UNIVERSITY OF CINCINNATI

Date:______

I, ______, hereby submit this work as part of the requirements for the degree of: in:

It is entitled:

This work and its defense approved by:

Chair: ______

The role of the PDZ protein, erbin, in Schwann cells

A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati

in partial fulfillment of the requirements for the degree of

DOCTORATE OF PHILOSOPHY (Ph.D.)

in the

Graduate Program in Neuroscience of the College of Medicine

February 20, 2005 by

Reshma Rangwala

B.S. Duke University, 1999 ABSTRACT

Erbin, a PDZ protein, was identified three years ago as an erbB2 interacting protein. It contains 16 leucine rich repeats in its amino terminus and a single PDZ domain in its carboxy terminus. It was originally believed to serve as a targeting protein; targeting the erbB2 receptor to the basolateral aspect of the . Since then studies have demonstrated that erbin can 1) modulate Ras/MAP kinase by interfering with Ras’ association with Raf

2) influence cell motility by modulating Rho signaling pathways 3) bind to a number of the p120 family of , β-, and the hemidesmosomal proteins β4 and eBPAG1 4) can influence the integrity of the neuromuscular junction. Furthermore, studies have demonstrated that erbin’s interactions with members of the p120 family of catenins are far more robust than its interaction with erbB2 or β-catenin. In light of the observation that erbin can modulate Rho signaling and can bind to p120 catenins, β- catenin, and erbB2 we were interested in determining the role of erbin in the peripheral nerve, and specifically in the Schwann cell. The data described here suggest that erbin co-localizes with E- and β-catenin in the paranode of the peripheral nerve and is, therefore, a putative component of the autotypic . Reduction in erbin expression in the Schwann using siRNA reveals that Schwann cells reduced in erbin expression exhibit 1) alterations in their prototypical bipolar morphology, 2) loss of cell- substrate adhesion 3) increased proliferation 4) a decrease in the hypophosphorylated form of merlin 5) decreased association between β-catenin and E-cadherin, β-catenin and merlin, and erbB2 and CD44 6) increased expression of phosphorylated β-catenin and E- cadherin 7) increased activation of ERK. Pharmacological inhibition of MEK, an ERK kinase, leads to a partial reversal of the alterations listed above suggesting that adherens junction instability following erbin reduction is MEK/ERK dependent. In vitro binding assays also demonstrate that EBP50, an ERM binding protein that contains two PDZ domains, directly interacts with erbin, thus serving as a bridge between merlin and β- catenin.

ACKNOWLEDGEMENTS

I would like to thank the following:

My parents, Yasmin and Abdulla, my sister, Fatima, my brother, Yusuf, and my brother-in law, Yousuf Zafar for their love and support throughout the years;

My thesis advisor: Dr. Larry Sherman, for being both a great friend and excellent advisor;

My thesis committee: Dr. Wallace Ip, Dr. Linda Parysek, Dr. Nancy Ratner, and Dr. Frank Sharp, for their advice and encouragement;

The members of the Sherman lab, past and present, for their much appreciated help and for creating a wonderful place in which to work;

My fellow students in the Neuroscience Program and PSTP, for the good times and great memories;

The Neuroscience Graduate Program, especially Dr. Mike Lehman and Deb Cummins, and the Physician Scientist Training Program, especially Dr. Les Myatt, Dr. Bob Colbert, Dr. Judy Harmony, and Terri Berning.

And last, but not least, my baby kittens Wilbur and Samuel, for staying up with me during the many sleepless nights.

TABLE OF CONTENTS

Table of Contents 1

Table of Figures 3

List of Abbreviations 5

Chapter 1: General Introduction 6

References 35 Figures 63 Figure legends 71

Chapter 2: Erbin regulates Ras-dependent dissociation of E-cadherin from β- catenin in Schwann cells 75

Abstract 76 Introduction 77 Results 80 Discussion 89 Materials and Methods 93 References 100 Figures 108 Figure legends 116

Chapter 3: Erbin mediates merlin’s interaction with β-catenin through an erbin- EBP50 direct association 122

Abstract 123 Introduction 124 Results 127 Discussion 136 Materials and Methods 140 References 145 Figures 154 Figure legends 159

Chapter 4: Erbin influences erbB2 function and its association with CD44 164

Abstract 165 Introduction 166

1 Results 169 Discussion 171 Materials and Methods 173 References 176 Figures 182 Figure legends 183

Chapter 5: General Discussion 184

References 197 Figures 203 Figure legends 204

2 TABLE OF FIGURES

Chapter 1

Figure 1. Schwann cell myelination 666

Figure 2. ERM and merlin regulation 67

Figure 3. The EBP50 protein 68

Figure 4. Distribution of E-cadherin in an “unrolled” Schwann cell, in the 69 paranodal channels and in a native Schwann cell

Figure 5. Homotypic and autotypic adherens junctions 70

Figure 6. Erbin structure and function 71

Figure 7. Direct interactions with erbin 72

Figure 8: Ras/MAP Kinase pathways 73

Chapter 2

Figure 1. Erbin co-localizes with β−catenin and E-cadherin, but not N-cadherin in 111 6 week old wild-type mouse sciatic nerve fibers

Figure 2. Association and co-localization of erbin and β-catenin in primary 112 Schwann cell cultures

Figure 3. Reducing erbin expression disrupts interactions between β-catenin and 113 E-cadherin

Figure 4. Schwann cells with reduced erbin expression exhibit increased 114 proliferation

Figure 5. MEK inhibition reverts the phenotypes induced by a reduction in erbin 115 expression

Figure 6. MEK inhibition rescues the erbin siRNA-induced dissociation of E- 116 cadherin from β-catenin

Figure 7. Reducing erbin expression leads to an increase in the levels of 117 phosphorylated β-catenin and cyclin D1

Figure 8: A model depicting the changes in E-cadherin-β-catenin interactions in

3 the absence and presence of erbin

Chapter 3

Figure 1. Association and co-localization of erbin and merlin in 157 primary Schwann cell cultures

Figure 2. Reduction in erbin expression disrupts interactions 158 between β-catenin and merlin

Figure 3. Reduction in erbin expression alters merlin 159 phosphorylation

Figure 4. MEK inhibition rescues the erbin siRNA-induced 160 dissociation of merlin from β-catenin and decreases the expression of the hypophosphorylated forms of merlin

Figure 5.Erbin is directly associated with EBP50 in the Schwann 161 cell

Chapter 4

Figure 1. An erbB2-CD44-merlin complex may be mediated 182 by erbin

Chapter 5

Figure 1. Diagram depicting an adherens junction “super complex” 203

4 List of Abbreviations CNS: central nervous system DLG: discs-large-1 EBP50: ezrin-radixin-moesin binding protein 50 ERK: extracellular signal-regulated kinase ERM: ezrin-radixin-moesin FAK: kinase FERM: four.1 ezrin-radixin-moesin GEF: guanine-nucleotide-exchange factors GFAP: glial fibrillary acidic protein KO: knockout LAP: LRR and PDZ LEF-1: lymphoid enhancer binding factor LRR: Leucine rich repeat MAP kinase: mitogen activated protein kinase MEF: mouse embryo fibroblast MERLIN: moesin-ezrin-radixin- like protein MBP: basic protein MDCK: Madine-Darby-Canine Kidney MMP: matrix metalloproteases NCAM: neural cell-adhesion molecule NHERF: N+/H+ exchanger regulatory factor NF2: neurofibromatosis 2 PAK: p-21 activated kinase PDZ: PSD 95/ Disc Large/ Zona occludens (ZO)1 PI3-kinase: phosphotidylinisitol 3-kinase PMP22: peripheral myelin protein PNS: peripheral nervous system PSD-95: postsynaptic density –95 siRNA: small interfering ribonucleotide TCF: T cell factor Wnt: wingless ZO-1: zonula occludens

5

Chapter 1 General Introduction

6 The myelin sheath is a unique and necessary adaptation of the nervous system.

In both the central nervous system (CNS) and the peripheral nervous system (PNS), the myelinated fibers are completely ensheathed by myelin except at small 1 micron gaps called the nodes of Ranvier (for review see Scherer, 1999). The myelin sheath effectively reduces the capacitance and increases the resistance across the internodal axonal membrane, thus facilitating saltatory conduction of the action potential down the length of the nerve (Ritchie, 1995). Multiple sclerosis, which arise as a consequence of autoimmune disturbances, the leukodistrophies, a group of genetically determined disorders of myelin formation and maintenance, and the toxometabolic demyelinating diseases such as central pontine myelinolysis are all diseases that specifically affect the formation, integrity, and/or function of the myelin sheath, and often lead to severe disability and, often, early death (Suzuki et al., 2001). It is, therefore, critical to better understand the molecular mechanisms underlying myelination to identify putative, therapeutic molecular targets. The goal of the work described in this dissertation is to understand the contribution of a recently identified intracellular protein, erbin, to the function of peripheral nerves.

Myelination and the development of nerve fibers was an active area of interest amongst the early neurohistologists. The whole internode, once formed, was described by Ranvier in 1872 as a liquid drop, encased within the Schwann cell, much like a fat droplet lies within an adipocyte. Moreover, many of the light microscopists hypothesized that the myelin sheath was a product of the axon, not the Schwann cell. (for review, see

Hildebrand et al., 1994) Because of imperfections in technique and subsequent

7 misinterpretations of the observations, this early view of how the Schwann cell, axon, and myelin interrelate markedly differs from our understanding today. It took the advent of the electron microscope for the structural relationship between axons, Schwann cells and myelin sheaths to be elucidated.

Peripheral nerve development

Nerve development is induced by both axonal and glial signals. Early in peripheral nerve development Schwann cells align along individual axonal fibers and establish domains that develop into myelin internodes. Internodes segregate nodes of Ranvier, a region of the axon where fibers are directly exposed to the extracellular milieu. As wrapping commences, the Schwann cell plasma membrane is compacted and circumferential layers of myelin are rapidly laid down. At the end of each internode myelin lamellae terminate in helically coiled paranodal channels that are continuous with the cytoplasm of the

Schwann cell soma. Within the internode, compact myelin layers become sequestered into many small “islands” by funnel-shaped spirals of cytoplasm known as Schmidt-

Lanterman incisures. (for review, see Scherer, 1999) The development of the node of

Ranvier, specifically, involves significant morphological changes in the myelinated

Schwann cell, and polarization of axonal molecules, cytoskeletal components such as the ERM (ezrin, radixin, moesin) proteins, and ion channels.

Although the role of axo-glial contacts in the clustering of voltage-gated sodium channels at the node remains controversial, recent studies suggest that Schwann cells reorganize and polarize microvillar components before the onset of myelination to the Schwann cell distal tips or “caps”, a region of dynamic growth cone-like behavior; the formation of

8 these caps are associated with the efficient clustering of membrane proteins at the node

(Gatto et al., 2003). (Figure 1)

Schwann cells myelinate peripheral nerves

Peripheral nerve development begins with the outgrowth of naked axonal sprouts.

Schwann cell precursors migrate into the nerves after axonal growth cones have begun

extending towards their targets (Carpenter and Hollyday, 1992; Bhattacharyya et al.,

1994). The timing of Schwann cell differentiation and the final number of Schwann cells

found in the adult nerve is influenced by a number of factors which influence aspects of

survival, differentiation and apoptosis.

Signals that influence Schwann cell proliferation are derived from the axon (for review

see, Jessen and Mirsky, 1999). One such factor includes members of the neuregulin

protein family. Neuregulin are encoded by alternatively spliced transcripts of the

neuregulin-1 gene (Burden and Yarden, 1997). These neuregulins are either membrane bound or soluble and function through the type 1 family of receptor tyrosine kinases which include the epidermal growth factor receptor (EGFR; also known as HER-1 and erbB1), erbB2 (HER-2/neu), erbB3 (HER-3), and erbB4 (HER-4) (Morrissey et al., 1995;

Vartanian et al., 1997; Rahmatullah et al., 1998; Klapper et al., 2000).

Recent evidence suggests that type 1 receptor heterodimerization and function is dependent upon the CD44 family of transmembrane glycoproteins (Sherman et al., 2001), a glycoprotein that has been implicated in a number of cellular functions including cell-

9 cell and cell-matrix contact, mediation of cellular proliferation, metastasis, and regulation

of growth factor signaling (Naor et al., 1997). Specifically, it has been found that the

CD44 ligand, hyaluronic acid, can induce erbB2 phosphorylation in a CD44-dependent

manner (Bourguignon et al., 1997). In Schwann cells CD44 mediates neuregulin-induced

erbB2/erbB3 heterodimerization and erbB2 phosphorylation (Sherman et al., 2000). The

relevance of these interactions in vivo is currently not known.

Upon differentiating into the myelinating form, Schwann cells generate survival factors

that signal in an autocrine loop and become independent of neuregulin for their survival.

Despite this independence, expression of neuregulin-1 in sensory and motoneurons

continues into adulthood. (Jessen and Mirsky, 1999) Furthermore, mature Schwann cells

express erbB2 and erbB3, although erbB2 expression is at reduced levels (Grinspan et al.,

1996).

As Schwann cells mature, they undergo extensive changes in gene expression and protein

synthesis. The myelin proteins, including Po, myelin basic protein (MBP), and peripheral

myelin protein (PMP22) are upregulated. Concomitantly, another group of proteins

including neural cell-adhesion molecule (NCAM), the neurotrophin receptor, p75, and

glial fibrillary acidic protein (GFAP), three proteins which are synthesized by immature

Schwann cells and mature non-myelinating Schwann cells, are down regulated in the myelinating subset. (Jessen et al., 1990) These axon –induced changes, however, are very much reversible, as in the specific case of nerve injury.

Schwann cell growth is regulated by merlin, a novel tumor suppressor

10 The ERM proteins, which are composed of ezrin, radixin, moesin, and merlin (moesin, ezrin, radixin like protein), are membrane--associated proteins that belong to the protein 4.1 superfamily. Merlin is the only member of the merlin/ERM subfamily that is known to function as a tumor suppressor; the mechanism by which it acts as such is currently unknown (for review see McClatchey, 2003). Like the ERM proteins, it localizes to the membrane-cytoskeleton junction, a unique place for a tumor suppressor, in addition to the cytoplasm.

Recent data suggest that merlin’s effect on cellular proliferation is dependent upon its ability to interact with CD44 (Morrisson et al., 2001), therefore placing CD44 as a central effector in Schwann cell development and growth via erbB2/erbB3 (Sherman et al., 2000) and proliferation via merlin. The interrelationship between erbB2/B3 and merlin, the effects of neuregulin on merlin function, and the degree of crosstalk between these signaling pathways are currently not known. Towards these ends, uncovering the mechanism by which merlin functions as a regulator of Schwann cell proliferation may prove significant in understanding the nature of the relationship between these two disparate signaling entities, their role in peripheral nerve development, and response to injury. By better understanding the structure, function, and signaling capacities of the

ERM proteins merlin’s mechanism may be elucidated.

ERM proteins

The ERM proteins crosslink the cytoskeleton with the plasma membrane. The most significant function of the Protein 4.1 family members is their structural stabilization of

11 the . The ERM proteins are concentrated in actin-rich surface structures

such as microvilli, filopodia and membrane ruffles (Franck et al., 1993; Amieva and

Furthmayr, 1995).

Members of the band 4.1 superfamily contain an amino-terminal four.one-ezrin-radixin-

moesin (FERM) domain that mediates interactions with integral membrane proteins

including the hyaluronic acid receptor, CD44 (Nunomura et al., 1997; Bretscher et al.,

2000).

ERM proteins can form intra- and intermolecular associations that are regulated by

protein phosphorylation or lipid interactions. Two intramolecular associations can occur:

one between the N-terminus and the C-terminus and the other within the N-terminus

domain itself (Magendantz et al., 1995). In addition to these intramolecular associations, the N-terminal most residues of the FERM domain can associate with the C-terminus of other ERM proteins (Gary and Bretscher, 1995; Magendantz et al., 1995). When the proteins are in a hypophosphorylated state, ERM proteins adopt a “closed” conformation that masks their binding sites for actin and CD44 (Hirao et al., 1996). Phosphorylation, mediated by Rho kinase (Matsui et al., 1998), separates the N- and C-termini resulting in an “open” form that permits interactions to occur with the actin cytoskeleton and other proteins (Pearson et al., 2000). (Figure 2)

ERM proteins also associate with several types of transporter molecules via PDZ domains. EBP50 (ERM Binding Protein-50) or NHERF1 (Na+/H+ exchanger regulatory

12 factor-1) is a homologue of a rabbit protein cofactor involved in renal brush-border

Na+/H+ exchange. EBP50 colocalizes with actin and ERM proteins in actin-rich structures and nodes of Ranvier, and can be immunoprecipitated with ERM proteins from placental microvilli (Reczek et al., 1997; Murthy et al., 1998; Nguyen et al., 2001). It is currently not known how EBP50 contributes to the ERM/merlin proteins’ function.

(Figure 3)

Merlin

Merlin is encoded by the neurofibromatosis 2 (NF2) gene. Its expression is absent in schwannomas from patients with neurofibromatosis type 2 (NF2), an autosomal dominant inherited cancer syndrome characterized by schwannomas of the eighth cranial nerve, as well as schwannomas of other nerves, meningiomas and ependymomas (Evans et al.,

2000; for review see Sun et al., 2002). This cancer syndrome affects approximately 1 in

35,000 individuals. While this syndrome is quite rare, development of sporadic schwannomas and meningiomas are amongst the most common nervous system tumors in humans and, as in NF2 patients, often show biallelic inactivation of the NF2 gene. In order to elucidate the role of this protein in tumor formation, mice with a targeted mutation in the NF2 gene have been generated. Heterozygous NF2-mutant mice develop malignant and metastatic tumors, including schwannomas, meningiomas, ependymomas, osteosarcomas, and hepatocellular carcinoma, suggesting a broad role for merlin in a variety of cell types (McClatchey et al., 1998). Complete inactivation of the NF2 gene results in embryonic lethality between days 6.5 and 7.0 (McClatchey et al., 1997) suggesting a role for this gene in embryogenesis and tissue differentiation.

13

The NF2 gene is located on chromosome 22 and encodes a 595-residue protein. Analysis of the predicted amino acid sequence reveals the presence of three domains: a FERM domain, a α-helical region, and a unique C-terminal domain (for review, see Baser et al.,

2003). Unlike the other ERM proteins merlin lacks a conventional actin-binding site in its

C-terminus; like the ERMs it contains alternative but functional actin-binding sites

within its FERM domain. This suggests that while a degree of homology exists between

the ERM proteins and merlin, their functions may still be quite unique (Brault et al.,

2001; Pelton et al., 1998; Xu and Gutmann, 1998).

Merlin’s function as a tumor suppressor is hypothesized to be dependent upon its ability

to form intramolecular associations, a phenomenon that is dependent upon its

phosphorylation status. The first association requires the binding of the N-terminus to the

C-terminus, whereas the second involves folding within the N-terminal domain itself

(Gutmann et al., 1999; Sherman et al., 1997). Folding of the merlin N-terminus is

required for the proper localization of the protein beneath the plasma membrane (Brault et al., 2001). The merlin N/C association is relatively weak and dynamic such that the C- terminus has a higher affinity for the N-terminus of ezrin than its own (Nguyen et al.,

2001), suggesting that it’s function as an adaptor-like protein for ezrin may be as or more significant than the functions attributed to it, alone.

Merlin directly associates with a number of proteins that may aid in its function as a putative tumor suppressor. One such protein, paxillin, binds to residues encoded by exon

14 2 of merlin and facilitates merlin localization to the cell membrane where it has been shown to interact with cell surface proteins such as CD44 and β1-integrin (Fernandez-

Valle et al., 2002; Obremski et al., 1998). CD44 interacts with several guanine- nucleotide-exchange factors (GEFs) of the Rho family GTPases (Bourguignon et al.,

2000; Bourguignon et al., 2001). The association between CD44 and specific GEFs leads to the activation of Rac 1. Under the appropriate conditions Rac 1 activation can result in increased Rho activation and altered ERM protein-plasma membrane associations (Hirao et al., 1996).

Although the precise mechanism by which merlin functions as a tumor suppressor has not been elucidated some regulatory pathways have been defined. Specifically, growth permissive conditions enable the activation of Rac and Rho, resulting in the phosphorylation of ERM proteins on C-terminal threonine or serine residues (Shaw et al.,

1998b). In the case of merlin, phosphorylation occurs by Rac1-dependent Pak activation, resulting in an “open” and therefore “inactive” form of merlin, which is incapable of negatively regulating cell growth (Morrison et al., 2001; Shaw et al., 2001; Kissil et al.,

2002; Xiao et al., 2002).

