake, S., et al., The tyrosine kinase regulator Cbl enhances the ubiquitination and degradation of the platelet-derived growth factor receptor alpha. Proe Natl Aead Sei USA, 1998.95(14): p. 7927-32. 86. Lee, P.S., et al., The Cbl protooncoprotein stimulates CSF-I receptor multiubiquitination and endocytosis, and attenuates macrophage proliferation. Embo J, 1999. 18(13): p. 3616-28. 87. Taher, T.E., et al., c-Cbl is involved in Met signaling in B cells and mediates hepatocyte growth factor-induced receptor ubiquitination. J Immunol, 2002. 169(7): p. 3793-800. Literature review 1-29

88. Terrell, J., et al., A function for monoubiquitination in the internalization of a G protein-coupled receptor. Mol Cell, 1998. 1(2): p. 193-202. 89. Shih, S.C., K.E. Sloper-Mould, and L. Hicke, Monoubiquitin carries a novel internalization signal that is appended to activated receptors. Embo J, 2000. 19(2): p. 187-98. 90. Mori, S., C.H. Heldin, and L. Claesson-Welsh, Ligand-induced ubiquitination of the platelet-derived growth factor beta-receptor plays a negative regulatory role in ils mitogenic signaling. J Biol Chem, 1993.268(1): p. 577-83. 91. Peschard, P., et al., Mutation of the c-Cbl TKB domain binding site on the Met

receptor tyrosine kinase converts if into a transforming protein. Mol Cell, 2001. 8(5): p. 995-1004. 92. Katzmann, DJ., M. Babst, and S.D. Emr, Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I Cell, 2001. 106(2): p. 145-55. 93. Jeffers, M., et al., Degradation of the Met tyrosine kinase receptor by the ubiquitin-proteasome pathway. Mol Cell Biol, 1997. 17(2): p. 799-808. 94. Hammond, D.E., et al., Down-regulation of MET, the receptor for hepatocyte growthfactor. Oncogene, 2001. 20(22): p. 2761-70. 95. Kerrnorgant, S., D. Zicha, and P.l Parker, Protein kinase C controls microtubule­ based traffic but not proteasomal degradation of c-Met. J Biol Chem, 2003. 278(31): p. 28921-9. 96. Herrnanson, M., et al., Platelet-derived growth factor and ils receptors in human glioma tissue: expression of mess enger RNA and protein suggests the presence of autocrine and paracrine loops. Cancer Res, 1992. 52(11): p. 3213-9. 97. Coltrera, M.D., et al., Expression of platelet-derived growth factor B-chain and the platelet-derived growth factor receptor beta subunil in human breast tissue and breast carcinoma. Cancer Res, 1995.55(12): p. 2703-8. 98. Hwang, R.F., et al., Inhibition of platelet-derived growth factor receptor phosphorylation by STI571 (Gleevec) reduces growth and metastasis of human pancreatic carcinoma in an orthotopic nude mouse model. Clin Cancer Res, 2003. 9(17): p. 6534-44. Literature review 1-30

99. Slamon, DJ., et al., Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science, 1987.235(4785): p. 177- 82. 100. Slamon, DJ., et al., Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science, 1989.244(4905): p. 707-12. 101. Nakajima, M., et al., The prognostic significance of amplification and overexpression of c-met and c-erb B-2 in human gastric carcinomas. Cancer, 1999.85(9): p. 1894-902. 102. Houldsworth, J., et a1., Gene amplification in gastric and esophageal adenocarcinomas. Cancer Res, 1990.50(19): p. 6417-22. 103. Wong, AJ., et al., Increased expression of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with gene amplification. Proc Natl Acad Sci USA, 1987.84(19): p. 6899-903. 104. Ro, J., et al., Amplified and overexpressed epidermal growth factor receptor gene in uncultured primary human breast carcinoma. Cancer Res, 1988.48(1): p. 161- 4. 105. Yamamoto, T., et al., High incidence of amplification of the epidermal growth factor receptor gene in human squamous carcinoma celllines. Cancer Res, 1986. 46(1): p. 414-6. 106. Fleming, T.P., et al., Amplification and/or overexpression of platelet-derived growth factor receptors and epidermal growth factor receptor in human glial tumors. Cancer Res, 1992.52(16): p. 4550-3. 107. Bargmann, C.L, M.C. Hung, and R.A. Weinberg, Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain ofp185. Cell, 1986.45(5): p. 649-57. 108. Aller, P., et al., Molecular dynamics (MD) investigations ofpreformed structures of the transmembrane domain of the oncogenic Neu receptor dimer in a DMPC bilayer. Biopolymers, 2005.77(4): p. 184-97. 109. Mulligan, L.M., et al., Specifie mutations of the RET proto-oncogene are related to disease phenotype in MEN 2A and FMTC. Nat Genet, 1994.6(1): p. 70-4. Literature review 1-31

110. Mulligan, L.M., et al., Germ-line mutations ofthe RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature, 1993. 363(6428): p. 458-60. 111. Donis-Keller, H., et al., Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC Hum Mol Genet, 1993.2(7): p. 851-6. 112. Wilkie, A.O., Bad bones, absent smell, seljish testes: the pleiotropic consequences of human FGF receptor mutations. Cytokine Growth Factor Rev, 2005. 16(2): p. 187-203. 113. Cappellen, D., et al., Frequent activating mutations of FGFR3 in human bladder and cervix carcinomas. Nat Genet, 1999.23(1): p. 18-20. 114. Santoro, M., et al., Activation of RET as a dominant transforming gene by germline mutations ofMEN2A and MEN2B. Science, 1995.267(5196): p. 381-3. 115. Hofstra, RM., et al., A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature, 1994.367(6461): p. 375-6. 116. Carlson, K.M., et al., Single missense mutation in the tyrosine kinase catalytic domain ofthe RET protooncogene is associated with multiple endocrine neoplasia type 2B. Proc Natl Acad Sci USA, 1994.91(4): p. 1579-83. 117. Schmidt, L., et al., Germline and soma tic mutations in the tyrosine kinase domain ofthe METproto-oncogene inpapillary renal carcinomas. Nat Genet, 1997. 16(1): p.68-73. 118. Miller, M., et al., Structural basis of oncogenic activation caused by point mutations in the kinase domain of the MET proto-oncogene: modeling studies. Proteins, 2001. 44(1): p. 32-43. 119. Furitsu, T., et al., Identification ofmutations in the coding sequence of the proto­ oncogene c-kit in a human mast cell leukemia cell line causing ligand­ independent activation of c-kit product. J Clin Invest, 1993.92(4): p. 1736-44. 120. Hirota, S., et al., Gain-of-function mutations of platelet-derived growth factor receptor alpha gene in gastrointestinal stromal tumors. Gastroenterology, 2003. 125(3): p. 660-7. Literature review 1-32

121. Hayakawa, F., et al., Tandem-duplicated Flt3 constitutively activa tes STAT5 and MAP kinase and introduces autonomous cell growth in IL-3-dependent cell lin es. Oncogene, 2000. 19(5): p. 624-31. 122. Mizuki, M., et al., Flt3 mutations fram patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways. Blood,2000. 96(12):p. 3907-14. 123. Nakao, M., et al., InternaI tandem duplication of the fit3 gene found in acute myeloid leukemia. Leukemia, 1996. 10(12): p. 1911-8. 124. Lee, J.H., et al., A novel germ line juxtamembrane Met mutation in human gastric cancer. Oncogene, 2000. 19(43): p. 4947-53. 125. Ma, P.C., et al., c-MET mutational analysis in small ceU lung cancer: novel juxtamembrane domain mutations regulating cytoskeletal functions. Cancer Res, 2003.63(19): p. 6272-81. 126. Gille, H., et al., A repressor sequence in the juxtamembrane domain of Flt-l (VEGFR-l) constitutively inhibits vascular endothelial growth factor-dependent phosphatidylinositol 3'-kinase activation and endothelial ceU migration. Embo J, 2000. 19(15):p.4064-73. 127. Binns, K.L., et al., Phosphorylation of tyrosine residues in the kinase domain and juxtamembrane region regulates the biological and catalytic activities of Eph receptors. Mol Cell Biol, 2000. 20(13): p. 4791-805. 128. Wybenga-Groot, L.E., et al., Structural basis for autoinhibition of the Ephb2 receptor tyrosine kinase by the unphosphorylated juxtamembrane region. Cell, 2001. 106(6): p. 745-57. 129. Griffith, J., et al., The structural basis for autoinhibition of FLT3 by the juxtamembrane domain. Mol Cell, 2004.13(2): p. 169-78. 130. Chan, P.M., et al., Autoinhibition of the kit receptor tyrosine kinase by the cytosolicjuxtamembrane region. Mol Cell Biol, 2003. 23(9): p. 3067-78. 131. Moriki, T., H. Maruyama, and LN. Maruyama, Activation of preformed EGF receptor dimers by ligand-induced rotation of the transmembrane domain. J Mol Biol, 2001. 311(5): p. 1011-26. Literature review 1-33

