CHARACTERIZATION OF THE INTERACTOME OF THE … · transplanted limbs grow to the original size of the donor but not that of the recipient ... YAP/TAZ on the same sites can also induce

CHARACTERIZATION OF THE INTERACTOME OF

THE HIPPO TUMOUR SUPPRESSOR PATHWAY

USING MASS SPECTROMETRY

By

Fang Yuan, B.Sc. (Hons.)

A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the

Requirements for the Degree Master of Science

University of Toronto

©Copyright 2013 by Fang Yuan

M. Sc. Thesis – Fang Yuan University of Toronto, Molecular Genetics

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MASTER OF SCIENCE

Department Molecular Genetics, University of Toronto

Convocation Date: November 2013

Title: Characterization of the Hippo tumour suppressor pathway using mass spectrometry

Author: Fang Yuan

Supervisor: Dr. Jeffrey Wrana

ABSTRACT

The Hippo signaling pathway offers an intrinsic mechanism to control organ sizes,

and dysfunction of this pathway can often lead to cancer. Great advancement has been

made in recent years into understanding this pathway. Despite all this invaluable

knowledge, much remains to be explored. Mass spectrometry offers an unbiased

approach to characterize the interactome of any protein of interest and is particularly

powerful for identifying potential novel regulators of signalling pathways. I therefore set

out to characterize the interactome of all the Hippo pathway main components using

mass spectrometry, with the goal of uncovering novel regulatory mechanism(s) of the

Hippo pathway. In the end, I was able to identify over 250 novel interactors of the Hippo

pathway in total. This study demonstrates the utility of mass spectrometry to identify

novel regulators of the Hippo pathway and characterization of one such interactor.


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ACKNOWLEDGEMENT

I owe a great debt of gratitude to those individuals whose contributions have made this

work possible. First, I would like to thank my supervisor, Dr. Jeffrey Wrana, whose constant

guidance, and wealth of experience and knowledge have helped shape me as a scientist. I

would also like to thank the members of my supervisory committee, Dr. David Kaplan and

Dr. Helen Mcneill for their invaluable advice and insights into my research.

Acknowledgement is also due to all the members of the Wrana lab who have contributed

directly or indirectly to this work and their willingness to partake in stimulating scientific

discussion and for their commitment to the research goals of our laboratory.

Finally, I would like to thank my family for supporting me in my decision to pursue a

career in research.


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TABLE OF CONTENTS

1. INTRODUCTION…………………………………………………………….……………1

1.1 The evolutionarily conserved tumor-suppressor Hippo pathway……………...…………..1

1.2 Cross-talk with other signaling pathways…………………………………….…………...5

1.3 Regulation of the Hippo pathway………………………………………………………7

1.4 Using mass spectrometry to identify novel regulators of the Hippo pathway in

mammals……………………………………………………………………………….10

2. MATERIALS AND METHODS…...……………………………………..………………13

2.1 Cell culture………………………………………………………………………………13

2.2 Generation of T-Rex FLP cell lines……………………………………………………...14

2.3 Affinity purification and tandem mass spectrometry……………………………………14

2.4 Bioinformatic analysis…………………………………………………………………...16

2.5 Plasmid and siRNA transfection, TEAD reporter assay………………………………...16

2.6 Immunofluorescence…………………………………………………………………….17

2.7 Primary cortical neuron culture………………………………………………………….17

3. RESULTS……………………………...……………………….………………..…….….18

3.1 Mass spectrometry………………………………………………………………………18

3.2 Protein-centric and function-centric clustering of the interactors……………………….20

3.3 Position of the tag, kinase activity and cell density do not seem to affect interactome of at

least certin proteins….......…………………………………………………………………...21

3.4 Identification and characterization of DOCK7 and its role in Hippo signaling………....22

3.5 Characterization of the role of DOCK7 in neuronal development………………………25

4．DISCUSSION…………………………………………………………………………….28


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4.1 Mass spectrometry……………………………………………………………………….28

4.2 DOCK7 failed to change Hippo signaling……………………………………………….29

4.3 Hippo signaling failed to change DOCK7 activity………………………………………31

5. CONCLUSION…………………………...………………………………………………31

6. REFERENCES…………………...………………………….……………………………34

7. FIGURES……………………………………..…………………………..………………43

8. TABLES……………………………….………………………………………….………52


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LIST OF FIGURES

Figure 1. The core Hippo pathway signaling cascade.

Figure 2. Characterization of HEK293 T-Rex FLP lines.

Figure 3 Summary of the mass spectrometry study.

Figure 4. The role of DOCK7 in regulating the Hippo pathway remains unclear.

Figure 5. The role of DOCK7 in regulating the Hippo pathway in Eph4 cells remains

inconclusive.

Figure 6. The role of MOB1 in regulating axon development in embryonic neurons remains

inconclusive.

Figure 7. The domain structure of LRCH proteins.


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LIST OF TABLES

Table 1. Summary of all the high-confidence interactors of the Hippo pathway main

components.

Table 2. Comparison of the intearctome between wild-type and kinase-dead variant of

MST1/2 and LATS1/2

Table 3. Comparison of the intearctome between N-term tagged and C-term tagged variant

of LATS1/2.

Table 4. Comparison of the intearctome between low and high cell density of core Hippo

pathway components

Table 5. Interactome of DOCK7


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LIST OF ABBREVIATIONS

ECM – Extracellular Matrix

GEF – Guanine Exchange Factors

MS – Mass Spectrometry

PPI – Protein-Protein Interaction

TBS – TEAD-binding-sequence

YAP/TAZ – YAP and TAZ


1

1. INTRODUCTION

1.1 The evolutionarily conserved Hippo tumor-suppressor pathway

Organ size is controlled by both extrinsic and intrinsic mechanisms. Extrinsic growth

factors, such as insulin-like growth factors, stimulate organ size, whereas lack of nutrients

significantly reduces organ size (Stanger, 2008). Intrinsic regulation has also been proposed

based on results from several limb transplant experiments in salamander, where the

transplanted limbs grow to the original size of the donor but not that of the recipient

(Stanger, 2008). The molecular mechanism of the intrinsic regulation of organ size had

remained largely unknown until the discovery of the Hippo tumor-suppressor pathway.

Early studies have shown that mutations in several evolutionarily-conserved genes can

cause overgrowth in a set of specific organs in both Drosophila and mouse without altering

cell fate or cell polarity. For example, the Hpo mutant in Drosophila causes enlargement of

imaginal discs epithelial tissues (Wu et al., 2003), and mutation in Mst1 or Mst2

(homologues of Hpo in mammal) results in liver enlargement and tumour development in

mouse (Dong et al., 2007). Other genes that cause similar phenotypes include the kinases

LATS1/2 (Tao et al., 1999), the adaptor proteins SAV1 (Callus et al., 2006) and MOB1A/B

(Praskova et al., 2008). Later work further revealed that LATS1/2 phosphorylates and

inhibits the function of the transcription co-activators YAP and TAZ (YAP/TAZ) (Zhao et al.,

2010b). Finally, overexpression of YAP/TAZ phenocopies mutations in the core Hippo

pathway components (Huang et al., 2005), thus revealing the last component of the Hippo

signaling pathway.


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The core Hippo signal cassette transduces signals in a fairly linear manner (Fig. 1)

(Zhao et al., 2010a). Upon activation, MST1/2 phosphorylate and activate LATS1/2 with the

aid of the adaptor proteins SAV1 (Chan et al., 2005). SAV1 interacts with MST1/2 through

the SARAH domains present in both SAV1 and MST1/2 (Callus et al., 2006). Activated

MST1/2 also phosphorylates MOB1A/B, resulting in an enhanced LATS1/2–Mob1

interaction which leads to a further increase in LATS1/2 activity (Bao et al., 2009). Activated

LATS1/2 in turn phosphorylates YAP/TAZ (Huang et al., 2005). Phosphorylation of

YAP/TAZ is inhibitory to their transcriptional functions because such phosphorylation

promotes 14-3-3 binding and subsequent cytoplasmic sequestration. Phosphorylation of

YAP/TAZ on the same sites can also induce their degradation by the E3 ubiquitin ligase

SCFβ–TRCP (Zhao et al., 2010b). When the Hippo pathway is inactive, YAP/TAZ are

dephosphorylated, dissociates from 14-3-3 and become more stable with subsequent

accumulation in the nucleus where they perform their functions as transcriptional co-

activators.

While in the nucleus, YAP/TAZ can bind to a variety of transcription factors under

different biological contexts, including RUNX2 (Cui et al., 2003), PAX8 (Di Palma et al.,

2009), TBX5 (Murakami et al., 2005), TTF-1 (Park et al., 2004), MyoD (Jeong et al., 2010),

PPARγ (Hong et al., 2005), P73 (Tomlinson et al., 2010), and TEADS (Mahoney et al.,

2005). RUNX2 was the first identified transcription factor that binds to YAP/TAZ using a

yeast two-hybrid system (Cui et al., 2003). RUNX2 promotes osteoblastic differentiation of

mesenchymal stem cell (MSC), which is enhanced by binding to YAP/TAZ. Later work

revealed that YAP/TAZ can also bind to the transcription factor PPARγ which promotes


3

adipocytic differentiation of MSC (Hong et al., 2005). Interestingly, such binding suppresses

the activity of PPARγ. Therefore, YAP/TAZ, PPARγ and RUNX2 together form a delicate

system in MSC where nuclear YAP/TAZ (inactive Hippo pathway) promotes osteoblastic

differentiation and cytoplasmic YAP/TAZ (active Hippo pathway) promotes adipocytic

differentiation. YAP/TAZ has also been shown to bind to and promote the activity of TBX5,

an essential transcription factor in cardiac and limb development (Murakami et al., 2005).

