Investigating the Relationship Between the Ret Receptor, Cbl, and Arhgef7 in Downregulation Of

INVESTIGATING THE RELATIONSHIP BETWEEN THE RET RECEPTOR,

CBL, AND ARHGEF7 IN DOWNREGULATION OF RET

HARVINDER KAUR

A thesis submitted to the Department of Pathology and Molecular Medicine

In conformity with the requirements for

the degree of Master of Science

Queen’s University

Kingston, Ontario, Canada

(July, 2008)

ISBN 978-0-494-44473-3

Abstract

The RET proto-oncogene encodes a receptor tyrosine kinase (RTK), with two

major isoforms, RET9 and RET51, which differ in their C-termini, and therefore recruit

different signaling complexes. RET plays an important role in cell growth, differentiation, migration and survival. Regulation of RET is critical for normal cellular

functioning, however, the biochemical mechanisms underlying the downregulation of

RET isoforms, are still not clear. Cbl (Casitas B-lineage Lymphoma) is an E3-ubiquitin

ligase that plays an essential role in mediating the degradation of RET. Recently, a

negative regulator of Cbl, ARHGEF7 (β-pix/ Cool-1) was found to prevent Cbl-catalyzed

deregulation of the Epidermal Growth Factor Receptor (EGFR).

In the current study, we characterized and further examined the association

between RET and Cbl. We showed that RET can associate with the c-Cbl and Cbl-b

homologues, in co-immunoprecipitations. Using far western assays and GST-pulldowns,

with the purified tyrosine kinase binding (TKB) domain of c-Cbl, we detected a potential

novel direct interaction between RET and c-Cbl. Previously, an indirect association

between RET and Cbl had been established, indicating that a bimodal interaction may

occur.

Furthermore, we proposed that ARHGEF7 may interfere with RET-Cbl

interactions, either by sequestring Cbl, so that it is unable to bind to RET, or by forming a

complex with both RET and Cbl, thereby blocking Cbl activation. Here, we investigated

the possibility of a complex formation using co-immunoprecipitations. We showed that

ARHGEF7 and c-Cbl can co-immunoprecipitate, but we could not detect either of the

ii RET isoforms in this complex. Further examination of a possible relationship between

RET isoforms, and ARHGEF7, showed that ARHGEF7 phosphorylation was dependent

on RET activation. However, in an in vitro kinase assay, we showed that this phosphorylation did not occur directly, but may occur indirectly through a pathway yet unknown.

Our data predicts that ARHGEF7 may modulate Cbl-binding to RET, and subsequently inhibit its degradation, in a manner similar to that seen for EGFR.

iii Acknowledgements

First of all I would like to thank Dr. Lois Mulligan for her support, patience, and

endless troubleshooting through this thesis project. Most of all, she has done a

tremendous job on editing every one of my essays, research proposal and the thesis,

which has helped me improve my writing greatly throughout these two years. She has

made sure that any work that was presented or written was the best it could be, by

constantly keeping me on track. Her endless capacity to help has been deeply

appreciated.

I would also like to thank all the past and present members of the Mulligan lab.

Special thanks go to Taran, Doug, Brandy, Shirley, Susan and Judy who have always been willing to help with science questions as well as experimental work. Taran, you have been a great friend and always inspired and helped me in every way through my masters.

Special thanks will also go to the Protein Function Discovery program in which I gained invaluable experience; I would especially like to thank Mr. Kim Munro for his great support as a teacher, and as a friend.

Finally, I would like to thank my family and Sunny for their constant comforting, encouragement and great support and love throughout this time. Thank you for

understanding what I do and for letting me follow my dreams. Last but not least, thank

you to all my friends who have been there for me and put a smile on my face. You all

mean a lot to me.

iv Table of Contents

Abstract ...... ii Acknowledgements ...... iv Table of Contents ...... v List of Figures and Tables ...... viii Abbreviations…………………………………………………………………………..viii Chapter 1 Introduction...... 1 1.1 Receptor Tyrosine Kinases…………………………………………………..1 1.2 Regulation of RTKs……………………………………………………….....2 1.2.1 The Lysosomal Pathway of RTK Degradation………………………………2 1.3 RET Receptor………………………………………………………………...3 1.4 RET Structure………………………………………………………………..5 1.5 RET Activation………………………………………………………………7 1.6 RET Signaling……………………………………………………………….7 1.7 RET Isoforms…………………………………………………………….…10 1.7.1 Differential Signaling of RET Isoforms……………………………………10 1.7.2 RET Isoforms and Differential Physiological Roles……………………….11 1.8 RET Regulation…………………………………………………………….12 1.9 The Proto-oncogene Cbl……………………………………………………13 1.9.1 A Bimodal Model for RTK and Cbl Interaction………………………....…16 1.10 Negative Regulation of Cbl by ARHGEF7………………………………...18 1.11 Rationale……………………………………………………………………20 1.12 Hypothesis………………………………………………………………….21 1.13 Objectives…………………………………………………………………..21

Chapter 2 Materials and Methods...... 24 2.1 Cell Lines …………………………………………………………………..24 2.2 Cell Culture…………………………………………………………………24 2.3 Constructs…………………………………………………………………..25 2.3.1 Plasmid DNA Isolations……………………………………………………28 2.4 Transient Transfections……………………………………………………..28 2.4.1 RET Expression and Activation…………………………………………….29 2.5 Protein Isolation…………………………………………………………….30 2.6 Protein Assay……………………………………………………………….30 2.7 Immunoprecipitations………………………………………………………30 2.8 Western blotting…………………………………………………………….31 2.8.1 Stripping Blots……………………………………………………………...34 2.9 In Vitro Kinase Assay……………………………………………………....34 2.10 Purification of Recombinant GST Proteins………………………………...35 2.10.1 GST Protein Binding on Beads…………………………………………….35 2.10.2 GST Protein Elution………………………………………………………...36 2.10.3 Analysis of Protein Purification………………………………..…………...36 v 2.11 Far Westerns………………………………………………………………..37

Chapter 3 Results ...... 39 3.1 Characterizing RET-Cbl Interaction………………………………………..39 3.2 Investigating Direct Binding of Cbl to RET9………………………………41 3.3 Investigating the Interactions Between RET, Cbl and ARHGEF7………...45 3.4 Investigating the Relationship Between RET and ARHGEF7……………..47 3.5 ARHGEF7 Phosphorylation is RET-ligand Dependent……………………50 3.6 RET Does Not Phosphorylate ARHGEF7 Directly………………………..54

Chapter 4 Discussion ...... 56 4.1 RET-Cbl Interaction………………………………………………………...56 4.2 Cbl Can Bind Directly to RET……………………………………………...58 4.3 Interactions Between the RET Receptor, Cbl, and ARHGEF7…………….59 4.4 RET- ligand Dependent Phosphorylation of ARHGEF7…………………...62 4.5 ARHGEF7 Phosphorylation by RET……………………………………….63 4.6 Summary and Conclusions…………………………………………………63 4.7 Significance and Future Directions…………………………………………64

References ...... 66

Appendix...... 73

vi List of Figures and Tables

Figures Page Figure 1.1 Mechanism of Endocytic Downregulation of RTKs, Using 4 EGFR as an Example Figure 1.2 Schematic Diagram of the RET Receptor Tyrosine Kinase 6 Figure 1.3 Diagram of RET Domains with Different Autophosphorylated 9 Tyrosines Figure 1.4 Cbl Interactions with RET Isoforms 14 Figure 1.5 Cbl Homologues 15 Figure 1.6 Schematic Diagram of ARHGEF7 Domains 19 Figure 1.7 A Model for The Effects of Phosphorylated ARHGEF7 on 23 RET Homeostasis

Figure 2.1 Intracellular RET Constructs 26

Figure 3.1 Both c-Cbl and Cbl-b Homologues Interact with RET 40 Figure 3.2 Purification of GST-Cbl-TKB 43 Figure 3.3 Direct Binding of Cbl-TKB to the Met and RET Receptors 44 Figure 3.4 RET9 and ARHGEF7 Do Not Interact in Co- 46 immunoprecipitations Figure 3.5 Interactions Between RET9, Cbl, and ARHGEF7 48 Figure 3.6 Interactions Between RET51, Cbl, and ARHGEF7 49 Figure 3.7 RET9 Activation is Required for ARHGEF7 Phosphorylation 51 Figure 3.8 RET51 Activation is Required for ARHGEF7 Phosphorylation 52 Figure 3.9 Timecourse Analysis of the GDNF-induced Phosphorylation of 53 ARHGEF7 Figure 3.10 RET Does Not Phosphorylate ARHGEF7 Directly 55

Figure 4.1 A Model of Cbl Binding to RET 60

Tables Table 2.1 Details of Constructs Used in This Study 27 Table 2.2 Details of Antibodies Used for Immunoblotting (IB), and 32 Immunoprecipitations (IP)

vii Abbreviations

ARHGEF7 (β-pix,Cool-1) Rho guanine nucleotide exchange factor 7 ATP adenosine triphosphate

bp base pair BSA bovine serum albumin

CC coiled coil C- terminal carboxyl terminus Cbl Casitas B-lineage lymphoma CMV cytomegalo virus CSF colony stimulating factor

DH Dbl homology DMEM Dulbecco’s modified eagle medium DNA deoxyribonucleic acid DOK downstream of tyrosine kinase

ECL electro chemiluminescence EDTA ethylenediaminetetraacetate EGF epidermal growth factor EGFR epidermal growth factor receptor ERK extracellular signal regulated kinase

FAK focal adhesion kinase FBS fetal bovine serum FKBP focal kinase binding protein FRS fibroblast growth factor receptor substrate

GBD GIT1 binding domain GDNF glial cell line-derived neurotrophic factor GFL glial cell line-derived neurotrophic factor family ligand GFRα GDNF family receptor alpha GIT1 G protein-coupled receptor kinase-interacting protein GST glutathione-S-transferase GRB growth factor receptor bound protein GTP guanosine triphosphate GEF guanine nucleotide exchange factor viii GPI glycosyl phosphatidyl inositol

HEK human embryonic kidney HIS histidine

IB immunoblot IP immunoprecipitation IPTG isopropylthio-β-D-galactoside IRS insulin receptor substrate

JNK Jun N-terminal kinase kD kilo Daltons

LB Luria- Bertani medium mA milli ampere MAPK mitogen-activated-protein-kinase MEN multiple endocrine neoplasia MEN 2B multiple endocrine neoplasia type 2B MVB Multivesicular bodies

NCK non catalytic region of tyrosine kinase N-terminus amino terminal

PAK p21 activated kinase PBS phosphate buffered saline PCR polymerase chain reaction PDGFR platelet-derived growth factor receptor PH plecstrin homology PI3K phosphatidylinositol 3 kinase PKA protein kinase A PLC-γ phospholipase C gamma PMSF phenylmethylsulphonylfluoride PRD proline rich domain PTB phosphotyrosine binding

RET rearranged in transfection RET-TET intracellular portion (amino acid 658 to C- terminus) of RET RING really interesting new gene

RPMI Roswell park memorial institute media ix RTK receptor tyrosine kinase rtTA reverse tetracycline transactivator

SDS sodium dodecyl sulfate SDS-PAGE SDS polyacrylamide gel electrophoresis SHC Src homology 2 domain-containing transforming protein C SH2 Src homology 2 SH3 Src homology 3 STAT signal transducer and activator of transcription

TBS tris-buffered saline TBST tris-buffered saline tween 20 TEMED N, N, N’, N’-tetramethylethylenediamine TET tetracycline TKB tyrosine kinase binding TRE tetracycline response element

UBA ubiquitin associated UBD ubiquitin binding domain UV ultra violet

V volt

WCL whole cell lysates WT wild type

x Chapter 1

Introduction

1.1 Receptor Tyrosine Kinases

Receptor tyrosine kinases (RTKs) are transmembrane proteins that have intrinsic

enzymatic activity (Reviewed in [1]), and are responsible for activating several signaling

pathways that control growth, proliferation, differentiation, survival, and migration of

cells (Reviewed in [2]). Signal transduction is a mechanism by which an extracellular

signal is passed into the cell. Growth factors act as extracellular ligands for RTKs,

inducing dimerization, and subsequent autophosphorylation of intracellular tyrosines.