132. McLaughlin, P.J., et al., Enhanced growth inhibition of squamous cell carcinoma of the head and neck by combination therapy of paclitaxel and opioid growth factor. Int J Oncol, 2005.26(3): p. 809-16. 133. Martin-Zanca, D., S.R. Hughes, and M. Barbacid, A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequences. Nature, 1986.319(6056): p. 743-8. 134. Golub, T.R, et al., Fusion of PDGF receptor beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5; 12) chromosomal translocation. Cell, 1994.77(2): p. 307-16. 135. Peschard, P. and M. Park, Escapefrom Cbl-mediated downregulation: a recurrent theme for oncogenic deregulation of receptor tyrosine kinases. Cancer Cell, 2003. 3(6): p. 519-23. 136. Mancini, A, et al., c-Cbl associates directly with the C-terminal tail of the receptor for the macrophage colony-stimulating factor, c-Fms, and down­ modulates this receptor but not the viral oncogene v-Fms. J Biol Chem, 2002. 277(17): p. 14635-40. 137. Ridge, S.A, et al., FMS mutations in myelodysplastic, leukemic, and normal subjects. Proc Nat! Acad Sci USA, 1990.87(4): p. 1377-80. 138. Roussel, M.F., et al., A point mutation in the extracellular domain of the human CSF-1 receptor (c-fms proto-oncogene product) activates its transforming potential. Cell, 1988.55(6): p. 979-88. 139. Wakioka, T., et al., APS, an adaptor protein containing Pleckstrin homology (PH) and Src homology-2 (SH2) domains inhibits the JAK-STAT pathway in collaboration with c-Cbl. Leukemia, 1999. 13(5): p. 760-7. 140. Herbst, R, S. Munemitsu, and A Ullrich, Oncogenic activation ofv-kit in volves deletion of a putative tyrosine-substrate interaction site. Oncogene, 1995. 10(2): p. 369-79. 141. Choi, O.R, et al., A single amino acid substitution in v-erbB confers a thermolabile phenotype to ts167 avian erythroblastosis virus-transformed erythroid ceUs. Mol Cell Biol, 1986.6(5): p. 1751-9. Literature review 1-34 Chapter 2 2-1

Chapter 2

Oncogenic activation of the Met receptor tyrosine kinase fusion protein,

Tpr-Met, involves exclusion from the endocytic degradative pathway

Hayley Hoi Lam Mak, Pascal Peschard, Tong Lin, Monica A.Naujokas, Dongmei Zuo and Morag Park. Dec 2006. Provisionally accepted, Oncogene. Chapter 2 2-2

Abstract

Many human cancers are associated with deregulation of receptor tyrosine kinases

(RTKs), which involves various molecular mechanisms. In addition to receptor activation, loss of negative regulation can also contribute to dysregulation of RTKs. Negative regulation of RTKs involves their intemalization and subsequent degradation in the lysosome. In general, RTK-derived oncoproteins activated following chromosomal translocations are no longer transmembrane proteins and would be predicted to escape lysosomal degradation. We addressed the importance of escape from the endocytic pathwayon oncogenic activation ofRTKs following chromosomal translocation using the

Tpr-Met oncogene as a model. Unlike the Met receptor, Tpr-Met is localized in the cytoplasm and lacks the binding site for Cbl, E3 ubiquitin ligases. To determine wh ether subcellular localization of Tpr-Met, and/or loss of its Cbl binding site, is important for its oncogenic activity, Tpr-Met variants, targeted to the plasma membrane with and without

Cbl recruitment were examined. Presence of a Cbl binding site and ubiquitination of cytosolic Tpr-Met oncoproteins does not alter their transforming activity, or protein st ab ility, and these proteins fail to enter the endocytic pathway. However, plasma­ membrane targeting allows Tpr-Met to enter the endocytic pathway and transformation as well as protein stability are decreased in a Cbl-dependent manner. We show the oncogenic activity of Tpr-Met is in part dependent on its ability to escape normal down­ regulatory mechanisms which provides a paradigm for many RTK-derived oncoproteins activated fOllowing chromosomal translocation. Chapter 2 2-3

Introduction

In normal cells, RTK activation is tightly regulated. Their inappropriate activation is associated with the development and progression ofmany human malignancies. Of the 58 genes known to encode RTKs, the deregulation of 30 has been associated with hum an tumors (reviewed Blume-Jensen & Hunter, 2001). In the past two decades, several mechanisms that deregulate RTKs, such as receptor amplification, chromosomal translocation and point mutations, have been identified (Blume-Jensen & Hunter, 2001;

Lamorte & Park, 2001; Rodrigues & Park, 1994). These gain-of-function mechanisms result in ligand-independent activation or enhanced catalytic activity ofRTKs in response to ligand. Consequently, the activation of downstream effector signaling increases and effectively promotes tumorigenesis. However, in addition to these positive mechanisms, there is growing evidence that escape from negative regulatory mechanisms is an important event in the deregulation ofRTKs (Bache et al., 2004; Peschard & Park, 2003)

In the absence ofligand, most RTKs are catalytically inactive. Binding of the ligand promotes receptor dimerizationloligomerization and induces a conformational change that triggers receptor kinase activity. RTK activation promotes their intemalization via c1athrin-coated pits and subsequently, many RTKs are down-regulated through lysosomal degradation. In addition recent publications have established that ubiquitination plays an important role in receptor down-regulation (reviewed Shtiegman & Yarden, 2003)

Protein ubiquitination is mediated by an enzymatic cascade composed of a ubiquitin-activating enzyme (El), a ubiquitin-conjugating enzyme (E2) and a ubiquitin­ protein ligase (E3). The presence of a polyubiquitin chain on many cytosolic and nuclear pro teins targets them for degradation by the 26S proteasome. In contrast, ubiquitination of many cell-surface receptors correlates with their intemalization and lysosomal Chapter 2 2-4 degradation. Evidence supports that RTKs are multi-monoubiquitinated as weIl as polyubiquitinated (Carter et al., 2004; Haglund et al., 2003; Huang et al., 2006; Mosesson et al., 2003).

Ubiquitin moieties can constitute binding sites for proteins that contain ubiquitin­ binding domains such as ubiquitin-interaction motif (UlM), ubiquitin-associated (UBA) and ubiquitin-conjugating-like (UBC-like) domains. The UIM-containing proteins, HRS and STAM, are thought to recruit ubiquitinated RTKs to ESCRT complexes that retain receptors in specialized microdomains of sorting endosomes characterized by a bilayered clathrin coat (Urbe et al., 2003). RTKs are then internalized in the endosomal lumen.

Sorting endosomes mature into multivesicular bodies (MVBs) where receptors remained trapped within internaI vesicles. Fusion ofMVBs with lysosomes leads to the degradation of internaI vesicles and their contents. EGF and Met R TK mutants that are not ubiquitinated are inefficiently sorted in the bilayered clathrin coat and escape lysosomal degradation (Abella et al., 2005; Grovdal et al., 2004; Haglund et al., 2003; Katzmann et al., 2002; Peschard et al., 2001; Waterman et al., 2002).

The Cbl family of ubiquitin-protein ligases (c-Cbl, Cbl-b and Cbl-3) plays an important role in the ligand-dependent ubiquitination ofmany RTKs (Thien & Langdon,

2005). Several receptors including EGFR, PDGFR, CSF-1R and Met (HGFR) are ubiquitinated following recruitment of c-Cb1 (Lee et al., 1999; Levkowitz et al., 1998;

Miyake et al., 1998; Peschard et al., 2001; Peschard et al., 2004). Cbl ubiquitin-protein ligases are modular proteins that contain a conserved N-terminal tyrosine kinase binding

(TKB) domain and a RING finger domain in addition to other protein interaction motifs

(Thien & Langdon, 2005). Where tested, the TKB domain interacts with specifie phosphotyrosine residues on RTKs, as well as on the cytoplasmic protein tyrosine kinases Chapter 2 2-5

(PTKs), Syk and ZAP?O. The RING finger domain recruits the E2 ubiquitin-conjugating enzyme, UbcH? Both domains are required for the transfer of ubiquitin residues to R TKs and PTKs (Lill et al., 2000; Miyake et al., 1999; Thien & Langdon, 2005; Waterman et al., 1999). The Met receptor is negatively regulated by intemalization and Met degradation is enhanced by recruitment of c-Cbl and Cbl-b (Abella et al., 2005; Peschard et al., 2001; Peschard et al., 2004). The c-Cbl TKB domain binds to a juxtamembrane tyrosine (1003) residue on the Met receptor, and this interaction is essential for ubiquitination and degradation of the Met receptor (Peschard et al., 2001).