Importantly, patients with Holt-Oram syndrome harbour various mutations in TBX5 which

result in impaired binding to YAP/TAZ, thus linking YAP/TAZ directly to human disease

progression. However, perhaps the most-studied binding partner of YAP/TAZ is the TEAD

family members (Mahoney et al., 2005). In fact, TEAD seems to be the primary mediator of

Hippo pathway in Drosophila (Zhao et al., 2008). Disrupting the interaction between

YAP/TAZ and TEAD, as well as TEAD knockdown, directly affects the YAP/TAZ-mediated

effects on cell proliferation, cell migration, and organ size control. Some transcriptional

targets of this complex have also been identified, including CTGF (Zhao et al., 2008), Cyr61

(Lai et al., 2011), ZEB1 (Liu et al., 2010b) and AREG (Zhang et al., 2009), all of which are

crucial in YAP/TAZ-induced cell proliferation and survival under different biological

contexts.

By inhibiting the functions of YAP/TAZ, the Hippo pathway has several important

biological functions. First and foremost, the Hippo pathway is a tumor suppressor pathway.

By sequestering YAP/TAZ in the cytoplasm, the Hippo pathway effectively inhibits the

transcription co-activator activity of YAP/TAZ and retards YAP/TAZ-mediated cell

proliferation, cell survival and cell migration. In both Drosophila and mouse models,


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mutations in different Hippo pathway components often result in tumor development (Dong

et al., 2007). Importantly, tumor sample analysis have revealed amplification of YAP in

breast cancer patients (Zender et al., 2006), mutation in NF2 (a Hippo pathway regulator) in

meningiomas patients (Striedinger et al., 2008), and epigenetic silencing of LATS1/2 in

breast cancer patients (Jiang et al., 2006), all pointing to a role of the Hippo pathway in

cancer progression. Related to this, nuclear YAP/TAZ has been shown to help cells maintain

a stem cell phenotype and confer cancer cells a more basal stem-cell-like property

(Cordenonsi et al., 2011). Activation, or re-activation in some cases, of the Hippo pathway,

limit the expansion or even promote the differentiation of the stem cell population. Lastly,

the Hippo pathway is a critical regulator of early mammalian development. During the early

stage of blastocyst formation, the zygote forms a fluid-filled epithelial vesicle that encloses a

compact group of cells (Stephenson et al., 2012). The outer cell layer is called trophectoderm

(TE) and the inner cell layer is called inner cell mass (ICM). The fate of the cells between

TE and ICM is dictated by expression of specific transcription factors. Specifically, OCT4 is

only expressed in ICM and CDX2 is only expressed in TE. Recently it’s been found that the

expression of CDX2 is absolutely dependent on the transcriptional activity of TEAD4

(Nishioka et al., 2009). Although TEAD4 is expressed in both TE and ICM, YAP/TAZ

remains in the nucleus and promotes the activity of TEAD4 only in TE, whereas YAP/TAZ

is sequestered in the cytoplasm in ICM. Not-surprisingly, the different localization of

YAP/TAZ between TE and ICM is largely dependent on its phosphorylation status by

LATS1/2. Overexpression of LATS in embryo led to cytoplasmic sequestration of

YAP/TAZ and significant reduction in CDX2 expression without affecting cell number. On

the other hand, LATS1/2 double-knockout mouse showed nuclear YAP and strong induction


5

of CDX2 even in inner cells. These data all point to a crucial role of the Hippo pathway in

early development.

1.2 Cross-talk with other signaling pathways

The Hippo pathway has been shown to regulate other signaling pathways. A prime

example is the TGFβ pathway and the similar BMP pathway. In both pathways, the TGFβ

superfamily ligands bind to the corresponding receptors, which then phosphorylates the

receptor-regulated SMAD (R-SMAD). Phosphorylated R-SMAD binds to SMAD4 and the

R-SMAD/SMAD4 complex accumulates in the nucleus where they co-modulate

transcription in co-operation with DNA-binding transcription factors to regulate target gene

expression. Our lab has shown that YAP/TAZ can bind to SMAD2/3, which is the R-

SMADS of the TGFβ branch, and dominantly controls the nucleo-cytoplasmic shuttling of

the SMAD2/3-SMAD4 complex (Varelas et al., 2008). In the absence of YAP/TAZ, or in

cells where YAP/TAZ is predominantly cytoplasmic (i.e. when the Hippo pathway is

activated), efficient accumulation of the SMAD complex in the nucleus is inhibited, which

leads to inhibition of the TGFβ-SMAD2/3 signaling pathway. Importantly, because the

TGFβ-SMAD2/3 signaling pathway plays an important role in cell migration and

particularly the epithelial-to-mesenchymal transition (EMT) process, the activity of the

Hippo pathway thus provides another guard against cell migration by sequestering

SMAD2/3 in the cytoplasm (Varelas et al., 2010b).

A somewhat similar mechanism also operates in the BMP signaling pathway. In this

pathway, activated BMP receptor phosphorylates SMAD1/5, which then binds to SMAD4

and translocates into the nucleus. YAP/TAZ has also been shown to bind phosphorylated


6

SMAD1 (Alarcon et al., 2009). However, this binding does not seem to control the

subcellular localization of the SMAD1/5-SMAD4 complex. Rather, YAP/TAZ translocates

with the SMAD1/5-SMAD4 complex into the nucleus and enhances their transcriptional

activity. Although the link between the BMP pathway and the Hippo pathway has not been

completely characterized, at least in Drosophila, inactivation of upstream Hippo pathway

components resulted in elevated Dpp (the Drosophila homologue of BMP) target gene

expression, presumably due to increased interaction between YAP/TAZ and SMAD1/5 in the

nucleus.

Furthermore, YAP/TAZ also serves as the cross-talk node between the Hippo pathway

and the WNT pathway. In the WNT pathway, the WNT proteins bind to the Frizzled receptor,

causing the receptors to activate CK1δ/ε which then phosphorylates the Dishevelled (DVL)

family proteins. Phosphorylation of DVL promotes the stabilization and the nuclear

translocation of the WNT pathway transducer β-catenin by blocking GSK-3-mediated

phosphorylation. Our lab has shown that TAZ can regulate WNT signaling via direct

interaction with the cytoplasmic protein DVL(Varelas et al., 2010a). TAZ, when

phosphorylated by the Hippo pathway and sequestered in the cytoplasm, competes with

CK1δ/ε kinases for binding to cytoplasmic DVL, resulting in decreased levels of

phosphorylated DVL and reduced WNT signaling. In addition, inactivation of Hippo

pathway components in Drosophila, which drives TAZ into the nucleus, led to reduced

interaction between DVL and TAZ and increased stability of Arm (Drosophila homologue of

β-catenin) and increased expression of WNT target genes. The Hippo pathway can thus

participate in the regulation of the WNT pathway through TAZ phosphorylation and


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subsequent inhibition of DVL phosphorylation.

1.3 Regulation of the Hippo pathway

Given the importance of the Hippo pathway, extensive research has been carried out to

understand how this pathway is regulated. Most of the early studies used Drosophila to

identify regulators of the Hippo pathway due to the ease of conducting genetic screening and

the prominent phenotype when the Hippo pathway is disrupted in this model organism.

These studies revealed that many polarity or cell-junction related proteins regulate the Hippo

pathway activity. For example, the atypical cadherin Fat (Ft) and Dachsous (Ds) have been

shown to control growth upstream of the Hippo pathway (Willecke et al., 2008).

Interestingly, it appears that the relative levels of Ft and Ds between neighbour cells control

the activity of the Hippo pathway. Thus Ft and Ds might provide a mechanism of non-cell-

autonomous regulation of the Hippo pathway. This discovery is particularly intriguing given

the fact that Ft and Ds are both major regulators of planar cell polarity (PCP), suggesting a

role for PCR in Hippo regulation. Another important complex is the Kibra-Merlin-Expanded

complex which is located at the sub-apical region of the cells (Hamaratoglu et al., 2006).

Disruption of this complex also leads to inactivation of the Hippo pathway. Importantly,

components of this complex physically interact with the core Hippo components. For

example, Expanded interacts with Yki (Drosophila homologue of YAP/TAZ) and Hpo

(Drosophila homologue of MST1/2), Kibra binds Wts (Drosophila homologue of LATS1/2),

and Merlin binds Sav. Therefore, it has been postulated that this complex might directly

regulate Hippo pathway depending on different polarity cues. Finally, protein complexes

controlling apical-basal polarity have also been shown to regulate the Hippo pathway


8

activity. These complexes include the apical aPkc-Bazooka-Par6 complex and the basal Lgl-

Scrib-Dlg complex (Grzeschik et al., 2010). Specifically, the two complexes work

antagonistically with each other where Lgl appears to activate the Hippo pathway and aPkc

seems to inhibit the Hippo pathway. Current studies are focusing on characterizing the

molecular link between these polarity proteins and the core Hippo pathway components.

Most of these polarity proteins have been shown to be functionally conserved in

mammals in terms of regulating the Hippo pathway. To identify novel regulators of the

Hippo pathway in mammals, our lab has shown using mass spectrometry that YAP/TAZ

interact physically with components of the Crumbs polarity complex (Varelas et al., 2010c).

Disruption of the Crumbs complex at high cell density leads to reduced phosphorylation of

YAP/TAZ and more nuclear accumulation of YAP/TAZ, suggesting that the physical

interaction between YAP/TAZ and the Crumbs complex is functionally linked to the

phosphorylation and cytoplasmic sequestration of YAP/TAZ. We believe that the Crumbs

complex serves as a cell-density-sensing platform that promotes the phosphorylation of

YAP/TAZ in mature epithelium. Using the same approach, other groups have reported that

YAP/TAZ also interacts with other polarity proteins such as AMOT (Zhao et al., 2011) and

α-catenin (Silvis et al., 2011), and showed that their interaction with YAP/TAZ are all

required for YAP/TAZ phosphorylation under different contexts. α-catenin, for example, is

particularly important in controlling epidermal proliferation.

