The Cytoplasmic Adaptor Protein Caskin Participates In

THE CYTOPLASMIC ADAPTOR PROTEIN CASKIN PARTICIPATES IN

LAR-MEDIATED MOTOR AXON GUIDANCE

YI-LAN WENG

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Thesis Advisor: Dr. Heather T. Broihier

Department of Neurosciences

CASE WESTERN RESERVE UNIVERSITY

May, 2011

We hereby approve the thesis/dissertation of

Yi-Lan Weng

Candidate for the Doctor of Philosophy degree *.

(Signed) Gary Landreth

(Chair of the committee)

Heather Broihier

Lynn Landmesser

Susann Brady-Kalnay

Jocelyn A. McDonald

(Date) 23 March 2011

*We also certify that written approval has been obtained for any proprietary material contained therein.

DEDICATION

To my beloved family who has always supported and encouraged me

TABLE OF CONTENTS

LIST OF TABLES 4

LIST OF FIGURES 5

ACKNOWLEDGMENTS 6

ABSTRACT 8

CHAPTER 1: INTRODUCTION

Summary 11

Drosophila neuromuscular system 12

Axon guidance in Drosophila 13

Tyrosine phosphorylation signaling and axon guidance 18

Structure and regulatory mechanisms of the LAR family 20

LAR signaling and synaptic plasticity 22

LAR signaling and axon guidance 24

LAR signaling and axonal regeneration 27

Physiological functions of LAR 29

Aims and significance 31

Figures 1.1-1.2 33

CHAPTER 2: THE CYTOPLASMIC ADAPTOR PROTEIN CASKIN MEDIATES LAR SIGNAL TRANSDUCTION DURING DROSOPHILA MOTOR AXON GUIDANCE

Abstract 38

Introduction 39

Materials and methods 41

Results 46

Discussion 61

Figures 2.1-2.7 68

Tables 2.1-2.2 87

CHAPTER 3: IDENTIFICATION OF CASKIN-INTERACTING PROTEINS AND THE REGULATORY MECHANISMS IN LAR-MEDIATED AXON PATHFINDING

Abstract 90

Introduction 91

Materials and methods 95

Results and discussion 98

Figures 3.1-3.3 108

Tables 3.1-3.3 113

CHAPTER 4: GENERAL DISCUSSION

Redundancy and complexity of LAR signaling 117

LAR signaling cascades in axon pathfinding 118

Caskin in neural development 122

Axonal regeneration 124

BIBLIOGRAPHY 127

LIST OF TABLES

2.1: ISNb phenotype in ckn LOF and GOF embryos 87

2.2: Genetic interactions between LOF mutations in Lar, ckn and ptp69D 88

3.1: Caskin-interacting proteins indentified by a yeast two-hybrid screen 113

3.2: GOF and LOF phenotypes in motor axon guidance genes 114

3.3: Pairwise interaction between Caskin and candidate proteins 115

LIST OF FIGURES

1.1: Motor axon projections in Drosophila embryo 34

1.2: Schematic diagrams of motor axon guidance phenotypes 36

2.1: Neuronal overexpression of CG12424 disrupts axon guidance, and

CG12424 RNA localizes to the embryonic neuropil 69

2.2: caskin gene structure and allele generation 71

2.3: ckn LOF alleles disrupt ISNb motor axon pathfinding 73

2.4: ckn and Lar interact genetically 75

2.5: Ckn and Lar interact physically 78

2.6: Ckn redistributes Larcyto in S2R+ cells, and Ckn and Liprin-α do not simultaneously bind Lar 81

2.7: Ckn exhibits physical and genetic interactions with Dock 85

3.1: Reduction in abl genetic dosage rescues ckn LOF phenotype 108

3.2: Abl-dependent tyrosine phosphorylation occurs on Caskin 110

3.3: Model of the interplay between Abl and Caskin during motor axon guidance 112

ACKNOWLEDGMENTS

First and foremost I would like to thank my advisor, Dr. Heather Broihier for her constant guidance and support in the past several years. I feel fortunate to have joined her laboratory and to have had the opportunity to work in such a friendly environment. Heather gave me freedom to pursue my own research interests and provided me valuable suggestions and advice. Most importantly, I admire her personality and enthusiasm for science. From Heather, I see a model for being a great person and a successful scientist.

I would also like to thank my committee chair, Dr. Gary Landreth, and my other committee members, Drs. Lynn Landmesser, Susann Brady-Kalnay, and Jocelyn

A. McDonald. Their helpful comments and constructive criticisms not only helped me to complete this thesis, but also made me a better scientist. In addition, I would like to thank our collaborator Dr. Aaron DiAntonio for providing the reagents from which my thesis was conceived.

Many thanks go to the previous and current members of the Broihier lab—

Jonathan Paek, Crystal Miller, Inna Nechipurenko, Chris Dejelo, and Becca

James for their support and friendship. In particular, I would like to thank Nan Liu for her encouragement and assistance. She treated me as a close friend to her family, making me feel at home in the United States. I also want to express sincere appreciation to Jared Cregg, whose help overcame many obstacles in my career journey. I benefited a considerable amount from his knowledge and

research expertise. In addition, I am grateful for the generosity of departmental

staff members and for people who have shared their ideas, reagents and equipment. Finally, I want to thank my family. Their love has been my strength and my shield in the face of adversity. Without their support and encouragement,

I could not have reached this achievement.

The Cytoplasmic Adaptor Protein Caskin Participates in LAR-Mediated Motor

Axon Guidance

Abstract

YI-LAN WENG

LAR (leukocyte common-antigen-related) family receptors, members of the receptor protein tyrosine phosphatase (RPTP) superfamily, are important in the formation, plasticity, and regeneration of neuronal circuits. Considerable progress has been made in identifying LAR-interacting proteins, which form the basis for the molecular relays that underly LAR function. However, our understanding of intracellular LAR signaling in axon guidance remains fragmentary. In my thesis work, I identified a novel LAR-interacting protein, Caskin, and elucidated its role in motor axon guidance in the developing Drosophila embryo.

Drosophila caskin encodes a neuronal scaffolding protein containing two SAM domains and a conserved C-terminal region. Pan-neuronal caskin overexpression leads to vast axon guidance defects in both the CNS and in motor axons, implicating Caskin as a regulator of growth cone behavior. To define Caskin’s function in axon guidance, ethyl methanesulfonate (EMS)- induced caskin loss-of-function (LOF) mutants were generated and characterized. caskin LOF mutants exhibit motor axon guidance defects identical to Dlar LOF mutants. Further biochemical and genetic examination demonstrated

Caskin interacts with Dlar via its first N-terminal SAM domain and relays LAR

signaling via its conserved C-terminal region. I examined downstream signaling

of Caskin in order to further understand how LAR signaling regulates the

cytoskeleton. I conducted a yeast two-hybrid screen to search for potential

binding partners that act through interaction with Caskin’s C-terminus. Through

this screen I found several novel Caskin-interacting proteins, some of which are

known to regulate the cytoskeleton.

Protein phosphorylation is important in motor axon guidance. An attractive model was previously proposed in which a common substrate, regulated by Abl kinase and LAR phosphatase, can provoke different axonal responses. Interestingly, I found that Caskin can become tyrosine phosphorylated in an Abl kinase-

dependent manner and that Caskin and Abl interact genetically in the regulation

of motor axon guidance. Further studies will seek to determine whether tyrosine

phosphorylation is important in Caskin’s function.

Recently, PTPsigma, a LAR family member in vertebrates, was identified as a

receptor for CSPGs, which are molecules that inhibit axonal regeneration. I found

that interactions between Caskin and LAR are conserved in vertebrates; mouse

Caskin 2 is capable of binding to LAR and PTPsigma. Taken together, my thesis

work reveals a function for Caskin in LAR-mediated motor axon guidance and

identifies a potential molecular target for intervention following nervous system

injury.