An oncogenic form of the Met receptor, Tpr-Met, was generated following a carcinogen-induced chromosomal rearrangement that fused a protein dimerization domain (Tpr) to the kinase domain of the Met receptor (Park et al., 1986). This results in the deletion of the juxtamembrane tyrosine-binding site for c-Cbl (Y1003), as well as the deletion of the extracellular domain of the reeeptor, rendering the Tpr-Met one op rote in eytop1asmie. The Tpr-Met oneoprotein is eonstitutive1y aetivated, but fai1s to bind e-Cb1 and is not ubiquitinated (Pesehard et al., 2001). This suggested that the inability to be targeted to the 1ysosoma1 degradative pathway 10ss and/or 10ss of Cbl-mediated ubiquitination and may eontribute to the oneogenie deregu1ation of Tpr-Met. Notab1y, a

Met reeeptor mutant that 1aeks on1y the e-Cbl TKB domain binding site (YI003F), has a prolonged half-life and is oneogenie in cell culture and tumorigenesis assays, identifying c-Cb1/Cbl-b and ubiquitination as important negative regulators for this receptor (Abella et al., 2005; Peschard et al., 2001; Peschard et al., 2004).

In addition to point mutations and overexpression, RTKs are frequently activated in human tumors following chromosoma1 translocation. In general, this fuses a protein dimerization domain with the cytosolic kinase domain of the receptor, resulting in Chapter 2 2-6 constitutive receptor dimerization and activation (Lamorte & Park, 2001). Over 25 RTK­ derived fusion pro teins have been identified in hum an tumors. In each case, the N­ terminal signal peptide, necessary for protein targeting to the plasma membrane, is deleted in the rearranged kinase and, with the exception of FIG-ROS (Charest et al.,

2003), where studied, these proteins are cytosolic (Lamorte & Park, 2001). Localization to the cytosol would preclude their entry in the endocytic pathway and hence, their lysosomal targeting and degradation. Hence, we predict that RTK-derived fusion proteins are more stable than their cognate receptors and that this feature contributes to their oncogemc activity. Here, we are formally testing this hypothesis using Tpr-Met as a model. Chapter2 2-7

Results

Addition of the Cbl TKB-binding site is required for Cbl-mediated ubiquitination ofTpr-Met.

We used the Tpr-Met oncoprotein as a model to address whether the exclusion of

RTK-derived oncoproteins, generated foIlowing chromosomal translocation, from endosomal-Iysosomal degradative pathways, contributes to their transforming activity. To test this, several Tpr-Met variants were generated. To examine a requirement for Cbl recruitment, we reintroduced the juxtamembrane domain containing the Cbl TKB binding site (YlO03) into Tpr-Met, (Tpr-Met-Juxta YI003), as weIl as a mutant that fails to recruit the Cbl TKB domain (Tpr-Met-Juxta YI003F) (Figure 1). To study the importance of uncoupling from the plasma membrane, Tpr-Met and Tpr-Met-Juxta constructs were targeted to the plasma membrane by fusion to the interleukin 2 alpha leader sequence and transmembrane domain (Tac) (Amaoutova et al., 2003; Naslavsky et al., 2003) (Figure 1).

FoIlowing transient transfections in HEK293, aIl Tpr-Met proteins were expressed and showed similar levels of tyrosine phosphorylation, demonstrating that the sequences added did not interfere with protein synthesis or activity (Figure 2, A and B).

Recruitment of c-Cbl and ubiquitination of the Met RTK requires YI003 in the juxtamembrane domain of Met (Peschard et al., 2001; Peschard et al., 2004). However, the importance of plasma membrane localization for Met ubiquitination has not been established. To test whether the Tpr-Met variants containing the juxtamembrane YI003 residue of Met can associate with Cbl, ceIls expressing an HA tagged c-Cbl TKB domain protein were co-transfected with cytoplasmic and membrane-targeted Tpr-Met and Tpr­

Met variants. The insertion of the juxtamembrane domain and YI003 is essential for the association of the Cbl-TKB domain, with either Tpr-Met-Juxta YI003, or membrane- Chapter 2 2-8 targeted Tpr-Met (Tac-Tpr-Met-Juxta YI003) (Figure 2). To examine the requirement for ubiquitination, Tpr-Met-Juxta variants were co-transfected with full-Iength Cbl. The Cbl

TKB domain-binding site, YI003, was essentiai for ubiquitination. In addition, both cytosolic and membrane-targeted Tpr-Met-Juxta variants were ubiquitinated to similar levels, indicating that their subcellular Iocalization did not affect ubiquitination (Figure 3).

Membrane targeting, but not ubiquitination, is required for entry of Tpr-Met into the endocytic pathway.

RTKs that are targeted for lysosomal degradation are first intemalized into early endosomes. Once ubiquitinated, RTKs are recognized by endocytic proteins that retain them in the sorting endosome, from which they can be targeted to the lysosome

(Katzmann et al., 2002). To establish if membrane targeting is sufficient to engage Tpr­

Met with the endocytic pathway, we examined the subcellular localization of non­ membrane-targeted and plasma membrane-targeted Tpr-Met variants by confocal microscopy following transient transfection of Cos 7 ceUs. Tpr-Met and aU cytoplasmic

Tpr-Met variants are diffusely dispersed in the cytoplasm and fail to localize with the early endosomal marker EEAI (Figure 4 and supplementary material). In contrast, plasma membrane targeting ofTpr-Met promotes entry into the endocytic pathway where

Tac-Tpr-Met localizes to EEAI positive endosomes (Figure 4 and supplementary material). Notably, Tac-Tpr-Met and Tac-Tpr-Met containing the juxtamembrane domain, showed similar localization to EEAI positive endosomes, demonstrating that ubiquitination of Tpr-Met is not required for endosomal localization (Figure 4 and supplementary material). Chapter 2 2-9

Membrane localization and ubiquitination decrease Tpr-Met protein stability.

To establish if subceUular localization and/or ubiquitination affect protein stability, we perfonned pulse-chase analyses on aU cytoplasmic and membrane-targeted Tpr-Met variants. The cytoplasmic Tpr-Met (TM) and Tpr-Met containing the juxtamembrane domain (TMJ) show similar half-lives of approximately 9 ho urs (Figure SA). Hence, the presence of the Cbl TKB binding site and ubiquitination of the Tpr-Met juxtamembrane­ containing variant does not affect the stability of pro teins localized in the cytoplasm. In contrast, membrane-Iocalized Tac-Tpr-Met pro teins containing the juxtamembrane domain and the YI003 residue, are more rapidly degraded (6-hr half-life) than Tac-Tpr­

Met proteins that lack the juxtamembrane domain or the Cbl TKB-binding site (YI003F)

(12-hr half-life) (Figure SB). This demonstrates that localization to the plasma membrane is insufficient to target Tpr-Met for rapid degradation and moreover, that the ability of

Tpr-Met to associate with Cbl proteins and become ubiquitinated is essential for its rapid degradation.

The transforming activity of plasma membrane-targeted Tpr-Met proteins is suppressed by Cbl.

To examine the consequence of Cbl-induced ubiquitination and escape from endosomal degradative pathways on the transfonning activity of Tpr-Met, we perfonned soft agar anchorage independent growth colony assays in Rat 1 fibroblasts with cytoplasmic and membrane-targeted Tpr-Met variants, in the presence or absence of CbI overexpression. We observed that Cbl expression had no effect on the colony-fonning activity of ceUs expressing the cytoplasmic Tpr-Met or cytoplasmic Tpr-Met-Juxta proteins that recruit Cbl and are ubiquitinated (Figure 6A). In contrast, the colony Chapter 2 2-10 fonning activity of cell expressing the plasma membrane-targeted Tac-Tpr-Met proteins was suppressed by Cbl recruitment and by Cbl overexpression (Figure 6B). Coexpression with CbI significantly suppresses the colony fonning ahility of cells expressing the Tpr­

Met variant containing the juxtamembrane YI003 residue (Tac-Tpr-Met-Juxta YI003,

Figure 6B) consistent with the reduced half-life of this variant in the presence of ChI

(Figure 5B).

