ABL TYROSINE KINASES MEDIATE INTERCELLULAR ADHESION By

ABL TYROSINE KINASES MEDIATE INTERCELLULAR ADHESION

Nicole L. Zandy

Department of Pharmacology and Cancer Biology Duke University

Date:______Approved:

______Ann Marie Pendergast, Supervisor

______Christopher Counter

______Mark Dewhirst

______Christopher Kontos

______Xiao‐Fan Wang

Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology and Cancer Biology in the Graduate School of Duke University

2008

ABSTRACT

ABL TYROSINE KINASES MEDIATE INTERCELLULAR ADHESION

Nicole L. Zandy

Department of Pharmacology and Cancer Biology Duke University

Date:______Approved:

______Ann Marie Pendergast, Supervisor

______Christopher Counter

______Mark Dewhirst

______Christopher Kontos

______Xiao‐Fan Wang

An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor in the Department of Pharmacology and Cancer Biology in the Graduate School of Duke University

2008

Abstract

Adherens junctions are calcium‐dependent cell‐cell contacts formed during

epithelial morphogenesis that link neighboring cells via cadherin receptors. Coordinated

regulation of the actin cytoskeleton by the Rho GTPases is required for the formation

and dissolution of adherens junctions, however the pathways that link cadherin

signaling to cytoskeletal regulation remain poorly defined. The Abl tyrosine kinases

been shown to modulate cytoskeletal reorganization downstream of various

extracellular signals including growth factor receptors and integrins.

Here we use pharmacological inhibition, genetic inactivation, and RNA

interference to identify the Abl family kinases as critical mediators of cadherin‐mediated

adhesion. Endogenous Abl family kinases, Abl and Arg, are activated and are required

for Rac activation following cadherin engagement, and regulate the formation and

maintenance of adherens junctions in mammalian cells. Significantly, we show that Abl‐

dependent regulation of the Rho‐ROCK‐myosin signaling pathway is critical for the

maintenance of adherens junctions. Inhibition of the Abl kinases in epithelial sheets

results in activation of Rho and its downstream target ROCK, leading to enhanced

phosphorylation of the myosin regulatory light chain. These signaling events result in

enhanced stress fiber formation and increased acto‐myosin contractility, thereby

disrupting adherens junctions. Conversely, Arg gain‐of‐function promotes adherens

junction formation through a Crk‐dependent pathway in cells with weak junctions.

These data identify the Abl kinases as a novel regulatory link between the

cadherin/catenin adhesion complex and the actin cytoskeleton through regulation of Rac

and Rho during adherens junction formation.

Unexpectedly, we identified a requirement for Abl and Crk downstream of Rac

in the regulation of adherens junctions. Therefore, Abl functions both upstream and

downstream of Rac in regulating adherens junctions, which suggests the possibility of a

positive feedback loop consisting of Abl‐Crk‐Rac.

Finally, we identified the Abl kinases as critical mediators of epithelial cell

response to HGF. Pharmacological inhibition of Abl kinase activity resulted in impaired

dissolution of adherens junctions downstream of HGF stimulation of the Met receptor.

Additionally, we observed decreased phosphorylation of the Met receptor itself, along

with Gab1 and Crk, downstream effectors of Met signaling. Taken together, these data

suggest a requirement for Abl kinases in both adherens junctions formation and

turnover.

Dedication

It is with a sense of profound loss and utter desolation that I dedicate this

dissertation in loving memory of my father, Leonard Marino Zandy (Sunday, August 6,

1944‐Friday, November 23, 2007).

Exactly five months have passed since I lost my first valentine, my career role

model, my sports buddy and “athletic” trainer, my TV gossip companion, my “project

manager,” my verbal sparring partner, my first styling “client,” my inspiration to achieve

without costing others and to give until it hurts, the foundation of my moral and ethical

beliefs, the sun in my solar system, the center of my family, and the platinum standard I

use to gauge the value of husband and father.

Just two days and five months ago, I was exhausted, in part due to our trip to

South Bend, but happily submitting my dissertation in plenty of time to finish dinner

before watching Duke win the Maui Invitational and looking forward to seeing Santa

Claus in the Macy’s Thanksgiving Day Parade the next day. On Thanksgiving Day, my

dad, mom, Kim, Pam, and I sat at the table eating turkey, roast “beast,” and mashed

potatoes, while Kurbain ate a cornucopia of food “donations.” It was a perfect meal.

On Friday, I woke up with a headache, but insisted I would not miss our family

tradition of me & mommy & daddy cutting down our Christmas tree‐I had to get it

decorated before I left to return to Durham to prepare for my thesis defense the

following week. We all got ready, went to our usual local tree farm, and almost too

easily found the “perfect tree,” our first Fraser Fir from PA in years, and brought it

home. The events that followed are too private to be shared: and just like that…he was

gone…

And in that instant, I learned infinitely more than the words that follow on these

hundred‐odd pages describing five years’ work convey. It is the wisdom from that

moment that I will continue to build on and share in his name.

I miss you, Daddy.

xoxo

Love, Nicole

vii

Contents

Abstract ...... iv

Dedication ...... vi

List of Figures ...... xii

1. Introduction ...... 1

1.1 Adherens junctions...... 1

1.1.1 Adherens junctions during development ...... 1

1.1.2 Adherens junctions in cancer...... 5

1.1.3 Adherens junction components...... 8

1.1.3.1 Cadherin family proteins...... 8

1.1.3.2 Catenin family proteins...... 9

1.1.3.3 Actin cytoskeleton...... 11

1.1.4 Regulation of adherens junctions...... 13

1.1.4.1 Adherens junction component binding ...... 14

1.1.4.2 Phosphorylation...... 15

1.1.4.3 Rho GTPases ...... 18

1.2 Cross‐talk between Adherens junctions and Receptor Tyrosine Kinases ...... 23

1.2.1 Interactions between adherens junctions and RTKs ...... 25

1.2.2 Met receptor tyrosine kinase...... 29

1.2.2.1 Cancer relevance ...... 29

1.2.2.2 Biological effects of Met activation...... 30

viii

1.2.2.3 Met signaling ...... 32

1.3 Abl family of non‐receptor Tyrosine Kinases...... 36

1.3.1 Abl and Arg...... 37

1.3.2 Regulation of Abl/Arg ...... 42

1.3.2.1 Physical...... 42

1.3.2.2 Protein modification ...... 44

1.3.2.3 Upstream signals regulating Abl tyrosine kinase activity ...... 45

1.3.3 Biological function of Abl/Arg ...... 48

1.3.3.1 In vivo roles of Abl and Arg...... 48

1.3.3.2 Abl kinases regulate response to growth factors and cell migration ...... 50

1.3.3.3 Abl family kinases in intercellular conversations ...... 52

2. Abl Tyrosine Kinases regulate cell‐cell adhesion via Rho GTPAses ...... 55

2.1 Introduction...... 55

2.2 Results ...... 57

2.2.1 Abl Tyrosine Kinases are Required for Cell‐Cell Adhesion...... 57

2.2.2 Abl Tyrosine Kinases are Required for the Maintenance of Adherens Junctions ...... 62

2.2.3 Cell‐cell Adhesion Leads to Abl Kinase Activation and Recruitment to Sites of Cell‐Cell Contact...... 65

2.2.4 Abl Kinases Regulate Rac Activation in Response to Cadherin Engagement via Crk/CrkL...... 69

2.2.5 Abl Kinases Regulate the Architecture of the Actin Cytoskeleton in Epithelial Cell Sheets via the Rho‐Rock Pathway...... 73

2.3 Discussion...... 79

3. A closer look at Abl‐Crk‐Rac...... 83

3.1 Background ...... 83

3.2 Results ...... 85

3.3 Discussion...... 90

4. Role for Abl kinases downstream of the Met receptor ...... 96

4.1 Background ...... 96

4.2 Results ...... 98

4.2.1 Abl kinases function downstream of the activated Met receptor tyrosine kinase for induction of cell scattering...... 98

4.2.2 Abl kinase inhibition alters signaling downstream of the activated Met receptor ...... 101

4.3 Discussion...... 106

5. Materials and Methods...... 110

5.1 Antibodies and Reagents...... 110

5.2 Cell Lines ...... 111

5.3 Retroviral Transduction...... 111

5.4 Cell lysis, immunoblotting, in vitro kinase assays, and RhoGTPase activation assays...... 112

5.5 Immunofluorescence...... 113

5.6 Calcium Switch Experiments...... 114

5.7 siRNA...... 114

5.8 Growth factor stimulation...... 115

6. Conclusion ...... 116

6.1 Delving deeper into signaling downstream of cadherins...... 118

6.1.1 Role for Abl and Crk...... 120

6.1.2 Abl and p190 RhoGAP...... 122

6.1.3 Abl and other downstream effectors ...... 127

6.2 Dissecting the role of Abl downstream of Met receptor activation...... 129

6.2.1 Abl and Met...... 130

6.2.2 Abl and other downstream targets ...... 132

6.3 Role of Abl downstream of other growth factors ...... 136

6.3.1 Abl and FGF ...... 137

6.3.2 Abl and VEGF ...... 140

6.4 Concluding remarks...... 143

References ...... 148

Biography...... 177

List of Figures

Figure 1: Schematic representation of adherens junctions...... 3

Figure 2: Functional domains of c‐Abl and Arg...... 40

Figure 3: Regulation of Abl kinase activity...... 47

Figure 4: Loss of Abl family kinases in fibroblasts disrupts N‐cadherin‐based adhesion...... 58

Figure 5: Inhibition of Abl kinase activity or protein impairs adherens junction formation in epithelial cells...... 60

Figure 6: Loss of Abl/Arg impairs intercellular adhesion...... 61

Figure 7: Inhibition of Abl kinase activity impairs accumulation of E‐cadherin‐catenin complexes at cell‐cell contacts...... 63

Figure 8: Cell‐cell adhesion regulates the activity of endogenous Abl kinases...... 66

Figure 9: Arg localizes to adherens junctions...... 68

Figure 10: Crk/CrkL function downstream of Abl‐mediated adherens junction formation in epithelial cells...... 70

Figure 11: Abl kinases regulate Rac activation in response to cadherin engagement...... 72

Figure 12: Abl kinases regulate the actin cytoskeleton and Rho activity in epithelial cell monolayers...... 74

Figure 13: Disruption of adherens junctions in response to inhibition of Abl kinase activity is reversed by inhibition of ROCK kinase activity...... 77

Figure 14: Model for Abl family kinase regulation of adherens junctions...... 78

Figure 15: Abl and Crk function downstream of Rac activation...... 87

xii

Figure 16: ArgPP expression rescues negative effects of Crk overexpression on adherens junctions...... 89

Figure 17: Proposed model for Abl‐Crk‐Rac signaling...... 92

Figure 18: Hyperactivation of Abl kinase activity in metastatic breast cancer cell lines. .95

Figure 19: Abl kinases act downstream of c‐Met following HGF stimulation...... 99

Figure 20: Abl kinase inhibition blocks tyrosine phosphorylation of Crk in response to Met activation...... 102

Figure 21: Inhibition of Abl kinase activity affects Met signaling downstream of HGF stimulation...... 104

Figure 22: Lack of evidence for role of Abl kinase modulation of p190 RhoGAP effect on adherens junctions...... 124

Figure 23: FGF2 stimulation activates endogenous Abl kinases...... 139

Figure 24: Abl kinases mediate intercellular adhesion in endothelial cells...... 142

xiii

1. Introduction

1.1 Adherens junctions

Adherens junctions are one of several types of specialized structures, which

allow cells to organize into tissues. Found at the basolateral surface connecting two

neighboring cells, they may line the contacts between cells continuously (as in epithelial

cells) or are arranged in a more discontinuous fashion (as in fibroblasts). In addition to promoting intercellular adhesion, adherens junctions promote the establishment of

apical‐basal polarity by providing a loose physical barrier and are required for the

formation of tight junctions (Vleminckx and Kemler, 1999).

1.1.1 Adherens junctions during development

Intercellular adhesion relies on the function and interaction of many cell

adhesion molecules, including classical cadherins, integrins, immunoglobulin‐type

adhesion molecules (Ig‐CAMs), and distinct pairs of membrane‐bound ligand/receptors

such as ephrins/Eph and semaphorins/plexins (Comoglio et al., 2004; Thiery, 2002;

Vleminckx and Kemler, 1999). The engagement of adhesion receptors leads to activation

of downstream signaling pathways resulting in dynamic remodeling of the cytoskeleton,

which is critical for their adhesive properties. Dynamic regulation of the actin

cytoskeleton is required for the formation and dissolution of intercellular adhesions

during tissue morphogenesis, cell migration, differentiation during gastrulation,

synaptogenesis, wound healing, and pathological processes such as tumor invasion and

metastasis (Thiery, 2002; Vasioukhin and Fuchs, 2001). Formation of cell‐cell contacts in

multicellular organisms is critically dependent on adherens junctions (Schock and

Perrimon, 2002). Impaired formation of adherens junctions is associated with developmental defects associated with loss of adhesion (Cavallaro and Christofori, 2004;

Thiery, 2002).

Adherens junctions are specialized structures formed during epithelial

morphogenesis which physically link the actin cytoskeletons of neighboring cells

through cadherin molecules, which bind to one another in a calcium‐dependent manner

(Schock and Perrimon, 2002) (Figure 1). The extracellular domains of the cadherins

dimerize and cluster, which triggers the indirect association of cadherin cytoplasmic

tails with the actin cytoskeleton. This indirect link is mediated by the binding of the

cadherin tail to β‐catenin, which in turn binds α‐catenin, which ultimately links the

cadherin/catenin complex to the actin cytoskeleton (Kobielak and Fuchs, 2004).

Epithelial (E)‐cadherin and the catenins play a critical role in promoting normal

epithelial morphogenesis. Their importance is demonstrated by the early embryonic

lethality of mice that carry null mutations of the corresponding genes (Jamora and

Fuchs, 2002). E‐cadherin expression is important for maintaining epithelial morphology,

α α - catenin β β - catenin

P120 P120 catenin catenin

Cadherin

P120 cat. β β - cat. α α - cat.

F Actin

Figure 1: Schematic representation of adherens junctions.

and its loss is implicated in the epithelial‐mesenchymal transitions (EMT) that play a

critical role during both normal embryonic development and tumor progression.

Deletion of E‐cadherin in murine embryonic stem cells inhibits cell aggregation and

induces lethality at the blastocyst stage (Larue et al., 1996). Homozygous mutant E‐

cadherin embryos fail to form epithelial trophectoderm, which is the first sign of tissue

differentiation in embryonic development (Ohsugi et al., 1997). This phenotype was not

rescued by expression of another cadherin family member, N‐cadherin, suggesting that

specific cadherins specify tissue type during development (Larue et al., 1996).

Conditional gene targeting of E‐cadherin in the developing mouse epidermis results in

perinatal lethality due to lack of a functional epidermal water barrier and loss of tight

junctions (Tunggal et al., 2005). Likewise, conditional genetic ablation of α‐catenin in the

mouse skin shows that loss of α‐catenin results in impaired adherens junction formation

in vivo (Vasioukhin et al., 2001). p120 ablation in the mouse embryonic salivary gland led

to reduced levels of E‐cadherin, as well as severe defects in adhesion, cell polarity, and

acinar development, which ultimately resulted in formation of intraepithelial neoplasia

(Davis and Reynolds, 2006).

In addition to directly affecting cellular morphogenesis and motility, intercellular

adhesion can also affect cell proliferation and apoptosis, which may regulate tissue

formation and remodeling during development (Vleminckx and Kemler, 1999). The roles

of a few cell adhesion molecules during mammalian development have been identified

by targeted gene inactivation in select tissues. Targeted inactivation of E‐cadherin in the

lactating murine mammary gland prevents terminal differentiation and induces

pervasive apoptosis (Boussadia et al., 2002). Conditional inactivation of the E‐cadherin

gene in thyroid follicular cells, resulted in impaired development of the thyroid gland,

however no loss of intercellular adhesion was observed, possibly due to compensation

from other cadherins (Cali et al., 2007). Catenins also affect functions other than

adhesion as targeted inactivation of α‐catenin in the skin epithelium of mice results in

hyperproliferation (Vasioukhin et al., 2001). Thus, adherens junction proteins are

required for multiple cellular processes, including intercellular adhesion, proliferation,

and survival.

1.1.2 Adherens junctions in cancer

Rarely do patients with solid, epithelial‐derived tumors die from the primary

tumor; rather, they die from a metastatic lesion. Just as epithelial tissues require

adherens junctions to maintain intercellular adhesion, so do tumors. Loss of intercellular

adhesion allows tumor cells to disseminate from the primary lesion, escape into the

bloodstream, and begin to grow at distal sites, thus initiating a metastatic growth.

Therefore, targeting the adherens junctions either by preventing dissolution in the

primary tumor or preventing adherens junction formation at the distal sites represents

an attractive therapeutic intervention to prevent death from cancer.

Well‐differentiated, minimally invasive tumors retain strong E‐cadherin

expression, however undifferentiated, invasive cancers with reduced intercellular

adhesion often show reduced expression of E‐cadherin (Hirohashi and Kanai, 2003). The

physiological consequence of E‐cadherin loss is that cells undergo an EMT resulting in a

change from uniform, compact organized cells to amorphous cells that spread out and

show reduced contact with one another (Birchmeier et al., 1996). Reduced E‐cadherin

expression has been linked to increased tumor grade and increased patient mortality

(Cavallaro and Christofori, 2004). E‐cadherin loss in tumors occurs through several

mechanisms including upregulation of transcription repressors, hypermethylation of the

cadherin promoter, mutations in the cadherin gene, signaling from oncoproteins,

proteolytic degradation by matrix metalloproteases (MMPs), and upregulation of

dysadherin, which has been shown to downregulate E‐cadherin (Cavallaro and

Christofori, 2004; Hirohashi and Kanai, 2003; Huber et al., 2005). The increased tyrosine

phosphorylaton of adherens junction components resulting from enhanced signaling

from oncoproteins, such as v‐Src, or increased growth factor receptor signaling will be

discussed in more detail later. β‐catenin is also frequently mutated in cancers, and may

play a role in enhanced proliferation through regulation of the Wnt pathway (Miyoshi

and Hennighausen, 2002). Reduced expression of E‐cadherin and α‐catenin have also

been observed in feline mammary tumors (Takauji et al., 2007). Likewise, reduced levels

of E‐cadherin and disruption of β‐catenin have been found in canine mammary tumors

(Restucci et al., 2007). The multiple levels at which cadherin‐mediated adhesion is

regulated and the frequency with which adhesion is disrupted in cancer demonstrate its

importance for tumor progression.

Cadherin switching during the course of tumor progression is a phenomenon

that has received increased attention. In several epithelial cancers, including breast and

prostate cancer, as well as melanoma, N‐cadherin expression has been found to be

upregulated (Hazan et al., 2004). Increased expression of N‐cadherin has been shown to

promote morphological changes suggestive of EMT as well as increasing motility and

invasiveness of breast cancer cells (Cowin et al., 2005). In addition to promoting cell

motility, the cadherin switch may program cancer cells to home to a new site where they

can adhere in an N‐cadherin dependent manner (Hazan et al., 2004). In the case of breast

cancer, this could explain the high incidence of metastases to bone and brain. In axon

guidance, the different cadherin types are thought to specify targets, e.g. N‐cadherin

expressing neurons are attracted to N‐cadherin, but repelled from E‐cadherin (Thiery,

2003). Because of its correlation to increased invasion and metastasis, N‐cadherin has

become an attractive therapeutic target and at least one peptide‐based inhibitor has

entered clinical testing (Mariotti et al., 2007) .

1.1.3 Adherens junction components

1.1.3.1 Cadherin family proteins

The classical cadherins consist of a family of single pass trans‐membrane

glycoproteins, which mediate calcium‐dependent homotypic interactions with cadherin

molecules on adjacent cells (Takeichi, 1994; Takeichi, 1995). Classical cadherins

participate in adherens junction complexes in multiple tissues, and are named after the

tissues in which they are most abundant. The most commonly studied mammalian

members include epithelial (E‐), neuronal (N‐), placental (P‐), and vascular endothelial

(VE‐), however multiple cadherins may be expressed in the same tissue. For example,

both N‐ and VE‐cadherin are expressed in endothelial cells and may act in concert to

modulate intercellular adhesion (Luo and Radice, 2005). Extracellular calcium is thought

to bind to the multiple extracellular cadherin‐motif subdomains, and stimulates cis

dimerization or cadherin clustering (Steinberg and McNutt, 1999). Highly conserved

HAV regions at the outermost cadherin‐motif (C1) are responsible for the trans‐

dimerization between cadherin molecules on neighboring cells (Koch et al., 1999; Pertz

et al., 1999). The strengthening of intercellular adhesions relies on both cis‐ and trans‐

interactions. Transcriptional control of E‐cadherin occurs through the binding of

transcriptional factors to the E‐box of the E‐cadherin promoter (Perez‐Moreno et al.,

2003). The transcription factors Snail and Slug bind to the E‐box to repress cadherin

promoter activation, are critical for tissue morphogenesis during development, and

promote EMT during tumor progression and metastasis (Perez‐Moreno et al., 2003).

Regulation of cadherin at the protein level occurs through several mechanisms, which

will be described in more detail below.

1.1.3.2 Catenin family proteins

The mammalian catenin family of proteins was initially identified nearly 20 years

ago and discovered in a complex with a cadherin family member (Ozawa et al., 1989).

Catenins were thought to provide a link from the cadherin cytoplasmic tail to the actin

cytoskeleton, and their name comes from the Latin catena for chain (Ozawa et al., 1989).

The family includes of β-catenin and γ‐catenin (plakoglobin), p120 catenin and δ‐

catenin, and α‐catenin. All catenin family members, with the exception of α‐catenin

contain armadillo repeats, so‐named for their homology to the Drosophila β‐catenin‐like

armadillo protein.

β‐catenin and plakoglobin bind directly to the catenin‐binding domain, a 30

amino acid sequence at the cytoplasmic tail of cadherin molecules and as such are

integral chains in the link to the cytoskeleton (Brunton et al., 2004). In addition to its role

in intercellular adhesion, β‐catenin participates in a number of signaling cascades, most

notably Wnt signaling, and is found in complexes in the cytoplasm and nucleus (Perez‐

Moreno and Fuchs, 2006). α‐catenin is linked indirectly to the cadherin complex by

binding β‐catenin. α‐catenin provides the link to the actin cytoskeleton and was initially assumed to do so through direct binding to actin or through intermediate actin binding

proteins like α‐actinin, formins, vinculin and others (Kobielak and Fuchs, 2004). While

α‐catenin may regulate the actin cytoskeleton by binding to intermediates, it may also

directly regulate actin‐filament organization by competing with the Arp2/3 complex for

binding to actin filaments, thereby suppressing Arp2/3‐mediated actin polymerization

(Drees et al., 2005). p120 catenin binds directly to the juxtamembrane region of cadherin,

and was initially identified as a protein tyrosine phosphorylated by Src kinase, and later

identified as a member of the catenin family (Brunton et al., 2004). Subsequently, p120

catenin has been identified as a “master regulator” of adherens junctions through

modulating the trafficking of cadherins (Reynolds, 2007). In addition to interacting with

the cadherin‐catenin adhesion complex, p120 catenin has been shown to bind to or affect

signaling downstream of numerous regulators of the actin cytoskeleton (Anastasiadis,

2007). While the catenins are required for intercellular adhesion through their

participation in the cadherin‐catenin adhesion complex, there is clearly a need for

further study of the indirect role these proteins may play through other signaling

cascades, which impinge on adherens junction regulation.

1.1.3.3 Actin cytoskeleton

The actin cytoskeleton consists of many actin filaments of various lengths and

acts to modulate the shape and motility of cells. It is highly dynamic and polymerization of actin monomers into filaments, followed by their cross‐linking into a 3‐D meshwork is

stimulated by a diverse set of physiological stimuli including stimulation by growth

factors and adhesion molecules. Several protein families have been identified which

directly modulate the polymerization and cross‐linking of actin, includingcomponents

of the Arp 2/3 complex and formin family proteins.

Cytoskeletal reorganization plays a critical role at each stage of adherens junction

formation. Both classes of actin nucleator proteins, the Arp2/3 complex and the formins

participate in de novo actin polymerization at various stages of adherens junction

formation (Kobielak et al., 2004; Kovacs et al., 2002; Verma et al., 2004). Early formation

of junctions is driven by the production of branched actin networks responsible for

generating lamellipodial protrusions as a result of actin nucleation through Arp2/3

(Kobielak and Fuchs, 2004; Welch and Mullins, 2002). In contrast the formin family

proteins nucleate unbranched actin filaments and are involved in the formation of the

linear actin cables that radiate from early cadherin/catenin‐containing puncta (Kobielak

et al., 2004; Wallar and Alberts, 2003). A recent report suggests that the mammalian

diaphanous‐related formin Dia1, a target of RhoA, is required for the reinforcement of

adherens junctions (Carramusa et al., 2007).

Actin polymerization provides the mechanical force which drives the formation

of adherens junctions (Vasioukhin et al., 2000). The initial assembly of cadherin‐

dependent intercellular adhesions is stimulated by lamellipodial or filopodial membrane

protrusions in adjacent cells (Ehrlich et al., 2002; Gavard et al., 2003; Kovacs et al., 2002;

Vasioukhin et al., 2000). The engagement of cadherin molecules on neighboring cells,

anchored to tips of lamellipodia and/or filopodia through the adhesion complex results

in clusters of puncta or spot adherens junctions containing cadherins/catenins and other

cytoskeletal proteins at sites of cell‐cell contact. Actin polymerization is stimulated at

these early adherens junctions resulting in the formation of linear, radial actin cables

which tether the adhesion complex at early junctions to the underlying cortical actin

ring. The rate‐limiting step in the early stage of junction formation is, in fact, tethering

the cadherin/catenin complex to the cortical actin cytoskeleton (Kobielak and Fuchs,

2004). Formation of these protrusive membrane structures requires the activity of Rho

family GTPases, Rac and Cdc42, (described in more detail later) which stimulate

formation of lamellipodia and filopodia, respectively (Etienne‐Manneville and Hall,

2002). Lamellipodia which form at puncta where cadherin molecules are engaged at

nascent cell adhesions are more persistent than at non‐contacting sites and also act to

reinforce cadherin clustering (Ehrlich et al., 2002). The persistence of lamellipodia

provides the force to close the “adhesion zipper,” resulting in a continuous line of

cadherin/catenin complexes that is accompanied by reorganization of the actin cytoskeleton from a perpendicular to a lateral orientation parallel to the sealed

membranes (Brunton et al., 2004). In addition to regulating adherens junction

formation, Arp 2/3‐dependent actin polymerization is also required for junction

disassembly (Ivanov et al., 2005; Vaezi et al., 2002; Vasioukhin et al., 2000). In this

regard, actin polymerization was shown to be required for dissolution of junctions in

response to removal of extracellular calcium (Ivanov et al., 2005). The actin cytoskeleton

also acts in concert with and regulates acto‐myosin contractility, microtubules and cell

tension during adherens junction formation and dissolution (Mege et al., 2006).

1.1.4 Regulation of adherens junctions

The formation and maintenance of adherens junction require localization of

members of the cell adhesion complex to the plasma membrane and binding of the

cadherins/catenins to one another. While there are many unknown factors regarding the

mechanisms through which localization and junctional integrity are regulated, several

levels of regulation have been revealed. The members of the cadherin‐catenin complex

regulate the localization of their binding partners; tyrosine phosphorylation of adherens

junction components regulates their localization and interactions; and the family of Rho

GTPases play a critical role in regulating intercellular adhesion.

1.1.4.1 Adherens junction component binding

The individual members of the adherens junction protein complex all play a role

in regulating the localization of the others. Cadherin localization to the plasma

membrane is required for localization of the catenins, and cadherin mutants lacking the

β‐catenin binding site fail to promote intercellular adhesion (Nagafuchi and Takeichi,

1988). β‐catenin binding to cadherins is required for adherens junction formation to

establish the link to α‐catenin. In this regard, the requirement for β‐catenin can be

overcome by expression of a chimeric cadherin molecule fused to α‐catenin (Imamura et

al., 1999; Ozawa, 1998). α‐catenin was traditionally believed to support intercellular

adhesion by providing the link from the cadherin‐catenin complex either directly to the

actin cytoskeleton or through intermediate actin‐binding proteins such as α‐actinin, vinculin, and ZO‐1 (Bershadsky, 2004). However, this view has been challenged by the

discovery that α‐catenin fails to bind actin while participating in a cadherin‐catenin

complex (Drees et al., 2005).

p120 catenin is essential for localization of cadherins and catenins to sites of cell‐

cell contact and acts to regulate cadherin trafficking (Xiao et al., 2007). Following

stimulation of adherens junction assembly, p120 catenin associates with kinesin and

essentially carries E‐cadherin to the plasma membrane along microtubules (Chen et al.,

2003). In addition to facilitating adherens junction formation, p120 catenin is required

for junctional stability. In all cell types examined, knockdown of p120 catenin led to

decreased levels of cadherins, including E‐, N‐, P‐, and VE‐cadherin and resulted in loss

of intercellular adhesion (Davis et al., 2003). In endothelial cells, p120 catenin binding to

the cytoplasmic tail of VE‐cadherin is sufficient to prevent internalization by clathrin‐

mediated endocytosis (Xiao et al., 2005). Conversely, selective uncoupling of p120

catenin from E‐cadherin increased the level of clathrin‐mediated endocytosis (Miyashita

and Ozawa, 2007). However, E‐cadherin expression in cadherin‐deficient cells is

sufficient to target p120 catenin to sites of cell‐cell contact, suggesting that the

localization of E‐cadherin and p120 catenin to the plasma membrane is interdependent

(Thoreson et al., 2000). Therefore, altered regulation of any individual member of the

cadherin‐catenin complex may affect the others in various cell types and tissues.

1.1.4.2 Phosphorylation

Tyrosine phosphorylation of adherens junction components plays a critical role

in the regulation of junction formation and stability. A wealth of data suggest that

phosphorylation of catenins downstream of activation of growth factors promotes the

dissolution of adherens junctions, while interactions with various protein tyrosine

phosphatases (PTPs) promote intercellular adhesion (Brunton et al., 2004; Steinberg and

McNutt, 1999). It is unknown whether catenins are direct substrates of tyrosine kinase

growth factor receptors or are phosphorylated by downstream activated nonreceptor

tyrosine kinases. Src is activated downstream of several growth factors, and cells

transformed with v‐Src exhibit elevated levels of β‐catenin tyrosine phosphorylation, for

example (Behrens et al., 1993; Esser et al., 1998). Tyrosine phosphorylation may not

necessarily be causatively linked to disruption of adherens junctions. For example, low‐

level Src tyrosine kinase activity is required for the formation of adherens junctions in

keratinocytes (Calautti et al., 1998). Likewise, tyrosine phosphorylation at sites of cell‐ cell contact is transiently increased during adherens junction formation in endothelial

cells, but is relatively low in confluent cells with mature junctions (Lampugnani et al.,

1997).

Cadherins (E‐, N‐, and VE‐), β‐catenin, and p120 catenin are all targets of

tyrosine phosphorylation (Brunton et al., 2004; Esser et al., 1998; Fujita et al., 2002; Siu et

al., 2007). Expression of v‐Src promotes the tyrosine phosphorylation of E‐cadherin

leading to decreased cell adhesion (Fujita et al., 2002). When phosphorylated, E‐cadherin

recruits Hakai, an E3 ubiquitin ligase similar to Cbl, which targets E‐cadherin for

ubiquitination and endocytosis (Fujita et al., 2002). Tyrosine phosphorylation of VE‐

cadherin appears to promote maintenance of a mesenchymal cell morphology by

inhibiting binding of β‐catenin and p120 catenin (Potter et al., 2005). The functional

consequence of phosphorylation of other cadherins remains an area of exploration.

β‐catenin contains several tyrosine residues, which are known to be

phosphorylated and lead to impaired intercellular adhesion. Abl‐mediated

phosphorylation of tyrosine 489 has been proposed to disrupt binding to N‐cadherin

(Rhee et al., 2007). Tyrosine phosphorylation of β‐catenin on residues 654 and 142

disrupts its association with E‐cadherin and α‐catenin, respectively (Brembeck et al.,

2004; Piedra et al., 2003; Roura et al., 1999). Src kinases have been shown to directly

phosphorylate tyrosine 654 however they do so inefficiently, suggesting that another

kinase downstream of Src family kinases may phosphorylate this site (Piedra et al.,

2003). In this regard, Fyn‐deficient keratinocytes exhibit decreased tyrosine

phosphorylation of both β‐catenin and p120 catenin (Calautti et al., 1998). p120 catenin

may function as a docking protein to recruit Fer, Fyn and Yes tyrosine kinases to the

cadherin complex, which results in the phosphorylation of tyrosine 142 on β‐catenin

(Piedra et al., 2003). Subsequently, an as yet unidentified tyrosine kinase (possibly Src,

Fyn, or Abl) would then be recruited to the complex and phosphorylate β‐catenin on tyrosine 654, thereby disrupting its association with E‐cadherin (Piedra et al., 2003). Fer

also serves to promote adhesion by phosphorylating PTP1B, which dephosphorylates

tyrosine 654 (Xu et al., 2004).

The relative importance of tyrosine phosphorylation of p120 catenin is unclear.

Tyrosine phosphorylation of p120 catenin is thought to be dispensable for its role in promoting adherens junction formation, however a recent report suggests that E‐ cadherin targeted to the plasma membrane induces serine/threonine phosphorylation of p120 catenin (Fukumoto et al., 2007; Mariner et al., 2004). In addition to its direct interaction with the cadherin‐catenin complex, p120 catenin may regulate intercellular adhesion through its interaction with RhoA. In this regard, phosphorylation at tyrosine

112 by Fyn was shown to inhibit association of p120 catenin with Rho, whereas phosphorylation at tyrosines 217 and 228 by Src promoted the p120 catenin‐Rho interaction (Castano et al., 2007). Future studies will undoubtedly reveal new insights into the positive and negative roles played by the tyrosine phosphorylation of adherens junction components.

1.1.4.3 Rho GTPases

Members of the family of RhoGTPases play a critical role in regulating the actin cytoskeleton, gene transcription, cell cycle progression, and membrane trafficking, and deregulation of RhoGTPase activity may contribute to tumorigenesis (Hall, 1998; Jaffe and Hall, 2002). The most well‐characterized family members are the small GTP‐binding proteins Rac1, Cdc42, and RhoA, although over 20 mammalian members have been identified. The phenotype of overexpression of activated forms of the Rho GTPases in

fibroblasts describes a general rule for what these proteins do: Rac1 mediates

lamellipodia formation and membrane ruffling, Cdc42 mediates filopodia formation,

and RhoA mediates stress fiber formation (Hall, 1998). RhoGTPases act indirectly

through intermediate regulators, including Pak (downstream of Rac and Cdc42) and

Rho‐activated kinase (ROCK, downstream of Rho) to effect cytoskeletal changes.