CHAPTER 1

INTRODUCTION

Summary

Axon pathfinding is essential for the establishment of neuronal circuits during development and the re-establishment of these circuits following central and peripheral injuries. One remarkable feature of developmental pathfinding is the ability of axons to undergo long-range guidance based on a discrete number of prototypic fasciculation or branching events. Together these events establish functional neuronal circuits.

Axons must interpret complex environments to parse out their final synaptic targets. Decades of studies reveal that such extracellular information is received by a variety of receptors on axon growth cones and transduced within the cell to evoke specialized axonal responses. Mechanistically, individual signaling cascades are not linear and do not converge on a single protein responsible for rearranging the cytoskeleton. Instead, each initial signaling event can diverge and branch in complex signaling cascades that elicit distinct cellular responses by coupling different sets of interacting proteins.

One regulatory mechanism underlying axon pathfinding is the control of phosphotyrosine signaling via tyrosine kinases and phosphatases. Of these molecules, receptor protein tyrosine phosphatases (RPTPs), which reverse phosphorylation reactions catalyzed by tyrosine kinases, have been implicated in the control of axon fasciculation, branching, and target recognition. Each member of the RPTP family conveys its own set of signals to direct axon pathfinding, and the combined signaling of this broad class of transmembrane phosphatases via

competition and heterophilic cooperation leads to distinct growth cone action. In addition to their role as regulators of developmental axon guidance, a recent study identified PTPsigma, a member of the RPTP family, as a receptor for chondroitin sulfate proteoglycan (CSPG), an inhibitory extracellular factor upregulated in central nervous system (CNS) lesions. PTPsigma knockout animals exhibit enhanced axonal regeneration following spinal cord injury.

Therefore, elucidation of how RPTP family members signal may allow identification of novel therapeutic targets in spinal cord repair.

Drosophila neuromuscular system

Studies on axon pathfinding utilize both vertebrate and invertebrate model systems to elucidate the molecular mechanisms of neural wiring. Cumulative evidence indicates axon signaling cascades responsible for forming neural connections are evolutionarily similar (Araujo and Tear, 2003). Because of the versatility in genetic manipulation, the ease of anatomical accession, and the high fidelity of stereotypic axonal projections, Drosophila has become an ideal system for studying axon pathfinding.

In particular, the Drosophila neuromuscular system serves as an excellent paradigm for studying motor axon guidance and synaptic differentiation

(Keshishian et al., 1996). Each abdominal hemisegment in the late stage 16

Drosophila embryo consists of approximately 36 motor neurons innervating 30 muscle fibers in a stereotyped pattern. A majority of motor axons initially exit the

CNS region via either the intersegmental nerve (ISN) or the segmental nerve

(SN), while only a few motor neurons extend axons through the transverse nerve

(TN). Once the ISN and SN arrive in the peripheral muscle field, they diverge into smaller axon bundles and innervate their corresponding muscle targets. The

ISNb, ISNd, and ISN emanate from the ISN nerve root while the SNa and SNd emerge from the SN nerve root (Fig. 1.1).

Of these five major axon pathways, the ISN projects to the most distal

(dorsolateral) muscle region whereas the ISNb defasciculates from the ISN and extends to the more proximal (ventrolateral) muscles. It has been proposed that these motor axons employ sophisticated mechanisms to regulate fasciculation and defasciculation at choice points.

To date, substantial progress in identifying the molecules responsible for axon guidance has been made by utilizing forward genetic and misexpression screening approaches in the Drosophila neuromuscular system (Araujo and

Tear, 2003; Jorgensen and Mango, 2002; Miller et al., 2008). These molecules regulate many aspects of axon pathfinding and usually have functionally relevant homologues in vertebrates. For example, vertebrate Semaphorin, Plexin and

RPTPs play remarkable similar roles to their Drosophila homologues in the regulation of different types of axon projections (Komiyama et al., 2007; Suto et al., 2005; Uetani et al., 2006). Mapping their genetic pathways in Drosophila will continue to provide a platform for understanding the molecular mechanisms of axon guidance in higher vertebrate systems.

Axon guidance in Drosophila

A most remarkable characteristic of axon pathfinding is the ability of axons to

travel long distances through complex environments to ultimately reach their

synaptic targets. This tremendous challenge is overcome by the allocation of

choice points formed by guidepost cells along axon trajectories (Chao et al.,

2009; Tessier-Lavigne and Goodman, 1996). Guidepost cells synthesize and

display guidance cues and therefore provide growth cones with directional

information. In order to receive and discriminate among these extracellular

guidance signals, the growth cone has evolved to encode a versatile receptor

system by which the growth cone links different extracellular signals to behavior.

Once growth cones reach a choice point, the integration of complex downstream

signaling drives axons to specify their paths toward future choice points. A

dedicated sorting mechanism separates axons into distinct fascicles en route to

their synaptic targets. A number of studies have shown that such defasciculation

processes are determined by a balance of adhesive and anti-adhesive molecules between axon-axon contacts as well as axon-environmental contacts (Fields and

Itoh, 1996; Tessier-Lavigne and Goodman, 1996; Van Vactor, 1998). Here I discuss the mechanisms regulating axon fasciculation which form the basis for my thesis work in determining the role of Caskin in axon pathfinding.

Cell adhesion molecules (CAMs) are essential for axon fasciculation. Most of these proteins belong to the immunoglobulin superfamily (IgSF) and mediate homo- or heterophilic interactions between cells. Of these IgSF proteins, vertebrate N-CAM and L1 are best characterized, regulating axon extension and fasciculation (Crossin and Krushel, 2000; Walsh and Doherty, 1997). Similar to

vertebrate N-CAM, the Drosophila homologue Fasciclin II (Fas II) functions in promoting adhesion between axons. Fas II is expressed in all motor neurons as well as a subset of interneurons in the CNS. Elevated Fas II levels perturb motor axon pathfinding and cause ISNb bypass phenotypes where ISNb axons remain in the common ISN pathway and do not enter the ventrolateral muscle field to innervate their synaptic targets (Lin et al., 1994) (Fig. 1.2). This FasII gain of function (GOF) phenotype is consistent with the effect of increased axon-axon adhesion, suggesting that Fas II acts as an adhesive molecule contributing attractive forces between axons. Although Fas II plays an important role in promoting axon fasciculation, mutations of FasII did not induce gross axon defasciculation. Overall axon architectures in both the CNS and the peripheral system are intact in FasII loss of function (LOF) mutants (Lin et al., 1994), suggesting that other CAMs or signaling pathways mediate axon adhesion to functionally compensate for the loss of Fas II.

Some molecules exert anti-adhesive effects to repel axons into smaller fascicles.

Genetic studies in Drosophila reveal several genes are required for the selective defasciculation of motor axons at certain choice points. One of these genes is beaten path (beat) (Fambrough and Goodman, 1996; Pipes et al., 2001; Vactor et al., 1993). beat encodes a transmembrane protein containing extracellular immunoglobulin-like (Ig) domains and has been shown to have motor axon expression. In beat LOF embryos, ISNb axons cannot defasciculate from ISN, and as a result, fail to innervate their target muscle group (Fig. 1.2). This result indicates that Beat acts with anti-adhesive function to promote axon

defasciculation. Since the beat LOF and FasII GOF phenotypes are nearly identical, it is very likely that Beat and Fas II antagonistically control axon adhesion. Consistent with this idea, the failure of defasciculation caused by increased axon adhesion in beat LOF phenotype can be partially rescued by reducing Fas-II expression (Fambrough and Goodman, 1996). These observations suggest Beat may counteract Fas II-mediated adhesion, although the mechanism has not been characterized.