To examine the importance of ubiquitination and subcellular localization in promoting the transfonning potential of Tpr-Met in vivo, stable Ratl cell populations expressing the plasma membrane targeted Tpr-Met variants al one or coexpressing Cbl were injected subcutaneously into nude mice. Tumor fonnation of aIl cell populations was observed as early as 5 days post-injection and initial measurements were comparable

(Figure 7). Tumor growth following injection of cells coexpressing Tac-Tpr-Met or Tac­

Tpr-Met-Juxta YI003F and Cbl was rapid and skin ulceration was observed earlier than with the mice injected with Tac-Tpr-Met-Juxta YlO03. However, as time progressed, tumors induced by cells coexpressing Cbl and the plasma membrane-targeted Tac-Tpr­

Met variant that recruits Cbl, showed reduced growth to 50% that of Tac-Tpr-Met with

Cbl (Figure 7A), even though the cells selected for injection expressed high levels of Tac­

Tpr-Met-Juxta YI003 (Figure 7B). Chapter 2 2-11

Discussion

Receptor endocytosis acts as an important mechanism that terminates receptor signalling following ligand activation via degradation of activated receptor complexes

(Katzmann et al., 2002). A large family of oncogenic RTKs are activated in human tumors following chromosomal rearrangement. These proteins are no longer associated with the plasma membrane and are localised to the cytoplasm. In general they are constitutively dimerized following the fusion of the kinase domain with a protein-protein dimerization domain, and this was considered sufficient for their oncogenic activation

(Rodrigues et al., 1993). In this report, we provide evidence that in addition to constitutive dimerization, the oncogenic activity of the Met RTK-derived oncoprotein,

Tpr-Met, is dependent on its ability to escape normal down-regulatory mechanisms involving receptor endocytosis. Our findings provide a paradigm for the large family of

RTK-derived oncoproteins activated in human cancers following chromosomal translocation.

We show that membrane targeting is essential for Tpr-Met to enter the endocytic pathway and for its localization to EEAI-postitive endosomes (Figure 4). However once engaged with the endocytic pathway, this is insufficient for efficient degradation, and a decrease in transforming efficiency of Tpr-Met pro teins is dependent on both membrane targeting and the ability to engage with the Cbl ubiquitin ligase (Figure 6). For example, the transforming activity of the membrane targeted Tac-Tpr-Met-Juxta proteins that engage with the CbI TKB domain is decreased by 60% when compared with Tac-Tpr-Met pro teins that fail to engage the Cbl TKB domain (Figure 6).

Ubiquitination plays an important role for the recruitment and trafficking of plasma membrane receptors within endocytic compartments and the targeting of activated Chapter 2 2-12 receptors for degradation in the lysosomes (Katzmann et al., 2002). The ubiquitin moieties attached to the receptors are thought to be recognized by endocytic proteins that contain ubiquitin-binding modules, and that promote both trafficking of the receptors and their inclusion into multivesicular bodies, leading to their lysosomal degradation

(Katzmann et al., 2002). We have shown that chromosomal translocation results in the deletion of the Cbl TKB binding site on Tpr-Met and, as a consequence, Tpr-Met is not ubiquitinated (Peschard et al., 2001). Although ubiquitination of this RTK-derived oncoprotein can be restored upon the addition of the juxtamembrane domain that contains the Y1003 residue (the c-Cbl TKB binding site) (Figure 3) this does not alter its transforming activity (Figure 6), nor protein stability (Figure 5), even when coexpressed with the ubiquitin E3 ligases, c-Cbl and Cbl-b. Hence ubiquitination of cytosolic Tpr-Met is not sufficient to promote enhanced degradation of this protein. Since several R TKs appear to be monoubiquitinated and K63-linked polyubiquitinated (Carter et al., 2004;

Haglund et al., 2003; Huang et al., 2006; Mosesson et al., 2003), Cbl-mediated ubiquitination of cytosolic RTKs activated following chromosomal translocations would not be predicted to target these proteins for degradation in the proteasome, since the latter recognizes specifically K48-linked chains of at least four ubiquitin residues (Thrower et al.,2000).

A previous study had indicated that the reinsertion of the Met juxtamembrane do main into Tpr-Met was sufficient to decrease the transforming activity ofthis protein (Vigna et al., 1999). This may reflect differences in the constructs generated, since the orientation of the Tpr dimerization domain with respect to the kinase domain can affect the efficiency of dimerization and catalytic activity of the enzyme (Rodrigues & Park, 1993).

Importantly, all of our constructs exhibited similar levels of autophosphorylation and Chapter2 2-13 phosphorylation of target proteins (Figure 2 and 3). Since the stability and transforming activity of a cytosolic Tpr-Met protein is unaffected by Cbl recruitment and ubiquitination, our data supports that deletion of the extracellular domain ofRTKs, which occurs following chromosomal rearrangment, is the key event that uncouples RTKs activated following chromosomal translocation from the normal degradative endocytic pathways. Once cytosolic, these R TK oncoproteins are not targeted for degradation following ubiquitination by CbI. This is distinct from transmembrane RTKs such as the

Met receptor that engage with the endocytic pathway, where loss of Cbl binding and ubiquitination is required to uncouple these proteins from degradation (Peschard et al.,

2003). In agreement with this, Tpr-Met displays a half-life of approximately 9 hrs (Figure

5A), while the half-life of an activated Met receptor is about an hour (AbeUa et al., 2005).

More than 25 different RTK-derived oncoproteins have been generated following chromosomal translocation in human tumors (Lamorte & Park, 2001). With one exception (Charest et al., 2003), aU of these proteins are no longer associated with the plasma membrane and would not engage with the endocytic degradative pathway.

Although the engagement with the endocytic pathway and ubiquitination is not sufficient to suppress transformation by the Tac-Tpr-Met oncoprotein, this results in a significant decrease in anchorage-independent growth (Figure 7). Since the majority of these RTK­ derived oncoproteins have fused a kinase domain with an upstream dimerization domain giving rise to a constitutive1y activated kinase in the absence of ligand, their ability to escape normal degradative pathways generates potent transforming genes.

In summary, we present a general mechanism for attenuation of R TK degradation through the formation ofRTK oncogenic variants that lack receptor extraceUular domains and fail to enter the endocytic degradative pathway. Targeting of one of these proteins, Chapter 2 2-14

Tpr-Met, to the plasma membrane engages this protein within the endocytic pathway where its transfoming activity is now attenuated by regulators of RTK degradation, such as ubiquitination. Further progress in understanding the molecular basis for deregulation of RTKs in human cancer will allow us to develop better strategies to target them efficiently. Chapter 2

Materials and Methods:

Cell Culture, Transfection: AIl cell lines were maintained in Dulbecco's modified

Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). For transient transfection assays, HEK293 cells were transfected using Lipofectamine Plus transfection reagent (Invitrogen) and Cos 7 cells were transfected with Fugene 6 transfection reagent following the manufacturer's instructions. Stable transfections were performed using

Fugene 6 transfection reagent (Roche).

Immunoprecipitation and Western Blotting: For coimmunoprecipitation experiments,

HEK 293 cells were lysed 48 hours post-transfection with 1% Triton lysis buffer (50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 2 mmol/L EGTA, 1.5 mmol/L MgCb, 1 mmol/L PMSF, 1 mmol/L NaV, 50 mmol/L NaF, 10 JLg/mL aprotinin, and 10 JLglmL leupeptin.). Cos 7 cells were lysed with RIPA lysis buffer (0.1 % SDS, 25 mmol/L Tris pH 8.0, 50 mmol/L NaCl, 0.5% NP-40, 0.5% NaDOC) which contains 1 mmol/L PMSF,

1 mmol/L NaV, 10 JLglmL aprotinin, 10 JLg/mL leupeptin. The antibodies indicated were incubated with equal amounts of protein for 1 hour at 4°C with rotation. After which, proteins collected with Protein A or Protein G Sepharose were washed three times with'

RIPA lysis buffer. Protein samples were subjected to SDS-PAGE and transferred to nitrocellulose membrane (Amersham Biosciences). Membranes were blocked with 5%

BSA in TBST (10 mmol/L Tris pH 8.0, 150 mmol/L NaCl, 2.5 mmol/L EDTA, 0.1%

Tween-20). Immunoreactive bands were visualized by enhanced chemiluminescence

(Amersham Biosciences). Chapter 2 2-16

Antibodies and Reagents: A polyclonal antibody was raised against a C-tenninal

~-

peptide of human Met receptor (Rodrigues et al., 1991) . HA monoclonal antibody was

purchased from Covance (Berkeley, CA). Anti-c-Cbl (sc-170) and anti-ubiquitin (P4D1)

were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phosho-Met

antibody (pY1234/35) was purchased from Cell Signaling Technology. EEA1 antibody is

from BD Biosciences (Mississauga, ON).

