Systematic Analysis of Protein-Protein Interactions Between the Erbb Family of Rtks and a Complete Set of Human Phosphatases

Systematic Analysis of Protein-Protein Interactions Between the ErbB Family of RTKs and a Complete Set of Human Phosphatases

Katelyn Dawn Darowski

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Biochemistry University of Toronto © Copyright by Katelyn D. Darowski 2014 ii

Systematic Analysis of Protein-Protein Interactions Between the ErbB family of RTKs and a Complete Set of Human Protein Phosphatases

Katelyn Darowski

Master of Science

Graduate Department of Biochemistry University of Toronto

2014 Abstract

This thesis has created a resource describing interactions between the ErbB family

receptor tyrosine kinases and human phosphatases using the Membrane Yeast Two

Hybrid system (MYTH). A total of 55 phosphatases form 109 unique interactions

between active and inactive ErbB family members. The 29 interactors of ErbB2 were

chosen for further study based on their rates of mutations across various cancer studies

reported on CBioPortal. PTPRB, TPTE2, TNS3, EYA1, and SSH1 had the highest

incidences of mutation and were analyzed for their effect on ERK1/2 phosphorylation.

While PTPRB could not be stably expressed in the system, the four other phosphatases

exhibited deregulated ERK1/2 phosphorylation. TPTE2 overexpression showed a

decrease in ERK1/2 phosphorylation time, as did SSH1, which also lowered maximum

ERK1/2 phosphorylation. TNS3 over-expression delayed the onset of ERK1/2

phosphorylation, while EYA1 lowered the maximum level of phosphorylation. This

thesis highlights new potential players in ErbB family and signaling regulation.

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Acknowledgments

I would first and foremost like to thank my supervisor Dr. Igor Stagljar, who has helped and supported me over the years, both in and out of science here at the University of Toronto. I would also like to thank my two committee members Dr. Stephane Angers, and Dr. Anne-Claude Gingras, both of whom have been a well of knowledge and were a wonderful addition of a new set of eyes on some old problems I had. I would also like to thank Dr. Oliver Ernst and Dr. Frank Sicheri for taking time out of their schedule to be a part of my examining committee.

I would like to acknowledge and thank all the past and present members of the Stagljar lab for both their help with my project through the years, and for being a wonderful bunch of people to work with, specifically Kate Sokolina, my graduate studies guide from the beginning, Zhong Yao who worked on this project by my side, Jamie Snider who never failed to aid in science, and Mehrab Ali who seemed to keep everything together. I would also like to thank Dr. Nicole St-Denis from Dr. Anne-Claude Gingras’ lab who helped both with phosphatases, co-ip attempts, cell culturing, and many questions.

Lastly, I would like to thank my family and friends for the endless support that I received not only through this degree, but also everything that came before. To my parents and sister for ‘editing’ and ‘proofreading’ my thesis, to Tiffany who is probably as invested in this thesis as I am, and to Carlos, who as an engineer probably knows way to much about kinases, phosphatases, and protein-protein interactions techniques. Thank you all for your love and support, which was beyond appreciated.

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Table of Contents

Acknowledgments ...... iii

Table of Contents ...... iv

List of Tables ...... vi

List of Figures ...... vii

List of Appendices ...... ix

Abbreviations ...... x

1 Chapter 1: Introduction ...... 1 1.1 Research Objectives ...... 1 1.2 Receptor Tyrosine Kinases ...... 1 1.2.1 ErbB Subfamily ...... 2 1.3 ErbB family Signal Transduction ...... 10 1.3.1 ERK ...... 10 1.4 Phosphatases ...... 13 1.4.1 Classifications ...... 14 1.5 Protein/Protein Interactions Research Tools ...... 16 1.5.1 MYTH ...... 17 1.5.2 MaMTH ...... 21 1.6 Pipeline of Work ...... 25

2 Chapter 2: Materials and Methods ...... 27 2.1 Prey Generation ...... 27 2.2 Bait Generation ...... 27 2.2.1 Bait Validation ...... 28 2.3 MYTH Screen ...... 29 2.4 Prey Validation ...... 30 2.5 MaMTH prey generation ...... 31 2.6 MaMTH Confirmation of Interactions ...... 31 2.7 Functionality in Signaling ...... 32 2.7.1 Over-expression of Phosphatases ...... 32

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2.7.2 Western Blot Protocol ...... 33 2.7.3 IMAGE J Analysis ...... 34

3 Chapter 3: Results ...... 35 3.1 MYTH Results ...... 35 3.2 MaMTH Confirmation ...... 39 3.3 Final Interactome ...... 43 3.4 Class Enrichment Analysis for Interactome ...... 45 3.5 Selecting Phosphatases to Further Investigate ...... 47 3.6 Effect on ERK Signaling Pathway ...... 48

4 Chapter 4: Discussion ...... 54 4.1 Analysis of MYTH and MaMTH Confirmed Interactome ...... 54 4.2 Analysis of Effect on ERK Signaling Pathway ...... 55 4.2.1 TPTE2 ...... 56 4.2.2 TNS3 ...... 57 4.2.3 EYA1 ...... 58 4.2.4 SSH1 ...... 59 4.3 Future Directions ...... 60 4.3.1 Pathway Activity Location ...... 60 4.3.2 Catalytic Activity ...... 61 4.3.3 Gene Knockdown Effects ...... 62 4.3.4 Gene Regulation Effects ...... 62 4.3.5 Other Pathways ...... 63 4.4 Summary and Conclusions ...... 64

References ...... 66

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List of Tables

Table 1 Transformation Template ...... 29

Table 2 Screening Plate Template ...... 30

Table 3 Phosphatase Gene Mutation Total in Cancer Studies ...... 47

Table 4 Phosphatase Library ...... 77

Table 5 Luciferase Reading Values ...... 87

Table 6 Enrichment Classification...... 89

Table 7 Summary of Cancer Study Results for Three Phosphatases ...... 90

Table 8 Bradford Assay Calculations ...... 93

Table 9 IMAGE J Values ...... 94

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List of Figures

Figure 1 RTK Subclass I: ErbB family Structure ...... 3

Figure 2 Ligand-Bound Structure of ErbB Family Members ...... 5

Figure 3 ErbB Family Dimerization ...... 6

Figure 4 ErbB family growth factors ...... 8

Figure 5 ErbB signaling pathways ...... 12

Figure 6 Phosphatase Classification ...... 15

Figure 7 MYTH System ...... 21

Figure 8 MaMTH System ...... 24

Figure 9 Pipeline of Work ...... 26

Figure 10 MYTH assay Raw Results ...... 35

Figure 11 Initial MYTH Interactome ...... 36

Figure 12 Identification of Frequent Flyers in MYTH Assay ...... 37

Figure 13 Refined Interactome ...... 38

Figure 14 Summary of MaMTH assay ...... 40

Figure 15 MaMTH Luciferase Signal Fold Change ...... 43

Figure 16 Final Interactome ...... 44

Figure 17 Phosphatase Class Enrichment ...... 47

Figure 18 ERK Pathway Stimulation Patterns based on Western Band Intensity ...... 53

Figure 19 MaMTH Expression Confirmation ...... 88 vii viii

Figure 20 Erk Analysis Westerns ...... 92

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List of Appendices

1 Appendix 1 ...... 77 1.1 Phosphatase Library ...... 77 1.2 Phosphatase Class Members ...... 80 1.3 MYTH Results ...... 84 1.4 Prey Validation ...... 84 1.5 MaMTH Confirmation ...... 84 1.6 Interaction Enrichment ...... 88 1.7 Phosphatase Ranking ...... 89 1.8 Western Analysis ...... 91 1.8.1 Concentration Determination ...... 92 1.9 IMAGE J Values ...... 93

2 Appendix 2 ...... 95 2.1 Strains Used ...... 95 2.1.1 Yeast ...... 95 2.1.2 Mammalian ...... 95 2.2 Plasmids Used ...... 95 2.3 Antibodies Used ...... 95 2.4 Cell culture care ...... 96 2.4.1 Cell Thawing ...... 96 2.4.2 Cell Freezing ...... 96 2.4.3 Splitting Cells ...... 96

3 Appendix 3 ...... 97 3.1 Media Recipes ...... 97 3.1.1 E.coli ...... 97 3.1.2 Yeast ...... 97 3.1.3 Mammalian ...... 98 3.2 Chemical Solution Recipes ...... 98

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Abbreviations

AR - amphiregulin Asp - aspartic acid BiFC - biomolecular fluorescence complementation BTC - betacellulin co-ip - co-immunoprecipitation Cub - C-terminal half of ubiquitin DNA - deoxyribonucleic acid DTT - Dithiothreitol DUB - deubiquitinating enzymes EGF - Epidermal Growth Factor EGFR - Epidermal Growth Factor Receptor EPR - epiregulin FRET - fluorescence resonance energy transfer GDP - Guanosine diphosphate GTP - Guanosine triphosphate H2AX - H2A histone family, member X hEGF - human Epidermal Growth Factor HER - human Epidermal Growth Factor Receptor HP-EGF - heparin-binding EGF KD - Kinase domain KDD - Kinase dead domain LiOAc - lithium acitate maMTH - mammalian membrane two hybrid MAPK - Mitogen Activated Protein Kinase mRNA - messenger ribonucleic acid MS - mass spectrometry MYTH - Membrane Yeast Two Hybrid NP-40 - nonyl phenoxypolyethoxylethanol 40 NRG - neuregulin Nub - N-terminal half of Ubiquitin NubG - Mutant N-terminal half of Ubiquitin NubI - Wild-type N-terminal half of Ubiquitin O/E – Over expression PEG - Polyethylene Glycol PH - Pleckstrin homology PI3K - Phosphoinositide 3-kinase PP - Protein Phosphatase PTP - Protein Tyrosine Phosphatase qPCR - Quantitative Polymerase Chain Reaction RTK - Receptor Tyrosine Kinase

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Ser - serine ssDNA - single stranded deoxyribonucleic acid TAP - tandem affinity purification TF - transcription factor TGF - transforming growth factor Thr -threonine TM - Trans-membrane domain TP - tyrosine phosphatase Tyr- tyrosine WT - wild-type

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1 Chapter 1: Introduction 1.1 Research Objectives

Receptor tyrosine kinases (RTKs) are a group of membrane-bound protein kinases that sense extracellular signals and transmit them into the cell through auto-phosphorylation and phosphorylation of tyrosine residues of other proteins. They play crucial roles in physiological and pathological processes such as tumorigenesis. Protein phosphatases (PPs) remove phosphates from RTKs as well as other proteins and are thus intimately involved in the regulation of RTK activities. Traditional biochemical approaches for studying transmembrane proteins such as RTKs are often cumbersome and arduous, mainly due to the complex biochemical nature of the proteins. The Membrane Yeast Two-Hybrid (MYTH) assay, originally developed by the Stagljar lab, is an ideal tool for studying this group of proteins in vivo (Snider 2010). To date, this technology has been successfully used to generate protein interaction maps for various types of membrane proteins (Paumi 2007, Lalonde 2008, Gisler 2008, Suter 2008, Snider 2010, Snider 2013).

The MYTH system, coupled with subsequent human cell culture analysis, offers a systems approach to qualifying interactors that may potentially lead to the identification of novel drug targets, contribute to therapeutic research, and shed new light on the mechanisms of transmembrane signaling.

The goal of this thesis was to map the interactions of all human phosphatases with the human ErbB (avian erythroblastic leukemia viral oncogene homolog) family of RTKs; EGFR, ErbB2, ErbB3, and ErbB4 using the MYTH assay. Several of these interactions were then confirmed and further validated in mammalian cells in order to analyze the effect they have on signaling downstream of the ErbB family member.

1.2 Receptor Tyrosine Kinases

Receptor Tyrosine Kinases (RTKs) are cell surface receptors with affinity for various hormones, cytokines, and growth factors that are responsible for the propagation, and movement of various signals between cells. In humans, they are a 58-member family in the 90-member

tyrosine kinase grouping divided based on their shared homology: a membrane-spanning region. In total there are 17 classes of receptor tyrosine kinases, since they all share a common evolutionary ancestor, they are classified by their sequence similarity of their kinase domain primarily, followed by further classification based on evolutionary conservation and sequence similarity of non-kinase domains (Hanks 1995, Manning 2002). Of the 17 classes of RTKs, the ErbB family makes up one class and contains: Epidermal Growth Factor Receptor (EGFR, ErbB1, HER1), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4). This classification was based on these four RTKs sharing an 88% sequence identity (EGFR and ErbB2), 77% sequence identity (EGFR and ErbB4), and 40% sequence identity (EGFR and ErbB3) in the catalytic domain (the low score between EGFR and ErbB3 is due to ErbB3 missing key catalytic residues which is further describes below) (Shih 2011). These four proteins also share a high degree of sequence identity outside of their catalytic domain (77% for both EGFR and ErbB2 and ErbB4, and 53% for EGFR and ErbB3) (Shih 2011).

1.2.1 ErbB Subfamily

As stated above, the ErbB subfamily of RTKs is comprised or four members: EGFR (ErbB1, HER1), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4). The first family member discovered was EGFR in 1978 by Cohen et al. (Carpenter 1979). Discovery of the other family members followed quickly, and were named for their homology to the erythroblastic leukemia viral oncogene (Yamamoto 1983, Downward 1984). This family of proteins has been implicated in various cellular processes such as cellular proliferation, differentiation, motility, and apoptosis (Yamamoto 1983, Downward 1984, Liu 1992, Lamaze 1995, Wang 2005, Park 2006, Lemmon 2010). Due to the wide range of processes in which these RTKs affect the cell, as well as the manner in which they do so, the understanding of the ErbB family roles and mechanisms are essential to determining not only the biology of these RTKs themselves, and RTKs as a whole, but the biology of signal transduction.

1.2.1.1 Structure and Function

Two separate groups concurrently elucidated the extracellular region of EGFR, and crystalized a dimer of EGFR with a bound ligand (Garrett 2002, Ogiso 2002, Stamos 2002).

This was followed by the crystallization of the other three family members, offering a way to compare structures of all four family members (Cho and Leahy 2002).

All four of the ErbB family members share structural similarity; each contain an intracellular kinase region, a membrane spanning domain, and an extracellular region which can be further divided into four separate domains (domains I through IV). Although the ErbB family members share considerable similarities, there are also several biochemical, biophysical, and biological differences (figure 1)(Garrett 2002, Ogiso 2002, Cho and Leahy 2002, Burgess 2003). ErbB2 has sequence variation in the extracellular region resulting in a different extracellular structure in comparison to its other three family members, rendering this RTK in a permanent ‘open’ or ‘activated’ state (further discussed below). ErbB3 has sequence variation within its intracellular kinase domain that renders that domain inactive. Finally, cells internalize ErbB family members at different rates after activation, dependent on the identity of the receptor, as well as the stimulus used for ErbB activation; EGFR is internalized at a much faster rate than the other family members (Eigenbrot 2008).

Figure 1 RTK Subclass I: ErbB family Structure: All four ErbB family members share a common structural organization. Each contains an intracellular region that houses the kinase

domain (KD), though ErbB3 has no catalytic activity (KDD). Each contains a trans-membrane region (TM), and an extracellular region, which contains four domains (I, II, III, IV). In the native state for EGFR, ErbB3, and ErbB4, domains II and IV interact holding the ErbB in an ‘inactive’ or ‘closed’ conformation. ErbB2 has natural peptide variants in the extracellular region allowing for stronger interactions between domains I and III causing ErbB2 to be in an ‘active’ or ‘open’ state.

The ErbB family, like most RTKs, is responsible for the transduction of signals from the extracellular to the intracellular region of the cell, and for this particular subclass of RTKs, these signals are received in the form of growth factors, which act as ligands for the ErbB receptors. Growth factors bind to the extracellular region of EGFR, ErbB3, and ErbB4; this binding results in a conformational change in the extracellular region of the RTK (figure 2). The growth factor ligand has affinity for both domain I and III in the extracellular region, and this affinity allows it to interact with both domains inducing a structural change which interrupts the interactions of domain II and IV. This change in structure allows the ErbB to be in an ‘active’ or ‘open’ conformation. This conformation is similar to the native conformation of ErbB2 (figure 2). The active or open confirmation is denoted by the release of domain II, which is the dimerization domain, from domain IV. This release of domain II allows for its interaction with a released domain II from a second ErbB family member.