The link between merlin phosphorylation and tumor suppressor status is based on a number of studies. Specifically, when cells are stimulated to undergo growth arrest due to cell-cell contact or by specific cues, i.e. hyaluronic acid, merlin and the ERM proteins exist in their hypophosphorylated states, resulting in molecules that are in their “closed” conformations (Morrison et al., 2001). Because the “closed”

15 conformation of merlin is also the active form of the protein, this model suggests that the

phosphorylation status of merlin is dependent upon growth arrest/proliferation signals

such as confluence and serum deprivation (Shaw et al., 1998a).

In light of observations that ERM/merlin and CD44 can be regulated by members of the

Rho family of small GTPases, which control actin cytoskeleton remodeling, that Rac-1

may serve as a potential regulator of E-cadherin/catenin association (Natale and Watson,

2002), and that merlin localizes to the plasma membrane/cytoskeleton interface it is not

surprising that recent evidence links merlin with adherens junction formation and

stabilization (Lallemand et al., 2003). Furthermore, unlike the ERM proteins, merlin localizes to the paranode with RhoA (Scherer and Gutmann, 1996). Studies performed in

NF2- null mouse embryo fibroblasts (NF2-null MEFs) indicate that loss of merlin leads

to the mislocalization of N-cadherin, β-catenin, and α-catenin; increased activation of

ERK (extracellular signal-regulated kinase), and uncontrolled cellular proliferation

(Lallemand et al., 2003). The mechanism by which merlin influences the formation and

stabilization of the adherens junction is not known, however, an intriguing hypothesis

may lie in its association to β-catenin via the PDZ protein, erbin. Like merlin, both β-

catenin and erbin localize to a specific regions in the peripheral nerve: the paranode, the

mesaxon, and Schmidt-Lanterman incisures.

Schwann cells help divide myelinated peripheral nerves into distinct domains

The peripheral nerve can be divided into specific domains: the internode, juxtaparanode,

paranode, and the . Compact myelin lamellae localize to the internode.

16 They are maintained by P0, an immunoglobulin gene superfamily member with strong adhesive properties (D’Urso et al., 1990; Filbin et al., 1990; Doyle and Colman, 1993) and, once formed, are extremely stable and relatively metabolically inactive. The juxtaparanode, a region that extends approximately 10-15 microns from the paranode

(Stolinski et al., 1981), contains the delayed rectifying potassium channels (Chiu and

Ritchie, 1980), two members of the Shaker-like family of potassium channels, and their associated β1 and β2 subunits (Wang et al., 1993; Rasband et al., 1998). At the nodes of

Ranvier, the that surrounds one Schwann cell continues uninterrupted to the adjacent Schwann cell. The nodal gap can be defined as that area under the basal lamina, between two adjacent Schwann cells. It contains the Schwann cell microvilli, as well as nodal gap substance, which is composed of extracellular matrix (Scherer, 1996). The microvilli contain F-actin (Trapp et al., 1989), and contain the inwardly rectifying potassium channels IRK1 and IRK3 (Mi et al., 1996).

Below the nodal axolemma is a dense undercoating as observed by transmission electron microscopy (Quick and Wax man, 1977). This dense undercoating represents voltage- gated sodium channels (Yasargil et al., 1982), molecular specializations that were first deduced by Ritchie and Rogart in 1977 to be highly enriched at the nodes (Ritchie and

Rogart, 1977). The high concentration of the sodium channels and Na+/K+ ATPase allows for the generation of the action potential in this region of the peripheral nerve.

In addition to these molecular specializations, other molecules also localize to the nodal axolemma including ankyrinG 480/270 kDa (Kordeli et al., 1990; Kordeli et al., 1995),

17 neurofascin, and Nr- (Nr-CAM). Ankyrin interacts with neurofascin, Nr-CAM, and the voltage –dependent sodium channels (Srinivasan et al.,

1988) and links them to the axonal cytoskeleton. The roles of neurofascin and Nr-CAM at the nodes have not been determined.

The paranode contains a unique set of adhesion molecules that serve to 1) tether terminal paranodal loops to the axon, and 2) mediate interactions between adjacent loops. These paranodal junctions play an important role in the process of myelination and saltatory conduction and is a site of early damage in several neuropathies and demyelinating disorders (for review, see Griffiths, 1996).

The role of the paranode in nerve function

The paranode has been strongly implicated in preventing the dissipation of the action potential from the nodal space. Autotypic adherens junctions localize to this region and mediate the interactions between adjacent myelin lamellae, PDZ proteins, and the ERM protein, merlin. In light of recent data indicating that merlin may control Schwann cell proliferation by influencing the formation and stability of the adherens junction

(Lallemand et al., 2003) the question arises as to whether the two functions attributed to merlin, i.e. cellular growth and adhesion in vitro and paranodal stabilization of adjacent lamellae, are related.

The axoglial , a point of strong attachment of myelinating glial cells to the axon, has several important functional roles: it provides partial electrical insulation to the

18 internodal region and it restricts the lateral mobility of the axonal membrane proteins

including ion channels (for review, see Denisenko-Nehrbass et al., 2002).

An intrinsic membrane glycoprotein known as paranodin or Caspr, localizes to the

paranodal axolemma (Einhaber et al., 1997; Menegoz et al., 1997). Paranodin/Caspr is a

member of the neurexin family, and has been postulated to bind to a ligand that is a

member of the neuroligan family (Einhaber et al., 1997; Menegoz et al., 1997). Because

the interactions between neuregulins and neuroligans are calcium mediated, a high

concentration of calcium is found at the axoglial junctions (Ellisman et al., 1980). The

cytoplasmic domain of paranodin/Caspr contains a region that can function as a binding domain for protein 4.1 (Menegoz et al., 1997) which, in turn, can bind to the actin cytoskeleton.

Proteins that are involved in cell motility and polarity also localize to the paranode including tubulin (Peters et al., 1991), actin and spectrin (Trapp et al., 1989), ankyrin

(Kordeli et al., 1990), -32 (Bergoffen et al., 1993), and certain

(Einheber et al., 1993). The sensitivity of the paranodal loops to calcium initially prompted Fannon et al., to examine whether might be involved in the formation and maintenance of the nodal architecture (Fannon et al., 1995).

The different domains of the peripheral nerve have distinct types of cell junctions

The cadherins are a superfamily of calcium dependent adhesion proteins that function in

cell recognition and segregation, morphogenetic regulation, and tumor suppression (for

19 reviews, see Magee and Buxton, 1991; Takeichi, 1991; Geiger and Ayalon, 1992;

Grunwald, 1993). The classical cadherins are the most well studied and include N- cadherin (neural cadherin), E-cadherin (epithelial cadherin), and P-cadherin (placental cadherin).

The cadherins are a family of single-span transmembrane-domain glycoproteins that function as specific cell-cell adhesion molecules (for review see Cavallaro and

Christofori, 2004). Cadherin-mediated cell-cell adhesion is mediated by homophilic protein-protein interactions between two cadherin molecules at the surface of their respective cells. This interaction is mediated by interactions between histidine-alanine- valine (HAV) domains, tryptophan residues and hydrophobic pockets in the most amino- terminal portion of the cadherin molecule (Grunwald et al., 1993). Cadherins show an exquisite specificity in their homophilic interactions by binding the same type of cadherin on an adjacent cell. The intracellular domain of classical cadherins interacts with various catenin proteins to form the cytoplasmic cell-adhesion complex (CCC) (Nagafuchi and

Takeichi, 1988). β-catenin and γ-catenin (also known as ) bind to the same conserved site at the carboxyl terminus of the cadherin molecule in a mutually exclusive manner (Zhurinsky et al., 2000). In contrast, the p120-catenins can interact with a number of sites in the cytoplasmic tail. The p120 catenin family which includes p0071, δ- catenin, and ARVCF (Hatzfeld and Nachtsheim, 1996), is defined as proteins with 10 armadillo repeats and localizes to the cell-cell border. Recent studies indicate that p120 plays a key role in regulating cadherin membrane trafficking in mammalian cells. These studies place the p120s at the hub of a cadherin-catenin regulatory mechanism that

20 controls cadherin plasma membrane levels in cells of both epithelial and endothelial origin. (for review, see Vincent et al., 2004) β-catenin and γ-catenin can bind to α-catenin

(Huber et al., 1997), a molecule that links the CCC to the actin cytoskeleton. An intact

CCC is necessary for strong cell-cell adhesion suggesting that the catenins can modulate, and are necessary for, the adhesive activity of the cadherins (Takeichi, 1991).

Using antibodies against E-cadherin, Fannon et al, demonstrated that E-cadherin is a major adhesive glycoprotein restricted to the noncompacted myelin domains in the peripheral nerve (Fannon et al., 1995). F-actin and β-catenin also co-localize to E- cadherin positive sites. These complexes are confined to the plasma membrane synthesized by a single Schwann cell; E-cadherin was never observed between two adjacent Schwann cells, or between Schwann cells and the axon. Moreover, they found that E-cadherin is excluded from myelin and does not participate in the compaction or adhesion of adjacent myelin lamellae in the internodal region of the nerve. Because they did not detect E-cadherin immunoreactivity in the axonal membrane, the authors hypothesized that E-cadherin and its associated proteins are essential components in the architecture of the Schwann cell cytoplasmic channel network. (Fannon et al., 1995)

(Figure 4)

In classical, so-called “homotypic” adherens junctions, E-cadherin in conjunction with one of its intracellular protein partners, β-catenin, plays a major role in the formation of homophilic complexes that serve to connect two adjacent membranes (Trapp et al., 1989;

Fannon et al., 1995; Gumbiner, 2000). Because E-cadherin mediates the formation of

21 adherens junctions between membrane lamellae of the same cell, they are referred to as

“autotypic” (Fannon et al., 1995) or “reflexive” (Balice-Gordan et al., 1998) adherens- type junction. (Figure 5) Mice lacking E-cadherin in peripheral nerves have a widened gap in the outer mesaxon between the two opposing membranes of the same Schwann cell. However, myelin integrity and the localization of β-catenin in the noncompacted regions of the nerve are maintained in these , suggesting that other factors are required for maintaining these junctional protein complexes. (Young et al., 2002)

Interestingly, at the node of Ranvier where there is no myelin, adjacent Schwann cells associate with each other via microvilli. This association is mediated by another calcium- dependent cell adhesion molecule, N-cadherin (Cifuentes-Diaz, et al., 1994; Wanner and

Wood, 2002). Moreover, studies also indicate that N-cadherin mediates the Schwann cell-axon interaction (Wanner and Wood, 2002).

Catenins play roles in cell adhesion and

In addition to its role in cellular adhesion, β-catenin has also been found to serve as vital component in signal processes during embryonic development and adult tissue homeostasis. Genetic and biochemical studies have identified β-catenin as central player in the wnt/wingless signal transduction cascade, a cascade that is important for pattern formation and axis determination during early development of invertebrate and vertebrate organisms (Kelly et al, 1995; Cox et al, 1996; Willert and Nusse, 1998; Huelsken et al,

2000). In the absence of wnt ligand, β-catenin is part of a supramolecular complex containing axin/conductin and the adenomatous polyposis coli (APC) tumor suppressor

22 protein and is phosphorylated by glycogen synthase kinase 3β (Rubinfeld, et al., 1996;

Yost, et al., 1996; Behrens et al., 1998; Ikeda et al., 1998) which targets the protein for proteosomal degradation (Orford et al., 1997). Once wnt binds to its receptor frizzled, a cascade of signaling events is triggered that leads to the inhibition of glycogen synthase kinase 3β-dependent phosphorylation of β-catenin (for review, see Sakanaka et al.,

2000). This stabilized, hypo-phosphorylated β-catenin accumulates both in the cytoplasm and the nucleus and interacts with the transcription factors lymphoid enhancer binding factor (LEF-1)/T cell factor (TCF) (van Noort, et al. 2002). The β-catenin-

LEF/TCF’s transcription complex controls the expression of myriad sets of target genes, including cyclin D1 and c-myc, that have been implicated in development and tissue formation in both invertebrates and vertebrate animals (for reviews, see Roose and

Clevers, 1999; Hecht and Kemler, 2000; Zhurinsky et al., 2000).

Inappropriate activation of the wnt pathways has also been implicated in cellular proliferation and tumor formation, and increased expression of c-myc and cyclin D1

(Korineck et al., 1997; Morin et al., 1997; Rubinfeld et al., 1997; for reviews, see Bienz and Clevers, 2000 and Polakis, 2000).

Loss of cadherin and/or catenin function often elicits reorganization of the cytoskeleton.

A key function in organizing the actin cytoskeleton is mediated by members of the Rho family of small GTPases. Recent studies indicate that p120-catenin promotes cell migration by recruiting activating Rac1 and cdc42 by inhibiting RhoA (Noren, et al.,

2000; Anastasiadis and Reynolds, 2000). Only cytosolic p120 –catenin is able to

23 modulate small GTPase activity, whereas this function is abolished by the binding of

p120-catenin to cadherins. Furthermore, studies have indicated that p120-catenin, like β-

catenin, can translocate to the nucleus (Daniel and Reynolds, 1999). The functional

implication of this translocation has not been determined.

E-cadherin’s role in tumorigenesis

A hallmark of tumor progression is loss of E-cadherin function (Birchmeier and Behrens,

1994). Loss of E-cadherin function can be caused by a number of factors including

mutations in the E-cadherin gene; down regulation of E-cadherin expression at the

transcriptional level; proteolytic degradation of E-cadherin by matrix metalloproteases

(MMPs); and tyrosine phosphorylation of E-cadherin, N-cadherin, β-catenin, γ-catenin, and p120-catenin resulting in the disassembly of the cytoplasmic cell adhesion complex

(for review, see Cavallaro and Christofori, 2004).

Localization of the autotypic adherens junction components to the paranode

Although the mechanism by which E-cadherin, β-catenin and the other components of

the autotypic adherens junction are localized to the noncompacted myelin domains is not

known, studies in P0 KO mice indicate that the localization of E-cadherin and β-catenin is

severely disrupted (Menichella et al., 2001). Specifically, neither E-cadherin nor β-

catenin localize to the paranode; instead they are found in small puncta throughout the

Schwann cell. Furthermore, only occasional adherens junction localize to the paranode

(Menichella et al., 2001). These observations suggest that P0 is necessary for the

formation of adherens junctions in the peripheral nerve and that the formation of these

24 complexes occurs after the compaction of the myelin sheath in the internodal region. In

contrast, the factors that influence nodal formation protein localization are, as suggested

above, influenced by Schwann cell tips or “caps,” regions enriched in microvillar

components. Schwann cell microvilli (Ichimura and Ellisman, 1991) are characterized by

expression of F-actin (Trapp et al., 1989), ERMs, and EBP50 (Melendez-Vasquez et al.,

2001; Scherer et al., 2001). Localization studies for the ERM proteins (excluding merlin)

and EBP50, suggest that these proteins localize to the nodes of Ranvier in peripheral

nerves (Melendez-Vasquez et al., 2001; Gatto et al., 2003). Both studies utilized

ankyrinG, a nodal component, as a marker for this region. In the case of EBP50,

however, EBP50 immunoreactivity extends beyond ankyrinG immunoreactivity,

indicating that there may be a subpopulation of EBP50 in the paranode. (see, Figure 3a)

LAP proteins may play roles in linking cell adhesion protein complexes to cell signaling

The classical apicobasal polarity seen in epithelial cells is by no means exclusive to this cell type; Schwann cells are similarly organized. Analogous to epithelial cells, the basal/abaxonal surface of the Schwann cell apposes the basal lamina, while the apical/adaxonal surface apposes the axon (for review, see Arroyo and Scherer, 2000).

The selective targeting and retention of proteins at either the apical or basolateral surfaces

of the cell underlie the ability of cells to transport solutes in a vectorial manner (Caplan,

2001). This polarity is orchestrated by a diverse set of protein families, one of which

includes the cell adhesion molecules described above. These molecules, however, cannot

in isolation establish cell-cell junctions; their function is dependent upon cytoplasmic

adaptor proteins that link the membrane proteins to the actin cytoskeleton (for review, see

25 Roh and Margolis, 2003). One such family of adaptor proteins includes the PSD

95/Discs Large/zona occludens (ZO)1 (PDZ) protein family. This family has been named such for the existence of a PDZ domain within each of their respective proteins structures. The PDZ domain is one of the most abundant protein domains found in multicellular eukaryotes, with approximately 320 PDZ domains found within the human genome alone.

PDZ domains were originally identified as approximately 90 amino acid repeats found in several structurally related proteins including the postsynaptic density –95 (PSD-95), the drosophila tumor suppressor protein discs-large-1 (DLG), the tight junction protein zona occludens (ZO-1), and the human erythrocyte membrane cytoskeletal protein p55 (Cho et al., 1992; Willott et al, 1993; Woods and Bryant, 1993).

PDZ proteins can be categorized into three main categories based upon their structure alone. The first category, or the multi-PDZ proteins contain anywhere from 2 to 10 PDZ domains. An example of such a protein is EBP50. The second, known as the MAGUKs

(membrane-associated guanylate kinases), include PSD-95, DLG, and ZO-1, and contain anywhere from one to three PDZ domains, one SH3 domain, and a guanylate kinase domain (GuK). The third category includes proteins that contain PDZ domains in addition to other protein domains, i.e. erbin (for review, see Zhang and Wang, 2003).

PDZ proteins typically associate with the extreme carboxy terminal residues of their ligands (Songyang et al., 1997), however recent studies indicate that some PDZ domains can heterodimerize with other PDZ domains (Hong et al, 2001; Sheng and Sala, 2001),

26 can bind to internal sequences, and can even associate with lipids. Moreover, PDZ- domain mediated interactions can sometimes be modulated by phosphorylation, and while the majority do not contain intrinsic enzymatic activity, some PDZ proteins contain recognizable catalytic domains (for review, see Zhang and Wang, 2003).

Erbin may link components of adherens junctions with signals that regulate Schwann cell proliferation and survival

The PDZ protein of interest here is erbin, originally described as an erbB2 interacting protein that contains 16 leucine rich repeats (LRR) and a single PDZ domain in its carboxy terminus. Because of this unique composition of domains, erbin has been categorized as a member of the LAP (for LRR and PDZ) protein superfamily (Borg et al.,

2000). In the original paper describing erbin, it was found that erbin and erbB2 colocalize to the lateral membrane of human intestinal epithelial cells (Borg et al., 2000). Mutations in the erbin binding site lead to the mislocalization of the receptor in these cells, suggesting that erbin plays a significant role in the localization and signaling of erbB2 in intestinal epithelial cells. (Figure 6)

It has recently been observed, however, that erbin has a higher affinity interaction with the class I p0071/δ-catenin peptide sequence (DSWV) than to the to the class II erbB2 peptide sequence (DVPV) (approximately 10 times less) (Jaulin-Bastard et al., 2002;

Laura et al., 2002). In fact, recent studies have demonstrated that the PDZ domain of erbin binds with high affinity to members of the p120-catenin family that are implicated in regulating cadherin turnover (Davis et al., 2003), including δ-catenin and ARVCF

27 (Laura et al., 2002), and with p0071 (also called plakophilin-4), another p120 family

member, in cell-cell junctions of epithelial cells (Jaulin-Bastard, et al., 2002; Izawa et al.,

2002; Ohno et al., 2002). Although the erbin PDZ domain may associate with β-catenin, it only does so in vitro with very low affinity (Laura et al., 2002). Collectively, these data suggest that erbin may play a role in stabilizing cell-cell junctions. (Figure 7)

Studies also suggests that the LRR may have a role in modulating a number of signaling processes, specifically, the G proteins Ras and Rho. In fact, studies involving erbin over expression and reduction indicate that it acts as a negative regulator of the Ras-Raf-ERK signaling pathway (Huang et al., 2003). Specifically, these authors demonstrate that while erbin binds to the carboxy terminus of erbB2 through its PDZ domain, it has no effect on erbB2 phosphorylation, or binding to the adaptor proteins Shc and Grb2 (Huang et al., 2003). Furthermore, erbin significantly impairs activation of ERK, but not Akt, by ligands that activate receptor tyrosine kinases, and that erbin inhibits ERK activation by active Ras but not by active Raf-1. Finally, they show that erbin inhibits the association of Ras to Raf both in vivo and in vitro.