Plasmid Construction: For the generation of cytoplasmic Tpr-Met variants, first the Tpr

dimerization domain was subcloned into the pXM mammalian expression vector using

XhoI and Not! sites with the following pnmers: forward

5'TCATCTCGAGCGGGGCGGTGAGGTG3' (XhoI site underlined) and reverse 5'­

CTCAAGAACTTGAATACTTAACAGCGGCCGCTACGTGAGTTCTTGAACTTATG

AATTGTCGCCGGCGATGCA3' (Not! site underlined). Vectors containing the Met wt

and YI003F cDNAs were used to amplify the cytoplasmic domain of Met starting at the

beginning of exon 14 usmg the 5'

TAGCAGCGGCCGCAGATCTGGGCAGTGAATTAGTTCGCTACG3' (N ot l site

underlined) forward primer and 5' CTGA TGCGGCCGCGCA TTGGTCCCTGGCCTG 3'

(Not! site underlined) reverse primer. Then, the Not! fragments were ligated into the

pXM vector containing Tpr. The same constructs were targeted to the membrane by

fusing a transmembrane protein, Tac, upstream of the Tpr dimerization domain. The

pBlueScript II SK (+)-TTC construct containing the Tac transmembrane domain was a

kind gift from Dr. Andre Veillette. The Tac fragment was amplified from the pBSK-TTC

vector by PCR by using the T7 univers al primer as the forward primer and the 3' Reverse

primer 5' -CTCAGGAATTCTCTCCGCTGCCAGGTGAGCCCAC-3' (EcoRI site Chapter 2 2-17 underlined). Tpr-Met fragments were amplified from the pXM vector by using 5'­

CCTGTAGAATTCGGCGGCGGCTCCGGAGGCGGTATGGCGGCGGTGTTGCAGC

AAGTC-3' (EcoRI site underlined) forward pnmer and 5'­

CCTGACCCGGGGTGTGGACTGTTGCTTTGACATAG-3' (Xma site underlined) reverse primer. The Tac and Tpr-Met fragments were subcloned into pXM using

XhoIJEcoRI and EcoRI/XmaI respectively.

Confocal Immunofluorescence microscopy: Cos 7 cells (4 x 10 4 cells per well) were plated on coverslips coated with Poly-D-Lysine in a 24-well plate. Tpr-Met variants were transiently transfected into Cos 7 cells with FuGene transfection reagent (Roche) 24 hours after plating. Twenty-four ho urs post-transfection, cells were fixed with 4% parafonnaldehyde (PF A, Fisher Scientific) in PBS for 20 minutes. Residual PF A were removed by extensively washing the coverslips three times with PBS and three times for

5 minutes with 100mM Glycine in PBS. Cells were then penneabilized with 0.25% Triton

X-I00/PBS and were blocked in blocking buffer for 30 minutes at room temperature (2% bovine serum albumin, 0.2% Triton X-I00, 0.05% Tween 20, PBS). Coverslips were incubated with primary and secondary antibodies, diluted in blocking buffer, for 1 hour and 40 min respectively. Confocal Images were taken with Zeiss 510 META scanning confocal microscope (Carl Zeiss Canada Ltd, Toronto, ON) with 100X objective and IX zoom. Images were analyzed with the LSM 5 image browser (Carl Zeiss, Missisaga, ON).

4 Soft agar assay: Rat 1 cells (3.3x 10 ) stably expressing Tpr-Met variants, with or without Cbl, were seeded into 0.3% soft agar on 60-mm plates on top of a 0.6% agar layer and grown at 3TC. The agar substrate included 10% FBS/DMEM and the cells Chapter 2 2-18 were fed every 5-7 days by adding more top layer. Pictures were taken using an Axiovert

Microscope with 2.5X objective and phase contrast setting. Colonies were counted after

21 days. Colonies under 0.27 mm were excluded because they were considered insignificant.

Metabolic labelling: HEK293 cells were plated on 100-mm plates 2 days prior to transfection. Cells were transfected with various Tpr-Met variants in the presence or absence of c-Cbl and Cbl-b. Twenty-four hours later, cells were incubated with cysteine and methionine-free medium for 1 ho ur, after which cells were incubated with complete media containing 0.1 mCi of 35 S-labeled methionine (Perkin Elmer) for two hours. Once labeling was completed, cells were washed and maintained in complete media for various time points. Cells were lysed with RIPA buffer, and lysates were pre-c1eared with Protein

A beads prior to immunoprecipitation. Protein samples were resolved on 8% SDS-PAGE and transferred to nitrocellulose membrane. The intensity of the radioactive bands was analyzed with the Typhoon 8600 Phosphoimager (Amersham Biosciences Corporation,

Piscataway, NJ).

In vivo tumorigenic assays: Rat 1 cells (5 x 105) were injected subcutaneously in nude mice. Tumor progression was analyzed by collecting data points every two days. At excision, tumors were frozen, ground in liquid nitrogen and lysed in 1% Triton lysis buffer. Protein samples were resolved by SDS-P AGE and the expression of the membrane targeted Tpr-Met variants were analyzed by immunoblotting with Met antibodies. Chapter 2 2-19

Acknowledgements:

We would like to thank Anie Monast for help with the mice. We are grateful to members of the Park Laboratory for helpful comments on the manuscript. This research was supported by an operating grant (MOP-11545) to MP from the Canadian Institutes of

Health Research. H.M. is a recipient of Canadian Institute Health Research Cancer

Consortium award, P. P. is a recipient of a Terry Fox research studentship from the

National Cancer Institute of Canada. M.P. is a senior scholar of the CIHR. The authors dec1are that they have no competing financial interests.

Supplementary information is available at Oncogene's website Chapter 2 2-20

References:

Abella JV, Peschard P, Naujokas MA, Lin T, Saucier C, Urbe S and Park M. (2005). Mol Cel! Biol, 25, 9632-45.

Amaoutova l, Jackson CL, A1-Awar OS, Dona1dson JG and Loh YP. (2003). Mol Biol Cel!, 14,4448-57.

Bache KG, S1agsvo1d T and Stenmark H. (2004). Emba J, 23,2707-12.

B1ume-Jensen P and Hunter T. (2001). Nature, 411, 355-65.

Carter S, Urbe Sand C1ague Ml (2004). J Biol Chem, 279,52835-9.

Charest A, Kheifets V, Park J, Lane K, McMahon K, Nutt CL and Housman D. (2003). Proc Nat! Acad Sei USA, 100,916-21.

Grovdal LM, Stang E, Sorkin A and Madshus IH. (2004). Exp Cel! Res, 300, 388-395.

Haglund K, Sigismund S, Polo S, Szymkiewicz l, Di Fiore PP and Dikic 1. (2003). Nat Cel! Biol, 5, 461-466.

Huang F, Kirkpatrick D, Jiang X, Gygi Sand Sorkin A. (2006). Mol CeU, 21, 737-48.

Katzmann DJ, Odorizzi Gand Emr SD. (2002). Nat Rev Mol Cel! Biol, 3, 893-905.

Lamorte L and Park M. (2001). Surg Oncol Clin NAm, 10,271-88, viii.

Lee PS, Wang Y, Dominguez MG, Yeung YG, Murphy MA, Bowtell DD and Stanley ER. (1999). Emba J, 18,3616-28.

Levkowitz G, Waterman H, Zamir E, Kam Z, Oved S, Langdon WY, Beguinot L, Geiger Band Yarden Y. (1998). Genes Dev, 12,3663-74.

Lill NL, Douillard P, Awwad RA, Ota S, Lupher ML, Jr., Miyake S, Meissner-Lula N, Hsu VW and Band H. (2000). J Biol Chem, 275, 367-77.

Miyake S, Lupher ML, Jr., Druker B and Band H. (1998). Proc Nat! Acad Sei USA, 95, 7927-32.

Miyake S, Mullane-Robinson KP, Lill NL, Douillard P and Band H. (1999). J Biol Chem, 274, 16619-28.

Mosesson Y, Shtiegman K, Katz M, Zwang Y, Vereb G, Szollosi J and Yarden Y. (2003). J Biol Chem, 278, 21323-6.

NaslavskyN, Weigert Rand Donaldson JG. (2003). Mol Biol Cel!, 14,417-31. Chapter 2 2-21

Park M, Dean M, Cooper CS, Schmidt M, O'Brien SJ, Blair DG and Vande Woude GF. (1986). Ce!!, 45, 895-904.

Peschard P, Fournier TM, Lamorte L, Naujokas MA, Band H, Langdon WY and Park M. (2001). Mol Ce!!, 8, 995-1004.

Peschard P, Ishiyama N, Lin T, Lipkowitz S and Park M. (2004). J Biol Chem, 279, 29565-71.

Peschard P and Park M. (2003). Cancer Cel!, 3, 519-23.

Rodrigues GA, Naujokas MA and Park M. (1991). Mol Ce!! Biol, 11,2962-70.

Rodrigues GA and Park M. (1993). Mol Cel! Biol, 13,6711-22.

Rodrigues GA and Park M. (1994). Curr Opin Genet Dev, 4, 15-24.

Shtiegman K and Yard en Y. (2003). Semin Cancer Biol, 13, 29-40.