Figure 2 Ligand-Bound Structure of ErbB Family Members: Upon ligand binding to domain I and III, EGFR, ErbB3, and ErbB4 adopt a conformational change which results in the interruption of the previous interactions between domain II and IV. The resulting conformational change allows the dimerization domain (II) to interact and dimerize with another ‘active’ or ‘open’ domain II on a second ErbB family member.

In order for an ErbB family member to be activated and propagate a signal, a ligand must first bind to ensure the freedom of domain II dimerization (figure 2), with the exception of ErbB2 whose natural variants allow it to be in a constitutively active conformation. Once an ErbB is in an active confirmation, it must then dimerize with another ErbB family member for signal transduction. Two ‘active’ or ‘open’ dimerization domains (II) facilitate this process (figure 3). ErbB family members have the ability to heterodimerize with other ErbB family members, or homodimerize, with the exception of ErbB3, which cannot form an active homodimer due to a lack of kinase activity. Since each ErbB member has a preference for different signaling pathways and propagates signals via different mechanisms, the results from mixing family members as heterodimers and homodimers provides numerous and different signal outputs and allows for variety and variation in stimulated signaling pathways. Of these possible combinations, ErbB2 and ErbB3 heterodimers are the most active (Tzahar 1996, Pinkas-

Kramarski 1996, Baselga 2009), and ErbB2 contains the strongest kinase activity, and is the preferred binding partner for all ErbB family members, most likely due to its permanently ‘active’ confirmation (Tzahar 1996, Graus-Porta 1997).

Upon dimerization of two ErbB members via domain II, a conformational change occurs in the ErbBs’ intracellular kinase domains. The conformational change is known as a ‘head to tale’ activation (Zhang 2006). This unique activation requires that the C terminal of one ErbB’s kinase domain interact with the N-terminal of the second ErbB’s kinase domain (Zhang 2006). The ‘head-to-tail’ confirmation is then responsible for the trans-auto-phosphorylation of tyrosine residues on the ErbB C-terminal tail. Depending on the identity of the ErbB partners, and the stimulus used, the phosphorylation pattern on the ErbB tails will vary. This variation is responsible for variety and specificity of downstream signaling pathways that are activated (further discussed below).

Figure 3 ErbB Family Dimerization: Upon ligand binding for EGFR, ErbB3, and ErbB4, the dimerization domain (II) is available for dimerization with another ‘open’ or ‘active’ domain (II)

from another ErbB family member. Upon ErbB dimerization, intracellular conformational changes occur, which allow the kinase domains to orientate themselves in a head-to-tail formation allowing for the trans-auto-phosphorylation of the tyrosine residues on the C-terminal tail of the ErbB partners. This phosphorylation occurs in a particular pattern that dictates the particular downstream signaling pathways to be activated.

In addition to variation in signal propagation pathways dictated by the ErbB family members that constitute the ErbB dimer, further specificity is added by the particular growth factor that is responsible for the activation of the ErbB member. Growth factors binding ErbB family members can be divided into three groups, classified on their specificity for each family member (figure 4). The first group can bind only EGFR and is comprised of: epidermal growth factor (EGF), Transforming growth factor alpha (TGF-α), and amphiregulin (AR). The second group has dual specificity for EGFR and ErbB4 and contains: betacellulin (BTC), heparin- binding EGF (HP-EGF), and epiregulin (EPR). The third group contains neuregulins (NRGs) and is further divided into two subgroups; the first of which can bind to ErbB3 and ErbB4 contains NRG1 and NRG2, and the second which binds to only ErbB4 contains NRG3 and NRG4. There are no known ligands for ErbB2, most likely due to its permanently active confirmation (figure 2).

Figure 4 ErbB family growth factors: The growth factors that bind to the ErbB family members can be divided into three groups: factors that only interact with EGFR (epidermal growth factor (EGF), Transforming growth factor alpha (TGF-α), and amphiregulin (AR)), factors have dual specificity for EGFR and ErbB4 (betacellulin (BTC), heparin-binding EGF (HP-EGF), and epiregulin (EPR)) and neuregulins (NRGs) which are further divided into two subgroups; the first of which can bind to ErbB3 and ErbB4 (NRG1 and NRG2), and the second which binds to only ErbB4 (NRG3 and NRG4).

The variety of specific pathways and responses that are possible with the variation of ErbB dimer members and various activating growth factors is enormous. This specificity is dictated and obtained by the phosphorylation pattern on the C-terminal tail of the ErbB with different residues attracting and binding different facilitator proteins depending on their phosphorylation. These recruited proteins then continue to transduce and propagate the signal through the cell in various ways, which are further discussed below.

After ErbB activation, and after a growth factor signal has been transduced and propagated, receptor dimers undergo endocytosis (Honegger 1987, Lamaze 1995, Sorkina 2002, Wang 2005, Zwang 2009). In the vesicle, and after fusion to the early endosome, ErbB can undergo ubiquitination, which targets the endosome to lysosomes and degrades the receptor (Simonsen 1998, Rubino 2000). Alternatively, the receptor can recruit Rab4 and Rab11 which target the receptors for recycling back to the plasma membrane as monomeric inactive receptors (van der Sluijs 1992, Ullrich 1996). Another interesting form of ErbB signaling is also found while the receptors are in the endosome; while in the early endosome EGFR, ErbB2, and ErbB4 have been shown to be translocated into the nucleus of the cell where they direct further gene expression (Ni 2001, Williams 2004, Giri 2005, Lo 2006, Rokicki 2010, Grandal 2012) adding another facet to the signaling pathways of the ErbB family members. The fate of a particular ErbB family member after activation depends on the dimer pair partner, along with the growth factor stimulus. These combine to create a unique phosphorylation pattern on the C-terminal tail of the ErbB receptor that not only dictates signal pathways to be activated, but the receptor fate as well.

1.2.1.2 Links to Disease

With so many avenues in which ErbB receptors can affect cellular growth and proliferation, it is not surprising that the ErbB family members have been found to play a part in various diseases, across various medical fields. For example, ErbB family members have been implicated in diseases such as pediatric intestinal inflammation, pulmonary disease, airway obstructions, schizophrenia like phenotypes, Behcet’s disease, and diabetes induced cardiac dysfunction to name a few (Anagnostis 2013, del Pino 2013, Xavier 2013, Akhtar 2013, Frey 2013). In this thesis, I closely examine the binding partners of ErbB2 since it is the preferred binding partner for other family members, suggesting that it plays a primordial role in all diseases affected by ErbB family members. Further evidence for its function in human cells also reside in the fact that it itself has been implicated in various types of cancers. In breast cancer subtypes in particular, ErbB2 has been found to heterodimerize and homodimerize at a much higher rate due to its over-expression (Yarden 2001, Stephens 2004, Moasser 2007, Koboldt 2012), being found to have 25-50 additional gene copies, and 40-100 fold more protein expression, resulting in approximately two million receptors on the cell surface in various cancer

samples (Venter 1987, Kallioniemi 1992, Lohrisch and Piccart 2001). ErbB2 gene and protein amplification have been shown to be an early event in tumor progression (Liu 1992, Ignatoski 2000, Stephens 2004, Park 2006), and a mutated ErbB2 has been found in 25-30% of all breast and ovarian cancers (Salmon 1987, Yan 2014).

1.3 ErbB family Signal Transduction

Upon ErbB activation, several downstream events occur to enable cellular signaling. ErbB family receptors activate three major signaling pathways: the JAK/STAT pathway, the AKT pathway, and the ERK pathway (figure 5). The varieties of additional signaling pathways, as well as combinations of pathways, that can be activated further add to the vastness and intricacy of the ErbB signaling cascades. There is much redundancy and cross-talk in all three pathways which leads to system robustness (Citri 2006, Keshet 2010, Mendoza 2011). That, coupled with system modularity, and the ‘bow-tie’ shape (a system with diverse options for both inputs and outputs regulated by a conserved core with high redundancy, crosstalk, and positive and negative feedback loops), provide for system bistability, or the option of the system to act in a switch-like manner, where addition or removal of a ligand or stimulus results in a change of the system from on to off (Kitano 2004, Oda 2005, Kholodenko 2006). All three signaling pathways are also heavily internally controlled. This is achieved via system buffering and numerous positive and negative feedback loops (Mimnaugh 1996, Citri 2003, Keshet 2010). The last important characteristic of these pathways is that they are compartmentalized. Original signaling takes place at the plasma membrane, but as the ErbB family members are endocytosed, they are able to further stimulate different signaling pathways, or different targets in signaling pathways, depending on their location (within the early endosome, late endosome, or nucleus). For the purpose of this thesis, it is the ERK pathways that will be further studied.

1.3.1 ERK

The first and primary substrates in the ERK pathway are the ErbB receptors themselves. As stated above, upon ligand binding, receptor dimers trans-autophosphorylate their C-terminal tails (Honegger 1989, Prigent 1994, Favelyukis 2001, Till 2002, Furdui 2006, Vermeer 2012). Depending on the ligand stimulus, as well as composition of the dimer pair, various residues within the C-terminal tail are phosphorylated (Pawson 1995, Schlessinger 2000). For the ERK

pathway to be activated, Grb2 must be recruited to the phosphorylated tyrosine residues, which triggers a positive feedback loop recruiting more Grb2 to the plasma membrane (Gu and Neel 2003, Teramoto 2004, Lemmon 2010). ErbB activation, with the aid of Grb2, also recruits Shc to the activated dimer (Teramoto 2004, McCubrey 2008, McCubrey 2009, Martelli 2010, Grandal 2012). These Shc and Grb2 proteins then further recruit SOS, which forces RAS to release its bound GDP in exchange for GTP rendering RAS activated (Wu 1993, Downward 2003). Once RAS in activated with GTP, it is able to recruit RAF to the membrane and activate it (Marais 1995). Active RAF is then able to Ser/Thr phosphorylate MEK1, a kinase which further phosphorylates ERK1/2 (Downward 2003, McMubrey 2008, McMubrey 2009, Lefloch 2009). From here, ERK1/2 target over 600 known substrates, such as other kinases such as Rsk and Msk1 (who further propagate signal) as well as transcription factors such as NFIL6, Ets-2, elk-1, CREB, Fos, and many more which directly bind to promoters to influence gene transcription (Davis 1995, Widmann 1999, Balan 2006, McCubrey 2008, McCubrey 2009, Martelli 2010). Genes activated by ERK1/2 signaling are, in part, the anti-apoptotic BCL-2 family genes such as mcl-1 (Davis 1995, Widmann 1999, Balan 2006, McMubrey 2008, McMubrey 2009, Martelli 2010, Steelman 2011). While ERK1/2 signaling increases transcription of the previously mentioned anti-apoptotic genes, it also down-regulates gene expression of the pro-apoptosis BCL-2 family genes such as BAD (Davis 1995, Widmann 1999, Balan 2006, McMubrey 2008, McMubrey 2009, Martelli 2010, Plotnikov 2011, Steelman 2011).

Figure 5 ErbB signaling pathways: Three major pathways are activated by ErbB signaling, the AKT pathway, the ERK1/2 pathway, and the STAT pathway, are shown above in a selective and simplistic manner. Green Arrows denotes activation of either a second protein, or downstream genes, while red arrows denote their inhibition. The very wavy arrows in the AKT and STAT pathway denote a longer signal transduction pathway to reach the signaling outcome. The focused pathway of this thesis is the ERK1/2 pathway. Upon receptor dimerization and phosphorylation, Shc and Grb2 are recruited to the phosphorylated tyrosine residues on the C- terminal tail. The recruitment of these two proteins allow for the further recruitment of SOS to the activated ErbB dimers. SOS activates RAS via the exchange of GDP to GTP allowing RAS to activate RAF. RAF then further activates MEK1/2, which phosphorylates ERK1/2 rendering ERK1/2 active. Active ERK1/2 has over 600 substrates including proteins such as Rsk and Msk1, as well as various transcription factors. The transcription factors activated by ERK1/2

promote expression of the anti-apoptosis genes in the BCL-2 family, and inhibit expression of pro-apoptosis genes in the BCL-2 family.

This summarizes the ERK1/2 pathway in a selective and simple manner. There is additional cross talk between other ErbB activated pathways, pathways activated by other receptors, housekeeping proteins, as well as numerous feedback loops that further add intricacy, and bistability to the signaling pathway.

1.4 Phosphatases

To complement protein kinases which add phosphate groups onto amino acids, are protein phosphatases (PPs) which remove them. Like protein kinases, protein phosphatases are used for effective communication between and within cells, to dictate cell shape, motility, signals for cell proliferation, differentiation, regulation of gene transcription, mRNA processing, and molecular transport. Though phosphatases and kinases have similar (signaling pathway regulation) yet opposite roles (addition versus removal of phosphatase groups), knowledge is far greater in understanding protein kinases. Regardless of this knowledge imbalance, it seems that RTKs and tyrosine phosphatases (PTPs) share similar targets of regulation due to a similar number of proteins in each class. Of the 90 human RTKs, 85 are active. This number is quite comparable to the 107 human PTPs, 11 of which are catalytically dead, 2 target mRNA, and 13 target inositol phospholipids, leaving 81 protein active members (Alonso 2004). A more recent investigation of phosphatases re-classified phosphatases into families based on structural similarity instead of the historic substrate based, and structure classification; this resulted in a PTP class comprised of 96 active members instead of the historic 107 (Li 2013). In addition to having similar numbers of RTK and PTP proteins, they also have comparable tissue distribution and expression, between 30-60% (Alonso 2004, Tonks 2006). This suggests that each kinase and phosphatase have specific duties with few redundancies, which is supported by several studies that show that unique phenotypes arise from knock-down of unique PPs in mouse models (Alonso 2004, Hasegawa 2004, Bottini 2004).

Since their discovery, research has been done to determine the specific roles of PPs in the cellular environment. They have been found to have active and specific roles in dephosphorylation in various areas of cellular biology, giving them an enormous area of regulation

(Fischer 1991, Walton and Dixon 1993, Tonks and Neel 1996, Mustelin 2002, Mustelin 2002, Mustelin and Tasken 2003, Tonks 2006, Sacco 2012, Sacco 2012).

Of particular interest to this thesis are the role phosphatases have in regulation of RTKs, specifically the ErbB family, and the signaling pathways they regulate or affect. Many phosphatases have been discovered to be tumor suppressors for cancers with mutations and amplifications of ErbB family members, or in other components of their signaling pathways. PTPN12 and PTPN23 are known to decrease cell invasion properties, while deletion of PTPRG, whose gene lies in a chromosomal hot-spot for deletion, is shown to promote cellular invasion and metastasis (Sun 2011, Julien 2011, Lin 2011). PTPRR, PTPRK, and PTPRJ have all been shown to be negative regulators of the ERK1/2 signaling pathway, a pathway that is frequently over-activated in cancer samples, as is the PI3K signaling pathway, which happens to be regulated by another phosphatase, PTEN; all of the above phosphatases are known tumor suppressors (La Forgia 1991, Panagopoulos 1996, van Niek and Poels 1999, Di Cristofano 2000, Pianetti 2001, Szedlacsek 2001, Liu 2002, She 2003, Vezzalini 2007, Tarcic 2009, Lin 2011). These studies all show that phosphatases are not simply housekeeping suppressors of kinases, but have active and specific roles in the regulation of various signaling pathways.

The numbers of PPs and RTKs and their potential interactions show the minimum level of intricacy that can be found within these systems. Complexity is further enhanced via the addition of various growth factors, other regulatory proteins and molecules, spatial and temporal distribution, and post-translational modifications leading to an extremely rich interaction profile (Alonso 2004, Tonks 2006, Karisch 2011, Sacco 2012).

1.4.1 Classifications

Unlike the ErbB family that evolved from a common ancestor, PPs evolved independently and have structurally different catalytic domains (Barford 1994, Su 1994, Yuvaniyama 1996, Fauman 1998). Due to these differences, they can be classified into two main groups, tyrosine phosphatases, or serine/threonine phosphatases (figure 6).

Serine/threonine phosphatases can be further divided into three categories: Asp-based phosphatases (with an aspartic acid as the catalytic residue), PPPs (which require a cation for catalytic activity), and PPMs (which require a magnesium cation)(Alonso 2004).