Phosphorylation occurs by translocation of γ-phosphates from adenosine triphosphate

(ATP) molecules, to tyrosine residues on the RTK [3], which subsequently act as docking sites for proteins involved in cellular signaling pathways [1].

RTKs are distinguished by a characteristic domain structure: a variable extracellular ligand binding domain is followed by an hydrophobic transmembrane domain. A highly conserved intracellular tyrosine kinase domain is responsible for transphosphorylation upon activation of the RTK [3, 4]. Lastly, an intracellular carboxy- terminal tail of the RTK is thought to be responsible for an increased range of substrate binding [3, 4].

The critical role of RTKs in various cellular processes demands tight regulation and proper homeostasis for normal function. It is therefore not surprising that

1 hyperactivity of RTKs, caused by activating mutations or impaired signal termination, is strongly associated with tumour progression (Reviewed in [5]).

1.2 Regulation of RTKs

Signal termination is controlled by a number of different mechanisms, with one major deactivation pathway being the downregulation of RTK activity (Reviewed in [5,

6]). One such form of regulation is carried out by a complex machinery of ubiquitin ligases that catalyze the targeted degradation of cellular proteins (Reviewed in [7]).

Monoubiquitination of the receptor involves ligand-induced internalization, and provides recognition signals for regulation of endosomal trafficking, followed by degradation in lysosomes. Polyubiquitination, on the other hand, tags the receptor for proteasomal degradation [5]. Recent research has proved that RTKs are monoubiquitinated at multiple sites [8], and that ubiquitination targets most RTKs for internalization and subsequent lysosomal degradation [5].

Thus, the homeostasis of RTKs can be controlled by two types of reversible modifications; phosphorylation of tyrosine residues recruits docking proteins and initiates signaling events, while the addition of the small protein, ubiquitin, targets the receptor for degradation (Reviewed in [5]).

1.2.1 The Lysosomal Pathway of RTK Degradation

Ligand-induced degradation of RTKs usually involves ubiquitination on lysine residues of autophosphorylated receptors [9], by ubiquitin ligases [5]. Endocytosis is 2

initiated by the assembly of clathrin-coated vesicles, which then detach from the plasma

membrane by fission, mediated by the GTPase Dynamin [10]. The clathrin coated vesicle

then forms an early endosome by fusion with internal vesicles, maturing into a late

endosome as it becomes progressively acidic and changes morphology, as well as

cytosolic localization [5]. At the early endosomal stage, the receptors may be recycled

back to the plasma membrane, or be targeted to lysosomes [11, 12]. Interestingly, some

RTKs can continue to signal after internalization, contributing to sustained cellular

signaling [13]. Further segregation in late endosomes involves invaginations of the

endosomal membrane to form internal vesicles, which characterizes the multivesicular

bodies (MVB) [8]. Ubiquitination acts as a signal for movement into the MVB pathway

by interactions with trafficking proteins that contain ubiquitin-binding domains (UBD)

[14]. Subsequent to segregation, the MVB fuses with the lysosome and delivers its internal contents for degradation by lysosomal hydrolases [8]. Note in this pathway, that internalization and degradation are distinct events; ubiquitination acts at the level of endosomal sorting, rather than as a required signal for clathrin-dependent internalization

[12, 14-16]. A simplified diagram for lysosomal degradation is shown in figure 1.1, using

EGFR as an example, as downregulation of this RTK is extensively studied [5].

1.3 RET Receptor

The RET (REarranged during Transfection) proto-oncogene encodes a receptor

tyrosine kinase involved in development of the kidney [17], and the enteric nervous

Figure 1.1 Mechanism of Endocytic Downregulation of RTKs, Using EGFR as an

Example. EGF binding triggers receptor autophosphorylation via receptor dimerization.

Cbl associates with activated EGFR, and is phosphorylated by it, which enables it to

catalyze ubiquitination (U) of the RTK at multiple sites. This facilitates endocytosis of the receptor via clathrin-coated pits. Clathrin polymerization, invagination of the membrane, and vesicle formation are enhanced and mediated by several proteins, omitted in the diagram for simplicity. The endocytic vesicle uncoats and fuses with an early endosome. Endosomal sorting of the RTK is mediated through ubiquitin. The RTK is probably dephosphorylated and deubiquitinated during this sorting step. Segregation within the early endosome, by formation of internal vesicles, forms multivesicular bodies

(MVB).The MVB then fuses with late endosomes or lysosomes, delivering its contents for subsequent degradation by lysosomal hydrolases. Note that several details are omitted in this diagram for generalization and simplicity. Modified from [5]. U: Ubiquitin, P:

Phosphate, MVB: Multivesicular body.

4 4a system [17, 18]. RET is primarily expressed in the kidney and neural crest derived cells; these include connective tissue around the brain and spinal cord, and parasympathetic and sympathetic ganglia [17]. RET activates several intracellular signaling cascades, which regulate cell survival, differentiation, proliferation, migration, chemotaxis, neurite outgrowth and synaptic plasticity (Reviewed in [19]). Mutations in RET are associated with a number of pathological conditions such as human papillary thyroid tumours, multiple endocrine neoplasia type 2, and Hirschprung disease; a congenital defect in the development of the enteric nervous system [20-24].

1.4 RET Structure

RET shares structural similarities with other RTKs, consisting of three domains: the extracellular ligand-binding domain that contains four cadherin-like repeats and a cysteine-rich region; a transmembrane domain; and an intracellular region containing the tyrosine kinase domain (Figure 1.2) [3, 25]. The extracellular domain possesses a unique domain with homology to the cadherin family, categorizing RET as the only member of the cadherin superfamily with a tyrosine kinase domain [25]. The cadherin-like domain can bind calcium molecules, and is required for rigidity of the receptor [25]. It is also essential to RET ligand and co-receptor binding [26], as well as for homodimerization of

RET monomers [25]. Following the cadherin-like domain, is a highly conserved cysteine- rich domain containing 27 cysteines, which are involved in the formation of intramolecular disulfide bonds, for formation of proper tertiary structure of RET [27].

Figure 1.2 Schematic Diagram of the RET Receptor Tyrosine Kinase. RET is composed of three domains: an extracellular ligand-binding domain, a single transmembrane-domain, and an intracellular kinase domain. The extracellular domain includes four cadherin-like domains, and a cysteine rich region. The intracellular domain includes a kinase domain, and a carboxy terminal domain. The three alternatively spliced isoforms of RET differ in their carboxy-terminal tails and are indicated as RET51,

RET43 and RET9.

6 6a

Mutations in the cysteine rich region can lead to constitutive activation or reduced receptor expression at the cell surface [28, 29]. The intracellular domain of activated RET contains multiple phosphorylated tyrosines that contribute to the activation of signalling pathways [30-32]. The phosphorylation sites of RET and their respective docking partners will be discussed below.

1.5 RET Activation

RET is unique amongst RTKs in its activation process, in that it requires a complex formation with co-receptors, in addition to a ligand [33, 34]. The RET ligands belong to the glial cell line derived neurotropic factor (GDNF) family of ligands (GFLs), which include four ligands: GDNF, neurturin, persephin and artemin [33-35]. These ligands preferentially bind to glycosyl phosphatidyl inositol (GPI)- anchored co-receptors of the GDNF family receptors α (GFRα1-4) [34]. GDNF binds to GFRα1, and the formation of this complex is thought to recruit RET into membrane lipid rafts, areas high in sphingolipid and cholesterol concentration, inducing homodimerization of RET [36].

Neurturin binds to GFRα2, artemin to GFRα3, and persephin to GFRα4; although some crosstalk between co-receptors and ligands has been observed [34, 35, 37, 38].

1.6 RET Signaling

The intracellular domain of activated RET contains multiple phosphorylated tyrosines which serve as docking sites for various Src-homology 2 (SH2) domain and

phosphotyrosine binding (PTB) domain-containing signaling molecules (Reviewed in

[19]). Such signaling molecules include STAT3 [39, 40], Grb7/10 [41, 42], NCK [43],

Src [44], phospholipase C γ (PLCγ) [45], Shc, FRS1,2, Dok1,2,4-6 and IRS1,2 [46-49].

Additionally, ENIGMA can interact in a phospho-independent manner through its LIM-

domain [50, 51]. RET contains eighteen tyrosine (Y) residues, of which Y687, Y752,

Y928, Y905, Y952, Y981, Y1015, Y1062, and Y1096 have been shown to activate downstream signaling pathways [30, 40-42, 50-58]. There are, however, fifteen known autophosphorylation sites, including Y806, Y809, Y826, Y864, Y900, Y1029, and

Y1090 in addition to the ones mentioned above [55, 56]. Crucial to RET signaling are

Y905 and Y1062. Phosphorylation of Y905 is required for RET kinase activity, and phopshorylated Y1062 is a major docking site for numerous adaptor molecules including

Shc, FRS1,2, Dok1,2,4-6 and IRS1,2 [46-49]. The current knowledge of RET phosphotyrosines and their respective docking partners is summarized in Figure 1.3.

Activated RET initiates a number of complex signaling cascades, including the

RAS/ERK, PI3K/AKT, and JNK pathways (Figure 1.3) [30, 31, 59]. Some of the best

characterized signaling pathways initiated downstream of RET, are the RAS/ERK and

PI3K pathways. Initiation of the RAS/ERK pathway is mediated by Shc, FRS1, 2, DOK

1,2,4-6, IRS2 and Grb7/10, leading to activation of the downstream MAP kinase cascades

[30, 46-49]. Activation of these signaling cascades can lead to cellular survival,

proliferation, differentiation and migration [60-62]. The PI3K pathway is another

essential target of RET, which is mediated by Shc and Src, leading to cell survival and

proliferation [63-65]. Activation of the RAS/ERK and PI3K pathways are also necessary

Figure 1.3. Diagram of RET Domains with Different Autophosphorylated Tyrosines.

Upon activation, several tyrosines in the intracellular domain of RET become autophosphorylated. These phosphotyrosines act as docking sites for various SH2- or

PTB-domain containing substrates. Phosphorylation of the substrates can then activate various downstream signaling pathways, such as the RAS-ERK and PI3K-AKT pathways. The figure also shows the two predominantly expressed RET isoforms, RET9 and RET51, and the additional tyrosine on RET51 which acts as a docking site for Grb2.

9 NCK

GRB2 CBL

CBL

for the migration of enteric neural crest cells [66]. Essential to this, is the phosphorylation

of Y687 and S696, which have been shown to play antagonistic roles in lamellipodia formation. Whereas phosphorylation of Y687 is believed to negatively regulate the activity of the Rac-1 guanine nucleotide exchange factor (GEF) and lamellipodia formation [66], phosphorylation of S697 by Protein Kinase A (PKA), allows for Rac-1 activation. It is thought that phosphorylation of S696 induces conformational changes in

the juxtamembrane region of RET, which lead to the inhibition of Y687 phosphorylation

[66].