Extracellular signal-regulated kinases (ERK) are a subfamily of mitogen –activated

protein kinases (MAPK) that play important roles in proliferation, differentiation and

apoptosis (Robinson and Cobb, 1997; Bar-Sagi and Hall, 2000). Following ligand

binding to a receptor tyrosine kinase receptor, the receptor is activated via

autophosphorylation on tyrosine residues (Ullrich and Schlessinger, 1990; Schlessinger,

2000). The phosphorylated tyrosine residues recruit adaptor proteins to the plasma

28 membrane. Grb2, one such adaptor, shuttles the guanyl nucleotide exchange factor

(SOS) to the plasma membrane where it facilitates the exchange of the GDP moiety for

GTP on Ras (Pawson and Scott, 1997). Ras-GTP, activated Ras, directly binds to Raf,

leading to the activation of the Raf molecule (Robinson and Cobb, 1997; Katz and

McCormick, 1997). Active Raf triggers the sequential activation of MEK, a MAPK

kinase, and ERK, ultimately leading to the phosphorylation of various regulatory proteins

including cyclin D1 (Kerkhoff and Rapp, 1998; Kerkhoff et al., 1998). (Figure 8 ) Cyclin

D1 expression can be upregulated by a number of pathways, specifically the wnt

pathway, as mentioned above, and by activation of the Ras/MAPK pathway (Westwick et

al., 1997; Westwick et al., 1998; Joyce et al., 1999; Shtutman et al., 1999; Tetsu and

McCormick, 1999). Furthermore, Rho-dependent pathways can interact with oncogenic

Ras and contribute to cell cycle abnormalities and increases in cylin D1 expression

(Welsh, 2004).

Since the identification of erbin three years ago, the functions attributed to erbin have increased many-fold. In light of data that implicate this protein in influencing both Ras and Rho GTPases, that control actin cytoskeleton remodeling and cadherin-catenin association (Natale and Watson, 2002), erbB2 localization and function, and its ability to bind to a number of cell adhesion molecules, including the p120 family of catenins and β-

catenin (see Figure 6), it can be hypothesized that this protein is an integral component in

peripheral nerve development, structure and integrity, and function. Erbin’s role in the

modulation of Rho signaling directly links this protein to ERM/merlin function, and

indirectly to the localization of nodal proteins to that region of the nerve (Gatto et al.,

29 2003). Because subpopulations of both EBP50 and merlin can directly associate with each other at the paranode of the peripheral nerve, this interaction may also contribute to the formation and stabilization of the adherens junction. Erbin’s ability to bind to p120 catenins and β-catenin suggests that this protein influences junctional complexes in the noncompacted myelin domains in the peripheral nerve, and its ability to bind to erbB2 ultimately places this protein as a significant factor in Schwann cell survival and differentiation.

As the data in chapters 2-4 suggest, erbin loss impacts both adherens junction integrity and function, and indirectly induces loss of contact- dependent growth inhibition, cellular proliferation, morphological changes, localization of adherens junction components, and the association between E-cadherin and β-catenin, merlin and β-catenin, and CD44 and erbB2. Because erbin has both a signaling component mediated by its sixteen leucine rich repeats and serves as an adaptor protein by directly binding to the proteins listed above, the question arises as to whether the changes observed after erbin loss is due to loss of structural integrity of the protein complex or is an indirect consequence of aberrant signaling (i.e. increased MAP kinase signaling), or both. Ras/MAPkinase has been implicated in phosphorylation of β-catenin and its subsequent degradation (Cavallaro and

Christofori, 2004) and can act induce cyclin D1 expression and indirectly effect cellular proliferation. Furthermore, like β-catenin, other members of the adherens junction, like

E-cadherin, are now being implicated in signaling events (Cavallaro and Christofori,

2004), suggesting that loss of cell-cell association mediated by the adherens junction may lead to activation of a multiple proliferation events separately induced by each of the

30 junctional components. The degree of influence that the catenins, cadherins, erbB2,

Ras/MAP kinase, and Rho proteins have on each other is not known, however, it is most

likely that these proteins do not stand in isolation; they are intimately associated with the

function of the others, suggesting that loss of erbin would not only affect nerve function, but could potentially influence processes such as cellular transformation, tumor formation, metastasis, cell survival, and apoptosis.

We are most interested in determining whether erbin mediates 1) the formation and stabilization of the autotypic adherens junction 2) merlin’s association with this specific

junctional complex via EBP50, and 3) a CD44-erbB2 interaction. As such the

overarching hypothesis of this thesis is that erbin stabilizes autotypic adherens junctions and links merlin with the junctional protein complex via an erbin EBP50 direct association.

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62

Figure 1:

Gatto et al., 2003., JCB 162: 489-498

63

Figure 2:

A)

Gautreau et al.,2002. Cur. Opin. Cell Biol.14: 104-109

B)

α-helix Charged FERM domain Proline rich terminus N C

64

Figure 3:

A)

PDZ 1 PDZ II ERM binding region N C

11 97 150 237 S289 358

β2AR PLCβ3 Ezrin CFTR H+ATPase Radixin Moesin PDGFR β -catenin Merlin

B)

Gatto et al., 2003., JCB 162: 489-498

65 Figure 4:

Fannon et al., 1995. J. Cell Biol. 129:189-202.

66 Figure 5:

A) B) C)

Myelin lamellae

E-cadherin

N-termini Paranode of E-cadherin E-cadherin

Myelin lamellae

β-catenin p120 α-catenin

actin

67 Figure 6:

adapted from Kolch, W. Sci STKE. 2003. 199:pe37.

68

Figure 7:

LRR PDZ eBPAG1 ERBIN Integrin β4 erbB2

β-catenin ARVCF p0071 δ-catenin

p120 proteins

69

Figure 8:

70 FIGURE LEGENDS

Figure 1: Model: Schwann cell myelination. Highly motile, premyelinating Schwann cells migrate along the axons. These Schwann cells contain numerous cell surface microvilli (red). Development of the mature Schwann cells occurs concomitantly with the

Schwann cells becoming more longitudinally oriented while retaining many cell surface microvilli. Upon induction, typically using serum and ascorbate, Rho stimulation then leads to the formation of dynamic Schwann cell cap structures, which are enriched in activated ERM proteins and other microvillar components (red). Elaboration of the myelin membrane (blue) occurs after axonal components are clustered and is associated with the formation of early nodes (green).

Figure 2: ERM and merlin regulation. a) Phosphorylation of the ERM carboxy-terminus leads to a weakened self-association and translocation to the membrane-cytoskeleton interface. The “active” forms of the ERM proteins directly interact with actin via their C- terminus and to transmembrane proteins through their N-terminus. Activation of RhoA leads to phosphorylation of the ERMs via the activation of an effector kinase. The ERM proteins can also negatively regulate Rho activity. b) Cartoon of the merlin molecule.

Merlin contains an N-terminal FERM domain and a charged C-terminus. Unlike the

ERM proteins, merlin does not contain an actin binding domain in its C-terminus. Like the ERM proteins, merlin contains a proline rich alpha helical domain.

71 Figure 3: A) Diagram of the EBP50 protein. EBP50 contains two PDZ domains, which mediate the interactions to a number of ion channels as well as to β-catenin. The

ERM/merlin proteins interact with EBP50 through the ERM binding domain located in the carboxy terminus of EBP50. The ERM/merlin proteins interact with EBP50 through their N-termini. B) Staining for the proteins EBP50, AnkG, and MBP (c-f) in teased peripheral nerve preparations. AnkG is a nodal specialization and therefore serves as a marker for the node. (f) Note that EBP50 staining encompasses axonal ankG, however,

EBP50 staining (red) extends past the ankG (green) staining, suggesting, that subpopulations of EBP50 may localize to the paranode.

Figure 4: Model of the distribution of E-cadherin in an “unrolled” Schwann cell, in the paranodal channels and in a native Schwann cell. A) In an unrolled Schwann cell, E- cadherin localizes to regions which form the outer boundaries of the cell. These include the outer mesaxon, the outer loop, the inner loop and the paranodal channel. E-cadherin also localizes to the internal portion of the cell in Schmidt-Lanterman incisures.

Therefore, E-cadherin localization is restricted to a cytoplasmic network which sequesters compact myelin into discrete domains. B) In a Schwann cell in its native

“rolled” conformation, E-cadherin proteins become aligned with each other, leading to the formation of junctional complexes that contribute to the overall stability of the cell.

The location of the E-cadherin in the outer mesaxon suggests that the outer mesaxon helically wraps around the cell and its orientation continues in the adjacent cells. C) E- cadherin localizes to densities in the paranodal channel. Electron microscopy of sciatic nerves from 7-day old mice and rats labeled with anti- P0 (a) or anti EcadEC5 (b-d). Anti

72 EcadEC5 intensely labels densities in the paranode (b, arrows). Note the extensive labeling of the densities in c. In d, the narrowing of the cytoplasm and the widening of the intracellular space is apparent. In a control P0 labeled paranode (a) myelin (arrows) is labeled, but the densities (arrowheads) remain unlabeled. Nars, 0.1 µm. (Fannon et al.,

1995)

Figure 5: Diagram of a homotypic and autotypic adherens junctions. A) Homotypic refers to the association of adjacent membranes from two different cells, while B) autotypic refers to the association of adjacent membranes produced from the same cell as found in the paranode of the peripheral nerve. The paranodal loops are produced from a single Schwann cell. C) Diagram of a typical autotypic adherens junction complex. The

N-terminal amino acids interact with the corresponding residues of an adjacent E- cadherin molecule. The cytoplasmic tail of E-cadherin associates with p120 catenin, β- catenin, and α-catenin, which in turn links the cadherin-catenin complex to the actin cytoskeleton

Figure 6: Erbin structure and function. Erbin is a 180 kDa protein and contains a PDZ domain in its carboxy terminus and 16 leucine rich repeats (LRR) in its amino terminus.

Erbin is involved in the regulation of Rho GTPases and Ras activation; and it directly binds to the erbB2 receptor kinase, to the p120 catenin family of proteins like arvcf and

δ-catenin, and to cell attachment proteins like β4-integrin. In light of erbin’s ability to modulate the Rho GTPases, which have been implicated in cell migration and regulation

73 of the ERM proteins and E-cadherin, it is possible that erbin may play a role in the regulation of adherens junctions.

Figure 7: Direct interactions with erbin. Erbin was originally identified as an erbB2 binding protein. Recently, studies demonstrate that erbin can also bind to the p120 proteins: ARVCF, δ-catenin, and p0071; to β-catenin, and to the hemidesmosomal proteins eBPAG1 and integrin β4.

Figure 8: Following ligand binding to a receptor tyrosine kinase receptor, the receptor is activated via autophosphorylation on tyrosine residues. The phosphorylated tyrosine residues recruit adaptor proteins to the plasma membrane. Grb2, one such adaptor, shuttles the guanyl nucleotide exchange factor (SOS) to the plasma membrane where it facilitates the exchange of the GDP moiety for GTP on Ras. Ras-GTP, activated Ras, directly binds to Raf, leading to the activation of the Raf molecule. Active Raf triggers the sequential activation of MEK, a MAPK kinase, and ERK, ultimately leading to the phosphorylation of various regulatory proteins including elk-1 and myc-1.

74

CHAPTER 2

Erbin localizes to autotypic adherens junctions and

regulates MAP kinase-dependent dissociation

of β-catenin from E-cadherin

75 ABSTRACT

E-cadherin-based autotypic adherens junctions play essential roles in peripheral nerve

development and maintenance. Here, we show that erbin, a PDZ protein implicated in

Ras-MAP kinase signaling, is a component of autotypic adherens junctions. Erbin is

expressed by Schwann cells in myelinated peripheral nerve fibers. Reduction of erbin

expression in Schwann cells using small interfering RNA (siRNA), disrupts cell-cell and cell-substrate interactions through a mechanism that includes the dissociation of β- catenin from E-cadherin but not from N-cadherin, as well as increased β-catenin phosphorylation, suggesting that erbin may stabilize adherens junction protein complexes. Loss of erbin also results in elevated levels of phosphorylated extracellular signal-regulated kinase (ERK). Disrupted cellular interactions in Schwann cells with reduced erbin expression can be reversed using a pharmacological inhibitor of ERK kinase (MEK). These data suggest that erbin plays a critical role in regulating MAP kinase-dependent signals that stabilize autotypic adherens junctions in Schwann cells.

76 INTRODUCTION

In the peripheral nervous system (PNS), myelination is achieved by Schwann cells that

wrap concentric layers of continuous, compact membrane around axons to form myelin

sheaths. Myelinated fibers are completely covered by these sheaths except at nodes of

Ranvier, where axons are exposed to the extracellular microenvironment. By reducing the

axonal capacitance and increasing the resistance, myelin reduces the current flow across the internodal axonal membrane, facilitating saltatory conduction. The compact myelin layers of peripheral nerve fibers are interspersed by non-compacted myelin domains that include Schmidt-Lanterman incisures, composed of Schwann cell cytoplasm, and the paranodes flanking the nodes of Ranvier (reviewed by Scherer and Arroyo, 2002).

Different domains of myelinated peripheral nerve fibers contain unique sets of Schwann cell membrane proteins that function to regulate Schwann cell-axon interactions, the clustering of potassium channels in the juxtaparanodal region (Rasband et al., 1998,

1999), and of sodium channels in the nodal region (Vabnick et al., 1996; reviewed by

Peles and Salzer, 2000; Scherer and Aroyyo, 2002).

Junctional specializations traditionally associated with epithelial cells (Mugnaini and

Schnapp, 1974; Fannon et al., 1995; Balice-Gordan et al., 1998; Spiegel and Peles, 2002) have been implicated in segregating functional domains of Schwann cells. In

“homotypic” adherens junctions, E-cadherin, a calcium-dependent cell adhesion molecule, and the intracellular protein β-catenin, play a major role in the formation of homophilic complexes that serve to connect two adjacent membranes of two cells (Trapp et al., 1989; Fannon et al., 1995; Gumbiner, 2000). In the non-compacted areas of

77 myelinated peripheral nerve, however, E-cadherin mediates the formation of adherens

junctions between membrane lamellae of the same cell and are referred to as “autotypic”

(Fannon et al., 1995) or “reflexive” (Balice-Gordan et al., 1998) adherens-type junctions.

Mice lacking E-cadherin in peripheral nerves have a widened gap in the outer mesaxon

between the two opposing membranes of the same Schwann cell (Young et al., 2002).

Interestingly, at the node of Ranvier, Schwann cell processes contact one another through

another calcium-dependent cell adhesion molecule, N-cadherin (Cifuentes-Diaz, et al.,

1994; Wanner and Wood, 2002). β-catenin can also serve as an intracellular partner for

N-cadherin, but it is unclear whether β-catenin or N-cadherin are required for the localization of junctional complexes at the node as compared to regions of non- compacted myelin.

Members of the PSD 95/Disc Large/zona occludens (ZO)1 (PDZ) protein family are present in cell junction protein complexes including adherens junctions. One such protein is erbin, originally described as an erbB2 interacting protein that contains 16 leucine rich repeats (LRR) and a single PDZ domain in its carboxy terminus (reviewed by Kolch,

2003). Because of this unique composition of domains, erbin is regarded as a member of the LAP (for LRR and PDZ) protein superfamily (Borg et al., 2000). Erbin has been implicated in regulating cell polarity and in basolateral targeting of its binding partners

(Legouis et al., 2003). The PDZ domain of erbin binds with high affinity to members of the p120-catenin family that are implicated in regulating cadherin turnover (Davis et al.,

2003), including δ-catenin and ARVCF (Laura et al., 2002). Erbin also interacts with p0071 (also called plakophilin-4), another p120 family member, in cell-cell junctions of

78 epithelial cells (Jaulin-Bastard, et al., 2002; Izawa et al., 2002; Ohno et al., 2002).

Although the erbin PDZ domain may associate with β-catenin, it only does so in vitro

with very low affinity (Laura et al., 2002). Collectively, these data suggest that erbin

may play a role in stabilizing cell-cell junctions.

Interestingly, erbin specifically inhibits ERK activation through its LRR domain (Huang et al., 2003). This effect appears to depend on indirect interactions between erbin and

active, GTP-bound Ras that disrupt the binding of Raf to Ras and subsequent activation

of ERK (Huang et al., 2003). As Ras activation has been linked to the disruption of

adherens junctions (Potempa and Ridley, 1998; Nam et al., 2002), it is possible that erbin

may influence both junctional protein complex localization as well as cellular signals that

stabilize interactions between proteins in these cell junctions. We report here that erbin is

associated with the autotypic adherens junctions of myelinated peripheral nerve fibers. It co-localizes with E-cadherin, β-catenin and δ-catenin but not N-cadherin in Schwann cells. Inhibiting erbin expression leads to aberrant cell-cell and cell-substrate interactions, increased cell proliferation, and abnormal activation of ERK signaling.

Furthermore, reducing in erbin expression disrupts the interaction between β-catenin and

E-cadherin, but not between β-catenin and N-cadherin. Inhibiting MEK activity partially reverses each of these phenotypes and reverses the dissociation of β-catenin from E- cadherin. Erbin may therefore play a significant role in regulating MAP kinase-dependent signals that lead to the disruption of autotypic adherens junctions in peripheral nerves.

79 RESULTS

Erbin co-localizes with β-catenin and E-cadherin, but not N-cadherin in peripheral nerve

fibers

In peripheral nerves, β-catenin is expressed in the outer cytoplasmic loops of

Schwann cells, in Schmidt-Lanterman incisures, and flanking axons at the paranodes,

with only diffuse expression at the node of Ranvier (Fannon et al., 1995). We therefore

tested whether erbin localized to the nodes or paranodes of teased sciatic nerve fibers by

double-labeling immunohistochemistry with anti-erbin and ant-β-catenin antibodies.

Erbin and β-catenin co-localized at incisures and in a portion of the paranode (Fig. 1, A

and B). Erbin was also weakly expressed at the abaxonal membrane, especially near

incisures (Fig. 1B, arrows, middle panel). Interestingly, erbin partially co-localized with

E-cadherin in a portion of the paranodes (arrowheads, Fig. 1C) and incisures (Fig. 1D) of

nerve fibers but not with N-cadherin (Fig. 1E), which, as previously reported, has a more

nodal localization at points of contact between two different Schwann cells (Wanner and

Wood, 2002; Cifuentes-Diaz et al., 1994; Fig 1E, arrow).

Erbin associates with E-cadherin junctional protein complexes in Schwann cells

In light of our finding that E-cadherin and β-catenin co-localize with erbin in

peripheral nerve fibers and previous studies showing that some catenin family members

that interact with E-cadherin can bind erbin (Laura et al., 2002), we tested if erbin is associated with E-cadherin in primary Schwann cell cultures. Under these conditions,

Schwann cell membranes come into contact with one another as they would in both homotypic and autotypic adherens junctions in vivo. Previous findings indicated that

80 Schwann cell-Schwann cell interactions are regulated predominantly by N-cadherin in

vitro (Wanner and Wood, 2002; Weiner et al., 2001; Letourneau et al., 1991) while E-

cadherin may mediate Schwann cell-Schwann cell interactions following nerve injury

(Hasegawa et al., 1996). E-cadherin and β-catenin co-immunoprecipitated with erbin

(Fig. 2A), consistent with the idea that erbin is associated with proteins in the Schwann

cells that mediate autotypic-type adherens junctions in peripheral nerves. N-cadherin, in contrast, failed to co-immunoprecipitate erbin (data not shown). The interactions with members of adherens junction protein complexes appeared to occur through erbin’s PDZ

domain, as only that portion of erbin containing the PDZ domain could precipitate β- catenin from Schwann cell lysates (Fig. 2B).

As the PDZ domain of erbin only binds β-catenin with very low affinity, we tested the possibility that Schwann cells express members of the p120 catenin family that associate with E-cadherin and bind erbin’s PDZ domain with high affinity (Laura et al., 2002).

Interestingly, by both Western blotting (Fig. 2C) and Immunocytochemistry (Fig. 2D), we found that Schwann cells express δ-catenin but not ARVCF. We therefore tested the possibility that erbin associates with δ-catenin in Schwann cells. Consistent with previous reports in other cell types, we found that δ-catenin co-immunoprecipitates with

E-cadherin and erbin in Schwann cells (Fig. 2C) and that both δ-catenin and β-catenin

co-localized with erbin, especially at the cell membrane at points of cell-cell contact (Fig.

2D), although all three proteins were also found in the cytoplasm. Furthermore, co-

localization only occurs at punctate domains along the plasma membrane, not throughout

the membrane. Because of the significant erbin immunoreactivity in the cytoplasm of

81 the cells, a substantial proportion of erbin in the Schwann cell does not co-localize with either β-catenin or δ-catenin. The role of cytoplasmic erbin has not been elucidated to date, however, in light of the co-localization detected at points of cell contact with proteins implicated in autotypic adherens junctions, and the immunoprecipitation data described above, it can be suggested that erbin is a part of the complex of proteins assembled at autotypic adherens junctions, likely through direct interactions with δ- catenin.