Thien CB and Langdon WY. (2005). Biochem J, 391, 153-66.

Thrower JS, Hoffman L, Rechsteiner M and Pickart CM. (2000). Emba J, 19,94-102.

Urbe S, Sachse M, Row PE, Preisinger C, Barr FA, Strous G, KIumperman J and CIague Ml (2003). J Ce!! Sei, 116, 4169-79.

Vigna E, Gramaglia D, Longati P, Bardelli A and Comoglio PM. (1999). Oncogene, 18, 4275-8l.

Waterman H, Levkowitz G, AIroy land Yarden Y. (1999). J Biol Chem, 274, 22151-4.

Waterman H, Katz M, Rubin C, Shtiegman K, Lavi S, EIson A, Jovin T and Yarden Y. (2002). Emba J, 21, 303-313. Chapter 2 2-22

Titles and Legends to figures:

Figure 1. Schematic diagram of the Met receptor, Tpr-Met and Tpr-Met variants.

As a consequence of chromosomal rearrangement, the oncogenic form of the Met

receptor, Tpr-Met, lacks the leader sequence, extracellular, transmembrane and 47 amino

acids of the juxtamembrane domain of the Met receptor, including YI003, the Cbl TKB

domain binding site. As a consequence Tpr-Met is localised to the cytosol. The 47 a~ino

acids of the juxtamembrane sequence from the Met receptor were added to Tpr-Met to

generate Tpr-Met-Juxta variants that contain YI003 or where YI003 was substituted for a

phenylalanine (YI003F). Additional constructs were targeted to the plasma membrane

using the leader sequence, extracellular and transmembrane domains (272 amino acids),

of the interleukin 2 alpha subunit (Tac). Previous studies have generated chimeras using

these Tac domains to study sorting signaIs (Amaoutova et al., 2003; Naslavsky et al.,

2003). The Tac sequences were fused upstream ofTpr-Met and Tpr-Met-Juxta variants.

".-.", Chapter 2 2-23

Figure 1

pY1OO3 pY1OO3 Yl003F

Tpr pY100l Y100lF

pY13S6

Met Tpr Met Tpr Mat Tpr Met TatTpr Met TacTprMet TacTpr Met Jum Y1003 Juxt:a y, OOlF Juxt:a YI 003 J uxt:a YlOOlF Chapter 2 2-24

Figure 2. Insertion of the juxtamembrane do main in Tpr-Met restores c-Cbl TKB domain-binding.

HEK293 cells transiently expressing Tpr-Met variants were co-transfected with HA-Cbl­

TKB and pro teins from lysates prepared 48 hrs post-transfection. Whole celllysates were immunoblotted for Met (A) and c-Cbl TKB (F). The same lysates were subjected to immunoprecipitation with Met and HA antibodies. Immunoprecipitated Met proteins were subjected to SDS-PAGE and the membrane was immunoblotted with (B) phosphotyrosine (pTyr) antibodies. Immunoprecipitation with HA complexes (c-Cbl TKB) were subjected to SDS-PAGE and was immunoblotted with (C) Met, (D) pTyr and (E)

HA antibodies. The following short forms are explained as TM: Tpr-Met, TMJ: Tpr-Met­

Juxta YI003, TMJYF:Tpr-Met-Juxta YI003F, TTM: Tac-Tpr-Met, TTMJ: Tac-Tpr-Met­

Juxta YI003, TTMJYF: Tac-Tpr-Met-Juxta YI003F. Chapter 2 2-25

Figure 2 +HA-CbITKB +HA-CbITKB

A ... TTMJ/TTMJYF ... TTM

... TMJ/TMJYF "" TM WCLBlotMet B ••

IP Met Blot pTyr

c -- TTMJ

""'TMJ IP HA Blot Met D ."'1 TTMJ

..... TMJ

.... IgG IP HA Blot pTyr E

IPHABlotHA F ••• ._.1 WCL Blot HA Chapter 2 2-26

Figure 3. Insertion of the juxtamembrane do main in Tpr-Met restores Cbl-induced ubiquitination of Tpr-Met.

Proteins from lysates prepared from HEK293 cells transiently transfected with Tpr-Met variants and Cbl and were subjected to immunoprecipitation with Met and HA antibodies.

Immunoprecipitated Met complexes were subjected to SDS-PAGE and were immunoblotted for (A) P4Dl (Uh), (B) pYI003, (C) Met and (D) pTyr antibodies.

Immunoprecipitated HA complexes were resolved by SDS-PAGE and immunoblotted for

(E) HA (c-Cbl and Cbl-b) and (F) pTyr. Whole celllysates were immunoblotted with Met antibodies to determine the level of expression of the Tpr-Met variants. Chapter 2 2-27

Figure 3

Tpl'-Met + Tpr-Met Jum Y1 003 - + - Tpr-MetJuxtaYl003F -- + Tac Tpr-Met --- + - - TacTpr-MeUumY1003 - - + - TllcTIPf'-MetJuxta Yl003F - - - + "lot A P4Dl (Ub)

B pV'OO3 ~~ IPMet c

D pTyr

E HA IPHA (CbO F pTyr

G WCl Met Chapter 2 2-28

Figure 4. Plasma membrane-targeting of Tpr-Met promotes its entry into the endocytic pathway.

Cos 7 cells plated on coverslips were transiently transfected with expression vectors encoding cytosolic and membrane targeted Tpr-Met variants. 24 hours later, coverslips were fixed with 4% PFA, stained with phospho-Met (Red) and EEAI (Green) antibodies.

Yellow staining corresponds to colocalization of Met and EEAI. Confocal images were taken with a lOOX objective and IX zoom. Bar represents IO um. Chapter 2 2-29

Figure 4

Phospho-Mat EEA1 Marge

Tpr-Met

Tac Tpr-Met

Tac Tpr-Met Juxta Yl003

Tac Tpr-Mat Juxta Yl 003F Chapter 2 2-30

Figure 5. Membrane localization and ubiquitination decrease Tpr-Met protein stability.

HEK293 cells were transiently transfected with expression plasmids encoding (A) (Left) cytoplasmic and (B)(Right) membrane-targeted Tpr-Met variants. 24 hours later, cells were cysteine- and methionine-starved for an hour, labeled with 35S-labeled cysteine and methionine for 2 hours and then chased with complete medium for the indicated times.

Cell lysates were pre-c1eared with protein A sepharose beads and later subjected to immunoprecipitation with Met antibodies. Samples were resolved using SDS- PAGE and were exposed to a phosphoimager screen for analysis. The phosphoimager was used to quantify the intensity of each radioactive band. Chapter 2 2-31

Figure 5

A B Chase (Hr) - 0 6 9 12 0 6 9 12 0 6 9 12 - 0 6 9 12 0 6 9 12 0 6 9 12

TM+Cbl TMJ+ Cbl TMJYF+Cbl TTM + Cbl TTMJ + Cbl TTMJYF + Cbl Cytoplasmic Membrane-targeted

__ TId+èbi ~12 TW+Obi< 1! 101~=;;;.=~< -.>-l'WYF • Obi 1 8v+~-"<~"ç-<"<~~--<""«:'~X«-««7::---1 :::~ln:M~YJ' t 1 20 20 1IV i ~ Ol+-~~~--~~~~ o 2 4 6 B 10 12 14 o 2 4 6 8 10 12 14 Tlme (Hr) Tlme(Hr) Chapter 2 2-32

Figure 6. The anchorage-independent growth of plasma membrane-targeted Tpr­

Met proteins is suppressed by CbI.

Cells from stable Rat 1 cell populations were generated from the focus forming assay and were plated in soft agar in 60 mm-dishes at a 3.3 x 104 cell density. A representative colony forming assay is shown. (A) (Top) Cytoplasmic Tpr Met and variants expressed with vector or coexpressed with CbI. (B) (Middle) Membrane-targeted Tpr-Met variants expressed with vector or co-expressed with CbI. Ten randomly chosen high-powered images of the colonies were taken after 25 days. We counted the number of colonies with a diameter equal or larger than 0.27 mm. The total number of colonies from three independent experiments performed in duplicate are plotted on a bar graph and the data represent the mean ± s.d. (C) (Bottom) Protein lysates were immunoprecipitated with Met antibodies (lmg) and were immunoblotted for Met. Chapter 2 2-33

Figure 6

/--"'"

A Tpr-Met Tpr-Met Tpr-Met JuxtaYl003 Juxta Yl003F

I:IVector Vector I+CbJ

+Cbl TM TMJ TMJYF Cytoplasmic Tpr-Met variants

B TacTpr-Met Tac Tpr-Met Tac Tpr-Met JuxtaYl003 Juxta Yl003F

160 140 +---~------i oVec:tor Vector ~ 120 i I+Cbl '6 100 Tl 80 '0 60 .. 40 20 +Cbl o TTM TTMJ TTMJYF Membrane-targeted Tpr-Met variants

c +Cbl +Cbl

IP Met Blot Met Chapter 2 2-34

Figure 7. The tumorigenic capacity of plasma membrane-localized Tpr-Met variants is suppressed by Cbl.