Rho GTPase activity is stimulated by extracellular signals transduced through

different classes of surface receptors including receptor tyrosine kinases, G‐protein‐

coupled receptors, cytokine receptors, and adhesion molecules (Kjoller and Hall, 1999).

Rho proteins are in an active conformation when bound to GTP and inactive when

bound to GTP. Rho guanine nucleotide‐exchange factors (Rho GEFs) promote the

activation of Rho GTPases by catalyzing the exchange of GDP for GTP. Over 60

mammalian GEF family members have been described, with varying degrees of

substrate selectivity. In addition to stimulating Rho activity, GEFs act to integrate

signals, serve as signaling scaffolds, and can regulate intermolecular actions between

Rho GTPases and other signaling cascades, e.g. heterotrimeric G proteins (Rossman et

al., 2005).

Rho GTPase‐activating proteins (Rho GAPs) function to activate the intrinsic

GTPase activity of the Rho proteins, thus promoting turnover to the inactive, GDP‐

bound state of the RhoGTPases. To date, over 70 Rho GAP family proteins have been

identified and cellular roles have been described in cell migration, cytokinesis,

angiogenesis, differentiation, endocytosis, and more; however few family members have

been studied in great detail (Tcherkezian and Lamarche‐Vane, 2007). p190 RhoGAP is a

large GAP that acts solely on RhoA. Its activity is regulated by both protein‐protein interactions (binding to p120 Ras GAP) and tyrosine phosphorylation (Src/Arg) (Bradley

et al., 2006; Bryant et al., 1995; Chang et al., 1995). p190 RhoGAP is known to regulate

cytoskeletal changes induced by signals including PDGF and semaphorins, receptor

signaling, integrin‐based adhesion, and intercellular adhesion (Barberis et al., 2005;

Bradley et al., 2006; Noren et al., 2003; Wildenberg et al., 2006).

Rho guanine nucleotide dissociation inhibitors (Rho GDIs) act at a different level

and regulate the cytosol/membrane‐bound state of Rho GTPases. RhoGDIs physically

interact with Rho GTPases to maintain a pool of inactive Rho GDP in the cytoplasm of resting cells by preventing the spontaneous dissociation of GDP from the GTPases

(Olofsson, 1999). In contrast to GEFs and GAPs, the Rho GDI family consists of only 3

family members. However, it is possible that other proteins which bind to Rho family

members could perform the same function. The biological function of GDIs remain

under‐studied, however GDIs have recently been implicated in tumor migration and

invasion as well as in bacterial infection of Clostridium difficile (Dransart et al., 2005). The

dearth of knowledge combined with the abundance of GEFs, GAPs, and GDIs denotes a

long road ahead for those studying the complex regulation of RhoGTPAses.

Rac1, Cdc42, and RhoA have been implicated in regulating the formation,

maintenance and turnover of cadherin‐based cell‐cell adhesion (Fukata and Kaibuchi,

2001). Rac has been shown to both positively and negatively regulate adherens junctions

in various contexts depending on cell type, level of activated Rac, maturity of the

adherens junction, and the type of cadherin (Lozano et al., 2003). Rac is rapidly

activated within minutes following stimulation of cadherin engagement and is

transiently recruited to nascent adherens junctions (Ehrlich et al., 2002). Both Rac and

Cdc42 regulate E‐cadherin trafficking to the membrane in MDCK cells, as expression of

dominant negative Rac and Cdc42 results in E‐cadherin accumulation at a distinct post‐

Golgi step prior to its association with p120 catenin (Wang et al., 2005). Rac and Cdc42

also act to retain E‐cadherin at sites of cell‐cell contact by blocking its endocytosis (Izumi

et al., 2004). Conversely, sustained activation of Rac in keratinocytes was sufficient to

disrupt adherens junctions and resulted in re‐localization of cadherin molecules from

the plasma membrane (Braga et al., 2000). Therefore, the role of Rac in modulating

adherens junctions likely depends on the complex integration of multiple cellular

signals.

Similarly, RhoA and its effector kinase ROCK, have been shown to to both

positively and negatively regulate adherens junctions (Lozano et al., 2003; Sahai and

Marshall, 2002). Confluent monolayers of epithelial cells show decreased levels of Rho

activity, and Rho activity is inhibited following the stimulation of cadherin engagement

(Noren et al., 2001). Cadherin engagement leads to enhanced tyrosine phosphorylation

of p190 RhoGAP, which likely stimulates its GAP activity (Noren et al., 2003). In this

regard, PP2 treatment, which pharmacologically inhibits the activity of both Src and Abl

kinases (Tatton et al., 2003), abolishes the binding of p190 RhoGAP to activated RhoA,

(Noren et al., 2003). Further, inhibition of RhoA activity by cadherin engagement is

blocked by expression of a dominant negative p190 RhoGAP (GAP‐deficient) (Noren et

al., 2003). p190 RhoGAP may act to regulate adherens junctions through interactions

with other molecules. Rac‐induced stimulation of adherens junctions in fibroblasts was

recently shown to result in binding of p190 RhoGAP to p120 catenin and their co‐

localization at sites of cell‐cell contact (Wildenberg et al., 2006). Rac‐induced adherens

junctions failed to form in fibroblasts lacking p190 RhoGAP (Wildenberg et al., 2006).

Similarly, RhoA has been reported to positively regulate adherens junction formation

and stability in keratinocytes (Perez‐Moreno et al., 2003).

In contrast, increased Rho activity may lead to increased cell contractility,

thereby increasing tensile stress at cell‐cell junctions, ultimately resulting in their

destabilization (Paszek et al., 2005). The functional consequence of activating RhoA and

its ability to both positively and negatively regulate cell‐cell adhesion may be explained

by context‐dependent activation of different downstream effectors. Activation of the

Rho effector diaphanous‐related formin Dia1 is required for the stability of adherens

junctions (Sahai and Marshall, 2002). However, activation of the ROCK kinase

downstream of active Rho results in acto‐myosin contractility and disruption of

adherens junctions (Sahai and Marshall, 2002). The consequences of Rho activation on

regulation of cell‐cell adhesion may only be understood in the context of integrating all of the other cellular signals feeding into the Rho pathway. Gaining a better

understanding of how individual Rho GEFs, GAPs, and GDIs regulate Rac, Cdc42, and

Rho control of intercellular adhesion may allow these apparent paradoxes to be

resolved.

1.2 Cross-talk between Adherens junctions and Receptor Tyrosine Kinases

Receptor tyrosine kinases (RTKs) are a special class of growth factor receptors

and have been shown to play a critical role in various cellular processes including cell

proliferation, survival, motility, and directed migration (Schlessinger, 2000). A role for

RTKs in the modulation of adhesion was first discovered in the cross‐talk between

growth factor receptors and integrin adhesion molecules (Brunton et al., 2004). More

recent evidence points to a critical role for RTKs in modulating changes in intercellular

adhesion through interactions with the cadherin‐based adherens junctions (Brunton et

al., 2004). Cross‐talk between RTKs and adherens junctions is particularly critical during

tumor progression, therefore understanding this phenomenon may lead to the discovery of novel therapeutic targets (Cavallaro and Christofori, 2004; Hazan et al., 2004).

The family of RTKs includes receptors which are commonly named for the

specific growth factor ligands to which they respond: epidermal growth factor receptor

(EGFR), platelet‐derived growth factor receptor (PDGFR), vascular endothelial growth

factor receptor (VEGFR), fibroblast growth factor receptor (FGFR). All RTKs possess a

kinase domain, which endows intrinsic protein tyrosine kinase activity, and a number of

tyrosine residues which may be phosphorylated and contribute to regulation

(Schlessinger, 2000). Individual RTKs are expressed preferentially in different cell types

and may act differently when stimulated in one cell type vs. another. However, they

share a common signaling mechanism and often activate a common set of downstream

signaling pathways.

RTKs are activated by ligand‐induced receptor dimerization leading to both

auto‐ and transphorylation of the receptor and resulting in enhanced tyrosine kinase

activity (Schlessinger, 2000). Phosphorylated tyrosines on the receptor cytoplasmic tail

serve as docking sites for the recruitment of additional signaling molecules containing

SH2 (Src homology 2) or PTB (phosphotyrosine binding) domains, and these molecules

are frequently substrates of the receptor (Schlessinger, 2000). Common signaling

modules activated downstream of growth factor receptor activation include the

Ras/MAP Kinase signaling cascade, phosphoinositol metabolism and downstream

signaling through the PI‐3 kinase pathway, and gene transcription through nuclear

translocation of STATs (Schlessinger, 2000). In addition, nonreceptor tyrosine kinases

(NRTKs) which are recruited to activated receptors may phosphorylate the RTK in trans

to alter signaling. In this regard, both Src and Abl are activated by EGFR activation, and

activated Src and Abl have been shown to phosphorylate EGFR (Tanos and Pendergast,

2006). Numerous studies suggest the aberrant cell signaling induced by hyperactivation

of RTKs and their downstream targets promotes tumor progression of many epithelial

cancers through effects on cell proliferation, survival, and motility (Christensen et al.,

2005; Normanno et al., 2006; Shinkaruk et al., 2003). RTKs also promote tumor progression by regulating cadherin‐based adhesion (Brunton et al., 2004; Cavallaro and

Christofori, 2004).

1.2.1 Interactions between adherens junctions and RTKs

Reciprocal signaling between adherens junctions and RTKs remains poorly

understood; however, three themes of cross‐talk have emerged. RTK signaling is

affected by the presence and strength of intercellular adhesion; RTKs regulate the

localization of adherens junctions components; and RTKs modulate the tyrosine

phosphorylation of adherens junctions components (Comoglio et al., 2003).

In a limited number of cases, cadherins have been shown to regulate signaling by

growth factor receptors. Stimulation of FGFR1 with FGF results in the cadherin‐

dependent co‐endocytosis of E‐cadherin and FGFR1, followed by nuclear translocation

of FGFR1 (Bryant and Stow, 2004). The effect of FGF is overridden with overexpression

of E‐cadherin or p120 catenin, which stabilizes protein levels of E‐cadherin (Bryant and

Stow, 2004). Conversely, stimulation of cadherin‐based adhesion can lead to association

of EGFR with E‐cadherin, which promotes the autophosphorylation of EGFR (Pece and

Gutkind, 2000). In this regard, cadherin engagement is required for signaling from the

EGFR through stimulation of both the MAPK cascade and Rac activation (Betson et al.,

2002; Pece and Gutkind, 2000). The effects of cadherin engagement on EGFR appear to

be dependent on cell density as activation of EGFR and EGFR‐dependent proliferation

are inhibited in cells with mature adherens junctions (Curto et al., 2007). However, cells

lacking NF2, which mediates cross‐talk between the EGFR and adherens junctions,

display persistent EGFR activation and fail to undergo growth arrest at high cell density

(Curto et al., 2007). This paradox whereby cadherin both promotes and arrests EGFR‐

mediated cell proliferation is a common theme in the cross‐talk between RTKs and

adherens junctions and points to the complexity of understanding interactions between

different signaling molecules.

There are numerous examples in which RTKs have been shown to regulate

adherens junctions through direct physical interactions with or indirect regulation of

cadherin molecules. For example, EGF stimulation of the EGFR has been shown to

increase endocytosis of E‐cadherin through caveolin, and chronic EGF stimulation can

decrease E‐cadherin expression, thereby increasing the invasive potential of tumor cells

(Lu et al., 2003). Likewise, both FGF1 and FGF2 stimulation result in E‐cadherin

internalization and re‐localization of β‐catenin from the cell membrane to the cytoplasm

and nucleus in NBT‐II cells (Billottet et al., 2004). Loss of E‐cadherin based cell‐cell

contacts in epithelial cells over the course of tumorigenesis leads to both morphological

changes and increased invasive and metastatic potential of the cells (Cavallaro and

Christofori, 2004).

Cadherin‐switching is a well‐documented process in various tumors of epithelial

origin where mesenchymal cadherins, especially N‐cadherin, are spontaneously

expressed following the loss of E‐cadherin (Bonitsis et al., 2006; Cavallaro et al., 2002;

Hazan et al., 2004). Cancer cells expressing N‐cadherin tend to be more invasive, but the

molecular mechanisms underlying this switch and its effects have yet to be defined.

Evidence from some epithelial cell lines suggests that N‐cadherin physically interacts

with FGFR to enhance FGF2 stimulated signaling and invasion (Suyama et al., 2002).

Additionally, a soluble fragment of N‐cadherin has been shown to promote angiogenesis

in an N‐cadherin‐ and FGFR‐dependent manner in endothelial cells (Derycke et al.,

2006). In neuronal cells, N‐cadherin has been shown to affect axon guidance through the

formation of an intermolecular complex with the Robo receptor, mediated by Abl (Rhee

et al., 2007; Rhee et al., 2002). Recently, Robo and its ligand Slit have been shown to be

upregulated in some breast and prostate cancers (Dallol et al., 2002; Latil et al., 2003). In

the brain, Slit mediates repulsion, whereas in breast cancer, it has been shown to

promote chemotaxis (McAllister, 2002; Prasad et al., 2004). The interplay between FGFR

and Robo with N‐cadherin warrants further study as both FGF and Slit have been

implicated in tumor angiogenesis (Fujiwara et al., 2006; Presta et al., 2005).

Activation of growth factor receptors can lead to tyrosine phosphorylation of

adherens junctions components. In endothelial cells, tyrosine phosphorylation of

adherens junction components, including VE‐cadherin, β‐catenin and p120 catenin, is

dynamically regulated during the formation and maturation of cell‐cell contacts (Esser et al., 1998; Lampugnani et al., 1997). VEGF stimulation leads to enhanced tyrosine

phosphorylation at endothelial cell‐cell contacts concomitant with increased tyrosine

phosphorylation of adherens junction components, including VE‐cadherin, β‐catenin,

and p120 catenin but not α‐catenin (Esser et al., 1998). This phosphorylation may be

important for VEGF‐induced cell permeability by promoting the dissolution of cell‐cell contacts. Similarly, activation of the Roundabout (Robo) receptor following binding of

its ligand Slit modulates adhesion by promoting tyrosine phosphorylation of β‐catenin

resulting in dissociation of β‐catenin from N‐cadherin (Rhee et al., 2002). In this context,

the enhanced phosphorylation of β‐catenin on tyrosine 489 has been reported to be

dependent on Abl kinase (Rhee et al., 2007). Tyrosine phosphorylation of β‐catenin is

enhanced following growth factor stimulation with EGF and IGF‐1, and p120 catenin is

phosphorylated in response to EGF and PDGF (Brunton et al., 2004). It is unclear

whether cadherins and catenins are direct substrates of the RTKs or whether they are

phosphorylated through other tyrosine kinases, such as Src or Abl.

1.2.2 Met receptor tyrosine kinase

1.2.2.1 Cancer relevance

Dynamic remodeling of cell‐cell adhesions plays a critical role in normal

development and is also required for tumor invasion and metastasis (Thiery, 2002).

Hepatocyte growth factor (HGF), also known as scatter factor (SF), was first

characterized as a physiological factor promoting cell scattering through the remodeling

of adherens junctions (Birchmeier et al., 2003; Stoker et al., 1987). Subsequently, HGF has

been identified as a critical mediator of tumor progression in various epithelial‐derived

tumors (Hurle et al., 2005; Jiang et al., 2005) and has been implicated in tumor

angiogenesis (Rosen et al., 1997). The receptor for HGF, Met, is upregulated in human

breast and prostate cancer patients and is known to promote metastasis (Birchmeier et

al., 2003; Knudsen and Edlund, 2004). Met is also upregulated in canine prostate cancer,

and the Met family member Stk, a homologue of RON, is upregulated in feline breast

carcinoma (De Maria et al., 2002; Liao et al., 2005). Met may also play a role in tumor

progression through upregulation of its activity. Recently, a somatic mutation was

identified in lung cancer patients, which leads to prolonged Met signaling (Kong‐Beltran

et al., 2006). HGF‐Met signaling has received increasing attention for its role in tumor

angiogenesis suggesting that Met may play a role in cancers in which it has not yet been

implicated (Lesko and Majka, 2008). The fact that increased Met activity and

upregulation of Met and its family members in spontaneous cancers in various species

have been observed underscores its importance as a potential therapeutic target.

1.2.2.2 Biological effects of Met activation

Acquisition of an invasive phenotype is a critical step in metastatic progression.

HGF acts at several levels to promote epithelial‐mesenchymal transition (EMT) by

stimulating breakdown of cell‐cell junctions and increasing cell motility and

invasiveness (Thiery, 2002). As the first phenotype attributed to HGF stimulation, cell

scattering is fairly well understood, however there are likely to be unidentified

components to the pathway, particularly linking Met activation to regulation of the

family of RhoGTPAses. Cell scattering in MDCK cells occurs in a step‐wise manner

following HGF stimulation. Within minutes after the Met receptor is activated, cortical

actin begins to break down and cells form lamellipodia and spread. At this point,

redistribution of β‐catenin to the cytoplasm from sites of cell‐cell contact is initiated and

the cells begin to detach from one another. On the order of hours following Met

stimulation, cells form actin stress fibers and begin to acquire a mesenchymal

morphology and become more motile. In addition to its well‐characterized role in cell

motility, migration, and invasion, HGF stimulation of the Met receptor also leads to

increased cell proliferation. However, a recent report suggests that the downstream

signals elicited by Met activation may differentiate between the two phenotypes:

proliferation is regulated by activation of the c‐myc pathway, whereas invasion is

mediated through activation of the MAPK pathway (Gao et al., 2005).

In a more physiologically relevant context, HGF can induce the formation of

branched tubules when epithelial cells are cultured within a three‐dimensional collagen

matrix in vitro (Rosario and Birchmeier, 2003; Stella and Comoglio, 1999). Initially,

individual cells seeded in a collagen matrix proliferate and form hollowed out spherical

cysts. When these cysts are stimulated with HGF, they form tubules in a step‐wise

manner (Pollack et al., 1997). First, individual cells undergo partial EMT and extend cell

processes. This is followed by the extension of cell chains from the cyst. The cells in the

chain then elongate and form cords and begin the process of redifferentiation in which

the cells reorganize into a hollowed‐out tube, projecting out of the cyst. In this regard,

prolonged stimulation with HGF leads to the step‐wise activation of signaling cascades,

which are critical at different stages of the process (OʹBrien et al., 2004). During the

initial phases of tube sprouting, cells undergo a partial EMT which is dependent on Erk

activation; during redifferentiation, MMP activation is required, however Erk activity is

maintained but is not required for this process (OʹBrien et al., 2004). An in vivo role for

HGF was demonstrated in tubule formation during liver and kidney regeneration, and

mammary gland formation (Rosario and Birchmeier, 2003).

1.2.2.3 Met signaling

A critical distinction between Met and other growth factor receptors is the

distinct program of invasive growth elicited downstream of its stimulation. This unique

activity may be due to the prolonged activation of signaling molecules such as Erk by

HGF compared to the more transient activation induced by growth factors such as EGF

(Comoglio and Trusolino, 2002). Additionally, the diversity in number and combination

of signaling molecules recruited directly to the Met receptor or indirectly through other

proteins may allow HGF stimulation to produce distinct biological responses in different

cellular contexts.

HGF binding to the Met receptor tyrosine kinase induces dimerization and

autophosphorylation of the receptor. Phosphorylation of tyrosine residues 1234 and

1235 in the catalytic domain are critical for Met activation (Longati et al., 1994; Rodrigues

and Park, 1994). Other phosphorylated tyrosine residues, including tyrosines 1349 and

1356, serve as docking sites for multiple SH2‐domain containing signaling molecules

which are recruited to the Met receptor (Ponzetto et al., 1994). Signaling molecules

recruited to Met include Grb2, PI3‐kinase, Src, PLC‐γ, Cbl, Crk, and Gab1. Gab1 is a

large adaptor molecule which is tyrosine phosphorylated and recruited to the Met receptor following Met activation (Weidner et al., 1996). Gab1 likewise contains a

number of tyrosine residues which, when phosphorylated, serve as docking sites for the

recruitment of additional signaling molecules and is required for Met signaling (Bardelli

et al., 1997; Sakkab et al., 2000).

Signal attenuation is less well understood but is mediated in part through Cbl‐

mediated downregulation of the Met receptor (Petrelli et al., 2002). Ubiquitination and

internalization of Met requires Cbl binding through its tyrosine kinase binding domain

to phosphorylated tyrosine 1003 of the Met receptor (Peschard et al., 2004). A ubiquitin‐

deficient Met receptor mutant in which this tyrosine has been mutated to phenylalanine

(Met Y1003F) shows increased receptor stability, prolonged activation of signaling

pathways, and enhanced transformation in response to HGF stimulation (Abella et al.,

2005). The finding that mutations in the Cbl‐binding region of Met have been observed

in multiple tumors supports a critical need for understanding the role of

phosphorylation of Met Y1003 (Peschard and Park, 2003).

Crk is one of several SH2‐domain‐containing adaptor molecules recruited to the

Met receptor by binding Gab1. Both Crk and its family member CrkL have been shown

to bind tyrosine phosphorylated YXXP motifs on Gab1 (Lamorte et al., 2002b; Sakkab et

al., 2000). Crk is required for the morphological changes induced in epithelial cells in

response to HGF treatment, and overexpression of Crk in MDCK cells promotes cell

spreading, induction of lamellipodial protrusions, activation of Rac, and dissolution of

adherens junctions, thus mimicking HGF‐mediated effects (Lamorte et al., 2002b).

Tyrosine phosphorylation of Crk increases in response to HGF treatment, and this

phosphorylation correlates with increased binding of Crk to p130Cas, Cbl, and Gab1

(Lamorte et al., 2002b). Crk was shown to be required for the sustained phosphorylation

of Gab1 at Y307 and sustained Met signaling in response to HGF treatment (Watanabe et

al., 2006). More recent data suggest that Crk participates in a negative regulatory

feedback loop with Met. In this regard, Met signaling induces Abl‐dependent tyrosine

phosphorylation of Crk at Y221, and this phosphorylation is required to attenuate Met

signaling in fibroblasts (Cipres et al., 2007). Thus, Crk appears to be required for

activation of both Met signaling and signal attenuation.

RhoGTPases regulate cytoskeletal rearrangements downstream of signaling by

numerous growth factors, including HGF. Both Rac and Cdc42 are activated and are

required for cell spreading following HGF stimulation of MDCK cells (Ridley et al.,

1995; Royal et al., 2000). Activation of Rac as well as the breakdown of junctions in

MDCK cells following Met activation is inhibited by treatment with a PI‐3 kinase

inhibitor, suggesting a critical role for the PI‐3 kinase pathway in some contexts (Royal

et al., 2000; Royal and Park, 1995). Rac activation is enhanced in cells overexpressing

Crk, and is required for Crk‐induced lamellipodia formation and cell spreading

(Lamorte et al., 2002b; Royal et al., 2000). A recent study suggests that Met

overexpression leads to Rac‐dependent generation of reactive oxygen species (ROS), and

that treatment with ROS scavengers blocks signaling to Erk (Ferraro et al., 2006).

Generation of ROS through Rac may occur through increased levels of of the Nox family

of NAD(P)H oxidases (Paravicini and Touyz, 2008), which has also been shown to

operate downstream of Abl kinase activation (Boureux et al., 2005). In addition, this

novel Met‐ROS pathway is required for the in vivo pro‐metastatic behavior of Met

(Ferraro et al., 2006). Less is known regarding a potential role for Rho signaling

downstream of Met activation, however its downstream target ROCK has been

implicated in one study (Royal et al., 2000). ROCK translocates to membrane ruffles in

response to HGF stimulation in MDCK cells and is required for formation of

lamellipodia, stress fiber formation, and reorganization of focal adhesions (Royal et al.,

2000). However, cell spreading occurs independently of ROCK (Royal et al., 2000). The

question of how signals are transduced from Met to the RhoGTPases remains open.

The mechanism through which Met activation results in adherens junction

dissolution is unclear, however, it may be due in part to regulation of adherens junction

components. HGF stimulation of Met results in increased tyrosine phosphorylation of β‐

catenin (Hiscox and Jiang, 1999; Monga et al., 2002). Tyrosine phosphorylation of β‐

catenin is known to alter its interaction with E‐cadherin and, in some cases, to disrupt

the cadherin‐catenin complex (Lilien and Balsamo, 2005). Further, Met has been shown

to interact physically with both β‐catenin and E‐cadherin (Birchmeier et al., 2003).

Significantly, expression of E‐cadherin in cells lacking E‐cadherin stimulated re‐

localization of the Met receptor from intracellular compartments to the plasma

membrane (Reshetnikova et al., 2007). A role for reciprocal signaling between the Met

receptor and E‐cadherin, particularly during tumor progression, remains unexplored.

1.3 Abl family of non-receptor Tyrosine Kinases

The founding member of the Abl protein family, Abl, was originally identified as

the cellular homolog of the v‐Abl oncogene product of the Abelson murine leukemia

virus (A‐MULV) (Goff et al., 1980; Wang et al., 1984). The Abl family of non‐receptor

tyrosine kinases consists of two mammalian members c‐Abl and Arg, (Goff et al., 1980;

Heisterkamp et al., 1983; Kruh et al., 1990), Drosophila Abl (D‐Abl) (Hoffman et al.,

1983), and C. elegans Abl (Goddard et al., 1986). Abl is perhaps best well‐known for its

role in human leukemias as part of the Philadelphia chromosome resulting from

chromosomal translocation of the two genes Bcr and Abl (Groffen et al., 1984; Melo,

1996). Both v‐Abl and BcrAbl are capable of inducing transformation in various cell

types, and have been shown to affect cell proliferation and survival pathways, as well as

cell adhesion and migration function (Pendergast, 2002). While there have been far

fewer studies describing the normal functions of cellular Abl and Arg, a growing body

of evidence suggests that normal functions also include regulation of cell proliferation,

survival, response to extracellular stresses, cytoskeletal rearrangments, and adhesion in

various tissues (Pendergast, 2002).

1.3.1 Abl and Arg

Abl and Arg show ~90% sequence identity in their SH3, SH2, and kinase

domains suggesting that they likely share binding partners and substrates (Pendergast,

2002) (Figure 2). The SH3 domain recognizes proline‐rich sequences with the consensus

PPXXXPPXXP, where P is proline and X any amino acid (Alexandropoulos et al., 1995;

Cicchetti et al., 1992; Ren et al., 1993; Rickles et al., 1994), while the SH2 domain

recognizes the YXXP sequence where Y is a phosphorylated tyrosine, X any amino acid,

and P proline (Songyang et al., 1993). The kinase domain preferentially phosphorylates

peptides with the consensus sequence I/VYXXP, where I is isoleucine, V valine, Y

tyrosine, X any amino acid, and P proline (Pendergast, 2002). Further highlighting the

conserved evolution of substrate specificity, the kinase domain of Drosophila Abl is

about 77% identical to that of mammalian Abl and Arg (Henkemeyer et al., 1988).

Abl family proteins diverge in the C‐terminal regions. In spite of the diversity,

there are several proline‐rich sequences downstream of the kinase domain which are

highly conserved among Abl and Arg (Pendergast, 2002) . Likewise, both Abl and Arg

contain actin‐binding domains and bundle F‐actin in vitro which may play a role in

modulating cytoskeletal rearrangements (Baum and Perrimon, 2001; Galkin et al., 2005;

Wang et al., 2001; Woodring et al., 2002). More recently, Arg has been shown to bind

microtubules and to play a role in lamellipodial dynamics through regulating cross‐

linking between actin bundles and microtubules (Miller et al., 2004). A key distinction

between Abl and Arg is the presence of three nuclear localization sequences (NLSs) in

Abl (Van Etten et al., 1989; Wen et al., 1996), which are not present in D‐Abl

(Henkemeyer et al., 1988). Abl also contains a functional nuclear export sequence (NES)

(Taagepera et al., 1998), which allows for nuclear‐cytoplasmic shuttling, as well as a

DNA‐binding domain (David‐Cordonnier et al., 1998; Kipreos and Wang, 1992; Miao

and Wang, 1996). This suggests that mammalian Abl has evolved and diverged from

Arg in response to a need for regulating some nuclear function not required in lower

organisms.

NLS Binding sites for:

b form DNA G-actin F-actin c-Abl V SH3 SH2 KINASE

NES a form

SH3 Domain Proline Binding Stretch Sites Binding sites for:

b form F-actin microtubules G-actin F-actin

Arg V SH3 SH2 KINASE

NES a form

32% 90% 94% 29% 56%

Figure 2: Functional domains of c‐Abl and Arg.

Adapted from Pendergast 2002.

Both Abl and Arg are expressed in a wide variety of fetal and adult tissues

(Courtney et al., 2000; Gertler et al., 1993; Koleske et al., 1998; Muller et al., 1982; OʹNeill

et al., 1997; Perego et al., 1991). Arg appears to be more highly enriched in the

mammalian brain and CNS in flies as well as in muscle, thymus, and spleen (Gertler et

al., 1993; Koleske et al., 1998). Abl appears to be preferentially expressed in cartilage,

adipocytes, and ciliated epithelium in adult tissues (OʹNeill et al., 1997). Abl and Arg

also exhibit similarities and differences in subcellular localization (Pendergast, 2002).

Whereas Abl has been shown to localize to the plasma membrane, cytosol, endoplasmic

reticulum, mitochondria, lipid rafts, actin cytoskeleton and the nucleus in response to

different stimuli (Frasca et al., 2001; Ito et al., 2001; Koleske et al., 1998; Lewis et al., 1996;

OʹNeill et al., 1997; Plattner et al., 1999; Van Etten et al., 1989; Westphal et al., 2000;

Wetzler et al., 1993; Zipfel et al., 2000), Arg is primarily observed in the cytoplasm and

cytoskeleton (Koleske et al., 1998; Wang and Kruh, 1996; Wang et al., 2001). More

recently, both exogenous Abl and BcrAbl have been shown to localize to nascent

adherens junctions when expressed in Drosophila (Stevens et al., 2007). The mechanism

by which fractions of the total pool of cellular Abl and Arg are dynamically re‐localized

to different compartments remains unclear, however localization studies often provide

the first clues to discovering novel normal cellular functions for Abl and Arg.

1.3.2 Regulation of Abl/Arg

The Abl kinases have been implicated in a wide variety of cellular processes as

described above. Because the Abl tyrosine kinases can be found throughout the cell, and

one or both are expressed in nearly every cell type, regulation of Abl kinase activity

must be tightly restricted. This regulation occurs at several levels include physical intra‐

and intermolecular interactions and protein modifications including phosphorylation

and ubiquitination, which act to regulate both basal and stimulated Abl kinase activity.

1.3.2.1 Physical

Activation of Abl kinases may occur through the binding of several proteins

involved in various signal cascades (Pendergast, 2002). In the context of Ras activation,

Rin1 was recently shown to bind Abl resulting in increased kinase activity (Hu et al.,

2005). Overexpression of the adaptor proteins Nck and Crk in 293T cells has been shown

to increase Abl kinase activity, and is dependent on the SH3 domains of both proteins,

however the exact mechanism is unknown (Shishido et al., 2001; Smith et al., 1999).

Overexpression of several other Abl‐interacting proteins, including Cbl, c‐Jun, and Abi

family proteins, increases Abl activity through the relief of inhibitory interactions (Dai

and Pendergast, 1995; Shi et al., 1995).

Intra‐ and intermolecular actions also play a key inhibitory role in regulating

cellular Abl kinase activity. In part, Abl is held in the inhibited state by a physical block

of the kinase domain by the SH3‐SH2 domain folding back over it. A recent analysis of

Abl crystal structure in the autoinhibited state revealed that a previously unseen N‐

terminal cap segment adjacent to the SH3‐SH2 domains is required to maintain the link

between SH3‐SH2 and the kinase domain (Nagar et al., 2006). Both deletion and

mutation of the Abl SH3 domain have been shown to stimulate Abl kinase activity in

vivo and result in enhanced tyrosine phosphorylation of Abl (Jackson and Baltimore,

1989). While the relative importance or interdependence of each interaction is unknown,

inhibitory interactions between the SH3 domain and the linker region connecting the

SH2 are important for the negative regulation of Abl kinase activity (Barila and Superti‐

Furga, 1998; Brasher et al., 2001). Additionally, binding of the Abl kinase domain to an

NH2‐terminal myristoyl residue provides an additional structural mechanism for

negative regulation (Nagar et al., 2003).

A number of proteins have been identified that negatively regulate Abl kinase

activity through binding to various domains. Proteins that inhibit Abl kinase activity

through binding to the SH3 domain include Pag/Msp23 and AAP1 (Wen and Van Etten,

1997; Zhu and Shore, 1996). The retinoblastoma protein, Rb, binds to the ATP‐binding lobe of the kinase domain of Abl in the nucleus (Welch and Wang, 1993). F‐actin has

been shown to inhibit Abl kinase activity by binding to the extreme carboxy terminus of

Abl (Woodring et al., 2002). The diversity of known physical methods of regulating Abl

activation and the possible existence of as‐yet unidentified methods highlight the critical

importance of modulating cellular levels of Abl activity.

1.3.2.2 Protein modification

In addition to physical regulation, protein modifications play a key role in

regulating Abl kinase activity. Tyrosine phosphorylation of Abl robustly stimulates its

catalytic activity. For example, Abl activity is increased 10‐ to 20‐fold in cells expressing

activated Src kinases (Plattner et al., 1999). The Src kinases directly phosphorylate Abl at

tyrosine 412, and phosphorylation of this site is required for enhanced Abl kinase

activity in the context of expression of activated Src Y527F (Plattner et al., 1999) Further,

autophosphorylation of Abl results in an 18‐fold increase in activity, which is reduced

by 90% when tyrosine 412 is mutated to phenylalanine (Brasher and Van Etten, 2000).

Mutation of a second tyrosine residue, tyrosine 245, also resulted in a 50% decreased

activation of wild type Abl suggesting this residue may also be important. The crystal

structure of the catalytic domain of c‐Abl in a complex with STI‐571, which is a small

molecule inhibitor of the Abl kinases, also suggests the critical importance of tyrosine

412 (Pendergast, 2002). In complex with STI‐571, tyrosine 412 in the activation loop of

Abl points inward decreasing accessibility to other kinases (Schindler et al., 2000).