Two other regulatory mechanisms on Hippo pathway have recently been identified,

the first one being the mechanotranduction pathway. The mechanotransduction pathway

describes how the cell responds to the mechanical changes in its surrounding environment,


9

for example the rigidity of the extracellular matrix (ECM). A recent study showed that the

mechanotransduction pathway re-organizes the cytoskeleton network to control the

localization of YAP/TAZ, which then controls specific biological outcome (Dupont et al.,

2011). By plating cells on ECM with different degrees of rigidity, the study showed that

YAP/TAZ localize in the nucleus on stiff ECM, whereas they localize in the cytoplasm on

soft ECM. Furthermore, by altering the cell shape, therefore altering the cytoskeleton

network, the study showed that YAP/TAZ localize in the nucleus when cells are spreading

whereas they localize in the cytoplasm when cells are confined into small regions.

Importantly, the study showed that manipulation of the localization of YAP/TAZ in

mesenchymal stem cells could alter their differentiation program, similar to how they

respond to ECM with different degrees of rigidity.

The most recent regulatory mechanism was characterized when specific extracellular

growth factors promote the nuclear accumulation of YAP/TAZ. These growth factors all

bind to specific transmembrane receptors which then lead to activation of specific signaling

pathways. For example, stimulation of various G-protein-coupled-receptors (GPCR) can lead

to either activation or inhibition of the Hippo pathway, which seems to depend on the

downstream signaling cascade of the particular GPCR (Yu et al., 2012). Specifically, upon

stimulation by glucagon and epinephrine, the Gs-coupled GPCR can lead to cAMP-

dependent activation of Hippo pathway. In contrast, upon stimulation by lysophosphatidic

acid and sphingosine 1-phosphate, the G12/13-coupled receptors can lead to inactivation of

the Hippo pathway through the small GTPase RHO. Other receptor-mediated Hippo pathway

regulation include the epidermal growth factor (EGF) which binds to the epidermal growth


10

factor receptor and subsequently inactivates the Hippo pathway via the PI3K signaling

pathway (Fan et al., 2013). Although the exact biochemical mechanisms of how the

activation of these signaling pathway leads to inactivation of the Hippo pathway is not

completely understood, these findings suggest that YAP/TAZ serves as one of the many

downstream effectors of these signaling pathways to promote cell proliferation.

1.4 Using mass spectrometry to identify novel regulator of the Hippo pathway in mammals

While many of the Hippo regulators were identified using genetic screens in

Drosophila, such method is less applicable in mammalian systems. Even when regulators

were identified, further work are needed to elucidate the precise molecular mechanism. Mass

spectrometry (MS) offers an unbiased approach to identify direct interactors and potential

regulators of the protein of interest(Liu et al., 2010a). The rationale here is that many

proteins do not function as independent units, but rather are part of large macromolecular

complexes of interacting proteins. Extensive protein-protein interaction (PPI) usually exists

in such complexes, and since most regulators require direct PPI, characterizing the entire

interactome surrounding a protein of interest can provide invaluable information regarding

how the protein is regulated. Furthermore, downstream biochemical characterization

becomes relatively easier compared to genetic screening since PPIs are already known from

the MS results. Even when compared to high-throughput PPI screening such as yeast-two

hybrid (Fromont-Racine et al., 1997) or the LUMIER technology (Barrios-Rodiles et al.,

2005), MS still offers two advantages. First, high-throughput PPI screening is often limited

by the size of the cDNA library whereas MS is capable of identifying proteome-wide

interactors. Second, traditional high-throughput PPI screening often involves over-


11

expressing both the target and the bait proteins. The over-abundance of the expressed

proteins may help catch transient interactions, but it may also lead to artificial interaction.

Most MS studies, on the other hand, were done in a semi-endogenous (i.e. generation of

stable cell lines expressing the bait protein) or endogenous manner (i.e. immunoprecipitating

endogenous protein using antibodies), which ensures that the interactors and/or the bait

proteins are in their fully processed and endogenous form, that the interactions are in the

native environment of the protein, and that multi-component complexes can be isolated in a

single step.

Of course, MS is not without shortcomings. Compared to binary interaction studies,

the interactors obtained from MS studies are ranked based solely on abundance. Thus it is

often hard to differentiate between non-specific interactors and true biological interactors

with low abundance. Taken into account that most MS studies are conducted without any

external stimulation in order to achieve an unbiased coverage of the interactome, it is very

likely that the characterized interactome will miss out on interactors that only bind

transiently with the protein of the interest under specific biological signals. Therefore, it is

often recommended to compare interactome of the same protein under different types of

external stimulation or different growth conditions in order to catch these transient

interactors. The problem is that we often don’t know beforehand what stimulus is

appropriate for the protein of interest in a particular biological system.

A general strategy to conduct MS studies is described as follows. Once the stable cell

line which expresses the protein of interest (usually with a tag) is generated, the surrounding

complex is purified by lysing the cells and immunoprecipitating the tag. Appropriate


12

negative controls can be conducted in parallel using cell lines expressing only the tag.

Purified protein complex may then be separated either before or after the trypsinization step

to achieve better resolution. The goal of trypsinization is to cleave individual proteins into

peptide fragments. Each protein generates unique pattern of fragments upon trypsinization.

The identity of the protein can thus be determined based on the masses of these fragments

using MS.

Several technical advancements have been made to improve MS data quality. First,

traditional stable cell lines usually have multiple random genomic integrations of the

expression vector, which may result not only in the disruption of endogenous gene

expression, but more importantly different genomic composition among lines expressing the

same protein. Furthermore, traditional cell lines constitutively express high levels of the

protein of interest, which may lead to artificial interaction. The T-Rex Flp-In cell line

provides solutions to these problems(Ward et al., 2011). It has a built-in TET-ON system that

allows expression of the protein only in the presence of tetracyclin or doxcyclin, thus

providing temporal control of the gene expression. In addition, the T-Rex line contains a

single stably integrated FRT site at a transcriptionally active genomic locus. In the presence

of the FLP flippase, the transfected expression vector is integrated only into the FRT site,

thus ensuring homogeneous levels of gene expression.

The second critical step in any MS study is the separation step. Even the most

sophisticated algorithm and the most powerful computer cannot identify every individual

protein in an immunoprecipitates when all peptide fragments are mixed together. Therefore,

pre-fractionation is required. A widely-used separation method is the reversed-phase


13

capillary liquid chromatography (RPLC)(Fekete et al., 2012), which can be applied after the

trypsinization step. The column itself is packed with hydrophobic carbon-silicon conjugates,

which binds preferably to hydrophobic peptides. When subjected to increasing amount of

polar solvents, the more hydrophilic peptides will move into the solvent phase earlier than

the more hydrophobic ones, thus creating separation.

The actual MS starts when the separated peptide is eluted from the tip of the RPLC

column. In a process referred to as electrospray ionization, a high electrical potential is

applied on the eluted peptide and takes only one electron away from the peptide thus making

the total charge on every single peptide equal to 1+. The fragments are then subjected to an

electric field in which all peptide fragments travel over a certain distance. The time-of-flight

depends solely on the mass of the peptide because the charge on the fragment is always 1+.

By recording the time-of-flight for each peptide, the mass of the peptide can be calculated

and the identity of the proteins can be determined by comparing the acquired MS spectrum

to appropriate databases such as MASCOT(van Gool and Hendrickson, 2012).

2. MATERIAL AND METHODS

2.1 Cell culture

HEK293T and Eph4 cells were grown at 37°C in Dulbecco's modified Eagle's medium

containing 10% fetal bovine serum under 5% CO2 in a humidified atmosphere and

maintained using standard procedures. Flp-InTM T-RExTM HEK293 cells were obtained from

Invitrogen. The cells were cultured in media containing 1μg/ml blasticidin and 250μg/ml


14

zeocin. Stable cell lines were maintained in presence of 1μg/ml blasticidin and 250μg/ml

hygromycin. Exogenous protein expression was induced by 16hrs of treatment with 1μg/ml

doxycyclin.

2.2 Generation of T-Rex FLP cell lines

All of the inducible expression cell lines were generated using the Flp-In T-REx system.

Constructs containing LATS1/2, MST1/2, MOB1A/B, SAV1, and RASSF5/6 were generated

by polymerase chain reaction and subsequently cloned into the pcDNA5-FRT-TO-FLAG

vector from Invitrogen. Cells were co-transfected with these constructs and the pOG44

vector (expressing FLP recombinase) into the HEK293 Flp-In T-REx host cell lines. Cells

that had integrated the expression plasmid were selected with 800μg/ml hygromycin B until

enough colonies were observed by naked eyes, and the colonies were pooled and maintained

as described above.