1.7 RET Isoforms

Alternative splicing of the RET proto-oncogene results in three isoforms: RET9,

RET43, and RET51. These isoforms differ in their cytoplasmic C-terminal tails of 9, 43,

and 51 residues, respectively [67, 68]. RET9 and RET51 are generally co-expressed and more predominant than RET43 [67, 69, 70]. The C-terminal tail of RET9 and RET51 are also more conserved across multiple species than that of RET43 [70, 71].

1.7.1 Differential Signaling of RET Isoforms

All three RET isoforms have Y1062 as the last common tyrosine residue, which acts as a docking site for PTB domain containing proteins [46-49] (Figure 1.3). However,

the unique C-terminal amino acids of RET isoforms can influence the binding of

substrates at Y1062, and thus, for RET9, Y1062 is also an SH2-domain binding site [47,

48, 50, 72, 73]. RET51 has two additional tyrosines, Y1090 and Y1096, of which Y1096

has been recognized as a docking site thus far [53, 74]. Phosphorylated Y1096 (pY1096)

serves as a binding site for the Grb2 adaptor protein which contributes to activation of the

PI3K and Ras/MAPK pathways [53]. In addition to the differential recruitment of

signaling complexes, when comparing phosphorylation patterns of RET isoforms, phosphorylation of Y905 and Y1062 is greater and more sustained in RET9 than RET51

[75, 76]. However, Y1015 is more phosporylated in RET51 [75].

Considering the differences in signaling and phosphorylation patterns of RET isoforms, it is not surprising that this results in distinct physiological roles attributed to each isoform (Reviewed in [19]).

1.7.2 RET Isoforms and Differential Physiological Roles

Relative RET9 and RET51 expression levels have been shown to change during embryonic and postnatal development [67, 69, 76]. The physiological role of the RET9 and RET51 isoforms has been demonstrated in mono-isoformic transgenic mouse studies

[17]. RET51 mono-isoformic mice show severe deficits in the enteric nervous system and kidney development, similar to RET null mice, whereas RET9 mono-isoformic mice have milder impairments and show normal embryonic and postnatal development. In addition, the expression of RET9 is able to rescue the effects of RET null mutations in mice, whereas RET51 is not, suggesting hypomorphicity of the RET51 allelle [76].

Conversely, RET51 is required for growth of mature sympathetic neurons, and has been shown to increase neurite outgrowth, and to induce differentiation to a greater extent than RET9 [48, 75, 77, 78]. Interestingly, RET51 has also been shown to be more

transforming than RET9 in in vitro assays [48, 78, 79]. These studies demonstrate how

alternative splicing can lead to functional differences between RET isoforms (Reviewed

in [19]). The molecular basis underlying the functional differences, however, is not fully

understood, and the paradox of how a hypomorphic allele can be accompanied by a

higher transforming ability, is yet to be elucidated.

1.8 RET Regulation

Recently, there has been an increased interest in the negative regulation of RTKs,

and its implications in potentiating oncogenesis (Reviewed in [5]). In addition to

hyperactivity of the receptors, any impairment in the negative regulation of RTKs can

also cause increased mitogenic signaling [5]. Many studies have shown that Cbl plays a major role in ligand-dependent downregulation of RTKs [80, 81]. Activated RTKs provide a docking site for Cbl, which recruits E2 ubiquitin ligases that catalyze the ubiquitination and subsequent deregulation of the RTKs [5]. As for other RTKs, RET is regulated carefully, and it has been demonstrated that negative regulation of RET is mediated by Cbl [74]. As a step to understanding the mechanisms by which RET is regulated, Scott et al. [74] showed that RET9 and RET51 are regulated differently, which may explain some of their functional differences. In this study, a higher turnover rate for

RET51 than for RET9 was seen, consistent with a higher degree of ubiquitination of

RET51. This may be attributed to the differences seen in the interactions between the

RET isoforms and Cbl, where a stronger association was seen between RET51 and Cbl

[74]. While the common residue, Y1062, was identified as a site of interaction for Cbl in

both RET isoforms, an additional Cbl-interaction site, Y1096, was identified for RET51

[74]. Both binding sites mediated an indirect interaction between RET and Cbl through adaptor proteins [7, 74]. In both RET9 and RET51, Shc docks at pY1062 [73], and recruits Grb2, which binds to the proline-rich domain of Cbl, such that a trimeric complex of Shc-Grb2-Cbl [74] binds to RET (Figure. 1.4). In RET51, the direct Grb2- binding site at Y1096 provides an additional mechanism by which Cbl binding can occur

(Figure. 1.4). The additional Cbl-binding site in RET51 may contribute to the more extensive ubiquitination pattern, the stronger association with Cbl, and the increased degradation of RET51, as compared to RET9 [74].

1.9 The Proto-Oncogene Cbl

Cbl (Casitas B-lineage Lymphoma) is an ubiquitously expressed protein with highest expression in hematopoetic cells and testis (Reviewed in [7, 82]). It was first discovered when the Cas- Br-M retrovirus, which induced hematopoetic tumours in mice,

was isolated. The oncogene responsible for the tumourigenesis was named v-Cbl and, since then, three mammalian homologues have been identified, namely the proto- oncogenes c-Cbl, Cbl-b and Cbl-3 (Figure 1.5) (Reviewed in [7]). All three isoforms show structural similarity, with highly conserved N-terminal regions, especially in the tyrosine kinase binding (TKB) domain and RING-finger domains (Figure 1.5) [7]. The

TKB region can bind directly to receptor tyrosine kinases, such as EGFR [83] , Met [84],

Kit [85], and PDGFR [86], and to cytoplasmic tyrosine kinases such as Src [87], Fyn

[88],

Figure 1.4. Cbl Interactions with RET Isoforms. A. Upon ligand activation (yellow) of

RET, phosphorylated Y1062 acts as a docking site for the adaptor molecule Shc, which further recruits Grb2 and Cbl. B. While the common residue, Y1062 is a site of interaction for Cbl in both RET isoforms, an additional Cbl-interaction site, Y1096 is found on RET51 [74], which can directly bind Grb2.

14 14a

Figure 1.5 Cbl Homologues (Modified from [7]). The figure shows three mammalian homologues of Cbl; c-Cbl, Cbl-b and Cbl-3, as well as the oncogene named v-Cbl which was first identified in the Cas-Br-M retrovirus. All Cbl proteins have a high degree of sequence homology between their TKB domains (N-terminal), which consists of a four- helix domain (4H), a Ca 2+ -binding EF domain (EF), and an SH-2 domain. v-Cbl can independently bind activated RTKs, and it is also oncogenic. Furthermore, a linker-region

(L) links the TKB domain to a RING-finger domain (RING). The mammalian homologues also have conserved proline-rich regions in their carboxy-terminals, and a

UBA domain, which refers to its homology with ubiquitin associated domains [7] .

15 15a

and Syk [88]. Upon binding to these tyrosine kinases, Cbl can be activated by

phosphorylation at Y700, Y731 and Y774 [89] . These phosphotyrosines can then act as dockings sites for SH2-domain containing proteins, such as Vav [90]. The RING finger

domain enables Cbl to act as an E3-ubiquitin ligase by recruiting the ubiquitin activating

enzymes, E1, and the ubiquitin conjugating enzymes, E2 [91]. Furthermore, c-Cbl and

Cbl-b, but not Cbl-3, contain proline-rich motifs, which enable them to bind to SH3-

domain containing proteins [7]. Lastly an ubiquitin-associated (UBA) domain is found in

the C-terminals of c-Cbl and Cbl-b [92]. UBA domains are characterized by hydrophobic

patches that recognize their binding partners, and have been shown to have affinity for

ubiquitin, especially in the Cbl-b homologue [92]. It was recently shown that the UBA

domain allows for both homo- and heterodimerization between the c-Cbl and Cbl-b homologues, and that this domain is required for efficient tyrosine phosphorylation of

Cbl [93]. An intact c-Cbl UBA domain was also required for efficient Met ubiquitination

[93].

1.9.1 A Bimodal Model for RTK and Cbl Interaction

In addition to the indirect Cbl binding sites on RTKs, direct binding sites for the

Cbl TKB-domain are found in EGFR [83], Met [84], Kit [85], and PDGFR [86]. A consensus for direct binding of the Cbl TKB domain (D/NXpYXXD/E/Φ) has previously been established from studies of other Cbl-binding tyrosine kinases [94]. It had recently been shown by Peschard et al. [95] that the Cbl TKB domain can bind directly to Met at

Y1003. Interestingly, the recognized Cbl TKB binding consensus sequence does not exist 16

in the Met kinase, but an atypical, conserved DpYR motif in the juxtamembrane domain

of the Met receptor was identified as a Cbl TKB-binding site [95]. These findings suggest that a general TKB-binding consensus sequence for c-Cbl is not limited to the previously identified sequence, and that the binding motif for Cbl-TKB may be less stringent than previously believed [95].

In a similar manner, the loss of the direct Cbl-TKB-binding site in other RTKs such as CSF-1R [96], EGFR [83], and Kit [85], results in an increased transforming activity (Reviewed in [97]). For instance, for EGFR it has been shown that substitutions of Y1045, which is the Cbl-TKB binding site, enhance the mitogenic response [80, 98].

This is due to the accelerated recycling of the mutant receptor and a concomitant defect in ligand-induced ubiquitination and endocytosis of EGFR [80, 98]. These observations suggest that loss of Cbl-TKB binding may be a common mechanism that contributes to full oncogenic activation of RTKs [80].

To further demonstrate the role of Cbl, a loss of c-Cbl and Cbl-b binding prevents ubiquitination, and thus degradation, of Met. Met mutants that have impaired ability to bind Cbl are transforming and tumorigenic, due to increased stability and sustained signaling by Met [84].

In view of the above discussion, the role of Cbl as an E3-ubiquitin ligase, and its interaction with tyrosine kinases, enables it to provide a powerful mechanism for maintaining homeostasis of RTK activity (Reviewed in [5]).

1.10 Negative Regulation of Cbl by ARHGEF7

One of the regulatory mechanisms for controlling RTK activity is through signal

termination. Events that reduce or block downregulation of RTK signaling may have

similar effects to mechanisms increasing RTK signaling, as has been described above for

a loss of Cbl binding [97]. Another such mechanism is mediated through the Rho Guanine

Nucleotide Exchange Factor 7 (ARHGEF7) [99, 100]. ARHGEF7, also known as the p21

activated kinase (PAK) interacting exchange factor, β-pix, or Cool-1 [101], is an ubiquitously expressed protein, with highest expression levels in skeletal muscle [102], and has two isoforms, p50 and p85 ARHGEF7, possibly arising from alternative splicing

[103, 104]. ARHGEF7 contains a Src homology 3 (SH3) domain, tandem Dbl homology

(DH) and plecstrin homology domains (PH), a GIT1 binding domain (GBD), as well as a coiled coil (CC) domain for oligomerization (Figure 1.6) [104]. The best characterized role of ARHGEF7 has been its involvement in cytoskeletal rearrangements, as it is a guanine nucleotide exchange factor (GEF) specific for Rho GTPases such as Cdc42, Rho and Rac [105, 106]. These proteins have been implicated in regulation of the actin cytoskeleton, as well as in the formation of focal adhesion complexes [107]. ARHGEF7 is widely expressed in these focal adhesion complexes, and recruits PAK to these sites, thereby inducing membrane ruffling through activation of Cdc42 and Rac1 [102].