Tyrosine phosphatases are further divided into four classes, each from a distinct evolutionary ancestor, based on the amino acid sequence of their catalytic domains (Alonso 2004). The first class is the largest; they have a cysteine based catalytic domain and are further divided, based on similar domain architecture and catalytic domain homology, into classical PTPs, which target only tyrosine residues, and dual specificity phosphatases (DSPs), which have the widest range of targets: tyrosine, threonine, serine, and/or phosphoinositides (Anderson 2001, Anderson 2004, Alonso 2004). The second class is also utilizes a cysteine based catalytic domain, is structurally related to bacterial arsenate reductases, has one of the highest rates of conservation (31% identical to S. cerevisiae, and 39% identical to B. subtilis), and targets only tyrosine residues (Alonso 2004). The third class, also with a cysteine based catalytic site, were all derived from bacterial rhodanese-like enzyme and comprise of three cell cycle regulators which have has the capacity to target tyrosine and threonine residues (Alonso 2004). The final group uses a different catalytic mechanism involving an aspartic acid and cation in the catalytic site, and has the capacity to dephosphorylate tyrosine, and possibly serine (Tootle 2003, Rayapureddi 2003, Li 2003, Alonso 2004). Phosphatase class members can be found in appendix 1.

Figure 6 Phosphatase Classification: Phosphatases are classified into different families based on their structure, composition of their catalytic domain, and phosphate targets. The colours of

specific phosphatase groups are used to easily identify family members in interactome maps presented in Results section 3. Phosphatase class members can be found in appendix 1.

1.5 Protein/Protein Interactions Research Tools

In the cellular and biological environment, cells nearly never are found in isolation, and cellular contents and processes are never found to work in isolation. Proteins are found with, or work with other proteins, biomolecules, and even drugs to perform their function. These interacting proteins, biomolecules or drugs can influence the function, rate, and targets of the other proteins they interact with, or can add another level of regulation to pathways affected by the protein in question. The advantages to this include: (1) reducing the likelihood of error that would arise from the transcription and translation of larger proteins; (2) altering the specificity of an enzyme for its substrate which can either further regulate the reaction dynamics, or can create multiple substrates for a single protein; (3) altering kinetics of enzymes’ reactions providing additional regulation and control; and (4) create a more efficient cell by having several smaller proteins be recruited to create several larger complexes, as opposed to have multiple large proteins present.

Due to the importance of protein interactions in almost every aspect of cellular processes, numerous methods have been developed to identify and characterize them. Some examples include the yeast two-hybrid (Fields and Songs 1989), biomolecular fluorescence complementation (BiFC) (Ventura 2011), co-immunoprecipitation (co-ips) (Kaboord and Perr 2008), LUMIER (Barrios-Rodiles 2005), and tandem affinity purification (TAP) and mass spectrometry (MS) (Babu and Krogan 2009). Though each of these studies offers a powerful way to identify protein-protein interactions, each has its limitations. For example, while the yeast two-hybrid system is scalable, low-tech and easy to use and manipulate, is known to present many false negatives (indicating that proteins do genuinely interact, but are not detected by the technology) or false positives (indicating that a protein that are found as interactors by the technology, but are not biologically genuine interactors) and can only detect interactions of proteins that can be transported into the yeast nucleus. BiFC allows for physiologically-relevant analysis to be done since protein interactions are looked at in vivo, at endogenous protein levels, and requires minimal cell perturbation. However it also produces an irreversible interaction excluding baits and preys from further interactions, is temperature dependent, and can provide

many false positives and negatives due to its sensitivity. Co-immunoprecipitation, though another easy and low-tech option, is biased toward sustained as opposed to transient protein interactions, however it is able to detect and identify non-tagged interacting proteins by MS allowing for a more complete interaction profile. All of these protein interaction detection techniques also share the fact that they are not ideally suited for the identification of the interactions of full-length integral membrane proteins, in the context of their native membrane environment. Integral membrane proteins are particularly difficult to characterize due to their biochemical complexity and hydrophobicity. Efforts to study integral membrane proteins using only their intra or extra-cellular domain have met with some success, although these approaches are less than ideal, and in certain cases have serious limitations. For instance, truncating proteins can alter the intra-protein bonds and change protein structure that would diminish the chances of finding native interacting partners. It also removes the proteins internal regulation; for example, with the ErbB family members the extracellular domain regulates the structure as well as binding partners and their binding locations in the intracellular domain. Proteins that are cleaved through their transmembrane domain allow for artificial binding partners to bind to the no longer regulated intracellular region. This cleavage and mis-regulation makes it impossible to determine all interacting partners on native structures in different cellular environments. In order to avoid the pitfalls of traditional protein interaction techniques, the MYTH system was developed (Johnsson 1994, Stagljar 1998, Stagljar 2002, Snider 2010).

1.5.1 MYTH

The MYTH system offers a qualitative way to identify novel protein-protein interactions of membrane bound proteins, overcoming many of the limitations associated with other methods. This design revolutionized the study of membrane proteins by characterizing the proteins in their native environment versus previous detergent treatment methods, and affinity purifications, and allows for screening of interactors in vivo. It offers a large-scale approach to quickly, and systematically test and determine interactions of membrane proteins from any organism. For this thesis MYTH was chosen for various reasons, predominantly because it allows for testing of potential ErbB and phosphatase interactions in a binary system. Due to the absence of human proteins in the yeast host that is used for MYTH, this binary system allows for less background noise while examining potential interactors. Furthermore, the activity of the ErbB receptors

testing can be easily manipulated and changed via mutations without interference from activating or in-activating signals that would be present in a mammalian system. In order for MYTH to be a viable option for qualifying protein-protein interactions, either the C or N-terminal tail of a membrane protein must be located in the cytosol of the cell, making MYTH impossible for some membrane proteins; even in the case of a tail being located in the cytosol, there are still some other limitations to the system. Selected baits may be ‘self-activating,’ indicating that the transcription factor is cleaved and activates selection genes in the absence of a protein interaction. These particular types of baits are recognized via the bait-dependancy test (further discussed in section 2.2.1) and are not able to be tested in the MYTH system. Also, there can also be steric interference from the MYTH tag used, limiting the number of identified protein- protein interactions; this can be avoided to a certain degree by varying the length of the linker used between the membrane protein and MYTH tag.

1.5.1.1 Yeast as a Model Organism

The MYTH assay is performed in S. cerevisiae, which possesses various properties that make it an ideal research tool and model organism. For example, S. cerevisiae contains complex internal cellular structures as well as various organelles similar to higher eukaryotes. The entire genome of S. cerevisiae has also been mapped, making for easy identification and tagging of genes for yeast protein-protein interactions. Perhaps the most relevant for this project is that the MYTH system can be used to identify protein-protein interactions for foreign organism proteins. This removes the need to postulate the connection between yeast homologue proteins and their interactors with their H. sapiens counterparts. It also provides a binary system for the testing of foreign proteins without the complication of other potential protein interactors, or competitors. Finally, the nature of S. cerevisiae growth makes it a quick and easy tool in a research laboratory; they have a short doubling time making them easy to culture in a lab, they can be easily transformed with plasmids containing foreign DNA, can express foreign protein, and finally can grow on various media allowing for easy manipulation of environment for assay selection. A limitation to using S. cerevisiae as a model system is that the protein interactions are artificial, and may not be representative of interactions present in another, or original organism. Also, proteins that require post-translational modification to achieve their natural

structure, or to facilitate binding of other proteins may not be properly processed resulting in a lack of full interaction data.

1.5.1.2 Technique

The premise of the MYTH system is that two putative interactors, each tagged with half of an ubiquitin molecule, can reconstitute the ubiquitin if they do indeed interact, and this reconstitution can be detected.

Ubiquitin is a highly expressed and conserved 76 amino acid protein found across all eukaryotic organisms. Ubiquitination is a post transcriptional modification adding the ubiquitin molecule onto the lysine residues of proteins and is used for various cellular processes such as proper protein localization, DNA repair, protein degradation, and cell cycle regulation (Kimura 2010). The removal of ubiquitin from molecules is a process known as deubiquitination and is carried out by deubiquitinating enzymes (DUBs), which cleave after the ubiquitin C-terminus.

This ubiquitin molecule can be stably truncated into two fragments, a 24 amino acid C- terminal end (Cub) and a 52 amino acid N-terminal end (Nub) that can be reconstituted to create a pseudo ubiquitin molecule. In order to prevent the spontaneous re-association of the Nub and Cub, a mutation is introduced into the N-terminal half: I13G. This mutation uses the terminology NubI for the wild-type N-terminal, and NubG for the mutated version. The change from the isoleucine to the glycine in the 13th position in the N-terminal half of ubiquitin prevents the spontaneous reconstitution of the two fragments. It is this prevention of reconstitution that allows the MYTH system to be used as a screening assay. If two interacting proteins are tagged with a Cub and

NubG respectively, the interacting tagged proteins will bring the two fragments of ubiquitin into close enough proximity to allow for DUBs to recognize the pseudo ubiquitin molecule. If DUBs do recognize ubiquitin, they are able to cleave off a transcription factor that was tethered to the

Cub domain. If this transcription factor is cleaved, it translocates to the nucleus allowing for the activation of the reporter genes. If the Cub and NubG tags are on two non-interacting proteins, ubiquitin is not reconstituted, DUBs are not recruited, the transcription factor remains attached to the tag and no reporter genes are activated.

In this system, three reporter genes are used: ADE2, HIS3, and lacZ. The activation of these reporter genes allows for growth of cells on media lacking histidine, and prevents formation of a pink coloration (which occurs in yeast cells not expressing ADE2 due to the accumulation of a biosynthetic intermediate). The third reporter gene causes to cells to become blue in colour when grown in the presence of X-Gal. In this way, two separate types of screening, one with minimal media, and a second with minimal media including X-Gal, can be used to detect interactions of proteins using the MYTH system.

The other important component in the MYTH assay is the α-mating sequence, which is fused to the N-terminus of the non-yeast bait protein. This allows for the use of MYTH on foreign membrane proteins. This sequence targets the bait (membrane or membrane-bound proteins) to the yeast membrane using the yeast machinery ensuring proper localization. Bait localization can be confirmed by the use of fluorescent microscopy. The same α-mating sequence can also be applied to preys that are membrane, or membrane-bound proteins. This allows for the testing of interactions between membrane and cytosolic, and between two membrane proteins. This system therefore offers an approach for identifying interactors of full- length human membrane proteins in a binary system.

Figure 7 MYTH System: A prey and bait protein, are fused to a NubG or Cub domain, the second of which also contains a transcription factor. If the bait and prey protein interact, then the NubG and Cub domains are brought into close enough proximity allowing for their reconstitution into a pseudo-ubiquitin molecule. This pseudo-ubiquitin model is recognized by cellular DUBs, which results in the release of a tethered transcription factor which then travels into the nucleus to activate the reporter genes, HIS3, ADE2, and lacZ thus allowing for yeast growth on selection media and detection of interactions.

1.5.2 MaMTH

The MaMTH system is a recent variation from the MYTH approach. Instead of testing putative interactors in yeast, it uses mammalian cells as a host, offering greater relevance for human protein interactions. It too operates on the premise of splitting ubiquitin and its reconstitution. It is also able to qualify protein-protein interactions of membrane bound proteins

in vivo, and in addition, it is also able to quantify the strength of these interactions. MaMTH has several of the same limitations that the MYTH system had: one of the membrane protein’s tail must be located in the cytosol, the MaMTH tag may still act as steric interference which can be alleviated to some extent by the length of the linker, and self-activation is still possible. The issues of an artificial environment and loss of post-translational modifications are avoided, though interactions are not tested with endogenous protein levels, instead being tested on transfected proteins.

1.5.2.1 Mammalian Cells as a Model

MaMTH uses mammalian cells as a host, which provides several new benefits. The system can use any cell line; I used HEK293 cells; a cell line obtained from embryonic kidney cells from H. sapiens. These cells are easy to grow in tissue culture, readily transfected, can express high levels of exogenous protein, and have been proven to produce excellent results within the MaMTH system (Petschnigg 2014). In addition, H. sapiens cells provide a more relevant setting for testing human proteins; other proteins that may be necessary to facilitate an interaction are present in the cellular environment, as well as present in physiological conditions. Due to this, more accurate results can be obtained due to testing being performed in a less artificial environment. It is also possible that more interactions can be detected due to co- binding proteins and scaffolding proteins being present to facilitate further interactions. Additionally, necessary posttranslational modifications can be applied to the proteins in question, which may not be possible in yeast.

Another benefit to MaMTH is that interactions can be tested in various cellular states. Cells can be treated with different drugs in various concentrations, in the presence or absence of of vital growth agents, analyzed in different cell cycle locations, as well as be subjected to numerous other conditions. Applying MaMTH through these conditions develops a protein- protein interaction pattern across all cellular states, providing an in-depth, and multi-faceted interactome.

1.5.2.2 Technique

As stated previously, MaMTH also uses the premise of splitting and reconstituting ubiquitin. The ubiquitin molecule is still truncated into two stable fragments, however in the

mammalian system, the two wild-type fragments are unable to spontaneously recombine to form a pseudo-ubiquitin molecule. The spontaneous reconstitution eliminates the need for the mutated

NubG in the MaMTH assay. This allows the wild-type NubI fragment to be used as the interactions testing tag.

As in MYTH, a prey and bait protein are tagged with the Nub and Cub domains respectively. Here however there is no α-mating sequence addition to the Cub tagged protein for the testing of human proteins. If the putative tagged interactors do indeed interact, the two wild type fragments of ubiquitin recombine to be recognized as an ubiquitin molecule to be cleaved by cellular DUBs. This cleavage allows for the release of transcription factors, which translocate to the nucleus. Once in the nucleus, the transcription factors activate the mammalian reporter genes.

The reporter gene in the MaMTH system is firefly luciferase. The amount of luciferase protein produced is directly correlated to the level of interaction between the bait and prey protein (assuming and testing for equal protein expression levels via western blots). In this way, the MaMTH system is not only able to identify protein-protein interactions, but also quantify their strength, to a degree, based on the luciferase signal produced.

After 24 hours of cellular growth, the luciferase signal is then measured in a luminometer. Results are measured as a fold change compared to a known non-interactor, or a negative control. If putative interactors have a luciferase signal that is two-fold or greater in comparison to the negative control, it is considered a positive hit (i.e. an interaction).

In this way, with this assay, protein-protein interactions can be tested in more relevant landscapes than what MYTH provided.

The other benefit of using MaMTH is that protein activity can be changed in a more pertinent way than protein mutation. For example, instead of mutation of the ErbB receptors to render activated or inactivated confirmations, cells in this assay instead can be starved or stimulated to create a more biologically representative activated or inactivated state. Similarly, other complex states and environments can also be better analyzed in this system because the system contains components that are found in a natural cell environment. Though not utilized in

this thesis, drugs can be tested for their efficiency in blocking protein-protein interactions by analyzing and comparing the strength of the luciferase signal between drug treated and untreated cells. Cells can be analyzed through different cell cycle progression stages to determine changes in protein binding based on cell cycle stage. Cells can be aged to detect changes in protein interactions at various cell ages. In short, the MaMTH system allows for a more thorough testing of many facets of protein interactions.

Figure 8 MaMTH System: A prey and bait protein, are fused to the Nub or Cub domain, the second of which also contains a transcription factor. If the bait and prey protein interact, then the

Nub and Cub domains can recombine to reconstitute an ubiquitin molecule. This pseudo ubiquitin model is then recognized by cellular DUBs, which results in the release of the transcription factor which then travels into the nucleus to activate the reporter gene, luciferase, whose signal is then measured and compared against a negative control to determine interactions.

1.6 Pipeline of Work

152 phosphatases were tested against 6 ErbB receptors in the MYTH system. I found that 55 phosphatases form 109 unique interactions, after removal of ‘frequent flyers’ via interactions with unrelated bait proteins. 35 of these interactions (all with ErbB2) were then further tested using the MaMTH system, yielding a 76% confirmation rate, or 28 confirmed interactions. Of the ErbB2 interactors, the top 5 phosphatases found to be mutated in cancers according to CBioPortal were further analyzed for their effect on ERK1/2 phosphorylation when overexpressed in a mammalian system.

Figure 9 Pipeline of Work: 152 phosphatases were tested in the MYTH system for interactions with 6 ErbB family members. After removal of ‘frequent flyers’ 55 phosphatases formed 109 unique interactions with ErbB family members. 27 of the 35 ErbB2 interactors were confirmed using MaMTH, and the 5 interacting phosphatases with the most cancer mutations were carried forward to determine their effect on ERK1/2 signaling.

2 Chapter 2: Materials and Methods

A list of all yeast strains, mammalian cell lines, plasmids, antibiotics, antibodies, media, and chemical solutions can be found in Appendix one through three.