2.3 Affinity purification and tandem mass spectrometry

Inducible FLAG-tagged HEK293 cell lines expressing Hippo pathway components were

generated using the Flp-In T-REx system and used for FLAG affinity purification. Cells were

grown to confluency and subsequently lysed in TNTE buffer (50mM Tris pH 7.4, 150mM

NaCl, 1mM EDTA, and 0.5% Triton X-100). Immunoprecipitation was carried out using

anti-Flag M2 beads (Sigma; A2220) for 1.5 hours, followed by 2 washes in lysis buffer and 3

washes in wash buffer (0.1% Triton X-100). Subsequently the beads were washed two times

with 20mM Tris buffer (pH 8.0). The bound proteins were then digested on beads at 37°C

overnight with trypsin. The trypsin was evaporated using SpeedVac and the digested proteins


15

were resuspended in 20μl of 2.5% Formic Acid (pH<3.0) then loaded into a 10cm of Zorbax

C18-packed column. A 100 min gradient of 0.1% formic acid to 0.1% formic

acid/acetonitrile was delivered in a split-flow manner by an Agilent 1100 Capillary HPLC. A

gradient of0-35% ACN was used over a 65 min peak collection window. Data were acquired

on a ThermoFisher LTQ linear ion trap, using a data-dependent method consisting of 1 MS

scan followed by 4 MS/MS scans. Acquired RAW files were converted to mgf format, which

were searched with the Mascot search engine (Matrix Sciences, London, UK) against the

NCBI data base, limited to human taxonomy with a precursor ion mass tolerance of 3.0 and a

fragmentation mass tolerance of 0.6. Methionine oxidation was allowed as a variable

modification, and trypsin specificity (with one missed cleavages allowed) was selected. The

results from the search were compared using an in-house program (Prohits) and proteins

unique to the bait samples (not present in control immunoprecipitation) with protein mascot

score >50, and >2 unique peptides were considered as confident hits.

2.4 Bioinformatics analysis

Protein-centric analysis was carried out using the CytoscapeTM software. Briefly, the names

of the bait and the prey proteins were listed side-by-side on Excel worksheets. The

worksheets were then imported into CytoscapeTM which visualizes the protein-protein

network by 1). seeking the common interactors among the bait proteins 2). connecting bait


16

proteins with their unique prey proteins 3). Connecting bait proteins with their shared prey

proteins.

Function-centric analysis was also carried out using the CytoscapeTM software using the

BiNGO plugin. Briefly, after importing the names of the bait and the prey proteins into

CytoscapeTM, BiNGO automatically searches the GO term for each protein according to the

Gene Ontology project. The complete protein list along with their GO terms was exported

back into Excel and the proteins were further categorized manually based on their GO terms.

The final function-centric cluster were generated using Excel.

2.5 Plasmid and siRNA transfection, TEAD reporter assay

Confluent cultures of Eph4 cells were trypsinized, resuspended in culture media, and then

counted. For low- and high-density cultures, 25,000 cells or 250,000 cells were plated in 24-

well dishes respectively and reverse-transfected using RNAiMAX (Invitrogen). For

transfection of HEK293T, calcium phosphate was used. For reporter assays, HEK293T cells

were seeded in 24-well dishes and transfected with cDNAs, pCMV-YFP, and 5xTBS-lux

reporters. Forty-eight hours after transfection, the cells were lysed. Luciferase activity in cell

lysates was determined and normalized to YFP activity.

2.6 Immunofluorescence


17

For immunofluorescence analysis of cell cultures, the samples were fixed with 4%

paraformaldehyde for 10min. Samples were permeabilized with 0.2% Triton X-100 in PBS

for 10min and blocked with 2% BSA-PBS for two hours prior to addition of primary

antibodies at 4°C overnight. The samples were then washed with 0.1% Tween-PBS,

incubated with secondary antibodies at room temperature for 1hr, washed again with 0.1%

Tween- PBS, and finally mounted. Quantification of fluorescence intensity was performed

on deconvolved, maximum projected Z-stacks acquired under non-saturating conditions.

Identical exposure times were used between different samples to ensure the linearity of the

fluorescence measurements using the VOLOCITY software.

2.7 Primary cortical neuron culture

Dissociated mouse cortical neurons from embryonic mouse brain tissue were first trypsinzed

at 37°C for 15min. 100,000 cells were then resuspended in Dulbecco's modified Eagle's

medium containing 10% fetal bovine serum and plated into a polylysine-coated glass

coverslips sitting in a 24-well plate. The next day, the media was changed to Neurobasal

media supplemented with 5% fetal bovine serum, B27 and GlutMAX. The cells were ready

for analysis 72 hours after plating.

3 RESULTS AND DISCUSSION

Despite the recent advancement in understanding the Hippo pathway, several questions


18

still remain. First, much less is known about how MST1/2 and LATS1/2 are regulated

compared to TAZ/YAP. It would be helpful to expand the studies to these important Hippo

pathway components as well. Second, are there any other cross-talk nodes within the Hippo

pathway in addition to TAZ/YAP? Third, most studies on the Hippo pathway focused on its

roles in cell proliferation and stem cell renewal in epithelial cells. Since the Hippo pathway

components are expressed in a large number of cell types, it’s likely that the Hippo pathway

has functions other than promoting cell proliferation in non-epithelial cell types. To answer

these questions, I decided to use mass spectrometry (MS) to conduct a systematic study on

the protein-protein interaction (PPI) network of the Hippo pathway components.

3.1 Mass spectrometry

I decided to generate stable cell lines that express the Hippo pathway main components

instead of immunoprecipitating endogenous proteins due to lack of good antibodies against

most of these proteins. The T-Rex HEK293 FLP cell line was chosen for the following

reasons. First, HEK293 cells shows density-dependent Hippo pathway activation (i.e.

nuclear YAP/TAZ at low density and cytoplasmic YAP/TAZ at high density) (Fig. 2a).

Second, HEK293 cells are among the easiest cells to obtain T-Rex FLP clones due to its high

transfection efficiency.

Using the T-Rex FLP system, I generated cell lines expressing all main Hippo pathway

components including MST1/2, LATS1/2, SAV, and MOB1A/B (Fig. 1). I also generated

lines expressing RASSF5, RASSF6 since they are known regulators of MST1/2 (Allen et al.,

2007; Hwang et al., 2007). For all the kinases included, I also generated lines expressing

their dominant-negative versions. The reason is that the dominant-negative kinase sometimes


19

can lock the substrate in a more stable fashion compared to their wild-type counterpart, thus

increasing the probability of catching substrates during the immunoprecipitation step. All of

these proteins were tagged with triple-FLAG tag at the N-terminal position. My negative

control was a cell line that expressed only the triple-Flag tag. I then confirmed that all cell

lines express the proteins in a doxcyclin-inducible manner (Fig. 2b). Figure 3a summarizes

my MS work-flow. Briefly, cells were allowed to grow to full confluency to maximize the

activation of the Hippo pathway. Next, the cells were treated with 1uM of doxycycline for 16

hours to induce gene expression. Cells were lysed at this point and were processed for MS.

A high-confidence list of interactors from any MS studies must include true biological

interactors of the bait protein and exclude non-specific binders which may result from non-

specific binding to the beads, non-specific binding to the tag, and/or artificial binding to the

bait protein during the lysis step with no biological function. To reduce the amount of these

“sticky proteins”, I first excluded highly abundant proteins such as heat shock proteins,

keratin, nucleoproteins, and ribosomal proteins, all of which have been shown to bind empty

beads in other MS studies (Liu et al., 2010a). To exclude proteins that bind to the FLAG tag,

I removed all interactors found in the control cell line which expresses only the triple-FLAG

tag. To further control the high confidence of the interactors, I kept the minimum number of

unique peptides above 2 and the minimum Mascot score above 50, adopted from other

similar MS studies. Unique peptide number tells the amount of protein in the

immunoprecipitated complex, and the Mascot score tells the confidence of the results

generated by the algorithm based on the MS spectra. The final list included interactors

detected in at least two of three biological repeats for each bait (Table 1) with the bold font


20

highlighting the bait proteins, and the blue shade highlighting the known interactors. Overall

the baits and their known interactors were highly enriched among the interactors, thus

indicating that my MS experiments are robust enough to identify the expected interactors.

3.2 Protein-centric and function-centric clustering of the interactors

The MS study on all Hippo pathway components yielded more than 250 high-

confident hits (Table 1). To gain more insight into the global interactome of the Hippo

pathway, I sought commonalities among the hits in both a protein-centric and function-

centric fashion. Protein-centric clustering was carried out with the aid of CytoscapeTM

software (Fig. 3b). The goal of this analysis is to generate PPI networks using the bait

proteins as nodes which allows for the identification of common interactors among different

Hippo pathway components that might reflect potential common regulatory complexes based

on the assumption that proteins are more likely to have common interactor(s) if they operate

in the same pathway. As a proof of principle, the STRIPAK phosphatase complex is

represented as a shared complex between MST1/2 and SAV1 (Fig. 3b, highlighted in pink)

(Ribeiro et al., 2010).

As a complementary approach, I also carried out a literature search to uncover

common cell biological relationships among these hits as well as functional clustering based

on GO terms using CytoscapeTM software. This exercise identified a number of molecular

targets, cellular processes, and signaling pathways that likely play important roles in Hippo

pathway regulation (Fig. 3c; Table 1). Several of these proteins have been previously

implicated in Hippo pathway regulation, for example the RASSF family members that

attenuate MST1/2 activity. In addition, this approach implicated a number of molecular


21

targets whose roles in the Hippo pathway regulation have not been established. For example,

the Scribble polarity complex component DLG5 was found to interact with MST1/2. The

Scribble polarity complex has been shown to regulate the Hippo pathway but the precise

molecular mechanism has not been demonstrated (Cordenonsi et al., 2011; Grzeschik et al.,

2010). Furthermore, previous work have focused more on how the Scribble complex acts on

YAP/TAZ and less on MST1/2. In fact, little is known about how MST1/2 are regulated

compared to the other Hippo pathway components. Thus, the DLG5-MST1/2 interaction

might reveal a novel regulatory mechanism of how the Scribble polarity complex regulates

MST1/2 in mammals. Another example is the guanine-exchange factor DOCK7 that interacts

with MOB1, whose characterization is described in the rest of this study.