Whether ARHGEF7 exhibits GEF activity remains controversial, however several studies do suggest some GEF activity [100, 102, 108]. Interestingly, ARHGEF7 can also heterodimerize with its homologue ARHGEF6 (α-pix/ Cool-2) [109], which exhibits strong GEF activity [110].

Figure 1.6. Schematic Diagram of ARHGEF7 Domains. ARHGEF7 contains Src homology 3 (SH3), Dbl homology (DH), Plecstrin homology (PH), G-protein coupled receptor kinase interactor-binding (GBD), and Coiled-Coil (CC) domains. The specific protein interactions these domains have been shown to mediate are also illustrated.

19 Y443 19a

Although initial studies of ARHGEF7 have focused on its role in cytoskeletal rearrangements [103, 109], recent studies have shown a novel role for ARHGEF7 as a negative regulator of downregulation [100]. It was discovered that ARHGEF7 is able to bind to Cbl through its SH3 domain [99]. Interestingly, ARHGEF7 can act as a modulator of EGFR degradation through either sequestration of Cbl in the cytosol [99], or through inhibiting EGFR-mediated activation of Cbl, when Cbl is bound to the receptor [100].

ARHGEF7 is activated by phosphorylation at Y443 in an EGF-dependent manner, which is mediated through the downstream cytoplasmic kinases Src, and the focal adhesion kinase, Fak [100]. Subsequent activation of the Rho GTPase Cdc42 leads to the formation of a trimeric complex with Cbl, depleting its cytosolic availability [100].

Formation of the trimeric complex leads to EGFR accumulation and increased cellular transformation [100]. Thus, ARHGEF7 can play a role in both metastasis and tumourigenesis; it can control cell spreading [111] and is required for cell motility [105], and it acts as a negative regulator of the downregulator Cbl [100]. ARHGEF7- mediated regulation of downregulation may therefore be a general mechanism for controlling the activity of RTKs, and perhaps an alternative mechanism contributing to oncogenesis.

1.11 Rationale

There is evidence that Cbl interacts indirectly with RET, and that it mediates its downregulation [74]. Cbl also plays a vital role in downregulation of other RTKs, by interacting in a bimodal manner through both direct and indirect interactions [83, 85]. For instance, for EGFR it has been shown that substitutions of Y1045, which is the Cbl-TKB

binding site, enhance the mitogenic response [80]. Based on data from other RTKs, we

might predict that a similar interaction may occur for RET and Cbl.

Further, it has been shown that ARHGEF7 can interact with Cbl upon

phosphorylation [100]. This phopshorylation has been shown to occur downstream of

EGFR, through Src and Fak [100]. It is possible that ARHGEF7 may also be

phoshorylated downstream of RET, in a manner similar to that seen for EGFR (Figure

1.7). Furthermore, ARHGEF7 has been shown to play a modulatory role in EGFR-Cbl interactions [100], and similar roles may exist for ARHGEF7 in modulating the relationship of Cbl and RET.

1.12 Hypotheses

In addition to the previously established indirect interaction between RET and

Cbl, we hypothesize that RET can interact directly with Cbl, through its TKB domain.

Cbl can also interact with ARHGEF7, possibly forming a complex with RET and

ARHGEF7. Thus, an interaction between RET and ARHGEF7 may be mediated through

Cbl. Further, we hypothesize that RET activation can lead to phosphorylation of

ARHGEF7, either directly, or indirectly.

We will address this hypothesis with the following objectives:

1.13 Objectives

1) To examine and characterize RET- Cbl binding.

2) To investigate the interactions between RET, Cbl and ARHGEF7, using both RET9 and RET51.

3) To look for a possible relationship between RET9 and RET51, and ARHGEF7.

Figure 1.7. A model For the Effects of Phosphorylated ARHGEF7 on RET

Homeostasis.

We predict that the phosphorylation of ARHGEF7 is initiated by GDNF binding. This may be mediated by Src and Fak. The effect of this phosphorylation is two-fold: 1) Upon activation, ARHGEF7 is able to bind to both Cdc42 and Cbl, sequestering Cbl and preventing it from catalyzing downregulation of RET. 2) An alternative pathway by which ARHGEF7 can block RET ubiquitination is by forming a stable complex with Cbl and RET. ARHGEF7 prevents Cbl from being phosphorylated by RET, thereby inhibiting Cbl activity. The diagram also illustrates the two RET isoforms and their interactions with Cbl (modified from [100]).

23 23a

Chapter 2

Materials and Methods

2.1 Cell Lines

TET-ON 293 cells were purchased from BD Biosciences Clonetech (Mississauga,

ON, CA). These cells are derived from human embryonic kidney (HEK), and have been stably transfected with the Reverse Tetracycline Transactivator (rtTA). HEK293 cells were provided by Dr. R. Deeley (Department of Pathology and Molecular Medicine,

Queen’s University). Stable cell lines (HEK293) expressing either full length RET9 or

RET51, together with GFRα1, were generated by S.Myers. A murine mammary carcinoma cell line, SP1, was generously provided by Dr. B. Elliott (Department of

Pathology and Molecular Medicine, Queen’s University).

2.2 Cell Culture

The HEK293, TET-ON 293, and SP1 cell lines, were cultured in Dulbecco’s modified Eagle’s Medium (DMEM) (Sigma Aldrich, Oakville, ON, CA), supplemented with 10% Fetal Bovine Serum (FBS) (Sigma Aldrich, Oakville, ON, CA), and 5% sodium pyruvate. Cells were maintained at 37ºC with 5% CO2, and medium was changed every

2-3 days. The cells were split upon reaching 80-90% confluency. SP1 cells were treated with TrypLE™ Express (Invitrogen, Burlington, ON, CA) for 2 minutes at 37ºC, before splitting.

2.3 Constructs

The full length RET9 and RET51 constructs generated in the CH269 vector [112], and in the pcDNA3.1 vector [113], and the GFRα1 constructs have previously been described [112]. Wildtype intracellular RET (RET-TET) constructs previously generated

(amino acids 653-1114), have a CMV promoter and a tetracycline- response element

(TRE), which controls expression, and is induced by doxycycline (Department of

Pathology and Molecular Medicine, Queen’s University) [13, 113, 114] (Table 2.1). The

RET-TET proteins contain a myristylation signal for membrane localization, and two modified focal kinase binding protein (FKBP) domains, for artificial dimerization upon induction with a synthetic dimerizer [13, 113, 114] (Figure 2.1)

The GST- tagged Cbl-N (Cbl TKB) construct was provided by Dr. M. Park

(Department of Oncology, McGill University) [84, 115]. HA-tagged c-Cbl was donated by Dr. W. Langdon (Department of Pathology, University of Western Australia) [116], and HA Cbl-b was acquired from Dr. S. Lipkowitz (National Cancer Institue, Bethesda)

[117]. FLAG-tagged ARHGEF7 was generously provided by Dr. A. Mak (Department of

Biochemistry, Queen’s University) [118]. The purified HIS- tagged ARHGEF7 protein was a gift from Dr. Zongchao Jia (Department of Biochemistry, Queen’s University)

[119], and the GST- RET protein was previously purified in our lab [113]. Table 2.1 summarizes the constructs used in this study.

Figure 2.1 Intracellular RET Constructs (RET-TET).

Intracellular RET constructs contain a TET responsive element (TRE), a cytomegalovirus promoter (CMV), a myristylation domain and two dimerization domains (FKBP). These domains are fused to the intracellular portion of RET encoding amino acids 653 to the C- terminus, depending on the RET isoform.

26 26a

Table 2.1 Details of Constructs Used in This Study

Construct Mutation/truncation Description References Name RET9 [13, 113, RET-TET9 N/A Wildtype- intracellular 114] RET9 N/A Wildtype- full length [112] RET51 N/A Wildtype- full length [112] ARHGEF7 FLAG- N/A Wildtype [118] ARHGEF7 SH3, DH, and PH Purified recombinant HIS-ARHGEF7 [119] domains ** protein Intracellular portion of Purified recombinant GST-RET [113] RET protein Cbl HA c-Cbl N/A Wildtype [116] HA Cbl-b N/A Wildtype [117] GST-Cbl-N TKB domain of Cbl Can bind directly to RTKs [84, 115]

* Wildtype refers to full length constructs with no mutations or truncations, unless otherwise indicated. ** HIS-ARHGEF7 was initially thought to range from aa 1-647, however during the course of this thesis revision, the sequence of the construct was not verified, and may not include the primary tyrosines Y443 [100], and Y542 [120].

2.3.1 Plasmid DNA Isolations

For large scale plasmid DNA isolations, plasmids were first transformed into

E.Coli DH5α using electroporation. Electroporation was carried out using a Biorad Gene

Pulser II® (Biorad Laboratories, Hercules, CA) in which an electroporation cuvette, containing 80µL of electrocompetent DH5α cells mixed with 1 µL of DNA, was placed.

The mixture was subjected to electroporation at 2500V, with a resistance of 200Ω and capacitance of 25µF. Upon electroporation, the cells were taken up in 250 µL of LB medium (appendix), and incubated at 37 ºC for 30 minutes. After incubation, the cells were grown on LB agar plates with 50 mg/mL of ampicillin overnight. A single colony was inoculated into LB medium (appendix) with 50 mg/mL ampicillin, and grown overnight at 37ºC with vigorous shaking. DNA isolation was performed using the Qiagen

Midiprep Kit (Qiagen, Mississauga, ON) according to the manufacturer’s instructions.

2.4 Transient Transfections

For tranfections, Lipofectamine Reagent 2000 (Invitrogen, Burlington, ON) was used. For transfections into 100mm dishes, 1.0 x 106 cells were plated and grown over night. 750µL of OPTI-MEM ® Reduced Serum Medium (Invitrogen, Burlington, ON), was pipetted into two sets of eppendorf tubes. A 3:1 ratio of Lipofectamine to DNA was used, with 3 µg of each DNA construct. DNA was pipetted into one set of eppendorf tubes, and lipofectamine was pipetted into the other set. After 5 minutes of incubation at room temperature, the contents of the tubes were combined, and incubated for an

additional 30 minutes. The transfection mixture was then pipetted dropwise onto the

plates, without removal of medium. The cells were grown for 48 hours, and the medium

was changed after approximately 6 hours. For RET- ligand dependent experiments, the cells were serum starved after 24 hours, by growing the cells in DMEM supplemented with 0.5% FBS.

For transfections into 6-well plates, 1.0 x 105 cells were plated, and 1/3 of the

reagent volumes, described above, were used per well. The DNA mass was also reduced

to 2 µg per well.

2.4.1 RET Expression and Activation

In the HEK293 TET-ON cell lines, which use the tetracycline responsive

inducible expression system, expression induction and RET activation involved two

steps. For induction of RET expression, the cells were treated with 1 µg/mL of

doxycycline (Sigma Aldrich, Oakville, ON, CA) 24 hours post transfection, and grown for

an additional 24 hours. RET activation was induced using 1 µmol/L of AP29187

dimerizer (ARIAD Pharmaceuticals, Cambridge, MA), for 30 minutes prior to protein

isolation [13, 113].

HEK293 cells that were either stably expressing the full length RET proteins and

GFRα1, or that were transiently transfected with these constructs, were treated with 100 ng/µL of GDNF (Pepro Tech, Inc., Rocky Hill, NJ), 30 minutes prior to protein isolation.