Stable Ratl cell populations expressing membrane targeted Tpr-Met variants expressed aione or with CbI were injected into nude mice. (A) (Top) Growth of tumor size was plotted against the number of days. The data represent mean ± s.d. (B)(Bottom) Whole celllysates (40ug) were immunoblotted for Met and Cbl (HA). Chapter 2 2-35

Figure 7

A

210,=:::","~-"~~~:"~;~:-·"~"~""""-~~~"-~···~·"·"~~·~: -····-TTM a!one 180 ...... TTM+Cbl "' E 150 : --TTMJ alone §. TTMJ + Cbl lU 120: --TTMJYF alone N 'in i --TTMJYF + Cbl ... 90 o § 60 1-

o '2 4 6 8 10 12 14 16 18 20 22 24 2û Days postinjection

B Tac Tpr-Met Tac Tpr-Met Tac Tpr- Met Juxta Y1 003 Juxta Yl003F + + - - + +

Met I,n. WCL Cbl (HA) 1 :=:=====i Tu bu lin Chapter 2 2-36

Supplemental Figure 1. Cytoplasmic Tpr-Met variants do not localize to EEAl­ positive endosomes.

Cos 7 cells plated on coverslips were transiently transfected with expression vectors encoding cytosolic and membrane-targeted Tpr-Met variants. 24 hours later, coverslips were fixed with 4% PFA, stained with phospho-Met (Red) and EEAI (Green) antibodies.

Yellow staining corresponds to co-localization of Met and EEA 1. Confocal images were taken with a 100X objective and IX zoom. Bar represents 10 um. Chapter2 2-37

Supplemental Figure 1

Phospho-Met EEAl Merge

Tpr-Met Juxta Yl 003

Tpr-Met luxt", Yl 003F General Discussion 3- 1

Chapter 3

General Discussion

/~, General Discussion 3- 2

Discussion

Over the last few years, the mechanism by which RTKs are downregulated has been extensively studied. It is weU documented that c-Cbl mediated ubiquitination plays an important role in the negative regulation of RTKs and that the presence of the c-Cbl TKB binding site in the targeted receptor is essential for the E3 ubiquitin ligase to mediate ubiquitination of the receptor. Previous work in the lab has demonstrated that a phosphorylated juxtamembrane YI003 residue in the Met receptor is required for Cbl to mediate its ubiquitination and downregulation [1]. My thesis work aimed to addresses whether the loss of Cbl TKB binding site in Tpr-Met through chromosomal translocation prevents it from being ubiquitinated and thereby contributes to its oncogenic activity. Tpr-Met is a constitutive active protein kinase which was first discovered to be encoded when hum an osteogenic sarcoma (HOS) ceUline was treated with the carcinogenic agent, MNNG [2].

1. Insertion of the Juxtamembrane do main of the Met receptor into Tpr-Met is required for its ubiquitination Recent studies have shown that polyubiquitination and monoubiquitination play different roles in the ceU [3]. Most cytoplasmic and nuc1ear pro teins are polyubiquitinated which targets them for proteasomal degradation whereas cell surface proteins, such as RTKs, are multimonoubiquitinated and are targeted for lysosomal degradation [4-6]. Recently, it has been shown that RTKs can be polyubiquitinated as weU, however the function remains to be defined [7]. Previous studies have shown that as a consequence of a chromosomal rearrangement event, the Tpr Met oncogene is localized in the cytoplasm and efficiently induces cell transformation in fibroblasts [8]. Since Tpr-Met lacks the direct binding site for Cbl (YI003), we hypothesize that the loss ofubiquitination in Tpr­ Met contributes to its oncogenic activity. To investigate this, Tpr Met variants which contain the missing region of the juxtamembrane domain, inc1uding the YI003 residue, were generated (Tpr-Met Juxta YI003; TMJ) (Figure 1). A YI003F mutant, which abrogates Cbl binding, was used as a control (Tpr-Met Juxta YI003F; TMJYF)(Figure 1). In parallel, membrane targeted Tpr-Met variants (Tac Tpr-Met; TTM, Tac Tpr-Met Juxta General Discussion 3- 3

YI003; TTMJ, Tac Tpr-Met Juxta YI003F; TTMJYF) have been generated to address whether subcellular Iocalization plays a role in the oncogenic activity of Tpr-Met. First 1 examined whether the insertion of the juxtamembrane domain of Met is sufficient to promote association with the TKB domain of c-Cbl since this interaction has been shown to be required for Cbl to exhibit its ligase activity. As expected, c-Cbl TKB domain (HA­ c-Cbl TKB) does not coimmunoprecipitate with Tpr-Met or Tpr-Met Juxta YlO03F, since they both lack a functional YI003. However, Tpr-Met Juxta YI003 coimmunoprecipitated with c-Cbl TKB domain. This confirms the importance of the juxtamembrane and especially the YI003 residue in the mediation of this interaction (Figure 2). c-Cbl and Cbl-b belongs to the same family of ubiquitin protein ligases. Initial studies have shown that c-Cbl acts as the ubiquitin ligase for RTKs. However, recent studies have shown Cbl-b may promote EGFR ubiquitination as well as ubiquitination of Met [9]. Thus, c-Cbl and Cbl-b are used together in this thesis work. Overexpression of c­ Cbl and Cbl-b promotes ubiquitination of Tpr-Met that contains the juxtamembrane domain that contains YI003 of Met (Figure 3). This confirms with previous studies which have shown that the juxtamembrane domain including YI003 of Met is crucial for c-Cbl to promote its ubiquitination [1].

2. Membrane targeting of Tpr-Met is sufficient for internalization Since Tpr-Met is cytoplasmic, incorrect subcellular Iocalization of this oncogene could be another potential mechanism to escape the de gradation machinery thus contributing to its oncogenic activity. Localization to the membrane might play an important role in targeting RTKs to Iysosomal degradation. Localization of Tpr-Met variants were examined by confocai microscopy to determine if membrane targeting of Tpr-Met allows these proteins to enter the endocytic pathway. We found that cytoplasmic Tpr-Met variants do not colocalize with the early endosomai marker, EEAI (Figure 4 and suppiementai data). All membrane targeted Tpr-Met variants, however, colocalize with EEAI (Figure 4). This suggests that without membrane targeting, oncoproteins escape from lysosomai degradation. General Discussion 3- 4

3. Tac-Tpr-Met Juxta YI003 has a shorter halflife To confirm that membrane targeting and ubiquitination play a direct role in Tpr­ Met degradation, l performed pulse chase analysis to determine the half life of cytoplasmic and membrane targeting Tpr-Met variants. My results show that cytoplasmic Tpr-Met variants have a very long half life

(~ I Oh) as compared to the Met receptor (~2h) [l, Figure 5 a]. Their higher stability means that these protein kinases continue to signal to downstream pathways which render them transforming. Results from Figure 2 and 3 demonstrate that the presence of the juxtamembrane do main containing YI003 is important for its association with the Cbl TKB domain and ubiquitination of the protein kinase. This modification alters the half life of the protein kinase as the pulse chase data shows that it has a half life of approximately 6 h (Figure 5b). When Tpr-Met and Tpr-Met Juxta YI003F are targeted to the membrane, their half life exceeds 10h (Figure 5b) while Tac Tpr-Met Juxta YI003 has a half life of approximately 6h. This demonstrates the importance of the juxtamembrane domain which is lacking in the oncogene during chromosomal translocation. Tac Tpr-Met and Tac Tpr-Met Juxta YI003F increased half life might be due to a delay in sorting them to the lysosomes because they are retained in the sorting endosome. Secondly, variants that lack the juxtamembrane domain containing the YI003 residue may undergo recycling which means it can be internalized, reach the multivesicular bodies but never the lysosome. Thus, reintroducing the juxtamembrane domain including the YI003 residue (Tac Tpr-Met Juxta YI003) and targeting the protein kinase up in the plasma membrane is sufficient to promote its degradation.