3.3 Position of the tag, kinase activity and cell density do not seem to affect interactome of at

least certain proteins

In the course of my MS studies I also assessed the kinase-dead variants of MST1/2 and

LATS1/2. As mentioned before, kinase-dead variants of certain enzymes sometimes can

“lock” the substrates in the binding pocket thus increasing the abundance of the substrate

peptides in the immunoprecipitates. Disappointingly, none of the dominant-negative kinases

co-immunoprecipitated more interactors compared to their wild-type counterpart (Table 2).

In some cases, the position of the tag might affect the interactome of the protein of

interest depending on how the protein folds. Therefore, I also tested the effects of having the

tag at either the N-terminal or the C-terminal for LATS1/2, both of which did not pull down

many polarity-related or signaling-related proteins. Unfortunately, I did not observe any

significant changes when comparing the MS results either (Table 3).


22

Lastly, I reasoned that the interactome of the Hippo pathway components might change

at different cell densities. It is possible that the core components are bound by inhibitors at

low cell density, or are bound by activators at high cell density. Thus I repeated MS studies

on MST1/2, LATS1/2, SAV and MOB1/2 at low cell density. However, no significant

changes were observed when comparing the MS results obtained from high cell density and

low cell density (Table 4). It might suggest that changing cell density may directly work on

YAP/TAZ instead of regulating the core Hippo pathway components.

3.4 Identification and characterization of DOCK7 and its role in Hippo signaling

As seen from Table 1 and 2, although many novel interactors were discovered from the

MS study, only a few are involved in signaling pathways and polarity regulation which are of

more interest to me. Dedicator of Cytokinesis 7 (DOCK7), along with its close homologue

DOCK6, is a novel interactor of MOB1. It is a member of the DOCK-C subfamily of the

DOCK family of guanine nucleotide exchange factors (GEFs) which function as activators

of small G proteins RAC1 and CDC42(Watabe-Uchida et al., 2006). DOCK7 possesses a

DHR2 domain which mediates G protein activation by stabilizing it in its nucleotide free

state. It also contains a DHR1 regulatory domain (Fig. 4a). The biological role of DOCK7

has been mainly demonstrated in the neuronal systems via regulating RAC1 and/or CDC42

activity. In one study, DOCK7 was shown to be required for axon formation in murine

embryonic hippocampal neurons (Watabe-Uchida et al., 2006). In another study, DOCK7

was shown to negatively regulate Schwann cell differentiation and myelination (Yamauchi et

al., 2011). Interestingly, the DOCK7 knockout mouse exhibits normal neuronal development

but with lighter skin pigmentation (Blasius et al., 2009), suggesting that loss of DOCK7


23

might be compensated by its close homologues DOCK6 and DOCK8.

Before beginning any further investigation on the DOCK7-MOB1 interaction, I first

confirmed this interaction manually. I overexpressed MOB1B and DOCK7 in HEK293T

cells, then immunopreciptated DOCK7, and immunoblotted for MOB1. This showed that

MOB1 interacted with DOCK7 (Fig. 4b). I then hypothesized that the interaction between

MOB1 and DOCK7 might be controlled to regulate DOCK7 activity. As mentioned in the

introduction, the interaction between LATS1/2 and MOB1 requires phosphorylation of two

evolutionarily-conserved threonine sites on MOB1 (T12 and T35 on MOB1B) by MST1/2

(Bao et al., 2009). I reasoned that these two sites might also be important for MOB1 to exert

its effects on DOCK7. To address these questions, I first confirmed that overexpressing

MST1 increased MOB1B T12 phosphorylation by blotting the lysates using an antibody

specific against the phosphorylated threonine sites (Fig. 4b). To examine whether

phosphorylation on these two threonine sites are required for the interaction between MOB1

and DOCK7, I co-expressed DOCK7 with either wild-type MOB1B or MOB1B(T12/35A)

mutant in HEK293T cells, then immunoprecipitated DOCK7, and found that

MOB1B(T12/35A) did not co-precipitate with DOCK7, suggesting that interaction of MOB1

with DOCK7 is phosphorylation dependent by MST1/2 (Fig. 4c). The data thus provided

evidences for a MST1/2-MOB1-DOCK7 signaling axis where activated MST1/2 regulates

DOCK7 activity via phosphorylating MOB1.

The next question I addressed was to characterize the functional role of this

interaction. Initially, I hypothesized that DOCK7 might regulate the Hippo signaling via

interacting with MOB1. I reasoned that MOB1 might serve as an adaptor protein for DOCK7


24

and DOCK7 might control a small GTPase to regulate Hippo signaling. To address this

possibility, I first tested the effects of DOCK7 on Hippo signaling in a simple but widely-

used reporter assay. In this assay, DOCK7 is co-transfected with a luciferase reporter driven

by TEAD-binding-sequence (TBS). HEK293T cells were used because of their high

transfection efficiency which maximizes luciferase signal production. As mentioned in the

introduction, YAP/TAZ binds strongly to TEAD and any change in YAP/TAZ localization

should affect the expression of the luciferase reporter. YFP driven by normal PCMV6

promoter was co-transfected to normalize transfection efficiency. Disappointingly,

expression of full-length DOCK7 (DOCK7-FL), DOCK7 that lacks the DHR2 catalytic

domain (DOCK7-Δ-DHR2, catalytic-dead), or the DHR2 domain of DOCK7 (DOCK7-

DHR2, lack regulatory domain) did not significantly alter the luciferase expression (Fig. 4d).

Transfection of wild-type or the T12/35A mutant form of MOB1 didn’t alter the luciferase

expression either (Fig. 4d). As a positive control, YAP/TAZ were strongly phosphorylated as

assessed by immunoblotting when LATS was overexpressed, indicating functional Hippo

signaling in HEK293T cells (Fig. 4e).

A more robust system is the well-polarized Eph4 murine mammary epithelial cell line

which exhibits density-dependent and polarity-controlled Hippo signaling (Varelas et al.,

2010b). To avoid the problems associated with the reporter assay, I first chose to examine the

localization of endogenous YAP/TAZ after changing the level of DOCK7 in Eph4 cells. I

first reduced DOCK7 expression by treating the cells with si-DOCK7 at either high or low

density, and then examined YAP localization using immunofluorescence. Si-Crumbs was

used as a positive control. Si-Dock7 did not significantly change YAP localization either at


25

high or low cell density (Fig. 5a), even though the knock-down efficiency is greater than

80% as measured by qPCR (Fig. 5b). I was not able to examine the protein level of DOCK7

using western blot after knockdown due to lack of good antibodies against DOCK7. Next, I

transiently transfected GFP-DOCK7 into Eph4 cells at either high or low density. Again,

DOCK7 expression did not significantly change YAP localization at either cell density level

(Fig. 5c).

3.5 Characterization of the role of DOCK7 in neuronal development

Because it was difficult to draw any definitive conclusions on the role of DOCK7 in

regulating the Hippo pathway, I decided to examine the DOCK7-MOB1 interaction from

another angle, that is, whether MOB1 regulates the function of DOCK7 in Hippo-

independent signalling pathway. There are at least two mechanisms for MOB1 to regulate

DOCK7. MOB1 can either regulate the localization of DOCK7, or it can regulate the GEF

activity of DOCK7. To examine the former possibility, one really needs to examine whether

double knockdown of MOB1A/B would affect the localization of endogenous DOCK7 using

immunofluorescence. However, due to lack of good antibody against DOCK7, it is difficult

to carry out such experiment at present (the localization of over-expressed GFP-DOCK7 do

not conform to that of the endogenous DOCK7) (Watabe-Uchida et al., 2006). To examine

whether MOB1 influences the GEF activity of DOCK7, I performed “PBD pull-down assay”

which assesses the level of endogenous RAC1-GTP (active RAC1). The premise of this

assay is that the p21 RAC1/CDC42 binding domain (PBD) of PAK binds specifically to the

active GTP-bound form of RAC1. HEK293T cells were transfected with various constructs

of DOCK7 and MOB1, and endogenous RAC1-GTP was precipitated from cell lysates using


26

the PAK-PBD protein beads. However, I was not able to obtain consistent results and most

experiments showed no significant change in endogenous RAC1-GTP level upon expressing

either DOCK7 or MOB1 or both (Fig. 6a and 6b).

Aside from the biochemical role of MOB1 in regulating DOCK7, it is more

important to examine whether the MOB1-DOCK7 interaction has any functional relevance

in any biological settings. As mentioned before, it has been reported that DOCK7 is required

for axon formation in developing neurons (Watabe-Uchida et al., 2006). Our lab has

developed a system to culture mouse embryonic cortical neurons in vitro from E14 mouse

embryos. Thus I decided to utilize this system to examine the biological role of the MOB1-

DOCK7 interaction. The system is described briefly below. Shortly after trypsinization,

50000 embryonic cortical neurons were electroporated with either plasmids or siRNA and

immediately plated. Shortly after plating, a neuron forms lamellipodia (stage 1), from which

several minor neurites sprout (stage 2). After 24hr, only one of these neurites elongates to

form the axon (stage 3), whereas several days later the remaining neurites form dendrites

(stage 4). Axons can then be immunostained using axon-specific markers. Embryonic

cortical neurons thus serve as an ideal system to study axon formation. The ideal experiment

is to treat the neurons with either si-DOCK7 or si-MOB1 and examine the effects on axon

formation using immunofluorescence. Again, due to lack of good antibodies against either

DOCK7 or MOB1, and due to the low transfection efficiency of siRNA in neurons, I was not

able to carry out this experiment. Therefore, I decided to investigate whether overexpressing

DOCK7 or MOB1 promotes axon formation. I thus generated GFP-tagged DOCK7 and

MOB1B constructs and attempted to express them in embryonic cortical neurons.