Negative regulation of Abl kinase activity following stimulation is critical to

restrict the signal duration and location. Evidence in fibroblasts lacking the PEST‐type

phosphotyrosine phosphatases (PTPases) suggest they act to dephosphorylate Abl

(Cong et al., 2000). Similarly, treatment of unstimulated cells with the protein tyrosine

phosphatase inhibitor orthovanadate results in enhanced tyrosine phosphorylation of

Abl suggesting that tyrosine phosphatases are responsible for maintaining relatively low

levels of endogenous Abl kinase activity (Cong et al., 2000; Dorey et al., 2001; Echarri

and Pendergast, 2001). Phosphorylation at tyrosines 245 and 412 also serves to regulate

protein stability as mutation of these tyrosines to phenylalanine results in increased

protein stability (Echarri and Pendergast, 2001). This effect is likely to be mediated

through ubiquitin‐mediated degradation. Inhibition of the 26s proteasome leads to both

enhanced tyrosine phosphorylation and enhanced protein stability of Abl (Echarri and

Pendergast, 2001). Together, dephosphorylation and ubiquitin‐mediated degradation act

to tightly control spatial‐temporal activation of stimulated and basal Abl kinase activity.

1.3.2.3 Upstream signals regulating Abl tyrosine kinase activity

Abl and Arg are activated by a multitude of extracellular and intracellular

signals and have been shown to participate in signaling cascades involving a number of

cellular processes (Pendergast, 2002) (Figure 3). Analysis of Abl kinase activity during

the progression of cells from quiescence to S phase revealed that the nuclear pool of Abl

is activated during S phase suggesting a role for Abl in cell cycle regulation (Welch and

Wang, 1993). Nuclear Abl is also activated in response to DNA damage (Kharbanda et

al., 1995; Liu et al., 1996) and may play a role in homologous recombination (Li et al.,

2002). Oxidative stress is another potent activator of Abl kinase activity. Treatment of

cells with hydrogen peroxide activates the cytoplasmic pool of Abl (Sun et al., 2000b;

Sun et al., 2000c). This process may be modulated by S‐glutathionylation of cysteine

residues on Abl induced by reactive oxygen species (Leonberg and Chai, 2007). The

functional consequences of Abl activation in this context are not well understood, yet

Abl may act in part by activating glutathione peroxidase 1 to protect cells against

damage induced by oxidative stress (Cao et al., 2003). Generation of reactive oxygen

species cancer by growth factor receptors, cytokines, and other stimuli, including Rac

activation, has been proposed to promote metastasis (Finkel, 2006; Wu, 2006). A

potential role for Abl kinases in this context remains untested.

Extracellular signals transmitted through receptor tyrosine kinases (discussed in

more detail below) and adhesion molecules also activate the Abl kinases (Lewis et al.,

1996; Pendergast, 2002). The Abl kinases are required for a variety of integrin‐dependent

cellular process including dendritic branching in response to laminin‐1 or Semaphorin

7A (Moresco et al., 2005), mammary epithelial cell adhesion (Hu et al., 2005), and

fibroblast spreading (Miller et al., 2004; Woodring et al., 2002; Woodring et al., 2004).

Arg has been shown to regulate adhesion‐dependent tyrosine phosphorylation of

p190RhoGAP in neuronal cells, which is critical for neuronal morphogenesis in the

Activated by: PDGF, EGF, NGF, Agrin TCR activation Bacterial Invasion Src family Oxidative stress DNA Damage Integrins Ub P P Ub Ub Ub c-Abl c-Abl

PIP2 Tyrosine Phosphatases E3

Deactivated by:

Degradation by the 26S proteasome

Figure 3: Regulation of Abl kinase activity.

Adapted from Pendergast 2002.

developing mouse brain (Hernandez et al., 2004). Arg‐dependent phosphorylation of

p190RhoGAP downstream of integrin engagement is required to inhibit Rho activity

locally at the cell periphery in order to allow fibroblasts to initiate cell spreading

(Bradley et al., 2006). However, arg‐/‐ fibroblasts have been shown to migrate more rapidly than fibroblasts due to increased actomyosin contractility (Peacock et al.,

2007).The critical need for spatial and temporal regulation of Abl kinase activity is

highlighted by the recent finding that Abl kinases stimulate Rac‐induced membrane

ruffling in response to adhesion and growth factor signaling, whereas overexpression of

Abl inhibits cell spreading in response to fibronectin (Jin and Wang, 2007; Sini et al.,

2004). Abl also plays a critical role in the regulation of axon guidance antagonizing

repulsive cue signaling by the Roundabout (Robo) receptor (Bashaw et al., 2000) and has

more recently been shown to affect adhesion by modulating cross‐talk between Robo

and N‐cadherin (Rhee et al., 2007; Rhee et al., 2002).

1.3.3 Biological function of Abl/Arg

1.3.3.1 In vivo roles of Abl and Arg

A role for Drosophila Abl (D‐abl) in cytoskeletal regulation was initially

identified in follicular epithelium: both D‐abl mutant flies and flies overexpressing high

levels of D‐abl displayed aberrant epithelial morphology with perturbation of the actin

cytoskeleton (Baum and Perrimon, 2001). D‐abl has also been implicated in the

regulation of adherens junctions. D‐abl mutants show delayed formation of adherens

junctions, decreased accumulation of Armadillo and α‐catenin at sites of cell‐cell contact,

and impaired dorsal closure during development, which is exacerbated by the additional loss of DE‐cadherin (Grevengoed et al., 2001). In this regard, D‐Abl was

shown to act at least in part by negatively regulating the subcellular localization of the

actin regulator Enabled (Ena). Loss of D‐Abl results in ectopic Ena and excessive actin

accumulation at the apical cortex , whereas ectopic expression of Abl or BcrAbl results in

decreased Ena function (Grevengoed et al., 2003; Stevens et al., 2007). Additionally, D‐

Abl mutants display mis‐localization of other known cytoskeletal regulators including

the Arp2/3 complex and the formin Diaphanous, and diaphanous mutations enhance the

Abl mutant phenotype (Grevengoed et al., 2003). Data in mice also support a role for Abl

family kinases in cytoskeletal reorganization. Abl/Arg double null embryos exhibit

delayed closure of the neural tube and defects in the actin cytoskeleton of neurepithelial

cells (Koleske et al., 1998). Abl family kinases are critical for development as mice

deficient in c‐Abl and Arg die by embryonic day 11 (E11) and show pervasive apoptosis

in all tissues (Koleske et al., 1998). In human leukemias, the kinase domain of Abl is

constitutively activated when portions of Abl are fused to Bcr in the Philadelphia

chromosomal translocation (Melo, 1996). Moreover, enhanced Arg expression has been

shown to correlate with metastatic progression in colorectal cancer, and Abl is

upregulated in pancreatic ductal carcinoma (Chen et al., 1999; Crnogorac‐Jurcevic et al.,

2002). Abl kinase activity has also recently been shown to be upregulated in human

breast cancer cell lines (Srinivasan and Plattner, 2006).

1.3.3.2 Abl kinases regulate response to growth factors and cell migration

Abl kinases have been identified as downstream components in several growth

factor receptor pathways, including Platelet‐derived Growth Factor (PDGF) and

Epidermal Growth Factor (EGF) (Plattner et al., 1999). PDGF stimulation results in

transient activation of Abl and Arg, and Abl kinase activity is required for membrane

ruffling and proliferation in response to PDGF (Plattner et al., 1999; Plattner and

Pendergast, 2003). In this case, phosphorylation of Abl occurs indirectly through Src

(Dorey et al., 2001; Plattner et al., 1999). However, whereas Abl is required to induce

chemotactic motility in response to PDGF, re‐expression of Arg fails to rescue the loss of

chemotaxis in Abl‐/‐Arg‐/‐ cells providing evidence of distinct roles for the Abl family

members (Plattner et al., 2004). Tyrosine phosphorylation of cortactin in response to

PDGF stimulation has recently been shown to be Abl‐dependent and represents one

mechanism through which Abl kinases promote the induction of dorsal ruffles (Boyle et

al., 2007). Loss of Abl/Arg also impairs Rac activation in response to growth factors,

which may be due to decreased tyrosine phosphorylation of the RacGEF Sos‐1 (Sini et

al., 2004). Further, Abl has been shown to be required for tyrosine phosphorylation of

WAVE‐3 in response to PDGF stimulation, which acts downstream of Rac to promote

actin nucleation leading to lamellipodial formation (Sossey‐Alaoui et al., 2007). The

defect in cell proliferation in response to PDGF stimulation in cells lacking Abl kinase

activity can also be attributed, at least in part, to inhibition of Rac activation (Boureux et

al., 2005). In this regard, cytoplasmic Abl is required for PDGF‐stimulated induction of

Myc and DNA synthesis downstream of a Rac/JNK and a Rac/Nox pathway (Boureux et

al., 2005). More recently, Abl kinases have been shown to promote invasion in metastatic

breast cancer cells (Srinivasan and Plattner, 2006).

While Abl kinases appear to play a positive role in PDGF‐stimulated cell

migration, other evidence points to negative effects on cell motility (Cipres et al., 2007;

Frasca et al., 2001; Kain and Klemke, 2001; Plattner et al., 2004). Abl‐/‐Arg‐/‐ MEFs

exhibit increased migration, which coincides with decreased tyrosine phosphorylation of

Crk at tyrosine 221 and increased Crk/CAS complex formation (Kain and Klemke, 2001).

Crk/CAS complex formation has been show to stimulate cell migration (Klemke et al.,

1998). HGF stimulation was shown to activate Abl kinase activity in thyroid cancer cells,

and treatment with STI571 promoted cell motility in response to HGF treatment (Frasca

et al., 2001). Inhibition of Abl kinase activity and overexpression of a Crk mutant lacking

the Abl‐specific tyrosine phosphorylation site resulted in enhanced motility in response

to HGF in fibroblasts expressing the Met receptor (Cipres et al., 2007). Taken together,

these studies suggest that Abl‐mediated phosphorylation of Crk may act downstream of

a diverse set of physiological stimuli to negatively regulate cell migration. These data

also point to the importance of cell‐type and context‐dependent effects in determining

the functional consequence of Abl kinase activation.

1.3.3.3 Abl family kinases in intercellular conversations

Evidence supporting a critical role for the Abl family of tyrosine kinases in

modulating cell signaling downstream of cell‐cell contact formation is rapidly

accumulating. Neuromuscular junction (NMJ) formation is stimulated when agrin

release from the nerve induces acetylcholine receptor (AChR) clustering in the

postsynaptic muscle (Sanes and Lichtman, 2001). Abl family kinases have been shown to

localize to the postsynaptic membrane of the developing NMJ following stimulation of

AChRs, and Abl kinase activity was required for agrin‐induced AChR clustering and enhancement of MuSK tyrosine phosphorylation (Finn et al., 2003). This positions the

Abl kinases to participate as mediators of a downstream signaling cascade, which is

required to bolster AChR clustering, possibly by participating in a positive feedback

loop with the MuSK receptor. The Abl kinases also play an important role in T cell receptor (TCR) signaling following formation of the immunological synapse. Abl kinases

are activated following TCR stimulation, and loss of Abl kinase activity results in

reduced tyrosine phosphorylation of several downstream targets including Zap70, LAT,

and PLC γ1 (Zipfel et al., 2004). The physiological consequences of Abl loss of function

include decreased IL‐2 production and cell proliferation in response to TCR stimulation,

again supporting a role for Abl kinases in signal amplification following the engagement of an MHC‐presenting cell with a T cell (Gu et al., 2007; Zipfel et al., 2004). Mice lacking

Abl kinases specifically in T cells are compromised in their ability to mount an immune

response to bacterial infection (Gu et al., 2007).

The previous examples highlight the role of Abl kinases in “normal” intercellular

signaling, however Abl signaling pathways may also be critical mediators of cell

signaling pathways induced during bacterial infection following bacterial cell

engagement with normal epithelial cells. During Shigella flexneri infection, Abl kinases

are recruited to the cell membrane at sites of bacterial entry and catalytically activated

(Burton et al., 2003). Abl kinase inhibition decreases bacterial cell uptake as well as cell‐

to‐cell spread and inhibits tyrosine phosphorylation of signaling molecules, including

Crk and N‐Wasp, required for these processes (Burton et al., 2005; Burton et al., 2003).

Rac activation downstream of Shigella infection was also shown to require Abl kinase‐

mediated phosphorylation of Crk (Burton et al., 2003). Recently, Abl has been shown to

be phosphorylated and activated by the bacterial CagA protein following Helicobacter

pylori infection (Poppe et al., 2007; Tammer et al., 2007). In this context, Abl kinase

activity is required for downstream cytoskeletal arrangements leading to enhanced cell

motility and sustained phosphorylation of the CagA protein (Poppe et al., 2007; Tammer

et al., 2007). Taken together, these data suggest that hijacking of the Abl kinase signaling

cascade to promote cytoskeletal re‐arrangement may be a critical component of

signaling from invasive bacteria to invasive host cells. Further, these reports suggest that

Abl kinases may function downstream of other receptors to regulate intercellular

conversations.

2. Abl Tyrosine Kinases regulate cell-cell adhesion via Rho GTPAses

This chapter appears in modified form in:

Proc Natl Acad Sci U S A. 2007 Nov 6;104(45):17686‐91

2.1 Introduction

Dynamic regulation of the actin cytoskeleton is required for the formation and

dissolution of intercellular adhesions during tissue morphogenesis (Schock and

Perrimon, 2002) and pathological processes such as tumor invasion and metastasis

(Paszek et al., 2005; Sahai and Marshall, 2002). The formation of cell‐cell contacts is

dependent on the assembly of adherens junctions (Schock and Perrimon, 2002).

Adherens junctions are specialized structures that link transmembrane cadherins in

neighboring cells to the actin cytoskeleton (Schock and Perrimon, 2002). The

extracellular domain of the cadherins cluster in a calcium‐dependent manner and trigger

association of the cadherin cytoplasmic domain with β‐catenin, which in turn binds to α‐

catenin, a protein that links the cadherin/catenin complex to the actin cytoskeleton

(Schock and Perrimon, 2002). Notably, disruption of the actin cytoskeleton, genetic

inactivation of actin regulatory proteins, or disrupting the link between the

cadherin/catenin complex and the actin cytoskeleton, all result in loss of adherens

junctions (Bershadsky, 2004; Ivanov et al., 2005; Vasioukhin et al., 2000). Adherens

junction formation, stability, and dissolution are processes regulated by Rho family

GTPases, which function in part by remodeling the actin cytoskeleton (Thiery, 2002).

However, little is known regarding the mechanisms that link cadherin‐mediated

signaling to regulation of Rho GTPases leading to dynamic reorganization of the actin

cytoskeleton.

Abl (Abl1) and Arg (Abl2) define a family of nonreceptor tyrosine kinases

characterized by unique carboxy‐terminal actin‐binding domains that can bundle actin

(Wang et al., 2001). The Abl kinases regulate a variety of cytoskeletal processes

downstream of receptor tyrosine kinases and integrins (Finn et al., 2003; Hernandez et

al., 2004; Kain and Klemke, 2001; Moresco et al., 2003; Plattner et al., 1999; Sini et al.,

2004). We have shown that Abl kinases play a role in the regulation of intercellular

signals at the neuromuscular junction (NMJ) and at sites of contact between invading

bacterial pathogens and mammalian host cells (Burton et al., 2003; Finn et al., 2003).

Genetic studies in Drosophila and mice support a role for Abl kinases in the regulation of

epithelial morphogenesis (Grevengoed et al., 2003; Grevengoed et al., 2001; Koleske et

al., 1998). Mice lacking Abl and Arg die before embryonic day 11 and display collapse of

the neural tube, which is secondary to disruption of the apical actin latticework in

neuroepithelial cells (Koleske et al., 1998). Based on the unique properties of the Abl

kinases we asked whether Abl and Arg regulate the formation and/or maintenance of

adherens junctions in mammalian cells, and sought to define a pathway whereby the

Abl kinases modulate these processes. Here we identify a novel role for Abl kinases in

the regulation of cell‐cell adhesion via Rho family GTPases.

2.2 Results

2.2.1 Abl Tyrosine Kinases are Required for Cell-Cell Adhesion

To determine whether the Abl kinases are involved in the regulation of cadherin‐

mediated cell‐cell adhesion, we first examined the consequences of genetic inactivation

of Abl and Arg on the formation of N‐cadherin‐dependent cell‐cell junctions in mouse

embryo fibroblasts (MEFs) (Yonemura et al., 1995). Abl‐/‐Arg‐/‐ MEFs were re‐

engineered to re‐express Abl and Arg (Burton et al., 2003; Plattner et al., 1999). Cells

expressing Abl and Arg displayed continuous cell‐cell junctions similar to wild type

(WT) MEFs as visualized by staining for N‐cadherin, β‐catenin and α‐catenin (Figure 4A,

top). In contrast, adherens junction proteins failed to accumulate at sites of cell‐cell

contact in Abl‐/‐Arg‐/‐ MEFs and displayed enhanced cytoplasmic staining (Figure 4A,

bottom). Loss of Abl and Arg did not alter the expression levels of adherens junction

proteins (Figure 4B). To assess the role of Abl kinases in the formation of adherens

junctions, cell monolayers were placed in low‐calcium media to dissolve pre‐existing

A N-cadherin β-catenin α-catenin B Abl/Arg

Null cells: vector +Abl/Arg N-cadherin

+ Abl/Arg β-catenin

α-catenin

Abl/Arg

β-tubulin vector

C Low calcium 30 min + calcium 4 h + calcium + Abl/Arg vector

Figure 4: Loss of Abl family kinases in fibroblasts disrupts N‐cadherin‐based adhesion.

Figure 4: Abl/Arg null MEFs transduced with retroviruses encoding Abl and Arg or vector alone were grown to near confluency and either fixed and stained (A) with antibodies to N‐cadherin, β‐catenin, or α‐catenin (red) and counterstained with DAPI (blue) or lysed (B) to evaluate adherens junction protein expression by blotting with the indicated antibodies. (C) Abl/Arg‐ or vector‐reconstituted null MEFs were grown to subconfluency and subjected to calcium switch to stimulate adherens junction formation. Cells were fixed and stained with antibody against N‐cadherin (red) and counterstained with DAPI (blue).

cell‐cell junctions and then switched to high‐calcium media to activate cadherin‐

mediated intercellular adhesion. Greater than 90% of cells expressing Abl/Arg formed

adherens junctions after 4 hours in high‐calcium media (Figure 4C, top). In contrast, Abl‐

/‐Arg‐/‐ cells displayed a disorganized N‐cadherin staining pattern and were unable to

form discrete adherens junctions even 4 hours after addition of high‐calcium medium

(Figure 4C, bottom). Taken together, these data reveal that Abl kinases are required for

the formation of adherens junctions in fibroblasts.

We next examined whether Abl kinase activity plays a role in the regulation of

epithelial cell‐cell junctions. Recently‐confluent NBT‐II rat epithelial cells were

stimulated to form adherens junctions by calcium switch in the presence or absence of

STI571, a pharmacological inhibitor of the Abl kinases (Kantarjian et al., 2002; Okuda et

al., 2001). In control cells, β‐catenin accumulated at sites of cell‐cell contact after incubation in high calcium media (Figure 5A, top). In contrast, cells treated with the Abl

kinase inhibitor failed to induce β‐catenin‐containing protrusions and to form adherens

junctions (Figure 5A, bottom). Abl kinases were markedly inhibited by 10μM STI571 as

determined by the reduction of endogenous CrkL phosphorylation on Y207, the Abl‐

specific site (Figure 5B). To determine whether Abl and Arg proteins are required for

intercellular adhesion in E‐cadherin‐expressing epithelial cells, Abl and Arg expression

was downregulated using RNA interference (RNAi) (Figure 5C). NBT‐II cells

A Low calcium 8 h + calcium B _ 10µM STI571: 1 h3 h 6 h IB: pCrkL Y207

control IB: CrkL 10µM STI571

C D control siRNA Abl siRNA β-catenin siGlo merge siRNA Control

Arg siRNA Abl/Arg siRNA Abl/Arg Abl/Arg siRNA

Figure 5: Inhibition of Abl kinase activity or protein impairs adherens junction formation in epithelial cells.

Figure 5: (A) Confluent NBT‐II cells were subjected to calcium switch in the absence or presence of 10μM STI571. Cells were fixed and stained with antibody against β‐catenin (red) and counterstained with DAPI (blue). (B) Tyrosine phosphorylation of the endogenous Abl substrate CrkL at the Abl‐specific site (Y207) is inhibited following treatment with 10μM STI571. (C) Anti‐β‐catenin immunostaining of NBT‐II cells transfected with control or Abl‐ and/or Arg‐directed siRNAs. (D) Anti‐β‐catenin immunostaining of MCF‐10A cells co‐transfected with siGLO RISC‐Free siRNA (to identify transfected cells) and with control or Abl‐ and Arg‐directed siRNAs. Images of β‐catenin (green) and siGLO oligo (red) were merged to indicate siRNA‐transfected cells. Scale bars in all figures represent 20 μm.

A E-cadherin siGlo merge B α-catenin siGlo merge Control siRNA Control Control siRNA Abl/Arg Abl/Arg siRNA Abl/Arg Abl/Arg siRNA

C siRNA: Control Abl/Arg Abl/Arg E-cadherin β-catenin α-catenin β-tubulin

Figure 6: Loss of Abl/Arg impairs intercellular adhesion.

Figure 6: (A) Anti‐E‐cadherin immunostaining of MCF‐10A cells co‐transfected with siGLO RISC‐Free siRNA and with control or Abl‐ and Arg‐directed siRNAs. Images of E‐cadherin (green) and siGLO oligo (red) were merged to indicate transfected cells. (B) Anti‐α‐catenin immunostaining of MCF‐10A cells co‐transfected with siGLO RISC‐Free siRNA and with control or Abl‐ and Arg‐directed siRNAs. Images of α‐catenin (green) and siGLO oligo (red) were merged to indicate transfected cells. (C) MCF‐10A cells were transfected with control or Abl‐ and Arg‐directed siRNAs and lysed 48 h post‐ transfection. Adherens junction protein levels were analyzed by Western blotting with the indicated antibodies.

transfected with Abl, Arg, or both Abl and Arg‐directed siRNAs showed decreased

levels of β‐catenin‐containing cell‐cell junctions. To determine whether this effect is

observed in other E‐cadherin‐expressing epithelial cells, Abl and Arg expression was

downregulated by RNAi in MCF‐10A epithelial cells (Figure 5D and 6C). MCF‐10A cells

transfected with Abl/Arg‐directed siRNAs showed decreased levels of E‐cadherin and

catenins at sites of cell‐cell contact or lacked cell‐cell contacts completely with no change

in adherens junction protein expression (Figure 5D and 6). Taken together, these data

support a critical role for Abl family protein and kinase activity in the formation of

epithelial adherens junctions.

2.2.2 Abl Tyrosine Kinases are Required for the Maintenance of Adherens Junctions

Adherens junctions in epithelial sheets are dynamic structures, which undergo

constant remodeling (Yamada et al., 2005). To test the involvement of Abl kinases in

adherens junction stability, Abl kinase activity was inhibited with STI571 after formation

of confluent monolayers of NBT‐II epithelial cells. Treatment with STI571 for 3 hours

resulted in relocalization of β‐catenin from adherens junctions to the cytoplasm (Figure

7A). After 6 hours of STI571 treatment, only 40% of the cells retained adherens junctions

as indicated by reduced β‐catenin staining at sites of cell‐cell contact (Figure 7A, B).

Adherens junction protein expression was unaffected by Abl kinase inhibition for up to

A B C

STI571: 0 h 3 h 6 h STI571: -6 h 100 E-cadherin 75 50 * β-catenin

-catenin 25 α-catenin β 0 junctions (%) junctions Cells with adherens Cells with actin STI571: -6 h D STI571:0 h 1 h 3 h 6 h E STI571: -1 h3 h6 h α-catenin

-catenin E-cadherin α β-tubulin

F IP: E-cadherin STI571:- 1 h 3 h 6 h

E-cadherin α-catenin E-cadherin

Figure 7: Inhibition of Abl kinase activity impairs accumulation of E‐cadherin‐ catenin complexes at cell‐cell contacts.

Figure 7: (A) Confluent NBT‐II cells were treated with 10μM STI571 for the indicated times, fixed and stained for β‐catenin (red) and counterstained with DAPI (blue). (B) β‐ catenin staining at sites of cell‐cell contact was analyzed in 4 independent experiments. *, significant decrease (p=.0004) in adherens junctions in STI571‐treated cells versus controls. (C) Recently‐confluent NBT‐II cells were untreated or treated with 10μM STI571 for 6 h. Total lysates were examined by immunoblotting with antibodies against the indicated adherens junction proteins. (D) Confluent MCF‐10A cells were grown to confluence and treated with 10μM STI571 for the indicated times. Cells were fixed and stained for the indicated adherens junction proteins. (E) Confluent MCF‐10A cells were untreated or treated with 10μM STI571 for the indicated times. Total lysates were examined by immunoblotting with antibodies against the indicated adherens junction proteins. (F) To evaluate integrity of the cadherin‐catenin complex in response to Abl kinase inhibition, the level of α‐catenin bound to E‐cadherin was assessed by immunoprecipitation of E‐cadherin from 500μg lysate and immunoblotting with an antibody against α‐catenin. Blots were stripped and reprobed with an antibody against E‐cadherin to demonstrate equal protein levels.

6 hours (Figure 7C). Similarly, STI571 treatment of MCF‐10A cells promoted

mislocalization of adherens junction proteins (Figure 7D) with no change in E‐cadherin

and α‐catenin protein levels (Figure 7E) or the integrity of the E‐cadherin/α‐catenin

complex (Figure 7F). Moreover, we did not observe any changes in the tyrosine

phosphorylation of components of the cadherin/catenin complex in the absence of Abl

kinases or after inhibition of Abl kinase activity (data not shown).

2.2.3 Cell-cell Adhesion Leads to Abl Kinase Activation and Recruitment to Sites of Cell-Cell Contact

To test whether Abl kinases are catalytically activated by cell‐cell adhesion, we

analyzed the phosphorylation of the Abl substrates CrkII or the related CrkL using

phosphospecific antibodies against the Abl‐specific sites, which have been shown to be

unphosphorylated in Abl‐/‐Arg‐/‐ MEFs or in cells lacking Abl kinase activity (Burton et al., 2003; Zipfel et al., 2004). Abl kinase activity was upregulated by 5 minutes following

calcium switch to induce cadherin‐mediated cell‐cell adhesion, and Abl activation was

sustained for 30 minutes in WT MEFs (Figure 8A). Similar kinetics were observed in

NBT‐II cells subjected to calcium switch using in vitro kinase assays with GST‐Crk to

measure Abl and Arg kinase activity (Plattner et al., 2003) (Figure 8B). To eliminate the

possible contribution of intercellular adhesion‐independent effects, we compared Abl

activation in sparse versus confluent cultures and found that Abl kinase activity is

+ Ca2+ A + Ca2+ B 0 min 30 min 60 min 1 min 2 min 5 min 10 min + EGTA 5 min 30 min IB: pCrk Y221 IP: Arg Fold: 1 1.63 1.94 Fold: 1 0.9 1.1 2.5 2.5 1.8 0.9 IB: Crk IP: Abl Fold: 1 0.9 0.7 1.6 1.6 1.8 0.7 IB: Abl

C Confluent cultures Sparse cultures D Ca2+: Hi LowHi Hi Hi Low Hi Hi 2.5 * 5 m 15 m 5 m 15 m 2 * IB : pCrkL Y207 1.5 Fold: 0.911 2.08 1.34 1.33 1 1.05 0.69 1 IB : CrkL 0.5 0 0 min 5 min 15 min

confluent cultures sparse cultures

Figure 8: Cell‐cell adhesion regulates the activity of endogenous Abl kinases.

Figure 8: (A) Activation of Abl and Arg kinases in response to calcium switch in wild‐ type (WT) MEFs was assessed by immunoprecipitation of Crk and immunoblotting with a phosphospecific antibody to Y221. Blots were stripped and reprobed with an anti‐Crk antibody to demonstrate equal loading. Results shown are representative of 3 independent experiments. (B) Confluent NBT‐II epithelial cells were subjected to calcium switch and Abl or Arg protein was immunoprecipitated as indicated using specific antibodies, and in vitro kinase assays were performed using GST‐Crk as substrate. Abl/Arg protein expression in whole cell lysates (bottom panel) was assessed by immunoblotting with 8E9 monoclonal antibody. (C) Abl/Arg kinase activity in response to calcium switch was assayed in confluent versus sparse epithelial cell cultures. (D) Abl kinase activity was quantitated using densitometry with pCrkL levels indexed against low calcium for each condition. Results represent three independent experiments. *, indicates p< 0.05. Fold‐changes in pCrk or pCrkL levels are indicated below the corresponding lanes.

unaltered in the absence or presence of calcium in sparse cultures (Figure 8C, D). In

contrast, Abl kinase activity increases in a time‐dependent manner after calcium switch

in confluent cell monolayers (Figure 8C, D). Thus, the regulation of Abl kinase activity

after calcium switch is dependent on cadherin‐mediated cell‐cell contacts.

To determine whether Abl and Arg are recruited to sites of cell‐cell contact to

participate in signaling, we generated MDCK cell lines expressing GFP‐tagged versions

of the Abl kinases and examined the localization of these kinases during adherens

junction formation. Arg accumulated at sites of cell‐cell contact and in the cytoplasm in both confluent cells and in cells stimulated to re‐form adherens junctions by switch to

high‐calcium medium (Figure 9A). Similar results were observed for GFP‐Abl (data not

shown). In confluent MCF‐10A cells, Arg also co‐localized with β‐catenin at sites of cell‐

cell contact (Figure 9C), and E‐cadherin and β‐catenin co‐immunoprecipitated with GFP‐

Arg (Figure 9B). Disruption of cadherin‐mediated cell‐cell adhesion (low calcium)

resulted in re‐distribution of Arg from adherens junctions to the cytoplasm (Figure 9A,

C). Following calcium switch to induce adherens junction formation, GFP‐Arg re‐

localized in lamellipodial extensions and at sites of nascent cell‐cell contacts (Figure 9C).

Together, these data further support a role for Abl and Arg in adherens junction

regulation as both proteins are recruited to sites of cell‐cell contact, are associated with

A B 15 min Hi calcium Low calcium + Hi calcium Vector GFP-Arg E-cadherin β-catenin

IP: GFP GFP

C 1 h + 6 h + Hi calcium Low calcium Hi calcium Hi calcium -catenin β GFP merge

Figure 9: Arg localizes to adherens junctions.

Figure 9: (A) MDCK cells expressing GFP‐tagged Arg were seeded onto glass coverslips, grown to confluence, and subjected to calcium switch. Cells were fixed and GFP fluorescence examined by microscopy. (B) MCF‐10A cells expressing vector or GFP‐ tagged Arg were grown to confluence and lysed in 1% Triton‐X‐100 lysis buffer. Anti‐ GFP antibody was used to immunoprecipitate Arg from 1mg pre‐cleared lysate, followed by immunoblotting for E‐cadherin or β‐catenin. Blots were stripped and re‐ probed for GFP. (C) MCF‐10A cells expressing GFP‐tagged Arg were seeded onto glass coverslips, grown to confluence, and subjected to calcium switch. Cells were fixed and stained for β‐catenin and GFP. Images of β‐catenin (red) and GFP (green) were merged to indicate co‐localization (yellow).

E‐cadherin/β‐catenin complexes and are activated in epithelial cells in response to

cadherin engagement.

2.2.4 Abl Kinases Regulate Rac Activation in Response to Cadherin Engagement via Crk/CrkL

To test whether Abl kinases are sufficient to promote adherens junction

formation, we expressed a constitutively active mutant form of Arg (ArgPP) (Plattner et

al., 2004) in HeLa cells. These cells express N‐cadherin and exhibit weak, immature

adherens junctions. ArgPP expression resulted in enhanced, continuous staining of β‐

catenin at sites of cell‐cell contact (Figure 10A). Thus, Abl kinases are required for

adherens junction formation and promote N‐cadherin‐mediated adhesion in cells with

relatively weak junctions.

Because formation of cell‐cell junctions correlates with increased

phosphorylation of Crk/CrkL proteins at the Abl‐specific sites, we next examined

whether Crk family proteins play a role in ArgPP‐induced adherens junction

strengthening by analyzing the consequences of Crk/CrkL siRNA‐mediated knockdown.

We observed a marked reduction in the integrity of adherens junctions in ArgPP‐

expressing cells transfected with Crk and CrkL siRNAs (Figure 10B, C). Similarly,

adherens junction integrity was markedly impaired in NBT‐II epithelial cells transfected

A vector ArgPP B vector ArgPP Control siRNA Crk/CrkL siRNA

C D control Crk/CrkL siRNA siRNA control siRNA: Crk/CrkL Crk CrkL β-tubulin

Figure 10: Crk/CrkL function downstream of Abl‐mediated adherens junction formation in epithelial cells.

Figure 10: (A) β‐catenin localization was visualized in vector‐ or ArgPP‐expressing HeLa cells. Expression of ArgPP results in enhanced, continuous staining of β‐catenin at sites of cell‐cell contact. (B) β‐catenin staining in vector‐ or ArgPP‐expressing HeLa cells transfected with control or Crk‐ and CrkL‐directed siRNA oligos. (C) The levels of Crk/CrkL proteins in lysates from HeLa cells transfected with control or Crk/CrkL‐ specific siRNAs were analyzed by blotting with the indicated antibodies; β‐tubulin was used as a loading control. (D) β‐catenin staining in NBT‐II cells transfected with control or Crk‐ and CrkL‐directed siRNA oligos.

with Crk and CrkL siRNAs (Figure 10D). Taken together these data reveal a critical role

for the Abl kinases in adherens junction formation in mammalian cells and suggest that

this effect may be mediated in part by Crk proteins.

The Rac GTPase has been implicated in the formation of adherens junctions

(Ehrlich et al., 2002; Noren et al., 2001). In agreement with previous findings (Noren et

al., 2001), we observed that Rac was activated by 5 min of cadherin engagement (Figure

11A). Basal levels of activated Rac were unchanged in confluent monolayers of WT or

Abl‐/‐Arg‐/‐ MEFs grown in complete calcium‐rich media (data not shown).

Significantly, Rac activation induced by adherens junction formation was delayed and

markedly reduced in Abl‐/‐Arg‐/‐ cells (Figure 11A, B), and in WT MEFs subjected to

calcium switch in the presence of the Abl kinase inhibitor STI571 (Figure 11C). Previous

studies have linked tyrosine phosphorylation of Crk to Rac activation following bacterial

infection and integrin engagement (Abassi and Vuori, 2002; Burton et al., 2003). We

found that overexpression of a Crk mutant lacking the Abl‐specific phosphorylation site

in MEFs resulted in decreased Rac activation in response to calcium switch during adherens junction formation (Figure 11D, E). The inhibitory effect of the Crk mutant

was similar to that induced by Abl kinase inhibition. Taken together, these data suggest

that Abl‐mediated phosphorylation of Crk is required for maximal activation of Rac

downstream of cadherin engagement.