Reducing erbin expression alters Schwann cell morphology and cell-cell interactions

To test the function of erbin in Schwann cells we utilized an siRNA strategy to reduce erbin expression in primary Schwann cell cultures. Using a previously described erbin siRNA construct (Huang et al., 2003) we found that we could reduce erbin expression in Schwann cells by 50-80% as determined by scanning densitometry of

Western blots probed with an erbin antibody (Fig. 3A). Neither an empty vector, an irrelevant siRNA (against GAPDH; not shown), nor a scrambled erbin siRNA (Fig. 3A, right panel) had any effect on erbin expression. Schwann cells with reduced erbin levels tended to pile up on top of one another, had a more flattened appearance than cells in control cultures, and failed to form typical cell-cell contacts in confluent cultures

(compare Fig. 3B and 3D with 3C and 3E). Many of the cells in the erbin siRNA-treated cultures became detached and floated in the culture dish.

The aberrant cell-cell contacts of cells treated with erbin siRNA are consistent with the hypothesis that the proteins expressed in Schwann cells and implicated in mediated

82 autotypic adherens junctions in the peripheral nerve may be rendered non-functional as a result of reduced erbin expression. To test this idea, Schwann cell cultures treated with either erbin siRNA or empty vector were immunoprecipitated with an E-cadherin antibody and immunoblotted for β-catenin. The association between β-catenin and E- cadherin (Fig. 3F) but not β-catenin and N-cadherin (Fig. 3G) was abrogated in cells with reduced erbin expression, suggesting that loss of erbin disrupts autotypic-like adherens junctions. We did not consistently observe a similar dissociation of δ-catenin from E- cadherin (data not shown). Reducing erbin levels had no affect on the total levels of β- catenin, δ-catenin, N-cadherin or E-cadherin at 55 hours post-transfection (Fig. 3A, H).

There were slight reductions in the levels of β-catenin, however, by 72 hours (data not shown).

The loss of E-cadherin-β-catenin cell junctions in cells with reduced levels of erbin was confirmed by immunocytochemical assays, where, unlike cells transfected with a control vector, β-catenin and E-cadherin became diffusely localized throughout erbin siRNA- treated cells and did not co-localize (compare Fig. 3I and 3J). The distribution of β- catenin tended to be cytoplasmic and peri-nuclear in these cells (Fig. 3J). Collectively, these findings are consistent with the notion that loss of erbin from E-cadherin protein complexes destabilizes autotypic but not homotypic adherens junctions in Schwann cells.

Schwann cells with reduced erbin expression lose their ability to adhere to their substrate

The aberrant cell-cell contacts and the significant number of detached cells in erbin siRNA treated cultures suggested that erbin loss not only affected cell-cell contact

83 but cell-substrate contact as well. Alternatively, loss of erbin could cause cells to detach as a result of cell death. To distinguish between these possibilities, Trypan blue exclusion assays and cell counts were performed on both attached cells and floating cells at 24, 48, and 55 hours post-transfection. By 55 hours, 40-60% of cells treated with erbin siRNA no longer adhered to their substrate as compared to controls (data not shown) but there was a 20-30% increase in the total number of live Schwann cells (determined by counting both attached and detached cells that excluded Trypan blue; Fig. 4A).

Consistent with this observation, we observed a dramatic increase in cyclin D expression in erbin siRNA-treated cultures (Fig. 4B). The vast numbers of detached cells found in the medium 55 hours post transfection were alive (Fig. 4C). However, 24 hours later, nearly all of these cells incorporated Trypan blue, suggesting that these cells died as a result of anoikis (data now shown). These data suggest that loss of erbin may result in a delay in anoikis.

To determine whether the floating cells had incorporated more erbin siRNA than those that were still attached, we repeated these experiments using a fluorophore-tagged erbin siRNA construct and determined that over 90% of the cells, both attached and floating, had taken up the siRNA 24 hours post transfection (data not shown). Moreover,

Immunocytochemistry revealed that at 50 hours post-transfection, erbin expression had been reduced to a similar degree in all the cells that had taken up the siRNA construct

(data not shown). Together, these data indicate that reducing the levels of erbin in

Schwann cells affects their ability to attach to their substrate and to other cells while promoting Schwann cell proliferation.

84

A MEK inhibitor rescues the effects of reduced erbin expression

Erbin has been implicated in regulating the activation of extracellular signal-

regulated kinases (ERKs) by interfering with the binding of Raf-1 to activated Ras

(Huang et al., 2003). Although ERK activation is not sufficient to induce the

proliferation of normal Schwann cells (Maurel and Salzer, 2000), it may promote

proliferation in conjunction with other cellular signals, especially cyclic AMP-mediated

pathways (Rahmatullah et al., 1998). Furthermore, blocking the MAP kinase pathway

abrogates the protection from anoikis afforded by β-catenin (Weng et al., 2002). Here,

we found that Schwann cells treated with erbin siRNA had a 3-4 fold increase in the

levels of phosphorylated ERK compared to controls (Fig. 5A). We therefore tested

whether the MEK inhibitor, U0126, could reverse the effects of reduced erbin expression.

Cells were treated with 25 µM U0126 for 24 hours following transfection with erbin siRNA. The MEK inhibitor completely blocked ERK phosphorylation in cells with reduced erbin expression (Fig. 5B). Furthermore, blocking MEK reversed the piling up phenotype induced by the loss of erbin and the loss of adhesion to the culture dish (Fig.

5C and 5D). Most cells, however, still appeared to have aberrant cell-cell contacts and did not retain their normal spindle-shaped morphology. Cell counts performed on these cultures further confirmed that there was an almost complete reversal of the overall increase in the number of live cells as well as in the number of live cells that had detached from the culture dish (Fig. 5E and 5F). Thus, blocking MEK activity can partially rescue cells from the effects of reduced erbin expression.

85 Inhibition of the MAP kinase pathway prevents the disruption of adherens junctions in

Schwann cells with reduced levels of erbin

Previous studies found that the activation of some members of the Ras family, i.e.

V12 Ras, can promote the dissociation of E-cadherin from β-catenin in other cell types

(Potempa and Ridley, 1998; Nam et al., 2002). To test this hypothesis, Schwann cells were grown in the presence of the erbin siRNA with either DMSO alone (as a vehicle control) or with U0126 as above; cell lysates were then immunoprecipitated with a β- catenin antibodies. In the presence of U0126, β-catenin remained associated with E- cadherin (Fig. 6), suggesting that upregulation of MAP kinase directly or indirectly effects E-cadherin’s association with β-catenin in Schwann cells. Adding U0126 to cells in the absence of the erbin siRNA had no influence on β-catenin-E-cadherin interactions

(Fig. 6).

Tyrosine phosphorylation of β-catenin disrupts its interaction with E-cadherin, leading to an increase in free, uncomplexed cytoplasmic β-catenin pools (Muller et al., 1999). The accumulation of cytoplasmic hypophosphorylated-β-catenin can result in its migration to the nucleus where, via interactions with the Tcf/Lef family of transcription factors, it can up-regulate the transcription of a number of genes typically activated by the Wnt pathway that promote cell proliferation, including cyclin D (He et al., 1998; Shtutman et al., 1999;

Tetsu et al., 1999; Lin et al., 2000). As loss of erbin results in E-cadherin-β-catenin dissociation, increased expression of cyclin D, and increased proliferation in Schwann cells, we tested the possibility that some of the phenotypes of Schwann cells with reduced levels of erbin were due to increased Wnt signaling. We immunoprecipitated β-catenin

86 from both control and erbin siRNA treated Schwann cell cultures and immunoblotted for phospho-tyrosine. Reducing erbin expression resulted in a significant increase in β- catenin tyrosine phosphorylation, consistent with its dissociation from E-cadherin (Fig.

7A). Treatment with U0126, however, only slightly reduced β-catenin phosphorylation, suggesting that the disruption of adherens junctions in these cells may be, at least in part, independent of elevated Erk activation. To test if loss of erbin resulted in increased β- catenin-dependent signaling, we used a transcriptional reporter assay utilizing the pTOPFLASH luciferase reporter construct that contains three copies of the Tcf/Lef- binding site (Morin et al., 1997). A mutant reporter, pFOPFLASH, which cannot bind the

Tcf/Lef transcription factor, was used as a negative control. Cells transfected with either a control vector or erbin siRNA demonstrated equal levels of TOPFLASH activity (data not shown) indicating that the β-catenin that becomes dissociated from E-cadherin in cells with reduced levels of erbin does not activate the canonical Wnt pathway.

We next tested the possibility that β-catenin that had dissociated from E-cadherin was being degraded. As mentioned above, although total β-catenin levels were unaltered at

55 hours following transfection with erbin siRNA, there was some reduction in β-catenin at 72 hours. We also found that cells with reduced erbin expression demonstrated decreased levels of phosphorylated or inactive glycogen synthase kinase 3β (GSK-3β)

(for review see, Pearl and Barford, 2002), which functions to promote β-catenin degradation through the ubiquitin-proteosome pathway (Fig. 7B). Collectively, these findings indicate that loss of erbin in Schwann cells results in the dissociation of β- catenin from E-cadherin and the subsequent degradation of β-catenin.

87 88 DISCUSSION

Our data are consistent with a model in which erbin stabilizes autotypic but not

homotypic adherens junctions in peripheral nerves by acting as a scaffold for adherens

junction protein complexes while simultaneously inhibiting MAP kinase signaling, likely

through a mechanism that involves blocking the association of Raf1 with activated Ras

(Huang et al., 2003). The scaffold function of erbin depends on its PDZ domain, which

interacts directly with members of the p120 catenin family (Laura et al., 2002). Erbin can,

therefore, integrate alterations in MAP kinase signaling that influence cell survival and

proliferation with signals that either maintain the stability of adherens junctions or

prevent their disruption. Furthermore, as a MEK inhibitor could rescue the effects of

reduced erbin expression on β-catenin-E-cadherin association but no β-catenin

phosphorylation, part of erbin’s function likely involves regulating MEK/ERK dependent

dissociation of E-cadherin adherens junctions but not the subsequent degradation of β- catenin.

In normal peripheral nerves, myelin-forming Schwann cells are quiescent and their autotypic adherens junctions presumably must remain stable to maintain optimal conduction velocities in their associated axons (Fannon et al., 1995). Although mice that lack E-cadherin do not appear to have altered myelin integrity, other proteins, such as N- cadherin, could compensate for the loss of E-cadherin and interact with β-catenin in junctional complexes to form interactions that support normal nerve conduction (Young et al., 2002). Furthermore, previous findings indicate that Schwann cell-Schwann cell

89 interactions are regulated predominantly by N-cadherin in vitro (Wanner and Wood,

2002; Weiner et al., 2001; Letourneau et al., 1991) while E-cadherin may mediate

Schwann cell-Schwann cell interactions following nerve injury (Hasegawa et al., 1996).

The process of myelination may induce expression of genes that are involved in the

association of adjacent lamellae but not involved in contact between adjacent Schwann

cell; i.e. E-cadherin. Consequently, the expression levels or localization of proteins in

vitro may not fully represent that which occurs in vivo. Based on our findings, we

predict that erbin is required to maintain stable adherens junctions at the paranode and

could, therefore, significantly influence the integrity of non-compacted myelin. This

function is also likely to be critical during nerve development as Schwann cells ensheath

and myelinate individual axons, establishing autotypic adherens junctions as nodal and

paranodal domains become established. Our findings could further implicate erbin in

regulating Schwann cell behaviors following peripheral nerve injury or other insults,

when Schwann cells must proliferate and undergo a series of changes in cell-cell and cell-

matrix adhesion during the course of Wallerian degeneration and regeneration, then re-

establish stable cell junctions and quiescence following repair (Stoll and Muller, 1999).

Indeed, E-cadherin is upregulated at points of Schwann cell-Schwann cell contact in peripheral nerves as they recover from nerve injury (Hasegawa et al., 1996). Future studies utilizing mice with Schwann cell-targeted mutations in the erbin gene will reveal

the contribution of erbin in each of these situations.

Interestingly, the phenotypes of cells with reduced erbin expression are very similar to

the phenotypes of cells that lack the merlin tumor suppressor protein (Lallemand et al.,

90 2003). Merlin is encoded by the neurofibromatosis 2 (NF2) gene and is highly homologous to the ezrin-radixin-moesin (ERM) family of proteins that link

transmembrane proteins to the actin cytoskeleton. Loss of merlin occurs in

schwannomas, benign Schwann cell tumors that arise spontaneously and in NF2 patients

(reviewed by Baser et al., 2003). Nf2-deficient mouse embryo fibroblasts (MEFs) pile up,

hyperproliferate, have defective cadherin-mediated cell-cell interactions, and exhibit

increased MEK signaling (Lallemand et al., 2003) suggesting that, like erbin, merlin may

be involved in the formation of adherens junctions through a mechanism that includes the

regulation of the Ras-MAP kinase pathway. Loss of merlin could therefore influence

erbin function, altering protein-protein interactions in adherens junction complexes, or

merlin and erbin may influence shared signaling cascades.

Erbin was originally identified as an erbB2 interacting protein (Borg et al., 2000). ErbB2

is a member of the EGF-family of receptor tyrosine kinases and is essential for Schwann

cell differentiation, growth and survival (Erickson et al. 1997; Britsch et al. 1998;

Woldeyesus et al. 1999; Morris et al. 1999; Garratt et al., 2000). ErbB2 can promote

activation of the Ras-MAP kinase pathway (Li et al., 2001) and is associated with E-

cadherin-β-catenin complexes through interactions with the carboxy terminal of β-

catenin (Kanai et al., 1995; Shibata et al., 1996). Tyr 654 in the β-catenin carboxy

terminal is at least one site whose phosphorylation has been implicated in the dissociation

of β-catenin from E-cadherin (Roura et al., 1999) and it is this residue that is most likely

phosphorylated as a result of the interactions between β-catenin and erbB2 (Bonvini et

al., 2001). Since loss of erbin results in elevated MAP kinase signaling, it is possible that

91 erbin regulates signaling between erbB2, E-cadherin and β-catenin in a MEK-dependent manner. This would place erbin in a position where it could regulate both signals that promote cell proliferation as well as signals that influence cell adhesion and cell shape. It is unclear, however, if erbin can influence erbB2 signaling through direct interactions, as the PDZ domain of erbin binds erbB2 with extremely low affinity (Laura et al., 2002).

Despite the fact that the MEK inhibitor we utilized in our studies, U0126, completely inhibited ERK phosphorylation, Schwann cells with reduced erbin levels still had increased β-catenin phosphorylation. In agreement with this observation, cells with reduced erbin that were treated with U0126 failed to form normal cell-cell contacts. It is possible that this was due to the chronic and total inhibition of MEK activity, which may be required for the establishment of normal cell adhesion (Eblen et al., 2004). However, erbin interacts with a number of other proteins implicated in cell-cell and cell-substrate adhesion, including other members of the catenin family, members of the Rho family of small GTPases, and β4-integrins (reviewed by Kolch, 2003). It is therefore likely that only some of the signaling pathways that are linked to erbin are dependent on Ras-MAP kinase activity, and that Schwann cells depend on erbin for additional functions related to cell adhesion.

92 MATERIALS AND METHODS

Cell Culture

Schwann cells were isolated from sciatic nerves of 3-day-old rat pups (Brockes et al.,

1979), purified by anti-Thy 1.1 immunoselection, and expanded for 6-7 passages on 10- cm plates coated with poly-l-lysine (Sigma) in DMEM supplemented with 10% FBS, 2

µM forskolin (Sigma), and 5 ng/ml recombinant human HRG-β1 (EGF domain) (R&D

Systems). All of the cultures used in these experiments were essentially free of fibroblasts.

Antibodies

We used the following antibodies: a polyclonal, monoclonal, and purified polyclonal against erbin (Borg et al., 2000); a monoclonal against phosphotyrosine (generously provided by B. Druker); polyclonals against ERK, E-cadherin, ARVCF, δ-catenin, cyclin

D1, GSK-3β and actin (Santa Cruz Biotechnology); a polyclonal against P-erk (Cell

Signaling); a polyclonal against c-myc (Research Diagnostics, Inc.); a monoclonal against β−catenin and N-cadherin (Transduction Laboratories); a monoclonal against phospho-GSK-3 (Y279/Y216) (Upstate Biotechnology); a monoclonal against E-cadherin

(BD Biosciences Pharmingen); a monoclonal against tubulin (NeoMarkers); Rabbit IgG,

Mouse IgG, and Goat IgG (Vector Laboratories); Horseradish peroxidase-conjugated

Rabbit and Mouse (BioRad); Horseradish peroxidase-conjugated goat (Santa Cruz

Biotechnology); Fluorescein isothiocyanate (FITC)-conjugated secondary antibodies,

Hoechst (Molecular Probes)

93

Immunoprecipitations

Total protein extracts were made in 85mM NaCl, 150 mM Tris-HCl, 1% Triton X-100, pH 7.5 supplemented with protease inhibitors. Lysates were kept on ice for 15 minutes and then cleared by centrifugation at 20,000g for 10 min. Approximately 100 µg of extract was precleared at 4° C for 30 min with 30 µl of Protein-A or Protein –A/G

(Oncogene) Sepharose beads and 1 µg of isotype- matched IgG. The supernatant was immunoprecipitated with 2.5 µg of the immunoprecipitating antibody and incubated at 4°

C overnight. The extract was then incubated with 35 µl of either Protein –A or Protein-

A/G Sepharose beads at 4° C for 2 hrs. Complexes were washed extensively with buffer

A (200 mM NaCl, 150 mM Tris-HCl, 1% Triton-X 100, pH 7.5), buffer B (100 mM

NaCl, 150 mM Tris-HCl, 1% Triton-X 100, pH 7.5), and buffer C (50 mM NaCl, 150 mM Tris-HCl, 1% Triton-X 100, pH 7.5) and were boiled in SDS sample buffer. SDS-

PAGE and western blotting was carried out according to standard protocols.

Immunostaining

Schwann cells were fixed in 4% paraformaldehyde for 10 min. Cultures were then washed extensively with 10% normal goat serum (NGS), 0.1% Triton X-100, in 1X PBS and nonspecific binding sites were blocked with the same buffer for 30 min at room temperature. Primary antibodies were diluted in blocking buffer and incubated on the cells for 1 hr. Polyclonal erbin antibodies (J-P Borg) were diluted 1:15 (antibody concentration not known) and monoclonal β-catenin antibodies were diluted 1:25. The

94 cells were washed extensively in the blocking buffer and secondary antibodies

conjugated to FITC or TRITC diluted in blocking buffer were added for 45 min. Cells

were washed and coverslips mounted with Fluoromount G (Southern Biotechnology

Associates, Inc.). We viewed the cells with a Leica confocal microscope (Leica TCS SP;

Leica Microsystems, Exton, PA).

Sciatic nerves were removed from adult mice and immediately placed in ice cold 4% paraformaldehyde for 30 min. They were then washed extensively in 1X PBS and teased on Superfrost Plus microscope slides and dried at room temperature. Non-specific sites were blocked with 10% NGS, 0.1% Triton X-100, in 1X PBS for 30 min at room temperature. Primary antibodies were diluted in the same blocking buffer and incubated on the tissues overnight at 4° C. Polyclonal erbin antibodies were diluted 1:10, monoclonal E- and N-cadherin antibodies were diluted 1:20, and monoclonal β-catenin antibodies were diluted 1:15. The tissues were washed extensively in the blocking buffer and secondary antibodies conjugated to FITC or TRITC diluted in blocking buffer were incubated on the tissue for 2 h. Cells were washed and coverslips mounted with

Fluoromount G (Southern Biotechnology Associates, Inc.). The tissues were viewed on an Axioskop 40 (Zeiss) microscope.

Inhibition of erbin expression by siRNA

The target region of siRNA was 540 nt downstream of the start codon, which contained approximately 50% G/C content. The nucleotide sequence was 5’ UAG ACU GAC CCA

GCU GGA A dTdT 3’ (nt 866-884), and was the same construct used by Huang, et al. to

95 decrease erbin expression in PC12 cells (Huang, et al., 2003). We searched the NCBI

sequence bank against this segment of DNA using the BLAST program, which confirmed

no match, suggesting the specificity of the target region by siRNA. The 21-nucleotide

RNAs were chemically synthesized by Integrated DNA Technologies, Inc. A scrambled

erbin siRNA was also constructed and used as a negative control. To demonstrate the

silencing effect of endogenous erbin expression by siRNA, 100% confluent Schwann cell

cultures grown in 10-cm dishes were transfected with the siRNA duplex using an amine-

based transfection reagent (Ambion). Briefly, 24 µl of the amine-based transfection

reagent was mixed with 665.5 µl of Opti-MEM (Gibco-Brl). Following a 15 minute

incubation at room temperature, 16.3 µl of 20 µM erbin-siRNA duplex was added and

incubated for an additional 10 minutes. The entire mix was overlaid onto the cells and

incubated overnight at 37 C and 7.5% CO2 before the addition of 7 ml of antibiotic free

medium. 55 hrs after transfection the cells were lysed and harvested in the same manner as described for Western blotting and subjected to immunoblotting for expression of erbin.