2.1 Prey Generation

Phosphatases in Gateway® entry vector (Appendix [1]) were acquired from Dr. Nicole St-Denis from Dr. Anne-Claude Gingras’ lab at the Lunenfeld-Tanenbaum Research Institute at

Mount Sinai Hospital. Phosphatases were transfered into both NubG and NubI MYTH prey expression vectors (Appendix [2]) by modifying Gateway® LR ClonaseTM II enzyme mix (Invitrogen 11791-020) protocol as follows: 2.5µL TE buffer, 0.5µL LR clonase, 1µL destination vector (150ng/µL), and 1µL entry vector (100ng/µL) left at room temperature for 2 hours followed by a 10 minute incubation at 37°C with 0.8µL of proteinase K. Competent DH5α E. coli (prepared in lab) was thawed from -80°C on ice. Once thawed the entire 5.8µL of the LR clonase reaction was added to 50µL of competent cells and left on ice for 15 minutes. This was followed by a 1 minute heat shock in a 42°C water bath, followed by 2 minutes on ice, after which 150µL of LB media (Appendix [3]) was added and cells were left to recover in a 37°C incubator. The entire solution was then plated onto 10 cm LB with ampicillin (Appendix [2,3]) plates and grown overnight in a 37°C incubator. Single colonies were then grown overnight in 2mL of LB media with ampicillin (Appendix [2,3]). Glycerol stocks were then created via the addition of 200µL of autoclaved 80% glycerol (Bioshop GLY002.4) and 800µL of the saturated LB media. Glycerol Stocks were then stored at -80°C for future use. The rest of the LB media was used for plasmid extraction and purification using mini-preps kits (FroggaBio PD300) and was performed as per manufacturers protocol, with the exception of the elution step: sterile double distilled Milli-Q water was used instead of elution buffer.

2.2 Bait Generation

EGFR, ErbB2, ErbB3, ErbB4, EFGR(L858R), and ErbB2(K753A) DNAs were already available in the Stagljar lab (EGFR (P00533), ErbB2 (P04626), and ErbB3 (P21860) unpublished data by Jasna Curak; ErbB4 (Q15303), EFGR(L858R), and ErbB2(K753A) unpublished data by Zhong Yao). DH5α E. coli transformations were performed on all these

RTKs for plasmid amplification, as well as preparation of E. coli glycerol stocks as outlined section 2.1. Attempts were made to create a kinase dead version of ErbB4, however this construct seemed to be toxic to yeast and would not produce any viable colonies

S. cerevisiae strain NMY51 (Appendix [2]) was streaked onto 10cm YPAD plates and incubated at 30°C for 72 hours. Single colonies were then grown in 5mL of YPAD media overnight at 30°C in an incubated shaker. The overnight sample was then used to inoculate 10mL of YPAD media to an OD600 of 0.15. This culture was grown for 4 to 5 hours (until OD600=0.6-0.7) at 30°C in a shaker then centrifuged for 5 minutes at 700xg. Supernatant was removed and the pellet was gently resuspended with sterile double distilled Milli-Q water, then centrifuged again for 5 minutes at 700xg. Supernatant was removed and pellet was resuspended in 1mL of water. Concurrently, a PEG/LiOAc/ssDNA solution was prepared by mixing 240µL of sterile 50% PEG (Bioshop Canada Inc. PEG225.1), 36µL of autoclaved 1M LiOAc (Bioshop LIA001.1), and 25µL of single stranded DNA (VWR 80601-118). 300µL of this PEG/LiOAc/ssDNA solution was added to 100µL of resuspended NMY51 cells along with 1µL of RTK plasmid DNA. This was done for each of the RTK plasmids. The mixture was incubated at 30°C for 30 minutes, then incubated at 42°C for 1 hour. Samples were then put on SD-L plates and grown for 72 hours at 30°C. Single colonies were then grown overnight shaking at 30°C in SD-L media, and 800µL of saturated media was added to 200µL of 80% glycerol (Bioshop GLY002.4) in cryogenic tubes (Corning Inc. 430658) and stored at -80°C for glycerol stocks for further use.

2.2.1 Bait Validation

EGFR, ErbB2, ErbB3, ErbB4, EFGR(L858R), and ErbB2(K753A) RTKs were all proven to be functional for the MYTH assay by Marta Wiezbicka in the Stagljar lab (unpublished data). Baits were used in the MYTH assay against two system negative controls (non-interactors) to test for auto-activation. MYTH assay data showed no growth on interaction selection plates indicating that the baits were not self-activating, showing that the ErbB family baits were viable for a MYTH screen.

2.3 MYTH Screen

NMY51 transformed with one of the ErbB family RTKs were streaked onto SD-L media from glycerol stocks and grown in 30°C for 72 hours. Single colonies were then used to inoculate 5mL SD-L media and was grown overnight at 30°C in a shaker. This culture was used to inoculate 40mL SD-L media at OD600=0.15 and this day culture was grown in a 30°C shaker until OD600=0.6-0.7 (for 4-5 hours) then centrifuged for 5 minutes at 700xg. Supernatant was removed and pellet was washed with 80mL of double distilled Milli-Q water and centrifuged for 5 minutes at 700xg. Pellet was resuspended in 4mL of double distilled Milli-Q water. Concurently, a PEG/LiOAc/ssDNA solution was prepared by mixing 9.6mL of sterile 50% PEG (Bioshop Canada Inc. PEG225.1), 1.44mL of autoclaved 1M LiOAc (Bioshop LIA001.1), and 1mL of single stranded DNA (VWR 80601-118). 120µL of this solution was added to 40µL of resuspended NMY51(RTK transformed), and 2µL of prey plasmid. This was done in a 96 well plate, set up as per table 1, in a way that each well contained a different prey plasmid (both NubG and NubI for the phosphatases, as well as for OstI and Fur4 as negative controls).

Table 1 Transformation Template: The template for the large-scale yeast transformation that was done for the MYTH assay. A single 96-well plate was used to transform 40 different preys in both the NubG and NubI form, as well as system controls. Each well contained a prey plasmid, resuspended NMY51 yeast, and a PEG/LiOAc/ssDNA solution

Plates were then floated in a 30°C water bath for 30 minutes, then transferred to a 42°C water bath for an hour. Plates were centrifuged for 10 minutes at 3700xg. 110µL of supernatant was removed and the remaining 52µL were put on SD-WL plates and grown at 30°C for 72 hours.

After three days of growth, three single colonies per biological sample from the prey transformation (table 1) were diluted into three separate wells of a second 96 well plate containing 100µL of double distilled Milli-Q water. This was performed for all samples, so that in totally, each prey (including the Fur4 and OstI) had three biological replicates of their NubG and well as NubI constructs. These plates were set up according to table 2:

Table 2 Screening Plate Template: All screening was completed in the same pattern as shown above for quick and easy reference. Each well either had a single yeast colony diluted in 100µL, or was empty. These plates were replicated onto the three screening media plates, which shared the sample template. In this fashion, each sample had three biological replicates for both the

NubG and NubI form, as well as the controls which were present on every plate.

This plate set up was then replicated onto the growth plate SD-WL, as well as the selection plates SD-WLAH, and SD-WLAH+X-Gal using 3µL of the diluted yeast colonies. All plates were then incubated for 72 hours at 30°C. At the end of the growth period plates were scanned to save the results.

This was performed in triplicate for each prey, providing three technical replicates, each containing 3 biological replicates. Preys that had inconsistent results were repeated for a fourth time.

2.4 Prey Validation

Preys that interacted with any of the baits were further tested against three unrelated yeast proteins, PDR10, VMRI, and NFTI present in the MYTH bait expression vector (Appendix [2]). These baits were obtained as glycerol stocks of transformed NMY51 previously used in the

Stagljar lab (Created by Jamie Snider, Snider 2013). This MYTH assay was performed as described in 2.3

2.5 MaMTH prey generation

Phosphatases that interacted with any of the ErbB receptors, as well as passed the prey validation, were prepared for confirmation as interactors via the MaMTH assay. The phosphatase entry vectors obtained from the Gingras lab were used again and put into the MaMTH expression vector (Appendix [2]) as per described in section 2.1. The only alterations are that the MaMTH assay requires only the NubI tagged construct.

2.6 MaMTH Confirmation of Interactions

MaMTH reporter line cells(Appendix [2]) were previously constructed in the Stagljar lab and were obtained from frozen stock. Mammalian cell care can be found in Appendix [2]. This method can also be found in Petchnigg 2014.

50 000 cells in 1mL of DMEM full media (Appendix [3]) were added to each well in a 24 well plate (BD Bioscience 353047). Cells were grown overnight and then transfected with ErbB2 bait and either an interacting prey, Shc as a positive control (petschnigg 2014), PecI as a negative control (Petchnigg 2014), or non-interacting phosphatases as additional negative controls. MaMTH transfection was via the calcium phosphate method where 1.8µL (100ng/µL) of bait, and 1.8µL (100ng/µL) of prey were mixed with 32.5µL of sterile double distilled Milli-Q water, 37.5µL of BES (Appendix [3]) and 3.75µL of calcium dichloride (Appendix [3]). After 6- 8 hours post transfection, transfection media was removed and cells were grown overnight in either full media, or minimal media depending on which state the RTK was in for prey interaction (either active, inactive, or both). Cells were harvested using 50µL of reporter lysis buffer (Promega E397A) with protease inhibitor (Roche 11 873 580 001). Plates were then gently rocked at 4°C for 15-20 minutes, followed by a freezing at -80°C for a minimum of 2 hours. Cell lysate was then stored at -20°C.

20µL of cell lysates was loaded into a 96 well plate and read on EG&G berthold’s microplate luminometerLB 96V using Z-term analysis. Interacting preys were ones that

produced a luciferase signal equal to or greater than 200% of the signal from Pec1, the negative non-interacting control. Samples were read and tested in three biological replicates.

A representative proportion of positive interactors, as well as all negative interactors were then tested for proof of bait and prey expression to confirm that they were not false negatives. Laemmli sample buffer (Appendix [3]) was added to samples from the luciferase screen which were run on a 8% Western gel for 70-80 minutes at 120V. Gels were transferred onto nitrocellulose membranes (Pall S80209) for 75 minutes at 0.3A. Membranes were blocked in 5% TBST milk for one hour at room temperature, and then incubated overnight at 4°C with anti- FLAG antibodies (Appendix [2]) and anti-V5 antibodies (Appendix [2]). Primary antibodies were then washed off with TBST and membranes incubated at room temperature for two hours with anti-mouse (Appendix [2]) and anti-rabbit (Appendix [2]) HRP- conjugated secondary. After washing of secondary antibodies, membranes were treated with Super Signal West Femto Max Sensitive Substrate (Thermo Scientific 34095) and exposed on Blue Ray film (VWR CA11006-128).

2.7 Functionality in Signaling

To elicit a biological relevance to interacting phosphatases, the following experiments were done in order to determine whether chosen phosphatases have an effect on ERK1/2 signaling. These experiments were performed in SKBR3 cells that could inducibly express a phosphatase of choice. Phosphatase plasmids were obtained from Dr. Nicole St-Denis in Dr. Anne-Claude Gingras’ lab at the Lunenfeld-Tanenbaum Research Institute at Mount Sinai Hospital.

2.7.1 Over-expression of Phosphatases

SKBR3 cells were transfected with FLAG-tagged phosphatases (Appendix [2]) of selected interacting partners of ErbB2 which were obtained from Dr. Nicole St-Denis at Dr. Anne-Claude Gingras’ lab at the Lunenfeld-Tanenbaum Research Institute at Mount Sinai Hospital. SKBR3 cells were seeded at 50 000 cells in 1ml of full media (Appendix [3]) per well in a 12-well plate (BD Bioscience 353043). Six of the 12 wells were further treated with tetracycline (Bioshop Canada Inc. TET701.25), in order to induce phosphatase expression. After 24 hours of growth, media was removed; cells were washed in PBS (Sigma D8537), and put into

minimum media (Appendix [3]), once again either with or without tetracycline (Bioshop Canada Inc. TET701.25). After 24 hours of starvation 1mg/mL of human epidermal growth factor (hEGF)(R&D systems 236EG) was added to the wells so that two time course of hEGF stimulation could be obtained, one for basal SKBR3 cell stimulation (non-TET-induced), and one for a phosphatase over-expression (TET-induced). hEGF stimulations were for 0, 2, 5, 10, and 60 minutes, with the sixth well remaining untreated. After stimulation, cells were harvested using 100µL of NP-40 lysis buffer (Appendix [3]). Plates were gently rocked at 4°C for 5 minutes and then moved to -80°C for a minimum of 2 hours. Cell lysate was stored at -80°C until ready for use in section 2.7.2.

2.7.2 Western Blot Protocol

Concentrations of cell lysate from section 2.7.1 were determined via Bradford assay. Bradford reagent (Biorad Laboratories Inc. 500-0006) was diluted 1:5 with double distilled Milli-Q water and 2µL of cell lysate was added to 200µL of diluted Bradford agent in a 96 well plate (BD Bioscience 353072). BSA standards were also added to 200µL of diluted Bradford reagent for the standard curve. Plates were read on the Spectra Max 190 by Molecular Devices using the SOFTmax Pro program.

Cell lysates were stored at -20°C in Laemmli sample buffer (Appendix [3]). Equal concentrations of cell lysate were loaded onto two identical 8% Western gels in a way that the over-expressed phosphatase lysate timecourse was run together first (with 0, 2, 5, 10, and 60 minutes), followed by the wild-type lystates (with 0, 2, 5, 10, and 60 minutes). Western gels were run for 70-80 minutes at 120V. Gels were then transferred onto nitrocellulose membrane (Pall S80209) for 75 minutes at a 0.3A.

Membranes were then blocked in 5% TBST milk for one hour at room temperature, and then incubated overnight at 4°C, one membrane with anti-ERK and anti-FLAG antibodies (Appendix [2]), and the other anti-phospho ERK antibodies (Appendix [2]). Primary antibodies were then washed off with TBST (two 10mL 10 minute washes) and then incubated at room temperature for two hours with anti-rabbit (Appendix [2]) HRP- conjugated secondary. Membranes were then treated with Super Signal West Femto Max Sensitive Substrate (Thermo Scientific 34095) and exposed on gel capture on Bio-imaging systems microchemi 4.2.

2.7.3 IMAGE J Analysis

Band analysis was done in order to quantify the changes in ERK1/2 phosphorylation pattern between replicates, as well as between phosphatase over-expressing, and wild-type cells. Analysis was done with ImageJ 1.46 version 10.2 and a sample calculation can be found in appendix [1]. Band intensity for phospho-ERK1/2 of each sample was measured. These values, across three replicates, for induced and uninduced samples were then plotted against the stimulation times to prove a quantitative stimulation profile for all samples.

3 Chapter 3: Results 3.1 MYTH Results

Six baits (EGFR, ErbB2, ErbB3, ErbB4, EFGR (L858R, constitutively activated), and

ErbB2 (K753A, constitutively inactivated)) were all validated using the NubG/I test by a laboratory technician in the Stagljar lab. This showed that there is no self-activation of any of the receptors on either –WLAH or X-Gal selection media, proving that all six baits are viable for further testing in the MYTH assay.

The six receptor baits were tested against all the phosphatases from the library I accumulated (appendix [1]) and a total of 89 phosphatases had 226 interactions with the six receptors. A subset of the raw results can be seen below in figure 10, while the complete results of this screen in total can be found in appendix [1]. The results from the initial screen were used to build an interactome, which can be seen in figure 11.

Figure 10 MYTH assay Raw Results: A subset of random MYTH assay results showing that all proteins tagged with the wild-type NubI domain can interact with target protein due to spontaneous ubiquitin reconstitution, resulting in growth across all selection media (-WLAH, X-

Gal). Preys tagged with NubG do not spontaneously reassociate yielding yeast that cannot grow on selection media (-WLAH, X-Gal) unless there is an interaction between bait and prey, as is the case for PPP5C, and UBLCP1.

Figure 11 Initial MYTH Interactome: Created from the results provided by the MYTH assay. Preys were tested against wild type ErbB receptors, as well as EGFR and ErbB2 mutants. Red lines denote interactions with inactive receptors, while green lines denote interactions with active receptors. Blue lines indicate interactions with both active and inactive receptors.

All 89 phosphatases were then tested against three unrelated yeast membrane proteins in an attempt to remove false positives. These raw results can be found in appendix [1]. 34 of the

89 phosphatases were identified as frequent flyers due to their interaction with one, two, or all three of the unrelated yeast proteins. An interactome showing which interactors are frequent flyers can be found in figure 12.