2.5 Protein Isolation

Proteins were harvested 48 hours post transfection, by removal of medium and

subsequent washing and lysis. The cells were first washed three times with 1X PBS pH

7.4 (appendix), lysed with 500 µL or 250 µL of 1x lysis buffer (appendix), for 100mm

and 6- well plates, respectively. The cells were gently removed from the plate using a cell

scraper and pipetted into eppendorf tubes. The lysis mixture was then rocked at 4ºC for

30 minutes before pelleting the cells at 13000g at 4ºC, using a Fisher Scientific Accuspin

Micro centrifuge. 50 µL of each lysate supernatant was mixed with Laemmli buffer

(appendix) in a 1:1 ratio, and 12.5 µL of each lysate was retained for protein assay.

2.6 Protein Assay

Protein assays were performed using a BCA Protein Assay Kit (Pierce

Biotechnology, Rockford, IL, US). Each sample was diluted in a 1:1 ratio with 1x lysis

buffer (appendix), and 25 µL was loaded in each well of a 96-well plate. The protein

concentration was determined from absorbance readings at 595 nm by an ELx 800 UV microplate autoreader (Bio-Tex Instruments Inc, USA).

2.7 Immunoprecipitations

400- 500 µg of lysates were used for immunoprecipitation with an antibody dilution of 1:50 per sample. The samples were incubated with the antibody for 2 hours at

4ºC on a rocker, and the complexes were collected on a 1:10 dilution of Protein A/G

beads (Santa Cruz Biotechnology, Santa Cruz,CA) for another 2 hours. The samples

were then washed three times with 1x lysis buffer, by spinning at 4000g for 2 minutes in

a Fisher Scientific Accuspin Micro centrifuge, and finally diluted in 60 µL of Laemmli

buffer (appendix).

2.8 Western blotting

Samples were heat-denatured at 99ºC for 5 minutes, together with a pre-stained

protein marker (New England Biolabs, Mississauga, ON), centrifuged at 13000g, and

kept on ice prior to SDS-PAGE on 10% gels. 10% SDS-PAGE resolving gels

(appendix) were made, and poured into a mini PROTEAN apparatus (Biorad

Laboratories Inc, Hercules, CA), and topped up with distilled water. 4% stacking gels

(appendix) were poured upon settling of the resolving gel, and removal of the distilled water. The gels were run using the Biorad mini PROTEAN running system, with 500 mL of running buffer (appendix) at 20 mA per gel, for the desired time.

Upon completion, the proteins on the gel were transferred onto a nitrocellulose membrane (Biorad Laboratories Inc, Hercules, CA) using a Biorad mini PROTEAN transfer apparatus, with transfer buffer (appendix) and 0.1% SDS, at 150 mA for 1-2 h, depending on the size of the proteins of interest. For larger proteins, the transfer was performed for a longer time period (2 hours).

After transfer, the nitrocellulose membrane was blocked in 5% milk in TBST

(appendix) for 1 hour at 37ºC with shaking. The membrane was then immunoblotted with appropriate antibodies (Table 2.2) at a dilution of 1:2000 or 1:1000, in 5% milk in TBST, either for 1h at 37ºC with shaking, or overnight at 4ºC. Secondary antibodies were used at

31 Table 2.2 Antibodies Used for Immunoblotting (IB), and Immunoprecipitations (IP).

Antibody Supplier Detection Type Use

Santa-Cruz 19 C-terminal RET9 (C19) Biotechnology Inc. amino acids of Primary IB, IP (Santa Cruz, CA, USA) RET9 Santa-Cruz 20 C-terminal RET51 (C20) Biotechnology Inc. amino acids of Primary IB, IP (Santa Cruz, CA, USA) RET51 Santa-Cruz Extracellular RET (H300) Biotechnology Inc. domain of full Primary IB (Santa Cruz, CA, USA) length RET FKBP domain of Affinity Bioreagents RET (FKBP) RET-TET Primary IB (Hornby, ON, Canada) constructs Cell Signaling pRET Technology (Danvers, pY905 of RET Primary IB MA, USA) Sigma-Aldrich Co. FLAG-tagged FLAG Primary IB, IP (Oakville, ON, Canada) proteins Santa-Cruz HA Biotechnology Inc. HA-tagged proteins Primary IB, IP (Santa Cruz, CA, USA) Santa-Cruz 15 C-terminal CBL Biotechnology Inc. amino acids of Primary IB, IP (Santa Cruz, CA, USA) human c-Cbl Santa-Cruz 19 C-terminal ARHGEF7 (β- Biotechnology Inc. amino acids of Primary IB, IP pix) (Santa Cruz, CA, USA) human ARHGEF7 Sigma-Aldrich Co. Tubulin Tubulin Primary IB (Oakville, ON, Canada) Santa-Cruz HIS (H-15) Biotechnology Inc. HIS-tagged proteins Primary IB (Santa Cruz, CA, USA) Santa-Cruz GST-tagged GST Biotechnology Inc. Primary IB proteins (Santa Cruz, CA, USA) Santa-Cruz C-terminal domain MET Biotechnology Inc. of mouse c-Met Primary IB, IP (Santa Cruz, CA, USA) p140

Antibody Supplier Detection Type Use

Santa-Cruz pTyr (pY99) Biotechnology Inc. pY residues Primary IB (Santa Cruz, CA, USA) Santa-Cruz Goat HRP Biotechnology Inc. Goat IgG Secondary IB (Santa Cruz, CA, USA) Cell Signaling Rabbit HRP Technology (Danvers, Rabbit IgG Secondary IB MA, USA) Cell Signaling Mouse HRP Technology (Danvers, Mouse IgG Secondary IB MA, USA)

a 1:2000 dilution for 1 hour at room temperature with gentle shaking. Prior to incubation with secondary antibodies, the membrane was quickly washed three times with distilled water, followed by five washes with TBST for 5 minutes each. Similarly, the membrane was washed after incubation with secondary antibody, prior to submersion in ECL

solution (Perkin Elmer, Boston, MA), and exposure to autoradiography (AGFA Curix

Ultra Medical X-Ray film, Electromedical Equipment, Richmond Hill, ON).

2.8.1 Stripping Blots

For stripping of blots after immunoblotting, 30 mL of stripping buffer (appendix)

was heated to 70 ºC in a microwave. 200 µL of β-mercaptoethanol was added to the

solution, and incubated with the membrane for 30 minutes at room temperature, with

gentle shaking. The membrane was washed five times for 5 minutes each with TBST, and subsequently blocked in 5% milk in TBST for 1 hour at 37ºC. Further immunoblotting

was then carried out as normal.

2.9 In Vitro Kinase Assay

In vitro kinase assays were performed as previously described [113, 121]. 5 µg of

each purified recombinant protein; dialyzed into 1X PBS, was mixed with 1X kinase

buffer (appendix), and 1 mM ATP. The samples were incubated at 30ºC for 30 minutes in

a Hybaid OmniGene PCR machine. The reactions were terminated by addition of SDS loading buffer (Laemmli), and boiled at 99ºC prior to loading onto 10% SDS-PAGE gels.

2.10 Purification of Recombinant GST Proteins

GST-tagged DNA constructs were transformed into BL21 electrocompetent cells,

and the cells were subsequently grown overnight in 10 mL inoculums of LB medium

(appendix) with 50 mg/mL ampicillin. The inoculums were added to 1 L 2YT (appendix)

media the next day, and grown at 37ºC for 6 hours. Protein expression was induced with

1mM IPTG, and the cultures were further incubated at 16ºC overnight with vigorous shaking.

The cultures were pelleted at 3000g using a Beckman Coulter centrifuge for 10 minutes, and the pellet was resuspended in 50 mL of 1X PBS (appendix). The cells were then lysed using a Branson Digital Sonifier for 8x 20 seconds, with 40 second pauses, at an amplitude of 65%, until the viscosity of the sonicate was decreased, and the colour had changed slightly. 2% Triton-X, and 1 mL of 100 mM PMSF was then added to the sample, and the mixture was rocked for 30 minutes at 4ºC. The sample was pelleted at

35000g in a Beckman Coulter centrifuge for 1 hour at 4ºC. The pellet was retained for later analysis on SDS-PAGE.

2.10.1 GST Protein Binding on Beads

The supernatant was incubated overnight at 4ºC, with 1mL of Glutathione

Sepharose beads (Amersham Biosciences, Uppsala, Sweden) per 100 mL of sonicated sample, in a beaker with a magnetic stirbar for gentle stirring. The beads were washed three times with 1xPBS (appendix) at 2500g for 2 minutes at room temperature in a

Fisher Scientific Accuspin Micro centrifuge, prior to incubation with the supernatant.

The proteins were purified the following day using the batch method, in which

centrifugation is used for washing and elution of the protein, as opposed to using an affinity tag purification column. The slurry with the supernatant and beads was transferred from the beaker to a 50 mL conical tube, and centrifuged at 2500g for 2 minutes at 4ºC using a SORVAL Legend RT centrifuge. The supernatant was removed, and the beads were washed 5 times with approximately 50 mL 1X PBS pH 7.4

(appendix). The supernatant and the wash buffer were retained for later analysis.

2.10.2 GST Protein Elution

Elution of the protein from the beads was performed with three to five aliquots of

1mL GST-elution buffer (appendix). For each elution, the beads were incubated with the

elution buffer for 10 minutes on ice, prior to centrifugation at 2000g at 4ºC. The

supernatants for each elution fraction were collected separately in 1.5 mL tubes, for

analysis on SDS-PAGE.

2.10.3 Analysis of Protein Purification

Finally, 25 µL of each of the pellet and supernatant after sonication, supernatant

that was incubated with beads, the collected washes, and each elution, was mixed with an

equal volume of Laemmli buffer. The samples were boiled at 99ºC for 5 minutes, and

separated on 10% SDS-PAGE. A BSA standard (New England Biolabs, Mississauga,

ON) of 1 mg/mL, diluted in a 1:1 ratio with Laemmli, was also run for approximate

determination of protein concentration, together with a pre-stained protein marker (New

England Biolabs, Mississauga, ON). The gel was coomassie stained for analysis, using a staining solution (appendix), and incubated at room temperature with rocking for approximately 1h. The coomassie stain was then removed using a destaining solution

(appendix), until desired clarity of the gel was reached. An image of the gel was obtained using an Alpha Innotech MultiImage™ Light Cabinet.

Upon determination of the cleanest elutions, with the highest protein concentration, the elutions were pooled and dialyzed into 1X PBS at 4ºC overnight.

Accurate protein concentrations were found using the BCA Protein Assay Kit (Pierce,

Rockford, IL), as previously described.

2.11 Far Westerns

For Far Western assays, transfections with desired constructs were carried out as described in section 2.4, in 100 mm plates. 50 μL of cell lysates were protein assayed and retained in Laemmli buffer. The remaining 500 μL (400-500 μg of protein) of the lysates were used for immunoprecipitations with the appropriate antibody in a 1:50 dilution. The immunoprecipitates and lysates were separated on SDS-PAGE, transferred and blocked, following procedures described previously. The blotting procedure differed from this point onwards, as instead of immunoblotting with an antibody; the membrane was blotted with 1μg/mL of the GST-fusion protein in 5% milk in TBST, for 2 hours at room temperature with gentle shaking [121]. The membrane was then washed with TBST at least ten times, for 5 minutes each, prior to immunoblotting with an α-GST antibody

(Santa Cruz Biotechnology, Santa Cruz, CA), for 1 hour at room temperature. The

37 membrane was washed extensively before incubation with the secondary antibody, and finally, the bound proteins were detected with the ECL detection kit, as described above.