In Vitro affinity precipitation assay

Expression of the recombinant GST fusion proteins was induced in DH5α with 1mM

isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 1 h at 37 C. We extracted proteins in

PBS containing DNase, lysozyme (Sigma), and Ethylene Glycol-bis(β-Aminoethyl

Ether)-N,N,N’,N’-Tetraacetic Acid (EGTA) by sonicating for 45 s and purified them on

glutathione-Sepharose beads (Amersham) according to the manufacturer’s

96 recommendations. Schwann cells were lysed as described for immunoprecipitation. We

incubated immobilized GST fusion proteins for 2 h at 4 C with 250 µg of lysates and

washed them sequentially with immunoprecipitation buffers B, C, and A before

immunoblotting analysis.

Cell Counts

Cell counts were performed in triplicate for empty vector vs. erbin siRNA transfected

cultures, and erbin siRNA plus DMSO vs. erbin siRNA plus MEK inhibitor, U0126

(Promega). Cells were seeded at 130,000 cells/10 cm culture dish in DMEM + 10% FBS

and growth factors. 24 hours after plating, cells were transfected with the erbin siRNA.

If MEK inhibitor was used in the protocol, it or DMSO was added approximately 24

hours before time of harvest. Cells were counted in a hemocytometer. Both cells floating

in the culture media and those that were still adhered to the culture dish were counted

using Trypan Blue (Sigma). Cells that had taken up the blue dye were considered dead,

while those that were clear were considered alive.

Transcriptional Reporter Assays

Schwann cell cultures grown in 100 mm dishes at approximately 90% confluency were

transfected with 0.5 µg of CMV-β-galactosidase (to provide an internal control for the normalization of all data for transfection efficiency) and 1 µg of either pTOPFLASH or pFOPFLASH (both generously provided by Dr. Kathleen Heppner-Goss, University of

Cincinnati) luciferase reporter constructs 24 hours after transfection with erbin siRNA or empty vector. Thirty hours later, cells were washed with PBS, and 500 µl of Promega

97 reporter lysis buffer was added to each well. Lysates were collected and assayed for luciferase activity using the dual luciferase reporter assay system (Promega) according to the manufacturer’s instructions.

98 Acknowledgements

The authors thank Anda Cornea for assistance with confocal microscopy, Katherine

Heppner-Goss for advice and reagents, and Steven Matsumoto, Nancy Ratner, Linda

Parysek, Wallace Ip, and Frank Sharp for helpful comments. This work was supported by grants from the National Institutes of Health (NS39550) to L.S. and from a University of Cincinnati Functional Genomics Fellowship to R.R.

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107

Figure 1

108 Figure 2

109 Figure 3

110 Figure 4

111 Figure 5

112

Figure 6

113 Figure 7

114 Figure 8

115 Figure legends

Figure 1: Erbin co-localizes with βcatenin and E-cadherin, but not N-cadherin in 6 week old mouse sciatic nerve fibers. Teased nerve fibers were double-labeled with antibodies against erbin and either βcatenin (A, B), E-cadherin (C, D), or N-cadherin (E). In myelinated fibers, erbin (green) co-localizes with β−catenin (red) both in a subset of paranodes (A) and incisures (B, arrowheads). There is also weak, patchy erbin expression at the mesaxonal membrane (B, middle panel, arrows). E-cadherin (green) also co- localizes with erbin (red) at paranodes (C; arrowheads) and incisures (D), although E- cadherin tends to have a broader distribution at the paranode. In contrast, N-cadherin

(green) has a more nodal (arrow) distribution and does not co-localize with erbin (red; E, arrowheads). Caliber of the nerve seems to alter antibody immunoreactivity, albeit slightly, hence the slight differences in protein localization. Scale bars = 10 µm.

Figure 2: Association and co-localization of erbin and β-catenin in primary Schwann cell cultures. (A) Immunoprecipitations using anti-erbin (left panel) or anti-E-cadherin (right

panel) antibodies from lysates of subconfluent Schwann cells followed were probed by

Western blot analysis (IB) using an anti-β-catenin, anti erbin, or anti-E-cadherin

antibody. Con = control immunoprecipitation with an irrelevant antibody; IP =

immunoprecipitation with the erbin antibody; lys = total cell lysate. (B) Glutathione S-

transferase (GST) deletion constructs comprising different portions of erbin’s carboxy tail

were used to determine the domain through which erbin associates with β-catenin.

Construct 914-1371 contains both the carboxy PDZ domain and an upstream LRR,

construct 914-1240 lacks the PDZ domain, and construct 1240-1371 lacks the LRR. The

116 Western blot shows that protein complexes containing β-catenin from Schwann cell

lysates associated with the GST-fusion proteins possessing the erbin PDZ domain but not to other domains. Lys = total cell lysate, as a control for β-catenin expression; GST =

pull-down assay with GST alone. (C) Western blots of total Schwann cell lysates (left

panel) were probed with antibodies to the p120 family members, δ-catenin and ARVCF.

Although δ-catenin could be detected, we were unable to detect ARVCF. The right panel

shows that δ-catenin and E-cadherin co-immunoprecipitated with erbin. (D) Confocal

images of cells double-stained for erbin (red) and either β-catenin or δ-catenin (green)

and Hoechst 33342 (blue; to label nuclei). Arrows indicate areas at cell membranes were

proteins co-localized. Arrowhead indicates an area of a cell where β-catenin and erbin

co-localize in the cytoplasm. Scale bars = 10 µm.

Figure 3: Reducing erbin expression disrupts interactions between β-catenin and E-

cadherin. (A) Western blot (left panel) showing expression of erbin and β-catenin in

Schwann cells treated with empty vector or erbin siRNA. Fifty-five hours post-

transfection, erbin expression was reduced by approximately 50% in cultures treated with the erbin siRNA while β-catenin levels remain unchanged. Transfection with a scrambled erbin siRNA construct (right panel) had not effect on erbin expression levels. Tubulin served as a loading control. (B-E) Low (B, C) and high (D, E) power phase contrast photomicrographs of Schwann cell cultures treated for 55 hours with (B, D) empty vector and (C, E) erbin siRNA. Note that cells with reduced levels of erbin piled-up on one another and tended to have more spread out morphology than cells in control cultures. (F)

Western blot probed for E-cadherin and β-catenin following immunoprecipitation with an

117 E-cadherin antibody. Note that very little β-catenin co-immunoprecipitates with E- cadherin in the presence of the erbin siRNA. (G) Western blot showing that β-catenin’s association with N-cadherin is not affected in cells with reduced erbin expression. (H) In the top three panels are Western blots of total cell lysates from the experiment in F and G, showing that the total expression levels of E-cadherin and N-cadherin remain unchanged in the presence of erbin siRNA. The bottom two panels are from a separate experiment, and show that total levels of δ-catenin also remain unchanged. Actin was used as a loading control. (I) A Schwann cells from a culture transfected with empty vector double labeled with anti-β-catenin (red) and anti-E-cadherin (green) antibodies. Note that E- cadherin and β-catenin co-localize in some regions of the cell membrane even when cells are not in contact with one another (arrow). In addition, there is some β-catenin expressed in the cytoplasm and nucleus. (J) Schwann cells from a culture transfected with erbin siRNA. Note that E-cadherin (green) is diffusely expressed and no longer co- localizes with β-catenin, while β-catenin is expressed at highest levels in peri-nuclear regions of the cytoplasm. Scale bars = 10 µm.

Figure 4: Schwann cells with reduced erbin expression exhibit slight increases in proliferation. (A) Both the cells floating in the culture media and those that were still attached to the substrate were harvested from empty vector and erbin siRNA transfected

Schwann cell cultures and counted following staining with Trypan Blue at 0, 24, 48, and

55 hours post-transfection. These cell counts revealed significantly (* = p<0.005 using a

Student’s T-test) increased cell numbers in erbin-reduced cultures, indicating increased cellular proliferation. (B) Western blot showing increased cyclin D expression in

118 Schwann cell cultures treated with erbin siRNA for 55 hours. (C). Analysis of the cells floating in the culture medium revealed that a far greater number of floating cells were present in the erbin siRNA transfected cultures as compared to control and that a majority of these cells were still alive at the time of harvest.

Figure 5: MEK inhibition reverts the phenotypes induced by a reduction in erbin expression. (A) Levels of phosphorylated and total ERK expression in empty vector and erbin siRNA transfected Schwann cells were analyzed by Western blot. Elevated levels of phosphorylated ERK were detected in erbin siRNA transfected cultures, while the levels of total ERK were unchanged. Blots were also labeled with an anti-actin antibody as a loading control. (B) The MEK inhibitor, U0126, or DMSO (as a vehicle control) was added to erbin siRNA transfected cultures approximately 30 hours prior to harvest

(at 55 hours post-transfection). Western blot analysis revealed that the inhibitor completely abolished the increase in phosphorylated ERK levels induced by erbin siRNA. (C and D) Phase-contrast photomicrographs of erbin siRNA transfected cultures treated with either DMSO or U0126. Cell-cell contact and cell morphology were partially rescued upon the addition of the MEK inhibitor. (E) Cell counts of Trypan Blue labeled cells revealed that the MEK inhibitor was able to abolish the increased proliferation observed in erbin siRNA transfected cultures. Furthermore, the overall increase in both the number of cells found floating in the culture medium and the total number of live cells was abolished upon the addition of the MEK inhibitor (F).

Figure 6: MEK inhibition rescues the erbin siRNA-induced dissociation of E-cadherin

119 from β-catenin. Schwann cells were treated with erbin siRNA or empty vector and subsequently treated with the MEK inhibitor, U0126, or DMSO. Schwann cells lysates were immunoprecipitated with a β-catenin antibody and immunoblotted for both β- catenin and E-cadherin. Cells treated with the MEK inhibitor maintained β-catenin-E- cadherin protein complexes. Total levels of β-catenin were not affected by the addition of the MEK inhibitor. Furthermore, the MEK inhibitor did not affect β-catenin-E- cadherin interactions in cells transfected with empty vector.

Figure 7: Reducing erbin expression leads to an increase in the levels of phosphorylated

β-catenin and cyclin D1. (A) Schwann cells were treated with erbin siRNA or empty vector and subsequently treated with the MEK inhibitor, U0126, or DMSO. Schwann cells lysates were immunoprecipitated with β-catenin and immunoblotted for both β- catenin and phospho-tyrosine. Control cultures treated with either vehicle or the MEK inhibitor exhibit low basal levels of phosphorylated β-catenin compared to cultures treated with erbin siRNA. The addition of the MEK inhibitor to cultures transfected with erbin siRNA reduced the levels of phosphorylated β-catenin by approximately 40% suggesting that the Ras-MAP kinase pathway is directly involved in the phosphorylation of β-catenin and its subsequent dissociation from E-cadherin. (B) Western blot showing that reducing erbin expression decreases the levels of GSK-3β phosphorylation (by 40-

50% as assessed by scanning densitometry) without affecting the total levels of GSK-3β in Schwann cells.

120 Figure 8: A model depicting the changes in E-cadherin-β-catenin interactions in the

absence and presence of erbin; not drawn to scale. Previous studies have shown that the

LRR domain of erbin is involved in blocking the association of Raf1 with active Ras, thus inhibiting MAP kinase signaling and phosphorylation of β-catenin. Based on our data, we predict that reduction in erbin levels or the inhibition of erbin-β-catenin interactions results in the dissociation of β-catenin from E-cadherin through a MAP kinase-dependent mechanism, resulting in the phosphorylation and subsequent degradation of β-catenin. It is unclear is p120 family members are similarly dissociated from E-cadherin. However, the loss of p120-E-cadherin interactions is predicted to result in E-cadherin degradation, and, at least at the time points that we evaluated following erbin siRNA transfection, there was no evidence of E-cadherin degradation

121

CHAPTER 3

ERBIN MEDIATES MERLIN’S ASSOCIATION WITH ADHERENS JUNCTION PROTEIN COMPLEXES THROUGH DIRECT INTERACTIONS WITH EBP50

122 ABSTRACT

The neurofibromatosis 2 (NF2) gene encodes merlin, an ezrin-radixin-moesin-(ERM)- related protein that functions as a tumor suppressor. Although the mechanism by which it acts as such is unknown, it has been postulated that its function as a tumor suppressor may involve the adherens junction complex. Here we show that erbin, a PDZ protein implicated in Ras-MAP kinase signaling and E-cadherin-β-catenin association, localizes with merlin in Schwann cells. Reduction of erbin expression in Schwann cells using small interfering RNA (siRNA), disrupts merlin’s association with β-catenin, but not its association with CD44 or EBP50. It also decreases the hypophosphorylated or active form of merlin. Loss of erbin results in elevated levels of phosphorylated extracellular signal-regulated kinase (ERK). The merlin-β-catenin dissociation and the elevated levels of phosphorylated merlin can be reversed using a pharmacological inhibitor of ERK kinase (MEK). Finally, we show a direct interaction between erbin and EBP50. These data suggest that erbin mediates merlin function in Schwann cells through an erbin-

EBP50 bridge.

123 INTRODUCTION

Neurofibromatosis type 2 (NF2) is a familial cancer syndrome that leads to the development of multiple tumors including ependymomas, meningiomas, and schwannomas, most predominantly of the vestibular branch of the eighth cranial nerve

(Gutmann, 1997; reviewed by Baser et al., 2003). The disease is caused by bi-allelic heterozygous mutations of the NF2 gene located on human chromosome 22. Genetic studies indicate that the NF2 gene protein product, merlin, acts as a tumor suppressor and regulates the growth and proliferation of a number of different cell types (McClatchey et al., 1997; McClatchey et al., 1998; Giovannini et al., 1999). Furthermore, inactivation of the NF2 gene and loss of merlin expression using genetically engineered mouse models results in increased incidence of tumor formation (McClatchey et al, 1998). The mechanism by which it regulates cellular growth and induces tumor formation when it is lost, however, is currently unknown.

The NF2 gene product, merlin, is highly homologous to the ezrin-radixin-moesin (ERM) protein family. Like the other ERM proteins, merlin localizes to the membrane: cytoskeleton interface, a highly unusual location for a tumor suppressor, as well as to the cytoplasm of the Schwann cell. Furthermore, merlin localizes to the adherens junction and physically interacts with adherens junctions’ components in wild-type mouse embryo fibroblasts (MEFs) (Lallemand et al., 2003). Utilizing Nf2-deficient MEFs, it was found that Nf2 deficiency led to piling up of cells, hyperproliferation, mislocalization of β- catenin, α-catenin, and N-cadherin, and defective cadherin-mediated cell-cell interactions

124 (Lallemand et al., 2003). Taken together, these data suggest that merlin is involved in the localization and function of the adherens junction protein complex.

Merlin mediates contact inhibition of growth through signals from the extracellular matrix. At high cell density, merlin becomes hypophosphorylated and inhibits cell growth in response to hyaluronic acid. Merlin's growth-inhibitory activity depends on a specific interaction with the cytoplasmic tail of CD44, a transmembrane glycoprotein and hyaluronic acid receptor. At low cell densities, merlin is phosphorylated, growth permissive, and exists in a complex with ezrin, moesin, and CD44. These data suggest that merlin and CD44 can form a molecular switch that can influence cell growth arrest or proliferation. (Morrison et al., 2001)

Members of the PSD-95 complex family are usually found at cell junctions. An integral component of all junctions, including adherens junctions, are members of the PSD

95/Disc Large/zona occludens (ZO)1 (PDZ) protein family. One such protein is erbin, originally described as an erbB2 interacting protein that contains 16 leucine rich repeats

(LRR) and a single PDZ domain in its carboxy terminus. Erbin has been implicated in regulating cell polarity and in basolateral targeting of its binding partners (Legouis et al.,

2003). Recent studies have demonstrated that erbin binds to a number of proteins, including p0071 (also called plakophilin-4) in of epithelial cells (Jaulin-

Bastard, et al., 2002), PSD-95 at postsynaptic membranes (Huang, et al., 2001), δ- catenin, and ARVCF (Laura et al., 2002), thus implicating it in adherens junction function. In addition to its role as a targeting protein, erbin also acts as a negative

125 regulator of the Ras-Raf-ERK signaling pathway (Huang et al., 2003; Rangwala et al.,

Chapter 2), suggesting that it may coordinate cell-cell interactions and intracellular

signaling.

As shown in the previous chapter, loss of erbin from primary Schwann cells induces many of the same changes seen in NF2-/- MEF’s. Schwann cells with reduced erbin levels tend to pile up on top of one another, have a more flattened appearance than cells in control cultures, and fail to form typical cell-cell and cell-substratum contacts

(Rangwala et al., Chapter 2). Consequently, the mechanism by which merlin influences adherens junction formation may be dependent on its association with erbin.

Here we show that loss of erbin in Schwann cells using an siRNA strategy influences merlin’s association with β-catenin, but not its association with CD44. Inhibition of the

MAP kinase pathway using a potent chemical inhibitor, reestablishes the basal associations between merlin and β-catenin, and merlin and ΕΒP50. Moreover, we found that the increased proliferation found in Schwann cell cultures treated with erbin siRNA influences the phosphorylation status of merlin by reducing the hypophosphorylated form of the protein which is believed to be the active form of the protein. Finally we found that merlin associates with erbin through the direct interactions with the ERM/merlin binding partner, EBP50. The mechanism by which erbin can influence merlin’s function as a tumor suppressor, its effect on adherens junction formation, and its localization to the plasma membrane may be explained by its direct association with EBP50.

126 RESULTS

Erbin mediates an association between merlin and adherens junction protein complexes

As merlin is known to localize to adherens junctions and to associate with β-catenin in

other cell types (Lallemand et al., 2003) we postulated that erbin may play a role in

linking merlin to the complex of proteins that constitute adherens junctions in Schwann

cells. In agreement with this idea and in light of our previous findings that erbin and β-

catenin localize to the paranode and Schmidt-Lanterman incisures of the peripheral nerve

(Rangwala et al., Chapter 2), the precise localization of merlin (Scherer and Gutmann,

1996), we found that merlin co-immunoprecipitated with erbin and β-catenin in Schwann cells grown in both confluent (not shown) and subconfluent densities (Fig. 1A, left panel). The proportion of merlin and β-catenin that associate with each other at confluent

or subconfluent cultures conditions is currently not known. Immunoprecipitations of

erbin from Schwann cell lysates indicate that while erbin associates with merlin, it does

not associate with ezrin, suggesting that erbin is not found in, and does not mediate the

function of, all ERM complexes (Fig. 1a, right panel).

The merlin antibody used in these studies (Santa Cruz, C-18), can cross-react with other

ERM proteins, like many of the other merlin antibodies available (see Fig. 1B). In

Schwann cells, specifically, ezrin, radixin, moesin, and merlin expression can be detected

(Scherer et al., 2001) With this caveat in mind, it is possible that the interactions detected

by immunoprecipitation may not, in fact, represent a specific merlin- interaction, but an interaction mediated by one or more of the ERMs. In order to determine such a possibility 1) immunoprecipitations between the ERMs and the possible merlin-

127 associated proteins should be performed and 2) transfect in a tagged form of merlin (i.e.

His-merlin) and subsequently use antibodies against the tag to determine associations

between merlin and its putative partners, i.e. erbin. Performing the immunoprecipitation in both directions as has been done, however, serves as one of the most reliable indicators of a true protein-protein interaction. These issues of nonspecificity are also of concern in the interpretation of immunohistochemical and immunocytochemical data. In order to reduce these effects, we used the highest possible dilutions of the Wa-30 antibody (1:300) in order to select for specific merlin immunoreactivity.

In light of the paranodal localization of merlin in the peripheral nerve, we were interested in determining whether merlin specifically associates with E-cadherin, a calcium- dependent cadherin molecule that localizes to regions of the paranode (Fannon et al.,

1995). Preliminary data (not shown) suggests that merlin associates with both E- and N- cadherin, not surprising in light of in vitro studies that demonstrate an association between merlin with N-cadherin in mouse embryo fibroblasts (Lallemand et al., 2003).

These data suggest that 1) different functional pools of merlin exist that mediate both E- and N-cadherin mediated adherens junctions 2) merlin is not exclusive to the autotypic adherens junction that localizes to the paranode of peripheral nerves, and 3) erbin may directly or indirectly regulate only a subpopulation of this tumor suppressor.