A Minutes: 05 15B C Rac-GTP 8 Minutes: 0515 Fold: 1 4.6 7.7 6 control

control * Total Rac 4 Fold: 1 2.52 3.27 2 STI Rac-GTP Rac-GTP 0 Fold: 1 1.34 2.8 Fold: 1 1.09 1.86 0 min 5 min 15 min

Abl/Arg null Total Rac Control Null

D E Minutes: 05 15 3 * Rac-GTP * 2 Fold: 1 2.03 1.95 1 control Total Rac 0 0 min 5 min 15 min Rac-GTP Control CrkYF Fold: 1 0.86 0.94

CrkYF Total Rac

Figure 11: Abl kinases regulate Rac activation in response to cadherin engagement.

Figure 11: (A) Rac activation in serum‐starved WT or Abl‐/‐Arg‐/‐ MEFs grown to confluency and subjected to calcium switch. (B) Rac activation was quantitated using densitometry with Rac‐GTP levels indexed against low calcium in each condition. Results represent three independent experiments. *, indicates p< 0.05. (C) Rac activation in serum‐starved WT or STI571‐treated MEFs grown to confluency and subjected to calcium switch. (D) Rac activation in serum‐starved WT MEFs expressing vector (control) or CrkYF was analyzed in cells grown to confluency and subjected to calcium switch. (E) Rac activation was quantitated using densitometry with Rac‐GTP levels indexed against low calcium in each condition. Results represent four independent experiments. *, indicates p< 0.05.

2.2.5 Abl Kinases Regulate the Architecture of the Actin Cytoskeleton in Epithelial Cell Sheets via the Rho-Rock Pathway

The Abl kinases regulate multiple cytoskeletal processes such as membrane

ruffling, chemotaxis, and cell spreading (Pendergast, 2002). Dynamic regulation of the

actin cytoskeleton is required for the formation, stability, and dissolution of adherens

junctions (Ivanov et al., 2005; Pendergast, 2002; Vaezi et al., 2002; Vasioukhin et al.,

2000). Therefore, we tested whether Abl/Arg kinase inhibition could alter the

cytoskeletal architecture of recently confluent cells. Epithelial cell clusters display thick

cortical bundles of actin at cell‐cell borders (Figure 12A). In contrast, cells treated with

the Abl kinase inhibitor showed decreased cortical actin, increased stress fiber

formation, accompanied by the appearance of gaps between cells, and increased

numbers of spiky membrane protrusions over time (Figure 12A). The enhanced

formation of stress fibers in STI571‐treated cells suggested that inhibition of Abl family

kinases may result in increased Rho activity. Indeed, cellular levels of Rho‐GTP

increased following STI571 treatment, which correlated with the appearance of stress

fibers (Figure 12A, B, C). To determine whether loss of Abl and Arg protein could

similarly affect cadherin‐mediated regulation of Rho activity, we examined Rho

activation in epithelial cells where Abl and Arg proteins were knocked down by RNAi.

In agreement with previous findings (Noren et al., 2001), cadherin engagement

AB STI571: 0 h 3 h 6 h STI571 (h): 00.51 3 Rho-GTP 11.761.81.4 Fold-change Total Rho

actin C 1.8 * 1.6 1.4 1.2 D 1 STI571: 0 h 1 h 3 h 0.8 0.6 0.4 0.2 0

pMLC 0 h 0.5 h 1h 3 h

E Hi Low Hi Ca2+ F 1.4 * Ca2+ Ca2+ 5 min 1.2 Rho-GTP 1 1.19 10.31Fold-change 0.8 0.6 siRNA Control Total Rho 0.4 0.2 Rho-GTP 0 1.37 11.31Fold-change 0 min 5 min

control siRNA Abl/Arg siRNA

Abl/Arg Abl/Arg siRNA Total Rho

Figure 12: Abl kinases regulate the actin cytoskeleton and Rho activity in epithelial cell monolayers.

Figure 12: Recently‐ confluent NBT‐II cells were treated with 10μM STI571 for the indicated times. (A) Staining with AlexaFluor 488‐conjugated phalloidin (green) and counterstained with DAPI (blue). (B) Lysates were analyzed for Rho activity using the EZ‐Detect Rho activation kit. Total Rho protein levels were analyzed by immunoblotting. (C) Rho activity was quantitated using densitometry with Rho levels indexed against the untreated condition. Results represent three independent experiments. *, indicates p< 0.05. (D) Cells were fixed and stained with a phosphospecific antibody to pMLC (red) and counterstained with DAPI (blue). (E) MCF‐ 10A cells were transfected with control or Abl‐ and Arg‐directed siRNA oligos, grown to confluence, and serum‐starved overnight. Cells were subjected to calcium switch and Rho activity was assayed activity using the EZ‐Detect Rho activation kit. (F) Rho activity was quantitated using densitometry with Rho levels indexed against the untreated condition. Results represent three independent experiments. *, indicates p< 0.05.

following switch from low to high calcium suppressed Rho activity in control epithelial

cells (Figure 12E, F). In contrast, Rho inhibition following cadherin engagement was not

observed in the Abl/Arg siRNA‐treated confluent monolayers (Figure 12E, F). Thus, the

Abl family kinases are required for cadherin‐dependent inhibition of Rho activity.

Rho activation may result in increased acto‐myosin contractility mediated

through phosphorylation of the myosin regulatory light chain (MLC) of myosin II by

Rho‐activated kinases (Bresnick, 1999). Inhibition of Abl family kinases resulted in a

time‐dependent increase of MLC phosphorylation and enhanced accumulation of

phospho‐MLC at the cell periphery, which correlated with increased formation of stress

fibers (Figure 12D). Similarly, Abl kinase inhibition resulted in enhanced

phosphorylation of MLC as assessed by immunoblotting to detect phospho‐serine 19 of

MLC (de Rooij et al., 2005) (data not shown).

Rho signaling has been shown to regulate both the formation and dissolution of

adherens junctions, and activation of the Rho effector ROCK has been shown to disrupt

adherens junctions in epithelial cells (Avizienyte et al., 2004; Bhowmick et al., 2001;

Braga et al., 1999; Paszek et al., 2005; Sahai and Marshall, 2002; Shewan et al., 2005; Vaezi

et al., 2002). To test whether the observed effects of Abl/Arg kinase inhibition are

dependent on ROCK kinase activity, we examined whether the phenotypes induced by

loss of Abl kinase activity could be reversed by inhibition of ROCK. Consistent with

previous findings (Sahai and Marshall, 2002), cells treated with the ROCK kinase

inhibitor Y27632 for 1 hour did not display adherens junctions defects, but did show a

marked decrease of stress fibers at the cell center (Figure 13A). Significantly, cells treated

with both STI571 and Y27632 failed to display any of the aberrant morphology

characteristic of STI571‐treated cells, with β‐catenin and actin remaining localized to

sites of cell‐cell contacts; also, stress fiber formation was inhibited in these cells (Figure

13A, B, C). Notably, whereas inhibition of the Abl kinases resulted in a 60% decrease in

the percentage of cells with mature, continuous adherens junctions, this effect was

blocked by inhibition of ROCK (Figure 13C). Further, dual inhibition of both Abl and

ROCK kinases abolished the increase in pMLC and the enhanced localization of MLC

protein to the cell membrane (data not shown). Thus, inhibition of Abl kinase activity leads to upregulation of Rho‐ROCK signaling, enhanced MLC phosphorylation, and

increased acto‐myosin contractility, thereby promoting the dissolution of cell‐cell

adhesions. Together, these data support a model whereby endogenous Abl kinases

normally function to modulate Rho‐ROCK activity and maintain cell‐cell contacts in

epithelial cell sheets (Figure 14).

AB actin β-catenin * 60

40 control 20 */**

fibers (%) fibers *

Cells with stress Cells with 0 control STI571 Y27632 STI571 + Y27632

10µM STI571 C

* 75 50 * 25 10µM Y27632

-catenin) 0 β Cells maturewith junctions cell-cell ( control STI571 Y27632 STI571 + Y27632 10µM STI571 10µM + 10µM Y27632

Figure 13: Disruption of adherens junctions in response to inhibition of Abl kinase activity is reversed by inhibition of ROCK kinase activity.

Figure 13: (A) Confluent NBT‐II cells were treated with 10μM STI571, 10μM Y27632, or both STI571 and Y27632 for 1 hour, fixed and stained with phalloidin (green) or anti‐β‐ catenin (red), and counterstained with DAPI (blue). (B) Stress fiber formation was analyzed by examining phalloidin staining. Results shown represent the mean of 4 independent experiments. *, significant increase (control vs. STI571 treatment p<.001) or decrease (control vs. Y27632 treatment p<.05, control vs. STI571 + Y27632 treatment p<.05) in stress fibers. ** , significant decrease (p<.001) in stress fibers in cells treated with both STI571 and Y27632 together compared to STI571‐treated cells. (C) The number of cells with mature adherens junctions marked by continuous β‐catenin staining at sites of cell‐cell contact was quantified in 4 independent experiments, in which 9 random fields were examined per condition. *, significant decrease (p<.001) or increase (p<.001) in adherens junctions in cells treated with STI571 in the absence or presence of Y27632.

α α - catenin β β - catenin

P120 P120 catenin catenin

Cadherin

RacGAP P P120 Y cat. P Crk/ Y CrkL Rac-GDP Rac-GTP β β - cat. Abl RacGEF α α - cat. ROS ? ? F Actin ? P P RhoGAP Enhanced ROCK MLC Contractility Rho-GDP Rho-GTP

RhoGEF

Figure 14: Model for Abl family kinase regulation of adherens junctions.

2.3 Discussion

Here we identify the Abl family of tyrosine kinases as critical mediators of

cadherin‐dependent adhesion in mammalian cells through regulation of Rho family

GTPases. We have employed genetic ablation and pharmacological inhibition to show

that Abl kinases are required for the formation of adherens junctions and that loss of Abl

kinases impairs Rac activation induced by cadherin engagement. Additionally, we show

that Abl gain‐of‐function promotes adherens junction formation in cells with weak

junctions and that Crk/CrkL proteins are required for this strengthening phenotype.

Significantly, we show for the first time that endogenous Abl kinases are

activated by cadherin engagement. We found that Abl and Arg are recruited to nascent

junctions and are present in E‐cadherin/β‐catenin protein complexes. Additionally, we

have shown that Abl kinase activity is required for the maintenance of adherens

junctions in epithelial cell sheets, as inhibition of these kinases results in the re‐

distribution of adherens junction components from sites of cell‐cell contact to the

cytoplasm. Abl kinases are not only required for maximal Rac activation induced by

cadherin‐mediated adhesion, but are likely to modulate cell‐cell adhesion via regulation

of other pathways. In this regard, we have shown that Abl‐mediated regulation of the

Rho‐ROCK‐myosin signaling pathway is essential for adherens junction stability.

Inhibition of the Abl kinases in epithelial sheets results in activation of Rho and its

downstream target ROCK, leading to enhanced phosphorylation of the myosin II

regulatory light chain. Activation of this signaling pathway leads to enhanced stress

fiber formation and increased acto‐myosin contractility, thereby disrupting adherens

junctions. These findings identify a novel role for the Abl tyrosine kinases as regulators

of intercellular adhesion through their ability to modulate both Rac and Rho GTPase‐

dependent pathways.

Notably, we showed that Abl kinases activated by cadherin engagement

phosphorylate Crk/CrkL proteins, and that both Abl and Crk family proteins are

required for adherens junction integrity in a pathway upstream of Rac. We and others

have shown that Crk phosphorylation on the Abl‐specific site is required for Rac

activation and membrane localization [Figure 11E, (Abassi and Vuori, 2002; Burton et al.,

2003)]. The mechanism by which Crk phosphorylation by Abl results in Rac activation

may involve the recruitment of the Crk‐binding protein DOCK180, a guanine nucleotide exchange factor (GEF) for Rac (Kiyokawa et al., 1998). Alternatively, Abl may activate

Rac by phosphorylating Sos‐1, which was reported to stimulate the Rac‐GEF activity of

Sos‐1 (Sini et al., 2004).

Abl kinases are required for both activation of Rac and inhibition of Rho at

adherens junctions, and it is possible that these events may be coordinately linked. Abl

may downregulate Rho signaling indirectly by activating a Rac‐ROS pathway (Nimnual

et al., 2003), or by directly regulating the activities of a RhoGAP or a RhoGEF. In this

regard, activated RacV12 was reported to inhibit Rho via p190 RhoGAP (Nimnual et al.,

2003), and p190 RhoGAP was shown to be phosphorylated in an Arg‐dependent manner

in response to integrin engagement in neuronal cells (Hernandez et al., 2004). However,

we have not detected any changes in p190 RhoGAP tyrosine phosphorylation in the

absence of Abl/Arg kinases or upon STI571 treatment in epithelial cells or fibroblasts

(data not shown). Nor did we observe any changes in complex formation between p190

RhoGAP and either p120 RasGAP, which is required for activating p190 RhoGAP

activity, or p120 catenin (data not shown). Moreover, in agreement with published data

(Wildenberg et al., 2006), we found that p190 RhoGAP does not localize to adherens

junctions in the cells analyzed here (data not shown). Thus, our data suggest that Abl

substrates other than p190 RhoGAP are likely to regulate adherens junction formation

and maintenance. We have however, identified the Crk/CrkL family of adaptors as

critical players in the Abl‐mediated regulation of adherens junctions. Future studies are

needed to identify all the GEFs and GAPs involved in the regulation of Rac and Rho

during adherens junction formation and stability in epithelial cells and fibroblasts.

Together our findings implicate the Abl family of tyrosine kinases as regulators

of intercellular adhesion in epithelial cells and fibroblasts, and support the model

depicted in Figure 14. These data and our previous results demonstrating that Abl

kinases are required for the formation of the neuromuscular junction, suggest that Abl

kinases may function to modulate adhesive interactions between the same or different

cell types. Changes in the activation of the Abl kinases may also modulate cell‐cell

adhesion during tumor progression and metastasis. In this regard, enhanced expression of Abl or Arg has been reported in a subset of metastatic colon carcinomas, pancreatic

ductal carcinoma, and renal medullary carcinoma (Chen et al., 1999; Crnogorac‐Jurcevic

et al., 2002; Simpson et al., 2005). Excessive Abl/Arg kinase activity may disturb the

proper balance of Rho and Rac activation and induce altered cytoskeletal reorganization,

thereby affecting cell‐cell adhesion.

3. A closer look at Abl-Crk-Rac

3.1 Background

While many of the players in the regulation of adherens junctions have been

identified, their individual roles and the way they interact with one another is often

controversial. This is particularly true for regulators of the actin cytoskeleton. For

example, activation of Rac, Rho, and Src kinases have all been implicated in both the formation and dissolution of adherens junctions (Calautti et al., 1998; Lilien and

Balsamo, 2005; Lozano et al., 2003; Sahai and Marshall, 2002; Yap and Kovacs, 2003). The

biological consequence of Rho signalling is thought to be determined by the choice of

downstream effector: activation of Dia promotes intercellular adhesion, while activation

of ROCK disrupts adhesion (Sahai and Marshall, 2002). However little is known

regarding the mechanism through which specific effectors are targeted.

Another unresolved question in adherens junction biology is how signaling to

promote actin polymerization is sustained throughout the process of junction formation.

Rac is activated following cadherin engagement and initially required to promote actin

polymerization, which may also facilitate cadherin clustering (Brunton et al., 2004).

Initial cell‐cell contacts resemble spots of adhesion and the process through which the

membranes are sealed with adherens junctions has been likened to zippering. Actin

polymerization is also required for this closing of the adhesion zipper, and, in fact, Rac

activation has been shown to occur in a biphasic manner downstream of cadherin

engagement (Noren et al., 2001). Recently, it was shown that rounds of Rac activation

and downregulation occur at the periphery of the contacting membranes between

neighboring cells in the process of epithelial cell‐cell adhesion (Yamada and Nelson,

2007). While it is possible that Rac is activated initially through one mechanism and later

activated again, it is also possible that Rac activation stimulates a positive feedback loop,

which is required for sustained signaling and actin polymerization during the

maturation of adherens junctions.

The Abl family of tyrosine kinases, Abl and Arg, are known to have a role in

modulating cytoskeletal responses (Pendergast, 2002). Abl and Arg participate in

signaling events leading to actin polymerization downstream of integrin engagement,

growth factor receptor signaling, bacterial infection, and repulsive cues in axon

guidance (Bashaw et al., 2000; Bradley et al., 2006; Burton et al., 2003; Plattner et al., 1999;

Sini et al., 2004). We recently showed for the first time that Abl kinases are activated

downstream of cadherin engagement and recruited to nascent cell‐cell contacts in

mammalian cells (Zandy et al., 2007). In this regard, Abl kinases are required for

maximal Rac activation following the stimulation of cell‐cell adhesion (Zandy et al.,

2007). This process is dependent on the Abl substrate Crk, which becomes tyrosine

phosphorylated during the formation of adherens junctions (Zandy et al., 2007).

Likewise, a requirement for Crk in Rac activation has been identified in regulation of

other cellular processes including signaling downstream of Shigella infection and the

Met receptor (Burton et al., 2003; Lamorte et al., 2002b).

3.2 Results

We previously showed that Abl kinases are sufficient to promote adherens

junction formation by expressing a constitutively active mutant form of Arg (Arg PP)

(Plattner et al., 2004) in HeLa cells (Zandy et al., 2007). ArgPP expression resulted in

enhanced, continuous staining of β‐catenin at sites of cell‐cell contact. Similarly, overexpression of activated Rac has been shown to promote adherens junction

formation in some epithelial cells (Wildenberg et al., 2006). Indeed, overexpression of

constitutively active Rac (RacV12) in HeLa cells phenocopied the effect of active Arg

expression. Thus, activated forms of both Abl kinases and Rac promote N‐cadherin‐

mediated adhesion in cells with relatively weak junctions (Figure 15A). We showed that

adherens junction strengthening in ArgPP‐expressing cells required the presence of the

Crk family proteins, Crk and CrkL (Zandy et al., 2007). To determine whether Crk

family proteins also play a role in RacV12‐ induced adherens junction strengthening, we examined the consequence of Crk/CrkL siRNA‐mediated knockdown. We observed a

marked reduction of adherens junctions in both RacV12‐ and ArgPP‐expressing cells

transfected with Crk and CrkL siRNAs (Figure 15A). These data reveal a critical role for

the Crk family of adaptors in adherens junction formation in mammalian cells and

suggest that they act downstream of both Abl kinases and Rac.

Because expression of RacV12 phenocopied the effect of ArgPP expression in

HeLa cells and Crk proteins were required downstream of both activated Rac and Abl

for strengthening of adherens junctions. We next asked whether the Abl kinases

may be required for RacV12‐induced enhancement of adherens junctions. To determine

whether Abl kinases are required for this phenotype, we examined the consequence of

Abl/Arg knockdown in HeLa cells expressing RacV12. Depletion of Abl/Arg or

treatment with the Abl kinase inhibitor STI571 (Gleevec) decreased the RacV12‐

enhanced localization of β‐catenin to sites of cell‐cell contact (Figure 15B, data not

shown). Expression of RacV12 led to increased phosphorylation of the Abl substrate

CrkL, which was reduced in cells with downregulated Abl/Arg protein (Figure 15C) or

cells treated with STI571 (data not shown) suggesting that enhanced Abl signaling

through Crk/CrkL family proteins may play a role in the observed phenotype.

Expression of constitutively active RacV12 has been shown to induce production of

reactive oxygen species (ROS) (Nimnual et al., 2003). Interestingly, ROS is a potent

activator of the Abl kinases, and promotes enhanced phosphorylation of Crk (Sun et al.,

2000a). Taken together, these data suggest that Crk family proteins may promote

A vector ArgPP RacV12 Control siRNA Crk/CrkL siRNA

B siRNA: control Abl/Arg C vector RacV12 control control

siRNA: Abl/Arg Abl/Arg vector Abl/Arg β-tubulin pCrkL Y207

RacV12 CrkL

Figure 15: Abl and Crk function downstream of Rac activation.

Figure 15: (A) β‐catenin staining in vector‐ , RacV12‐, or ArgPP‐expressing HeLa cells transfected with control or Crk‐ and CrkL‐directed siRNA oligos. (B) β‐catenin staining was examined in vector‐ or RacV12‐expressing HeLa cells transfected with control or Abl‐ and Arg‐directed siRNA oligos. (C) HeLa cells were lysed 48 h post‐transfection. Abl/Arg protein levels and CrkL phosphorylation and expression were examined using Western blotting.

adherens junction formation and strengthening downstream of active Rac and Abl

kinases.

Whereas our data suggest a positive role for Crk in regulating intercellular

adhesion, overexpression of Crk has been shown to promote the dissolution of adherens

junctions in other contexts (Lamorte et al., 2002a; Lamorte et al., 2002b; Suzuki et al.,

2005). We reasoned that tyrosine phosphorylation of Crk may hold the key to resolving

this paradox. We observed that enhanced phosphorylation of Crk downstream of ArgPP

and RacV12 expression was correlated with adherens junction strengthening. If Crk

overexpression led to an increase in unphosphorylated Crk levels due to limiting

amounts of active Abl kinases, the prediction is that unphosphorylated Crk may

promote the dissolution of cell‐cell junctions, and that perhaps overexpression of ArgPP

might rescue the inhibitory effect of Crk overexpression on cell‐cell adhesion.

To test this hypothesis, we overexpressed Crk in NBT‐II cells and, indeed, observed reduced β‐catenin staining at sites of cell‐cell contact and enhanced cell spreading, which

phenocopied the effect of STI571 inhbition of Abl kinase activity (Figure 16). In contrast,

cells co‐expressing Crk and ArgPP retained β‐catenin at adherens junctions and showed

reduced cell spreading (Figure 16). These data support our hypothesis that Arg‐

mediated tyrosine phosphorylation of Crk promotes intercellular adhesion, while

unphosphorylated Crk promotes the destabilization of intercellular adhesions.

vector ArgPP

vector

Crk

Figure 16: ArgPP expression rescues negative effects of Crk overexpression on adherens junctions.

Figure 16: β‐catenin staining in vector‐ or Crk‐expressing NBT‐II cells, co‐expressing vector‐ or ArgPP.

3.3 Discussion

We showed that Abl kinases activated by cadherin engagement phosphorylate

Crk/CrkL proteins and function upstream of Rac (Zandy et al., 2007). We and others

have shown that Crk phosphorylation on the Abl‐specific site is required for Rac

activation and membrane localization (Abassi and Vuori, 2002; Burton et al., 2003;

Zandy et al., 2007). The functional significance of Crk phosphorylation during adherens

junction formation is likely due to its role in regulating Rac activity, possibly through

regulating its localization to sites of cell‐cell contact. However, the fact that Rac

activation also leads to Crk‐dependent formation of adherens junctions suggest the role

of Crk is complex. In isolated fibroblasts, tyrosine phosphorylation has been shown to

disrupt Crk/CAS complexes, which stimulate cell migration (Klemke et al., 1998).

Conversely, STI571 treatment or Abl/Arg knockdown decrease tyrosine phosphorylation

of Crk at tyrosine 221, which may lead to increased Crk/CAS complex formation as in

Abl‐/‐Arg‐/‐ fibroblasts (Kain and Klemke, 2001). Future studies are needed to examine

how RacV12 and ArgPP expression affect the intermolecular complexes containing Crk

and to determine their relevance to adherens junction regulation.

Moreover, we discovered that ArgPP expression rescues the destabilizing effect

of Crk overexpression on adherens junctions. This suggests that there is a critical

difference in the biological activity of phosphorylated vs. unphosphorylated Crk. Our

results also raise the question of how levels of phosphorylated Crk are “measured” by

the cell: is there an absolute amount of tyrosine phosphorylation of Crk required to

promote adherens junction formation? how does the balance of phosphorylated versus

unphosphorylated Crk affect adherens junctions? and does Crk phosphorylation lead to changes in Rac/Crk localization or duration of signaling?

We have also demonstrated a requirement for Crk/CrkL in RacV12‐induced and

ArgPP‐induced strengthening of adherens junctions. We showed that expression of

constitutively active Rac leads to increased Abl kinase activity and tyrosine

phosphorylation of Crk. Thus, Abl‐Crk signaling may function not only upstream but

also downstream of Rac in a positive feedback loop (Figure 17). Abl activation

downstream of cadherin engagement could promote Rac activation and localization to

nascent cell‐cell contacts. Heightened Rac activity could then reinforce the initial wave

of Abl kinase activation and sustain Crk phosphorylation, leading to recruitment of

more Rac to sites of cell‐cell contact. This positive feedback loop may be required to

generate enough force from actin‐based lamellipodia to hold the cells together during

both the formation and maturation of adherens junction. Follow‐up studies using

fluorescent‐tagged Arg, Crk, and Rac mutants may shed some light on the hierarchical

requirement for each of these molecules in the regulation of one another during

adherens junction formation and maintenance.

α α - catenin β β - catenin

Cadherin

Rac-GTP ROS (H2O2) P β β - cat. Y Crk/ α α - cat. CrkL Y P Abl

Decreased ? ? Rho-GDP contractility & RhoGAP stabilization of cell- cell junctions RhoGEF

Figure 17: Proposed model for Abl‐Crk‐Rac signaling.

The mechanism through which Rac activation leads to increased Abl kinase

activity may be due to generation of ROS in cells. Expression of constitutively active

RacV12 promotes production of ROS (Nimnual et al., 2003). Moreover, ROS was

reported to activate Abl kinases and to promote tyrosine phosphorylation of Crk (Sun et

al., 2000a). Thus, the Rac‐ROS pathway may strengthen adherens junctions by activating

Abl kinases and promoting Crk tyrosine phosphorylation (Figure 17). Generation of

ROS is commonly induced in cancer cells through hyperactivation of growth factor

signaling pathways and Rac activation. In this regard, hyperactivation of the Met receptor leading to Rac‐induced generation of ROS is required for the in vivo pro‐

metastatic behavior of Met (Ferraro et al., 2006). This raises the intriguing possibility that

Abl‐Crk signaling may play a role in this process as well.

Rac activation has been shown to promote both the formation and dissolution of

adherens junctions, dependent on cellular context (Lozano et al., 2003). Our novel

finding that Abl kinase activity is enhanced in the presence of high levels of Rac activity

suggest that Abl kinases may also have opposite effects on adherens junctions. In this

regard, high levels of Abl kinase activity have been detected in metastatic vs. non‐

metastatic breast cancer cells, and blocking Abl kinase activity impaired invasion

(Srinivasan and Plattner, 2006). Similarly, we have observed elevated levels of Abl

kinase activity in metastatic vs. non‐metastatic mouse mammary tumor cell lines (Figure

18). These data raise the larger question of how the biological consequences of elevated

Abl kinase or Rac activity may result in distinct outcomes leading to either formation or

dissolution of adherens junctions. Factors involved in this regulation may include

integration of various signals, levels of signaling, spatio‐temporal regulation, and

downstream effectors. We have identified a positive feedback loop between Abl‐Crk‐Rac

in the context of intercellular adhesion, and this feedback loop may function in other

cellular processes mediated by Abl and Rac signaling, including cell migration,

endocytosis, and processes that require cytoskeletal reorganization.

A 67NR 4T1 4TO7 66c14 IB: pCrk IP: CrkL IB: CrkL B 4T1 4TO7 66c14 67NR IB: Abl/Arg IB: β-tubulin

Figure 18: Hyperactivation of Abl kinase activity in metastatic breast cancer cell lines.

Figure 18: Mouse mammary tumor cell lines 4T1, 4TO7, 66c14, and 67NR were grown to similar confluency (70%) and lysed in 1% Trition lysis buffer in the presence of protease and phosphatase inhibitors. (A) To assess Abl/Arg activity levels in each cell type, lysates were immunoprecipitated with antibody to the Abl substrate CrkL followed by immunoblotting with a phospho Crk antibody which recognizes CrkL Y207, the Abl‐ specific site. (B) Total levels of Abl/Arg protein are shown and β‐tubulin is shown as a loading control. Abl kinase activity is lowest in the non‐metastatic line 67NR and highest in the metastatic cell line 4T1. Interestingly, Abl kinase activity is higher in the 4T1 cell line than in the 66c14 cell line. Unlike the 66c14 cells, the 4T1 cells metastasize in the absence of lymphatics. Abl family kinases may play a role in this process.

4. Role for Abl kinases downstream of the Met receptor

4.1 Background

Dynamic remodeling of cell‐cell adhesions is critical for morphogenetic changes

that occur during normal development and is also required for tumor invasion and

metastasis (Thiery, 2002). Hepatocyte growth factor (HGF) was first characterized as a

physiological factor promoting cell scattering remodeling adherens junctions leading to

dissociation of intercellular contacts (Birchmeier et al., 2003; Stoker et al., 1987).

Subsequently, HGF has been identified as a critical mediator of tumor progression in

various epithelial‐derived tumors (Hurle et al., 2005; Jiang et al., 2005) and is also

implicated in tumor angiogenesis (Rosen et al., 1997). The receptor for HGF, Met, is

upregulated in human breast and prostate cancer patients and is known to promote

metastasis (Birchmeier et al., 2003; Knudsen and Edlund, 2004).

HGF stimulation of Met induces dimerization and autophosphorylation of the

receptor. Autophosphorylation of tyrosine residues 1234 and 1235 in the catalytic

domain is critical for Met activation (Longati et al., 1994; Rodrigues and Park, 1994).

Other phosphorylated tyrosine residues, including tyrosines 1349 and 1356, serve as

docking sites which recruit multiple SH2‐domain containing signaling molecules to the

Met receptor (Ponzetto et al., 1994). Signaling molecules recruited to Met include Grb2,

PI3‐kinase, Src, PLC‐γ, Cbl, Crk, and Gab1. Gab1 is a large adaptor molecule which is

tyrosine phosphorylated and recruited to the Met receptor following Met activation

(Weidner et al., 1996). Gab1 also contains a number of tyrosine residues which, when

phosphorylated, serve as docking sites for the recruitment of additional signaling

molecules and are required for Met signaling (Bardelli et al., 1997; Sakkab et al., 2000).

Both Crk and its family member CrkL have been shown to bind tyrosine phosphorylated

YXXP motifs on Gab1 (Lamorte et al., 2002b; Sakkab et al., 2000). Crk is required for the

morphological changes induced in epithelial cells in response to HGF treatment, and

overexpression of Crk in MDCK cells promotes cell spreading, induction of

lamellipodial protrusions, activation of Rac, and dissolution of adherens junctions, thus

mimicking HGF‐mediated effects (Lamorte et al., 2002b).

The Abl family of nonreceptor tyrosine kinases, Abl and Arg, participate in

signaling cascades leading to a number of cellular responses including regulation of cell

proliferation, survival, response to extracellular stresses, cytoskeletal rearrangments,

and adhesion in various tissues (Pendergast, 2002). Previously, Abl kinase has been

implicated in regulating signaling downstream of the PDGF and EGF growth factor

receptors (Plattner et al., 1999; Tanos and Pendergast, 2006). Abl kinases activate several

downstream molecules, including Crk, Rac, Cdc42, Wave, and Abi1 (Bierne et al., 2005;

Bosse et al., 2007; Burton et al., 2003) which are also activated following HGF stimulation

of the Met receptor (Royal et al., 2000). HGF‐stimulated collagen invasion of MDCK cells

has been linked to several signaling molecules, including N‐WASP and Rac, which

operate downstream of Abl kinases in other signaling pathways (Burton et al., 2005;

Burton et al., 2003; Miao et al., 2003; Yamaguchi et al., 2002). Further, Abl kinase activity

is upregulated in a number of metastatic vs. non metastatic breast cancer cell lines, and

has been shown to mediate invasion (Srinivasan and Plattner, 2006). These observations

led us to investigate whether Abl kinases play a role in modulating the cellular response

to Met activation.

4.2 Results

4.2.1 Abl kinases function downstream of the activated Met receptor tyrosine kinase for induction of cell scattering

The Abl kinase has been shown to regulate motility induced by Met activation

(Abassi and Vuori, 2002; Frasca et al., 2001). To test the hypothesis that Abl kinases

function downstream of HGF and its receptor, the Met tyrosine kinase to induce

breakdown of cell‐cell junctions, we first examined whether HGF treatment of MDCK

epithelial cells could induce activation of the endogenous Abl family kinases. Using

phosphorylation of the Abl substrate Crk as a readout, we observed that the endogenous

Abl kinases are markedly activated as early as 5 minutes after HGF stimulation (Figure

19A). HGF‐induced activation of the Abl kinases persists after 15 minutes of HGF

stimulation. To test whether Abl kinases play a role in HGF‐induced morphogenesis,

A 20 ng/mL HGF untreated 15 min 5 min IB: pCrk IP: CrkL IB: CrkL

B STI571: None +10 µM STI571 HGF: - 15 min HGF 4 h HGF - 15 min HGF 4 h HGF

β-catenin

actin

Figure 19: Abl kinases act downstream of c‐Met following HGF stimulation.

Figure 19: (A) MDCK cells were starved for 16h in 0.25% FBS DMEM prior to stimulation with 20 ng/mL HGF. Cells were lysed in 1% Triton lysis buffer supplemented with protease and phosphatase inhibitors. Abl kinase activity was assessed by immunoprecipitation of CrkL followed by immunoblotting with a phospho‐ specific antibody recognizing Y207 in CrkL, the Abl‐specific site of tyrosine phosphorylation. The blot was stripped and re‐probed for CrkL to demonstrate equivalent protein levels. (B) MDCK cells were seeded onto glass coverslips and starved for 4h. Cells were pre‐treated with vehicle control or with 10μM STI571 for 1h to inhibit endogenous Abl kinase activity. Cells were stimulated with 20 ng/mL HGF for the indicated times and were fixed and stained for β‐catenin (upper panels) and actin (lower panels). In the absence of HGF stimulation, there was no difference between control and STI571‐treated cells. At 15m following HGF stimulation, STI571‐treated cells spread more quickly than control cells. By 4h, cell spreading was similar in both groups. In control cells, cell‐cell contacts dissolved in response to HGF as evidenced by relocalization of β‐catenin to the cytoplasm and extensive remodeling of cortical actin. However, in STI571‐treated cells, β‐catenin remained localized to sites of cell‐cell contact and thick bundles of cortical actin remained in many cells.

Abl kinase activity was inhibited with STI571 prior to stimulation of MDCK epithelial

cells with HGF. HGF stimulation results in morphological changes that lead to cell

scattering and epithelial‐mesenchymal transition (EMT). Cell‐cell adhesion is disrupted

after 4 hours of HGF stimulation (Figure 19B). In contrast, inhibition of the endogenous

Abl kinases with STI571 blocks the dissolution of adherens junctions (stained with β‐

catenin) and inhibits actin remodeling at sites of cell‐cell contact in the presence of HGF

(Figure 19B). Similar effects were observed in A431 epithelial cells (data not shown).