27

Unfortunately, no GFP expression was observed after several attempts. Upon trouble-

shooting, I suspected that the PCMV promoter of my vector is not strong enough to drive

high expression in primary neurons. Thus I re-cloned my GFP-tagged DOCK7 and MOB1B

constructs into a pCAG-promoter driven vector which is a stronger promoter than pCMV in

many primary cells. Significant GFP expression was observed three days after transfection

(Fig. 6c). I then quantified the axon number per neuron by staining them with Tau-1 (axon

specific antibody) and MAP2 (general neurite marker). Neither DOCK7 nor MOB1

expression produced any significant effects on axon number in cortical neurons (Fig. 6d).

One possibility of producing no observable phenotypes in neurons is that MOB1

controls DOCK7 activity outside of the context of axon formation, or that MOB1 controls

DOCK7 via RAC1-independent avenues. Because the function of DOCK7 is not extensively

studied, I decided to perform mass spectrometry on DOCK7 in order to identify novel

interactors and potential regulators. Flag-tagged DOCK7 was expressed in HEK293T cells

and mass spectrometry was performed on the anti-Flag precipitate. Proteins from the LRCH

family were enriched in the precipitates (Table 3). Very little is known about this family of

proteins. The Drosophila Lrch protein contains a repetition of protein-interaction LRR domain

at the N-term and a single actin-binding CH domain at the C-term (Foussard et al., 2010) (Fig.

7). Depletion of Lrch in Drosophila leads to defects in mitosis. Furthermore, genetic variation

of one of the human homologues, LRCH1, is associated with osteoarthritis (Spector et al.,

2006). Thus, it is possible that MOB1B controls cytoskeleton via regulating the interaction

between DOCK7 and the LRCH family proteins, and they may play a role in the development

of osteoarthritis.


28

4 DISCUSSION

4.1 Mass spectrometry

I picked nine proteins from the Hippo signaling pathway as the basis of my mass

spectrometry study. Out of all the hits identified, one might notice that only a few are

involved in polarity complexes which have been traditionally shown to regulate Hippo

signaling. One explanation is that the regulatory mechanism of the Hippo pathway in

HEK293 cells is not well understood. Seeing that the HEK293 cells are not a typical

columnar epithelial cell line, it is likely that the Hippo pathway in HEK293 cells is not

mainly regulated by the polarity complexes. Thus, HEK293 cells might not be the ideal

system to uncover novel polarity-related regulators of the Hippo pathway. In fact, to address

the aforementioned problem of poorly-assembled polarity complexes in HEK293 cells, I

explored the possibility of implementing the T-Rex FLP system in other more-epithelial-like

cell lines, for example the Eph4 murine mammary epithelial cells, but failed to obtain any

surviving clones upon selection, most likely due to poor transfection efficiency in Eph4

cells.

4.2 DOCK7 failed to change Hippo signaling

In this study, I decided to pursue the novel hit DOCK7 to study its role in Hippo

pathway signaling. I first confirmed that DOCK7 interacts with MOB1A/B. Furthermore, I

showed that the interaction depends on two evolutionarily-conserved residues that are

phosphorylated by MST1/2. This interesting data suggests that in additional to the canonical

Hippo signaling branch where MST1/2 stimulates the activity of LATS1/2, there might be

another non-canonical signaling branch where MST1/2 stimulates the activity of DOCK7.


29

Disappointingly, most other data was negative, especially ones concerning the

functions of DOCK7 and its relationship with the Hippo pathway. I first hypothesized that

DOCK7 regulates the Hippo signaling. The first such experiment was the TEAD-reporter

assay in which over-expression of neither DOCK7 nor MOB1 resulted any change in

luciferase expression (Fig. 4d). In this case, the limitations of this assay system must be

taken into account before making any conclusions from these negative data. First, as

mentioned before, the regulation of Hippo pathway in HEK293T cells differs from that in

epithelial cell lines due to poorly assembled polarity complexes. One can imagine that if

DOCK7 operates only within the context of the polarity arm of Hippo regulation, then any

effects it might have on the Hippo pathway will be diminished in HEK293T cells. Second,

the artificial reporter system used in this assay contains five concatamerized TEAD binding

sites, which may render the system too sensitive to detect any change in YAP/TAZ level even

when Hippo signaling is altered (i.e. signal saturation). Indeed, expressing LATS1 in this

system reduced the reporter signals by a mere ~40% (Fig. 4d). Therefore, if DOCK7

activates the Hippo pathway (thus reducing the nuclear YAP/TAZ level), such effects might

not be detected by this system.

I then switched to the more robust Eph4 cells and examined the endogenous

localization of YAP/TAZ when changing the level of DOCK7. Again, the results were

negative (Fig. 5). However, it is still difficult to draw a definitive conclusion on whether

DOCK7 truly regulates the Hippo pathway given the negative data. First, as mentioned

before, despite great knockdown at the RNA level, how much the protein level was reduced

remains unknown due to lack of good antibodies. Especially at high cell density where cell


30

division rate is low and the protein turnover rate is reduced, the levels of some junctional

protein remain comparatively high despite greatly reduced RNA level. Seeing that DOCK7

has been shown to localize at junctions, it is possible that the reduction at the protein level of

DOCK7 is too insignificant to produce a phenotype even after greater than 80% RNA

knockdown. Second, close homologues of DOCK7, for example DOCK6 and DOCK8,

might compensate the loss of DOCK7 even assuming the protein level of DOCK7 was

reduced significantly. Both proteins are GEF proteins and DOCK8 have also been shown to

bind MOB1(Mou et al., 2012). Therefore, double or even triple knockdown against DOCK7

and its two homologues in Eph4 cells need to be carried out in order to definitively conclude

whether DOCK7 truly has any effects on Hippo signaling. Lastly, it is possible that

endogenous DOCK7 in Eph4 cells is already working at full capacity. If so, any additional

DOCK7 transfected into the system will not produce any significant effects because the

system is already saturated.

4.3 Hippo signaling failed to change DOCK7 activity

Since no definitive conclusions can be drawn on whether DOCK7 regulates the

Hippo signaling, I asked whether the Hippo signaling regulates the activity of DOCK7.

Again, few positive data was generated. First, I wasn’t able to show that DOCK7 acts as a

GEF protein in a biochemical assay as reported in other literature (Fig. 6a). One possibility

of seeing no phenotype is that this particular experiment examines the total endogenous level

of RAC1-GTP from all cells, but only a fraction of cells are transfected with the DOCK7 or

MOB1 vectors. One can imagine that even if the RAC1-GTP level did increase in these


31

transfected cells, it might not be strong enough to influence the total level of RAC1-GTP.

Therefore, it might be better in this case to co-transfect with exogenous RAC1 and examine

the exogenous level of RAC1-GTP.

Neither DOCK7 nor MOB1 produced any significant changes in the axon assay

either (Fig. 6). One possible reason could be that in the original paper, overexpressed

DOCK7 was shown to promote axon formation in embryonic hippocampal neurons (Watabe-

Uchida et al., 2006), whereas I used the embryonic cortical neurons. The subtle differences

between these two biological systems might result in different biological roles of DOCK7.

5 CONCLUSION

Through my MS studies, I identified more than 250 interactors for the main Hippo

pathway components. Protein-centric and function-centric analyses of these interactors

allowed me to infer proteins and processes that likely are important for the regulation of the

Hippo pathway, including several proteins which have not previously been shown to play roles

in Hippo regulation. For example, the Scribble complex DLG5 was identified as a novel

interactor of MST1/2. It may therefore be interesting to learn whether the Scribble complex

plays a role in regulating the Hippo pathway in mammals.

Among the novel interactors identified, I decided to pursue DOCK7 which interacts with

MOB1A/B. DOCK7 is a GEF protein for small G protein RAC1 and CDC42. After manually

confirming the DOCK7-MOB1 interaction using western blot, I further showed that this

interaction is regulated by a MST1/2-dependent phosphorylation event on two threonine

residues on MOB1. I was not able to conclude whether MOB1 regulates the GEF activity of


32

DOCK7, most likely due to the limitation of the PBD pull-down assay.

I initially hypothesized that DOCK7 might be a novel regulator of the Hippo pathway.

Disappointingly, neither using a reporter assay system in HEK293T cells nor examining

endogenous YAP/TAZ localization in the more epithelial-like Eph4 cells did I observe any

change in Hippo signaling upon changing the level of DOCK7. Possible explanations include

insufficient knockdown at protein level, or compensation from close homologues DOCK6 and

DOCK8.

I next hypothesized that MOB1 might regulate DOCK7 in Hippo-independent functions.

DOCK7 has been shown to promote axon formation. Utilizing an in vitro embryonic cortical

neuron system, I was not able to show that expressing either DOCK7 or MOB1 has any effects

on axon growth. It is possible that hippocampal neurons were used instead of cortical neurons

in the original study where they showed the axon-promoting effects of DOCK7. The subtle

differences between cortical neurons and hippocampal neurons may be why I did not see any

phenotype.

Another possibility of producing no observable phenotypes in neurons is that MOB1B

controls other DOCK7 activity. MS performed on DOCK7 revealed that DOCK7 interacts

with proteins from the LRCH family. These proteins are involved in cytoskeleton

reorganization. Thus, it is possible that MOB1B controls cytoskeleton via regulating the

interaction between DOCK7 and the LRCH family proteins independent of RAC1.

I would also like to use this opportunity to comment on some improvements and future

experiments that could be carried out. As mentioned in the Discussion section, a huge

weakness in my study is the lack of a good DOCK7 antibody. Without it, no solid conclusions

can be drawn from any experiments involving knocking down endogenous DOCK7, which is


33

one of the most important experiments to demonstrate any biological functions of DOCK7. In

addition to definitively show that the knockdown worked at the protein level, a good DOCK7

antibody will also allow us to visualize the change in endogenous localization of DOCK7 in

cells where transfection efficiency is low and western blot might not be representative of the

transfected cells (i.e. primary neurons). Therefore, a good DOCK7 antibody must be acquired

before performing any future experiments.