Figure 12 Identification of Frequent Flyers in MYTH Assay: Positive interactors from the initial MYTH screen where further tested against unrelated yeast baits to determine their propensity to interact with unrelated proteins. Preys that interacted with the yeast baits are highlighted in light red and termed frequent flyers, while green preys did not interact with unrelated baits. Red lines denote interactions with inactive receptors, while green lines denote interactions with active receptors. Blue lines indicate interactions with both active and inactive receptors.

A refined version of the interactome from figure 12 was then made which excluded the 34 phosphatases that were identified as frequent flyers, removing false positives. This refined version of the interactome generated by MYTH has 55 phosphatases which provide 109 interactions with EGFR, ErbB2, ErbB3, ErbB4, EGFR(L858R), and ErbB2(K753A) and can be seen in figure 13.

Figure 13 Refined Interactome: This interactome was created as a summary of the MYTH assay results, from the main screen and frequent flyer test. Here, the frequent flyers are removed and the protein interactions are further annotated based on phosphatase classification via node colour (using the colouring defined in section 1.4.1, which also provides more detail on

classification). Red lines denote interactions with inactive receptors, while green lines denote interactions with active receptors. Blue lines indicate interactions with both active and inactive receptors.

3.2 MaMTH Confirmation

The MaMTH assay was able to confirm 76% of the ErbB2 interactions detected in the MYTH assay (figure 14), with no particular bias towards specific phosphatases. Phosphatases were either tested in minimal or full media based on their interaction profile from the MYTH screen. Three phosphatases (CDC14B, PPP3CC, and PTPN20B) were tested twice in the MaMTH system due to two plasmids being present in the phosphatase library. Three phosphatases did not express in the MaMTH system, which led to 8% of the interactions not being tested. Figure 15 shows the fold change in luciferase signal from samples compared to the negative control. A fold change of two or greater constitutes an interaction.

Not Tested 8% Not Conirmed 16%

Conirmed 76%

Figure 14 Summary of MaMTH assay: This pie chart summarized the findings in the MaMTH assay. 76% of the ErbB2 interactors identified in MYTH were able to be confirmed MaMTH. Subsets of these positive results were further tested to ensure differences in protein expression did not lead to false positives. 8% of all proteins selected for MaMTH validation were not expressing and were unable to be tested in the MaMTH system. The last 16% were true negative hits that did not give a luciferase signal that was two-fold greater than the negative control, Pec19, but were expressed properly.

Figure 15 MaMTH Luciferase Signal Fold Change: The above graph represents the results from the MaMTH assay. Cells were grown and transfected with ErbB2 bait and a phosphatase either in full or minimal media (as per section 2.6). After growth and incubation, cells were lysed and measured for their luciferase signal on EG&G berthold’s microplate luminometer LB 96V. The signal from the negative interactor Pec19 was used as a standardized baseline, and any preys that produced a luciferase signal two-fold greater than Pec19 is considered a positive interaction.

In order to confirm expression of baits and prey in the mammalian cells used for the MaMTH assay, and therefore validate the results obtained, westerns were performed on all the negative samples, and a representative selection of the positive samples. Westerns can be seen in appendix 1. Results from samples that did not express the bait, the prey, or both proteins were excluded and the MaMTH assay was attempted again for that sample. Zorana Tubic, a summer student, performed westerns to verify expression of MaMTH constucts.

3.3 Final Interactome

Data from section 3.1(MYTH assay and validation of preys) and 3.2(MaMTH validation) was combined with a list obtained from Max Kotlyar in Igor Jurisica’s lab, which provided all documented interactors of EGFR family proteins, to determine the amount of novel and previously reported interactions the MYTH assay detected. A final interactome (provided in figure 16) shows both which interactions are consistent with previous results, and how the various phosphatases are classified.

Figure 16 Final Interactome: This final interactome provides an in depth analysis of novel and confirmed interactions (by MaMTH and database), and provided the data need to determine the next stages of the project. Preys are coded to show phosphatase class via node colour (reviewed in, and using the same colouring scheme as in section 1.4.1. Red lines denote interactions with inactive receptors, while green lines denote interactions with active receptors. Blue lines indicate interactions with both active and inactive receptors.

3.4 Class Enrichment Analysis for Interactome

In an attempt to determine if there is any binding pattern between any ErbB family members with a particular phosphatase classes (appendix 1), an enrichment of initial interactions was looked at (figure 17). Enrichment was calculated using Fisher’s exact test (sample calculation can be found in appendix 1). Samples for each particular ErbB family member’s interactions in every classification listed in section 1.4.1 (or appendix 1) were tested for both positive and negative enrichment against the global ErbB family member’s interactions. There is a positive enrichment for serine/threonine phosphatases for ErbB2 (p=0.0237) as well as for PTENs with ErbB3 (p=0.0139). EGFR is negatively enriched for myotubularins (p=0.0066), as well as its encompassing parents: DSPs (p=0.0041), class I (p=0.0290), and tyrosine phosphatases (p=0.0220).

* p < * < 0.05 p

* *

Figure 17 Phosphatase Class Enrichment: An analysis of interacting phosphatases for all four ErbB family members. Phosphatase interactions are sorted based on the classification found in section 1.4.1 (listed in appendix 1). Categories include major classifications (tyrosine phosphatases, & serine/threonine phosphatases) as well as their sub-families (resulting in two Asp-based labels the first within the tyrosine phosphatase family, the second within the serine/threonine phosphatase family). The asterisks denotes either positive enrichment, as is the case for ErbB3 and the PTEN phosphatase class, as well as ErbB2 and serine/threonine phosphatase; or negative enrichment, as is the case for EGFR and myotubularins (p=0.0066), as well as its encompassing parent classifications DSPs (p=0.0041), class I (p=0.0290), and tyrosine phosphatases (p=0.0220).

3.5 Selecting Phosphatases to Further Investigate

A subset of phosphatases were chosen in order to examine their effects in more detail. In order to accomplish this, all ErbB2 interacting phosphatases were checked for incidences of mutations across all 43 cancer studies found in CBioPortal on August 22nd 2013 (Gao 2013, Cerami 2012). CBioPortal provided a percentage of cases in a particular study on a particular cancer that had a mutated gene of interest. These percentages were then totaled across all cancer studies and ranked (table 3). The five phosphatases that had the highest percentage of incidences were the five phosphatases that were further investigated. They are: PTPRB, TPTE2, TNS3, EYA1and SSH1. The full table can be seen in appendix 1.

Table 3 Phosphatase Gene Mutation Total in Cancer Studies: CBioPortal (Gao 2013, Cerami 2012) was used to accumulate data on the incidences of phosphatase gene mutation across various cancer studies. The percentage of incidences per each individual case was then added across all cancer studies to yield total mutation percentage. Based on that number, phosphatases were ranked one through 20, with the top five phosphatases, PTPRB, TPTE2, TNS3, EYA1, and SSH1, one through five respectively, chosen to be further investigated. Total Mutation Percentage Rank CDC14B 14.7 20 CTDSP1 9.4 27 CTDSP2 10.3 25 DUSP18 14.1 17

DUSP22 25.4 12 DUSP8 14.1 18 EYA1 53.4 4 MTM1 41.7 7 MTMR14 19.5 15 MTMR2 18.7 16 MTMR4 42.9 8 PHLPP1 38 9 PPEF1 37.8 11 PPM1F 13.5 21 PPP1CA 7.7 24 PPP1CC 12 22 PPP3CC 21.3 14 PTP4A3 9.7 26 PTPMT1 7.4 28 PTPN12 38.2 10 PTPN20B 1.2 29 PTPN7 13.4 19 PTPRB 111.3 1 PTPRR 48.8 6 RNGTT 23.9 13 SSH1 46 5 STYX 11.8 23 TNS3 55 3 TPTE2 66.7 2 3.6 Effect on ERK Signaling Pathway

The top 5 phosphatase from the previous section (3.5) were overexpressed in SKBR3 cells(ErbB2 overexpression breast cancer cell line) to determine if there would be a biological response to aid in determining the role these phosphatases play in ErbB cell signaling. The western gels seen in appendix 1 show differences in ERK1/2 phosphorylation between wild type, and phosphatase overexpressed samples. A sample of Bradford assay numbers, and calculations for equal loading can be found in appendix 1. The results show that that all samples have mis- regulated ERK1/2 phosphorylation when any of the four phosphatases (TPTE2, TNS3, EYA1, and SSH1) were overexpressed. This is also supported by IMAGEJ analysis, which can also be seen in figure 18. PTPRJ was chosen as a positive control since it has previously been shown to negatively regulate ERK1/2 signaling in an expression dependent manner (Tarcic 2009). Tarcic

et al described a increase in ERK1/2 phosphorylation when PTPRJ was knocked down with siRNA, while I conversely show that overexpression of PTPRJ delays the onset, and the intensity of ERK1/2 phosphorylation. The effects of PTPRB overexpression (not shown) were not measurable since it was not well expressed in the chosen system, perhaps due to its large size. The other four phosphatases did express properly. TPTE2 overexpression shows a significant decrease of ERK1/2 phosphorylation, only at the ten-minute time point, leading to an earlier decay of ERK1/2 signaling in comparison to non-overexpressed cells. TNS3 overexpression haves the least effect on ERK1/2 signaling with only a small yet significant decrease in ERK1/2 signaling at the 2 minute time point suggesting the TNS3 overexpression delays the onset of ERK1/2 signaling. EYA1 overexpression behaves similarly to PTPRJ overexpression, where ERK1/2 signaling is significantly less intense that non-overexpressed samples at both the two and five minute time point. SSH1 shows a combination of effects including lowering ERK1/2 signaling intensity, as well as showing an earlier decay of ERK1/2 signaling. In summary, four of the five chosen phosphatases do have a negative effect on ERK1/2 signaling when overexpressed in mammalian cells, and one of the five could not be tested.

Figure 18 ERK Pathway Stimulation Patterns based on Western Band Intensity: Phosphatases of interest were chosen to complete further studies for the effect they have on the ERK1/2 pathway (TPTE2, TNS3, EYA1, and SSH1). The ERK1/2 phosphorylation profile was obtained for both a wild type, as well as phosphatase overexpressed cells at various time points (0 min, 2 min, 5 min, 10 min, and 60 min). Above graphs show the ERK1/2 phosphorylation profile for both the individual triplicates of each run in the first panel (WT 1-3 in dark blue, red, and green, and O/E 1-3 in purple, light blue and orange) as well as the average ERK1/2 phosphorylation profile for both wild-type (blue) and phosphatase overexpression (red) cells in the second panel. Asterisks denote a statistically significant difference in the phosphorylation levels of ERK1/2 between the wild type and phosphatase-overexpressed samples with p≤0.05 when using a two-tailed t-test.

4 Chapter 4: Discussion 4.1 Analysis of MYTH and MaMTH Confirmed Interactome

The interactome elucidated from this thesis provides considerable new information regarding the phosphatase interactors of the ErbB family members, and may serve as a foundation for further studies, several of which are described below in section 4.3, to explore the roles these interactors play in cellular biology, processes, and disease. Of 144 potential ErbB family interacting phosphatases, I identified a total of 55. The 55 unique phosphatases identified as interactors formed 109 unique ErbB interactions. The data and knowledge presented in this thesis will allow for a quick reference to phosphatases that should be further investigated for their role and interactions with ErbB family members. With this knowledge, phosphatases are, and can still be, prioritized as interactors and non-interactors, which drastically lowers the number of phosphatases that should be analyzed to further understand the reasons and mechanisms behind their binding with ErbB family members. The comprehension of the mechanism behind phosphatase and ErbB interactions will not only deepen our understanding of signaling biology, but would also open up new avenues in which to examine new potential drug targets for diseases and malfunctions in the pathways ErbBs and these 55 phosphatases affect. The mechanisms of current drugs that target these pathways, would be better understood, older drugs can be repurposed into new designs for better potency, and off-target effects could be better elucidated with the knowledge of the particular cross-talk that occurs between the various ErbB signaling pathways.

The positive enrichment of the serine/threonine group of phosphatases for ErbB2, as well as the PTEN group of phosphatases for ErbB3, may indicate a specialized function for those particular groups to regulate their particular target RTK.

The negative enrichment between EGFR and myotubularins (as well as its encompassing groups: DSPs, class I, tyrosine phosphatases) may indicate a lack of involvement of EGFR in phosphoinositide biology since myotubularins specifically de-phosphorylate PI(3)P. This PI(3)P activity may be regulated in part by the other three ErbB family members, and not EGFR. The negative enrichment for EGFR and DSPs, class I, and tyrosine phosphatases may be a result of

the lack of interactions and negative enrichment from the myotubularins groupings which are encompassed by each of these larger groups (section 1.4.1)

The lack of significant differences across the other phosphatase families, for the other ErbB family members, suggests that no specific phosphatase class, or small group of classes preferentially bind to ErbB receptors, and are solely responsible for ErbB regulation. This furthers the notion that different combinations of stimulus, ErbB members in the dimer, and recruited proteins each regulate similar signaling pathways, though in various unique ways such as unique protein binding profiles, proving that these signaling pathways have redundancies in them to safeguard against potential mutations and problems. This redundancy is found across all classes of phosphatases, with phosphatases in each class interacting with the same protein (ErbBs) to perform similar but different roles via similar, but different biological processes. For example, a phosphatase in one class may directly bind to, and dephosphorylate an ErbB family member to end signal transduction, while another may bind to the ErbB family member and dephosphorylate a second ErbB-bound protein to prevent signaling. In this way, the same outcome is achieved, however their biology is unique. In addition to providing the same outcome via different means, there is also the case where there is a distinct outcome reached via similar means. Such is the case in the phosphatase over-expression experiments which analyzed ERK1/2 phosphorylation, in each case a phosphatase was over-expressed, though the overexpression of different phosphatases resulted in a different ERK1/2 phosphorylation profile; certain phosphatases can post-pone ERK1/2 signaling, while over expression of others lowers ERK1/2 signaling intensity.

4.2 Analysis of Effect on ERK Signaling Pathway

In order to follow-up on the previous interaction experiments, specific phosphatases had to be identified as highly mutated in cancer studies, suggesting they play a role in the progression of the disease state. This was done by ranking the interacting phosphatases of ErbB2 based on the number of incidences they have been found to be mutated in various cancer studies as described on CBioPortal. From that analysis, five proteins, TPTE2, TNS3, SSH1, EYA1, and PTPRB were ranked as the highest mutated phosphatases across several cancer studies. Four of these five phosphatases when over-expressed were found to affect the phosphorylation pattern of ERK1/2, and the fifth, PTPRB was not able to properly express in this system. Possible

explanations for each phosphatase’s outcome, as well as possible mechanisms are discussed below.

4.2.1 TPTE2

TPTE2, phosphatidylinositiol 3,4,5-triphosphate 3-phosphatase is a lipid phosphatase named for its homology to TPTE and PTEN (a known tumor suppressor) (Walker 2001, She 2003, Tarcic 2009). It has been shown to have lipid phosphatase activity, and is localized to intra-cellular membranes (Walker 2001). Due to its similarity to PTEN, studies have looked for TPTE2 mutation in cancer to determine if it too could be a tumor suppressor protein (In particular, in testicular cancer where TPTE2 is most highly expressed), however no mutations of TPTE2 were found across several samples (Walker 2001, Tapparel 2003).

Regarding TPTE2’s biological role, it is hypothesized that TPTE2 may be regulated by TPTE, which has no phosphatase activity and regulates TPTE2 as a competitive substrate binder (Tapparel 2003). It has been previously shown that TPTE2 has no effect on PKB phosphorylation, which is a required component in the AKT pathway, suggesting that TPTE2 plays no dominant role in AKT signaling (Walker 2001). In addition, the N-terminal domain of TPTE2 was looked at in isolation, and upon over-expression of this domain in both HeLa and HEK293 cells, growth and proliferation of cells is halted, and apoptosis is induced (Mishra 2011, Mishra 2012). The induction of apoptosis is facilitated via the activation of caspase 3 (Mishra 2011, Mishra 2012). Caspase 3 is normally present as an inactive 32kDa protein, and during apoptosis, it is cleaved into its 17-19kDa and ~12kDa active forms which facilitate cell death (Henson 2006, Mishra 2011, Mishra 2012). Caspase 3 is a known member of the death receptor pathway, which is also known to be regulated by ErbB signaling (Henson 2006). Upon activation of the ERK1/2 pathway, activated downstream proteins and transcription factors facilitate the inhibition of caspase 3 cleavage, maintaining its inactivity and preventing apoptosis (Henson 2006).