In addition to cell adhesion molecules, several signaling pathways regulate axon adhesion. Cumulative evidence indicates that Semaphorin/Plexin signaling can induce repulsive action that causes defasciculation (Bashaw, 2004) (Fig. 1.2). To this end, several downstream effectors mediating Semaphorin/Plexin repulsive guidance events have been identified. These include MICAL, Nervy, and Gyc76c

(Ayoob et al., 2004; Ice et al., 2005; Terman and Kolodkin, 2004; Terman et al.,

2002). Genetic studies in Drosophila indicate that the guanylyl cyclase catalytic activity of Gyc76c is required for Sema-mediated axonal repulsion. These data suggest a more general model where cGMP levels act as important secondary messengers controlling axon fasciculation. Additional studies will be required to elucidate how cGMP signaling pathways affect axon adhesion.

Receptor protein tyrosine phosphatases (RPTPs) are also involved in regulating axon defasciculation in subsets of motor axons. Within the Drosophila RPTP gene family there are five neural RPTPs (Dlar, Ptp10D, Ptp52f, Ptp69D, and

Ptp99a). These RPTPs elicit axon branching at choice points through combinatorial signaling, however their genetic interactions are highly complex

(Desai et al., 1997; Jeon et al., 2008; Schindelholz et al., 2001). Particularly, LAR and PTP69D are necessary for steering ISNb axons away from the ISN (Desai et al., 1996; Krueger et al., 1996). Several lines of evidence indicate that tyrosine phosphorylation signaling may control axon adhesion; for example, ectopic expression of Abl tyrosine kinase results in failure of ISNb defasciculation similar to that of LAR phosphatase LOF mutants (Wills et al., 1999a) (Fig. 1.2).

Nevertheless, the substrates of Abl and LAR remain elusive.

In addition to guidance molecules of the axolemma, target-derived attractants play a pivotal role in axon defasciculation. In Drosophila, sidestep (side) encodes a transmembrane protein in the peripheral muscles and is capable of directing motor axons to their synaptic targets (Sink et al., 2001). In embryos lacking Side protein expression, subsets of motor axons cannot enter the ventral muscle region because they form tight fasciculated axon bundles at the exit point. As a consequence, side LOF mutants display ISNb bypass phenotype identical to that in beat LOF or in FasII GOF mutants (Fig. 1.2). Surprisingly, reducing axonal adhesion by decreasing Fas II protein level cannot rescue side LOF phenotype.

These data suggest that Side induces axon defasciculation via a Fas II- independent mechanism.

Recently, Siebert et al. provided compelling evidence that Beat is a neuronal receptor for muscle-derived Side (Aberle, 2009; Siebert et al., 2009; Zinn, 2009).

Using time-lapse imaging techniques, Beat-expressing axons were found to track

Side along axon trajectories. Side is spatiotemporally expressed in a variety of intermediate choice points. After Beat-expressing axons contact a Side-

expressing choice point, Side expression is down regulated in the choice point tissue leading axons toward the next Side-expressing choice point. In this manner, subsets of motor axons (Beat-positive axons) diverge from primary fascicles and follow a migratory track toward their final targets established by

Side-expressing intermediates.

Tyrosine phosphorylation signaling and axon guidance

Protein phosphorylation is a key regulatory step in modulating signaling cascades in different cellular contexts. Phosphorylation can control enzymatic activity and regulate binding sites on proteins, leading to formation of diverse signaling complexes. A number of studies have shown that spatial and temporal changes of the growth cone phosphotyrosine proteome contribute to axon pathfinding behaviors. For example, phosphotyrosine signals in filopodia correlate with filopodial initiation and extension. Local application of PP2, a Src kinase family inhibitor, to growth cones leads to asymmetric distribution of phosphotyrosine proteins, subsequently inducing axon turning (Robles et al.,

2005). Additionally, protein tyrosine phosphatases (PTPs) are vital for axon outgrowth and guidance. Blocking PTP activity can lead to inhibition of nerve growth factor-directed neurite outgrowth in PC12 cells (Chiou and Westhead,

1992). Together these studies suggest that tyrosine phosphorylation may act as a general mechanism allowing dynamic filopodia and specific growth cone behaviors.

Consistent with this idea, genetic studies in both vertebrate and invertebrate nervous systems reveal that many axon guidance molecules encode protein tyrosine kinases and phosphatases. Eph receptors are the largest subfamily of receptor tyrosine kinases (RTKs) and are essential for the formation of topographic maps in the visual system and dorsal-ventral innervation of motor axons in limb muscles (Luria et al., 2008; McLaughlin and O'Leary, 2005). A considerable amount of work has been done to establish the forward signaling of the Eph family. Upon ligand binding, the cytoplasmic region of activated Eph receptors becomes tyrosine phosphorylated and serves as a site for SH2 domain proteins. To date, a number of Eph interacting adaptors have been identified, including Fyn and Src tyrosine kinases, Nck and Crk adaptors, and Abl and Arg kinase (Poliakov et al., 2004; Yu et al., 2001).

A considerable amount of evidence in different cellular contexts indicates that these Eph binding partners have important roles in the regulation of cytoskeleton dynamics. For example, Src kinase can phosphorylate focal adhesion kinase

(FAK) to propagate signaling. Activated FAK further phosphorylates cytoskeleton binding proteins, such as N-Wiskott Aldrich syndrome protein (N-WASP), talin, paxillin, and Crk-associated substrate (Cas) family proteins, leading to cytoskeletal remodeling. Thus, Eph receptors signal through these cytoskeletal- associated protein complexes in a phosphorylation-dependent manner to control axon pathfinding.

Likewise, phosphorylation cascades act downstream of other guidance receptors to transduce extracellular signals into changes in growth cone morphology.

Recent studies have shown that FAK and Src are required for mediating netrin- induced axon outgrowth. Both kinases are found to associate with the Netrin receptor DCC (deleted in colorectal cancer), causing tyrosine phosphorylation of the receptor in response to Netrin engagement (Li et al., 2004; Liu et al., 2004).

Functional axon guidance machinery demands an appropriate balance of intracellular tyrosine phosphorylation levels. Receptor protein tyrosine phosphatases reverse tyrosine phosphorylation catalyzed by tyrosine kinases and play important roles in regulating axon growth and targeting (Johnson and

Van Vactor, 2003). In Drosophila, five RPTPs are expressed in CNS axons, but little is known about how their downstream binding partners control axon pathfinding. It is possible that ligand-engaged RPTPs transduce extracellular signals into changes in growth cone morphology by modifying growth cone phosphotyrosine levels.

Structure and regulatory mechanisms of the LAR family

The human genome encodes 21 different RPTPs. Based on their domain architecture and sequence homology, these RPTPs can be further divided into 8 subfamilies (Alonso et al., 2004). LAR, PTPsigma, and PTPdelta together make up the LAR (receptor type IIA) subfamily of RPTPs. These receptors share common features: an extracellular region composed of three immunoglobulin-like

(Ig-like) domains and four to eight fibronectin type III domains (FNIII) based on alternative splicing. Similar to other RPTPs, LAR family members contain a single transmembrane spanning region followed by two intracellular tandem

phosphatase subunits, domains D1 and D2. The first membrane-proximal D1 domain is thought to contribute the majority of the receptor catalytic activity as inactivation of the D1 domain results in abolishment of enzyme activity (Krueger et al., 2003; Streuli et al., 1990). In contrast, the C-terminal D2 domain functions as an accessory binding site for protein interactions. Most LAR interacting proteins, including Abl, Ena, Trio, and Liprin-alpha, associate with the D2 domain

(Debant et al., 1996; Serra-Pages et al., 1998; Wills et al., 1999a).

Dimerization of RPTP family proteins has been proposed as a mechanism of regulation, where dimerization inhibits phosphatase activity. Structural analysis of

RPTPalpha has revealed that a small wedge domain at the N terminus of the D1 domain can insert into the catalytic site of the reciprocal D1 domain, resulting in joint occlusion of the active sites (Bilwes et al., 1996). However, crystallization of

LAR intracellular tandem phosphatase domains (D1 and D2) does not support this model of wedge-mediated catalytic inhibition. The LAR intracellular crystal structure indicates that the D2 domain prevents LAR dimerization via steric hinderance (Nam et al., 1999). It is likely that LAR family catalytic activity in the

D1 domain is inhibited by the reciprocal D2 domain; the PTPdelta D2 domain is capable of binding PTPsigma D1 and LAR D1, and reduces their catalytic activity by 50% (Wallace et al., 1998). However, whether such in vitro heterogeneous associations between LAR proteins confer biological function remains elusive.