Recent data indicate that merlin’s ability to regulate cellular proliferation may be mediated by the adherens junction (Lallemand et al., 2003), and that conditions of serum starvation and confluency can alter the phosphorylation and, therefore, activation of this

128 protein (Shaw et al., 1998). We were interested in determining whether erbin’s

association with components of the adherens junction complex was also affected by these

factors. We immunoprecipitated confluent and subconfluent Schwann cell cultures in the

presence or absence of neuregulin, a Schwann cell mitogen and survival factor, and found

that erbin more robustly associates with β-catenin in subconfluent cultures than confluent cultures, regardless of neuregulin stimulation (Fig. 1C, top panel ). Erbin associates with the activated form of erbB2 (P-B2) specifically in confluent cultures treated with neuregulin (Fig. 1C , lower panel). Heavy chain IgG was present in equal amounts in all of the immunoprecipitations (data not shown) suggesting that each of the experimental groups was equally loaded, however, immunoblots against erbin would serve as a better loading control. Furthermore, immunoblots using erbB2 antibodies would determine whether confluency and/or neuregulin stimulation effected erbin’s association with the inactive form of the receptor tyrosine kinase. These data suggest that factors such as confluence and growth stimulation via neuregulin can influence erbin’s interaction with at least two of its known interacting partners, and that the interactions between erbin and

β-catenin and erbin and p-erbB2 are not constitutive.

Immunocytochemistry data indicate (Fig. 1d) that while β-catenin localizes to the plasma

membrane, cytoplasm and nucleus of rat Schwann cells grown in vitro in isolated (bottom

panel) and contacting Schwann cells (top panel), there is more cytoplasmic and nuclear

β-catenin staining in contacting Schwann cells as compared to the isolated Schwann cell;

merlin localization did not differ significantly between the two states. Furthermore,

merlin co-localized with β-catenin at punctate domains along the cell membrane near the

129 cell body and in Schwann cell processes regardless of confluency. However, the greatest

degree of co-localization occurred at points of cell-cell contact. This finding is similar to

δ-catenin-erbin and erbin-β-catenin co-localization in Schwann cells (Rangwala et al.,

Chapter 2), suggesting that merlin may be involved in junctional complexes mediated by

β-catenin, δ-catenin, and erbin.

The above data suggest that the merlin-erbin association is more robust in the

subconfluent state. Preliminary evidence indicates that merlin and erbin do localize at

punctate domains along the cell membrane near the cell body and in processes (Figure

1e). However, in order to determine whether confluency and/or the presence of

neuregulin can influence the association between these two proteins, similar

immunoprecipitation and co-localization studies comparing confluent and subconfluent

cultures, as described above, should be performed.

Erbin mediates merlin’s association with β-catenin

As we have previously reported, we were able to reduce erbin expression in Schwann cells using a erbin siRNA construct initially described by Huang et al (Huang et al.,

2003). We found that we could reduce erbin expression in Schwann cells by 50-80% as determined by scanning densitometry of Western blots probed with an erbin antibody

(Fig. 2a). Neither an empty vector, an irrelevant siRNA (against GAPDH), nor a scrambled erbin siRNA had any effects on erbin expression. Furthermore, cells treated with erbin siRNA had no significant influence on the levels of CD44 while the expression

130 of an intracellular merlin binding partner, EBP50, was slightly elevated (greater than or

equal to 2 fold) (Fig. 2b, left)

The phenotypic changes that occur in Schwann cells as a result of a loss of erbin are

reminiscent of cells that have either targeted or spontaneous mutations in the

neurofibromatosis type 2 (NF2) gene. We found that like NF2 loss, erbin reduction leads

to a dramatic mislocalization of adherens junction proteins (Rangwala et al, Chapter 2).

This mislocalization directly affects β-catenin’s association with merlin; when we

immunoprecipitated merlin from cells treated with erbin siRNA little or no β-catenin co-

immunoprecipitated with merlin (Fig. 2b, right). In order to ensure this was not a

spurious result, we performed co-immunoprecipitation assays between merlin and CD44

and found that CD44 still bound to merlin in cells with reduced levels of erbin.

ERM-binding-protein-50 (EBP50) is an adaptor protein that links merlin and ERM

proteins to membrane proteins and other partners via two PDZ like domains (reviewed by

Bretscher et al., 2000). While we found that the levels of EBP50 in erbin reduced

cultures increased by approximately 2- fold compared to control (Fig. 2b, left), the

association between EBP50 and β-catenin did not change (Fig. 2c), not surprising in light of the fact that a direct association between β-catenin-EBP50 has been described in hepatocytes (Shibata et al., 2003). This suggests a stable and constitutive association between EBP50 and β-catenin in Schwann cells that is independent of erbin.

Reducing erbin expression alters merlin phosphorylation

131 Merlin’s putative role as a tumor suppressor protein has been linked to its phosphorylation status. Phosphorylation of this protein is regulated by high cell density, growth factor withdrawal, and cell: ECM detachment. (Shaw et al., 1998; Morrison et al.,

2001; Shaw et al., 2001; Xiao et al., 2002; Kissil et al., 2002) In Schwann cells, merlin appears to suppress cell growth only when it is in the hypophosphorylated state, thus in confluent Schwann cell cultures, the hypophosphorylated band is the predominant form, although lesser amounts of the hyperphosphorylated band still exist (Morrison et al.,

2001).

Merlin migrates as a doublet regardless of erbin expression, however, in the presence of erbin siRNA, there was decreased expression of the band with faster mobility, or the hypo-phosphorylated form of merlin, surprising in light of the fact that the cultures were transfected when they were approximately 95% confluent. When Schwann cell lysates were treated with λ-protein phosphatase, the merlin band with slower mobility was abolished confirming that this band represents a phosphorylated form of merlin (Fig. 3A).

These data further indicate that erbin may directly or indirectly influence merlin activity as a tumor suppressor via proteins involved in cell-cell junctions. Erbin has been implicated in regulating the activation of extracellular signal-regulated kinases (ERKs) by interfering with the binding of Raf-1 to activated Ras (Huang, et al., 2003). Consistent with this notion, PC12 cells reduced in erbin reduction had increased expression of activated ERK. We show here that erbin reduction in Schwann cells also leads to increased ERK activation (Fig. 3B)

132 A MEK inhibitor reestablishes the association between β-catenin and merlin

As was previously described (Rangwala et al., Chapter 2) the addition of the MEK

inhibitor, U0126, to Schwann cells treated with erbin siRNA reversed the piling up

phenotype and the increased proliferation induced by the loss of erbin and the loss of

adhesion to the cells’ substrate. Cell-cell contact and the normal spindle-shaped

morphology, however, were not reestablished following the addition of the MEK

inhibitor. Surprisingly, we also found that the MEK inhibitor reestablished the

association between erbin and E-cadherin, an association that is abrogated by the loss of

erbin. (Rangwala, et. al., Chapter 2) We therefore tested whether the addition of the MEK

inhibitor to cells treated with erbin siRNA could also reestablish the abrogated merlin-β-

catenin association. As Figure 4B, left, reveals, the association between merlin and β-

catenin is, indeed, reestablished following the inhibition of MEK. The MEK inhibitor

also decreased expression of the hyperphosphorylated or inactive form of merlin. In fact,

we found that erbin reduction induced both a phosphorylated and hyperphosphorylated

form of merlin (Figure 4B, right, top two bands—see arrows), and that inhibition of

MEK abrogated expression of the hyperphosphorylated form of merlin. This suggests that inhibition of the Ras-MAP kinase pathway may inhibit the cellular proliferation following erbin reduction (Fig. 4B, right). In order to conclusively determine whether these top two bands are phosphorylated isoforms of the proteins, lambda phosphatase studies, similar to the ones described earlier, would have to be performed.

133

Erbin associates with merlin through direct interactions with EBP50

One possible way that merlin could be linked to erbin and the adherens junction complex

is via EBP50. This adaptor protein links merlin and ERM proteins to membrane proteins

and other partners via two PDZ like domains (reviewed by Bretscher et al., 2000). Erbin

and EBP50 co-localize in both isolated Schwann cells as well as in contacting cells at

discrete points along the plasma membrane, at cell tips, along processes, and in the

cytoplasm (Figure 5 a and b). Like erbin-β-catenin, and β-catenin-merlin, there was also

substantial co-localization between erbin and EBP50 at points of cell-cell contact (Figure

5b), suggesting that that both of these proteins may be involved in a junctional complex

that mediates cell contact. We also found substantial co-localization between erbin and

EBP50 at Schwann cell tips (Figure 5c). EBP50 localization at the tips of Schwann cells has been implicated in nodal formation and localization of ion channels to this domain of the peripheral nerve. EBP50 immunoreactivity was also detected in other regions of the cell, a finding similar to that observed by Gatto et al. (Gatto et al., 2003). Our finding that erbin localizes to the tips suggests that a subpopulation of erbin may localize to the node of peripheral nerves and functions independently of the autotypic adherens junction.

Immunoprecipitations of whole Schwann cell lysates using antibodies against erbin following by Western blot analysis against EBP50 indicate that these two proteins associate in the Schwann cell (Figure 5d), and the domain adjacent to the PDZ domain of erbin precipitates EBP50 (Figure 5e). Data not shown indicate that approximately 50% of erbin associates with EBP50 in the Schwann cell. To test if EBP50 binds directly to erbin, we performed in vitro binding assays. Consistent with the precipitation

134 experiments using cell lysates, we found that EBP50 bound to the domain of erbin encompassing amino acids 914-1240, adjacent to but not including the PDZ domain (Fig.

5f). Using a series of EBP50 deletion constructs (Fig. 5g), we found that erbin specifically binds to the carboxy tail of EBP50 but not to either of its two PDZ like domains (Fig. 5h).

135 DISCUSSION

Despite the cloning of the NF2 gene more than a decade ago and the presence of NF2

null mouse models, the mechanism by which the putative tumor suppressor, merlin,

functions as such is still unknown. Recent evidence indicates that its role in the

regulation of growth and proliferation may be dependent on cells’ ability to contact

inhibit via the adherens junction complex. Merlin has been shown to co-localize with the

adherens junction components β-catenin, α−catenin, and E-cadherin in mouse embryo fibroblasts, and to influence their localization to the plasma membrane (Lallemand et al.,

2003). Here we show that in Schwann cells, a primary cell type to be affected by merlin loss in vivo, merlin co-localizes with β−catenin and possibly erbin at the plasma membrane and at points of cell-cell contact. The putative association between merlin and

this junctional complex is most probably influenced by the adaptor protein, erbin, as

indicated by the decreased association between merlin and β-catenin in Schwann cells

with reduced levels of erbin, and the mislocalization of β-catenin and merlin in the cell.

We have previously demonstrated that erbin also influences E-cadherin localization to the

plasma membrane, and its association with β−catenin by using an siRNA strategy

(Rangwala et al, Chapter 2). Using this same model system, we have extended our study

to merlin and have demonstrated that erbin also influences merlin’s association with its

binding partner, EBP50, but not its association with CD44. As erbin loss induces

activated ERK (Huang et al, 2003; Rangwala et al., Chapter 2), we used the potent

inhibitor, U0126, to inhibit the MAP kinase pathway, and demonstrated that erbin loss

coincident with MAP kinase inhibition reestablishes merlin’s association with β-catenin,

suggesting that loss of adherens junction integrity occurs in a Ras dependent manner.

136 Furthermore, we have found that the increased proliferation seen in Schwann cell cultures treated with erbin siRNA (Rangwala et al., Chapter 2) leads to an increase in the hyper- phosphorylated form of merlin, surprising in light of the fact that these cultures were treated with the siRNA construct when they were high density. Normally these culture conditions favor the hypophosphorylated, presumably, active form of merlin. As we described previously, however, a significant effect due to erbin reduction is loss of cell- substrate adhesion (Rangwala et al., Chapter 2). This effect is manifested by the significant increase in cellular detachment from the culture dish, an effect not due to cell death. Consequently, the loss of contact inhibition between the cells as a result of cellular detachment may be the cause for the induction of the hyperphosphorylated form of merlin.

While a link between merlin and adherens junction formation and localization has been established, the means by which they influence each other has not. One explanation may lie in our finding that erbin and EBP50 directly interact within Schwann cells, thus bridging E-cadherin/β-catenin with merlin. Although EBP50 has been shown to directly interact with β-catenin in hepatocytes (Shibata et al., 2003), theoretically bridging merlin to β-catenin, loss of erbin directly destabilizes this merlin-β-catenin association

suggesting that erbin may be involved in the stabilization of this complex.

We cannot rule out the possibility that the association between EBP50 and β-catenin are

independent of each of their roles in the formation and function of the adherens junction.

Multiple, functional pools of EBP50 and β-catenin exist in the Schwann cell. EBP50 has

137 been implicated in influencing the role of a number of ion channels including the cystic fibrosis and the Na+/H+ ion pump, and other ERM proteins

(Bretscher et al., 2000). Furthermore, the large cytoplasmic and perinuclear pools of

EBP50 in Schwann cells suggests that it may have a role outside of regulating these plasma membrane associated proteins. Because we show that associations between erbin and β-catenin (see Chapter 2), merlin and β-catenin, and erbin and EBP50 are most robust at points of cell-cell contact it is likely that these proteins are members of a junctional complex, specifically of the adherens junction type.

We have demonstrated previously that loss of erbin as a result of siRNA treatment induces phosphorylation of β-catenin, cytoplasmic accumulation of the protein, an increase in cyclin D1 expression and cellular proliferation (Rangwala et al., Chapter 2).

Consequently, the stable interaction between β-catenin and EBP50 following erbin siRNA treatment may reflect the induction of EBP50 expression and stabilization of other transmembrane proteins, and may not reflect the role that they each have in the integrity of the adherens junction.

Erbin was originally described as an erbB2 interacting protein (Borg et al., 2000). ErbB2 is a member of the EGF-family of receptor tyrosine kinases and is essential for Schwann cell differentiation, growth and survival (Erickson et al. 1997; Britsch et al. 1998;

Woldeyesus et al. 1999; Morris et al. 1999; Garratt et al., 2000). At least part of the survival signal that is transduced by erbB2 in Schwann cells involves activation of the mitogen-activated protein kinase (MAPK) pathway (Li et al., 2001). Since loss of both

138 erbin and merlin result in elevated MAPK signaling, and since a MEK inhibitor can revert virtually all of the phenotypes seen in Schwann cells with reduced erbin expression

(Rangwala et al., Chapter 2), it is possible that erbin regulates signaling between erbB2, merlin and β-catenin in a MEK-dependent manner. Indeed, in C. elegans the Ras/ERK and Wnt/β-catenin signaling pathways cooperate to induce P12 and vulval cell fates

(Freeman and Bienz, 2001; Howard and Sundaram, 2002; Tuli et al., 2003).

Furthermore, oncogenic Ras has been implicated in the dysregulation of E-cadherin, which can be blocked by the use of a MAP kinase inhibitor (Schmidt, et al., 2003), suggesting that signaling by erbB2, merlin and β-catenin/E-cadherin, each influenced by erbin, may converge upon Ras. It has also been demonstrated that merlin associates with erbB2 in Schwann cells through interactions with paxillin (Fernandez-Valle et al., 2002).

Recent data also indicate that merlin may also indirectly interact with erbB2 through its interactions with CD44, which mediates erbB2 activation in Schwann cells (Sherman et al., 2000).

Together, these data suggest that in addition to stabilizing protein-protein interactions between junctional protein complexes and merlin, erbin may also potentiate cell signaling that influences Schwann cell growth and survival through erbB2 activation of MEK and

β-catenin signaling. Such protein complexes could play significant roles in coordinating changes in cell adhesion and cell growth.

139 MATERIALS AND METHODS

Cell Culture

Schwann cells were isolated from sciatic nerves of 3-day-old rat pups (Brockes et al.,

1979), purified by anti-Thy 1.1 immunoselection, and expanded for 6-7 passages on 10- cm plates coated with poly-l-lysine (Sigma) in DMEM supplemented with 10% FBS, 2

µM forskolin (Sigma), and 5 ng/ml recombinant human HRG-β1 (EGF domain) (R&D

Systems). All of the cultures used in these experiments were essentially free of fibroblasts.

Antibodies

We used the following antibodies: a polyclonal, monoclonal, and purified against erbin

(all generously provided by J-P Borg); a polyclonal and purified against EBP50 (both generously provided by A. Bretscher); polyclonals against merlin (c-18), erb B2, erk, and E-cadherin (Santa Cruz Biotechnology);a polyclonal against merlin (Wa-30; generously provided by D. Gutmann) a polyclonal against P-erk (Cell Signaling); a monoclonal against β−catenin and N-cadherin (Transduction Laboratories); a monoclonal against E-cadherin (BD Biosciences Pharmingen); a monoclonal against CD44, 5G8

(Serotec, Ltd.); a monoclonal against tubulin (NeoMarkers); Rabbit IgG, Mouse IgG, and

Goat IgG (Vector Laboratories); Horseradish peroxidase-conjugated Rabbit and Mouse

(BioRad); Horseradish peroxidase-conjugated goat (Santa Cruz Biotechnology);

Fluorescein isothiocyanate (FITC)-conjugated secondary antibodies, Hoechst (Molecular

Probes)

140

Immunoprecipitations

Total protein extracts were made in 85mM NaCl, 150 mM Tris-HCl, 1% Triton X-100, pH 7.5 supplemented with protease inhibitors. Lysates were kept on ice for 15 minutes and then cleared by centrifugation at 20,000g for 10 min. Approximately 100 µg of extract was precleared at 4 C for 30 min with 30 µl of Protein-A or Protein –A/G

(Oncogene) Sepharose beads and 1 µg of isotype- matched IgG. The supernatant was immunoprecipitated with 2.5 µg of the immunoprecipitating antibody and incubated at 4

C overnight. The extract was then incubated with 35 µl of either Protein –A or Protein-

A/G Sepharose beads at 4 C for 2 hrs. Complexes were washed extensively with buffer

A (200 mM NaCl, 150 mM Tris-HCl, 1% Triton-X 100, pH 7.5), buffer B (100 mM

NaCl, 150 mM Tris-HCl, 1% Triton-X 100, pH 7.5), and buffer C (50 mM NaCl, 150 mM Tris-HCl, 1% Triton-X 100, pH 7.5) and were boiled in SDS sample buffer. SDS-

PAGE and western blotting was carried out according to standard protocols.

Immunostaining

Schwann cells were fixed in 4% paraformaldehyde for 10 min. Cultures were then washed extensively with 10% normal goat serum (NGS), 0.1% Triton X-100, in 1X PBS and nonspecific binding sites were blocked with the same buffer for 30 min at room temperature. Primary antibodies were diluted in blocking buffer and incubated on the cells for 1 hr. Merlin antibodies (Wa-30) were used at a 1:300 dilution; erbin antibodies

(J-P Borg were used at a 1:15 dilution); β-catenin antibodies were used at a 1:20 dilution;

EBP50 antibodies were used at a 1:15 dilution. The cells were washed extensively in the

141 blocking buffer and secondary antibodies conjugated to FITC or TRITC diluted in

blocking buffer were added for 45 min. Cells were washed and coverslips mounted with

Fluoromount G (Southern Biotechnology Associates, Inc.). We viewed the cells with a

Leica confocal microscope (Leica TCS SP; Leica Microsystems, Exton, PA).

Inhibition of erbin expression by RNAi

The target region of siRNA was 540 nt downstream of the start codon, which contained

approximately 50% G/C content. The nucleotide sequence was 5’ UAG ACU GAC CCA

GCU GGA A dTdT 3’ (nt 866-884), and was the same construct used by Huang, et al. to

decrease erbin expression in PC12 cells (Huang et al., 2003). We searched the NCBI sequence bank against this segment of DNA using the BLAST program, which revealed no match, suggesting the specificity of the target region by siRNA. The 21-nucleotide

RNAs were chemically synthesized by Integrated DNA Technologies, Inc. To demonstrate the silencing effect of endogenous erbin expression by siRNA, 100% confluent Schwann cell cultures grown in 10-cm dishes were transfected with the siRNA duplex using an amine-based transfection reagent (Ambion). Briefly, 24 µl of the amine- based transfection reagent was mixed with 665.5 µl of Opti-MEM (Gibco-Brl).

Following a15 minute incubation at room temperature, 16.3 µl of 20 µM erbin siRNA duplex was added and incubated for an additional 10 minutes. The entire mix was overlaid onto the cells and incubated overnight at 37 C and 7.5% CO2 before the addition of 7 ml of antibiotic free medium. 55 hrs after transfection the cells were lysed and harvested in the same manner as described for Western blotting and subjected to immunoblotting for expression of erbin.

142 In Vitro affinity precipitation assay

Expression of the recombinant GST fusion proteins was induced in DH5α with 1mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 1 h at 37 C. We extracted proteins in

PBS containing DNase, lysozyme (Sigma), and Ethylene Glycol-bis(β-Aminoethyl

Ether)-N,N,N’,N’-Tetraacetic Acid (EGTA) by sonicating for 45 s and purified them on glutathione-Sepharose beads (Amersham) according to the manufacturer’s recommendations. Schwann cells were lysed as described for immunoprecipitation. We incubated immobilized GST fusion proteins for 2 h at 4 C with 250 µg of lysates and washed them sequentially with immunoprecipitation buffers B, C, and A before immunoblotting analysis.