Chapter 3

Results

3.1 Characterizing RET- Cbl Interaction

Many RTKs have been shown to interact with the c-Cbl and Cbl-b homologues

(Reviewed in [80]). ARHGEF7 has also been shown to interact with both Cbl homologues [99, 100]. An extensive study on RET and c-Cbl interaction was previously performed [74], and it was also shown that Cbl-b recruits RET into lipid rafts [122].

However, because a comparative binding between the c-Cbl and Cbl-b homologues, and

RET, had not previously been done, we wished to investigate whether there were major differences in binding of the Cbl homologues to RET (Figure 3.1). This would help to determine which Cbl homologue to use in further experiments.

For this study, HEK293 cells stably expressing RET9 and GFRα were used, in which HA-tagged c-Cbl or Cbl-b were overexpressed (Figure 3.1 B). We used a control in which only RET9 and GFRα were expressed, without transfection of Cbl, in addition to a negative control, consisting of parental HEK293 cells. All cells were treated with the

RET ligand, GDNF, for 30 minutes prior to protein isolation, and cell lysates were subjected to immunoprecipitation with an HA antibody. Expression of RET was confirmed by immunoblotting with an α- RET9 antibody (C19), and of Cbl proteins using the HA antibody (Figure 3.1 B). As RET has a mature, glycosylated form, and an immature non-glycosylated form, two bands of approximately 175 kD and 155 kD were seen [123] (Figure 3.1 B, top panel). The Cbl proteins also differ in size, Cbl-b is

Figure 3.1 Both c-Cbl and Cbl-b Homologues Interact with RET

A. A schematic showing the essential domains of Cbl (Figure 1.5), and the differences in size between the c-Cbl and Cbl-b proteins (adapted from [7]) B. Western blot analysis of c-Cbl and Cbl-b binding to RET9. HEK293 cells stably expressing RET9 and GFRα were

used, in which HA-tagged c-Cbl or Cbl-b were overexpressed. HEK293 cells that did not

stably express RET9 were used as a negative control. All cells were treated with the RET

ligand, GDNF, and cell lysates were subjected to immunoprecipitation. Expression of

RET was ascertained by immunoblotting with an α- RET9 antibody (C19), and of Cbl

proteins using the HA antibody. Activated RET was assessed using an antibody against

phosphotyrosine residue, pY905. IP: immunoprecipitation, WCL: whole cell lysates.

40 A

40a B approximately 128kD in size, as compared to c-Cbl which is approximately 120 kD

(Reviewed in [7]) (Figure 3.1 A,B). Subsequent to immunoprecipitation with the HA antibody, Cbl binding to RET9 was detected in the co-immunoprecipitate with Cbl.

Activation state of RET was also assessed, using an antibody specific to phosphorylated

Y905 on RET (Figure 3.1 A, bottom panel).

Our results show that both the c-Cbl and Cbl-b homologues were able to interact with RET9. As major differences in binding between the homologues were not seen, c-

Cbl was used in further studies. c-Cbl will be referred to as Cbl here after, unless otherwise indicated.

3.2 Investigating Direct Binding of Cbl to RET9

Scott et al. [74] demonstrated that c-Cbl can bind indirectly to RET through Grb2 at phosphorylated Y1062 [74]. Based on data from other RTKs, such as Met [84], it was reasonable to investigate whether a direct interaction between the TKB domain of c-Cbl and RET may occur. To test this, the recombinant GST-tagged TKB domain of c-Cbl, also denoted Cbl-N, was purified (Figure 3.2 A). The purified proteins were coomassie stained for determination of purity (Figure 3.2 B), and can be seen as a 62 kD band.

Purified GST- Cbl TKB was used to perform a far western assay with Met, in order to determine the functionality of the protein, and to replicate previous data published on direct interation of Cbl and Met [84] (Figure 3.3 A). Immunoprecipitation of Met was performed with lysates from a murine carcinoma cell line, SP1, which overexpresses the

Met receptor [124], and HEK293 cells, which served as a negative control. The purified

GST Cbl- TKB was used as a probe, and detection of the direct binding was ascertained

using an α-GST antibody. A band at 145 kD was visualized, corresponding to the size of

Met (Figure 3.3 A, B). The nitrocellulose membrane was then stripped and reprobed with

the Met antibody, to confirm that the band seen upon detection with α- GST, was the Met

receptor. As expected, and previously published [84], our results showed that the TKB

portion of Cbl can bind directly to Met (Figure 3.3 B).

Having confirmed the functionality of the purified protein, a similar procedure

was followed to investigate RET9 binding to Cbl- TKB (Figure 3.3 C). HEK293 Tet-ON

cells, which carry a tetracycline response element for induction of RET9 expression, were

used in this experiment. RET expression was induced using doxycycline, and a dimerizer was added to induce activation of RET. No induction of expression was carried out in the negative control, which consisted of HEK293 Tet-ON cells. Both samples were subjected to immunoprecipitation for RET9, and the western blots were incubated with the GST- tagged Cbl- TKB domain. Immunoblotting with an α-GST antibody detected a band of 83

kD; the expected size of the intracellular RET9 protein [13, 113, 114]. The membrane

was stripped and immunoblotted with the RET9 antibody (C19), to confirm that the

appearing band of 83 kD was RET. In addition, to ensure that the GST-tag was not by

itself binding to RET; purified GST alone was used in a far western assay with RET9

expressing HEK293 Tet-ON cells. Our results showed that GST alone could not bind to

RET, however the GST-tagged Cbl TKB domain showed direct binding (Figure 3.3 C).

Figure 3.2 Purification of GST- Cbl- TKB

A. Schematic diagram showing the structure of c-Cbl, and Cbl-TKB (Cbl-N) proteins.

GST-Cbl-TKB consists of the tyrosine kinase binding (TKB) domain of c-Cbl, fused to a

GST- tag. B. Coomassie staining of the purified samples. The image shows the 62 kD

Cbl-TKB protein after washing and elutions. The 26 kD GST-tag is also indicated. P=

pellet after sonication, FT= flowthrough, or supernatant after centrifugation, B1, B2= beads, proteins on the bead prior to elution, E1- E6= pooled elution fractions.

43 A

B P FT B1 E1-E3 B2 E4-E6 43a

Cbl- N 62kD Æ

GST 26kD Æ

Figure 3.3 Direct Binding of Cbl- TKB to the Met and RET Receptors

A. Diagram showing the basic principle of a far western assay, using Met as an example.

Immunoprecipitated Met is on the nitrocellulose membrane. Purified GST- Cbl- TKB is allowed to bind to the receptor, and detected using an α- GST antibody. This results in bands appearing at the same molecular weight as Met. B. Functional validation of GST-

Cbl-TKB in a far western assay with Met. SP1 cells overexpressing the Met receptor were used. HEK293 cells were used as a negative control (-). Met was immunoprecipitated from the lysates, using an anti- Met antibody, and purified GST Cbl-

TKB was used as a probe. Detection of the direct binding was performed using an α-GST antibody. The nitrocellulose membrane was then stripped and reprobed with the Met antibody. C. A far western assay was performed with the RET receptor. HEK293 Tet-

ON cells, expressing RET9, were used. RET expression was induced in one of the samples (RET9), whereas the negative control was not induced (-). Lysates were immunoprecipitated with an anti-RET antibody and subjected to a far western assay with

GST- Cbl-TKB, and with purified GST alone.

44 A 44a

B C

3.3 Investigating the Interactions Between RET, Cbl, and ARHGEF7

Upon establishment of RET- Cbl interactions, the relationship between the RET receptor, Cbl, and ARHGEF7 was investigated. According to our hypothesized model,

RET, ARHGEF7, and Cbl may form a complex (Figure 3.4 A). To exclude that there may be direct interactions between RET and ARHGEF7, we looked for co- immunoprecipitation of the two, by immunoprecipitating for ARHGEF7 (Figure 3.4 B).

In addition, confirmation of binding between ARHGEF7 and c-Cbl was performed.

HEK293 cells stably expressing full length RET9 and GFRα were used, in which FLAG- tagged ARHGEF7 was overexpressed alone, or together with HA-tagged c-Cbl. The negative control consisted of HEK293 cells alone. RET was activated using GDNF, and all samples were subjected to co-immunoprecipitation for ARHGEF7. Expression of each of the proteins was detected in the lysates (Figure 3.4 B, top panels). The bottom panels in Figure 3.4 B show the ARHGEF7 immunoprecipitates, which were immunoblotted for

RET9 or Cbl. Our results detected no interaction between RET and ARHGEF7; however

ARHGEF7 and c-Cbl interaction was confirmed, as previously published [99].

To further investigate the interactions between RET, Cbl and ARHGEF7, we examined both RET9 and RET51, and their interactions with Cbl and ARHGEF7. In this experiment, HEK293 cells stably expressing either RET9 or RET51, together with GFRα, were used. The negative control was HEK293 cells that did not stably express RET.

FLAG-tagged ARHGEF7 and HA- Cbl were either transiently co-expressed, or singly expressed. For RET9, Cbl was immunoprecipitated with both the HA antibody, and the

Cbl-specific antibody for precipitation of HA- Cbl (Figure 3.5 B). Detection of all three

Figure 3.4 RET9 and ARHGEF7 Do Not Interact in Co-Immunoprecipitation

A. A hypothetical model of interactions between RET9 and ARHGEF7. The figure shows that an antibody (red) specific for ARHGEF7 is used to detect interactions with Cbl and

RET. B. Western blot analysis of co-immunoprecipitation with ARHGEF7. HEK293 cells stably expressing full length RET9 and GFRα were used, in which FLAG-tagged

ARHGEF7 was overexpressed alone, or together with HA-tagged c-Cbl. The negative control consisted of HEK293 cells. RET was activated using GDNF, and all samples were subjected to immunoprecipitation for ARHGEF7. Expression of each of the proteins is shown in lysates (top panels), using C19 for detection of RET9, αHA for detection of

HA-Cbl, and αFLAG for detection of ARHGEF7. Tubulin was used as a loading control.

The bottom panels show the anti-ARHGEF7 immunoprecipitates, which were immunoblotted for RET9 and Cbl.

46 A RET9

Y1062 SHC GRB2 CBL ARHGEF7 P

46a proteins is shown in the top panels in Figure 3.5 B. Samples immunoprecipitated for Cbl with the HA antibody, were subjected to immunoblotting for RET9 and ARHGEF7. The results showed that c-Cbl and RET9 interact. Strong interaction was seen between c-Cbl and ARHGEF7, using both tag-specific and protein specific antibodies. Similar results were obtained when studying RET51 (Figure 3.6 B). Using the Image J software, we performed spot densitometry analysis of the results. The ratios of tubulin to RET in the immunoprecipitates were compared, as well as the ratios of RET in the lysates (WCL) to

RET in the IPs. Taking the levels of tubulin and RET into account, densitometry analysis of the blots confirmed that there was no difference in the binding of RET to Cbl, when

ARHGEF7 was overexpressed (data not shown). In conclusion, whereas interaction between RET and ARHGEF7 could not be detected when immunoprecipitating for

ARHGEF7, interactions between RET and Cbl, and ARHGEF7 and Cbl, were observed when immunoprecipitating for Cbl. This showed that Cbl can interact with both RET and

ARHGEF7.