Notably, LAR family members can undergo a cleavage event to yield two noncovalently associated subunits, the extracellular E-subunit and the transmembrane P-subunit. This proteolytic processing is mediated by Furin

protease and occurs at the extracellular penta-arginine site in the juxtamembrane region of the LAR family (Serra-Pages et al., 1994). Several lines of evidence suggest that alpha-secretase and gamma-secretase are responsible for receptor shedding (Haapasalo et al., 2007). Furthermore, these cleavage events release the LAR P-subunit from its membrane and allow its entry into the nucleus.

Increasing evidence demonstrates that nuclear LAR P-subunit retains phosphatase activity and can dephosphorylate its target substrate, beta-catenin, to regulate transcription activity. Nonetheless, the functional relevance of receptor shedding and nuclear LAR P-subunit has yet to be determined.

LAR signaling and synaptic plasticity

Synaptic adhesion molecules participate in the stabilization of initial contact between axons and dendrites. These heterophilic trans- interactions between pre- and postsynaptic adhesion molecules further induce bi-directional signaling to promote synapse maturation (Dalva et al., 2007; Shen and Scheiffele, 2010).

A growing body of evidence indicates LAR family proteins and NGL-3 (netrin-G ligand-3) mediate synaptic organization (Kwon et al., 2010; Woo et al., 2009).

NGL-3 encodes a transmembrane protein mainly localized to postsynaptic sites.

The extracellular region of NGL-3 comprises nine leucine-rich repeats that mediate interactions with LAR family proteins, while the cytoplasmic region of

NGL-3 binds to PSD-95, a scaffolding/adaptor protein required for excitatory synapse formation. Therefore, upon binding with LAR family proteins, PSD-95 may recruit and assemble a postsynaptic complex to NGL-3 for synaptic differentiation. In contrast, LAR promotes presynaptic differentiation via

interaction with Liprin-alpha. During this process Liprin-alpha functions as an adaptor protein, positioning various synaptic proteins such as ERC/ELKS, RIM and CASK, at the center of synaptic pathways to assemble active zones (Olsen et al., 2005; Stryker and Johnson, 2007).

Analogous to vertebrate excitatory synapse formation, Drosophila LAR acts through Liprin-alpha to conduct presynaptic differentiation. Genetic ablation of either LAR or Liprin-alpha causes synaptic abnormality at the Drosophila neuromuscular junction, including a reduction in synapse size and complexity as well as impairment of neurotransmission (Kaufmann et al., 2002). A number of experiments indicate that phosphatase activity is necessary for synapse development (Hofmeyer and Treisman, 2009; Johnson et al., 2006). For example, Drosophila Syndecan (Sdc) and Dallylike (Dlp), two physiologically relevant ligands of LAR, have opposing effects on LAR phosphatase activity upon ligand binding. LAR binding with Syndecan (Sdc) results in increased phosphatase activity and synaptic growth whereas inhibition of LAR phosphatase activity by Dlp promotes active zone morphogenesis. Thus, the phosphatase activity of LAR is thought to be tightly regulated in the maintenance of synaptic stability. Alterations in LAR activity might underlie changes in synaptic number and transmission, leading to an induction of synaptic plasticity. The substrate(s) and mechanisms accounting for two divergent developmental processes

(synaptic growth versus active zone morphogenesis) regulated by the same receptor are still elusive.

Notably, the LAR activity-dependent repression of active zone maturation in

Drosophila is not conserved in mammalian hippocampal neurons. In contrast to reduction of Drosophila LAR phosphatase activity promotes active zone maturation, overexpressing a LAR phosphatase-dead variant of D1 domain inhibits active zone morphogenesis and leads to 40-50% reduction in active zone number (Dunah et al., 2005).

Although sequence homology of LAR family proteins suggests functional redundancy, these three receptors function uniquely in some developmental processes. Recently, postsynaptic TrkC was shown to induce presynaptic differentiation via a neurotrophin-independent mechanism. Screening for corresponding presynaptic receptors using a TrkC ectodomain probe indentified

PTPsigma as a TrkC trans-synaptic binding partner. This TrkC-PTPsigma interaction demonstrates PTPsigma’s functional specificity in excitatory synaptic development among LAR family members as TrkC is unable to bind to LAR or

PTPdelta (Takahashi et al., 2011).

LAR signaling and axon guidance

The process of axon navigation begins with a set of guidance receptors recognizing and binding to various guidance cues. Such receptor engagement is known to induce signaling cascades inside growth cones, which ultimately leads to axonal extension, orientation, or retraction. The extracellular features of the

LAR subfamily of RPTPs are consistent with those of cell adhesion molecules, suggesting that they participate in axon-axon and axon-ECM adhesion during

axon navigation. An extensive effort has been made to identify ligands for LAR family proteins. Surprisingly, several physiological ligands are found to be sugar chains. For example, using a receptor affinity probe combined with biochemical analyses, heparan sulphate (HS) chains on HSPGs were identified to associate with the first Ig-like domain of LAR family receptors (Aricescu et al., 2002; Fox and Zinn, 2005). Numerous studies in different model organisms have documented the role of HSPGs in axon outgrowth and targeting (Inatani et al.,

2003; Irie et al., 2002; Johnson et al., 2004). For example, mice lacking HS glycosyltransferase in the central nervous system exhibit servere neural defects, including misprojection of commissural axons in the forebrain. Given that the

LAR family and HSPGs share overlapping expression patterns in the developing nervous system and they exhibit physical interactions, HSPGs may potentially function as a key class of guidance cues to LAR family proteins. In addition, the laminin-nidogen complex, which functions in stimulating axon outgrowth, interacts in a GST-pull down assay with LAR via the FNIII domain 5 (FN5)

(O'Grady et al., 1998), raising a possibility that LAR-laminin binding is permissive for axon growth.

How LAR family proteins regulate growth cone behavior remains elusive.

However, it appears that Rho family GTPases signal downstream of LAR family members to alter actin dynamics. Trio, another LAR binding partner, may provide such a molecular link between LAR family proteins and Rho GTPases (Debant et al., 1996). Trio consists of two Rho GEF domains that activate Rac1 and RhoA respectively. Several studies have together established roles for Rac1 and RhoA

in growth cone motility. In general, Rac1 promotes axon growth whereas RhoA functions to inhibit axon growth. It is likely that Trio, once recruited to the LAR family proteins, can differentially activate Rac1 or RhoA dependent on receptor state. In support of this notion, Dlar and rac1 have synergistic genetic interactions and their defective mutants exhibit similar motor axon guidance defects in Drosophila (Kaufmann et al., 1998).

In addition, several cytoskeleton regulators, including Abl and Ena, have been found to physically interact with LAR family proteins and regulate axon targeting.

Abl is a non-receptor tyrosine kinase and its catalytic activity is crucial for the control of cytoskeleton and cell migration. Many lines of evidence indicate that activated Abl is localized to focal adhesions and membrane ruffles, where it phosphorylates substrates that regulate actin dynamics (Colicelli, 2010;

Woodring et al., 2003). For example, Michael et al. demonstrated that tyrosine phosphorylation of Lamellipodin (Lpd), a member of the MRL family of Ras effector proteins, by Abl kinase leads to the recruitment of Ena/VASP at the cell’s leading edge. Because Ena/VASP is capable of inducing elongation of actin filaments, the formation of Lpd-Ena/VASP complex by Abl might be a general regulatory mechanism for membrane protrusions in different cell types (Michael et al., 2010).