Combining the knowledge presented in this thesis: that TPTE2 binds only activated ErbB2, and the over-expression of TPTE2 results in quenching of ERK1/2 phosphorylation, with the knowledge obtained from Mishra 2011, and Mishra 2012, I can hypothesize a mechanism of action between TPTE2 and ErbB2 to be further investigated. In a non-stimulated cell TPTE2 is

controlled and regulated in a way that facilitates its ability to activate the pro-apoptotic caspase 3 pathway. Upon ErbB2 stimulation, anti-apoptotic pathways are stimulated, and pro-apoptotic pathways, such as the caspase 3 pathway, are inhibited. The inhibition of the caspase 3 pathway is facilitated by the recruitment of TPTE2 to ErbB2, preventing TPTE2 from activating caspase 3. Upon ErbB2 inactivation, TPTE2 is released, due to its inability to bind inactive ErbB2, and then able to activate and regulate the caspase 3 apoptotic pathway.

This also could explain its effect on ERK1/2 signaling (figure 18). We see a substantial reduction in ERK1/2 phosphorylation levels after 10 minutes of EGF stimulation compared to the control sample. If we continue with the hypothesis that TPTE2 binds ErbB2 in an activated state to prevent it from activating caspase 3 (and subsequent apoptosis), we can assume that the overexpression of TPTE2 results in far too many TPTE2 phosphatases in comparison to TPTE2 binding locations on ErbB2. Extra TPTE2 phosphatases, which are stericly unable to bind ErbB2, are instead unregulated, and continue to promote caspase 3 cleavage and activation resulting in pro-apoptosis functions. Since there is an abundance of cross-talk between both pro- apoptosis and proliferative signaling pathways within a cell, it is very likely that the activation of the pro-apoptosis caspase 3 pathway by TPTE2 is connected to and down-regulates the proliferative EGF induced ERK1/2 pathway. In short, the overexpression of TPTE2 would result in the activation of the caspase 3 apoptotic pathway, which, through various cross-talk pathways, inactivates the ERK1/2 anti-apoptotic pathway, resulting in a quicker de-phosphorylation of ERK1/2 in comparison to wild-type cells.

4.2.2 TNS3

TNS3, or tensin-3 is expressed on cellular membranes, particularly at cell junctions, which regulates cellular migration (Cui 2004, Katz 2007). TNS3 is expressed in many tissues including the heart, skeletal muscles, liver, lung, and spleen (Cui 2004). It has also been linked to EGF signaling; EGF signaling induces tyrosine phosphorylation of TNS3 with the aid of the Src protein (a protein in the JAK/STAT pathway), which increases TNS3’s affinity for ErbB family members (Cui 2004). In regards to TNS3’s specific role in ErbB signaling, it has been found that knockdown of TNS3 results in increased cell migration, and in addition to this, CTEN (a phosphatase that is shown to have opposite roles from those of TNS3) expression is correlated to an increase in HER2 breast cancer metastasis. Together, this suggests that TNS3 and CTEN

act as a switch that can contribute to breast cancer metastasis (Katz 2007). Further implicating TNS3 as a tumor suppressor is a study done by Maeda et al 2006, showing that TNS3 is expressed in normal thyroid tissues, but is absent in thyroid cancer tumor samples. In addition to TNS3 being implicated in cancers and as a tumor suppressor, it has also been suggested that TNS3 plays a role in ErbB signal transduction by acting as a platform for the disassembly of ErbB signaling complexes (Cui 2004, Maeda 2006, Katz 2007).

Though there is a small, yet statistically significant, change in the phosphorylation pattern of ERK1/2 at the two-minute mark upon overexpression of TNS3, there isn’t much deviation at any other time points in the assay, suggesting that TNS3 does not affect ERK1/2 signaling. It would be interesting to do a similar experiment on the JAK/STAT pathway and to see how overexpressing TNS3 would affect this pathway, since the phosphorylation of TNS3 has been shown to be dependent on Src presence. This could link TNS3 to a known ErbB signaling pathway not investigated in the scope of this thesis. In addition, a knockdown of TNS3 protein could yield more interesting results. If I could determine if TNS3 knockdown resulted in prolonged ERK1/2 signaling, or JAK/STAT signaling, I could corroborate the idea that TNS3 is vital for the dissociation of ErbB signaling complexes due to it having a physical interaction with ErbB2. Proving this could also aid in the understanding of why it is absent in thyroid tumors, but not normal cell samples. This would further suggest TNS3 acts as a negative regulator for the dissociation of proteins needed for ErbB cellular signaling, and its loss could promote longer signaling processes and transduction.

4.2.3 EYA1

There are not nearly as many links to EYA1 and ErbB signaling in comparison to the TPTE2, TNS3, and SSH1. It has been proven to have a necessary role in regulating developmental genetic cascades as a transcriptional co-factor (Fougerousse 2002, Cook 2009). In a more closely related area, it was shown to have phosphatase activity that works to dephosphorylate the H2AX histone, and this dephosphorylation correlates with a response to repairing DNA damage or cellular apoptosis (Fougerousse 2002, Li 2003, Rayapureddi 2003, Tootle 2003, Cook 2009). EYA1 has been shown to bind and dephosphorylate tyrosine residues of H2AX in vivo, and that this particular de-phosphorylation leads to cellular apoptosis, but only when the cell is undergoing a response to DNA damage (Cook 2009). With no DNA damage

signals, EYA1 does not bind H2AX, which retains its phosphorylated tyrosine residues (Cook 2009).

It is not immediately clear based upon the information known about this protein why an interaction between EYA1 and inactive ErbB2 was detected in this study, or why the overexpression of this protein results in a delayed ERK1/2 signaling response. It could be due to the intricate cross-talk between various cellular pathways, or a second unknown pathway the EYA1 protein is also involved in. There could also be a link between EYA1 and ErbB signaling similar to what was hypothesized with TPTE2, that it is recruited to ErbB2 in order to prevent it from executing its pro-apoptosis role while the ErbB pathway is activating anti-apoptosis pathways. Regardless, much more research needs to be done on the role EYA1 plays in cellular processes and its role in ErbB interactions and signaling.

4.2.4 SSH1

To date, SSH1, slingshot homologue 1, has been implicated in actin rearrangement (Niwa 2002, Endo 2003, Ohta 2003, Nishita 2004). It is present diffusely throughout the cell, and upon stimulation, or entrance of cells into telophase, becomes co-localized with and binds f-actin (Niwa 2002, Kaji 2003, Endo 2003, Ohta 2003, Nagata-Ohashi 2004, Nishita 2004). It has been implicated in playing crucial roles for proper cell motility and extension, and in preventing the formation of multi-nuclear cells due to improper cellular division (Niwa 2002, Endo 2003, Ohta 2003, Nagata-Ohashi 2004, Nishita 2004, Nishita 2005). It has also been proven that SSH1’s role is stimulus based, as is evident in its co-localization with f-actin upon stimulation with NRGs, an ErbB family ligand (Niwa 2002, Nagata-Ohashi 2004).

Though there is no previous knowledge or clear link between SSH1 and ErbB2 interaction, I would propose investigating if SSH1 plays a role in receptor endocytosis, which could be supported by its role in actin reorganization, and the role actin plays in endocytosis. ERK1/2 analysis shows that over-expression of SSH1 results in quicker quenching of ERK1/2 signaling; I suggest that this is due to an increased rate of receptor endocytosis, which leads to a reduced ERK1/2 stimulation time. In order to test this further, experiments would have to be performed on the endocytosis rate of ErbB receptors upon SSH1 knock-down and overexpression to determine if this particular phosphatase regulates ErbB activity via endocytosis.

4.3 Future Directions

The next logical step to build upon the work presented in this thesis involves several important follow up experiments that should be done for the particular phosphatases I chose to examine in more details, as well as experiments that should be done on other phosphatases to expand the breadth of the results. These are in short: (1) examining where in the ERK1/2 pathway the phosphatase is executing its effect (at the ErbB binding site, or on an additional ErbB binding protein etc.), (2) all ERK1/2 experiments repeated with catalytically dead phosphatases to determine if the catalytic activity is responsible for results, or if the phosphatase acts as a scaffold or recruiting protein, and if so what is recruited, (3) the knockdown of endogenous phosphatases to check for a similar yet opposite (complementary) effect in ERK1/2 phosphorylation levels in comparison to the results from section 3.6, (4) an expansion to look at effects on the other signaling pathways, such as the JAK/STAT and AKT pathways, which are the other two major pathways which are heavily regulated by RTKs and PPs, and (5) determine how the changes in ERK1/2 phosphorylation as a result of phosphatase mis-regulation affect gene expression, particularly genes involved in proliferation and pro-apoptosis signaling.

4.3.1 Pathway Activity Location

Based on the experiments completed in this thesis, now I know that i) which particular phosphatases interact with which receptors, and ii) the overexpression of four of those phosphatases results in various and specific changes in ERK1/2 signaling, a known signaling pathway downstream of RTKs. As discussed in the introduction however, the ERK1/2 pathway involves many different proteins, both kinases and phosphatases, downstream of the ErbB family RTKs under investigation. I would like to determine if the phosphatases that interact with ErbB2 and also affect ERK1/2 signaling, TPTE2, TNS1, EYA1, and SSH1, do so by modifying phosphate groups on ErbB2, or on some other protein in the ERK1/2 cascade.

Using specific mutants in the ERK1/2 pathway (figure 5) to disrupt signaling I could easily test this. For example, overexpressing a mutant of Shc, one of the first proteins to be recruited to an active RTK to start the ERK1/2 signaling pathway, and causes it to be over- activated (i.e. constantly phosphorylated), would create an increase in the signaling pathway. The Shc mutant should perpetually activate the ERK1/2 cascade independently of the receptor

leading to permanently phosphorylated ERK1/2. If one of the overexpressed phosphatases (TPTE2, TNS3, EYA1, or SSH1) executed its effect upstream of Shc in the signaling cascade (i.e. only on the RTK), we should see no significant difference in ERK1/2 phosphorylation regardless of phosphatase overexpression. If however the phosphatase executed its effect either on Shc or downstream of Shc, then an overexpression of the phosphatase should partially correct the hyperactive Shc, and result in a difference in ERK1/2 phosphorylation. In the same way, hyperactive or hypoactive proteins in the ERK1/2 cascade can be used to pinpoint a location of activity for the phosphatase tested by examining if the over-expression of the phosphatase in question produces a change in ERK1/2 phosphorylation levels. By working with different proteins in the ERK1/2 pathway, a specific protein in the signaling cascade can be elucidated for the target protein of the phosphatase in question that results in the difference of ERK1/2 signaling.

4.3.2 Catalytic Activity

This thesis has identified interacting RTKs and phosphatases, however the nature of this interaction in unknown. I suggest determining if the interaction between RTK and phosphatase is dependent on the phosphatases catalytic activity by performing the MYTH screen with catalytically dead phosphatase preys. In addition to determining if the interaction is dependent on the catalytic activity, I also suggest performing the overexpression and ERK1/2 phosphorylation level analysis with catalytically dead mutants. This experiment could yield more information about the process in which these phosphatases effect signaling pathways. For example, if a catalytically dead mutant mis-regulated ERK1/2 phosphorylation levels in the same way its catalytically active counterpart did, then we could hypothesize that this particular phosphatase acts as a recruiter or scaffold for other proteins, since the phosphatase activity is not necessary. If however the catalytically dead mutant did not mis-regulate the ERK1/2 phosphorylation levels in a similar way to its active counterpart, we can then hypothesize that this particular phosphatase’s phosphatase activity is require and regulated the phosphorylation of ERK1/2.

If it is determined that catalytic activity is not necessary, then new experiments can be introduced to further elucidate the mechanism of action for the phosphatase in question. Screens can be done to determine if, and how many other proteins bind to that phosphatase assuming it

acts as a scaffold, or perhaps a cell can sense when this particular phosphatase is mis-regulated and has a defense mechanism to counteract any negative effects that would be a result of the loss of catalytic activity in the particular phosphatase. Depending on the results of these experiments, more information on how the interaction between RTK affects various different cellular regulation mechanisms would be elucidated.

4.3.3 Gene Knockdown Effects

As with all overexpressed systems, a change seen via overexpression should yield a similar, but opposite change with the knockdown of the same protein. Once all above experiments have been performed on an overexpressed system (effects on signaling and signaling location) the same experiments should be performed using a knockdown model. For example, TPTE2, when overexpressed, quenches ERK1/2 signaling faster than its normally regulated counterpart. Based on that, when TPTE2 is knocked down, we should theoretically see prolonged ERK1/2 signaling.

If we however did not see this effect, then we could continue to investigate why that is and what the true role of TPTE2 is. For example, if I determined TPTE2 phosphatase activity is not necessary for ERK1/2 signaling, the fact that prolonged ERK1/2 signaling was not seen could be due to another protein which can act the same way as TPTE2 (scaffold like), or replaces TPTE2 when the cell detects a disruption in TPTE2 regulation. The results of all these proposed experiments, when combined, could shed an enormous amount of light on the intricacies of RTK/PP signaling.

4.3.4 Gene Regulation Effects

Another investigation that should be completed is whether these changes in ERK1/2 phosphorylation patterns produce a biological effect, particularly on gene expression, or if there is another system whose purpose is to regulate ERK1/2 signaling (or compensate for ERK1/2 signaling if the signaling is reduced/shut-down/mis-regulated) if something changes upstream (such as overexpressing or losing a key phosphatase or that phosphatase’s phosphatase ability). This would not only show that the effect has a biological outcome, but if it did not, we would have the building blocks to identify or discover a new regulation mechanism or pathway that regulates the ERK1/2 pathway.

To this end, the analysis of the up or down regulation of ERK1/2 target genes such as MCL1, BCL1, c-FLIP, NFκ1 and CREB (Davis 1995, Widmann 1999, Downward 2003, Balan 2006, McCubray 2008, McCubray 2009, Martelli 2010, Steelman 2011), all anti-apoptosis genes, would be taking the biological significance and outcome one step further. By using qPCR, the transcript numbers of key regulation genes can be seen and quantified for basal versus overexpressing, knock-out, or catalytically dead phosphatase samples. This knowledge will further solidify the links between ERK1/2 pathway outcome with respect to RTK/PP interactions.

4.3.5 Other Pathways

The above experiments would provide a fairly extensive base of knowledge on the effects the five phosphatases chosen will have on the ERK1/2 pathway, where the effect is localized to, and via which mechanism. However the ERK1/2 pathway is one of many in the cell, and even when narrowing down the scope to the pathways RTKs and PPs are known to interact with, there are still several more such as the JAK/STAT, AKT, and death receptors which are equally affected by RTK signaling. In order to have a complete understanding of the signaling cascades between RKT and PP interactions, these pathways must also be investigated.

Since the beginning of the ERK1/2 signaling investigation, a new technique has been identified as a mean to quickly and quantitatively look at various pathways within a single experiment, the Cignal Reporter Assay (available through Qiagen). In short, you are able to test up to 45 different signaling pathways (such as MAPK/ERK, MAPK/JNK, cAMP/PKA, PI3K/AKT, Myc/Max, along with others previously implicated in RTK/PP signaling) for changes in transcription factor response by monitoring changes in luciferase signal. For example, there is a 96-well format that has 10 different signal pathways, each of which can be investigated under 8 different conditions. For each signal pathway experiment, there are two wells, one to remain untreated, and a second to be treated. In this case, the untreated would be wild type cells exposed to hEGF (or other stimulus) for two, five, or ten minutes, while treated would be either phosphatase overexpressing cells, phosphatase knock-down, or catalytically dead treated with hEGF (or other stimulus) for two, five, or ten minutes. After performing the experiment, the readout would show changes between the treated and untreated cells as a

luciferase signal. In this way the changes in downstream gene activation would be able to be monitored for each of our situations. This would reduce the time it took to investigate other high interest phosphatases, as well as the time it takes to investigate multiple signaling pathways for a single phosphatase of interest.