3.4 Investigating the Relationship Between RET and ARHGEF7

As RET interacts with Cbl [74], and Cbl interacts with ARHGEF7 [99], it was plausible to believe that there was some relationship between RET and ARHGEF7. We tested whether ARHGEF7 could be phosphorylated downstream of both RET9 and RET51, in

HEK293 cells in which we overexpressed FLAG-tagged ARHGEF7. Cells were serum starved for 24 hours, and RET activation was induced by treatment with GDNF for 30 minutes. Expression of RET9 and RET51, and ARHGEF7, was detected in cell lysates

Figure 3.5 Interactions between RET9, Cbl and ARHGEF7

A. A hypothetical model of interactions between RET9, Cbl and ARHGEF7. The figure shows that an antibody (red) specific for Cbl was used in immunoprecipitations for

detection of interactions with ARHGEF7 and RET B. Western blot analysis of Cbl

interaction with RET9 and ARHGEF7. FLAG-tagged ARHGEF7 and HA- Cbl were

transiently expressed in HEK293 cells stably expressing RET9 and GFRα. Tubulin was

used as a loading control. Samples were immunoprecipitated with the HA antibody, and

the Cbl-specific antibody, for detection of ARHGEF7. RET9 binding was assessed in the

immunoprecipitates for the HA-tag of Cbl. The results showed that Cbl interacts with

both RET9 and ARHGEF7.

48 A RET9

Y1062 SHC GRB2 CBL ARHGEF7 P

48a

Figure 3.6 Interactions between RET51, Cbl, and ARHGEF7

A. A hypothetical model of possible interactions between RET51, Cbl and ARHGEF7.

The figure shows that an antibody (red) specific for Cbl was used in the

immunoprecipitation for detection of interactions with ARHGEF7 and RET. B. Western blot analysis of Cbl interaction with RET51 and ARHGEF7. HEK293 cells stably expressing RET51 and GFRα were used. FLAG-tagged ARHGEF7 and HA- Cbl were either transiently co-expressed, or singly expressed. Detection of all three proteins in the lysates is shown in the top panels. Tubulin was used as a loading control. Cbl was immunoprecipitated with an HA antibody, and the immunoprecipitates were subjected to immunoblotting with either anti-RET51 (C20), or anti-FLAG antibodies for detection of

ARHGEF7 binding. The results showed that Cbl interacts with both RET51 and

ARHGEF7.

49 A RET51

Y1062 SHC GRB2 CBL P ARHGEF7 Y1096 GRB2 CBL P ARHGEF7 B

49a

(Figures 3.7 A and 3.8 A). The lysates were then immunoprecipitated for ARHGEF7, and

phosphorylation was assessed using a general phosphotyrosine specific antibody, pY99.

Using this antibody, a band of approximately 74 kD was seen, which corresponded to the

size of ARHGEF7. The blot was stripped and reprobed with the FLAG antibody to

confirm that the phospho-specific band was ARHGEF7, and with an anti-RET antibody

(Figure 3.7 A), to confirm that there was no interaction between RET and ARHGEF7.

These experiments showed that ARHGEF7 can be phoshorylated downstream of both

RET9 and RET51, in response to GDNF.

3.5 ARHGEF7 Phosphorylation is RET-ligand Dependent

As our results showed that phosphorylation of ARHGEF7 was GDNF-induced,

we investigated whether the phosphorylation was time dependent, in order to establish an optimal time of GDNF treatment for full induction of ARHGEF7 phosphorylation. We used HEK293 cells stably expressing RET9, transiently transfected with FLAG-tagged

ARHGEF7, and subsequently serum starved for 24 hours. The cells were treated with

GDNF for 0, 5, 10 and 15 minutes. Phosphorylation of ARHGEF7 was assessed using the phosphotyrosine specific antibody, pY99 (Figure 3.9, top panel). RET expression in the lysates is shown in the bottom panel. We saw an increase in ARHGEF7 phosphorylation upon treatment with GDNF, which was maximal by 15 minutes. This showed that phosphorylation of ARHGEF7 was dependent on RET activation, and correlated with

RET phosphorylation [13].

Figure 3.7 RET9 Activation is required for ARHGEF7 Phoshorylation

A. Western blot analysis of ARHGEF7 phosphorylation downstream of RET9. HEK293

cells stably expressing RET9, co-transfected with FLAG-ARHGEF7, were subjected to

serum starvation, and treatment with GDNF for 30 minutes prior to protein isolation. The

samples were immunoprecipitated for ARHGEF7 using αFLAG, and subjected to western blotting. Phosphorylation was assessed using a phosphotyrosine-specific antibody (pY99), and phosphorylation of ARHGEF7 is indicated. RET9 did not co- immunoprecipitate with ARHGEF7. B. The figure illustrates the question asked in this

experiment, whether RET9 activation can lead to ARHGEF7 phosphorylation.

51 A B 51a

Figure 3.8 RET51 Activation is required for ARHGEF7 Phoshorylation

A. Western blot analysis of ARHGEF7 phosphorylation downstream of RET51. HEK293

cells stably expressing RET51, co-transfected with FLAG-ARHGEF7, were subjected to

serum starvation, and treatment with GDNF for 30 minutes prior to protein isolation. The

samples were immunoprecipitated for ARHGEF7 using αFLAG, and subjected to western blotting. Phosphorylation was assessed using a phosphotyrosine-specific antibody (pY99), and phosphorylation of ARHGEF7 is indicated. RET51 phosphorylation is not detected. B. The figure illustrates the question asked in this experiment, whether RET51 activation can lead to ARHGEF7 phosphorylation.

52 A B 52a

Figure 3.9 Timecourse Analysis of the GDNF-induced Phoshorylation of ARHGEF7

Western blot analysis of a timecourse experiment, assessing ARHGEF7 phosphorylation

downstream of RET9. HEK293 cells expressing ARHGEF7 and RET9 were treated with

GDNF for the indicated times, and phosphorylation of ARHGEF7 was assessed using a phosphotyrosine-specific antibody (pY99). An increase in ARHGEF7 phosphorylation was seen upon treatment with GDNF over time.

53 53a

IB: RET (H300)

3.6 RET Does Not Phosphorylate ARHGEF7 Directly

We established that activation of RET was required for ARHGEF7

phosphorylation, but no interaction between RET9, or RET51, and ARHGEF7 was seen.

In this experiment, we investigated whether RET could phosphorylate ARHGEF7 directly. We used purified recombinant proteins; GST-tagged RET9 [113], and HIS- tagged ARHGEF7 [119] (Figure 3.10 A), in an in vitro kinase assay [113, 121].

Activation of RET was carried out by the addition of ATP to the reaction mixture [113,

121], and samples containing either GST- RET alone, HIS- ARHGEF7 alone, or both

together, were subjected to the same treatment. Subsequent western blotting showed the

presence of ARHGEF7. For assessment of phosphorylation, the general phosphotyrosine

specific pY99 antibody was used. Our results showed that RET was phosphorylated, by

the presence of bands of 74 kD. However, phosphorylation of ARHGEF7 was not seen,

indicating that RET does not directly phosphorylate ARHGEF7 (Figure 3.10 B)

Figure 3.10 RET Does not Phosphorylate ARHGEF7 Directly

A. Structure of recombinant GST-tagged RET [113], and HIS-tagged ARHGEF7 [119] .

B. Western blot analysis of an in vitro kinase assay with RET and ARHGEF7. Purified recombinant HIS -ARHGEF7 was subjected to an in vitro kinase assay using purified

GST- RET9, and immunoblotted with an anti- phosphotyrosine antibody (pY99). The results showed that RET cannot directly phosphorylate ARHGEF7.

55 A 1 665 1072 Kinase domain RET WT GST Kinase domain GST-RET

HIS HIS-ARHGEF7 55a

Chapter 4

Discussion

The RET receptor tyrosine kinase (RTK) is required for development of the kidney and neural crest derived cell types (Reviewed in [19]). RET can activate several intracellular signaling cascades which regulate cell survival, differentiation, proliferation, migration, chemotaxis, neurite outgrowth, and synaptic plasticity [19]. Alternative

splicing of the RET proto-oncogene results in isoforms with distinct physiological roles

partly due to the recruitment of different signaling complexes [19], and also differential

regulation [74]. RET downregulation is thought to be mediated by Cbl [74]. Although a

few studies have been done on RET and Cbl relationships [74, 122], the exact mode of

binding of Cbl to RET, and RET downregulation, is not fully understood. In this study,

we have focused on examining the interactions between RET and Cbl, and more

importantly, the interaction of Cbl with an antagonist, ARHGEF7, and its relationship to

the RET receptor.

4.1 RET- Cbl Interaction

Our results showed that RET9 could interact with both c-Cbl and Cbl-b

homologues (Figure 3.1). c-Cbl and Cbl-b have been shown to have overlapping, yet

distinct roles in signaling and development (Reviewed in [80]). Although c-Cbl and Cbl-

b interact with the same spectrum of RTKs, there are a number of differences in their

interactions with other regulatory proteins, as well as in their roles in mediating

ubiquitination of their substrates (Reviewed in [80]). The Cbl proteins have highly

conserved regions, with high sequence similarity in the TKB domain, encompassing the

RING domain (Figure 3.1A). The C-terminal regions, however, are less conserved [80].

The UBA domains were initially thought to have affinity for ubiquitin, however, recent

studies have shown that only the UBA domain of Cbl-b, can interact with ubiquitin [92].

In light of the differences in the role of Cbl homologues [80], it was reasonable to investigate the binding of c-Cbl and Cbl-b to RET, to identify any major differences in binding, as well as to determine which Cbl homologue to use in ongoing experiments.

The studies were done in HEK293 cells, which is a biologically relevant cell line to use, as RET is expressed in the kidney [17, 18]. Furthermore, Cbl is endogenously expressed in this cell line [125, 126]. We determined that RET could interact with both Cbl homologues. Although we were not able to quantitate relative binding, large differences in binding were not seen. Three factors influenced our choice of c-Cbl for further experiments: firstly, more extensive studies had been done on the interactions between

RET and c-Cbl [74]. Secondly the studies done on the interactions between EGFR, Cbl and ARHGEF7 [99, 100], on which we based our ARHGEF7 related models and experiments, used c-Cbl, although Cbl-b has also been shown to interact with ARHGEF7

[127]. Lastly, we had several c-Cbl constructs available for use in our laboratory.

Intriguingly, during the course of our investigation, Peschard et al. [93] showed that the UBA domains can mediate both homo- and heterodimerization between Cbl

homologues, indicating that c-Cbl and Cbl-b may have overlapping and complementary

functions in the regulation of RTKs, including RET [74, 122].

4.2 Cbl Can Bind Directly to RET

Met and EGFR serve as paradigms for Cbl-mediated negative regulation of RTKs.

It has previously been shown that Cbl can bind to Met and EGFR through Grb2 [84, 128].

Peschard et al. [95] established Y1003 as a Cbl TKB binding site on Met. Thus, Cbl can

bind to RTKs directly via its TKB domain and indirectly through Grb2 via its proline rich

region [83-86, 95]. This bimodal model of Cbl-RTK binding is thought to enhance the

stability of the interaction ([84], Reviewed in [129]).