Inhibition of the Abl kinases specifically inhibits HGF‐induced morphological changes,

as in the absence of HGF stimulation no difference is observed between control and

STI571‐treated cells. Thus, the data show that endogenous Abl tyrosine kinase activity is

required for HGF‐induced remodeling of adherens junctions and the actin cytoskeleton.

HGF stimulation of Met results in increased tyrosine phosphorylation of β‐

catenin (Hiscox and Jiang, 1999; Monga et al., 2002). Tyrosine phosphorylation of β‐

catenin on residues 654 and 142 disrupts its association with E‐cadherin and α‐catenin,

respectively (Brembeck et al., 2004; Piedra et al., 2003; Roura et al., 1999).

Phosphorylation at Y654 has been linked to disruption of adherens junctions and

dissociation of the cadherin‐catenin complex (Piedra et al., 2001). We have shown that

expression of activated Abl results in phosphorylation of β‐catenin at Y654 (unpublished

data). We reasoned that STI571 inhibition of Abl kinase activity may block Abl‐mediated

phosphorylation of β‐catenin at Y654, thereby stabilizing the link between the cadherin‐

100

catenin complex and blocking HGF‐induced adherens junction remodeling. However,

we observed no change in levels of overall tyrosine phosphorylation of β‐catenin or of

phosphorylated Y654 (data not shown) suggesting that Abl regulates the physiological

consequence of HGF stimulation in MDCK cells through alternative pathways.

4.2.2 Abl kinase inhibition alters signaling downstream of the activated Met receptor

Tyrosine phosphorylation of Crk downstream of Met activation in MDCK cells

has been previously reported, and Crk was postulated to be a direct substrate of the Met

receptor (Lamorte et al., 2000; Sakkab et al., 2000). Our data showing that tyrosine

phosphorylation on the Abl‐specific site of Crk suggested that Abl may be responsible

for Crk phosphorylation. To test this, we treated MDCK cells with HGF in the presence

or absence of STI571 and examined phosphorylation of Crk. We observed increased

tyrosine phosphorylation of Crk in HGF stimulated cells; however, phosphorylation was

not induced in the presence of the Abl kinase inhibitor (Figure 20). A direct role for Abl

in Crk phosphorylation is further supported by the recent finding that HGF‐induced

phosphorylation of Crk in HeLa cells is inhibited by both STI571 treatment and

expression of a dominant negative, kinase‐dead form of Abl (Abassi and Vuori, 2002).

Abl has previously been identified in a screen for binding partners of Met

(Weidner et al., 1996). Recently, our lab demonstrated that Abl associates with EGFR and

101

20 ng/mL HGF 0 min 5 min 15 min 30 min control control contol control +STI +STI +STI +STI

IB: pCrkL Y207

IB: Crk

Figure 20: Abl kinase inhibition blocks tyrosine phosphorylation of Crk in response to Met activation.

Figure 20: MDCK cells were starved for 16h in 0.25% FBS DMEM prior to stimulation with 20 ng/mL HGF. Cells were pre‐treated with vehicle control or with 10μM STI571 for 1h to inhibit endogenous Abl kinase activity. Cells were lysed in 1% Triton lysis buffer supplemented with protease and phosphatase inhibitors. Abl kinase activity was assessed by immunoblotting with a phospho‐specific antibody recognizing Y207 in CrkL, the Abl‐specific site of tyrosine phosphorylation. The blot was stripped and re‐ probed for CrkL to demonstrate equivalent protein levels.

102

directly phosphorylates tyrosine residues in the EGFR cytoplasmic tail to regulate

endocytosis (Tanos and Pendergast, 2006). We hypothesized that Abl may play a similar

role in Met receptor regulation. To test this, we stimulated MDCK cells with HGF in the

presence or absence of STI571 and examined phosphorylation of the Met receptor using

phospho‐specific antibodies. Autophosphorylation of tyrosine residues 1234 and 1235 in

the catalytic domain is critical for Met activation (Longati et al., 1994; Rodrigues and

Park, 1994). We observed significantly decreased phosphorylation of both Y1234/1235 in

the presence of Abl kinase inhibition (Figure 21). This data suggests that while

autophosphorylation of Y1234/1235 following dimerization of Met molecules may

initiate signaling, Abl‐mediated transphosphorylation of Y1234/1235 may be required

for sustained signaling. Phosphorylation of tyrosine 1003 of the Met receptor is required

for Cbl‐mediated ubiquitination and internalization of Met (Peschard et al., 2004). We

examined phosphorylation of Y1003 in response to HGF stimulation in the presence or

absence of STI571 and observed a reduction in tyrosine phosphorylation in the presence

of Abl kinase inhibition suggesting that Y1003 is another potential target of the Abl

kinases (Figure 21). A Met receptor mutant in which this tyrosine has been mutated to phenylalanine (Met Y1003F) shows increased receptor stability, prolonged activation of

signaling pathways, including the MAPK pathway, and enhanced transformation in

response to HGF stimulation (Abella et al., 2005). We examined the effect of Abl kinase

inhibition on Erk activation following HGF treatment and observed both elevated levels

103

20 ng/mL HGF 0 min 5 min 15 min 30 min control control contol control +STI +STI +STI +STI IB: pY1003 Met

IB: pY1234/1235 Met

IB: Met

IB: pErk

IB: Erk

IP: Gab1 IB: ptyr

Figure 21: Inhibition of Abl kinase activity affects Met signaling downstream of HGF stimulation.

Figure 21: MDCK cells were starved for 16h in 0.25% FBS DMEM prior to stimulation with 20 ng/mL HGF. Cells were pre‐treated with vehicle control or with 10μM STI571 for 1h to inhibit endogenous Abl kinase activity. Cells were lysed in 1% Triton lysis buffer supplemented with protease and phosphatase inhibitors. Tyrosine phosphorylation of Met was evaluated using phospho‐specific antibodies to Y1234/1235 and Y1003. Erk activation was assessed using a phospho‐specific antibody. Total protein levels for Met and Erk are indicated. Tyrosine phosphorylation of Gab1 was assessed by immunoprecipitation of Gab1 followed by immunoblotting with antibody to phosphotyrosine.

104

of pErk as well as prolonged phosphorylation (Figure 21) similar to the effect of the Met

Y1003F mutant (Abella et al., 2005). This data is consistent with the finding that pErk is

elevated in thyroid cancer cells treated with STI571 and HGF (Frasca et al., 2001). Thus,

Abl kinases alter phosphorylation of the Met receptor in response to HGF stimulation,

possibly through direct phosphorylation, and alter downstream signaling.

Gab1 is a large adaptor molecule recruited to the Met receptor following Met

activation, which contains 6 YXXP sites that become tyrosine phosphorylated following

HGF stimulation (Weidner et al., 1996). A number of tyrosine residues, when

phosphorylated, serve as docking sites for the recruitment of additional signaling

molecules and play a critical role in Met signaling (Bardelli et al., 1997; Sakkab et al.,

2000). The Crk adaptor protein is protein is recruited to tyrosine 307 of Gab1 and is

required for sustained Gab1 phosphorylation and physiological response to HGF

stimulation in synovial sarcoma cells (Watanabe et al., 2006). We and others have

identified Crk phosphorylation downstream of HGF stimulation as an Abl‐mediated

process in other cell types; therefore, we hypothesized that Abl may play a role in

regulating Gab1 phosphorylation downstream of Met activation. To test this, we

examined HGF‐stimulated phosphorylation of Gab1 in the presence or absence of

STI571. We observed a nearly complete loss of Gab1 tyrosine phosphorylation in

response to HGF in the presence of Abl kinase inhibition, with no change in total Gab1

suggesting that Abl kinases are required for Gab1 phosphorylation (Figure 21). Abl‐

105

mediated phosphorylation of Gab1 may occur directly or indirectly through Abl

regulation of Met and/or Crk tyrosine phosphorylation in response to HGF treatment.

4.3 Discussion

Here we identify the Abl family of tyrosine kinases as critical mediators of HGF‐

stimulated dissolution of adherens junctions in epithelial cells. Pharmacological

inhibition of Abl kinase activity blocks re‐localization of β‐catenin to the cytoplasm and

remodeling of cortical actin in response to Met receptor activation. Further, we show that Abl kinase activity is increased in response to HGF stimulation resulting in the

tyrosine phosphorylation of Crk at the Abl‐specific site. We observed no detectable

changes in the levels of tyrosine phosphorylation of β‐catenin in cells treated with HGF

and the Abl kinase inhibitor STI571, suggesting that the effect of Abl is not due to direct

regulation of the cadherin‐catenin complex. We have previously shown that Abl is

required for adherens junction formation (Zandy et al., 2007); thus, we have

demonstrated a requirement for Abl in both dissolution and formation of adherens

junctions. This is not unsurprising considering that Rac activation leading to actin

polymerization and Src kinase activity have been shown to be required for both

processes (Avizienyte et al., 2002; Calautti et al., 1998; Ehrlich et al., 2002; Noren et al.,

2001; Royal et al., 2000). A simple explanation may involve the degree to which Abl

kinase activity is activated: cadherin‐activation results in activation of the Abl kinases,

but at a relatively low level compared to activation following HGF stimulation. Perhaps

106

a low basal level of Abl kinase activity is required for the maintenance of adherens

junctions, but high levels of Abl kinase activity may result in the phosphorylation of

different targets which play a negative role in regulating adherens junctions. In this

regard, a recent report demonstrated similar requirement for Src kinase activity

(McLachlan et al., 2007).

Significantly, we show for the first time that Met receptor phosphorylation and

activation is impaired in the absence of Abl kinase activity. Interestingly, a previous

report found that STI571 treatment of thyroid cancer cells led to increased tyrosine

phosphorylation of the Met receptor in response to HGF stimulation (Frasca et al., 2001).

This suggests that the consequences of Abl kinase inhibition may be context‐dependent.

While we examined immortalized “normal” canine kidney cells, this report used highly

motile cancer cells, in which other growth factor receptor signaling pathways or

downstream targets may be upregulated. Another difference may be the presence of

strong adherens junctions in MDCK cells as compared to the cells examined in the other

study. In this regard, Abl may act to integrate signaling from adhesion receptors, such as

E‐cadherin, and the Met receptor with adhesion playing a role in determining the consequence of Met activation and vice versa.

In addition, we showed decreased phosphorylation of Gab1, a critical regulator

of Met signaling. Crk has been shown to be required for tyrosine phosphorylation of

Gab1, and phosphorylation of Y307 of Gab1 was nearly eliminated in cells treated with

107

the Abl kinase inhibitor. Several possibilities exist to explain this result: Gab1 may be

phosphorylated directly by Abl or Gab1 may be phosphorylated by the Met receptor,

itself, or by another downstream kinase. In this regard, we showed that Abl kinase

inhibition decreased phosphorylation of Met at the autophosphorylation site

Y1234/1235, which is correlated with its activity. Alternatively, Abl‐mediated

phosphorylation of Crk may be required for Gab1 activity. This hypothesis is supported

by the finding that knockdown of Crk in synovial sarcoma cell lines led to decreased

phosphorylation of Gab1 as well as impaired cell scattering in response to HGF

stimulation (Watanabe et al., 2006). Our observation that Abl kinase inhibition also

inhibits the cell scattering response to HGF suggests that tyrosine phosphorylation of

Crk rather than the presence or absence of Crk protein determines cellular response to

HGF stimulation.

Our finding that Abl kinase inhibition leads to decreased phosphorylation of Met

at Y1003 and increased phosphorylation of Erk in response to HGF stimulation suggests

that Abl may play an additional role in signal attenuation. Cbl is recruited to Y1003 of

the Met receptor phosphorylation and is required for monoubiquitination of the Met

receptor, thereby promoting lysosomal degradation (Abella et al., 2005). The mutant Met

receptor Y1003F displays sustained signaling, including enhanced, prolonged

phosphorylation of Erk, thus mimicking the effect of Abl kinase inhibition. Future

studies will be directed at evaluating the contribution of Abl kinases to the various steps

108

in this process and to determine whether, similar to Met Y1003F, inhibition of Abl kinase

activity promotes transformation (Abella et al., 2005). In this context, Abl may

participate in a negative regulatory feedback loop with the Met receptor. In agreement

with this hypothesis, a recent study suggested that Abl‐mediated phosphorylation of

Crk acts as a negative regulator of cell motility downstream of Met signaling in HeLa

cells stimulated with HGF or in fibroblasts expressing the Tpr‐Met oncoprotein (Abassi

and Vuori, 2002).

Taken together, these results suggest that Abl may play both a positive and

negative regulatory role in Met receptor signaling. One possible explanation for this

phenomenon is that Abl kinase activation of different targets downstream of Met

activation is governed through spatio‐temporal regulation. Alternatively, the biological

consequence of Abl kinase activity may be determined by cell type. Cells with weak

intercellular adhesions, which display enhanced motility in response to HGF stimulation

may utilize Abl kinase activity to attenuate Met signaling. While cells with strong

intercellular adhesions may require Abl kinase activity to activate and sustain Met

signaling. In this regard, Abl kinases have been shown to play a role in cellular

processes downstream of diverse signaling molecules including growth factor receptors,

integrins, Src family kinases, and reactive oxygen species (Pendergast, 2002). Perhaps

these pathways converge on Abl, whose job it is to interpret the complex message and

pass it along to elicit the proper functional consequence.

109

5. Materials and Methods

5.1 Antibodies and Reagents

Antibodies and chemical reagents used were: N‐cadherin, β‐tubulin, actin, all

mouse monoclonal (Sigma), pCrkL Y207, pCrk Y221, pMLC, all rabbit polyclonal (Cell

Signaling Technology), E‐cadherin 61045 and 610182, β‐catenin, p120 catenin, p190

RhoGAP, Crk, all mouse monoclonal (BD Transduction Labs), α‐catenin, mouse

monoclonal (Zymed), Abl K12, CrkL, Met sc‐162, all rabbit polyclonal (Santa Cruz), Abl

8E9, mouse monoclonal (BD Pharmingen), Rac, Rho, both mouse monoclonal(Pierce),

GFP (Roche), Gab1 06‐579 rabbit polyclonal (Upstate Biotechnology), anti‐pY1003 Met

AF4059 and anti‐pY1234/2345‐Met AF2480, both rabbit polyclonal (R & D Systems),

CY3‐donkey anti‐mouse, CY3‐donkey anti‐rabbit, CY2‐donkey anti‐mouse (Jackson

ImmunoResearch), Alexa488‐conjugated phalloidin, protein A‐Sepharose, protein G‐

Sepharose, HRP‐linked protein A, ECL western blotting reagents (Amersham), HRP‐

linked goat anti‐mouse IgG (Santa Cruz), HRP‐linked goat anti‐rabbit IgG, DAPI

(Molecular Probes), Y27632 (Calbiochem), human basic FGF 100‐18B (PeproTech),

mouse VEGF V4512 and human HGF H1404 (Sigma). Antibody to Arg was produced by

immunizing rabbits with a peptide specific for a unique C‐terminal region (Finn et al.,

2003). STI571 was a gift from B. Druker (Oregon Health Sciences University). The pK1

110

Arg GFP plasmid was obtained from A. Koleske (Yale University). pK1 ArgPP (Plattner

et al., 2004), MigR1 RacV12 and MigR1 CrkYF (Burton et al., 2003) plasmids were

generated as described.

5.2 Cell Lines

Abl‐Arg double null mouse embryo fibroblasts (MEFs) were obtained from A.

Koleske (Yale University) and reconstituted with Abl and Arg as previously described

(Finn et al., 2003; Plattner et al., 2003). MEFs, HeLa, NBT‐II, MDCK , MCF‐7, A431, and

PY‐4‐1 cells were maintained in Dulbeco’s modified eagle medium (DMEM)

supplemented with 10% fetal bovine serum (FBS) (Koleske et al., 1998; Plattner et al.,

2003). MCF‐10A human epithelial cells were maintained in DMEM/F12 supplemented

with 5% donor horse serum, hEGF (20 ng/mL), hydrocortisone (500 ng/mL), insulin (10

μg/mL), and cholera toxin (100 ng/mL). 67NR, 66c14, 4TO7 and 4T1 cells were grown in

DMEM supplemented with 10% FBS, 1mM non‐essential mixed amino acids, and 2mM

L‐glutamine.

5.3 Retroviral Transduction

Transduction of MDCK, HeLa, MCF‐10A, and MEF cells was performed as

described (Grove et al., 2004; Reya et al., 2003). Viral supernatant was harvested from

transfected 293T cells and target cells were infected for 4‐6 h. After 48h, cells were either

111

plated for experiments or sorted for GFP. Cells transduced with GFP‐Arg infected cells

were subjected to FACS sorting to select a heterogeneous GFP‐positive population.

5.4 Cell lysis, immunoblotting, in vitro kinase assays, and RhoGTPase activation assays

Cells were lysed in either Triton lysis buffer (1% Triton‐X‐100, 150 mM NaCl, 50

mM Tris pH 7.5) or RIPA buffer (0.5 M NaCl, 1% NP‐40, 0.5% sodium deoxycholate,

0.1% SDS, 50 mM Tris pH 8.0) plus inhibitors (1mM phenylmethylsulfonyl fluoride,

2mM sodium pyrophosphate, 1 mM sodium orthovanadate, 25 mM β‐glycerophosphate,

and 1 μg/mL each of leupeptin, aprotinin, and pepstatin A). In vitro kinase assays were

performed as previously described (Plattner et al., 1999). Rac/Rho activation assays

were performed according to manufacturer’s instructions using the EZ‐Detect Rac or

Rho activation kit (Pierce). Briefly, cells were lysed in lysis/binding/wash buffer plus

inhibitors (1mM phenylmethylsulfonyl fluoride, Results of 3‐4 independent

experiments were used for quantitation of data. ImageJ Software was used to quantify

relative band intensity normalized to the time 0 timepoint. Control and experimental

conditions at each timepoint were compared and analyzed for statistical significance

using ANOVA followed by the paired Student’s T test (two‐tailed analysis, Excel).

112

5.5 Immunofluorescence

Cells plated on glass coverslips were fixed with 4% (w/v) PFA for 10 min,

permeabilized by the addition of 0.25% TritonX‐100 for 5 min, and incubated in block

(2% BSA in PBS). Antibodies were diluted in block as follows: N‐cadherin (1:100), E‐

cadherin (1:200), β‐catenin (1:300), α‐catenin (1:100), 488 phalloidin (1:1000), p190 (1:200),

pMLC pAb (1:100), CY3 donkey anti‐mouse secondary (1:1000), CY3 donkey anti‐rabbit

secondary (1:1000), CY2 donkey anti‐mouse secondary (1:600), DAPI (1:25,000).

Fluorescence microscopy was carried out at 40x or 63x using a Zeiss Axioskop

microscope equipped with a Hamamatusu ORCA‐ER digital camera or at 40x or 63x

using a Zeiss Axiovert 200M equipped with AxioCamMRm high resolution. Images

were analyzed using Metamorph software (Universal Imaging Corp.) or AxioVision 4.5

(Zeiss). Statistical analysis of adherens junction integrity in untreated vs. STI571‐treated

cells was performed using the paired Student’s T test (two‐tailed analysis, Excel or

Graphpad software). All other statistical analyses using multiple conditions were

performed using one‐way ANOVA analysis followed by Bonferroni’s multiple

comparison test (Graphpad software).

113

5.6 Calcium Switch Experiments

MEFs, NBT‐II, MCF‐10A, or MDCK cells were grown to confluency, washed with

PBS, and DMEM containing 10% FBS and 2mM EGTA was added overnight. The

following day, medium was replaced with DMEM containing 10% FBS. For Abl kinase

activity assays, NBT‐II cells were seeded onto glass coverslips, grown to subconfluency,

washed with PBS, and medium was replaced with KBM‐2 containing bovine pituitary

extract (BPE), human epidermal growth factor (hEGF), insulin, hydrocortisone, GA‐

1000, epinephrine, transferrin, and 30 μM calcium. The following day, medium was

replaced with KBM‐2 as described above containing 1.8 mM calcium for the indicated

times.

5.7 siRNA

Cells were transfected with Oligofectamine (Invitrogen) and control or the

indicated duplex oligonucleotides according to the manufacturer’s instructions.

siControl Non‐Targeting siRNA Pool (D‐001206‐13) , siGENOME SMARTpool reagents

human Abl1(M‐003100), rat Abl1 (M‐090649‐00), human Abl2(M‐003101), rat Abl2 (M‐

082758‐00), and ON‐TARGETplus Duplex reagents human CrkL (J‐012023‐07), human

Crk (J‐010503‐07), rat CrkL (J‐087587‐12) and rat Crk (J‐090657‐09) were purchased from

Dharmacon. Cells were lysed 48 h following transfection. For immunostaining, siGLO

114

RISC‐Free siRNA (Dharmacon) was co‐transfected to identify cells containing control or

Abl1 and Abl2 siRNAs.

5.8 Growth factor stimulation

MDCK or A431 cells were grown to subconfluency in 100mm plates or on glass

coverslips and starved O/N in DMEM containing 0.25% FBS. Using conditioned

medium, cells were pre‐treated or not with 10 μM STI571 for 1 h, followed by treatment

with 20 ng/mL HGF for the indicated times. PY‐4‐1 cells were treated with 50 ng/mL

VEGF, and MEFs and MCF‐7 cells were treated with 20 ng/mL FGF‐2 in a similar

manner.

115

6. Conclusion

Regulation of intercellular adhesion is tightly controlled during development

and contributes to a number of biological processes including epithelial morphogenesis,

vasculogenesis, and neural development (Thiery, 2003). Conversely, deregulation of

intercellular adhesion plays a critical role during cancer progression, contributing to

EMT, and promoting escape, invasion, and metastasis (Thiery, 2002). As is often the

case, signaling pathways that play a role in normal development have been identified as

being hijacked by tumor cells in order to grow and disseminate.

The cadherin‐based cell adhesion complex, adherens junction, plays a critical role

in development, and loss of adherens junctions has been implicated in tumorigenesis. In

fact, loss of or mislocalization of members of the cadherin‐catenin complex has been

identified in a variety of epithelial‐derived cancers (Christofori, 2003). Tumors may also

have elevated levels of tyrosine kinase activity as well as deregulated signaling through

Rho GTPases, which are two of the primary mechanisms through which adherens

junctions are regulated (Steinberg and McNutt, 1999). We have described the role of the

Abl family of tyrosine kinases as critical mediators of intercellular adhesion, particularly

through regulation of the Rho GTPases (Zandy et al., 2007). We propose that further

understanding this pathway in the context of normal and cancer cells will provide

insight into potential new therapeutic interventions.

116

One area of adherens junction biology that is poorly understood is the

mechanism through which cross‐talk between growth factor receptors and cadherins is

regulated. Reports of interactions between EGFR and E‐cadherin, VEGFR and VE‐

cadherin, and FGFR and N‐cadherin pepper the cadherin literature, yet the significance and generalizability of these findings remains unclear (Brunton et al., 2004). We and

others have previously shown that Abl kinases are activated downstream of PDGF and

EGF stimulation and play a role in cytoskeletal reorganization (Boyle et al., 2007;

Plattner et al., 1999; Sini et al., 2004; Tanos and Pendergast, 2006). We have also

described the role of Abl in regulating signaling downstream of cadherin molecules

(Zandy et al., 2007). Therefore, the Abl kinases, acting downstream of both RTKs and

cadherins, appear to be perfectly positioned to integrate signaling from the two inputs

and to regulate cross‐talk between them.

The effect of Met receptor activation on adherens junctions is probably the most

well‐characterized RTK/cadherin interaction. Met and E‐cadherin have been shown to

interact physically, and HGF stimulation of Met has been shown to promote

relocalization of β‐catenin to the cytosol, thereby disrupting adherens junctions

(Birchmeier et al., 2003). We have now shown that Abl kinases are required for

disruption of adherens junctions downstream of Met activation and play a role in

modulating Met signaling. While the mechanism through which Abl acts is at this time

unclear, our data suggest that spatio‐temporal regulation of Abl kinase activity is

117

important and that the biological consequence of Abl activation is likely to be cell‐type‐

and context‐dependent. This novel finding raises the exciting possibility that the Abl

kinases act downstream of other RTK/cadherin interactions to integrate signaling and

regulate cytoskeletal changes.

6.1 Delving deeper into signaling downstream of cadherins

The role of Abl kinases in regulating adherens junctions was first suggested by

loss of function studies in Drosophila and recently corroborated by the finding that

exogenous Abl localizes to adherens junctions in Drosophila (Grevengoed et al., 2001;

Stevens et al., 2007). Our studies have confirmed that the Abl kinases play a critical role in promoting adherens junction formation and maintenance and have extend this

finding to mammalian cells. We provide the first evidence of cadherin activation of Abl

kinases in both fibroblasts (N‐cadherin) and epithelial cells (E‐cadherin), suggesting that

the relatively conserved cytoplasmic tail of cadherins is responsible for this effect. We

also showed that Abl kinases are recruited to nascent sites of cell‐cell contact and are

found in complex with E‐cadherin and β‐catenin. At this time, the mechanism of Abl

activation and recruitment to the cadherin‐catenin complex remains unclear. Our lab has

previously shown that Src is required for activation downstream of PDGF stimulation

(Plattner et al., 1999). Src has recently been shown to be activated downstream of

cadherin engagement, and is postulated to function upstream of PI 3‐kinase activation

118

(McLachlan et al., 2007). Therefore, the role of Src kinases in Abl activation downstream

of cadherins should be investigated.

Another avenue for future study is evaluating the requirement for individual

domains in the Abl kinases. Our finding that pharmacological inhibition and siRNA‐

knockdown of Abl and Arg result in the same phenotype suggest a critical role for the

Abl kinase domain in regulating cadherin signaling, however it is unknown whether

kinase activity is required for localization to sites of cell‐cell context or interaction with

the adhesion complex. Dissecting the role of SH3 and SH2 domain‐mediated protein‐

protein interactions as well as contributions from the F‐actin binding domain of the

cytoplasmic tail may shed some light on this process. Re‐introduction of both Abl and

Arg into cells lacking both Abl family members may also reveal individual roles for

each. In this regard, Abl, but not Arg, was shown to rescue PLC‐γ-mediated increased chemotaxis in response to PDGF in Abl‐/‐Arg‐/‐ cells, in spite of the fact that both

kinases are activated and form complexes with members of the PDGFR signaling

components (Plattner et al., 2004).

To date, the bulk of evidence has suggested that tyrosine kinases directly target

members of the cadherin‐catenin complex, particularly β‐catenin, to regulate adherens

junctions (Brunton et al., 2004). Additionally, tyrosine phosphorylation was thought to

primarily play a negative regulatory role (Brunton et al., 2004). Our studies provide

evidence of positive regulation of cadherin‐based adhesion through a tyrosine kinase,

119

whose effects do not involve directly targeting the cadherin‐catenin complex, but rather

downstream effectors. We showed that Abl regulates the activity of Rho GTPases

downstream of cadherin engagement. This represents a significant contribution to

understanding how signals are transduced through cadherin molecules to the Rho

GTPases resulting in actin polymerization. Further, this represents another pathway in

which Abl acts upstream of Rac to promote its activation. In this regard, Abl is required

for Rac activation downstream of both growth factor receptor stimulation and bacterial

infection (Burton et al., 2003; Sini et al., 2004). The inhibitory effect of the Abl kinases on

Rho signaling has also been observed in the context of integrin engagement (Bradley et

al., 2006). The mechanism through which Abl modulates Rho activity remains unclear.

Abl may inhibit Rho through intermediates like p190 RhoGAP, which has been

identified as a substrate for Arg (Bradley et al., 2006; Hernandez et al., 2004) or Abl‐

mediated activation of Rac may lead to inhibition of Rho activity (Nimnual et al., 2003;

Wildenberg et al., 2006). It seems likely that the Abl kinases contribute to regulation of

Rac and Rho activity in other signaling cascades and should be examined.

6.1.1 Role for Abl and Crk

Our studies led to the unexpected observation that Abl and Crk promote

adherens junction formation and stability by acting both upstream and downstream of

activated Rac. This is the first evidence of a positive feedback loop downstream of

cadherin engagement, which could help explain the sustained activation of Rac during

120

this process. We found that Abl phosphorylates Crk in response to cadherin

engagement, and that Abl activity and Crk phosphorylation are required to stimulate

Rac activation in this context. Phosphorylation of Crk is required for Rac localization to

the plasma membrane and activation in other contexts, however the exact mechanism

through which it acts is unknown (Abassi and Vuori, 2002; Burton et al., 2003). The Rac

GEF DOCK180 is an attractive candidate downstream of Crk (Kiyokawa et al., 1998).

Alternatively, Abl kinases may promote Rac activation independently of Crk. In this

regard, Abl‐mediated activation of Rac downstream of growth factor signaling requires

direct phosphorylation of the Rac Gef Sos‐1 (Sini et al., 2004).

The significance of Crk tyrosine phosphorylation was highlighted by the finding

that overexpression of Crk, which led to increased levels of unphosphorylated Crk,

promoted the disruption of adhesion. This phenotype was rescued by co‐expression with ArgPP, which suggested that the overabundance of unphosphorylated Crk was

responsible for the dissolution of adherens junctions. Therefore, evaluating the

biological consequence of loss of Crk should be accompanied by studies examining its

phosphorylation. Tyrosine phosphorylation of Crk has been shown to alter complex

formation with other molecules, including p130 Cas, and this regulation has been

implicated in regulating cell migration and focal adhesion formation (Stupack et al.,

2000; Takino et al., 2003). The combination of knocking down Crk family proteins using

RNAi and expressing Crk WT or the mutant Crk Y221F will allow the signaling

121

contributions of unphosphorylated vs. phosphorylated Crk to be dissected. Our findings

also suggest the possibility that unphosphorylated Crk may play a dominant role over

phosphorylated Crk as the levels of phosphorylated Crk did not decrease over baseline

in cells overexpressing Crk.

We also observed that expression of constitutively active RacV12 resulted in

increased Abl kinase activity and Crk tyrosine phosphorylation. This data suggests that

one way in which actin polymerization may be sustained throughout the hours‐long

process of adherens junction formation is through this feedback loop. It also highlights

the difficulty of studying this dynamic process. The only way to truly understand the

hierarchy of signaling is to dissect the signaling pathway in a temporal manner. This

could be done by examining the timing of recruitment/activation of Abl‐Crk‐Rac via live

imaging. The mechanism through which Rac activation stimulates Abl activity is

unknown, but may occur through Rac‐mediated generation of ROS. Reactive oxygen

species have been shown to promote Abl kinase activation and tyrosine phosphorylation

of Crk (Sun et al., 2000b). Future studies using ROS scavengers will be helpful in

determining the potential significance of this pathway.

6.1.2 Abl and p190 RhoGAP

Whereas p190 RhoGAP is an Arg substrate and is implicated in Arg‐mediated

regulation of neural development and integrin engagement (Bradley et al., 2006;

Hernandez et al., 2004), our studies do not support a role for p190 RhoGAP in the

122

regulation of adherens junctions downstream of the Abl kinases in epithelial cells or

fibroblasts. p190‐A RhoGAP was recently shown to function with p120 catenin in the

regulation of cell‐cell adhesion in mouse embryo fibroblasts (MEFs) expressing

constitutive active Rac‐V12 (Wildenberg et al., 2006). We found no evidence that p190‐A

(endogenous or exogenously expressed) localizes to adherens junctions in the epithelial

or fibroblast cell lines we studied (Figure 22B). Thus, we expressed activated DA‐Rac in

fibroblasts (MEFs) and did not observe detectable p190 RhoGAP localization at cell‐cell

junctions in these cells or in MEFs expressing activated Arg, which we have shown

induces strengthening of adherens junctions similar to DA‐Rac (data not shown).

Moreover, we detected no change in junctional integrity of p190‐A null MEFs and

pEGFP‐p190‐A reconstituted null MEFs in the absence or presence of DA‐Rac or

activated Arg‐PP (Figure 22A). However, we observed on unexplained and puzzling

finding in p190‐A null MEFs, which had been reconstituted with pEGFP‐p190A: in all

cells reconstituted with p190, tyrosine phosphorylation of CrkL at the Abl‐specific site

was elevated as compared to control cells (Figure 22C). Additionally, we observed

increased phosphorylation of CrkL Y207 in MEFs expressing both constitutively active

Arg and Rac, further corroborating evidence observed in epithelial cells (Figure 22C).

This suggests the intriguing possibility that p190 RhoGAP may play some role in the

Abl‐Crk‐Rac positive feedback loop we have postulated.

123

A IF: β-catenin B IF: p190 RhoGAP p190 -/- +pEGFP p190 WT +pEGFP p190 WT NBT-II cells vector vector

C p190 -/- + p190 WT DA-Rac ArgPP vector DA-Rac vector DA-Rac ArgPP IB: pCrkL Y207

IB: Abl/Arg ArgPP E NBT-II NBT-II D MCF-10A Abl/Arg STI571 Control siRNA: Abl1/2 Control Cntrl 0 h 3 h siRNA: Abl/Arg IP: P190 ptyr IP: P120 IB: p190 RhoGAP RhoGAP p190 RasGAP IB: p120 RasGAP IP: P190 p120 IB: p190 RhoGAP catenin WCL RhoGAP IB: p120 RasGAP p190

Figure 22: Lack of evidence for role of Abl kinase modulation of p190 RhoGAP effect on adherens junctions.

124

Figure 22: (A) p190 RhoGAP null or pEGFP p190RhoGAP‐reconstituted MEFs were obtained from Jeff Settleman (Harvard). MEFs were transduced with retroviruses encoding DA‐Rac, ArgPP, or vector alone, grown to near confluency and fixed and stained with antibody against β‐catenin (red) and counterstained with DAPI (blue) to evaluate adherens junctions. (B) pEGFP p190RhoGAP‐reconstituted MEFs obtained from the Settleman lab (left panel) or NBT‐II epithelial cells (right panel) were fixed and stained with antibody against p190 RhoGAP (red) and counterstained with DAPI (blue) to assess p190 localization. (C) p190‐null and pEGFP p190RhoGAP‐reconstituted MEFs obtained from the Settleman lab expressing DA‐Rac, ArgPP, or vector alone were lysed in 1% Triton‐X‐100 lysis buffer containing protease and phosphatase inhibitors. A phospho‐specific antibody to CrkL Y207 was used to assess levels of CrkL phosphorylation at the Abl‐specific site, and 8E9 was used to evaluate Abl/Arg protein expression. (D) MCF‐10A cells (left panels) were transfected with control or Abl‐ and Arg‐directed siRNAs or were treated or left untreated with 10 μM STI571. NBT‐II cells (right panels) were transfected with control or Abl‐ and Arg‐directed siRNAs. Cells were lysed in 1% Triton‐X‐100 lysis buffer containing protease and phosphatase inhibitors. p120 RasGAP was immunoprecipitated from 500 μg lysate, followed by immunoblotting for p190 RhoGAP. Blots were stripped and re‐probed for p120 RasGAP. The indicated antibodies were used to detect p120 RasGAP and p190 RhoGAP in cell lysates. (E) NBT‐II cells were transfected with control or Abl‐ and Arg‐directed siRNAs and lysed in 1% Triton‐X‐100 lysis buffer containing protease and phosphatase inhibitors. p190 RhoGAP was immunoprecipitated from 1 mg lysate, followed by immunoblotting for phosphotyrosine or p120 catenin as indicated. Blots were stripped and re‐probed for p190 RhoGAP.