Direct binding assay

Expression of the full length GST-tagged EBP50 protein was induced in DH5α cells with1 mM IPTG for 1 h at 37 C. The proteins were purified on Glutathione-Sepharose beads (Amersham) according to the manufacturer’s recommendations. The GST-erbin constructs were purified as described above. The GST-EBP50 was digested with trypsin

(Amersham) and was repurified to obtain GST-free EBP50. We mixed the GST-free

EBP50 with each of the GST-tagged erbin proteins in a 10:1 ratio in 0.5 ml of 150 mM

NaCl, 1% Triton X-100, 50 mM Tris-HCl, pH 7.4 and incubated them overnight at 4 C.

The beads were isolated and washed sequentially with immunoprecipitation buffers B, C, and A before immunoblotting analysis. Binding was assessed by immunoblotting for

EBP50 with specific antibodies. Binding of erbin to specific domains of EBP50 was determined by the same protocol except erbin construct GST-914-1371 was digested with trypsin, and the untagged 914-1371 protein incubated with each of the GST-tagged

143 EBP50 proteins. Binding was assessed by immunoblotting for erbin with specific antibodies.

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153

154

155

156 (c-18)

157

158 FIGURE LEGENDS

Figure 1: Association and co-localization of erbin and merlin in primary Schwann cell

cultures. (A) Immunoprecipitation of merlin (c-18) from subconfluent, Schwann cell

lysates followed by Western blot analysis (IB) using anti-erbin, β-catenin, and merlin

antibodies reveals that merlin associates with β-catenin and erbin in Schwann cells. The

amount of lysate was underloaded as compared to the immunoprecipitation reaction,

consequently, the degree of association between the two proteins can not be extrapolated

from the intensity of the bands. Immunoprecipitation of erbin followed by Western blot

analysis reveals that erbin does not associate with ezrin. Con = control

immunoprecipitation with an irrelevant antibody; IP = immunoprecipitation with the erbin antibody; lys = total cell lysate. (B) Western analysis of total Schwann cell lysates comparing the specificity of two merlin antibodies: Wa-30 (generously provided by D.

Guttmann) and C-18 (Santa Cruz). As the blot indicates, both antibodies detect five

bands. (C) Immunoprecipitation of erbin from confluent (hi) or subconfluent (lo)

Schwann cell cultures grown in the presence of absence of neuregulin (NRG) followed

by Western blot analysis using anti-β-catenin or anti-phosho-erbB2 antibodies. Erbin

associates with β-catenin more robustly in subconfluent cultures regardless of neuregulin

stimulation. In contrast, erbin exclusively associates with the activated form of erbB2 (p-

erbB2) in confluent cultures stimulated with neuregulin. (D) Schwann cells were cultured

in the presence of growth factors until they were either 50% confluent or approximately

90% confluent. Confocal images of cells double-stained for merlin (red) and β-catenin

(green) (left) reveal that merlin and β−catenin localize to punctate domains on the cell

membrane near the cell body and at processes. There was less cytoplasmic β−catenin

159 staining in the isolated Schwann cell as compared those cells that were contacting each other. Furthermore, there was substantial merlin-β−catenin localization at points of cell- cell contact (arrowhead). (E) Schwann cells were cultured in the presence of growth factors until they were approximately 50% confluent. Confocal images of cells double stained for erbin (green) and merlin (red). Erbin co-localizes with merlin at the cell membrane. There is also substantial cytoplasmic staining of erbin. Erbin is also localizes at the tips of Schwann cells where it only partially co-localizes with merlin. Scale bars =

10 µm.

Figure 2: Reduction in erbin expression disrupts interactions between β-catenin and merlin. (A) Western blot showing expression of erbin and actin in Schwann cells treated with empty vector or erbin siRNA or a scrambled erbin siRNA construct. Fifty-five hours post transfection, erbin expression was not affected in an irrelevant GAPDH siRNA, vector alone, or scrambled siRNA transfected cultures. Erbin expression in erbin siRNA treated Schwann cell cultures was reduced by approximately 50%. Actin served as a loading control. (B) (left) Western blot probed for CD44, β-catenin, and merlin following immunoprecipitation with a merlin antibody. Note that very little β-catenin co- immunoprecipitates with merlin in the presence of the erbin siRNA. Merlin’s association with CD44, however, is affected by erbin reduction. (right) Western blot of total cell lysates from the experiment in B (left) showing that while erbin expression is decreased by approximately 50%, the total expression levels of CD44, merlin and β-catenin remain unchanged in the presence of erbin siRNA. EBP50 expression increases by approximately 50%. Actin expression served as a loading control. (C) Western blot

160 probed for EBP50 following immunoprecipitation with a β-catenin antibody. β-catenin’s association with EBP50 is not affected by erbin loss.

Figure 3: Reduction in erbin expression alters merlin phosphorylation. (A) In the presence of erbin siRNA, the band with slower mobility, or the hyper-phosphorylated form of merlin, is in far greater abundance than the band with faster mobility as compared to control. Schwann cell lysates were also treated with λ-protein phosphatase.

The band with the slower mobility was abolished confirming that this band represents the phosphorylated form of merlin. (B)Levels of phosphorylated and total ERK expression in empty vector and erbin siRNA transfected Schwann cells were analyzed by Western blot.

Elevated levels of phosphorylated ERK were detected in erbin siRNA transfected cultures while the levels of total ERK were unchanged.

Figure 4: MEK inhibition rescues the erbin siRNA-induced dissociation of merlin from

β-catenin and decreases the expression of the hypophosphorylated forms of merlin. (A)

The MEK inhibitor, U0126, or DMSO (as a vehicle control) was added to erbin siRNA transfected cultures approximately 30 hours prior to harvest (at 55 hours post- transfection). Western blot analysis revealed that the inhibitor completely abolished the increase in phosphorylated ERK levels induced by erbin siRNA. (B, left) Schwann cells were treated with erbin siRNA or empty vector and subsequently treated with the MEK inhibitor, U0126, or DMSO. Following incubation, Schwann cells lysates were immunoprecipitated with a merlin antibody and immunoblotted for β-catenin, merlin and

EBP50. Cells treated with the MEK inhibitor maintained β-catenin-merlin, and merlin-

161 EBP50 protein complexes. Total levels of β-catenin were not affected by the addition of the MEK inhibitor. (B, right) Cells treated with the MEK inhibitor had decreased expression of the hyperphosphorylated band (top arrow) of merlin.

Figure 5: Erbin is directly associated with EBP50 in the Schwann cell (A) Schwann cells were cultured in the presence of growth factors until they were approximately 50% confluent. Confocal images of cells double-stained for EBP50 (red) and erbin (green) reveal that erbin and EBP50 localize to punctate domains on the cell membrane near the cell body (arrowhead) and at Schwann cell tips (arrow). Erbin and EBP50 are also found in the cytoplasm of the cell body where the two proteins do not co-localize. Scale bars = 10 µm. (B) Similar co-localization studies were performed on 90% confluent

Schwann cell cultures. Substantial co-localization is present at points of cell-cell contact

(arrow). (C) High power magnification of a Schwann cell tip. Discrete populations of erbin and EBP50 co-localize in this domain of the Schwann cell.

(D)Immunoprecipitation of Schwann cell lysates with anti-erbin antibody followed by

Western blot analysis reveal that erbin and EBP50 associate in the Schwann cell. The amount of lysate was substantially underloaded as compared to the immunoprecipitation reaction, consequently, the degree of association between the two proteins can not be extrapolated from the intensity of the bands. (E) Western blot showing that EBP50 from

Schwann cell lysates bound specifically to the GST-fusion proteins not containing the erbin PDZ domain. Lys = total cell lysate, as a control for EBP50 expression; GST = pull-down assay with GST alone. (F) Direct in vitro binding assay showing that EBP50 binds to the GST-fusion protein not containing the erbin PDZ domain. (G) Glutathione

162 S-transferase (GST) deletion constructs comprising different portions of EBP50 were used to determine the domain through which erbin interacts with EBP50. The panel of constructs are comprised of full-length EBP50 (full-length), PDZ 1 alone (1+), PDZ 1 and 2 (1+2), and the carboxy tail (COOH). (H) Direct in vitro binding assay showing that erbin binds to the GST-fusion protein containing the carboxy tail of EBP50.

163

CHAPTER 4 ERBIN MAY LINK ERB B2 AND CD44

164 ABSTRACT

The receptor tyrosine kinase, erbB2, is vital for Schwann cell differentiation and survival.

CD44 has been implicated in neuregulin-induced erbB2 phosphorylation and erbB2- erbB3 heterodimerization. Here we show that erbin, a PDZ protein that directly binds to erbB2 and localizes the receptor to the lateral membrane of intestinal epithelial cells, and associates with CD44 and activated erbB2. Reduction of erbin expression in Schwann cells using small interfering RNA (siRNA) dissociates erbB2 from CD44, and leads to a modest decrease in the total levels of erbB2. We also show that erbB2 and merlin associate in the Schwann cell, raising the possibility that CD44, erbB2, and merlin may be present in the same complex and, therefore, influence each other’s function.

165 INTRODUCTION

Schwann cells are a subset of glial cells derived from the neural crest (for review, see

Jessen and Mirsky, 1999). After the growth cones of axons have begun extending

towards their targets, Schwann cell precursors migrate into the nerves (Carpenter and

Hollyday, 1992; Bhattacharyya et al., 1994). The timing of Schwann cell differentiation

and the final number of Schwann cells found in the adult nerve is influenced by a number

of factors which influence aspects of survival, differentiation and apoptosis.

Signals that influence Schwann cell proliferation are derived from the axon (for review

see, Jessen and Mirsky, 1999). One such factor includes members of the neuregulin

protein family. Neuregulin are encoded by alternatively spliced transcripts of the

neuregulin-1 (Burden and Yarden, 1997). These neuregulins can either be membrane bound or soluble and bind to and function through the transmembrane receptor tyrosine kinases erbB2 and erbB3 (Morrissey et al., 1995; Vartanian et al., 1997; Rahmatullah et al., 1998). These receptors can form homodimers or heterodimers depending on the specific type of ligand and the receptors that are expressed by the cell (Klapper et al.,

2000). Both erbB3 and erbB4 bind to neuregulin with high affinity; erbB2 lacks an extracellular binding domain and is the preferred dimerization partner of all type 1 receptors.

Knockout studies indicate that neuregulin-1, erbB2 and erbB3 are essential for Schwann cell differentiation and survival as well as axonal survival (Lee et al., 1995; Meyer et al.,

1995; Kramer et al., 1996; Erickson et al., 1997; Riethmacher et al., 1997; Liu et al.,

166 1998; Morris et al., 1999; Wolpowitz et al., 2000; Garratt et al., 2000), while erbB2 and

erbB4 signal primarily in the myocardium (Alroy and Yarden, 1997; Adlkofer, 1999).

These conclusions have been drawn from the following observations: 1) the neuregulin-1

(Meyer et al., 1995; Kramer et al., 1996; Liu et al., 1998; Wolpowitz et al., 2000), erbB2

(Lee et al., 1995; Erickson et al., 1997; Garratt et al., 2000), and erbB4 (Gassmann et al.,

1995) knockouts (KOs) are embryonic lethal due to the arrest of trabeculation of the heart

ventricle 2) trabeculation occurs normally in erbB3 KO animals however severe PNS

pathology is present (Riethmacher et al., 1997; Erickson et al., 1997), which consists of a

complete lack of Schwann cells and Schwann cell-precursors 3) erbB3 KO mice lose

most of their sensory and motoneurons during the second half of embryonic

development, suggesting that the loss of neurons is specifically due to the loss of trophic

support provided by the Schwann cell, Schwann cell precursors, and early Schwann cells.

(Riethmacher et al., 1997) 4) the progeny of erbB2 KOs that have been crossed with

animals expressing erbB2 under the control of a heart specific promoter, although viable,

have PNS pathology similar to that found in the erbB3 KOs. These data, taken together,

highlight the significance of an erb2/erbB3-neuregulin system in peripheral nerve

development.

As mentioned previously, erbB2 and erbB3 function in the peripheral nervous system is via heteromerization of the two receptors (Woldeyesus et al., 1999; Morris et al., 1999).

Although erbB2 has no known ligand and no extracellular binding domain, neuregulin can bind to erbB3. ErbB3 has no intrinsic kinase activity (Guy et al., 1994) and is, therefore, dependent upon erB2 for signal transduction. Neuregulin-erbB3 association

167 facilitates erbB2/B3 heterodimerization, thus leading to activation of the erbB2 receptor

(Sliwkoski et al., 1994).

Additional studies involving antibodies against neuregulin-1 or erbB2 further demonstrate the Schwann cell’s absolute requirement for a functional erb2/erbB3 receptor system and neuregulin. The use of inactivating antibodies against neuregulin reduces the axon-driven proliferation, indicating that an axonally bound neuregulin-1 isoform serves as the growth promoting signal (Morrissey et al., 1995). Knockout studies indicate that neuregulin-1, erbB2 and erbB3 are essential for Schwann cell differentiation and survival as well as axonal survival (Lee et al., 1995; Meyer et al., 1995; Kramer et al., 1996; Erickson et al., 1997; Riethmacher et al., 1997; Liu et al., 1998; Morris et al.,

1999; Wolpowitz et al., 2000; Garratt et al., 2000), underscoring the significance of the erb2/erbB3-neuregulin system in peripheral nerve development.

It has recently been demonstrated that erb2/B3 heterodimerization is facilitated by CD44, a transmembrane glycoprotein that has been implicated in cell-cell and cell-matrix adhesion, cellular migration, and growth factor signaling (Naor et al., 2002). In fact, reduction of CD44 expression inhibits erbB2/B3 heterodimerization following the addition of neuregulin and activation of erbB2 as assessed by expression of phospho- erbB2 (Sherman et al., 2000).

We have investigated the possibility that erbin, an erbB2 binding protein originally studied in intestinal epithelial cells (Borg et al., 2000), may influence erbB2 activation

168 and its association with CD44 following erbin reduction. Erbin was originally found to

colocalize with erbB2 at the lateral membrane of human intestinal epithelial cells (Borg

et al., 2000). Mutations in the erbin binding site lead to the mislocalization of the

receptor in these cells, suggesting that erbin plays a role in the localization of this

receptor and possibly in its signaling (Borg et al., 2002). Erbin RNAi studies performed

in PC12 cells further indicate that erbin reduction leads to increased activation of ERK in

the presence of neuregulin (Huang et al., 2003) Taken together, these data support the idea that erbB2 function and heterodimerization with erbB3 is regulated by this PDZ containing protein, erbin .

Using an erbin siRNA approach, we have observed that following erbin reduction

CD44’s association with erbB2 is abrogated, suggesting that erbin mediates the association and function of erbB2 through its interaction with CD44.

169 RESULTS—All data shown are preliminary

ErbB2 expression is high during perinatal Schwann cell proliferation and decreases as

Schwann cell maturation is completed (Cohen et al., 1992; Jin et al., 1993). Studies concerning erbin expression during development have not been performed, however, we found that erbin expression is not dependent upon Schwann cell confluence (data not shown). In light of the observation that both of these proteins are expressed in the

Schwann cell, and that in vitro binding assays suggest that erbin directly associates with erB2 (Borg et al., 2000), we were interested in determining whether this interaction also occurs in the Schwann cell. We cultured Schwann cells in the presence of HRGβ1, a neuregulin, previously shown to promote Schwann cell survival, proliferation, and development (Morrissey et al., 1995). Lysates were then immunoprecipitated erbin antibody and analyzed by Western blotting. We found that erbin associates with the phospho- or activated form of erbB2 (Figure 1).

In order to determine if erbin influences erbB2 function, we used a RNA interference

(RNAi) approach to reduce erbin expression in Schwann cells. The 21 nucleotide RNAi were used previously by Huang et al. (Huang et al., 2003) in order to demonstrate the effect of erbin loss in PC12 differentiation. The siRNA or vector alone was transfected into approximately 90% confluent Schwann cell cultures in the absence of neuregulin.

Fifty-five hours following transfections, Schwann cell lysates were collected and analyzed by Western blot. Erbin expression decreased by approximately 60% as compared to control, while erbB2 expression decreased by approximately 30%. CD44 and actin expression remain unchanged, suggesting that erbin siRNA did not

170 nonspecifically affect protein expression. We also found that both activated ERK and c- jun expression were increased as a result of erbin reduction (Figure 1b), findings that are consistent with data suggesting that erbin modulates the Ras-MAP kinase signaling pathway (Huang et al., 2003). These lysates were immunoprecipitated with erbB2 antibodies and analyzed by Western blotting. We found that erbB2’s association with

CD44 and β-catenin is abrogated in the presence of erbin siRNA (Figure 1c), suggesting that these associations may be influenced by activated Ras/MAP kinase signaling.

Merlin is a member of the ezrin-radixin-moesin (ERM) family of proteins that localize to the membrane-actin cytoskeleton interface. It is encoded by the neurofibromatosis-2

(NF2) gene and functions as a tumor suppressor (for review, see McClatchey, 2003).

Merlin mediates contact inhibition of growth though signals from the extracellular matrix, specifically, through CD44’s interaction with hyaluronic acid, a component of the extracellular matrix (Morrison et al., 2001). Merlin’s growth-inhibitory activity depends on its specific interaction with the cytoplasmic tail of CD44 (Morrison et al., 2001). In light of erbB2 and merlin’s dependence on CD44 for their respective functions, we were interested in determining whether they were present in the same complex. Confluent

Schwann cell cultures were immunoprecipitated with either merlin or erbB2 antibodies and analyzed by Western blots. These data indicate that erbB2 associates with β-catenin and CD44, and merlin associates with erbB2 (Figure 1d).

171 DISCUSSION

Our studies indicate that erbin associates with the activated form of erbB2 and it mediates CD44’s interaction with erbB2. Further studies will assess whether erbB2/B3 heterodimerization is affected following erbin loss and whether the addition of neuregulin can prevent CD44-erbB2 disassociation. We also demonstrated that erbB2 and merlin associate in the Schwann cell, suggesting that CD44 may not only influence their respective functions, but that they may be part of a complex that directly influences each other. In light our data that erbin and EBP50 directly associate (Rangwala et al. Chapter

2), this complex may be stabilized not only by CD44 but through this erbin-EBP50 bridge.

CD44 constructs that lack the merlin binding domain could be used to assess whether erb2 function as assessed by erbB2 phosphorylation and erb2/B3 heterodimerization is dependent upon merlin. Studies of this nature would suggest that erbB2 and merlin signal through similar signaling pathways, and that Schwann cell survival and differentiation, traditionally viewed as being controlled by the neuregulin-erbB2/B3 signaling system was directly associated with Schwann cell proliferation as mediated by merlin.

Recent observations suggest that erbB2 and merlin may, in fact, share common signaling components. ErbB2 signals through the PI3K/AKT/Bad signaling pathways (Li et al.,

2001), erbin associates with active Ras and interferes with Ras’ association with Raf, and merlin can inhibit the activation of the Ras/ERK pathway (Lim et al., 2003). This

172 common signaling component suggests that erbB2 and merlin are directly influenced each other or through the actions of erbin and/or CD44.

173 Materials and Methods

Cell Culture

Schwann cells were isolated from sciatic nerves of 3-day-old rat pups (Brockes et al.,

1979), purified by anti-Thy 1.1 immunoselection, and expanded for 6-7 passages on 10- cm plates coated with poly-l-lysine (Sigma) in DMEM supplemented with 10% FBS, 2

µM forskolin (Sigma), and 5 ng/ml recombinant human HRG-β1 (EGF domain) (R&D

Systems). All of the cultures used in these experiments were essentially free of fibroblasts.

Antibodies

We used the following antibodies: a polyclonal, monoclonal, and purified against erbin; a polyclonal against erbB2, c-jun, ERK, and actin (Santa Cruz Biotechnology); a polyclonal against P-erk (Cell Signaling); a monoclonal against β−catenin (Transduction

Laboratories); a monoclonal against tubulin (NeoMarkers)a monoclonal against CD44,

5G8 (Serotec, Ltd.); Rabbit IgG and Mouse IgG, (Vector Laboratories); Horseradish peroxidase-conjugated Rabbit and Mouse (BioRad); Horseradish peroxidase-conjugated goat (Santa Cruz Biotechnology).

Immunoprecipitations

Total protein extracts were made in 85mM NaCl, 150 mM Tris-HCl, 1% Triton X-100, pH 7.5 supplemented with protease inhibitors. Lysates were kept on ice for 15 minutes

and then cleared by centrifugation at 20,000g for 10 min. Approximately 100 µg of

extract was precleared at 4 C for 30 min with 30 µl of Protein-A or Protein –A/G

174 (Oncogene) Sepharose beads and 1 µg of isotype- matched IgG. The supernatant was

immunoprecipitated with 2.5 µg of the immunoprecipitating antibody and incubated at 4

C overnight. The extract was then incubated with 35 µl of either Protein –A or Protein-

A/G Sepharose beads at 4 C for 2 hrs. Complexes were washed extensively with buffer

A (200 mM NaCl, 150 mM Tris-HCl, 1% Triton-X 100, pH 7.5), buffer B (100 mM

NaCl, 150 mM Tris-HCl, 1% Triton-X 100, pH 7.5), and buffer C (50 mM NaCl, 150

mM Tris-HCl, 1% Triton-X 100, pH 7.5) and were boiled in SDS sample buffer. SDS-

PAGE and western blotting was carried out according to standard protocols.