4.4 Summary and Conclusions

In summary, a comprehensive interactome was constructed for all human phosphatases against the wild type ErbB family receptors, as well as mutated EGFR and ErbB3, providing an active and inactive interactome for these two receptors, using the MYTH assay. The interactome for ErbB2 and its interacting partners was confirmed via MaMTH at a rate of 76%, with 8% of interactors not being able to be tested. Five ‘top interactors,’ PTPRB, TPTE2, TNS3, EYA1, and SSH1, were chosen from the ErbB2 interactome due to their high prevalence of mutation in cancer genomes. These five proteins were further investigated to determine their role in the ERK1/2 signaling pathway. To this end, phosphatases were overexpressed in a controlled system to determine the outcome on ERK1/2 phosphorylation. PTPRB was unable to be tested due to expression problems, while the other four proteins all had an effect on ERK1/2 signaling: TPTE2 quenched signaling faster than wild-type, TNS3 slightly postponed signaling initiation, EYA1 decreased the intensity of signal, and SSH1 both decreased signal intensity and quenched the signaling faster.

These results will prove to be invaluable in decoding the relationships between RTKs, particularly the ErbB family members, and human phosphatases. The interactome created is a reference that can be pulled upon to confirm interaction of proteins studied by other labs, as well as will provide key information as to what phosphatases should be further investigated for their role in signaling, or in disease states. The knowledge of the effects of overexpressing certain phosphatases (TPTE2, TNS3, EYA1, and SSH1) provides invaluable information on not only how wild-type levels of this protein probably interact with and affect RTK/PP/ERK1/2 signaling, but also provides an insight as to what mechanism is being improperly regulated in cancers with mutations in these particular proteins’ genes. Armed with this knowledge, and the potential knowledge that would be obtained by conducting the experiments presented in section 4.3, a full mechanism of action can be ascertained for both wild-type and diseased cellular states, and from

the knowledge, drugs and treatments to correct dysfunctional phosphatase activity could be easier to identify.

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1 Appendix 1 1.1 Phosphatase Library

Phosphatases were obtained from Dr. Nicole St-Denis from Dr. Anne-Claude Gingras’ lab at the Lunenfeld-Tanenbaum Research Institute at Mount Sinai Hospital. Below in table 4 is a summary of all phosphatases tested, along with their OF number (vector ID numbers from OpenFreezer, Olhovsky 2011), vector bacterial resistance, insert size, and vector size. Note that table 4 is alphabetical order, however for section 1.2 the order of the phosphatases is based on the order they were received.

Table 4 Phosphatase Library: A summary of all phosphatases tested, along with OF number (vector ID numbers from OpenFreezer, Olhovsky 2011), vector resistance, insert size (bp), and vector size (bp). Phosphatase OF Number Bacterial Resistance Insert Size Total size ACP1 V7433 Spectinomycin 477 3270 CDC14A V6837 Spectinomycin 993 3786 CDC14B V99163 Spectinomycin 1538 4314 CDC14B V99163 Spectinomycin 1538 4314 CDC25A V107842 Kanamycin 1595 4125 CDC25B V6838 Spectinomycin 1743 4536 CDC25C V113640 Kanamycin 1445 3975 CDKN3 V6977 Spectinomycin 519 3312 CTDSP1 (SCP1) V6979 Spectinomycin 786 3579 CTDSP2 (SCP1) V7010 Spectinomycin 816 3609 CTDSPL (SCP3) V103606 Spectinomycin 839 3615 CTDSPL2 (HSPC129) V108647 Kanamycin 1421 3951 Dullard (CTDNEP1) V108053 Kanamycin 755 3285 DUPD1 (DUSP27) V7012 Spectinomycin 663 3456 DUSP1 V7128 Spectinomycin 1104 3897 DUSP10 V6970 Spectinomycin 1449 4242 DUSP11 NA Kanamycin 993 5755 DUSP12 V107689 Kanamycin 1043 3573 DUSP13 V7274 Ampicillin 617 5800 DUSP14 V106303 Kanamycin 617 3147 DUSP15 V120726 Spectinomycin 719 3495 DUSP16 V7129 Spectinomycin 1989 4782 DUSP18 V93516 Spectinomycin 578 3354 DUSP19 V106130 Kanamycin 674 3204 DUSP2 V113655 Kanamycin 968 3498 DUSP21 V7014 Spectinomycin 573 3366 DUSP22 V108316 Kanamycin 575 3105 DUSP23 V7508 Spectinomycin 451 3243

DUSP23 V7508 Spectinomycin 451 3243 DUSP24 (STYXL1) V108343 Kanamycin 962 3492 DUSP26 (Putative) V7016 Spectinomycin 636 3429 DUSP3 V111609 Kanamycin 578 3108 DUSP4 V6971 Spectinomycin 1185 3978 DUSP5 V112552 Kanamycin 1178 3708 DUSP6 V107413 Kanamycin 1166 3696 DUSP7 V7018 Spectinomycin 963 3756 DUSP8 V7327 Spectinomycin 1878 4671 DUSP9 V7135 Spectinomycin 1155 3948 EYA1 V7439 Spectinomycin 1779 4572 EYA2 V7436 Spectinomycin 1545 4338 EYA3 V7495 Spectinomycin 1608 4384 EYA4 V7440 Spectinomycin 1851 4644 ILKAP V107723 Kanamycin 1199 3729 Laforin (EPM2A) V7331 Spectinomycin 996 3789 MTM1 V7020 Spectinomycin 1812 4605 MTMR1 V7329 Spectinomycin 1092 3885 MTMR10 V6973 Spectinomycin 1002 3795 MTMR11 V7428 Ampicillin 1964 7147 Mtmr12 (Mouse!) V7437 Spectinomycin 1581 4374 MTMR14 V7429 Ampicillin 1976 7159 MTMR2 V7022 Spectinomycin 1932 4725 MTMR3 V6972 Spectinomycin 3597 6390 MTMR4 V7136 Spectinomycin 3588 6381 MTMR6 V92752 Kanamycin 1883 4124 MTMR7 V7024 Spectinomycin 1983 4776 MTMR8 V7026 Spectinomycin 2115 4908 MTMR9 V7427 Ampicillin 1670 6853 PDP1 V6649 Spectinomycin 1614 4413 PDP2 V6651 Spectinomycin 1590 4389 PHLPP1 V6653 Spectinomycin 3618 6417 PPEF1 (PP7α) V112829 Kanamycin 1985 4515 PPEF2 (PP7β) V94512 Spectinomycin 2303 5079 PPM1A V106690 Kanamycin 1169 3699 PPM1B V6656 Spectinomycin 1440 4239 PPM1D V105971 Kanamycin 1838 4368 PPM1E V6658 Spectinomycin 2271 5073 PPM1F V7028 Spectinomycin 1362 4155 PPM1G V107074 Kanamycin 1661 4191 PPM1H V103476 Spectinomycin 1586 4362 PPM1J V7030 Spectinomycin 1017 3810 PPM1K V6660 Spectinomycin 1119 3918 PPM1L V6662 Spectinomycin 1083 3882 PPM1M V7108 Spectinomycin 744 3537 PPP1CA V7774 Spectinomycin 1010 3786 PPP1CB V6667 Spectinomycin 984 3783 PPP1CC V113009 Kanamycin 995 3525 PPP2CA V7221 Spectinomycin 930 3723 PPP2CB V7222 Spectinomycin 930 3723 PPP3CA V6669 Spectinomycin 1536 4335

PPP3CA V93054 Kanamycin 1553 4083 PPP3CB V7137 Spectinomycin 1578 4371 PPP3CB V7137 Spectinomycin 1578 4371 PPP3CC V6673 Spectinomycin 1539 4338 PPP3CC V6673 Spectinomycin 1539 4338 PPP4C V106871 Kanamycin 944 3474 PPP5C V6675 Spectinomycin 1500 4299 PPP5C V6675 Spectinomycin 1500 4299 PPP6C V7218 Spectinomycin 918 3711 PPTC7 V7110 Spectinomycin 915 3708 PTEN V107527 Kanamycin 1232 3762 PTP4A1 V105639 Kanamycin 542 3072 PTP4A2 V92943 Kanamycin 2642 4883 PTP4A3 V6967 Spectinomycin 447 3240 PTPDC1 V7139 Spectinomycin 2265 5058 PTPMT1 V7424 Ampicillin 629 5812 PTPN1 V7520 Spectinomycin 1308 4101 PTPN11 V7431 Spectinomycin 153 2946 PTPN12 V7501 Spectinomycin 2343 5119 PTPN13 V7142 Spectinomycin 7401 10194 PTPN14 V7444 Spectinomycin 3564 6357 PTPN18 V7497 Spectinomycin 1054 3829 PTPN2 V7493 Spectinomycin 1161 3937 PTPN20A V7573 Spectinomycin 681 3474 PTPN20B V7430 Ampicillin 722 5905 PTPN20B V94234 Spectinomycin 722 3498 PTPN21 V7499 Kanamycin 3535 6096 PTPN22 V7441 Spectinomycin 2259 5052 PTPN23 V7446 Spectinomycin 4911 7704 PTPN3 V7442 Spectinomycin 2742 5535 PTPN4 V7443 Spectinomycin 2781 5574 PTPN5 V7438 Spectinomycin 1698 4491 PTPN6 V7425 Ampicillin 1814 6997 PTPN7 V7739 Spectinomycin 1200 3993 PTPN9 V7426 Ampicillin 1802 6985 PTPRA V7574 Spectinomycin 2379 5172 PTPRB V7570 Spectinomycin 5991 8784 PTPRC V7575 Spectinomycin 3456 6249 PTPRD V7569 Spectinomycin 4518 7311 PTPRE V7561 Spectinomycin 2133 4929 PTPRF V7572 Spectinomycin 5691 8487 PTPRG V7566 Spectinomycin 4335 7128 PTPRH V7563 Spectinomycin 3345 6138 PTPRJ V7576 Spectinomycin 4011 6804 PTPRK V7567 Spectinomycin 4338 7131 PTPRN V7562 Spectinomycin 2253 5046 PTPRO V7564 Spectinomycin 3648 6441 PTPRR V7505 Spectinomycin 1239 4015 PTPRS V7568 Spectinomycin 4503 7296 PTPRT V7577 Spectinomycin 1380 7173 PTPRU V7503 Spectinomycin 4302 7078

Ptprz1 (Mouse!) V7571 Spectinomycin 6936 9729 RNGTT V7768 Spectinomycin 1811 4587 SBF1 (MTMR5) V112437 Kanamycin 5705 8235 SSH1 V7123 Spectinomycin 3150 5943 SSH2 V103633 Spectinomycin 4313 7089 SSH3 V7125 Spectinomycin 1026 3819 SSU72 V107910 Kanamycin 605 3135 STYX V7032 Spectinomycin 672 3465 TAB1 (MAP3K7BP1) V6665 Spectinomycin 1515 4314 TENC1 V6969 Spectinomycin 4230 7023 TIMM50 V7138 Spectinomycin 1371 4164 TNS1 V7530 Spectinomycin 5208 8001 TNS1 V7530 Spectinomycin 5208 8001 TNS3 V7445 Spectinomycin 4338 7131 TPTE V106135 Kanamycin 1622 4152 TPTE2 V6968 Spectinomycin 1236 4029 UBLCP1 V7034 Spectinomycin 957 3750 1.2 Phosphatase Class Members

A comprehensive list of the class and families particular phosphatases belong to according to Alonso et al. 2004. PTPRA PTPRB PTPRC PTPRD PTPRE PTPRF PTPRG PTPRH PTPRJ PTPRK PTPRN PTPRN2 PTPRO PTPRR PTPRS PTPRT Receptor PTPRU Like PTPRZ1

Non- PTPN1 Receptor PTPN2

Like PTPN3 PTPN4 PTPN5 PTPN6 PTPN7 PTPN9 PTPN11 PTPN12 PTPN13 PTPN14 PTPN18 PTPN20A/B PTPN21 PTPN22 PTPN23

DUSP1 DUSP2 DUSP4 DUSP5 DUSP6 DUSP7 DUSP8 DUSP9 DUSP10 MKPs DUSP16

DUSP3 DUSP11 DUSP12 DUSP13 DUSP14 DUSP15 DUSP18 DUSP19 DUSP21 DUSP22 Atypical DUSP23 DSPs DUSP24

DUSP26 DUSP27 EPM2A RNGTT STYX

SSH1 SSH2 Slingshots SSH3

PTP4A1 PTP4A2 PRLs PTP4A3

CDC14A CDC14B CDKN3 CDC14s PTPDC1

PTEN TENC1 TNS1/3 TPTE PTENs TPTE2

MTM1 MTMR1 MTMR2 MTMR3 MTMR4 MTMR5 MTMR6 MTMR7 MTMR8 MTMR9 MyoTubularins MTMR10

MTMR11 MTMR12 MTMR14

LMPTP ACP1

CDC25A CDC25B CDC25 CDC25C

EyA1 EyA2 Eya3 EyA Eya4

PPEF1 PPEF2 PPP1CA PPP1CB PPP1CC PPP2CA PPP2CB PPP3CA PPP3CB PPP3CC PPP4C PPP5C PPP PPP6C

ILKAP PDP1 PDP2 PHLPP1 PPM1A PPM1B PPM PPM1D

PPM1E PPM1F PPM1G PPM1H PPM1J PPM1K PPM1L PPM1M PPTC7 TAB1

CTDSP1 CTDSP2 CTDSPL CTDSPL2 TIMM50 SSU72 UBLCP1 PTPMT1 Asp-based CTDNEP1

1.3 MYTH Results

Summary of the raw data (yeast colonies) can be accessed via the Stagljar lab as computer back ups of raw results of this thesis (Katelyn/Grad Project/pictures/MYTH results).

1.4 Prey Validation

Summary of the raw data (yeast colonies) can be accessed via the Stagljar lab as computer back ups of raw results of this thesis (Katelyn/Grad Project/pictures/MYTH results).

1.5 MaMTH Confirmation

The raw luciferase readings, along with their respective fold change, from the MaMTH confirmation can be seen summarized in table below.

Table 5 Luciferase Reading Values: This table shows the raw luciferace readings obtained for each of the active and inactive phosphatases tested, for all three technical replicates along with their relative fold change. These were the values used in all MaMTH confirmation graphs and figures (figure).

Replicate 1 Replicate 2 Replicate 3 Average Standard Phosphatase Luc. Sig. Fold Δ Luc. Sig. Fold Δ Luc. Sig. Fold Δ Fold Δ Deviation CDC14B (INACTIVE) 637 4.04 691 2.30 637 5.59 3.98 1.64 CDC14B (INACTIVE) 1778 3.21 483 2.96 263 2.31 2.83 0.47 CTDSP1 (INACTIVE) 1653 12.07 1749 9.02 1331 9.72 10.27 1.60 CTDSP2 (INACTIVE) 368 2.19 229 2.01 620 2.39 2.20 0.19 DUSP18 (INACTIVE) 1237 2.15 310 2.09 549 2.11 2.12 0.03 DUSP22 (INACTIVE) 748 4.37 903 3.16 903 3.47 3.67 0.63 DUSP8 (INACTIVE) 137 1.02 161 1.09 130 0.94 1.02 0.08 EYA1 (INACTIVE) 662 2.32 452 3.25 662 2.55 2.71 0.49 MTM1 (ACTIVE) 454 3.17 476 3.58 823 2.26 3.00 0.68 MTMR14 (INACTIVE) 125 0.93 117 0.88 156 1.05 0.96 0.09 MTMR2 (ACTIVE) 1829 5.02 2972 5.16 1092 6.39 5.52 0.75 MTMR2 (INAVTIVE) 794 5.80 896 6.45 1783 6.86 6.37 0.54 MTMR4 (ACTIVE) 1085 3.62 760 4.66 236 2.07 3.45 1.30 PHLPP1 (ACTIVE) 151 1.13 150 1.13 175 1.26 1.17 0.08 PPEF1 (INACTIVE) 151 1.13 154 1.16 188 1.35 1.21 0.12 PPM1F (INACTIVE) 1986 14.29 10844 18.83 3754 14.44 15.85 2.58 PPP1CA (INACTIVE) 360 2.54 870 2.90 581 3.69 3.04 0.59 PPP1CC (INACTIVE) 704 4.82 692 3.57 569 3.33 3.91 0.80 PPP3CA (INACTIVE) 905 3.44 633 3.17 464 3.39 3.33 0.15 PPP3CC (INACTIVE) 598 3.67 856 2.35 648 4.44 3.49 1.06 PPP3CC (INACTIVE) 938 6.85 1520 4.20 1474 4.91 5.32 1.37 PTP4A3 (ACTIVE) 304 0.88 128 0.90 120 0.84 0.87 0.03 PTPMT1 (INACTIVE) 582 3.10 551 2.93 568 2.18 2.74 0.49 PTPN20B (ACTIVE) 818 2.73 1242 3.43 331 2.90 3.02 0.37 PTPN20B (ACTIVE) 942 2.71 566 2.92 602 2.05 2.56 0.45 PTPN20B (INACTIVE) 821 2.74 1001 2.77 433 2.17 2.56 0.34 PTPN20B (INACTIVE) 826 2.75 602 3.10 662 2.56 2.80 0.28 PTPN7 (ACTIVE) 326 2.30 406 2.96 569 2.85 2.70 0.36 PTPN7 (INACTIVE) 1199 6.18 779 4.02 563 3.45 4.55 1.44 PTPRB (ACTIVE) 134 1.00 135 1.02 164 1.11 1.04 0.06 PTPRR (ACTIVE) 169 1.26 127 0.95 134 0.96 1.06 0.17 RNGTT (ACTIVE) 603 4.25 509 2.62 1465 2.64 3.17 0.93 SSH1 (INACTIVE) 313 2.34 444 3.19 2967 5.15 3.56 1.44 TNS3 (ACTIVE) 386 2.30 607 2.07 607 2.31 2.22 0.14 TPTE2 (ACTIVE) 301 1.79 321 1.61 266 1.01 1.47 0.41 Pec1 (negative cnt) 134 1.00 133 1.00 139 1.00 1.00 Shc1 (positive cnt) 18383 137.19 17798 133.82 13317 116.82 129.27 10.92

A summer student, Zorana Tubic, performed the western confirmation of expression for the MaMTH system. Her results are shown below in figure 19. Whole westerns can be accessed in the Stagljar lab.