125

We also investigated whether loss of Abl kinases induced by knockdown of

Abl/Arg, or treatment with the Abl kinase inhibitor STI571 affected p190 RhoGAP tyrosine phosphorylation or the formation of P190 protein complexes with either p120

RasGAP or p120 catenin. In response to integrin engagement, p190 RhoGAP is phosphorylated by Arg which promotes its binding to p120 RasGAP (Bradley et al.,

2006; Hernandez et al., 2004). We did not observe any changes in tyrosine phosphorylation of p190 RhoGAP in epithelial cells lacking Abl kinases or in the formation of p190/p120 RasGAP or p190/p120 catenin complexes in response to cadherin engagement in the absence of Abl kinases compared to control cells (Figure 22, E). Thus, together our data suggest that Abl targets other than p190 RhoGAP mediate the Abl‐ dependent regulation of adherens junctions. Similarly, loss of p190 RhoGAP is dispensable for PDGF‐induced formation of dorsal ruffles, a process mediated by Abl kinases (Boyle et al., 2007; Plattner et al., 1999). Thus, whereas Arg and p190 RhoGAP activities are linked in response to integrin engagement, their functions appear to be uncoupled in other processes such as growth factor induced dorsal ruffle formation and cadherin‐dependent adherens junctions.

However, the possibility exists that Abl may signaling through p190 RhoGAP regulates adherens junctions in other contexts. It is possible that the contribution of Abl‐ mediated p190 phosphoryation was undetectable in the cells we studied, but could play a more prominent role in other cell types. For example, Src‐mediated phosphorylation of

126

p190 RhoGAP has been shown to regulate EGFR signaling (Haskell et al., 2001a; Haskell

et al., 2001b). Therefore, the contribution of Src to p190 RhoGAP phosphoryation may

obscure Abl‐mediated changes, particularly when they occur at such a specialized site as

the adherens junction. Similarly, the p190 RhoGAP contribution to adherens junction

regulation may be especially prominent when cadherin signaling is particularly strong.

For example, induction of p190 RhoGAP phosphorylation was initially observed by

directly activating cadherins using the soluble cadherin extracellular domain (Noren et

al., 2003; Wildenberg et al., 2006).

6.1.3 Abl and other downstream effectors

Our data reveal a critical role for Crk phosphorylation downstream of Abl

activation in regulating adherens junction formation and stability. However, there are

certainly other candidate effector molecules, which may act as Abl targets downstream

of cadherin signaling. In cells expressing constitutively activated ArgPP, we observed

slightly elevated levels of p120 catenin at tyrosine 228 (data not shown).

Phosphorylation of Y228 on p120 catenin by Src has been shown to promote binding of

p120 catenin to Rho, thereby promoting Rho inhibition (Castano et al., 2007). This

suggests a potential mechanism through which expression of ArgPP leads to

strengthening of intercellular adhesions. Arg‐mediated tyrosine phosphorylation of

p120 catenin could lead to enhanced binding and inhibition of Rho activity. Rho

inhibition is required for the formation of cadherin‐mediated cell contacts, and our data

127

suggest that Abl normally functions to maintain low levels of Rho activity in confluent

cells. We did not detect any difference in p120 catenin phosphorylation in Abl loss of

function studies, suggesting that p120 catenin may only be a substrate for Abl kinases

when they are overexpressed or hyperactivated.

Cortactin represents another possible Abl‐mediated substrate downstream of

cadherin engagement. Recently, Arg was shown to phosphorylate cortactin in response

to PDGF stimulation, and cortactin has been shown to function downstream of E‐

cadherin and N‐cadherin to promote intercellular adhesion (Boyle et al., 2007; El Sayegh

et al., 2004; Helwani et al., 2004). Both loss and gain of function studies with Abl and

Arg will be required to evaluate whether cortactin acts downstream of the Abl kinases to

regulate adhesion.

Finally, Arg was recently shown to phosphorylate myosin IIb (Boyle and

Koleske, 2007). We have observed that Abl kinase inhibition leading to Rho activation

promotes phosphorylation of MLC leading to enhaced acto‐myosin contractility, thereby

disrupting adherens junctions. The biological consequences of Arg‐mediated

phosphorylation of myosin IIb are unknown at this time, but future studies may reveal a

role for this phosphorylation in adherens junction regulation. In this regard, myosin IIb was found to interact with N‐cadherin and β‐catenin in the spinal canal of mice, and

genetic ablation of myosin IIb resulted in loss of intercellular adhesion (Ma et al., 2007).

128

Our studies have revealed a significant novel role for the Abl kinases in the

positive regulation of adherens junctions. We have identified Crk as a direct Abl

substrate, which is required for adherens junction formation and strengthening.

However, gaps in our knowledge regarding the mechanism of Abl kinase activation and

the identity of other potential Abl targets remain.

6.2 Dissecting the role of Abl downstream of Met receptor activation

Data from Chapter 4 reveal a novel role for the Abl kinases in mediating the

disruption of adherens junctions downstream of Met activation. We add Met to a list of

tyrosine kinase growth factor receptors, including PDGFR and EGFR, which activate Abl

kinases (Plattner et al., 1999; Sini et al., 2004). Previous studies have similarly shown that

Abl kinase activity is upregulated following HGF stimulation, and the Abl kinase has

been shown to negatively regulate motility induced by Met activation (Abassi and

Vuori, 2002; Frasca et al., 2001). In contrast, our findings suggest a positive role for the

Abl kinases in regulating Met signaling. We hypothesize that differences in cell‐type,

level and duration of Abl kinase activation, inputs from other signaling pathways, and

different downstream effectors play a role in the Abl‐dependent regulation of Met

signaling.

129

6.2.1 Abl and Met

One of our most interesting findings is that Abl kinase inhibition results in

decreased phosphorylation of the Met autophosphorylation sites Y1234/1235. While this

may be an indirect effect, we propose that Abl directly phosphorylates the Met receptor

and contributes to a positive feedback loop, which contributes to sustained signaling.

Abl was previously identified in a screen for binding partners of Met (Weidner et al.,

1996), and binding may be required to allow Abl to directly phosphorylate Met or may

induce a conformational change which sustains Met kinase activity. Our lab has recently

shown that Abl kinases can directly phosphorylate the EGFR, and that phosphorylation

leads to sustained signaling and decreased endocytosis (Tanos and Pendergast, 2006). In

the case of EGFR, this leads to hyperactivation of signaling pathways (Tanos and

Pendergast, 2006). However, HGF stimulation of the Met receptor is known to lead to

sustained signaling as compared to stimulation of other growth factor receptors,

therefore it is possible that aberrant signaling in the case of EGFR is similar to normal

signaling by Met. It is also unknown whether Met directly phosphorylates Abl leading

to its activation or whether other signaling molecules may modulate the effects of Met

activation on Abl. In this regard, PLC‐γ is recruited to the Met receptor following HGF

stimulation and promotes Abl activation downstream of PDGF stimulation (Plattner et

al., 2004). Future studies will be directed at furthering an understanding of the

interaction between Abl and Met in this system. Expression of kinase dead mutants of

130

Abl and Met in the presence of wild type Met and Abl, respectively, will shed light on

whether Abl directly phosphorylates Met in response to HGF stimulation and vice versa.

Additional studies using GFP‐tagged Abl or Arg in combination with live cell imaging

can be used to assess whether Abl kinases are, as we expect, recruited to the Met

receptor following HGF stimulation, and allow us to evaluate the duration of this

interaction. Additionally, we will use biochemical means to evaluate Met‐Abl binding.

Kinase‐dead mutants will also be useful to determine whether kinase activation is

required for binding. The Met receptor contains multiple tyrosines, which when

phosphorylated allow SH2‐domain containing proteins to bind (Ponzetto et al., 1994).

The use of both tyrosine to phenylalanine mutant Met receptors and Abl kinases lacking

functional domains, such as SH2 may allow the binding regions on both proteins to be identified. Further, the contribution of phosphorylation sites and specific domains to

recruitment to the plasma membrane and phosphorylation and activation of both Abl

and Met could be determined.

Phosphorylation of tyrosine 1003 of the Met receptor is required for Cbl‐

mediated ubiquitination and internalization of Met (Peschard et al., 2004). We observed reduced phosphorylation of Y1003 in response to HGF stimulation in the presence of

Abl kinase inhibition suggesting that Y1003 is another potential target of the Abl

kinases. This finding is particularly interesting because mutations in this region of the

Met receptor have been implicated in tumorigenesis, therefore suggesting a role for the

131

Abl kinases in these tumors (Kong‐Beltran et al., 2006). In vitro data support a critical

role for Y1003 in cancer. A Met receptor mutant in which this tyrosine has been mutated

to phenylalanine (Met Y1003F) shows increased receptor stability, prolonged activation

of signaling pathways, and enhanced transformation in response to HGF stimulation

(Abella et al., 2005). Met Y1003F fails to bind Cbl, which prevents its trafficking to the

lysosome for degradation (Abella et al., 2005). Further, Met Y1003F results in sustained

signaling through the MAPK pathway and increased and prolonged phosphorylation of

Erk. Our data also showed that Abl kinase inhibition decreased phosphorylation of Met

at Y1003 and increased phosphorylation of Erk. We hypothesize that Abl phosphorylates

the Met receptor downstream of HGF stimulation and normally acts to promote

turnover of Met. We expect that inhibition of Abl kinases will phenocopy expression of

Met Y1003F, and future studies will examine this hypothesis. We expect that loss of Abl

kinases will result in increased stability of the Met receptor, and expect to see enhanced,

prolonged cytoplasmic staining of both Met and Erk. We also expect Abl kinase

inhibition or knockdown of Abl/Arg to prevent binding of Cbl with Met and the subsequent monoubiquitination of the Met receptor.

6.2.2 Abl and other downstream targets

Tyrosine phosphorylation of Crk in response to HGF stimulation was nearly

eliminated in cells treated with the Abl kinase inhibitor. Previously, Crk has been shown

to be required for disruption of adherens junctions following Met activation, and Crk

132

overexpression leads to increased Rac activation and cell spreading in MDCK cells in the

absence of Met activation (Lamorte et al., 2002a; Lamorte et al., 2002b). This effect was

similar to what we observed in response to STI571 inhibition of Abl kinase activity and

Crk overexpression in NBT‐II cells, thus suggesting that unphosphorylated Crk is

responsible for these biological effects. Crk phosphorylation in response to HGF

stimulation, is in fact, transient suggesting that the timing and duration of Crk

phosphorylation may be critical for Met signaling. For example, early signaling events

downstream of Met activation may require phosphorylation of Crk. In this regard,

tyrosine phosphorylation of Crk has been shown to be required for Rac recruitment to

the plasma membrane, and this may be the case in early Met signaling (Abassi and

Vuori, 2002). That overexpression of Crk resulted in enhanced cell spreading (Lamorte et

al., 2002b) and inhibition of Abl kinase activity enhances motility in response to HGF

(Cipres et al., 2007; Frasca et al., 2001) suggests a negative role for Abl‐Crk in

modulating Met signaling. These responses are not immediate, and unphosphorylated

Crk may be required for late signaling events downstream of Met activation. Future

studies which carefully examine the phosphorylation status of Crk localization and its

effects on Rac activity as well as phenotypic consequences will be required to re‐

evaluate the role of Crk phosphorylation. Alternatively, the different effects of Crk may

be dependent on other factors such as cell type or strength of intercellular adhesion. We

have demonstrated that the Abl‐Crk‐Rac signaling module acts downstream of cadherin

133

engagement, therefore, the contribution of cadherin signaling may alter signaling

downstream of Met activation.

Gab1 is a large adaptor molecule recruited to the Met receptor following Met

activation. Gab1 contains 6 YXXP sites that become tyrosine phosphorylated following

HGF stimulation and serve as docking sites for the recruitment of additional SH2

domain‐containing proteins to the Met signaling complex (Weidner et al., 1996). The Crk

adaptor protein is recruited to phosphorylated tyrosine 307 of Gab1 and is required for

sustained Gab1 phosphorylation and physiological response to HGF stimulation in

synovial sarcoma cells (Watanabe et al., 2006). Our studies reveal a role for Abl kinases

in modulating Gab1 phosphorylation, and the numerous YXXP sites in Gab1 make it yet

another candidate substrate for Abl downstream of Met activation. If Abl is not recruited

directly to Met, Gab1 also represents a likely intermediate which might recruit Abl to

allow it to participate in Met signaling. Supporting this possibility, BcrAbl has been

shown to associate with the closely related Gab2 protein in chronic myelogenous

leukemia lines (Samanta et al., 2006). We examined total phosphorylation of Gab1, but

use of phosphospecific antibodies or tyrosine to phenylalanine mutants of Gab1 may

reveal which tyrosines are phosphorylated by Abl directly or indirectly. It is also unclear

at this time whether Abl directly phosphorylates Gab or whether Gab1 phosphorylation

is affected indirectly through Abl‐mediated effects on the Met receptor or Crk. Future

studies will be directed at examining the functional consequences of loss of Abl kinases

134

with regard to Gab binding to Met and recruitment of SH2 domain‐containing proteins,

including Crk. If Abl is found to directly phosphorylate specific sites, studies using Gab1

tyrosine to phenylalanine mutants could be use to examine the biological consequences

of Abl loss of function on Gab1 signaling.

Abl may also function to regulate interactions between β‐catenin and E‐cadherin downstream of Met activation. HGF stimulation of Met results in increased tyrosine

phosphorylation of β‐catenin (Hiscox and Jiang, 1999; Monga et al., 2002).

Phosphorylation at Y654 has been linked to disruption of adherens junctions and

dissociation of the cadherin‐catenin complex (Piedra et al., 2001). While we have observed no change in tyrosine phosphorylation of β‐catenin in response to HGF

stimulation in our studies, that does not eliminate the possibility that in some contexts

Abl phosphorylation of β‐catenin regulates cell scattering downstream of Met. Another

possible explanation of how Abl kinase inhibition blocks adherens junction turnover is

that endocytosis and turnover of the Met receptor and E‐cadherin are somehow linked.

If Abl kinase inhibition mimics the effect of increased Met Y1003F stability, and Met is

not degraded, it is possible that E‐cadherin recycling may also be blocked. However,

rather than remaining in the cytoplasm, E‐cadherin may be recycled back to the plasma

membrane. Very little is known regarding the mechanisms through which the Met

receptor regulates adherens junctions, therefore highlighting the significance of our

finding that Abl kinases act downstream of both Met receptor and cadherins.

135

While our preliminary findings support a critical role for the Abl kinases in

regulating Met signaling, the Met signaling cascade is highly complex and contains

many candidate Abl kinase substrates to study. Once a clear understanding of how the

Abl kinases affect the response to HGF stimulation in one cell type, it will be important

to examine the generalizability to other cell types. For cells in which Abl appears to act

differently, factors such as cell type, cell confluence, and presence or absence of other

signals which stimulate Abl kinase activity, including other growth factor receptors and

cadherins will need to be examined. Additionally, while we have examined disruption

of adherens junctions in response to Met, we have not examined other physiological

responses to HGF such as motility, invasion, transformation, angiogenesis, or

tumorigenesis (Birchmeier et al., 2003). Our finding that Abl kinases play a role in

adherens junction remodeling in response to HGF have opened the door to studying the

potential role for Abl kinases as master regulators integral for interpreting signaling

inputs and allowing cross‐talk between growth factor receptors and cadherins.

6.3 Role of Abl downstream of other growth factors

Our lab and others have shown that the Abl kinases are activated downstream of

various growth factors and participate in signaling downstream of PDGFR and EGFR

(Boyle et al., 2007; Plattner et al., 1999; Sini et al., 2004; Tanos and Pendergast, 2006). Our

data from Chapter 5 suggest that the Abl kinases function as critical intermediaries in

modulating communication between the activated Met receptor and the cadherin‐

136

catenin complex. While HGF‐induced disruption of adherens junctions is perhaps the

most well‐characterized and well‐understood example of cross‐talk between RTKs and

the cadherin‐based adhesion complex, other growth factor receptors have been

implicated in adherens junction remodeling. For example, our lab has observed a role for the Abl kinases in modulating endocytosis of the EGFR, whereby expression of

constitutively active AblPP blocks EGF stimulated endocytosis (Tanos and Pendergast,

2006). EGF stimulation of the EGFR has been shown to increase caveolin‐mediated

endocytosis of E‐cadherin (Lu et al., 2003). We have observed that expression of ArgPP results in strengthening of N‐cadherin‐based cell adhesion in HeLa cells (Zandy et al.,

2007). Taken together, these data suggest the possibility that cross‐talk from the EGFR to

cadherins may be responsible for this phenotype if E‐cadherin endocytosis is dependent

on endocytosis of the EGFR and suggests an area for future study.

6.3.1 Abl and FGF

N‐cadherin and FGFR have been shown to act cooperatively to promote FGF2

stimulated signaling, resulting in increased invasion and angiogenesis (Derycke et al.,

2006; Suyama et al., 2002). Abl kinases have been implicated in negatively regulating N‐

cadherin‐based adhesion in neurons by mediating cross‐talk between Robo and N‐

cadherin (Rhee et al., 2007; Rhee et al., 2002). We propose that the Abl kinases act as

master regulators at the intersection of RTK and cadherin signaling, thus we expect the

137

Abl kinases may play a role in modulating FGFR/N‐cadherin cross‐talk in epithelial

cells.

We have previously established a role for Abl kinases in regulating N‐cadherin‐

based intercellular adhesion (Zandy et al., 2007). To evaluate the possibility that Abl acts

downstream of the FGFR as well, we stimulated WT or Abl‐/‐Arg‐/‐ MEFs with FGF2 and examined phosphorylation of Crk at the Abl‐specific site. We observed increased

Abl kinase activity by 5 minutes in WT cells, with no phosphorylation of Crk in cells

lacking Abl kinases (Figure 23A). To test whether this pathway functions in epithelial

cells, we stimulated MCF‐7 cells with FGF2, and again observed elevated levels of Abl

kinase activity 15 minutes following stimulation (Figure 23B). Taken together, these data

support our hypothesis that Abl kinases are perfectly positioned to integrate signaling

between the FGFR and N‐cadherin. Further, many of the same signaling pathways in

which Abl has been implicated play a critical role in modulating FGF2 signaling,

including Erk, Grb2, and PLCγ. Future experiments will be directed at uncovering which

physiological processes are mediated by Abl kinases (proliferation, migration, invasion,

and/or angiogenesis) and dissecting the role of Abl kinases in the FGF2 stimulated

signaling pathway. We surmise that Abl may play a role in these processes and possibly

also in cadherin switching during tumorigenesis in epithelial cells. An alternative, and

equally exciting, possibility is that Abl kinases may also modulate angiogenesis through

mediating FGF2 signaling in endothelial cells. Our novel finding that Abl kinases are

138

A 20 ng/mL FGF2 0 min 5 min 10 min 20 min null null null null Abl/Arg Abl/Arg Abl/Arg Abl/Arg

IB: pCrk

IP: Crk IB: Crk

B 10 min 0 min 5 min 20 min

IB: pCrk

IP: Crk IP: IB: Crk

Figure 23: FGF2 stimulation activates endogenous Abl kinases.

Figure 23: (A) Abl/Arg null MEFs transduced with retroviruses encoding Abl and Arg or vector alone cells were were starved for 16h in 0.25% FBS DMEM prior to stimulation with 20 ng/mL FGF2. Cells were lysed in 1% Triton lysis buffer supplemented with protease and phosphatase inhibitors. Abl kinase activity was assessed by immunoprecipitation of Crk followed by immunoblotting with a phospho‐specific antibody recognizing Y221 in Crk, the Abl‐specific site of tyrosine phosphorylation. The blot was stripped and re‐probed for Crk to demonstrate equivalent protein levels. (B) MCF‐7 cells were starved for 16h in 0.25% FBS DMEM prior to stimulation with 20 ng/mL FGF2 in the presence or absence of 10 μM STI571. Cells were lysed in 1% Triton lysis buffer supplemented with protease and phosphatase inhibitors. Abl kinase activity was assessed by immunoprecipitation of Crk followed by immunoblotting with a phospho‐specific antibody recognizing Y221 in Crk, the Abl‐specific site of tyrosine phosphorylation. The blot was stripped and re‐probed for Crk to demonstrate equivalent protein levels.

139

activated by FGF2 opens the door for studies of Abl’s involvement in yet another

pathway critically involved in tumorigenesis and tumor progression.

6.3.2 Abl and VEGF

VEGF stimulation induces multiple effects in endothelial cells, including increased proliferation and vessel development. However, VEGF stimulation also can

increase vascular permeability. One of the ways in which VEGF stimulation may

promote vessel leakiness is by disrupting VE‐cadherin‐based intercellular adhesion. In

this regard, VEGF stimulation of VEGFR has been shown to increase tyrosine

phosphorylation of members of the cadherin‐based adhesion complex in endothelial

cells, including VE‐cadherin, β‐catenin, and p120 catenin (Esser et al., 1998). Tyrosine

phosphorylation of both E‐cadherin and β‐catenin have been shown to disrupt

intercellular adhesion in other cell types (Brembeck et al., 2004; Fujita et al., 2002; Piedra

et al., 2001; Roura et al., 1999). Thus, VEGF‐induced tyrosine phosphorylation of

adherens junction components may lead to disruption of cadherin‐based adhesion,

thereby promoting vascular permeability.

We identified a role for the Abl kinases in positively regulating intercellular

adhesion in fibroblasts in epithelial cells (Zandy et al., 2007), but have also observed a

negative role for Abl kinases downstream of Met activation. These observations led us to

hypothesize that Abl kinases may likewise play both a positive role downstream of

140

cadherin engagement and a negative role downstream of growth factor stimulation in

regulating VE‐cadherin based intercellular adhesion in endothelial cells. To examine

whether Abl kinase activity is required for the formation of adherens junctions in

endothelial cells, we performed a calcium switch to induce cadherin engagement in the presence or absence of STI571 inhibition of Abl kinase activity (Figure 24). We observed

reformation of adherens junctions by 2 h post‐switch in control cells; however, β‐catenin

remained cytosolic in cells treated with the Abl kinase inhibitor (Figure 24A). This

supports a positive role for Abl kinases in promoting VE‐cadherin‐based intercellular adhesion. To examine the other side of our hypothesis, we first needed to determine

whether Abl was activated by VEGF stimulation. Using phosphorylation of Crk at the

Abl‐specific site as a readout, we examined Abl kinase activation in response to VEGF

stimulation in PY‐4‐1 cells and observed elevated levels of Abl kinase activity at 5 and 15

min (Figure 24B). Together, these data place the Abl kinases in a position to modulate

the negative effects of VEGF stimulation on adherens junctions. In fact, VEGF

stimulation of PY‐4‐1 cells resulted in relatively rapid re‐localization of β‐catenin from

sites of cell‐cell contact to the cytoplasm. In contrast, β‐catenin remained at sites of cell‐

cell contact in cells treated with the Abl kinase inhibitor STI571 supporting the

hypothesis that Abl kinases might act to promote disruption of endothelial adherens

junctions downstream of VEGF stimulation. The signaling events which govern VEGF‐

induced vascular permeability remain poorly understood, but likely involve many of the

141

A Hi Calcium Low Calcium 30 min +calcium 2 h +calcium control + 10 µM STI571 µM + 10

BC50 ng/mL VEGF : -5 min15 min 50 ng/mL VEGF : - 5 min15 min IB: pCrkL Y207 IB: CrkL control IP: CrkL IB: CrkL (WCL) + 10µM STI571

Figure 24: Abl kinases mediate intercellular adhesion in endothelial cells.

Figure 24: (A) PY‐4‐1 cells were grown to subconfluency and subjected to calcium switch to stimulate adherens junction formation in the presence or absence of 10 μM STI571. Cells were fixed and stained with antibody against β‐catenin (red) and counterstained with DAPI (blue). (B) PY‐4‐1 cells were starved for 16h in 0.25% FBS DMEM prior to stimulation with 50 ng/mL VEGF. Cells were lysed in 1% Triton lysis buffer supplemented with protease and phosphatase inhibitors. Abl kinase activity was assessed by immunoprecipitation of CrkL followed by immunoblotting with a phospho‐ specific antibody recognizing Y207 in CrkL, the Abl‐specific site of tyrosine phosphorylation. The blot was stripped and re‐probed for CrkL to demonstrate equivalent protein levels. (C) PY‐4‐1 cells were seeded onto glass coverslips and starved for 4h. Cells were pre‐treated with vehicle control or with 10μM STI571 for 1h to inhibit endogenous Abl kinase activity. Cells were stimulated with 50 ng/mL VEGF for the indicated times and were fixed and stained for β‐catenin.

142

same players implicated in the Met signaling cascade. Therefore, understanding the role

of the Abl kinases in these pathways may help reveal a universal signaling module used

in various cell types to translate signals from growth factor receptors and adhesion

molecules into biological consequences.

6.4 Concluding remarks

Our data point to a positive role for the Abl kinases in regulating adherens

junction formation and maintenance downstream of cadherin engagement in numerous

mammalian cells, including epithelial cells, fibroblasts, and endothelial cells. Further, we

have identified downstream signaling effectors involved in this regulation and have

proposed other molecules which might be involved. Conversely, we have identified a

negative role for the Abl kinases in promoting the dissolution of adherens junctions

downstream of growth factor receptor interaction and identified potential signaling

molecules which may contribute to Abl’s effect. At this point, we take a step back to

consider the big‐picture relevance of these findings.

The Abl kinases have now been identified to play a positive role in promoting

adherens junction formation in Drosophila, C. elegans, and mammals (Grevengoed et

al., 2001; Sheffield et al., 2007; Zandy et al., 2007), suggesting the role of Abl kinases on

junctions is evolutionarily conserved. It is unknown whether the Abl kinases act

downstream of growth factor receptors to regulate adherens junctions in lower

organisms, however a role for Abl in regulating cross‐talk between N‐cadherin and the

143

Robo receptor in Drosophila has been described (Rhee et al., 2002). This finding lends

further support to our hypothesis that Abl‐mediated regulation of adherens junctions

was wired early in evolution and positions Abl at the center of regulating

communication between these different signaling inputs.

We have identified the Abl kinases as regulators of intercellular adhesion using

tissue culture models, so what does this mean for the 3‐D organism? Tightly regulated

control of intercellular adhesion is imperative for numerous processes that occur during

development including formation of neural networks, organogenesis, and tissue

specification (Thiery, 2003). Thus, the loss or deregulation of Abl kinases during

development would have disastrous consequences for the embryo. Future studies using

tissue‐specific Abl/Arg knockout mice may help elucidate the specific deficits which

arise in the absence of Abl/Arg. On the other hand, deregulation of adherens junctions

plays a critical role in promoting tumor progression and metastasis, and, therefore,

ultimately death from cancer. The fact that we have found both positive and negative

regulatory roles for the Abl kinases in regulating intercellular adhesion suggest caution

in the use of Abl kinase inhibition as a means of cancer treatment. However, we propose

that further research to gain a better understanding of when, where, and how Abl

participates in adherens junction regulation in different types and stages of cancer will

open the door for the use of small molecule inhibitors of Abl kinase activity to treat

selected patients.

144

In this regard, Gleevec (also known as imatinib or STI571), the small molecule

Abl kinase inhibitor, is the poster child for successful targeted molecular therapy.

Gleevec is the front‐line treatment for patients with chronic myelogenous leukemia

resulting from the BcrAbl chromosomal translocation (Kantarjian et al., 2002). What do our findings mean for patients currently taking Gleevec? It is critical to understand the

normal function of Abl kinases in cells to predict potential unwanted side effects. That

Abl kinase inhibition may lead to impaired formation and maintenance of highly

dynamic adherens junctions suggests that patients treated with Gleevec would likely experience minimal adverse effects if they are otherwise healthy. Most adult tissues are

fully developed, and adherens junctions are not likely undergoing constant turnover.

However, it is possible that in epithelial tissues like skin or gastric epithelia, in which

cells are continually shed and will need to form adherens junctions to maintain tissue

architecture, that high concentrations of Gleevec could impair these processes,

potentially leading to blistering of the skin or gastrointestinal disorders. Moreover,

recent reports have shown that a subset of patients treated with Gleevec exhibit

cardiotoxicity, and Gleevec‐treated mice develop left ventricular contractile dysfunction

(Kerkela et al., 2006). The potential unpleasantness of these potential side effects are

seemingly minimal in contrast to the probably loss of life from not taking Gleevec.

Our findings also point to several different potential therapeutic indications for

Abl kinase inhibitors for the treatment of solid tumors. In this regard, Abl kinase

145

inhibitors could potentially be used as adjuvant therapy to weaken intercellular

adhesion in the tumor. This could help promote penetration of other drugs, including

biologics, into tumors or could help cells of the immune system gain access to the tumor

to attack and destroy its cells. Recent studies using the Abl kinase inhibitor, imatinib,

support this hypothesis. Treatment of NSCLC cells with imatinib resulted in both

increased tumor oxygenation and increased delivery of chemotherapeutic agents in a

xenograft model (Vlahovic et al., 2007; Vlahovic et al., 2006). Abl kinase inhibiton may

also prevent escaped tumor cells from forming new junctions in distal sites, therefore

preventing the formation of metastases. We have observed effects of Abl kinase

inhibition in multiple cell types, suggesting that Abl kinase inhibitors could be used to

target epithelial, fibroblast, and endothelial cells, which comprise the tumor

microenvironment. That Abl kinases act to regulate adherens junction in multiple cell types also suggests the potential importance of finding better ways to target small

molecule inhibitors specifically to cells of interest. It will be critically important to

continue research directed at discovering novel methods of controlled drug delivery to

prevent adverse effects.

The success of Gleevec in treating CML has also led to the research and

development of new small molecule inhibitors targeting the Abl kinases. It will be

important to test the effects on adherens junctions of these novel compounds, which

include dasatinib a potent dual Bcr‐Abl and Src inhibitor, which is FDA‐approved for

146

imatinib‐resistent CML; nilotinib, a selective, potent Bcr‐Abl inhibitor; bosutinib (SKI‐

606) and INNO‐406 (NS‐187), which are both Src‐Abl inhibitors(Jabbour et al., 2007).

Further, our finding that Abl kinases act downstream of FGF, VEGF, and HGF

stimulation suggests that inhibition of Abl kinases may represent an important

therapeutic target for preventing tumor angiogenesis. The discovery that Abl kinases

regulate adherens junction biology, in part by acting downstream of growth factor

receptors, has thus opened a floodgate for future research into examining the Abl

kinases as therapeutic targets to prevent progression and metastasis of tumors derived

from a host of epithelial tissue types.

147

References

Abassi, Y. A., and Vuori, K. (2002). Tyrosine 221 in Crk regulates adhesion‐dependent membrane localization of Crk and Rac and activation of Rac signaling. EMBO Journal 21, 4571‐4582.

Abella, J. V., Peschard, P., Naujokas, M. A., Lin, T., Saucier, C., Urbe, S., and Park, M. (2005). Met/Hepatocyte growth factor receptor ubiquitination suppresses transformation and is required for Hrs phosphorylation. Mol Cell Biol 25, 9632‐9645.

Alexandropoulos, K., Cheng, G., and Baltimore, D. (1995). Proline‐rich sequences that bind to Src homology 3 domains wiht individual specificities. Proc Natl Acad Sci U S A 92.

Anastasiadis, P. Z. (2007). p120‐ctn: A nexus for contextual signaling via Rho GTPases. Biochim Biophys Acta 1773, 34‐46.

Avizienyte, E., Fincham, V. J., Brunton, V. G., and Frame, M. C. (2004). Src SH3/2 domain‐mediated peripheral accumulation of Src and phospho‐myosin is linked to deregulation of E‐cadherin and the epithelial‐mesenchymal transition. Molecular Biology of the Cell 15, 2794‐2803.

Avizienyte, E., Wyke, A. W., Jones, R. J., McLean, G. W., Westhoff, M. A., Brunton, V. G., and Frame, M. C. (2002). Src‐induced de‐regulation of E‐cadherin in colon cancer cells requires integrin signalling. Nat Cell Biol 4, 632‐638.

Barberis, D., Casazza, A., Sordella, R., Corso, S., Artigiani, S., Settleman, J., Comoglio, P. M., and Tamagnone, L. (2005). p190 Rho‐GTPase activating protein associates with plexins and it is required for semaphorin signalling. J Cell Sci 118, 4689‐4700.

Bardelli, A., Longati, P., Gramaglia, D., Stella, M. C., and Comoglio, P. M. (1997). Gab1 coupling to the HGF/Met receptor multifunctional docking site requires binding of Grb2 and correlates with the transforming potential. Oncogene 15, 3103‐3111.

Barila, D., and Superti‐Furga, G. (1998). An intramolecular SH3‐domain interaction regulates c‐Abl activity. Nat Genet 18, 280‐282.

Bashaw, G. J., Kidd, T., Murray, D., Pawson, T., and Goodman, C. S. (2000). Repulsive axon guidance: Abelson and Enabled play opposing roles downstream of the roundabout receptor. Cell 101, 703‐715. 148

Baum, B., and Perrimon, N. (2001). Spatial control of the actin cytoskeleton in Drosophila epithelial cells. Nature Cell Biology 3, 883‐890.

Behrens, J., Vakaet, L., Friis, R., Winterhager, E., Van Roy, F., Mareel, M. M., and Birchmeier, W. (1993). Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E‐cadherin/beta‐catenin complex in cells transformed with a temperature‐sensitive v‐SRC gene. J Cell Biol 120, 757‐766.

Bershadsky, A. (2004). Magic touch: how does cell‐cell adhesion trigger actin assembly? Trends in Cell Biology 14, 589‐593.

Betson, M., Lozano, E., Zhang, J., and Braga, V. M. (2002). Rac activation upon cell‐cell contact formation is dependent on signaling from the epidermal growth factor receptor. J Biol Chem 277, 36962‐36969.

Bhowmick, N. A., Ghiassi, M., Bakin, A., Aakre, M., Lundquist, C. A., Engel, M. E., Arteaga, C. L., and Moses, H. L. (2001). Transforming growth factor‐beta1 mediates epithelial to mesenchymal transdifferentiation through a RhoA‐dependent mechanism. Molecular Biology of the Cell 12, 27‐36.