Inhibition of erbin expression by RNAi

The target region of siRNA was 540 nt downstream of the start codon, which contained

approximately 50% G/C content. The nucleotide sequence was 5’ UAG ACU GAC CCA

GCU GGA A dTdT 3’ (nt 866-884), and was the same construct used by Huang, et al. to

decrease erbin expression in PC12 cells (Huang et al., 2003). We searched the NCBI sequence bank against this segment of DNA using the BLAST program, which revealed no match, suggesting the specificity of the target region by siRNA. The 21-nucleotide

RNAs were chemically synthesized by Integrated DNA Technologies, Inc. To demonstrate the silencing effect of endogenous erbin expression by siRNA, 100% confluent Schwann cell cultures grown in 10-cm dishes were transfected with the siRNA duplex using an amine-based transfection reagent (Ambion). Briefly, 24 µl of the amine- based transfection reagent was mixed with 665.5 µl of Opti-MEM (Gibco-Brl).

Following a15 minute incubation at room temperature, 16.3 µl of 20 µM erbin siRNA duplex was added and incubated for an additional 10 minutes. The entire mix was

175 overlaid onto the cells and incubated overnight at 37 C and 7.5% CO2 before the addition of 7 ml of antibiotic free medium. 55 hrs after transfection the cells were lysed and harvested in the same manner as described for Western blotting and subjected to immunoblotting for expression of erbin.

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181 Figure 1

182 FIGURE LEGENDS

Figure 1: An erbB2-CD44-merlin complex may be mediated by erbin. (A)

Immunoprecipitation of erbin from subconfluent Schwann cell lysates followed by

Western blot analysis (IB) using an anti-phospho-erbB2 antibody reveals that erbin associates with phospho-erbB2 in Schwann cells. Con=control immunoprecipitation with an irrelevant antibody; IP = immunoprecipitation with the erbin antibody. (B-E) erbin

RNAi should be replaced by erbin siRNA. (B) Western blot showing expression of erbin, erbB2, CD44, c-jun, phosphor-ERK, total ERK, and actin expression in Schwann cells treated with empty vector or erbin siRNA. Fifty-five hours post- transfection, erbin expression was reduced by approximately 60% while erbB2 expression was reduced by approximately 30% in cultures treated with the erbin siRNA. Expression of phospho-

ERK and c-jun were significantly increased. CD44 and actin levels remain unchanged.

(C) Western blot probed for erbB2, CD44, and β-catenin following immunoprecipitation with an erbB2 antibody. Note that very little CD44 and β-catenin co-immunoprecipitate with erbB2 in the presence of erbin siRNA. (D) Immunoprecipitation of erbB2 and merlin from subconfluent Schwann cell lysates followed by Western blot analysis using

β-catenin and CD44 antibodies reveal that erbB2 associates with β-catenin and CD44

(left) and merlin associates with erbB2 (right) in Schwann cells.

183

CHAPTER 5 GENERAL DISCUSSION

184 Among the most fundamental questions in neurobiology pertains to how signals concerning our internal and external environments are relayed throughout the body. A significant component of this relay system lies in the myelin sheath, which effectively insulates many of the nerves of the central and peripheral nervous systems. The myelin sheaths of the central and peripheral nervous systems are remarkably similar despite being formed from two rather different cell types: Schwann cells in the PNS and oligodendrocytes in the CNS. The unique cell types utilized in these two compartments are central to elucidating the differences found in the formation of the myelin sheaths during development, the response to it following injury, and the myriad diseases inflicting this nerve specialization. The research described here focuses on the Schwann cell, and specifically an adherens junction complex that regulates aspects of development, growth, and proliferation of this cell.

The data described suggest that erbin is a component of Schwann cell autotypic junctions and is intimately involved in controlling cell-cell and cell-substrate interactions and proliferation. Furthermore, by regulating ERK activation erbin can indirectly influence the formation, stabilization, and function of the adherens junction protein complexes. By utilizing an siRNA approach, erbin levels within the Schwann cell were significantly reduced and specifically resulted in 1) loss of E-cadherin and β-catenin immunoreactivity at the plasma membrane 2) an increase in β-catenin phosphorylation 3) a decreased association between β-catenin and E-cadherin, β-catenin and merlin, and

CD44 and erbB2, and an increased association between merlin and EBP50 4) aberrant cellular morphology 5) loss of cell-cell and cell-substrate contact 6) increase in cellular

185 proliferation 7) increase in the hyperphosphorylated or inactive form of merlin 8)

increase in phosphorylated ERK signaling and cyclin D1 levels. These effects were

partially reversed by the MEK inhibitor, U0126, suggesting that MAPK signaling could

be the underlying cause of these aberrant phenotypes.

The significance of the data described above is multifold. The cadherin’s association

with its respective catenin is extremely strong and traditionally believed to occur

independent of any adaptor proteins. The observation that reduction in erbin expression

in the Schwann cell can abrogate the association between E-cadherin and β-catenin

suggests that erbin either 1) directly affects the cadherin-catenin association by

structurally stabilizing the adherens junction complex or 2) that it is a negative regulator

of the cadherin-catenin association that is indirectly mediated by the Ras/MAP kinase

pathway. The latter hypothesis suggests that the loss in E-cadherin-β-catenin association following erbin reduction is an indirect consequence of downstream signaling events.

The data described above do not resolve these two conflicting hypotheses, however, our observation that the MEK inhibitor, U0126, can reverse a number of the phenotypes induced by erbin loss, suggests that aberrant signaling is certainly a factor. Our observation that the MEK inhibitor, alone, had relatively no affect on Schwann cell morphology, cell-substrate or cell-interaction, and cellular proliferation suggests that the candidate kinase that phosphorylates β-catenin is upstream of the kinase involved in the activation of ERK. Studies that could assess its role in the Ras-MAP kinase pathway and its contribution, if any, to ERM/merlin regulation would further tie together these disparate signaling pathways and position erbin as an integral regulator of a number of

186 diverse phenomena including nerve development and regeneration following injury, cell proliferation, and tumorigenesis.

Erbin’s role in mediating cellular migration

Many of the cellular phenotypes observed in erbin reduced cultures are strikingly similar to those seen in transformed cells. Transformed cells are highly migratory and often have increased lamellipodia, focal adhesions, and membrane ruffles. Our data indicate that erbin reduction results in increased membrane ruffling, interesting in light of observations that implicate Rac, a member of the Rho GTPases, in the formation of membrane ruffles and lamellipodia and erbin in the modulation of the Rho GTPases (for review see, Kolch, 2003; Ridley et al., 1999). These proteins play a significant role in the remodeling of the cytoskeleton required for cellular migration. Of the approximately 20

Rho family members that have been identified to date, RhoA, Rac1, and cd42 have been the most extensively studied (Etienne-Manneville and Hall, 2000). Cdc42 is involved in establishing cellular polarity during cell migration (Etienne-Manneville and Hall, 2000).

Rac1 initiates cell migration by initiating lamellipodia extension at the leading edge of the cell (Ridley et al., 1992; Nobes and Hall, 1999), and its function is mediated, in part by, by its ability to activate the p21-activated kinases (PAKs). RhoA promotes the formation of focal adhesions and actin stress fibers by activating its effectors mDia and the Rho-kinases, ROCK1 and ROCK2 (Ridley and Hall, 1992; Leung et al., 1996;

Amano et al., 1997).

187 Merlin and the ERMs can be regulated by members of the Rho family of GTPases. In fact, recent studies indicate that merlin is regulated by Rac1 (Shaw et al, 2001; Kissil et al., 2002; Xiao et al., 2002). In mammalian cells, Rac1 activation results in the phosphorylation of the C-terminus of merlin, disrupted self association, and inactivation of its tumor suppressor activity. NF2-deficient fibroblasts manifest features of increased

Rac1 activity, indicating a bi-directional regulation between merlin and Rac1 activation

(Shaw et al., 2001). These data taken together, suggest, that erbin may be intimately involved in the regulation and function of the ERM/merlin proteins, and directly or indirectly influence cellular migration. However, studies specifically focused on the relationship between erbin and the Rho family of proteins, specifically Rac-1, and studies assessing the migratory capabilities of erbin reduced Schwann cells would further implicate erbin in these cellular processes.

Merlin has also been reported to directly interact with the focal-adhesion component, paxillin, in a complex that contains integrin-β1 and erbB2 (Fernandez-Valle et al., 2002).

Focal adhesions are regulated by RhoA and PAK1; specifically, RhoA has been implicated in the formation of focal adhesions (Ridley and Hall, 1992; Amano et al.,

1997) while PAK1 activation has been implicated in their disassembly (Manser et al.,

1997; Frost et al., 1998). The phenotypes observed in erbin reduced Schwann cells indicate that paxillin’s association to merlin, and focal adhesion regulation and formation, may be influenced by erbin. Studies focusing on these interactions would further implicate a feed-forward regulation between erbin, erbB2, and merlin.

188 Recent data suggest that erbin inhibits Erk activation by active Ras, but not by active Raf-

1. Furthermore, erbin associates with active Ras, but not inactive Ras or Raf. Consistent with these findings, erbin interferes with the interaction between Ras and Raf both in vivo and in vitro. (Huang et al., 2003) These data suggest that either Raf-1 induced activation of Erk is independent of Erk activation following erbin reduction or that erbin acts upstream of Raf-1. Use of constitutively activated Raf-1 mutants could differentiate between these two possibilities. Such a study would further substantiate the role of this pathway in adherens junction formation and stabilization.

Cross talk between erbB2, β-catenin, and merlin signaling pathways

Our data indicate that erbin loss results in β-catenin phosphorylation through a

MEK/Erk dependent manner, a pathway through which erbB2 also signals. This suggests one of two possibilities: 1) that loss of erbB2 mediation following erbin reduction directly effects β-catenin and adherens junction function, or 2) erbB2 and β-catenin are linked to common signaling pathways via MEK. Several lines of evidence also link merlin function to growth –factor receptor signaling. In fact genetic screens in

Drosophila have revealed that mutations in the EGFR (epidermal growth factor receptor) signaling pathway enhance merlin dominant-negative and loss-of-function mutations

(LaJeunesse et al., 2001), and in humans aberrant EGFR signaling has been associated with tumorigenesis (DeClue et al., 2000). Furthermore, cross-talk between wnt cascades and the Ras-ERK cascades have been described in a number of model systems including

C. elegans and Drosophila. Recent studies have demonstrated that in C. elegans the

Ras/ERK and Wnt/β-catenin signaling pathways cooperate to induce P12 and vulval cell

189 fates in a Hox-dependent manner. (Freeman and Bienz, 2001; Howard and Sundaram,

2002; Tuli et al., 2003) Therefore, erbin’s role in potentiating neuregulin’s signals via

erbB2 may have a very direct role in β-catenin function. Taken together, these data

suggest that there is a significant amount of cross-talk between the merlin, erbB2, and β-

catenin signaling pathways, and that they all may converge upon erbin. What is currently

not known are the common mediators within the pathways, and environmental influences

such as confluency, serum-starvation, growth factor starvation, and composition of the

extracellular matrix that may serve to regulate these pathways. Also an interesting

question that remains to be answered is whether erbB2 and β-catenin influence p-21

activated kinase-2, the kinase that is thought to phosphorylated merlin serine 518 (Kissil

et al., 2002; Xiao et al., 2002).

Does merlin function independent of erbin?

A discrepancy between the phenotypes observed NF2-/- MEFs and those observed

in erbin reduced Schwann cell cultures is that the association between β-catenin and E- cadherin is not affected by merlin loss. This suggests the following: that the pathway(s) involved in Erk activation as a result of merlin loss do not lead to the phosphorylation of

β-catenin, there are significant differences in the two cell types, and/or merlin and erbin act upon different levels of the same pathway. In light of these different possibilities it would be interesting to determine whether inhibition of MEK in NF2-/- MEFs would also

abrogate the phenotypes seen in these cells. Such a study would highlight the extent to

which erbin and merlin share common signaling components, and would help determine

whether merlin function is dependent upon erbin.

190

In addition, the use of a constitutively active merlin mutant (mutant S518A) (Shaw et al.,

2001) concomitant with erbin siRNA may determine erbin directly regulates merlin

function. As described above, erbin loss results in aberrant cellular proliferation as well

in an increase in the hyperphosphorylated or inactive form of merlin. This is surprising

in light of the fact that the Schwann cell cultures are transfected with the erbin siRNA

when they are fully confluent, a condition that favors growth inhibition and therefore, an

active or hypophosphorylated form of merlin. However, the loss of cell-substrate

adhesion following the addition of erbin siRNA results in cellular detachment, thus

effectively mimicking a subconfluent culture condition that supports an inactive form of

merlin. Thus the questions remains as to whether a constitutively active form of merlin

could abrogate the cellular phenotypes observed as a result of erbin loss. Results of this

nature would suggest that merlin loss is the primary cause for tumor formation, and that

merlin’s association with β-catenin via erbin is not the mechanism by which it acts as a

tumor suppressor.

An adherens junction “super complex”

As suggested in the data described, the function, formation, and stabilization of the adherens junctions requires far more proteins than traditionally believed. Our hypothesis that merlin’s association with β-catenin is mediated by an erbin-EBP50 direct

interaction suggests that these proteins are integral in the formation and stabilization of

the traditional adherens junction complex. Furthermore, it implies that that integrity of

this entire “super complex” is necessary for the function of the adherens junction and that

191 mutations that compromise the integrity of this complex could mediate the same pathogenesis observed in merlin inactivated tumors.

A significant percentage of schwannomas do not arise as a result of merlin inactivation

(Jacoby et al., 1994; Jacoby et al., 1996). Our hypothesis as outlined above suggests that mutations in erbin and/or EBP50 could also mediate tumor formation. Because erbin has both a structural and signaling role, mutations in the signaling domains of erbin and/or

EBP50 that do not lead to a loss in the complex integrity may also induce tumor formation. Studies determining whether mutations in these proteins can lead to cellular transformation would further substantiate the merlin-adherens junction hypothesis and would go far in explaining the cause of both sporadic and neurofibromatosis-2 related schwannomas that do not result from merlin inactivation.

What role does erbin’s LRR play in the formation, stabilization, and function of the autotypic adherens junction?

In addition to its PDZ domain erbin contains 16 LRRs (leucine rich repeat). This unique composition of domains has been detected in other LAP (LRR and PDZ) proteins.

In fact, members of this protein family contain 16 LRRs at their amino termini and none, one or four PDZ domains at their carboxy termini. Localization studies indicate that LAP proteins are found at the basolateral membrane or associated with junctions in polarized epithelial cells of worms, flies, and humans (Borg et al., 2000).

192 The family includes four mammalian proteins, one Drosophila melanogaster protein

(Scribble), and one Caenorhabditis elegans protein (LET-413). Scribble and Let-413 mutants manifest improper cell-cell junctions and aberrant cellular polarity (Bilder and

Perrimon, 2000; Borg et al., 2000; Legouis et al., 2000). Deletion and mutational analyses indicate that the LRR domain but not the PDZ domain of the LAP proteins is crucial for basolateral membrane targeting (Legouis et al, 2003). This property, however, is specific to the LAP proteins as non-LAP proteins do localize to the basolateral membrane despite having a closely related LRR.

Despite the data indicating the LRR’s role in basolateral targeting, our studies focused on the PDZ portion of the protein because of its role in mediating the association to erbB2 and β-catenin. In light of the studies on the LAP mutants Scribble and Let-413, we cannot conclusively state that the LRR’s do not mediate the adherens junction integrity in the Schwann cell. To this end, deletion constructs made of the full-length protein may reveal the domains involved in polarity and signaling, and whether erbin localizes to the adherens junction by virtue of its association with β-catenin, alone. Of course, the localization studies described above indicate that large cytoplasmic pools of erbin exist within the cell, suggesting that erbin may have functions that are independent of cell junctions. Functional analysis of the individual domains of erbin may shed light upon the many disparate functions of the protein as a whole.

cAMP and Ras

193 A significant amount of cross-talk exists between cAMP activation of protein kinase A (PKA) and the Ras/MAP kinase pathway. Specifically, PKA can inhibit the activation of Ras (for review see, Waltereit and Weller, 2003). Forskolin, an inducer of cAMP production, is added to our Schwann cell growth media to induce cellular growth and proliferation. In the erbin siRNA studies described above, neither neuregulin nor forskolin were added to the media in light of original observations that the phenotypic alterations observed upon erbin reduction were abrogated in the presence of these mitogens. One explanation of this effect is that forskolin indirectly inhibits activation of

Ras. Inhibition of this pathway would certainly protect the cell from the Ras-induced phosphorylation of β-catenin and E-cadherin, and subsequent degradation of these proteins. Although it is not known whether erbin can directly influence the PKA or PKC pathways, both of these pathways influence the Ras/MAP kinase pathways at the level of

Raf (Liebmann, 2001); erbin acts upstream at the level of Ras-Raf association (Huang et al., 2003), suggesting that these pathways’ influence on the Ras/MAPkinase could abrogate any effects observed upon erbin loss.

An erbin knockout mouse model

Our in vitro model of erbin expression knockdown in Schwann cells has raised a

tantalizing array of possibilities in terms of the proteins role in tumor formation, cell-cell

and cell-substrate contact, proliferation, and development. Towards that end, a

transgenic mouse knockout model may shed light upon the broader roles for this protein

in vivo. A complete knockout of this protein would not doubt prove lethal because of our

data indicating that erbin loss has direct consequences on β-catenin function and wnt

194 signaling. However, erbin loss specifically in the Schwann cell may determine whether erbin loss leads to cellular transformation and subsequent tumor formation. If, in fact, erbin regulates merlin function, tumor formation would be suspected.

Moreover, erbin’s regulation of the erbB2 receptor tyrosine kinase suggests that loss of this protein from Schwann cells would lead to axonal loss during development and similar phenotypes observed in tissue specific erbB2 and erb3 KO models. Construction of a tissue specific erbin knockout model utilizing promoters that came on at time points following precursor development but before myelination is completed, may differentiate erbin’s regulation of erbB2 versus its regulation of β-catenin. ErbB2 loss led to the loss of sensory and motoneurons only during the second half of embryonic development

(Morris et al., 1999). Myelination, however, is not complete until postnatal day 2-3 (for review see, Jessen and Mirsky, 1999). Consequently, constructing a mouse model that could differentiate erbin’s effect on these two proteins may also differentiate erbin’s role in regulating Schwann cell survival and differentiation, and its role Schwann cell myelination, compaction, and formation of the nodal structure.

Transgenic models would also prove helpful in elucidating erbin’s role during nerve regeneration following injury. After axotomy of the sciatic nerve, distal axons of the proximal stump and the fibers of the distal stump degenerate. E-cadherin, however, is still detectable at the outer mesaxons of the myelinated axons as long as they remain morphologically intact. The E-cadherin facilitates Schwann cell proliferation and migration. E-cadherin is also expressed at points of Schwann cell contact with each other

195 and later with sprouting axons. (Hasegawa et al., 1996) This data suggests that erbin loss

would impede the formation of cell-axon, cell-cell and intralammellar associations, and

that mice lacking erbin in their Schwann cells would have reduced regenerative

capabilities.

The data described here suggests that erbin has a significant role in all aspects of the

Schwann cell, and, thus, indirectly on nerve function. It serves as a foundation for more in depth studies specifically aimed at elucidating erbin’s role in cellular transformation and tumor formation; nerve development and regeneration; the function of merlin, E- cadherin, β-catenin, and erbB2; and the extent to which each of these proteins influence

of the function of the others.

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202 Figure 1:

203 Figure legend

Figure 1: Diagram depicting an adherens junction “supercomplex.” Note: cartoon not drawn to scale. Erbin’s association with merlin is mediated through its interaction with EBP50. Previous studies have shown that the LRR domain of erbin is involved in blocking the association of Raf1 with active Ras, thus inhibiting MAP kinase signaling and phosphorylation of β-catenin. Based on our data, we predict that the activated Ras- MAP kinase signaling, which occurs as a result of erbin reduction, leads to the dissociation of β-catenin from E-cadherin through Ras-dependent phosphorylation of β- catenin and E-cadherin and their subsequent degradation. Merlin’s association with β- catenin, and erbB2’s association with both CD44 and β-catenin are disrupted as a result of this phosphorylation event.

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