Furthermore, choosing to work on the primary neurons right from the start might not be

the wisest choice in retrospect. Working on these primary neurons are expensive, time

consuming, and requires more than a fair amount of experience. As suggested by my

committee member Dr. Kaplan, there are many neuronal cell lines available that mimic the

axonal growth in primary neurons. If I could’ve restarted over on this project, I would’ve

definitely tested the effect of DOCK7 on these cell lines first before moving on to the primary

neurons.

Lastly, I want to emphasize that DOCK7 is not the only novel hit identified from the

mass spectrometry study. DLG5, for example, is another interesting novel interactor of

MST1/2. In fact, DLG5 is a polarity-related protein, and polarity complexes have always been

implicated in Hippo pathway regulation. One can imagine that DLG5 is a novel regulator of

MST1/2. Similar experiments could be applied to study how DLG5 regulates the Hippo

signaling, using assays such as the TEAD reporter assay, or examining the endogenous

YAP/TAZ localization upon DLG5 knocking down in Eph4 cells.

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42

6. FIGURES

Figure 1. The core Hippo pathway signaling cascade. Upon activation, MST1/2 phosphorylate

and activate LATS1/2 with the aid of the adaptor proteins SAV1. Activated MST1/2 also

phosphorylate MOB1A/B, resulting in an enhanced LATS1/2–Mob1 interaction which leads

to an enhanced LATS1/2 activity. Activated LATS1/2 in turn phosphorylate YAP/TAZ.

Phosphorylation of YAP/TAZ is inhibitory to their transcriptional functions because such

phosphorylation promotes 14-3-3 binding and subsequent cytoplasmic sequestration.

Phosphorylation of YAP/TAZ on the same sites can also induce their degradation by the E3

ubiquitin ligase SCFβ–TRCP. When the Hippo pathway is inactive, YAP/TAZ become more

stable and translocate into the nucleus to perform their functions as transcriptional co-

activators. YAP/TAZ are known to bind a variety of transcriptional factors including TEAD,

Smad2/3, p73, and β-catenin, most of which have been implicated in promoting tumor

development, thus making the Hippo pathway a tumor suppressor pathway. (Zhao, 2010)


43

Figure 2. Characterization of HEK293 T-Rex FLP lines. (A) HEK293 cells were grown into

indicated density and stained with anti-YAP/TAZ antibody. (B) Dox-inducible expression of

bait proteins in all cell lines generated. All cell lines were tagged at N-terminal by triple-Flag

tag unless otherwise stated. C: C-terminal triple-Flag tag.


44

Figure 3. Summary of the mass spectrometry study. (A) HEK293 cell lines expressing triple-

Flag tagged Hippo components were generated. Cells were lysed after 16 hours incubation of

1uM doxycycline. Anti-Flag affinity purification was performed on three independent

biological samples. Proteins were identified using mass spectrometry and the list was filtered

using the parameters described in the text. (C) Protein-protein interaction map generated for

all bait proteins using Cytoscape. Circles represent proteins and lines represent interactions.

Blue circles represent bait proteins and green circles represent interactors. Red lines

represent known interactions and blue lines represent novel interactions. The STRIPAK

complex is highlighted in pink. (D) Functional clustering pie-chart of all interactors. All

interactors were annotated using the GO terms and the pie chart was generated based on the

percentage of proteins in each category. Representative proteins in each category were listed.


45

Figure 4. The role of DOCK7 in regulating the Hippo pathway remains unclear. (A)

Schematics of different DOCK7 constructs. (B) MST1 stimulates MOB1 phosphorylation.

HEK293T cells were transfected with the indicated plasmids. After 48 hours, the cells were

lysed and endogenous p-MOB1T12 was probed using the antibody described. The ratio of p-

MOB1T12 in the pull-down fraction relative to the MOB1 in the total lysate of the control

cells was set to one; other ratios were normalized to this value. (C) DOCK7 binds

specifically to phospho-Mob1. HEK293T cells were co-transfected with HA-MOB1B WT or

HA-MOB1B[Thr12Ala/Thr35Ala], and Flag-DOCK7. After 48 hours, the cells were lysed

and subjected to anti-Flag immunoprecipitation, and the presence of MOB1B was assessed

by anti-HA immunoblotting. (D) DOCK7 did not change luciferase level in the TEAD

reporter assay. HEK293T cells were co-transfected with PCMV-YFP, 5xTBS-lux, and the

indicated vectors. After 48 hours, the cells were lysed and the luciferase level were measured

and normalized to the YFP level. (E) LATS2, but not LATS2KD increases phosphorylation

of TAZ. HEK293T cells were co-transfected with indicated plasmids and blotted using

indicated antibodies.


46


47

Figure 5. The role of DOCK7 in regulating the Hippo pathway in Eph4 cells remains

inconclusive. (A) Eph4 cells were treated with indicated siRNA in either low or high density

and were stained with anti-YAP antibody. The knockdown efficiency is shown in (B). (C)

Eph4 cells were transfected with indicated plasmids in either low or high density and were

stained with anti-YAP antibody.


48


49

Figure 6. The role of MOB1 in regulating axon development in embryonic neurons remains

inconclusive. (A) The PBD-pull down assay was not able to conclude the effects of MOB1B

on DOCK7 GEF activity. HEK293T cells were transfected with the indicated plasmids. After

48 hours, the cells were lysed and endogenous RAC1-GTP was pulled down using the PAK-

PBD beads. Immunoblots from one such experiment is shown. Relative level of active

RAC1 was measured from three independent experiments and is shown in (B). (C)

Embryonic cortical neurons transfected with the indicated plasmid were stained 72hrs later

with anti-Tau1 and anti-MAP2 antibodies. (D) Percentage of polarized neurons, identified as

ones containing only one tau-1-positive extension, is shown for different plasmids.


50

Figure 7. The domain structure of LRCH proteins. All LRCH proteins contain repetition of

LRR domains and one or more CH domains.


51

7. TABLES

Table 1. Summary of all the high-confidence interactor of the Hippo pathway main components.

Bait=hLATS1 Total Peptide Number Annotation

Gene R1 R2 R3

LATS1 114 105 73 Bait

MOB1B 6 3 Known interactor

FBXO3 5 5 2 Transcription factor

STAU1 2 2 RNA binding

TET2 2 4 2 DNA methylase

Bait=hLATS2 Total Peptide Number

Gene R1 R2 R3

LATS2 390 409 398 Bait

CAD 50 54 27 Metabolism

PDP1 22 4 Metabolism

MOB1B 12 3 5 Known interactor

USP9X 7 8 4 ubiquitin

PRKDC 6 1 8 DNA damage

CPVL 6 3 2 Metabolism

UTRN 5 4 5 Cytoskeleton

STXBP4 5 7 3 Transport

YWHAE 4 3 3 14-3-3

ANAPC1 3 2 2 Cell cycle

YWHAZ 2 3 14-3-3

BAG4 2 2 Chaperone

Bait=hMOB1a Total Peptide Number

MOB1A 30 78 54 Known interactor

LATS1 21 47 47 Known interactor


STAU1 3 2 2 RNA binding


DOCK7 2 17 27 Polarity

STK3 8 5 Known interactor

CAD 3 5 Metabolism

TET2 2 2 DNA methylase

TMOD2 2 2 Cytoskeleton

Bait=hMOB1b Total Peptide Number

Gene R1 R2 R3

MOB1b 55 65 42 Bait



STK3 4 12 Known interactor

STAU1 3 3 RNA binding


52


YWHAE 3 4 14-3-3

Bait=hRASSF5 Total Peptide Number

Gene R1 R2 R3

RASSF5 131 120 184 Bait

STK3 82 85 126 Known interactor


MAP1S 45 31 48 Cytoskeleton


KCTD12 5 2 6 Apoptosis

FBXW11 3 3 3 Transcription factor

PDCD6 2 5 6 Apoptosis

PHGDH 2 2 Metabolism

SUGT1 5 9 Metabolism

Bait=mRassf6 Total Peptide Number

Gene R1 R2 R3



RASSF6 9 9 8 Bait


AIFM1 5 4 9 Apoptosis

PSMD1 5 5 Protease

PSMD12 4 4 4 Protease


PSMC4 3 5 5 Protease

CCT2 3 3 2 Metabolism

PSMC5 3 4 Protease



PSMC3 3 9 Protease

Bait=hSAV Total Peptide Number

Gene R1 R2 R3


SAV1 57 83 99 Bait




ATAD3A 20 7 Transcription

IRS4 13 10 5 Signaling

SEC16A 7 9 7 Transcription

PHGDH 7 5 Metabolism

AKAP8L 6 15 11

UBR5 6 3 Ubiquitin

SON 5 4 13 Metabolism


53

RANBP9 5 3 3 Cytoskeleton

PSMD3 4 2 Protease

PCBP2 3 4 3 DNA binding

FANCI 3 4 DNA binding

NUP93 3 3 Transport

EMD 3 3 Metabolism

SSR4 3 2 Transprot

C20orf11 2 8 6

FAM83H 2 3 3 Metabolism

PPP1CA 2 3 Phosphatase

DSP 2 2 3 Polarity

CCT2 6 2 Chaperone

YWHAE 5 5 14-3-3

CCT3 4 3 Chaperone

IQGAP3 4 2 Cytoskeleton

ZNF281 2 6 DNA binding

TCP1 2 6 Metabolism

ASPM 2 4 Cell cycle

YWHAZ 2 3 14-3-3

MOV10 2 3 Metabolism

ATP2A2 2 3 cytoskeleton

YWHAG 2 2 14-3-3

SLC25A3 2 2 Transport

PSMC4 2 2 Proteasome

MATR3 2 2 Cytoskeleton

LARP4B 2 2 Transcription

CCT7 2 2 Metabolism

Bait=hSTK3 Total Peptide Number

Gene R1 R2 R3

STK3 404 472 252


RASSF2 24 24 39 Known interactor

DLG5 22 9 Polarity


LTK 8 3 Receptor

SAV1 8 12 8 Known interactor

PRKRIR 4 7 Kinase inhibitor

MAP1S 3 10 4 Cytoskeleton



SLMAP 9 13 Known interactor

STRN3 7 8 Known interactor

STRN 7 10 Known interactor

PPP2R1A 4 4 Known interactor


54

MOB4 3 4 Known interactor

FGFR1OP2 2 4 Known interactor

SCIN 2 3 Lipoprotein

TET2 2 3 DNA methylase


PPP2CA 2 3 Known interactor

Bait=hSTK4 Total Peptide Number

Gene R1 R2 R3

STK4 524 407 397 Bait





SAV1 10 8 4 Known interactor


STRN 7 11 Known interactor



55

Table 2. Comparison of the intearctome between wild-type and kinase-dead variant of

MST1/2 and LATS1/2.