Bierne, H., Miki, H., Innocenti, M., Scita, G., Gertler, F. B., Takenawa, T., and Cossart, P. (2005). WASP‐related proteins, Abi1 and Ena/VASP are required for Listeria invasion induced by the Met receptor. J Cell Sci 118, 1537‐1547.

Billottet, C., Elkhatib, N., Thiery, J. P., and Jouanneau, J. (2004). Targets of fibroblast growth factor 1 (FGF‐1) and FGF‐2 signaling involved in the invasive and tumorigenic behavior of carcinoma cells. Mol Biol Cell 15, 4725‐4734.

Birchmeier, C., Birchmeier, W., and Brand‐Saberi, B. (1996). Epithelial‐mesenchymal transitions in cancer progression. Acta Anat (Basel) 156, 217‐226.

Birchmeier, C., Birchmeier, W., Gherardi, E., and Vande Woude, G. F. (2003). Met, metastasis, motility and more. Nat Rev Mol Cell Biol 4, 915‐925.

Bonitsis, N., Batistatou, A., Karantima, S., and Charalabopoulos, K. (2006). The role of cadherin/catenin complex in malignant melanoma. Exp Oncol 28, 187‐193.

Bosse, T., Ehinger, J., Czuchra, A., Benesch, S., Steffen, A., Wu, X., Schloen, K., Niemann, H. H., Scita, G., Stradal, T. E., et al. (2007). Cdc42 and phosphoinositide 3‐kinase drive

149

Rac‐mediated actin polymerization downstream of c‐Met in distinct and common pathways. Mol Cell Biol 27, 6615‐6628.

Boureux, A., Furstoss, O., Simon, V., and Roche, S. (2005). Abl tyrosine kinase regulates a Rac/JNK and a Rac/Nox pathway for DNA synthesis and Myc expression induced by growth factors. J Cell Sci 118, 3717‐3726.

Boussadia, O., Kutsch, S., Hierholzer, A., Delmas, V., and Kemler, R. (2002). E‐cadherin is a survival factor for the lactating mouse mammary gland. Mech Dev 115, 53‐62.

Boyle, S. N., and Koleske, A. J. (2007). Use of a chemical genetic technique to identify myosin IIb as a substrate of the Abl‐related gene (Arg) tyrosine kinase. Biochemistry 46, 11614‐11620.

Boyle, S. N., Michaud, G. A., Schweitzer, B., Predki, P. F., and Koleske, A. J. (2007). A critical role for cortactin phosphorylation by Abl‐family kinases in PDGF‐induced dorsal‐wave formation. Curr Biol 17, 445‐451.

Bradley, W. D., Hernandez, S. E., Settleman, J., and Koleske, A. J. (2006). Integrin signaling through Arg activates p190RhoGAP by promoting its binding to p120RasGAP and recruitment to the membrane. Mol Biol Cell 17, 4827‐4836.

Braga, V. M., Betson, M., Li, X., and Lamarche‐Vane, N. (2000). Activation of the small GTPase Rac is sufficient to disrupt cadherin‐dependent cell‐cell adhesion in normal human keratinocytes. Mol Biol Cell 11, 3703‐3721.

Braga, V. M., Del Maschio, A., Machesky, L., and Dejana, E. (1999). Regulation of cadherin function by Rho and Rac: modulation by junction maturation and cellular context. Molecular Biology of the Cell 10, 9‐22.

Brasher, B. B., Roumiantsev, S., and Van Etten, R. A. (2001). Mutational analysis of the regulatory function of the c‐Abl Src homology 3 domain. Oncogene 20, 7744‐7752.

Brasher, B. B., and Van Etten, R. A. (2000). c‐Abl has high intrinsic tyrosine kinase activity that is stimulated by mutation of the Src homology 3 domain and by autophosphorylation at two distinct regulatory tyrosines. J Biol Chem 275, 35631‐35637.

150

Brembeck, F. H., Schwarz‐Romond, T., Bakkers, J., Wilhelm, S., Hammerschmidt, M., and Birchmeier, W. (2004). Essential role of BCL9‐2 in the switch between beta‐cateninʹs adhesive and transcriptional functions. Genes Dev 18, 2225‐2230.

Bresnick, A. R. (1999). Molecular mechanisms of nonmuscle myosin‐II regulation. Current Opinion in Cell Biology 11, 26‐33.

Brunton, V. G., MacPherson, I. R., and Frame, M. C. (2004). Cell adhesion receptors, tyrosine kinases and actin modulators: a complex three‐way circuitry. Biochim Biophys Acta 1692, 121‐144.

Bryant, D. M., and Stow, J. L. (2004). The ins and outs of E‐cadherin trafficking. Trends Cell Biol 14, 427‐434.

Bryant, S. S., Briggs, S., Smithgall, T. E., Martin, G. A., McCormick, F., Chang, J. H., Parsons, S. J., and Jove, R. (1995). Two SH2 domains of p120 Ras GTPase‐activating protein bind synergistically to tyrosine phosphorylated p190 Rho GTPase‐activating protein. J Biol Chem 270, 17947‐17952.

Burton, E. A., Oliver, T. N., and Pendergast, A. M. (2005). Abl kinases regulate actin comet tail elongation via an N‐WASP‐dependent pathway. Mol Cell Biol 25, 8834‐8843.

Burton, E. A., Plattner, R., and Pendergast, A. M. (2003). Abl tyrosine kinases are required for infection by Shigella flexneri. EMBO J 22, 5471‐5479.

Calautti, E., Cabodi, S., Stein, P. L., Hatzfeld, M., Kedersha, N., and Paolo Dotto, G. (1998). Tyrosine phosphorylation and src family kinases control keratinocyte cell‐cell adhesion. Journal of Cell Biology 141, 1449‐1465.

Cali, G., Zannini, M., Rubini, P., Tacchetti, C., DʹAndrea, B., Affuso, A., Wintermantel, T., Boussadia, O., Terracciano, D., Silberschmidt, D., et al. (2007). Conditional inactivation of the E‐cadherin gene in thyroid follicular cells affects gland development but does not impair junction formation. Endocrinology 148, 2737‐2746.

Cao, C., Leng, Y., Huang, W., Liu, X., and Kufe, D. (2003). Glutathione peroxidase 1 is regulated by the c‐Abl and Arg tyrosine kinases. J Biol Chem 278, 39609‐39614.

Carramusa, L., Ballestrem, C., Zilberman, Y., and Bershadsky, A. D. (2007). Mammalian diaphanous‐related formin Dia1 controls the organization of E‐cadherin‐mediated cell‐ cell junctions. J Cell Sci 120, 3870‐3882.

151

Castano, J., Solanas, G., Casagolda, D., Raurell, I., Villagrasa, P., Bustelo, X. R., Garcia de Herreros, A., and Dunach, M. (2007). Specific phosphorylation of p120‐catenin regulatory domain differently modulates its binding to RhoA. Mol Cell Biol 27, 1745‐ 1757.

Cavallaro, U., and Christofori, G. (2004). Cell adhesion and signalling by cadherins and Ig‐CAMs in cancer. Nat Rev Cancer 4, 118‐132.

Cavallaro, U., Schaffhauser, B., and Christofori, G. (2002). Cadherins and the tumour progression: is it all in a switch? Cancer Lett 176, 123‐128.

Chang, J. H., Gill, S., Settleman, J., and Parsons, S. J. (1995). c‐Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation. J Cell Biol 130, 355‐368.

Chen, W. S., Kung, H. J., Yang, W. K., and Lin, W. (1999). Comparative tyrosine‐kinase profiles in colorectal cancers: enhanced arg expression in carcinoma as compared with adenoma and normal mucosa. International Journal of Cancer 83, 579‐584.

Chen, X., Kojima, S., Borisy, G. G., and Green, K. J. (2003). p120 catenin associates with kinesin and facilitates the transport of cadherin‐catenin complexes to intercellular junctions. J Cell Biol 163, 547‐557.

Christensen, J. G., Burrows, J., and Salgia, R. (2005). c‐Met as a target for human cancer and characterization of inhibitors for therapeutic intervention. Cancer Lett 225, 1‐26.

Christofori, G. (2003). Changing neighbours, changing behaviour: cell adhesion molecule‐mediated signalling during tumour progression. Embo J 22, 2318‐2323.

Cicchetti, P., Mayer, B. J., Thiel, G., and Baltimore, D. (1992). Identification of a protein that binds to the SH3 region of Abl and is similar to Bcr and GAP‐rho. Science 257, 803‐ 806.

Cipres, A., Abassi, Y. A., and Vuori, K. (2007). Abl functions as a negative regulator of Met‐induced cell motility via phosphorylation of the adapter protein CrkII. Cell Signal 19, 1662‐1670.

152

Comoglio, P. M., Boccaccio, C., and Trusolino, L. (2003). Interactions between growth factor receptors and adhesion molecules: breaking the rules. Curr Opin Cell Biol 15, 565‐ 571.

Comoglio, P. M., Tamagnone, L., and Giordano, S. (2004). Invasive growth: a two‐way street for semaphorin signalling. Nat Cell Biol 6, 1155‐1157.

Comoglio, P. M., and Trusolino, L. (2002). Invasive growth: from development to metastasis. J Clin Invest 109, 857‐862.

Cong, F., Spencer, S., Cote, J. F., Wu, Y., Tremblay, M. L., Lasky, L. A., and Goff, S. P. (2000). Cytoskeletal protein PSTPIP1 directs the PEST‐type protein tyrosine phosphatase to the c‐Abl kinase to mediate Abl dephosphorylation. Mol Cell 6, 1413‐1423.

Courtney, K. D., Grove, M., Vandongen, H., Vandongen, A., LaMantia, A. S., and Pendergast, A. M. (2000). Localization and phosphorylation of Abl‐interactor proteins, Abi‐1 and Abi‐2, in the developing nervous system. Mol Cell Neurosci 16, 244‐257.

Cowin, P., Rowlands, T. M., and Hatsell, S. J. (2005). Cadherins and catenins in breast cancer. Curr Opin Cell Biol 17, 499‐508.

Crnogorac‐Jurcevic, T., Efthimiou, E., Nielsen, T., Loader, J., Terris, B., Stamp, G., Baron, A., Scarpa, A., and Lemoine, N. R. (2002). Expression profiling of microdissected pancreatic adenocarcinomas. Oncogene 21, 4587‐4594.

Curto, M., Cole, B. K., Lallemand, D., Liu, C. H., and McClatchey, A. I. (2007). Contact‐ dependent inhibition of EGFR signaling by Nf2/Merlin. J Cell Biol 177, 893‐903.

Dai, Z., and Pendergast, A. M. (1995). Abi‐2, a novel SH3‐containing protein interacts with the c‐Abl tyrosine kinase and modulates c‐Abl transforming activity. Genes Dev 9, 2569‐2582.

Dallol, A., Da Silva, N. F., Viacava, P., Minna, J. D., Bieche, I., Maher, E. R., and Latif, F. (2002). SLIT2, a human homologue of the Drosophila Slit2 gene, has tumor suppressor activity and is frequently inactivated in lung and breast cancers. Cancer Res 62, 5874‐ 5880.

David‐Cordonnier, M. H., Hamdane, M., Bailly, C., and DʹHalluin, J. C. (1998). The DNA binding domain of the human c‐Abl tyrosine kinase preferentially binds to DNA

153

sequences containing an AAC motif and to distorted DNA structures. Biochemistry 37, 6065‐6076.

Davis, M. A., Ireton, R. C., and Reynolds, A. B. (2003). A core function for p120‐catenin in cadherin turnover. J Cell Biol 163, 525‐534.

Davis, M. A., and Reynolds, A. B. (2006). Blocked acinar development, E‐cadherin reduction, and intraepithelial neoplasia upon ablation of p120‐catenin in the mouse salivary gland. Dev Cell 10, 21‐31.

De Maria, R., Maggiora, P., Biolatti, B., Prat, M., Comoglio, P. M., Castagnaro, M., and Di Renzo, M. F. (2002). Feline STK gene expression in mammary carcinomas. Oncogene 21, 1785‐1790.

de Rooij, J., Kerstens, A., Danuser, G., Schwartz, M. A., and Waterman‐Storer, C. M. (2005). Integrin‐dependent actomyosin contraction regulates epithelial cell scattering. Journal of Cell Biology 171, 153‐164.

Derycke, L., Morbidelli, L., Ziche, M., De Wever, O., Bracke, M., and Van Aken, E. (2006). Soluble N‐cadherin fragment promotes angiogenesis. Clin Exp Metastasis 23, 187‐201.

Dorey, K., Engen, J. R., Kretzschmar, J., Wilm, M., Neubauer, G., Schindler, T., and Superti‐Furga, G. (2001). Phosphorylation and structure‐based functional studies reveal a positive and a negative role for the activation loop of the c‐Abl tyrosine kinase. Oncogene 20, 8075‐8084.

Dransart, E., Olofsson, B., and Cherfils, J. (2005). RhoGDIs revisited: novel roles in Rho regulation. Traffic 6, 957‐966.

Drees, F., Pokutta, S., Yamada, S., Nelson, W. J., and Weis, W. I. (2005). Alpha‐catenin is a molecular switch that binds E‐cadherin‐beta‐catenin and regulates actin‐filament assembly. Cell 123, 903‐915.

Echarri, A., and Pendergast, A. M. (2001). Activated c‐Abl is degraded by the ubiquitin‐ dependent proteasome pathway. Curr Biol 11, 1759‐1765.

Ehrlich, J. S., Hansen, M. D., and Nelson, W. J. (2002). Spatio‐temporal regulation of Rac1 localization and lamellipodia dynamics during epithelial cell‐cell adhesion. Developmental Cell 3, 259‐270.

154

El Sayegh, T. Y., Arora, P. D., Laschinger, C. A., Lee, W., Morrison, C., Overall, C. M., Kapus, A., and McCulloch, C. A. (2004). Cortactin associates with N‐cadherin adhesions and mediates intercellular adhesion strengthening in fibroblasts. J Cell Sci 117, 5117‐ 5131.

Esser, S., Lampugnani, M. G., Corada, M., Dejana, E., and Risau, W. (1998). Vascular endothelial growth factor induces VE‐cadherin tyrosine phosphorylation in endothelial cells. J Cell Sci 111 ( Pt 13), 1853‐1865.

Etienne‐Manneville, S., and Hall, A. (2002). Rho GTPases in cell biology. Nature 420, 629‐635.

Ferraro, D., Corso, S., Fasano, E., Panieri, E., Santangelo, R., Borrello, S., Giordano, S., Pani, G., and Galeotti, T. (2006). Pro‐metastatic signaling by c‐Met through RAC‐1 and reactive oxygen species (ROS). Oncogene 25, 3689‐3698.

Finkel, T. (2006). Intracellular redox regulation by the family of small GTPases. Antioxid Redox Signal 8, 1857‐1863.

Finn, A. J., Feng, G., and Pendergast, A. M. (2003). Postsynaptic requirement for Abl kinases in assembly of the neuromuscular junction.[see comment]. Nature Neuroscience 6, 717‐723.

Frasca, F., Vigneri, P., Vella, V., Vigneri, R., and Wang, J. Y. (2001). Tyrosine kinase inhibitor STI571 enhances thyroid cancer cell motile response to Hepatocyte Growth Factor. Oncogene 20, 3845‐3856.

Fujita, Y., Krause, G., Scheffner, M., Zechner, D., Leddy, H. E., Behrens, J., Sommer, T., and Birchmeier, W. (2002). Hakai, a c‐Cbl‐like protein, ubiquitinates and induces endocytosis of the E‐cadherin complex. Nature Cell Biology 4, 222‐231.

Fujiwara, M., Ghazizadeh, M., and Kawanami, O. (2006). Potential role of the Slit/Robo signal pathway in angiogenesis. Vasc Med 11, 115‐121.

Fukata, M., and Kaibuchi, K. (2001). Rho‐family GTPases in cadherin‐mediated cell‐cell adhesion. Nature Reviews Molecular Cell Biology 2, 887‐897.

155

Fukumoto, Y., Shintani, Y., Reynolds, A. B., Johnson, K. R., and Wheelock, M. J. (2007). The regulatory or phosphorylation domain of p120 catenin controls E‐cadherin dynamics at the plasma membrane. Exp Cell Res.

Galkin, V. E., Orlova, A., Koleske, A. J., and Egelman, E. H. (2005). The Arg non‐receptor tyrosine kinase modifies F‐actin structure. J Mol Biol 346, 565‐575.

Gao, C. F., Xie, Q., Su, Y. L., Koeman, J., Khoo, S. K., Gustafson, M., Knudsen, B. S., Hay, R., Shinomiya, N., and Vande Woude, G. F. (2005). Proliferation and invasion: plasticity in tumor cells. Proc Natl Acad Sci U S A 102, 10528‐10533.

Gavard, J., Lambert, M., Grosheva, I., Marthiens, V., Irinopoulou, T., Riou, J. F., Bershadsky, A., and Mege, R. M. (2003). Lamellipodium extension and cadherin adhesion: two cell responses to cadherin activation relying on distinct signalling pathways. Journal of Cell Science 117, 257‐270.

Gertler, F. B., Hill, K. K., Clark, M. J., and Hoffmann, F. M. (1993). Dosage‐sensitive modifiers of Drosophila abl tyrosine kinase function: prospero, a regulator of axonal outgrowth, and disabled, a novel tyrosine kinase substrate. Genes Dev 7, 441‐453.

Goddard, J. M., Weiland, J. J., and Capecchi, M. R. (1986). Isolation and characterization of Caenorhabditis elegans DNA sequences homologous to the v‐abl oncogene. Proc Natl Acad Sci U S A 83, 2172‐2176.

Goff, S. P., Gilboa, E., Witte, O., and Baltimore, D. (1980). Structure of the Abelson murine leukemia virus genome and the homologous cellular gene: Studies with cloned viral DNA. Cell 22, 777‐785.

Grevengoed, E. E., Fox, D. T., Gates, J., and Peifer, M. (2003). Balancing different types of actin polymerization at distinct sites: roles for Abelson kinase and Enabled. Journal of Cell Biology 163, 1267‐1279.

Grevengoed, E. E., Loureiro, J. J., Jesse, T. L., and Peifer, M. (2001). Abelson kinase regulates epithelial morphogenesis in Drosophila. Journal of Cell Biology 155, 1185‐1198.

Groffen, J., Stephenson, J. R., Heisterkamp, N., De Klein, A., Bartram, C. R., and Grosveld, G. (1984). Philadelphia chromosomal breakpoints are clustered within a limited region, Bcr, on chromosome 22. Cell 36, 93‐99.

156

Grove, M., Demyanenko, G., Echarri, A., Zipfel, P. A., Quiroz, M. E., Rodriguiz, R. M., Playford, M., Martensen, S. A., Robinson, M. R., Wetsel, W. C., et al. (2004). ABI2‐ deficient mice exhibit defective cell migration, aberrant dendritic spine morphogenesis, and deficits in learning and memory. Molecular and Cellular Biology 24, 10905‐10922.

Gu, J. J., Zhang, N., He, Y. W., Koleske, A. J., and Pendergast, A. M. (2007). Defective T cell development and function in the absence of Abelson kinases. J Immunol 179, 7334‐ 7343.

Hall, A. (1998). Rho GTPases and the actin cytoskeleton. Science 279, 509‐514.

Haskell, M. D., Nickles, A. L., Agati, J. M., Su, L., Dukes, B. D., and Parsons, S. J. (2001a). Phosphorylation of p190 on Tyr1105 by c‐Src is necessary but not sufficient for EGF‐ induced actin disassembly in C3H10T1/2 fibroblasts. J Cell Sci 114, 1699‐1708.

Haskell, M. D., Slack, J. K., Parsons, J. T., and Parsons, S. J. (2001b). c‐Src tyrosine phosphorylation of epidermal growth factor receptor, P190 RhoGAP, and focal adhesion kinase regulates diverse cellular processes. Chem Rev 101, 2425‐2440.

Hazan, R. B., Qiao, R., Keren, R., Badano, I., and Suyama, K. (2004). Cadherin switch in tumor progression. Ann N Y Acad Sci 1014, 155‐163.

Heisterkamp, J., Stephenson, J. R., Groffen, J., Hansen, P. F., de Klein, A., Bartram, C. R., and Grosveld, G. (1983). Localization of the c‐abl oncogene adjacent to a translocation point in chronic myelocytic leukemia. Nature 306, 329‐342.

Helwani, F. M., Kovacs, E. M., Paterson, A. D., Verma, S., Ali, R. G., Fanning, A. S., Weed, S. A., and Yap, A. S. (2004). Cortactin is necessary for E‐cadherin‐mediated contact formation and actin reorganization. J Cell Biol 164, 899‐910.

Henkemeyer, M. J., Bennett, R. L., Gertler, F. B., and Hoffmann, F. M. (1988). DNA sequence, structure, and tyrosine kinase activity of the Drosophila melanogaster Abelson proto‐oncogene homolog. Mol Cell Biol 8, 843‐853.

Hernandez, S. E., Settleman, J., and Koleske, A. J. (2004). Adhesion‐dependent regulation of p190RhoGAP in the developing brain by the Abl‐related gene tyrosine kinase. Current Biology 14, 691‐696.

Hirohashi, S., and Kanai, Y. (2003). Cell adhesion system and human cancer morphogenesis. Cancer Sci 94, 575‐581.

157

Hiscox, S., and Jiang, W. G. (1999). Hepatocyte growth factor/scatter factor disrupts epithelial tumour cell‐cell adhesion: involvement of beta‐catenin. Anticancer Research 19, 509‐517.

Hoffman, F. M., Fresco, L. D., Hoffman‐Falk, H., and Shilo, B.‐Z. (1983). Nucleotide sequences of the Drosophila src and abl homologs: Conservation and variability in the src family oncogenes. Cell 35, 393‐401.

Hu, H., Bliss, J. M., Wang, Y., and Colicelli, J. (2005). RIN1 is an ABL tyrosine kinase activator and a regulator of epithelial‐cell adhesion and migration. Curr Biol 15, 815‐823.

Huber, M. A., Kraut, N., and Beug, H. (2005). Molecular requirements for epithelial‐ mesenchymal transition during tumor progression. Curr Opin Cell Biol 17, 548‐558.

Hurle, R. A., Davies, G., Parr, C., Mason, M. D., Jenkins, S. A., Kynaston, H. G., and Jiang, W. G. (2005). Hepatocyte growth factor/scatter factor and prostate cancer: a review. Histol Histopathol 20, 1339‐1349.

Imamura, Y., Itoh, M., Maeno, Y., Tsukita, S., and Nagafuchi, A. (1999). Functional domains of alpha‐catenin required for the strong state of cadherin‐based cell adhesion. J Cell Biol 144, 1311‐1322.

Ito, Y., Pandey, P., Mishra, N., Kumar, S., Narula, N., Kharbanda, S., Saxena, S., and Kufe, D. (2001). Targeting of the c‐Abl tyrosine kinase to mitochondria in endoplasmic reticulum stress‐induced apoptosis. Mol Cell Biol 21, 6233‐6242.

Ivanov, A. I., Hunt, D., Utech, M., Nusrat, A., and Parkos, C. A. (2005). Differential Roles for Actin Polymerization and a Myosin II Motor in Assembly of the Epithelial Apical Junctional Complex. Molecular Biology of the Cell 16, 2636‐2650.

Izumi, G., Sakisaka, T., Baba, T., Tanaka, S., Morimoto, K., and Takai, Y. (2004). Endocytosis of E‐cadherin regulated by Rac and Cdc42 small G proteins through IQGAP1 and actin filaments. J Cell Biol 166, 237‐248.

Jabbour, E., Cortes, J., OʹBrien, S., Giles, F., and Kantarjian, H. (2007). New targeted therapies for chronic myelogenous leukemia: opportunities to overcome imatinib resistance. Semin Hematol 44, S25‐31.

158

Jackson, P., and Baltimore, D. (1989). N‐terminal mutations activate the leukemogenic potential of the myristoylated form of c‐Abl. Embo J 8, 449‐456.

Jaffe, A. B., and Hall, A. (2002). Rho GTPases in transformation and metastasis. Adv Cancer Res 84, 57‐80.

Jamora, C., and Fuchs, E. (2002). Intercellular adhesion, signalling and the cytoskeleton. Nat Cell Biol 4, E101‐108.

Jiang, W. G., Martin, T. A., Parr, C., Davies, G., Matsumoto, K., and Nakamura, T. (2005). Hepatocyte growth factor, its receptor, and their potential value in cancer therapies. Crit Rev Oncol Hematol 53, 35‐69.

Jin, H., and Wang, J. Y. (2007). Abl tyrosine kinase promotes dorsal ruffles but restrains lamellipodia extension during cell spreading on fibronectin. Mol Biol Cell 18, 4143‐4154.

Kain, K. H., and Klemke, R. L. (2001). Inhibition of cell migration by Abl family tyrosine kinases through uncoupling of Crk‐CAS complexes. J Biol Chem 276, 16185‐16192.

Kantarjian, H., Sawyers, C., Hochhaus, A., Guilhot, F., Schiffer, C., Gambacorti‐Passerini, C., Niederwieser, D., Resta, D., Capdeville, R., Zoellner, U., et al. (2002). Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia.[see comment][erratum appears in N Engl J Med 2002 Jun 13;346(24):1923]. New England Journal of Medicine 346, 645‐652.

Kerkela, R., Grazette, L., Yacobi, R., Iliescu, C., Patten, R., Beahm, C., Walters, B., Shevtsov, S., Pesant, S., Clubb, F. J., et al. (2006). Cardiotoxicity of the cancer therapeutic agent imatinib mesylate. Nat Med 12, 908‐916.

Kharbanda, S., Ren, R., Pandey, P., Shafman, T. D., Feller, S. M., Weichselbaum, R. R., and Kufe, D. W. (1995). Activation of the c‐Abl tyrosine kinase in the stress response to DNA‐damaging agents. Nature 376, 785‐788.

Kipreos, E. T., and Wang, J. Y. (1992). Cell cycle‐regulated binding of c‐Abl tyrosine kinase to DNA. Science 256, 382‐385.

Kiyokawa, E., Hashimoto, Y., Kobayashi, S., Sugimura, H., Kurata, T., and Matsuda, M. (1998). Activation of Rac1 by a Crk SH3‐binding protein, DOCK180. Genes & Development 12, 3331‐3336.

159

Kjoller, L., and Hall, A. (1999). Signaling to Rho GTPases. Exp Cell Res 253, 166‐179.

Klemke, R. L., Leng, J., Molander, R., Brooks, P. C., Vuori, K., and Cheresh, D. A. (1998). CAS/Crk coupling serves as a ʺmolecular switchʺ for induction of cell migration. J Cell Biol 140, 961‐972.

Knudsen, B. S., and Edlund, M. (2004). Prostate cancer and the met hepatocyte growth factor receptor. Adv Cancer Res 91, 31‐67.

Kobielak, A., and Fuchs, E. (2004). Alpha‐catenin: at the junction of intercellular adhesion and actin dynamics. Nat Rev Mol Cell Biol 5, 614‐625.

Kobielak, A., Pasolli, H. A., and Fuchs, E. (2004). Mammalian formin‐1 participates in adherens junctions and polymerization of linear actin cables. Nat Cell Biol 6, 21‐30.

Koch, A. W., Bozic, D., Pertz, O., and Engel, J. (1999). Homophilic adhesion by cadherins. Curr Opin Struct Biol 9, 275‐281.

Koleske, A. J., Gifford, A. M., Scott, M. L., Nee, M., Bronson, R. T., Miczek, K. A., and Baltimore, D. (1998). Essential roles for the Abl and Arg tyrosine kinases in neurulation. Neuron 21, 1259‐1272.

Kong‐Beltran, M., Seshagiri, S., Zha, J., Zhu, W., Bhawe, K., Mendoza, N., Holcomb, T., Pujara, K., Stinson, J., Fu, L., et al. (2006). Somatic mutations lead to an oncogenic deletion of met in lung cancer. Cancer Res 66, 283‐289.

Kovacs, E. M., Goodwin, M., Ali, R. G., Paterson, A. D., and Yap, A. S. (2002). Cadherin‐ directed actin assembly: E‐cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts. Curr Biol 12, 379‐382.

Kruh, G. D., Perego, R., Miki, T., and Aaronson, S. A. (1990). The complete coding sequence of arg defines the Abelson subfamily of cytoplasmic tyrosine kinases. Proc Natl Acad Sci U S A 87.

Lamorte, L., Kamikura, D. M., and Park, M. (2000). A switch from p130Cas/Crk to Gab1/Crk signaling correlates with anchorage independent growth and JNK activation in cells transformed by the Met receptor oncoprotein. Oncogene 19, 5973‐5981.

160

Lamorte, L., Rodrigues, S., Naujokas, M., and Park, M. (2002a). Crk synergizes with epidermal growth factor for epithelial invasion and morphogenesis and is required for the met morphogenic program. J Biol Chem 277, 37904‐37911.

Lamorte, L., Royal, I., Naujokas, M., and Park, M. (2002b). Crk adapter proteins promote an epithelial‐mesenchymal‐like transition and are required for HGF‐mediated cell spreading and breakdown of epithelial adherens junctions. Molecular Biology of the Cell 13, 1449‐1461.

Lampugnani, M. G., Corada, M., Andriopoulou, P., Esser, S., Risau, W., and Dejana, E. (1997). Cell confluence regulates tyrosine phosphorylation of adherens junction components in endothelial cells. J Cell Sci 110 ( Pt 17), 2065‐2077.

Larue, L., Antos, C., Butz, S., Huber, O., Delmas, V., Dominis, M., and Kemler, R. (1996). A role for cadherins in tissue formation. Development 122, 3185‐3194.

Latil, A., Chene, L., Cochant‐Priollet, B., Mangin, P., Fournier, G., Berthon, P., and Cussenot, O. (2003). Quantification of expression of netrins, slits and their receptors in human prostate tumors. Int J Cancer 103, 306‐315.

Leonberg, A. K., and Chai, Y. C. (2007). The functional role of cysteine residues for c‐Abl kinase activity. Mol Cell Biochem 304, 207‐212.

Lesko, E., and Majka, M. (2008). The biological role of HGF‐MET axis in tumor growth and development of metastasis. Front Biosci 13, 1271‐1280.

Lewis, J. M., Baskaran, R., Taagepera, S., Schwartz, M. A., and Wang, J. Y. (1996). Integrin regulation of c‐Abl tyrosine kinase activity and cytoplasmic‐nuclear transport. Proc Natl Acad Sci U S A 93, 15174‐15179.

Li, Y., Shimizu, H., Xiang, S. L., Maru, Y., Takao, N., and Yamamoto, K. (2002). Arg tyrosine kinase is involved in homologous recombinational DNA repair. Biochem Biophys Res Commun 299, 697‐702.

Liao, A. T., McMahon, M., and London, C. A. (2005). Characterization, Expression and Function of c‐MET in Canine Spontaneous Cancers. Veterinary and Comparative Oncology 3, 49.

161

Lilien, J., and Balsamo, J. (2005). The regulation of cadherin‐mediated adhesion by tyrosine phosphorylation/dephosphorylation of beta‐catenin. Curr Opin Cell Biol 17, 459‐465.

Liu, Z. G., Baskaran, R., Lea‐Chou, E. T., Wood, L. D., Chen, Y., Karin, M., and Wang, J. Y. (1996). Three distinct signalling responses by murine fibroblasts to genotoxic stress. Nature 384, 273‐276.

Longati, P., Bardelli, A., Ponzetto, C., Naldini, L., and Comoglio, P. M. (1994). Tyrosines1234‐1235 are critical for activation of the tyrosine kinase encoded by the MET proto‐oncogene (HGF receptor). Oncogene 9, 49‐57. Lozano, E., Betson, M., and Braga, V. M. (2003). Tumor progression: Small GTPases and loss of cell‐cell adhesion. Bioessays 25, 452‐463.

Lu, Z., Ghosh, S., Wang, Z., and Hunter, T. (2003). Downregulation of caveolin‐1 function by EGF leads to the loss of E‐cadherin, increased transcriptional activity of beta‐ catenin, and enhanced tumor cell invasion. Cancer Cell 4, 499‐515.

Luo, Y., and Radice, G. L. (2005). N‐cadherin acts upstream of VE‐cadherin in controlling vascular morphogenesis. J Cell Biol 169, 29‐34.

Ma, X., Bao, J., and Adelstein, R. S. (2007). Loss of cell adhesion causes hydrocephalus in nonmuscle myosin II‐B‐ablated and mutated mice. Mol Biol Cell 18, 2305‐2312.

Mariner, D. J., Davis, M. A., and Reynolds, A. B. (2004). EGFR signaling to p120‐catenin through phosphorylation at Y228. J Cell Sci 117, 1339‐1350.

Mariotti, A., Perotti, A., Sessa, C., and Ruegg, C. (2007). N‐cadherin as a therapeutic target in cancer. Expert Opin Investig Drugs 16, 451‐465.

McAllister, A. K. (2002). Conserved cues for axon and dendrite growth in the developing cortex. Neuron 33, 2‐4.

McLachlan, R. W., Kraemer, A., Helwani, F. M., Kovacs, E. M., and Yap, A. S. (2007). E‐ cadherin adhesion activates c‐Src signaling at cell‐cell contacts. Mol Biol Cell 18, 3214‐ 3223.

Mege, R. M., Gavard, J., and Lambert, M. (2006). Regulation of cell‐cell junctions by the cytoskeleton. Curr Opin Cell Biol 18, 541‐548.

162

Melo, J. V. (1996). The molecular biology of chronic myeloid leukaemia. Leukemia 10, 751‐756.

Miao, H., Nickel, C. H., Cantley, L. G., Bruggeman, L. A., Bennardo, L. N., and Wang, B. (2003). EphA kinase activation regulates HGF‐induced epithelial branching morphogenesis. Journal of Cell Biology 162, 1281‐1292.

Miao, Y. J., and Wang, J. Y. (1996). Binding of A/T‐rich DNA by three high mobility group‐like domains in c‐Abl tyrosine kinase. J Biol Chem 271, 22823‐22830.

Miller, A. L., Wang, Y., Mooseker, M. S., and Koleske, A. J. (2004). The Abl‐related gene (Arg) requires its F‐actin‐microtubule cross‐linking activity to regulate lamellipodial dynamics during fibroblast adhesion. J Cell Biol 165, 407‐419.

Miyashita, Y., and Ozawa, M. (2007). Increased internalization of p120‐uncoupled E‐ cadherin and a requirement for a dileucine motif in the cytoplasmic domain for endocytosis of the protein. J Biol Chem 282, 11540‐11548.

Miyoshi, K., and Hennighausen, L. (2002). β-Catenin: a transforming actor on many stages. Breast Cancer Research 5, 63‐68.