The bimodal model of Cbl-RTK binding could also be implicated in Cbl binding

to RET. Based on data from other RTKs, we might predict that RET-Cbl interactions may

include both a direct RET-Cbl-TKB interaction that stabilizes and orients the proteins,

and the previously established indirect associations between the Cbl proline- rich region

and Grb2 [74]. As of yet, there are no convincing data to rule out direct binding of Cbl to

RET. It should also be noted that the RET9 Y1062F mutant, and the RET51

Y1062/1096F double mutant, which abrogate indirect binding of Cbl, did not show a

complete loss of Cbl binding in the study published by Scott et al. [74]. In light of these

observations, it was plausible to investigate whether a direct interaction may occur

between RET and Cbl. To test this hypothesis, we first used the Met receptor as a control

for the functionality of the purified GST- Cbl- TKB protein in a far western assay [84]

(Figure 3.3 B). A similar experiment was performed with RET, and our data showed that the TKB domain of c-Cbl could bind directly to RET, as was seen for Met. This may

indicate that the complexity of Cbl binding to RET may have been understated previously

[74], and the exact mode of how this binding occurs is yet to be elucidated. Unpublished

58 data in our lab indicate Y952 on RET as a possible direct interaction site for Cbl- TKB

(Andrew et al., unpublished), as this tyrosine lies within the consensus sequence for Cbl

TKB binding (pYXXP) [94]. An hypothetical model of the interaction between RET9 and Cbl is shown in Figure 4.1. In future studies, it will be interesting to pursue an understanding of the phosphotyrosines on RET that mediate this direct binding.

Further, it will be interesting to investigate the functional outcomes of a loss of direct Cbl binding on RET ubiquitination and RET-mediated oncogenesis, in order to understand its significance. Met mutants that have impaired ability to bind Cbl-TKB are transforming and tumorigenic, due to increased stability and sustained signaling by Met [84]. In a similar manner, the loss of the direct Cbl-TKB-binding site in other RTKs results in an increased transforming activity [97]. These observations suggest that loss of a Cbl-TKB binding site may be a common mechanism that contributes to full oncogenic activation of

RTKs. Similar studies using tyrosine mutants of RET will be interesting to pursue, once the exact binding site on RET for Cbl TKB has been established.

4.3 Interactions Between the RET Receptor, Cbl, and ARHGEF7

Recent studies have demonstrated that negative regulation of the Cbl-induced degradation of EGFR can be mediated by ARHGEF7 [99, 100]. ARHGEF7 was previously known to be a guanine nucleotide exchange factor (GEF) that could activate

Figure 4.1. A model of Cbl binding to RET. A. We predict that Cbl can bind directly through its TKB- domain at Y952 on RET, and indirectly through an interaction between

Grb2 and the proline-rich domain (PRD) of Cbl, to further stabilize the interaction. B. As

RET51 has an additional Grb2- binding site at Y1096, we hypothesize that an interaction between the PRD of Cbl and Grb2, in the absence of Shc, is a more stabilizing interaction.

60 60a

Rho GTPases, in particular Cdc42, in pathways leading to cytoskeletal rearrangements

[130]. However, these findings provided evidence of novel roles of ARHGEF7 as an antagonist of Cbl [99, 100] The existence of a trimeric complex involving Cdc42,

ARHGEF7, and Cbl was shown to sequester Cbl away from EGFR. However, an alternative pathway by which ARHGEF7 could block Cbl-catalyzed downregulation of

EGFR, without forming a trimeric complex with Cdc42, was proposed [100]. Feng et al. showed that ARHGEF7 coimmunoprecipitated with EGFR and Cbl, without binding

Cdc42 [100].

We investigated the possibility of ARHGEF7 and RET co-immunoprecipitation upon overexpression in HEK293 cells (Figure 3.4 B). However, if complex formation was to occur between RET, Cbl and ARHGEF7, we did not expect to see interaction between RET and ARHGEF7, due to the involvement of Shc and Grb2 in this hypothetical interaction [74]. The binding of ARHGEF7 would be too far downstream of

RET, to be able to detect interaction between the two in co-immunoprecipitations. To test this hypothesis, and to rule out any possible closer interaction, we immunoprecipitated for ARHGEF7, and RET binding was not detected (Figure 3.4 B).

Notably, as Cbl binding to ARHGEF7 was also assessed in this experiment, we showed that ARHGEF7 and c-Cbl interact. This was confirmed in multiple experiments (Figures

3.4, 3.5, 3.6). These results together showed that Cbl can interact with RET, and with

ARHGEF7. However, this does not necessarily indicate a simultaneous interaction between the three proteins, as these interactions may occur in different pools in the cell;

we may be identifying cytosolic Cbl with ARHGEF7, and also Cbl which is bound to

RET, independently.

4.4 RET- Ligand Dependent Phoshorylation of ARHGEF7

Feng et al. showed that tyrosine phosphorylation of ARHGEF7 is necessary for its interaction with Cbl downstream of the EGF receptor, and that this phosphorylation

was EGF- induced [100]. As we had observed interaction between ARHGEF7 and Cbl

subsequent to RET activation, it was conceivable that RET activation may induce

ARHGEF7 phosphorylation. We therefore conducted RET-ligand dependent experiments, in which ARHGEF7 phosphorylation was evaluated downstream of both

RET9 and RET51 (Figures 3.7 B, 3.8 B, 3.9 B). We showed that ARHGEF7 phosphorylation is GDNF- dependent, and we therefore predict that it requires kinase activity of each RET isoform (Figures 3.7B, 3.8B.). Note that RET did not co- immunoprecipitate with ARHGEF7, as observed when immunoblotting with the RET9

(C19) antibody (Figure 3.7 B). This agrees with previous results in which ARHGEF7 and

RET9 interaction was not detected (Figure 3.4 B).

For further assessment of the ligand- dependency of ARHGEF7 phosphorylation, induced by GDNF treatment, a time course experiment was performed (Figure 3.9). The time frame chosen, zero to fifteen minutes, was based on peak phosphorylation of

ARHGEF7 seen after 10 minutes with EGF treatment [100], and on optimal RET activity after 15 minutes of GDNF treatment [13]. As seen for EGF treatment [100], ARHGEF7 phosphorylation increased upon GDNF treatment over time. Thus, this provided

optimized experimental conditions in future experiments where ARHGEF7 phosphorylation is essential.

4.5 ARHGEF7 Phosphorylation by RET

We investigated whether RET could mediate the phosphorylation of ARHGEF7

directly. We used recombinant RET protein consisting of the intracellular regions, including the kinase domain, and the C-terminal tail of RET9, whereas ARHGEF7 consisted of the SH3, DH and PH domains, encompassing Y443 which is the primary site

of phosphorylation on ARHGEF7 [100] (Figure 3.10 A). GST- RET is fully functional

and capable of substrate phosphorylation [113], and the functionality of HIS- ARHGEF7

had previously been demonstrated [119]. Our in vitro kinase assay showed that, although

phosphorylation of RET was seen, no phosphorylation was detected for ARHGEF7

(Figure 3.10 B). More importantly, these results suggested that other RET substrates are

responsible for the GDNF-stimulated phosphorylation of ARHGEF7, such as Src [44],

and Fak [100, 131].

4.6 Summary and Conclusions

The role of Cbl as an E3-ubiquitin ligase, and its interaction with tyrosine kinases,

enables it to provide a powerful mechanism for maintaining homeostasis of RTK activity.

It is important to try to utilize the knowledge gained of Cbl and its binding to RET and

the Cbl antagonist, ARHGEF7, and the interplay between all three proteins, to elucidate

mechanisms of RET regulation in future studies.

Our study provides a base for exploring an array of possible means of RET

regulation, as well as for comparing RET9 and RET51 isoforms. In summary, we showed that RET can interact with two important Cbl homologues, c-Cbl and Cbl-b. A potential

novel direct interaction between RET and c-Cbl was detected, whereas previously, only

an indirect association between RET and Cbl had been established [74]. Moreover, we

showed that c-Cbl interacts with ARHGEF7; however, a complex formation between all

three proteins could not be confirmed. Our work has provided novel insight establishing a

relationship between RET and ARHGEF7. We showed that RET activation was required

for ARHGEF7 phosphorylation, and that ARHGEF7 phosphorylation increases upon

RET-ligand treatment. Phosphorylation was not mediated directly by RET, and RET and

ARHGEF7 association was also not detected.

4.7 Significance and Future Directions

Our work has raised many interesting questions to be explored in future research

in order to increase the understanding of RET regulation. As mentioned, the exact mode

of direct Cbl binding to RET should be elucidated, as well as functional consequences of

a loss of this binding. More importantly, forthcoming studies should address the pathway

responsible for ARHGEF7 phosphorylation. In the case of EGFR, ARHGEF7 is activated by phosphorylation at Y443 in an EGF-dependent manner, which is mediated through the downstream cytoplasmic kinases, Src and the focal adhesion kinase, Fak [100]. It has previously been shown that RET can activate Src and Fak [44, 131]. The involvement of

Src in mediating the phosphorylation of ARHGEF7, and the functional consequences of

this event in relation to Cbl- RET binding will be of interest in our understanding of RET

regulation. Such studies can include the use of Src inhibitors [132] in order to assess the

phosphorylation of ARHGEF7 when Src is inhibited. Alternatively, the SYF-/- cell line,

which is a Src -/-, Yes -/-, Fyn -/- cell line, can be used to study the effect of Src

knockout on ARHGEF7 phosphorylation. Similarly a Fak -/- cell line should be used to determine the role of Fak in mediating ARHGEF7 phosphorylation, as was performed by

Feng et al. [100].

In summary, it is now evident that a clear balance exists between positive and

negative regulation of RTK signaling which is critical for normal cell homeostasis.

Dissecting the molecular mechanisms involved in the regulation of RTKs such as RET,

holds wide implications in medicine, particularly in the development of novel therapeutic

approaches. Understanding the regulation of RET mediated by Cbl and ARHGEF7 can

explain the functional differences between RET isoforms as well as help provide insight

into RET-mediated oncogenesis. However, the functional consequences of a loss of RET-

Cbl interaction on RET ubiquitination, internalization, and degradation in vivo are still

challenges for future research.

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Appendix 1: Composition of Solutions

SDS- PAGE Gels TBST 10 mM Tris pH 7.5 10% Resolving Gel 100 mM NaCl 10 % acrylamide 0.1 % Tween 20 0.031 M Tris-HCl pH 8.8 0.1 % SDS Gel Running Buffer 0.1 % ammonium persulfate 25 mM Tris 0.05 mM TEMED 190 mM glycine 0.001% SDS

4% Stacking Gel Transfer Buffer 1.7 % acrylamide 25 mM Tris 0.01 M Tris-HCl pH 6.8 190 mM glycine 0.1 % SDS 0.2 % methanol 0.1 % ammonium persulfate 0.09 nM TEMED 10X Lysing Buffer 10X TBS 200 mM Tris-HCl, pH 7.8 100 mM Tris pH 7.5 1.5 M NaCl 1M NaCl 10 % Igepal 20 mM Na2EDTA

1X Lysing buffer 20 mM Tris- HCl, pH 7.8 Kinase Buffer (used in in vitro kinase 0.15 M NaCl assay) 1% Igepal 10 mM Tris-HCl 2 mM Na2EDTA 5 mM MgCl2

Laemmli Buffer (2X) 250 mM Tris-HCl pH 6.8 10X PBS 4% SDS 1.4 M NaCl 10% glycerol 0.03 M KCl 0.006% bromophenol blue 0.04 M Na2PO4 2% Beta-mercaptoethanol 0.02 M KH2PO4 pH to 7.4 GST Elution Buffer 1.5 M Tris-HCl pH 8.0 100 mM glutathione 1M NaCl

Coomassie Blue Stain 0.2% coomassie blue 50% methanol 7% acetic acid

Coomassie Blue Destain 5% methanol 7% glacial acetic acid

LB Medium 1% Bacto-tryptone 0.5% Bacto-yeast 1% NaCl pH 7.0

LB Agar As per medium Before autoclaving, 1.5% Bacto-agar added

2YT Medium 1.6% bacto-tryptone 1% yeast extract 0.5% NaCl pH 7.0