It has been proposed that LAR and Abl kinase control motor axon guidance in a phosphorylation-dependent manner; the phosphorylation state of Abl and LAR substrate(s) may result in different signal complexes and distinct growth cone responses. Drosophila Ena/VASP has been characterized as a common

substrate of Abl and LAR; its phosphorylation is reciprocally regulated by Abl and

LAR activity (Wills et al., 1999a). Biochemical analysis has revealed six tyrosine residues of Ena can undergo Abl-dependent phosphorylation (Gertler et al.,

1995). Surprisingly, mutation of these tyrosine residues does not affect Ena’s function because the non-phosphorylated Ena variant is still capable of rescuing axon guidance defects in ena LOF mutants. This suggests that Abl-dependent phosphorylation may occur on additional unknown substrate(s).

LAR signaling and axonal regeneration

Injured axons in the adult mammalian central nervous system (CNS) fail to regenerate. The lack of CNS axon regeneration is thought to result from extracellular molecules limiting axon growth and intrinsic barriers limiting regeneration capacity. Several extracellular molecules including myelin- associated glycoprotein (MAG), NOGO, oligodendrocyte myelin glycoprotein

(OMgp), and chondroitin sulfate proteoglycans (CSPGs) are enriched in CNS lesions and thought to act through cognate receptors to arrest growth cone advance (Giger et al., 2010; Silver and Miller, 2004).

CSPGs are composed of a core protein that is decorated with chondroitin sulfate sugar modifications. Numerous studies have implicated the chondroitin sulfate side chains in inhibition of axon growth. Enzymatic removal of chondroitin sulfate side chains from the core protein by chondroitinase ABC (ChABC) promotes axonal regeneration and functional recovery after spinal cord injury (Bradbury et

al., 2002). Due to the complexity of these sugar side chains, the existence of

specific neuronal CSPG receptors has been debated for decades.

Recently, Shen et al. identified PTPsigma as the first known receptor for CSPGs

(Shen et al., 2009). Deletion of PTPsigma allows modest regeneration of axons into a CSPG-rich injury site, suggesting that CSPGs might act partially through axonal PTPsigma. Biochemical assays demonstrate that the first Ig-like domain

of PTPsigma is responsible for its interaction with the negatively charged CS

moiety of CSPGs. When a cluster of lysine residues (K-K-X-K-K) in the first Ig-

like domain is deleted from PTPsigma, or when chondroitinase ABC is applied to

CSPGs, PTPsigma fails to bind CSPGs. Given that all LAR family members

share the consensus binding sequence for chondroitin sulfate, it is possible that

both LAR and PTPdelta also engage CSPGs and limit axon regeneration after

injury.

How CSPGs initiate growth collapse through PTPsigma is unknown. It remains to

be understood whether phosphatase activity or alternative intracellular signaling

of PTPsigma is necessary for transduction of the initial binding event. Several

lines of evidence suggest PKC and Rho mediate cytoskeleton instability in

response to CSPG binding (Monnier et al., 2003; Sivasankaran et al., 2004).

Interestingly, axon growth inhibition by CSPGs can be reduced by introducing

either PKC or RhoA inhibitors. Because Trio is a LAR binding protein and

contains RhoGEF activities, it is possible that Trio plays a pivotal role in this

context by activating RhoA.

Physiological functions of LAR

Five RPTPs (DLAR, PTP19D, PTP69D, PTP99A, and PTP52F) are expressed in the developing Drosophila nervous system. Phenotypic characterization of these neural RPTPs reveals their involvement in choice point decisions and in synapse maturation. In triple mutant embryos lacking DLAR, PTP69D and PTP99A, ISNb axons stall at exit junctions of the ISN, indicating that these RPTPs are required for defasciculation at early choice points (Desai et al., 1997). Both DLAR and

PTP69D are important for the entry of ISNb axons into ventrolateral muscles. In both Dlar and ptp69D LOF mutants, motor axons exhibit a bypass phenotype where ISNb axons fail to defasciculate from the ISN and innervate their target muscles (Desai et al., 1996; Krueger et al., 1996). Despite their similar structural homologies, genetic studies on compound mutants of Dlar, ptp69A and ptp99A indicate that these RPTPs control axon targeting in both synergistic and antagonistic manners. For example, whereas ptp69D mutations augment the

ISNb bypass phenotype of Dlar mutants, loss of ptp99A restores the axonal abnormality in Dlar mutants. These results support the idea that a combination of these RPTPs confers the specificity of growth cone decision-making at each choice point.

Phosphorylation signaling is known to play a crucial role in regulating growth cone behavior. Genetic studies in Drosophila have shown that overexpression of abl causes an ISNb bypass phenotype similar to that found in Dlar LOF mutants.

In addition, half reduction of abl genetic dosage rescues Dlar LOF mutant phenotype (Wills et al., 1999a). These data suggest that Abl kinase and DLAR

steer ISNb axons to their muscle targets via reciprocal mechanisms.

Interestingly, although the enzyme activity of LAR is essential for motor axon

guidance, it is dispensable for axon targeting in the Drosophila visual system

(Hofmeyer and Treisman, 2009). Thus, it is very likely LAR employs distinct signaling mechanisms to control different axon projections. A comprehensive understanding of LAR signaling awaits the identification of LAR’s substrates and binding partners.

In mammals, LAR family members exhibit overlapping, but not identical, expression patterns in the developing nervous system (Chagnon et al., 2004).

Briefly, LAR has broader expression whereas PTPdelta and PTPsigma have expression restricted to cell types of the CNS and PNS, including the cerebral cortex and motor pools within the spinal cord. Both PTPsigma and PTPdelta deficient mice exhibit striking developmental disorders including motor dysfunction as well as spatial learning impairment (Elchebly et al., 1999; Uetani et al., 2000; Wallace et al., 1999). In contrast, LAR knockout mice have only minor neural defects in cholinergic innervation of the dentate gyrus (Van Lieshout et al., 2001; Yeo et al., 1997). Because LAR family members have high sequence homology and overlapping expression patterns, they often function

redundantly. For example, while Dlar deletion causes motor axon guidance

defects in Drosophila, mice lacking either PTPdelta or PTPsigma do not exhibit

gross defects in axon targeting. However, PTPdelta and PTPsigma double

knockout mice exhibit a severe loss of motor neurons and aberrant axonal

innervation of the diaphragm, indicating their compensatory roles during neural

development (Stepanek et al., 2005; Uetani et al., 2006).

The analysis of LAR family mutants in injury models demonstrates their importance in axon regeneration. For example, LAR deletion reduces axon growth rate after sciatic nerve lesion (Van der Zee et al., 2003; Xie et al., 2001).

Conversely, PTPsigma is inhibitory to axon regeneration. Studies have shown

that PTPsigma deletion enhances axon regeneration after optic nerve injury,

sciatic nerve injury, and injury to the dorsal columns of the spinal cord (McLean

et al., 2002; Sapieha et al., 2005; Shen et al., 2009). Thus, the ability of LAR and

PTPsigma to convey divergent effects warrants further study. While nothing is

known about the role of PTPdelta in regeneration, structural and sequence

homology to LAR and PTPsigma point to a functional importance.

Aims and significance

In my thesis work I sought to understand how LAR signaling promotes

cytoskeletal rearrangement. Previous studies have shown that Pak (p21-

activated kinase) is a downstream effector of Rac GTPase, and Rac/Pak

signaling contributes to cytoskeletal remodeling required for cell motility and axon

pathfinding (Kreis and Barnier, 2009). However, it remains elusive whether Rac

acts primarily through Pak to regulate LAR-dependent target recognition.

Tyrosine phosphorylation signaling acts as a regulatory mechanism antagonizing

LAR function. As discussed above, several lines of evidence suggest that the

phosphorylation states of a common substrate(s) catalyzed by Abl and LAR

contribute to different axonal responses. In this hypothesis, Ena was recognized as a potential target; the phosphorylation of Ena is reciprocally regulated by Abl and LAR, and Ena is required for motor axon guidance. Nonetheless, we cannot exclude the possibility that other substrates exist.