Monga, S. P., Mars, W. M., Pediaditakis, P., Bell, A., Mule, K., Bowen, W. C., Wang, X., Zarnegar, R., and Michalopoulos, G. K. (2002). Hepatocyte growth factor induces Wnt‐ independent nuclear translocation of beta‐catenin after Met‐beta‐catenin dissociation in hepatocytes. Cancer Research 62, 2064‐2071.

Moresco, E. M., Donaldson, S., Williamson, A., and Koleske, A. J. (2005). Integrin‐ mediated dendrite branch maintenance requires Abelson (Abl) family kinases. J Neurosci 25, 6105‐6118.

Moresco, E. M., Scheetz, A. J., Bornmann, W. G., Koleske, A. J., and Fitzsimonds, R. M. (2003). Abl family nonreceptor tyrosine kinases modulate short‐term synaptic plasticity.[erratum appears in J Neurophysiol. 2003 Aug;90(2):1362]. Journal of Neurophysiology 89, 1678‐1687.

Muller, R., Slamon, D. J., Tremblay, J. M., Cline, M. J., and Verma, I. M. (1982). Differential expression of cellular oncogenes during pre‐ and postnatal development of the mouse. Nature 299, 640‐644.

163

Nagafuchi, A., and Takeichi, M. (1988). Cell binding function of E‐cadherin is regulated by the cytoplasmic domain. Embo J 7, 3679‐3684.

Nagar, B., Hantschel, O., Seeliger, M., Davies, J. M., Weis, W. I., Superti‐Furga, G., and Kuriyan, J. (2006). Organization of the SH3‐SH2 unit in active and inactive forms of the c‐Abl tyrosine kinase. Mol Cell 21, 787‐798.

Nagar, B., Hantschel, O., Young, M. A., Scheffzek, K., Veach, D., Bornmann, W., Clarkson, B., Superti‐Furga, G., and Kuriyan, J. (2003). Structural basis for the autoinhibition of c‐Abl tyrosine kinase. Cell 112, 859‐871.

Nimnual, A. S., Taylor, L. J., and Bar‐Sagi, D. (2003). Redox‐dependent downregulation of Rho by Rac.[see comment]. Nature Cell Biology 5, 236‐241.

Noren, N. K., Arthur, W. T., and Burridge, K. (2003). Cadherin engagement inhibits RhoA via p190RhoGAP. J Biol Chem 278, 13615‐13618.

Noren, N. K., Niessen, C. M., Gumbiner, B. M., and Burridge, K. (2001). Cadherin engagement regulates Rho family GTPases. Journal of Biological Chemistry 276, 33305‐ 33308.

Normanno, N., De Luca, A., Bianco, C., Strizzi, L., Mancino, M., Maiello, M. R., Carotenuto, A., De Feo, G., Caponigro, F., and Salomon, D. S. (2006). Epidermal growth factor receptor (EGFR) signaling in cancer. Gene 366, 2‐16.

OʹBrien, L. E., Tang, K., Kats, E. S., Schutz‐Geschwender, A., Lipschutz, J. H., and Mostov, K. E. (2004). ERK and MMPs sequentially regulate distinct stages of epithelial tubule development. Dev Cell 7, 21‐32.

OʹNeill, A. J., Cotter, T. G., Russell, J. M., and Gaffney, E. F. (1997). Abl expression in human fetal and adult tissues, tumours, and tumour microvessels. J Pathol 183, 325‐329.

Ohsugi, M., Larue, L., Schwarz, H., and Kemler, R. (1997). Cell‐junctional and cytoskeletal organization in mouse blastocysts lacking E‐cadherin. Dev Biol 185, 261‐271.

Okuda, K., Weisberg, E., Gilliland, D. G., and Griffin, J. D. (2001). ARG tyrosine kinase activity is inhibited by STI571. Blood 97, 2440‐2448.

Olofsson, B. (1999). Rho guanine dissociation inhibitors: pivotal molecules in cellular signalling. Cell Signal 11, 545‐554.

164

Ozawa, M. (1998). Identification of the region of alpha‐catenin that plays an essential role in cadherin‐mediated cell adhesion. J Biol Chem 273, 29524‐29529.

Ozawa, M., Baribault, H., and Kemler, R. (1989). The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. Embo J 8, 1711‐1717.

Paravicini, T. M., and Touyz, R. M. (2008). NADPH oxidases, reactive oxygen species, and hypertension: clinical implications and therapeutic possibilities. Diabetes Care 31 Suppl 2, S170‐180.

Paszek, M. J., Zahir, N., Johnson, K. R., Lakins, J. N., Rozenberg, G. I., Gefen, A., Reinhart‐King, C. A., Margulies, S. S., Dembo, M., Boettiger, D., et al. (2005). Tensional homeostasis and the malignant phenotype. Cancer Cell 8, 241‐254.

Peacock, J. G., Miller, A. L., Bradley, W. D., Rodriguez, O. C., Webb, D. J., and Koleske, A. J. (2007). The Abl‐related gene tyrosine kinase acts through p190RhoGAP to inhibit actomyosin contractility and regulate focal adhesion dynamics upon adhesion to fibronectin. Mol Biol Cell 18, 3860‐3872.

Pece, S., and Gutkind, J. S. (2000). Signaling from E‐cadherins to the MAPK pathway by the recruitment and activation of epidermal growth factor receptors upon cell‐cell contact formation. Journal of Biological Chemistry 275, 41227‐41233.

Pendergast, A. M. (2002). The Abl family kinases: mechanisms of regulation and signaling. Advances in Cancer Research 85, 51‐100.

Perego, R., Ron, D., and Kruh, G. D. (1991). Arg encodes a widely expressed 145 kDa protein‐tyrosine kinase. Oncogene 6, 1899‐1902.

Perez‐Moreno, M., and Fuchs, E. (2006). Catenins: keeping cells from getting their signals crossed. Dev Cell 11, 601‐612.

Perez‐Moreno, M., Jamora, C., and Fuchs, E. (2003). Sticky business: orchestrating cellular signals at adherens junctions. Cell 112, 535‐548.

Pertz, O., Bozic, D., Koch, A. W., Fauser, C., Brancaccio, A., and Engel, J. (1999). A new crystal structure, Ca2+ dependence and mutational analysis reveal molecular details of E‐cadherin homoassociation. Embo J 18, 1738‐1747.

165

Peschard, P., Ishiyama, N., Lin, T., Lipkowitz, S., and Park, M. (2004). A conserved DpYR motif in the juxtamembrane domain of the Met receptor family forms an atypical c‐Cbl/Cbl‐b tyrosine kinase binding domain binding site required for suppression of oncogenic activation. Journal of Biological Chemistry 279, 29565‐29571.

Peschard, P., and Park, M. (2003). Escape from Cbl‐mediated downregulation: a recurrent theme for oncogenic deregulation of receptor tyrosine kinases. Cancer Cell 3, 519‐523.

Petrelli, A., Gilestro, G. F., Lanzardo, S., Comoglio, P. M., Migone, N., and Giordano, S. (2002). The endophilin‐CIN85‐Cbl complex mediates ligand‐dependent downregulation of c‐Met. Nature 416, 187‐190.

Piedra, J., Martinez, D., Castano, J., Miravet, S., Dunach, M., and de Herreros, A. G. (2001). Regulation of beta‐catenin structure and activity by tyrosine phosphorylation. Journal of Biological Chemistry 276, 20436‐20443.

Piedra, J., Miravet, S., Castano, J., Palmer, H. G., Heisterkamp, N., Garcia de Herreros, A., and Dunach, M. (2003). p120 Catenin‐associated Fer and Fyn tyrosine kinases regulate beta‐catenin Tyr‐142 phosphorylation and beta‐catenin‐alpha‐catenin Interaction. Mol Cell Biol 23, 2287‐2297.

Plattner, R., Irvin, B. J., Guo, S., Blackburn, K., Kazlauskas, A., Abraham, R. T., York, J. D., and Pendergast, A. M. (2003). A new link between the c‐Abl tyrosine kinase and phosphoinositide signalling through PLC‐gamma1. Nature Cell Biology 5, 309‐319.

Plattner, R., Kadlec, L., DeMali, K. A., Kazlauskas, A., and Pendergast, A. M. (1999). c‐ Abl is activated by growth factors and Src family kinases and has a role in the cellular response to PDGF. Genes and Development 13, 2400‐2411.

Plattner, R., Koleske, A. J., Kazlauskas, A., and Pendergast, A. M. (2004). Bidirectional signaling links the Abelson kinases to the platelet‐derived growth factor receptor. Molecular & Cellular Biology 24, 2573‐2583.

Plattner, R., and Pendergast, A. M. (2003). Activation and signaling of the Abl tyrosine kinase: bidirectional link with phosphoinositide signaling. Cell Cycle 2, 273‐274.

166

Pollack, A. L., Barth, A. I., Altschuler, Y., Nelson, W. J., and Mostov, K. E. (1997). Dynamics of beta‐catenin interactions with APC protein regulate epithelial tubulogenesis. J Cell Biol 137, 1651‐1662.

Ponzetto, C., Bardelli, A., Zhen, Z., Maina, F., dalla Zonca, P., Giordano, S., Graziani, A., Panayotou, G., and Comoglio, P. M. (1994). A multifunctional docking site mediates signaling and transformation by the hepatocyte growth factor/scatter factor receptor family. Cell 77, 261‐271.

Poppe, M., Feller, S. M., Romer, G., and Wessler, S. (2007). Phosphorylation of Helicobacter pylori CagA by c‐Abl leads to cell motility. Oncogene 26, 3462‐3472.

Potter, M. D., Barbero, S., and Cheresh, D. A. (2005). Tyrosine phosphorylation of VE‐ cadherin prevents binding of p120‐ and beta‐catenin and maintains the cellular mesenchymal state. J Biol Chem 280, 31906‐31912.

Prasad, A., Fernandis, A. Z., Rao, Y., and Ganju, R. K. (2004). Slit protein‐mediated inhibition of CXCR4‐induced chemotactic and chemoinvasive signaling pathways in breast cancer cells. J Biol Chem 279, 9115‐9124.

Presta, M., DellʹEra, P., Mitola, S., Moroni, E., Ronca, R., and Rusnati, M. (2005). Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine Growth Factor Rev 16, 159‐178.

Ren, R., Mayer, B. J., Cicchetti, P., and Baltimore, D. (1993). Identification of a ten‐amino acid proline‐rich SH3 binding site. Science 259, 1157‐1161.

Reshetnikova, G., Troyanovsky, S., and Rimm, D. L. (2007). Definition of a direct extracellular interaction between Met and E‐cadherin. Cell Biol Int 31, 366‐373.

Restucci, B., Maiolino, P., Martano, M., Esposito, G., De Filippis, D., Borzacchiello, G., and Lo Muzio, L. (2007). Expression of beta‐catenin, E‐cadherin and APC in canine mammary tumors. Anticancer Res 27, 3083‐3089.

Reya, T., Duncan, A. W., Ailles, L., Domen, J., Scherer, D. C., Willert, K., Hintz, L., Nusse, R., and Weissman, I. L. (2003). A role for Wnt signalling in self‐renewal of haematopoietic stem cells. Nature 423, 409‐414.

Reynolds, A. B. (2007). p120‐catenin: Past and present. Biochim Biophys Acta 1773, 2‐7.

167

Rhee, J., Buchan, T., Zukerberg, L., Lilien, J., and Balsamo, J. (2007). Cables links Robo‐ bound Abl kinase to N‐cadherin‐bound beta‐catenin to mediate Slit‐induced modulation of adhesion and transcription. Nat Cell Biol 9, 883‐892.

Rhee, J., Mahfooz, N. S., Arregui, C., Lilien, J., Balsamo, J., and VanBerkum, M. F. (2002). Activation of the repulsive receptor Roundabout inhibits N‐cadherin‐mediated cell adhesion. Nat Cell Biol 4, 798‐805.

Rickles, R. J., Botfield, M. C., Weng, Z., Taylor, J. A., Green, O. M., Brugge, J. S., and Zoller, M. J. (1994). Identification of Src, Fyn, Lyn, PI3K and Abl SH3 domain ligands using phage display libraries. Embo J 13, 5598‐5604.

Ridley, A. J., Comoglio, P. M., and Hall, A. (1995). Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cells. Mol Cell Biol 15, 1110‐1122.

Rodrigues, G. A., and Park, M. (1994). Autophosphorylation modulates the kinase activity and oncogenic potential of the Met receptor tyrosine kinase. Oncogene 9, 2019‐ 2027.

Rosario, M., and Birchmeier, W. (2003). How to make tubes: signaling by the Met receptor tyrosine kinase. Trends Cell Biol 13, 328‐335.

Rosen, E. M., Lamszus, K., Laterra, J., Polverini, P. J., Rubin, J. S., and Goldberg, I. D. (1997). HGF/SF in angiogenesis. Ciba Found Symp 212, 215‐226; discussion 227‐219.

Rossman, K. L., Der, C. J., and Sondek, J. (2005). GEF means go: turning on RHO GTPases with guanine nucleotide‐exchange factors. Nat Rev Mol Cell Biol 6, 167‐180.

Roura, S., Miravet, S., Piedra, J., Garcia de Herreros, A., and Dunach, M. (1999). Regulation of E‐cadherin/Catenin association by tyrosine phosphorylation. Journal of Biological Chemistry 274, 36734‐36740.

Royal, I., Lamarche‐Vane, N., Lamorte, L., Kaibuchi, K., and Park, M. (2000). Activation of cdc42, rac, PAK, and rho‐kinase in response to hepatocyte growth factor differentially regulates epithelial cell colony spreading and dissociation. Molecular Biology of the Cell 11, 1709‐1725.

Royal, I., and Park, M. (1995). Hepatocyte growth factor‐induced scatter of Madin‐Darby canine kidney cells requires phosphatidylinositol 3‐kinase. J Biol Chem 270, 27780‐27787.

168

Sahai, E., and Marshall, C. J. (2002). ROCK and Dia have opposing effects on adherens junctions downstream of Rho. Nature Cell Biology 4, 408‐415.

Sakkab, D., Lewitzky, M., Posern, G., Schaeper, U., Sachs, M., Birchmeier, W., and Feller, S. M. (2000). Signaling of hepatocyte growth factor/scatter factor (HGF) to the small GTPase Rap1 via the large docking protein Gab1 and the adapter protein CRKL. J Biol Chem 275, 10772‐10778.

Samanta, A. K., Lin, H., Sun, T., Kantarjian, H., and Arlinghaus, R. B. (2006). Janus kinase 2: a critical target in chronic myelogenous leukemia. Cancer Res 66, 6468‐6472.

Sanes, J. R., and Lichtman, J. W. (2001). Induction, assembly, maturation and maintenance of a postsynaptic apparatus. Nat Rev Neurosci 2, 791‐805.

Schindler, T., Bornmann, W., Pellicena, P., Miller, W. T., Clarkson, B., and Kuriyan, J. (2000). Structural mechanism for STI‐571 inhibition of abelson tyrosine kinase. Science 289, 1938‐1942.

Schlessinger, J. (2000). Cell signaling by receptor tyrosine kinases. Cell 103, 211‐225.

Schock, F., and Perrimon, N. (2002). Molecular mechanisms of epithelial morphogenesis. Annu Rev Cell Dev Biol 18, 463‐493.

Sheffield, M., Loveless, T., Hardin, J., and Pettitt, J. (2007). C. elegans Enabled exhibits novel interactions with N‐WASP, Abl, and cell‐cell junctions. Curr Biol 17, 1791‐1796.

Shewan, A. M., Maddugoda, M., Kraemer, A., Stehbens, S. J., Verma, S., Kovacs, E. M., and Yap, A. S. (2005). Myosin 2 is a key rho kinase target necessary for the local concentration of e‐cadherin at cell‐cell contacts. Molecular Biology of the Cell 16, 4531‐ 4542.

Shi, Y., Alin, K., and Goff, S. P. (1995). Abl‐interactor‐1, a novel SH3 protein binding to the carboxy‐terminal portion of the Abl protein, suppresses v‐abl transforming activity. Genes Dev 9, 2583‐2597.

Shinkaruk, S., Bayle, M., Lain, G., and Deleris, G. (2003). Vascular endothelial cell growth factor (VEGF), an emerging target for cancer chemotherapy. Curr Med Chem Anticancer Agents 3, 95‐117.

169

Shishido, T., Akagi, T., Chalmers, A., Maeda, M., Terada, T., Georgescu, M. M., and Hanafusa, H. (2001). Crk family adaptor proteins trans‐activate c‐Abl kinase. Genes Cells 6, 431‐440.

Simpson, L., He, X., Pins, M., Huang, X., Campbell, S. C., Yang, X. J., Perlman, E. J., and Bergan, R. C. (2005). Renal medullary carcinoma and ABL gene amplification. Journal of Urology 173, 1883‐1888.

Sini, P., Cannas, A., Koleske, A. J., DiFiore, P. P., and Scita, G. (2004). Abl‐dependent tyrosine phosphorylation of Sos‐1 mediates growth‐factor‐induced Rac activation. Nature Cell Biology 6, 268‐274.

Siu, R., Fladd, C., and Rotin, D. (2007). N‐cadherin is an in vivo substrate for protein tyrosine phosphatase sigma (PTPsigma) and participates in PTPsigma‐mediated inhibition of axon growth. Mol Cell Biol 27, 208‐219.

Smith, J. M., Katz, S., and Mayer, B. J. (1999). Activation of the Abl tyrosine kinase in vivo by Src homology 3 domains from the Src homology 2/Src homology 3 adaptor Nck. J Biol Chem 274, 27956‐27962.

Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., and et al. (1993). SH2 domains recognize specific phosphopeptide sequences. Cell 72, 767‐778.

Sossey‐Alaoui, K., Li, X., and Cowell, J. K. (2007). c‐Abl‐mediated phosphorylation of WAVE3 is required for lamellipodia formation and cell migration. J Biol Chem 282, 26257‐26265.

Srinivasan, D., and Plattner, R. (2006). Activation of Abl tyrosine kinases promotes invasion of aggressive breast cancer cells. Cancer Res 66, 5648‐5655.

Steinberg, M. S., and McNutt, P. M. (1999). Cadherins and their connections: adhesion junctions have broader functions. Current Opinion in Cell Biology 11, 554‐560.

Stella, M. C., and Comoglio, P. M. (1999). HGF: a multifunctional growth factor controlling cell scattering. Int J Biochem Cell Biol 31, 1357‐1362.

Stevens, T. L., Rogers, E. M., Koontz, L. M., Fox, D. T., Homem, C. C., Nowotarski, S. H., Artabazon, N. B., and Peifer, M. (2007). Using Bcr‐Abl to Examine Mechanisms by which Abl Kinase Regulates Morphogenesis in Drosophila. Mol Biol Cell.

170

Stoker, M., Gherardi, E., Perryman, M., and Gray, J. (1987). Scatter factor is a fibroblast‐ derived modulator of epithelial cell mobility. Nature 327, 239‐242.

Stupack, D. G., Cho, S. Y., and Klemke, R. L. (2000). Molecular signaling mechanisms of cell migration and invasion. Immunol Res 21, 83‐88.

Sun, X., Majumder, P., Shioya, H., Wu, F., Kumar, S., Weichselbaum, R., Kharbanda, S., and Kufe, D. (2000a). Activation of the cytoplasmic c‐Abl tyrosine kinase by reactive oxygen species. Journal of Biological Chemistry 275, 17237‐17240.

Sun, X., Majumder, P., Shioya, H., Wu, F., Kumar, S., Weichselbaum, R., Kharbanda, S., and Kufe, D. (2000b). Activation of the cytoplasmic c‐Abl tyrosine kinase by reactive oxygen species. J Biol Chem 275, 17237‐17240.

Sun, X., Wu, F., Datta, R., Kharbanda, S., and Kufe, D. (2000c). Interaction between protein kinase C delta and the c‐Abl tyrosine kinase in the cellular response to oxidative stress. J Biol Chem 275, 7470‐7473.

Suyama, K., Shapiro, I., Guttman, M., and Hazan, R. B. (2002). A signaling pathway leading to metastasis is controlled by N‐cadherin and the FGF receptor. Cancer Cell 2, 301‐314.

Suzuki, M., Mimuro, H., Suzuki, T., Park, M., Yamamoto, T., and Sasakawa, C. (2005). Interaction of CagA with Crk plays an important role in Helicobacter pylori‐induced loss of gastric epithelial cell adhesion. J Exp Med 202, 1235‐1247.

Taagepera, S., McDonald, D., Loeb, J. E., Whitaker, L. L., McElroy, A. K., Wang, J. Y., and Hope, T. J. (1998). Nuclear‐cytoplasmic shuttling of C‐ABL tyrosine kinase. Proc Natl Acad Sci U S A 95, 7457‐7462.

Takauji, S. R., Watanabe, M., Uyama, R., Nakagawa, T., Miyajima, N., Mochizuki, M., Nishimura, R., Sugano, S., and Sasaki, N. (2007). Expression and subcellular localization of E‐cadherin, alpha‐catenin, and beta‐catenin in 8 feline mammary tumor cell lines. J Vet Med Sci 69, 831‐834.

Takeichi, M. (1994). The cadherin cell adhesion receptor family: roles in multicellular organization and neurogenesis. Prog Clin Biol Res 390, 145‐153.

171

Takeichi, M. (1995). Morphogenetic roles of classic cadherins. Curr Opin Cell Biol 7, 619‐ 627.

Takino, T., Tamura, M., Miyamori, H., Araki, M., Matsumoto, K., Sato, H., and Yamada, K. M. (2003). Tyrosine phosphorylation of the CrkII adaptor protein modulates cell migration. J Cell Sci 116, 3145‐3155.

Tammer, I., Brandt, S., Hartig, R., Konig, W., and Backert, S. (2007). Activation of Abl by Helicobacter pylori: a novel kinase for CagA and crucial mediator of host cell scattering. Gastroenterology 132, 1309‐1319.

Tanos, B., and Pendergast, A. M. (2006). Abl tyrosine kinase regulates endocytosis of the epidermal growth factor receptor. J Biol Chem 281, 32714‐32723.

Tatton, L., Morley, G. M., Chopra, R., and Khwaja, A. (2003). The Src‐selective kinase inhibitor PP1 also inhibits Kit and Bcr‐Abl tyrosine kinases. J Biol Chem 278, 4847‐4853.

Tcherkezian, J., and Lamarche‐Vane, N. (2007). Current knowledge of the large RhoGAP family of proteins. Biol Cell 99, 67‐86.

Thiery, J. P. (2002). Epithelial‐mesenchymal transitions in tumour progression. Nature Reviews Cancer 2, 442‐454.

Thiery, J. P. (2003). Cell adhesion in development: a complex signaling network. Curr Opin Genet Dev 13, 365‐371.

Thoreson, M. A., Anastasiadis, P. Z., Daniel, J. M., Ireton, R. C., Wheelock, M. J., Johnson, K. R., Hummingbird, D. K., and Reynolds, A. B. (2000). Selective uncoupling of p120(ctn) from E‐cadherin disrupts strong adhesion. J Cell Biol 148, 189‐202.

Tunggal, J. A., Helfrich, I., Schmitz, A., Schwarz, H., Gunzel, D., Fromm, M., Kemler, R., Krieg, T., and Niessen, C. M. (2005). E‐cadherin is essential for in vivo epidermal barrier function by regulating tight junctions. Embo J 24, 1146‐1156.

Vaezi, A., Bauer, C., Vasioukhin, V., and Fuchs, E. (2002). Actin cable dynamics and Rho/Rock orchestrate a polarized cytoskeletal architecture in the early steps of assembling a stratified epithelium. Developmental Cell 3, 367‐381.

172

Van Etten, R. A., Jackson, P., and Baltimore, D. (1989). The mouse type IV c‐abl gene product is a nuclear protein, and activation of transforming ability is associated with cytoplasmic localization. Cell 58, 669‐678.

Vasioukhin, V., Bauer, C., Degenstein, L., Wise, B., and Fuchs, E. (2001). Hyperproliferation and defects in epithelial polarity upon conditional ablation of alpha‐ catenin in skin. Cell 104, 605‐617.

Vasioukhin, V., Bauer, C., Yin, M., and Fuchs, E. (2000). Directed actin polymerization is the driving force for epithelial cell‐cell adhesion. Cell 100, 209‐219.

Vasioukhin, V., and Fuchs, E. (2001). Actin dynamics and cell‐cell adhesion in epithelia. Curr Opin Cell Biol 13, 76‐84.

Verma, S., Shewan, A. M., Scott, J. A., Helwani, F. M., den Elzen, N. R., Miki, H., Takenawa, T., and Yap, A. S. (2004). Arp2/3 activity is necessary for efficient formation of E‐cadherin adhesive contacts. J Biol Chem 279, 34062‐34070.

Vlahovic, G., Ponce, A. M., Rabbani, Z., Salahuddin, F. K., Zgonjanin, L., Spasojevic, I., Vujaskovic, Z., and Dewhirst, M. W. (2007). Treatment with imatinib improves drug delivery and efficacy in NSCLC xenografts. Br J Cancer 97, 735‐740.

Vlahovic, G., Rabbani, Z. N., Herndon, J. E., 2nd, Dewhirst, M. W., and Vujaskovic, Z. (2006). Treatment with Imatinib in NSCLC is associated with decrease of phosphorylated PDGFR‐beta and VEGF expression, decrease in interstitial fluid pressure and improvement of oxygenation. Br J Cancer 95, 1013‐1019.

Vleminckx, K., and Kemler, R. (1999). Cadherins and tissue formation: integrating adhesion and signaling. Bioessays 21, 211‐220.

Wallar, B. J., and Alberts, A. S. (2003). The formins: active scaffolds that remodel the cytoskeleton. Trends Cell Biol 13, 435‐446.

Wang, B., and Kruh, G. D. (1996). Subcellular localization of the Arg protein tyrosine kinase. Oncogene 13, 193‐197.

Wang, B., Wylie, F. G., Teasdale, R. D., and Stow, J. L. (2005). Polarized trafficking of E‐ cadherin is regulated by Rac1 and Cdc42 in Madin‐Darby canine kidney cells. Am J Physiol Cell Physiol 288, C1411‐1419.

173

Wang, J. Y. J., Ledley, F., Goff, S. P., Lee, R., Groner, Y., and Baltimore, D. (1984). The mouse c‐abl locus: Molecular cloning and characterization. Cell 36, 349‐356.

Wang, Y., Miller, A. L., Mooseker, M. S., and Koleske, A. J. (2001). The Abl‐related gene (Arg) nonreceptor tyrosine kinase uses two F‐actin‐binding domains to bundle F‐ actin.[comment]. Proceedings of the National Academy of Sciences of the United States of America 98, 14865‐14870.

Watanabe, T., Tsuda, M., Makino, Y., Ichihara, S., Sawa, H., Minami, A., Mochizuki, N., Nagashima, K., and Tanaka, S. (2006). Adaptor molecule Crk is required for sustained phosphorylation of Grb2‐associated binder 1 and hepatocyte growth factor‐induced cell motility of human synovial sarcoma cell lines. Mol Cancer Res 4, 499‐510.

Weidner, K. M., Di Cesare, S., Sachs, M., Brinkmann, V., Behrens, J., and Birchmeier, W. (1996). Interaction between Gab1 and the c‐Met receptor tyrosine kinase is responsible for epithelial morphogenesis. Nature 384, 173‐176.

Welch, M. D., and Mullins, R. D. (2002). Cellular control of actin nucleation. Annu Rev Cell Dev Biol 18, 247‐288.

Welch, P. J., and Wang, J. Y. (1993). A C‐terminal protein‐binding domain in the retinoblastoma protein regulates nuclear c‐Abl tyrosine kinase in the cell cycle. Cell 75, 779‐790.

Wen, S.‐T., and Van Etten, R. A. (1997). The PAG gene product, a stress‐induced protein with antioxidant properties, is an Abl SH3‐binding protein and a physiological inhibitor of c‐Abl tyrosine kinase activity. Genes Dev 11, 2456‐2467.

Wen, S., Jackson, P., and Van Etten, R. A. (1996). The cytostatic function of c‐Abl is controlled by multiple nuclear localization signals and requires the p53 and Rb tumor suppressor gene products. Embo J 15, 1583‐1595.

Westphal, R. S., Soderling, S. H., Alto, N. M., Langeberg, L. K., and Scott, J. D. (2000). Scar/WAVE‐1, a Wiskott‐Aldrich syndrome protein, assembles an actin‐associated multi‐kinase scaffold. Embo J 19, 4589‐4600.

Wetzler, M., Talpaz, M., Van Etten, R. A., Hirsh‐Ginsberg, C., Beran, M., and Kurzrock, R. (1993). Subcellular localization of Bcr, Abl, and Bcr‐Abl proteins in normal and leukemic cells and correlation of expression with myeloid differentiation. J Clin Invest 92, 1925‐1939.

174

Wildenberg, G. A., Dohn, M. R., Carnahan, R. H., Davis, M. A., Lobdell, N. A., Settleman, J., and Reynolds, A. B. (2006). p120‐catenin and p190RhoGAP regulate cell‐ cell adhesion by coordinating antagonism between Rac and Rho.[see comment]. Cell 127, 1027‐1039.

Woodring, P. J., Litwack, E. D., OʹLeary, D. D., Lucero, G. R., Wang, J. Y., and Hunter, T. (2002). Modulation of the F‐actin cytoskeleton by c‐Abl tyrosine kinase in cell spreading and neurite extension. Journal of Cell Biology 156, 879‐892.

Woodring, P. J., Meisenhelder, J., Johnson, S. A., Zhou, G. L., Field, J., Shah, K., Bladt, F., Pawson, T., Niki, M., Pandolfi, P. P., et al. (2004). c‐Abl phosphorylates Dok1 to promote filopodia during cell spreading. J Cell Biol 165, 493‐503.

Wu, W. S. (2006). The signaling mechanism of ROS in tumor progression. Cancer Metastasis Rev 25, 695‐705.

Xiao, K., Garner, J., Buckley, K. M., Vincent, P. A., Chiasson, C. M., Dejana, E., Faundez, V., and Kowalczyk, A. P. (2005). p120‐Catenin regulates clathrin‐dependent endocytosis of VE‐cadherin. Mol Biol Cell 16, 5141‐5151.

Xiao, K., Oas, R. G., Chiasson, C. M., and Kowalczyk, A. P. (2007). Role of p120‐catenin in cadherin trafficking. Biochim Biophys Acta 1773, 8‐16.

Xu, G., Craig, A. W., Greer, P., Miller, M., Anastasiadis, P. Z., Lilien, J., and Balsamo, J. (2004). Continuous association of cadherin with beta‐catenin requires the non‐receptor tyrosine‐kinase Fer. J Cell Sci 117, 3207‐3219.

Yamada, S., and Nelson, W. J. (2007). Localized zones of Rho and Rac activities drive initiation and expansion of epithelial cell‐cell adhesion. J Cell Biol 178, 517‐527.

Yamada, S., Pokutta, S., Drees, F., Weis, I., and Nelson, W. (2005). Deconstructing the Cadherin‐Catenin‐Actin Complex. Cell 123, 889‐901.

Yamaguchi, H., Miki, H., and Takenawa, T. (2002). Neural Wiskott‐Aldrich syndrome protein is involved in hepatocyte growth factor‐induced migration, invasion, and tubulogenesis of epithelial cells. Cancer Research 62, 2503‐2509.

Yap, A. S., and Kovacs, E. M. (2003). Direct cadherin‐activated cell signaling: a view from the plasma membrane. Journal of Cell Biology 160, 11‐16.

175

Yonemura, S., Itoh, M., Nagafuchi, A., and Tsukita, S. (1995). Cell‐to‐cell adherens junction formation and actin filament organization: similarities and differences between non‐polarized fibroblasts and polarized epithelial cells. Journal of Cell Science 108, 127‐ 142.

Zandy, N. L., Playford, M., and Pendergast, A. M. (2007). Abl tyrosine kinases regulate cell cell adhesion through Rho GTPases. Proc Natl Acad Sci U S A.

Zhu, J., and Shore, S. K. (1996). c‐ABL tyrosine kinase activity is regulated by association with a novel SH3‐domain‐binding protein. Mol Cell Biol 16, 7054‐7062.

Zipfel, P. A., Grove, M., Blackburn, K., Fujimoto, M., Tedder, T. F., and Pendergast, A. M. (2000). The c‐Abl tyrosine kinase is regulated downstream of the B cell antigen receptor and interacts with CD19. J Immunol 165, 6872‐6879.

Zipfel, P. A., Zhang, W., Quiroz, M., and Pendergast, A. M. (2004). Requirement for Abl kinases in T cell receptor signaling.[see comment]. Current Biology 14, 1222‐1231.

176

Biography

Nicole Lynn Zandy

Born to Leonard M. and Patricia A. Zandy on April 7, 1977 in Altoona, PA

Education

Ph.D., Molecular Cancer Biology Duke University, Durham, NC May 2008

B.S., cum laude, Biological Sciences and Psychology University of Pittsburgh, Pittsburgh, PA August 1999

Publications

Zandy, N.L. and Pendergast, A.M. “The Abl‐Crk‐Rac signaling module positively regulates adherens junctions.” Cell Cycle, submitted

Zandy, N.L., Playford, M., and Pendergast, A.M. “Abl tyrosine kinases regulate cell‐cell adhesion through Rho GTPases.” Proc Natl Acad Sci U S A. 2007 Nov 6; 104 (45): 17686‐ 91

Presentations

“Regulation of cell‐cell adhesion by Abl tyrosine kinases,” Nicole L. Zandy and Ann Marie Pendergast, Selected Oral Presentation, 2006 Salk Institute Meeting “Protein Phosphorylation and Cell Signaling”

“Role of Abl kinases in epithelial morphogenesis,” Nicole L. Zandy and Ann Marie Pendergast, Poster and Selected Oral Presentation, 2005, FASEB Summer Research Conference “Growth Factor Receptor Tyrosine Kinases in Mitogenesis, Morphogenesis & Tumorigenesis”

Fellowships

Breast Cancer Research Program Predoctoral Traineeship Award, Department of Defense, BC050405, 4/1/2006‐2/29/2008

177

Predoctoral fellowship, PhRMA Foundation, 1/1/2006‐4/1/2006

Other activities

2007 Graduate Student Symposium Planning Committee, Duke University

2006-present Independent Animal Rescue, Inc., Durham, NC 2007-present President 2006-2007 Volunteer Coordinator, Director, Fundraising Team Leader

2003-present Graduate and Professional Student Council Basketball Committee, Duke University 2007-present Treasurer 2005-2006 Food Chair 2004 Entertainment Co-Chair

2004-2006 Gordon G. Hammes Award Selection Committee, Duke University Medical Center 2005-2006 Chair

178