4. Membrane targeting of Tpr-Met and ubiquitination is required to reduce the transforming ability of Tpr-Met In order to assess the effect of membrane targeting and juxtamembrane insertion on the biological signal of the Tpr-Met oncogene, l performed a focus forming assay to test the uncontroUable growth and soft agar assays to test anchorage independent growth. Stable ceU lines of cytoplasmic and membrane targeted Tpr-Met variants alone or coexpressed with Cbl from the focus forming assay were used for the soft agar assay. Cytoplasmic Tpr-Met variants induce uncontrollable growth at a higher rate than the General Discussion 3- 5 membrane targeted variants. This agrees with the pulse chase data suggesting that they are stable and can readily transduce downstream signaling pathways. Once these protein kinases are targeted to the membrane, the number of foci in the focus forming assay as well as the number and size of colonies in the soft agar assay was drastically reduced, even when 20 fold more of Tac Tpr-Met variants was used as compared to cytoplasmic Tpr-Met variants. In addition, coexpression of c-Cbl and Cbl-b did not affect the transforming ab il ity of cytoplasmic Tpr-Met or Tac Tpr-Met, which lack 47 aa in the juxtamembrane. However, it decreased Tac Tpr-Met Juxta Y1003 and to a lesser degree Tac Tpr-Met Juxta Y1003F. This suggests that Cbl activity is localized to the membranelendocytic pathway since only mutants which reach that compartment and have the ability to bind c-CbllCbl-b are affected. Moreover, we performed a tumorigenic assay to characterize the rate of tumor growth of cells stably expressing membrane targeted Tpr-Met variants (Tac Tpr-Met, Tac Tpr-Met Juxta YI003, Tac Tpr-Met Juxta Y1003F) injected subcutaneously into nude mice. As predicted, the rate of tumor growth is much faster for the two membrane targeted Tpr-met variants that does not contain the juxtamembrane domain which inc1udes the YI 003 residue. A recent paper in our lab has demonstrated that the Met Y1003F mutant has a prolonged half life when compared to the wild type Met receptor and this may be due to retenti on of the Y1003F mutant in the multivesicular body, rendering it unable to be delivered to the lysosome for degradation [10]. They have also demonstrated that the Y1003F mutant is able to induce tumor formation in nude mice at a higher rate than the normal Met receptor [10]. My data coincides to this study as the y showed that the ubiquitination deficient Met Y1003F mutant receptor can be intemalized, undergo endosomal trafficking with kinetics to the Met wt receptor, yet it is inefficiently targeted for degradation [10]. Accordingly, another group c1aims that the insertion of the juxtamembrane domain into Tpr-Met is sufficient to impair the transforming ability ofTpr-Met despite its comparable kinase activity [11]. In that study, they generated similar Tpr-Met variants. They propose that the transforming ability of the Tpr-Met variant containing the juxtamembrane domain weakly associates with the adaptor protein Grb2 which can initiate downstream SOS-Ras pathway as well as Gab 1 which allows recruitment of General Discussion 3- 6 transducers such as PI3Kinase known to control cell motility and invasion. We performed similar experiments but have obtained different results. This may reflect the different cell types used for these assays as well as the different constructs. However, our detailed analysis would support a role for loss ofubiquitination in the oncogenic activation ofTpr­ Met. Loss of RTK tight regulation will lead to prolonged activation of downstream effector pro teins which may lead to tumorigenesis. This data confirms that ubiquitination is required to downregulate these constitutively active protein kinases. When this regulation is lost, downstream signaling readily induces tumor growth.

5. Proposed Mechanism My thesis work proposes that the localization and ubiquitination are both involved

III the oncogenicity of Tpr-Met. However, the question of how the localization of ubiquitinated protein kinase affects its degradation remains to be addressed. l've shown that the cytoplasmic Tpr-Met-Juxta Yl003 and membrane targeted Tpr-Met Juxta YI003 are both ubiquitinated when coexpressed with the Cbl E3 ubiquitin ligase. But it seems that the ubiquitin conjugated to the membrane targeted Tpr-Met-Juxta YI003 serves as a signal which leads to its rapid degradation whereas this is not the case for the cytoplasmic Tpr-Met Juxta YI003. One possible mechanism that would explain the above could be that the Lys 63 ubiquitin chains attached to the cytoplasmic Tpr-Met-Juxta YI003 fail to be recognized by the proteasomal degradation machinery. Since it has been widely accepted that proteins targeted for the proteasomal pathway are conjugated with ubiquitin linked through Lys 48, a protein like the cytoplasmic Tpr-Met-Juxta YI003 conjugated with ubiquitin linked through Lys 63, would not be degraded by the proteasome. Thus, the type of linkage that conjugates ubiquitin moieties to a protein plays an important role in its degradation. Another factor that might answer the above question could be due to the localization of the accessory proteins. When membrane targeted Tpr-Met-Juxta YI003 becomes ubiquitinated, accessory proteins in the membrane are brought into close proximity with the protein kinase to facilitate its entry into the endocytic pathway leading to its degradation. Even though the cytoplasmic Tpr-Met-Juxta YI003 contains Lys 63 ubiquitin chains, entry into the endocytic pathway from the cytoplasm would be difficult General Discussion 3-7

as accessory proteins localized in the membrane are required. The proposed mechanism is ~ .. illustrated in Figure 1.

Early Endosomo

PROTEASOME LYSOSOME J Endoproteins(~~

Figure 1. An illustration showing the proposed mechanism. Cytoplasmic Tpr-Met­ Juxta YI003 cannot be degraded through the lysosomal pathway as well as the proteasomal pathway due to its localization and the type of ubiquitin linkage conjugated to the protein kinase. This is not the case for membrane targeted Tpr-Met-Juxta YI 003 as the ubiquitin conjugated to it can associate with accessory proteins in the membrane and the endosome which leads to its degradation.

6. Summary and perspectives Several groups have proposed the absence of the binding site for the c-Cbl TKB domain in many RTK-derived oncoproteins leads to their oncogenic activity but have not tested this hypothesis. In a recent review, Peschard et al have suggested that many RTK­ derived oncoproteins through chromosomal translocation have lost the c-Cbl TKB binding site [12]. Most ofthem become cytoplasmic after this chromosomal translocation. The study is the first to confirm that RTK-derived oncoproteins which result from chromosomal translocation, escape downregulation thereby leading to their oncogenic activity. Many RTK-derived oncoproteins are associated with diseases such as acute myeloblastic leukemia and glioblastomas [12]. It is necessary to understand the molecular General Discussion 3- 8 mechanism by which R TKs escapes downregulation so therapeutic approaches could be developed. Since most RTK-derived oncoproteins become cytoplasmic after chromosomal translocation, the use of kinase inhibitors is one approach to target these RTK-derived oncoproteins. Another approach in targeting RTK-derived oncoproteins is the use of antisense oligonucleotides (ODNs), antisense RNA, triple helix or small interfering RNAs which inhibit their transcription and translation. These two strategies would prove useful for the design oftherapeutic drugs targeted to RTKs in cancer. General Discussion 3- 9

References

1. Peschard, P., et al., Mutation of the c-Cbl TKB domain binding site on the Met receptor tyrosine kinase converts it into a transforming protein. Mol Cell, 2001. 8(5): p. 995-1004. 2. Cooper, C.S., et al., Molecular cloning of a new transforming gene from a chemically transformed human cellline. Nature, 1984.311(5981): p. 29-33. 3. Passmore, L.A. and D. Barford, Getting into position: the catalylic mechanisms of protein ubiquitylation. Biochem J, 2004. 379(Pt 3): p. 513-25. 4. Hicke, L., Protein regulation by monoubiquitin. Nat Rey Mol Cell Biol, 2001. 2(3): p. 195-201. 5. Hershko, A. and A. Ciechanoyer, The ubiquitin system. Annu Rey Biochem, 1998. 67: p. 425-79. 6. Pickart, C.M. and D. Fushman, Polyubiquitin chains: polymerie protein signais. CUIT Op in Chem Biol, 2004.8(6): p. 610-6. 7. Huang, F., et al., Differentiai regulation ofEGF receptor internalization and degradation by multiubiquitination within the kinase domain. Mol Cell, 2006. 21(6): p. 737-48. 8. Fixman, E.D., et al., Efficient cell transformation by the Tpr-Met oncoprotein is dependent upon tyrosine 489 in the carboxy-terminus. Oncogene, 1995. 10(2): p. 237-49. 9. Ettenberg, S.A., et al., Cbl-b-dependent coordinated degradation ofthe epidermal growthfactor receptor signaling complex. J Biol Chem, 2001. 276(29): p. 27677- 84. 10. Abella, J.V., et al., Met/Hepatocyte growthfactor receptor ubiquitination suppresses transformation and is required for Hrs phosphorylation. Mol Cell Biol, 2005.25(21): p. 9632-45. 11. Vigna, E., et al., Loss of the exon encoding the juxtamembrane domain is essential for the oncogenic activation ofTPR-MET. Oncogene, 1999. 18(29): p. 4275-81. General Discussion 3-10

12. Peschard, P. and M. Park, Escapefrom Cbl-mediated downregulation: a recurrent theme for oncogenic deregulation of receptor tyrosine kinases. Cancer CeU, 2003. 3(6): p. 519-23.