Our identification of Caskin provides novel insight into LAR-mediated axon guidance. We found that Caskin is capable of serving as a molecular link between LAR and Dock/Nck in vitro. As Dock/Nck is known to interact with Pak, we were surprised to find that dock and pak do not exhibit genetic interaction with caskin in motor axon guidance. This suggests that Dock/ Pak complex does not participate in LAR signaling, and alternatively, suggests that Rac1 acts through other cytoskeletal regulators.

In addition, I found that Caskin is tyrosine phosphorylated in the presence of Abl and that Abl and Caskin exhibit genetic interactions in motor axon guidance. I propose that Abl-dependent phosphorylation is likely to abolish interactions between Caskin and its downstream effector(s), leading to breakdown of the signaling relay. Further analysis of how Caskin’s phosphorylation code regulates function may allow us to develop phosphomimetic or dominant-negative Caskin variants that alter LAR signaling in vivo.

Figure 1.1

Figure 1.1: Motor axon projections in Drosophila embryo

Motor axons exit the ventral nerve cord (VNC, bottom) through the intersegmental nerve (ISN) and the segmental nerve (SN) roots. These axons further branch into smaller fascicles and innervate their muscle targets. The main

ISN divides into ISN (purple), ISNb (blue), and ISNd (green), while the SN separates into SNa (red) and SNc (black). RP motor neurons (RP1,3,4,5) project axons that innervate with the 6, 7, 12 and 13 muscle clefts.

Figure 1.2

Figure 1.2: Schematic diagrams of motor axon guidance phenotypes

ISNb axons normally defasciculate from the main ISN nerve and enter the ventral muscle field. Alteration of axonal adhesivity or defasciculation signals causes motor axon bypass phenotype in which ISNb axons remain in the common ISN pathway and project to the dorsal muscle field.

CHAPTER 2

THE CYTOPLASMIC ADAPTOR PROTEIN CASKIN MEDIATES LAR SIGNAL TRANSDUCTION DURING DROSOPHILA MOTOR AXON GUIDANCE

Abstract

The multiprotein complexes that receive and transmit axon pathfinding cues

during development are essential to circuit generation. Here, we identify and

characterize the Drosophila sterile α-motif (SAM) domain-containing protein

Caskin, which shares homology with vertebrate Caskin, a CASK

(calcium/calmodulin-dependent serine protein kinase)-interacting protein.

Drosophila caskin (ckn) is necessary for embryonic motor axon pathfinding and interacts genetically and physically with the leukocyte common antigen-related

(LAR) receptor protein tyrosine phosphatase. In vivo and in vitro analyses of a panel of ckn loss-of-function alleles indicate that the N-terminal SAM domain of

Ckn mediates its interaction with LAR. Similarly, Liprin-α is a neuronal adaptor protein that interacts with LAR via a SAM domain-mediated interaction. We present evidence that LAR does not bind Caskin and Liprin-α concurrently, suggesting they may assemble functionally distinct signaling complexes on LAR.

Furthermore, a vertebrate Caskin homolog interacts with LAR family members, arguing that the role of ckn in LAR signal transduction is evolutionarily conserved. Last, we characterize several ckn mutants that retain LAR binding yet display guidance defects, implying the existence of additional Ckn binding partners. Indeed, we identify the SH2/SH3 adaptor protein Dock as a second

Caskin-binding protein and find that Caskin binds LAR and Dock through distinct domains. Furthermore, whereas ckn has a nonredundant function in LAR- dependent signaling during motor axon targeting, ckn and dock have mostly

overlapping roles in axon outgrowth in the CNS. Together, these studies identify

caskin as a neuronal adaptor protein required for axon growth and guidance.

Introduction

The Drosophila neuromuscular system is an excellent paradigm to decipher the

molecular signals orchestrating the precise matching between individual

motorneurons and their muscle partners. A number of guidance cues and

receptors coordinately regulate motor axon pathfinding assuring the high fidelity

of this process. The axon must integrate these disparate signals as it navigates

through its environment. Multidomain adaptor proteins promote such integration

since they serve as platforms to facilitate communication between signal

transduction cascades. The leukocyte common antigen-related (LAR)-related subfamily of receptor protein tyrosine phosphatases (RPTPs type IIA) are conserved regulators of axon pathfinding and synaptogenesis. This subfamily includes the Drosophila receptors LAR and PTP69D, and the vertebrate receptors LAR, protein tyrosine phosphatase σ (PTPσ), and PTPδ (Ensslen-Craig and Brady-Kalnay, 2004; Johnson and Van Vactor, 2003). Family members contain three Ig-like domains and a variable number of fibronectin-III (FNIII) domains extracellularly, and two intracellular phosphatase domains. The membrane-proximal D1 phosphatase domain (D1) confers most if not all of the catalytic activity of the receptor, whereas the membrane-distal D2 domain is catalytically inactive and may contribute to LAR family function via interaction with downstream signaling components. These receptors exhibit neuronal expression patterns, and loss-of-function (LOF) mutants display defects in axon

targeting and synapse formation (Desai et al., 1996; Kaufmann et al., 2002;

Krueger et al., 1996). Heparin sulfate proteoglycans (HSPGs) are binding partners of LAR family members in axon pathfinding and synaptogenesis (Fox and Zinn, 2005; Johnson et al., 2006). In vertebrates, PTPσ is a neuronal receptor for chondroitin sulfate proteoglycan (CSPG) and inhibits axon regeneration after CNS injury (Fry et al., 2010; Shen et al., 2009). On the intracellular side, LAR activity in some contexts requires phosphatase activity, whereas in other contexts its function is independent of catalytic activity

(Hofmeyer and Treisman, 2009; Johnson et al., 2006; Prakash et al., 2009), suggesting a diversity of downstream signaling pathways. Indeed, a number of

LAR-interacting proteins have been identified (Bateman et al., 2000; Ensslen-

Craig and Brady-Kalnay, 2004; Johnson and Van Vactor, 2003). LAR function in synaptic maturation requires Liprin-α, a sterile α-motif (SAM) domain-containing adaptor protein that interacts with LAR in vertebrates and invertebrates

(Kaufmann et al., 2002; Serra-Pages et al., 1995; Serra-Pages et al., 1998).

Drosophila Dock and vertebrate Nck are neuronal SH2/SH3 containing adaptor proteins that link guidance receptors to cytoskeletal remodeling (Li et al., 2001).

Given the widespread expression of Dock in the embryonic CNS and its central position linking guidance receptors to the actin cytoskeleton, it is notable that motor axons in dock LOF mutant embryos display only subtle defects (Desai et al., 1999), raising the possibility of compensation or redundancy. Dock/Nck interact directly with a number of receptors, including Robo, DSCAM (Down syndrome cell adhesion molecule), and the insulin receptor (Fan et al., 2003;

Schmucker et al., 2000; Song et al., 2003) and bind directly to cytoskeletal

effectors such as p21-activated protein kinase (Pak) and WASP (Wiskott–Aldrich

syndrome protein), thereby presumably linking receptor activation to cytoskeletal

rearrangement (Hing et al., 1999; Rivero-Lezcano et al., 1995). Of particular relevance, Caskin has been identified as a potential Nck interactor (Balazs et al.,

2009; Fawcett et al., 2007). Caskin was first identified as a novel protein binding the CaM kinase domain of CASK (Tabuchi et al., 2002). Vertebrate Caskins are

predicted scaffolding proteins with multiple ankyrin repeats, an SH3 domain, and

two SAM domains, suggesting that Caskin is a component of a multiprotein

complex. Here, we present genetic, cell-biological, and biochemical evidence

arguing that Caskin is a LAR-binding partner and is required for LAR signal

transduction in Drosophila motor axon guidance.