Bait=hLATS1 Total Peptide Number (WT) Total Peptide Number (KD)

Gene R1 R2 R3 R1 R2 R3

LATS1 114 105 73 100 88 150

MOB1B 6 3 4 4 2

FBXO3 5 5 2 5 3

STAU1 2 2 1 2 2

TET2 2 4 2 3

Bait=hLATS2 Total Peptide Number (WT) Total Peptide Number (KD)


LATS2 390 409 398 250 400 401

CAD 50 54 27 48 34 36

PDP1 22 4

MOB1B 12 3 5 8 15 12

USP9X 7 8 4

PRKDC 6 1 8 4

CPVL 6 3 2 1 3 3

UTRN 5 4 5 7 2 2

STXBP4 5 7 3

YWHAE 4 3 3 5 1

ANAPC1 3 2 2 2 2 2

YWHAZ 2 3 2 1 1

BAG4 2 2

Bait=hSTK3 Total Peptide Number (WT) Total Peptide Number (KD)


STK3 404 472 252 288 387 347

STK4 283 300 148 300 305 199

RASSF2 24 24 39 30 33 40

DLG5 22 9 11 19 15

RASSF1 9 36 9 19 18

LTK 8 3

SAV1 8 12 8 5 11 11

PRKRIR 4 7 3 7 3

MAP1S 3 10 4 4 15 1

RASSF3 3 4 3 1 1 2

RASSF4 2 5 12 3 7

SLMAP 9 13 8 8 11

STRN3 7 8 1 4 9

STRN 7 10 3 1 10

PPP2R1A 4 4 5 6

MOB4 3 4 2 5 4

FGFR1OP2 2 4 2


56

SCIN 2 3 4 5 3

TET2 2 3 5 4

STRN4 2 4 1 5 3

PPP2CA 2 3 2 1 3

Bait=hSTK4 Total Peptide Number (WT) Total Peptide Number (KD)


STK4 524 407 397 499 600 577

STK3 188 171 92 158 151 192

RASSF2 51 40 46 41 48 37

RASSF4 12 6 26 14 16

RASSF1 10 10 3

SAV1 10 8 4 11 4 4

RASSF5 5 4 3 15 4

STRN 7 11 1 3 9

STRN3 5 3 1 5 4


57

Table 3. Comparison of the intearctome between N-term tagged and C-term tagged variant

of LATS1/2.

Bait=hLATS1 Total Peptide Number (N-Term) Total Peptide Number (C-Term)


LATS1 114 105 73 99 153 88

MOB1B 6 3 3

FBXO3 5 5 2 6 1 2

STAU1 2 2 3 3 1

TET2 2 4 2 2 1

Bait=hLATS2 Total Peptide Number (N-Term) Total Peptide Number (C-Term)


LATS2 390 409 398 299 444 345

CAD 50 54 27 20 27

PDP1 22 4 21 19 19

MOB1B 12 3 5 11 13 8

USP9X 7 8 4 9 5

PRKDC 6 1 8 1 1

CPVL 6 3 2 3 2 2

UTRN 5 4 5 4 1

STXBP4 5 7 3 1 1 1

YWHAE 4 3 3 5 5 6

ANAPC1 3 2 2 2 3 5

YWHAZ 2 3

BAG4 2 2 1 1 3


58

Table 4. Comparison of the intearctome between low and high cell density of core Hippo

pathway components.

Bait=hLATS1 Total Peptide Number (Hi) Total Peptide Number (low)


LATS1 114 105 73 99 101 133

MOB1B 6 3 3 1 2

FBXO3 5 5 2 4 3 4

STAU1 2 2 3 1 1

TET2 2 4 2 1 2

Bait=hLATS2 Total Peptide Number (Hi) Total Peptide Number (low)


LATS2 390 409 398 378 345 501

CAD 50 54 27 40 44 38

PDP1 22 4 19 9 21

MOB1B 12 3 5 7 11 11

USP9X 7 8 4 8 8

PRKDC 6 1 8

CPVL 6 3 2 4 4 1

UTRN 5 4 5 6 5 5

STXBP4 5 7 3 4 4 6

YWHAE 4 3 3 2 4

ANAPC1 3 2 2 5 2

YWHAZ 2 3 1 3

BAG4 2 2 2 3 3

Bait=hMOB1a Total Peptide Number (Hi) Total Peptide Number (low)

MOB1A 30 78 54 28 44 64

LATS1 21 47 47 42 55 51

LATS2 4 12 8 7 19 19

STAU1 3 2 2 2 1

PRKDC 2 6 4 3 4 6

DOCK7 2 17 27 11 3

STK3 8 5 2 7 6

CAD 3 5 4 2

TET2 2 2 1 1 1

TMOD2 2 2 3 1 1

Bait=hMOB1b Total Peptide Number (Hi) Total Peptide Number (low)


MOB1b 55 65 42 70 61 66

LATS1 66 49 45 49 55 51

LATS2 10 16 27 25 33 7

STK3 4 12 5 6 7

STAU1 3 3

PRKDC 3 12 9 4 1 8


59

YWHAE 3 4 3

Bait=hSAV Total Peptide Number (Hi) Total Peptide Number (low)


PRKDC 63 44 15 14 26 41

SAV1 57 83 99 60 66 44

STK3 52 145 57 45 71 48

STK4 44 136 103 39 46 49

CAD 30 58 33 40 38 53

ATAD3A 20 7 21 18

IRS4 13 10 5 14 8 24

SEC16A 7 9 7 3 4 3

PHGDH 7 5

AKAP8L 6 15 11 5 5 14

UBR5 6 3 1 1

SON 5 4 13 3 4 3

RANBP9 5 3 3 3 2

PSMD3 4 2 4

PCBP2 3 4 3 1

FANCI 3 4 2 5

NUP93 3 3 3 3 6

EMD 3 3 4 7 1

SSR4 3 2

C20orf11 2 8 6 4

FAM83H 2 3 3 1 1 1

PPP1CA 2 3 4 2 1

DSP 2 2 3 4 1

CCT2 6 2 1 2

YWHAE 5 5 2 4 4

CCT3 4 3 2 2

IQGAP3 4 2 1 1 1

ZNF281 2 6

TCP1 2 6 1 2

ASPM 2 4 3 3 4

YWHAZ 2 3 3

MOV10 2 3 4

ATP2A2 2 3 4 3

YWHAG 2 2 1 2

SLC25A3 2 2 2 2

PSMC4 2 2

MATR3 2 2 1 4

LARP4B 2 2 4 3 2

CCT7 2 2 1 2 1

Bait=hSTK3 Total Peptide Number (Hi) Total Peptide Number (low)



60

STK3 404 472 252 514 300 445

STK4 283 300 148 199 244 287

RASSF2 24 24 39 40 31 35

DLG5 22 9 19 26 18

RASSF1 9 36 9 3

LTK 8 3 2 4 6

SAV1 8 12 8 7 9 9

PRKRIR 4 7 4 4 4

MAP1S 3 10 4

RASSF3 3 4 3 2 3 1

RASSF4 2 5 12 1 1 1

SLMAP 9 13 7 9 10

STRN3 7 8 2 5

STRN 7 10 2 3

PPP2R1A 4 4

MOB4 3 4 1 4

FGFR1OP2 2 4 5 2 2

SCIN 2 3 2

TET2 2 3 1

STRN4 2 4 4 1

PPP2CA 2 3 2

Bait=hSTK4 Total Peptide Number (Hi) Total Peptide Number (low)


STK4 524 407 397 444 500 517

STK3 188 171 92 190 148 200

RASSF2 51 40 46 70 80 64

RASSF4 12 6 26 20 16 22

RASSF1 10 10 3 14 18 13

SAV1 10 8 4 14 8

RASSF5 5 4 3

STRN 7 11 1 2

STRN3 5 3 4 3 3


61

Table 5. Interactome of DOCK7

Bait=hDOCK7 Total Peptide Number Annotation

Gene R1 R2 R3

DOCK7 1202 999 1178 Bait

LRCH3 8 8 9 Cytoskeleton

LRCH2 6 7 Cytoskeleton

LRCH4 6 6 5 Cytoskeleton

LRCH1 5 5 Cytoskeleton

AMOT 3 2 Polarity

TET2 3 2 2 Transcription

CDK12 2 2 1 Cell cycle

BAG5 2 2 Chaperone

YWHAZ 2 2 14-3-3

PRKDC 2 2 DNA damage

Documents

CHARACTERIZATION OF THE INTERACTOME OF THE … · transplanted limbs grow to the original size of the donor but not that of the recipient ... YAP/TAZ on the same sites can also induce