Figure 19 MaMTH Expression Confirmation: The above westerns are proof of expression for selected phosphatases that were tested for an interaction with ErbB2 in the MaMTH assay. Cell lysates were stored after luciferase signal was read, and samples were then run on an 8% acrylamide gel that was blotted for α-FLAG or α-V5. The α-FLAG antibody recognized the phosphatase preys, which ran at the indicated size, and the α-V5 antibody recognized the ErbB2 bait, at approximately 200kDa. The positive luciferase signal, along with positive proof of expression further confirmed positive hits, while lack of luciferase signal, as well as proof of expression confirmed that the non-hits were true non-interactors instead of false negatives.

1.6 Interaction Enrichment

Enrichment was tested for each of the ErbB family members against the remaining ErbB family members across all phosphatase classes (section 1.4.1, or A.1.2). Calculations were based on the Fisher’s exact test with two-tailed p-values being used. Sample Calculations can be found below in table 6.

Table 6 Enrichment Classification: example calculations using a two-tailed Fisher exact test for the tyrosine phosphatase class of human phosphatases against each of the ErbB family members. Interaction numbers are pulled from MYTH data.

1.7 Phosphatase Ranking

The Summary chart of the phosphatase rankings is shown below. CBioPortal (www.cbioportal.org; Gao 2013, Cerami 2012) compares across 43 different cancer studies and provides the percentage of cases in which a particular gene is mutated. I then added the percentages of mutated cases across all genes, to find the five phosphatases which were most

mutated across all cancers studied. Below in table 7 is a summary of all cancer studies and their corresponding percentage of mutations for three phosphatases, CDC14B, CTDSP1, and CTDSP2, along with the total percentage of mutations across all cancer studies, and the phosphatases overall rank. The complete summary is accessible via the Stagljar lab.

Table 7 Summary of Cancer Study Results for Three Phosphatases: CBioPortal (Gao 2013, Cerami 2012) was used to accumulate data on the incidences of phosphatase gene mutation across various cancer studies, the results of which are shown here for three phosphatases, CDC14B, CTDSP1, and CTDSP2. The percentage of incidences per each individual case was then added across all cancer studies to yield total mutation percentage. Based on that number, phosphatases were ranked one through 20, with the top five phosphatases chosen to be further investigated. A full summary can be accessed via the Stagljar lab.

Percentage of Cases Mutated Cancer Study Reference CDC14B CTDSP1 CTDSP2 Acute Myeloid Leukemia TCGA, NEJM 2013 Acute Myeloid Leukemia TCGA, provisional Adenoid Cystic Carcinoma MSKCC, Nature Genetics 2013 Bladder Cancer MSKCC, JCO 2013 Bladder Urothelial Carcinoma TCGA, provisional Brain Lower Grade Giloma TCGA, provisional 0.9 0.5 Breast Invasive Carcinoma British Colombia, Nature 2012 Breast Invasive Carcinoma Broad, Nature 2012 0.5 Breast Invasive Carcinoma Sanger, Nature 2012 Breast Invasive Carcinoma TCGA, Nature 2012 0.4 Breast Invasive Carcinoma TCGA, provisional 0.1 0.3 Cancer Cell Line Encyclopedia Novartis/Broad, Nature 2012 Cervical Squamous Cell Carcinoma TCGA, provisional 2.6 Colon and Rectum Adenocarcinoma TCGA, Nature 2012 1.8 0.9 Colon and Rectum Adenocarcinoma TCGA, provisional 1.8 0.9 Colorectal Cancer Genetech, Nature 2012 2.8 Gliobastoma TCGA, Nature 2008 Gliobastoma Multiforme TCGA, provisional Head and Neck Squamous Cell Carcinoma TCGA, in preparation 0.4 0.8 Head and Neck Squamous Cell Carcinoma TCGA, provisional 0.4 0.8 Kidney Renal Clear Cell Carcinoma TCGA, in press 0.5 Kidney Renal Clear Cell Carcinoma TCGA, provisional 0.7 Kidney Renal Papillary Cell Carcinoma TCGA, provisional Lung Adenocarcinoma Broad, Cell 2012 0.5

Lung Adenocarcinoma TCGA, provisional 0.4 0.4 Lung Adenocarcinoma TSP, Nature 2008 Lung Squamous Cell Carcinoma TCGA, Nature 2012 1.1 1.1 0.6 Lung Squamous Cell Carcinoma TCGA, provisional 1.1 1.1 0.6 Ovarian Serous Cystadenocarcinoma TCGA, Nature 2011 Ovarian Serous Cystadenocarcinoma TCGA, provisional 0.3 Prostate Adenocarcinoma MSKCC, Cancer Cell 2010 Prostate Adenocarcinoma TCGA, provisional 1.2 Sarcoma MSKCC, Nature Genetics 2010 Skin Cutaneous Melanoma TCGA, provisional 0.4 0.4 0.9 Stomach Adenocarcinoma TCGA, provisional 0.5 1.4 0.5 Thyroid Carcinoma TCGA, provisional Uterine Corpus Endometrial Carcinoma TCGA, provisional 2 0.4 Uterine Corpus Endometrioid Carcinoma TCGA, Nature 2013 2 0.4 Kidney Chromophobe TCGA, provisional Liver Hepatocellular Carcinoma TCGA, provisional Lymphoid Neoplasm Diffuse Large B-cell Lymphoma TCGA, provisional Pancreatic Adenocarcinoma TCGA, provisional Sarcoma TCGA, provisional Total Mutation Percentage 14.7 9.4 10.3 Rank 20 27 25 1.8 Western Analysis

Figure 20 shows the westerns for the phosphatase overexpression and ERK1/2 analysis. The full un-cropped western blots from the phosphatase overexpression/ERK1/2 analysis experiments can be found in the Stagljar lab.

Figure 20 Erk Analysis Westerns: Raw results of ERK1/2 and Phos-ERK1/2 analysis.

1.8.1 Concentration Determination

A sample calculation, from Bradford readings, to loading amounts can be found below in table 8. Values for other samples can be found in lab book 2 in the Stagljar’s lab.

Table 8 Bradford Assay Calculations: This table shows the sample calculations for the first replicate of the PTPRK over-expression experiment. BSA samples are done with every phosphatase sample. The concentration of each sample is then used to calculate volumes needed for equal, and maximum loading.

1.9 IMAGE J Values

IMAGE J values for all samples can be found table 9 below. These are the values that were used to create the graphs in figure 18.

Table 9 IMAGE J Values: These are the raw values obtained from IMAGE J using the Western blots seen in figure 20, which were also used for the graphs in figure 18. These show the results for each of the three westerns for the four chosen phosphatases as well as the control phosphatase.

2 Appendix 2 2.1 Strains Used

2.1.1 Yeast Strain Genotype Source/Reference NMY51 MATa his3delta200 trp1-901 leu2-3, 112 ade2 Condamine, Le Texier et al. LYS2::(lexAop)4-HIS3 ura3::(lexAop)8-lacZ 2010 (lexAop)8-ADE2 GAL4

2.1.2 Mammalian Strain ATCC # Description maMYTH CRL-11268 HEK293Tcells stable lentiviral transfection of 5x GAL4UAS- luciferase SK-BR-3 HTB-30 Human breast adenocarcinoma cell line 2.2 Plasmids Used Use Description Phosphatase Phosphatase flanked by attL regions (for gateway® recombination) Entry Vector MYTH Prey pGPR3N : attR regions (for gateway® recombination) in frame with NubG vector or NubI portion of ubiquitin, ampicillin resistance, TRP1 gene (Dual Systems Biotech) MYTH Bait pTMBVα: attR regions (for gateway® recombination) in frame with Cub vector portion of ubiquitin, and yeast alpha mating factor (for proper localization), kanamycin resistance, LEU2 gene (Dual Systems Biotech) MaMTH Prey pCMV: attR regions (for gateway® recombination), in frame with NubI, Vector CMV-promoter (Dual Systems Biotech) MaMTH Bait attR regions (for gateway® recombination), in frame with Cub-GAL4, V5 Vector tag, CMV-promoter (Dual Systems Biotech) Overexpression FLAG-tagged phosphatases, vector with ampicillin resistance Vectors 2.3 Antibodies Used Antibody Details Company Catalogue # α-tubulin Mouse monoclonal IgM Santa Cruz Biotechnology Inc. Sc-8035 α-FLAG Mouse monoclonal IgG Sigma-Aldrich F3165 α-p44/42 Rabbit monoclonal IgG Cell Signaling Technology 4695 MAPK α-phospho- Rabbit monoclonal IgG Cell Signaling Technology 4370

p44/42 MAPK α-mouse Sheep IgG GE Healthcare Life Science NXA931 horseradish peroxidase linked α-rabbit Donkey IgG GE Healthcare Life Science NA934V horseradish peroxidase linked 2.4 Cell culture care

2.4.1 Cell Thawing

Frozen tube of cell is quickly thawed in a 37°C water bath, as soon as pellet is completely thawed, the tube is sterilized with 70% ethanol and contents are transferred into a 10cm dish (BD Bioscience 3530033) with 10mL of full media. Cells are grown for 24 hours and media is then changed to full media again. Cells are grown to ~90% confluent and then used as described.

2.4.2 Cell Freezing

Cells that are 80-90% confluent are trypsinized with 100µL 0.025% trypsin (Life Technologies 25200-056) in 2mL of PSB (Sigma Life Science D8537). Cells are resuspended in 2mL of 2x freezing medium (Appendix[]) and quickly aliquoted into four cryotubes (Corning Inc. 431386) which are put on ice immediately. These cells are then slowly frozen to -80°C using an isopropanol filled container.

2.4.3 Splitting Cells

Cells are grown to 80-90% confluence, at which time media is aspirated off, cells are rinsed with 2mL of PBS (Sigma Life Science D8537), and trypsinized with 100µL 0.025% trypsin (Life Technologies 25200-056) in 2mL of PSB (Sigma Life Science D8537). Cells are then resuspended in 8mL of full media, and 1mL of the confluent cell mixture is transferred into a new 10cm dish with 9mL of full media (1:10 splitting). At this concentration, cells grow to 80%-90% within three to four days. Splitting ratios were varied when cells were need on specific days. SKBR3 cells were split 1:5 due to their extremely slow growth when lacking in cell/cell contact.

3 Appendix 3 3.1 Media Recipes

3.1.1 E.coli Media Components Company LB • 20g LB Broth Lennox • Bioshop Canada Inc. LBL405.1 *for solid media 20g of agar was *Bioshop Canada Inc. AGR001.1 added **for antibiotic resistance, drug **Ampicillin (Bioshop Canada Inc. was added after cooled from AMP201.100), Kanamycin (Bioshop autoclave Canada Inc. KAN201.25), Spectinomycin (Bioshop Canada Inc. SPE 201.10)

3.1.2 Yeast Media Components Company SD • 6.7g yeast nitrogenous base • Bioshop Canada Inc. YNB406.500 • 20g D-glucose • Bioshop Canada Inc. GLU501.5 • 100mL 10x dropout in 900mL of ddH2O Milli-Q *for solid media 20g of agar was * Bioshop Canada Inc. AGR001.1 added 10x dropout • 400mg adenine • Bioshop Canada Inc. ADS201.100 • 200mg arginine • Bioshop Canada Inc. ARG006.100 • 200mg histidine • Bioshop Canada Inc. HIS200.100 • 300mg isoleucine • Bioshop Canada Inc. ISO910.100 • 1000mg leucine • Bioshop Canada Inc. LEU222.100 • 300mg lysine • Bioshop Canada Inc. LYS101.100 • 1500mg valine • Bioshop Canada Inc. VAL201.100 • 1500mg methionine • Bioshop Canada Inc. MET222.100 • 500mg phenylalanine • Bioshop Canada Inc. PHA302 • 2000mg threonine • Bioshop Canada Inc. THR002.25 • 400mg tryptophan • Bioshop Canada Inc. TRP100.100 • 300mg tyrosine • Bioshop Canada Inc. TYR333.100 • 200mg uracil • Bioshop Canada Inc. URA241.100 in 1L of ddH2O Milli-Q *for selective media SD-L leucine was not added; for SD-WL leucine and tryptophan were not added; for SD-WLAH leucine tryptophan, adenine, and

histidine were not added YPAD • 20g yeast extract • Bioshop Canada Inc. YEX401.1 • 40g peptone • Bioshop Canada Inc. PEP403.1 • 40g D-glucose • Bioshop Canada Inc. GLU501.5 • 40mg adenine sulphate • Bioshop Canada Inc. ADS201.100 in 1L ddH2O Milli-Q • *for solid media 20g of agar • * Bioshop Canada Inc. AGR001.1 was added

3.1.3 Mammalian Media Components Company Full • DMEM • Wisent Inc. 319-005-CL • 10% FBS • Wisent Inc. 080150 • 1% penicillin/ streptomycin • Wisent Inc. 450-201-EL Minimal • DMEM • Wisent Inc. 319-005-CL • 1% penicillin/ streptomycin • Wisent Inc. 450-201-EL 2X Freezing • 20% DMSO • Sigma D2650 • 80% FBS • Wisent Inc. 080150 3.2 Chemical Solution Recipes Solution Components Company X-Gal • 100mg X-Gal powder • Bioshop Canada Inc. XGA001.5 in 1mL N,N-dimethyl formamide Bioshop Canada Inc. DMF 451 NP-40 Lysis • 50mM Hepes-NaOH pH8.0 • Bioshop Canada Inc. HEP005.500 Buffer • 100mM KCL • Bioshop Canada Inc. POC888.1 • 2mM EDTA • Bioshop Canada Inc. EDT002.1 • 0.1% NP40 • Bioshop Canada Inc. NON505.500 • 10% glycerol • Bioshop Canada Inc. GLY002.4 • 1mM PMSF (added fresh) • Santa Cruz 3597 • 1mM DTT (added fresh) • Bioshop Canada Inc. STT001.10 • 1mM Protease Inhibitor • Roche 11 873 580 001 (added fresh) in ddH2O Milli-Q 6x Laemmli • 180mM tris HCl pH 6.8 • Bioshop Canada Inc. TRS001.5 Sample Buffer • 9% SDS • Bioshop Canada Inc. SDS001.500 • 30% glycerol • Bioshop Canada Inc. GLY002.4 • 0.15% bromophenol blue • Signma-Aldrich B8026 • 10% β-mercaptoethanol • Bioshop Canada Inc. MER002.100 (added fresh) in ddH2O Milli-Q Luciferase • 20M tricine • Sigma T0377 Substrate • 1.07mM • Sigma M5671 (MgCO3)MG(OH)25H2O

• 2.67mM MgSO4 • Sigma 208094 • 0.1mM EDTA • Bioshop Canada Inc. EDT002.1 • 33.3mM DTT • Bioshop Canada Inc. STT001.10 • 270µM coenzyme A • Sigma C3144 • 470µM D-luciferin • Sigma L9504 • 530µM ATP • Sigma A9187 in ddH2O Milli-Q *added in this order