Materials and Methods

Ethyl methanesulfonate reversion mutagenesis

yw; GS1205 males were mutagenized with 22 mM ethyl methanesulfonate (EMS)

and crossed to homozygous yw;; elavGAL4 virgins. As ckn overexpression

causes embryonic lethality, we selected viable F1 individuals, which may contain

mutations in the ckn locus. The revertants were balanced over CyO and

segregated from the elavGAL4 driver. The ckn genomic coding region was PCR

amplified and sequenced in each mutant. In GS1205-A, C, K, and Y mutants, ckn

cDNA was amplified by reverse transcription–PCR and cloned into pGEM-T Easy

vector (Promega) for DNA sequencing and construct generation.

Immunohistochemistry

Embryo fixation, antibody staining, and RNA in situ hybridization procedures

were performed as described (Miller et al., 2008). The following primary

antibodies were used: monoclonal antibody (mAb) ID4 (anti-Fasciclin II; 1:10;

Developmental Studies Hybridoma Bank), mAb BP102 (1:50; Developmental

Studies Hybridoma Bank), and mAb anti-β-gal (1:500; Promega). For RNA in situ

hybridization, digoxigenin (DIG)-labeled RNA probe was made by Roche DIG

RNA Labeling kit (SP6/T7) using full-length caskin cDNA as template. For S2R+

cell immunofluorescence, protein expression constructs were generated in

Gateway-based vectors (Drosophila Gateway Collection, Carnegie Institution of

Washington, Baltimore, MD) and transiently transfected into cells by FuGENE

HD (Roche). After 48 h transfection, cells were fixed with 4% formaldehyde/PBS

for 15 min, washed three times with PBS, and permeabilized in blocking buffer

(3% BSA, 0.1% Triton X-100 in PBS) for 30 min at room temperature. Cells were

incubated with primary antibodies: rat anti-hemagglutinin (HA) (3F10; Roche) at

1:500, mouse anti-Flag (Sigma-Aldrich) at 1:1000, and rabbit anti-green fluorescent protein (GFP) (Invitrogen) at 1:500 for 2 h at room temperature or

4°C overnight. Appropriate Alexa Fluor secondary antibodies (1:500) (Invitrogen) were applied for 1 h at room temperature.

Microscopy and data analysis

Embryos were filleted in 70% glycerol using a Leica MZ9 dissecting microscope

and analyzed on a Zeiss Axioplan 2 microscope with a 63X oil-immersion

objective. Images were captured on an AxioCam MRc camera. Brightness and contrast were adjusted in Adobe Photoshop. Fluorescence images were obtained on a Zeiss Axio Imager.ZI confocal microscope. Statistical analyses were performed using Fisher’s exact test.

Plasmids

Full-length cDNA clones for liprin-α (LD33094), abl (GH109917), caskin

(LD09801), Lar (LD45391), and ena (SD08336) were obtained from the

Drosophila Genomics Resource Center. Mouse caskin 2 (clone ID 5704642), ptprD (clone ID 4223353), ptprF (clone ID 6409440), and ptprS (clone ID

6834684) expressed sequence tag clones were purchased from Open

Biosystems. pDEST-GBKT7 and pDEST-GADT7 (Rossignol et al., 2007) for yeast two-hybrid assays were obtained from Arabidopsis Biological Resource

Center DNA Stock Center. pRS426GPD (Alberti et al., 2007) from Addgene was used for yeast three-hybrid assay. Dock and its truncated forms fused to LexA

(Fan et al., 2003) are from Greg Bashaw (The University of Pennsylvania,

Philadelphia, PA). To generate UAS transgenic flies, corresponding cDNA was amplified from transcription start to stop by PCR and subcloned into pUAST or pTHW vectors (HA-tagged; Carnegie Institution of Washington) by the Gateway system (Invitrogen).

For yeast two-hybrid and three-hybrid assays, GAL4BD constructs (bait) were made in pGBKT7 or in pDEST-GBKT7. Similarly, GAL4AD constructs (prey) were generated in either pACT2 or pDEST-GADT7. The bridge constructs were

generated by adding nuclear localization sequence (MPKKKRK) to the N termini

of Ckn or LAR and subcloned into pAG426GPD vector. For glutathione S-

transferase (GST) and Ni-NTA pull-down experiments, PCR fragments of Lar-

cyto (1442–2029 aa), Lar D1 (1442–1728 aa), Lar D2 (1729–2029 aa), and full-

length Dock were cloned into the pGEX vectors to express GST fusion protein.

Full-length Caskin was cloned into pET29a vector to generate His-tagged fusion

protein.

Yeast two-hybrid and three-hybrid assay

Yeast transformation was performed by the lithium acetate method. Interactions

were scored after 3 d at 30°C. In yeast two-hybrid assays, bait constructs

(pGBKT7 or pDEST-GBKT7 derivatives) were transformed into strain PJ694a

whereas prey constructs (pACT2 or pGADT7-DEST derivatives) were transformed into strain SL3004. Drop tests were performed by plating 5 ul of serial dilutions of mid-log-phase cultures on selective plates. For domain mapping experiments, Dock bait constructs were introduced into yeast strain

PJ694a. Caskin GAL4AD and pSH18–34, a LexA-responsive LacZ reporter,

were introduced into yeast strain SL3004 by cotransformation. GST and Ni-NTA

pull-down assay. GST and His-tagged fusion proteins were expressed in

Escherichia coli and purified on glutathione-Sepharose and Ni-NTA agarose

beads, respectively. In the GST pull-down assay, HA-Caskin overexpression

embryos were homogenized on ice in embryo lysis buffer for 15 min. The lysates

were centrifuged at 15,000 X g for 20 min and the supernatant was incubated

overnight at 4°C with 5 µg of GST-bound or GST-fusion protein-bound

glutathione-Sepharose beads. Beads were washed three times with ice-cold embryo lysis buffer and boiled in 2X sample buffer. Protein samples were resolved by SDS polyacrylamide gel and subjected to Western blot analysis using anti-HA antibody (12CA5; Roche). In Ni-NTA pull-down assays, 5 ug of

His-tagged Caskin fusion protein immobilized on Ni-NTA agarose beads was incubated with GST-Lar in binding buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris, pH 8.0, 30 mM imidazole) at 4°C for 2 h. Beads were washed three times with cold binding buffer and subjected to Western blot analysis using anti-

GST antibody (GE Healthcare).

Immunoprecipitation

Drosophila S2 cells were transiently transfected with the indicated expression plasmids by FuGENE HD (Roche) and harvested after 48 h. Cells were lysed in immunoprecipitation (IP) buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1%

Triton X-100, and 1X protease inhibitor (Roche)) on ice for 15 min and cleared by centrifugation. Lysates were then incubated with anti-FLAG M2 mAb (Sigma-

Aldrich) prebound to protein G beads for 2 h at 4°C. Beads were washed three times with cold IP buffer. Proteins were eluted in 2X sample buffer and subjected to SDS-PAGE.

Results

Neuronal CG12424 overexpression alters CNS and motor projections

To identify genes required for neuronal development, we took advantage of the

GAL4/UAS misexpression system and screened a collection of UAS-containing

P-element lines for elavGAL4-dependent defects in CNS fate or axon guidance

(Viquez et al., 2006; Miller et al., 2008). Axon development in these lines was

characterized with 1D4 antibody, which labels a subset of ipsilateral axons within

the CNS and all motor axon projections (Van Vactor et al., 1993). We identified

one line, GS1205, in which 1D4-positive axons inappropriately cross the CNS

midline (data not shown). Inverse PCR analysis indicated that the line contains a

P-element inserted in an untranslated 5’ exon of CG12424. To determine

whether CG12424 overexpression causes the observed midline phenotype, we

generated UAS-CG12424 transgenic lines. In elavGAL4/UAS-CG12424

embryos, 1D4-positive axons frequently cross and circle the midline (Fig. 2.1B,

arrows). These observations demonstrate that CG12424 overexpression

interferes with midline repulsion. We next evaluated motor axon outgrowth in

elav>CG12424 embryos. We focused this analysis on the “b” branch of the

intersegmental nerve (ISN), which innervates four ventrolateral muscles in the

embryo and larva. In wild type, ISNb-extending axons defasciculate from the

primary ISN root at a ventral choice point called the exit junction, enter their

muscle field, and grow dorsally to their stereotyped targets (Fig. 2.1C). In

elav>CG12424 embryos, ISNb axons frequently stall in the ventrolateral muscle

field and fail to innervate their targets (Table 2.1). Given the severity of the

midline defect, however, this phenotype was difficult to interpret. To evaluate

whether overexpression of CG12424 more specifically alters motor axon

behavior we characterized motor projections in Hb9GAL4/UAS-CG12424 embryos,

as Hb9-GAL4 is expressed in ventrally and laterally projecting motorneurons and

significant midline defects are not observed when misexpressing CG12424 with

this driver (Broihier and Skeath, 2002). Hb9Gal4/UAS-CG12424 embryos also

exhibit ISNb pathfinding errors. In 55% (n =198) of hemisegments, ISNb axons

stall in the ventrolateral muscle field (Fig. 2.1D). These experiments demonstrate that overexpression of CG12424 disrupts axon guidance in two distinct contexts, suggesting that it interacts with guidance machinery.

CG12424 RNA localizes to the embryonic CNS neuropil

To determine whether endogenous CG12424 regulates axon behavior, we

characterized its expression pattern. In wild type embryos, CG12424 RNA

expression appears mostly restricted to the CNS axon scaffold (Fig. 2.1E). We

do not observe appreciable embryonic CG12424 expression outside the CNS.

This expression pattern is observed with an antisense CG12424 RNA probe, but

not with a sense probe, suggesting the signal is specific. As an additional test of

specificity, we evaluated CG12424 RNA expression in embryos homozygous for

a deficiency of the CG12424 locus, Df(2R)BSC330. Neuropil expression of

CG12424 RNA is not observed in these embryos (Fig. 2.1I), demonstrating that

the signal highlights CG12424 RNA. To verify that the observed expression is

axonal, embryos were colabeled with CG12424 RNA and BP102, a monoclonal

antibody recognizing all CNS axons. This experiment confirms that CG12424

RNA is expressed in longitudinal and commissural axons at stage 16 (Fig. 2.1F–

H). The presence of CG12424 RNA in neuronal processes rather than cell bodies suggests it is actively localized, consistent with a role for CG12424 in axon guidance.

caskin gene structure and allele generation

Sequence analysis indicates that CG12424 codes for a predicted cytoplasmic

adaptor protein containing two SAM domains and an EVH1-binding motif (Fig.

2.2A). The SAM domains in CG12424 have a high degree of sequence homology

to the SAM domains in vertebrate Caskin family members (Fig. 2.2A). Caskin, or

CASK-interacting protein, is a neuron-specific protein identified from rat brain

extract as a binding partner of the synaptic calcium/calmodulin-dependent serine

kinase CASK (Tabuchiet al., 2002). CG12424 also contains a highly conserved

20 aa sequence at its C terminus present in vertebrate Caskins (Fig. 2.2A, CTD).

In addition to two SAM domains, vertebrate Caskin homologs contain N-terminal

ankyrin repeats and an SH3 domain not present in CG12424. A BLAST search of

the Drosophila genome using the ankyrin repeats of vertebrate Caskin identifies

CG4393 as the most closely related fly gene to the N-terminal sequences of

vertebrate Caskin. To determine whether CG4393 may be functionally related to

vertebrate Caskin, we assayed embryonic CG4393 expression by in situ

hybridization. We do not detect expression of CG4393 in the embryonic CNS

(data not shown), indicating that CG4393 is unlikely to contribute to CNS

development. Based on sequence analysis and shared binding partners of

vertebrate Caskins and CG12424 (see below), we have named CG12424, caskin

(ckn).

The overexpression phenotype, RNA expression profile, and sequence homology

of ckn suggest that it participates in neuronal development or function. To test

this hypothesis, we generated eight EMS-induced LOF alleles via reversion of

lethality associated with elavGAL4-dependent ckn overexpression. All ckn

alleles, including null alleles, are homozygous viable. Allele sequencing revealed

mutations within coding sequence in all alleles (Fig. 2.2B). Notably, the two

missense mutations fall in conserved domains- cknA contains an arginine-to-

glutamine substitution in the first SAM domain and cknY has an alanine-to-valine substitution in the C-terminal domain (CTD) (Fig. 2.2C). These alleles strongly

suggest these conserved sequences are required for ckn function in vivo.

caskin LOF alleles disrupt ISNb motor axon pathfinding

With alleles in hand, we investigated whether ckn homozygous mutant embryos

exhibit defects in CNS development. After verifying that the parental GS1205 P-

element line does not display appreciable CNS or motor axon guidance errors

(Table 2.1), we asked whether the overall axonal architecture of the CNS

neuropil is disrupted in ckn mutants. Overt defects in the axonal scaffold are not

observed using BP102 (data not shown). In contrast, ckn mutant embryos display

consistent motor axon projection defects, exhibiting classic ISNb “bypass”

phenotypes, where ISNb axons fail to innervate the ventrolateral muscle (VLM)

field (Fig. 2.3B, D, F, Table 2.1). ISNb-projecting axons in ckn mutants appear to

defasciculate normally from the primary ISN branch at the exit junction, but fail to

enter the ventral muscle field, instead bypassing their targets as they extend

parallel to the primary ISN fascicle. Frequently, mistargeted ISNb axons reach

back from the dorsal edge of muscle 12 to innervate the VLM field (Fig. 2.3C,

arrows). In the majority of affected nerves, ISNb axons are visible as a distinct

fascicle next to the ISN (“parallel bypass”) (Fig. 2.3B, arrow). Both weaker and

stronger ISNb bypass phenotypes are also observed. “Partial parallel bypass”

was scored when a subset of ISNb axons correctly enters the muscle field,

whereas “fusion bypass” was scored if ISNb axons are tightly bundled with ISN

axons and cannot be distinguished at the light-microscope level (Fig. 2.3D,

arrow; Table 2.1). These phenotypes demonstrate that ckn participates in ISNb

pathfinding, perhaps by contributing to a signaling pathway(s) mediating ISNb

target recognition. We quantified the guidance errors in ckn homozygous LOF

embryos and in embryos carrying each allele over Df(2R)BSC330, a small

deficiency for the region (Table 2.1). All alleles display ISNb bypass phenotypes

over the deficiency, indicating that the molecular lesions identified in the ckn

locus are responsible for the observed phenotypes. Three alleles (cknK, cknD, and cknF) behave as genetic nulls as the bypass frequency does not differ

between homozygotes and allele/Df ( p < 0.05 for each) (Table 2.1). Null alleles

display significant ISNb bypass phenotypes—35% of axons exhibit bypass in

cknD homozygotes, 39% in cknF, and 41% in cknK. Interestingly, cknL codes for

an early nonsense mutation yet retains some activity (Table 2.1, Fig. 2.2B), suggesting there is read-through of the mutation or that an alternative

downstream start methionine is used. The penetrance and nature of ISNb bypass phenotypes in null ckn alleles is similar to that of Lar mutants (Desai et al., 1997;

Krueger et al., 1996). This demonstrates that ckn is an important regulator of

ISNb guidance and raises the possibility that ckn functions with Lar. As the ckn alleles were generated on a chromosome with a UAS-containing GS P-element immediately upstream of the coding region, we tested whether GAL4-dependent neuronal overexpression of mutant proteins interferes with axon pathfinding. As discussed above, overexpression of wild-type Ckn leads to ISNb stall in the ventrolateral muscle field but does not give bypass phenotypes (Table 2.1). We analyzed motor axon behavior in embryos with neuronal overexpression of four missense mutants: Ckn-A, Ckn-C, Ckn-K, and Ckn-Y. Interestingly, overexpression of these mutants has quite different effects on motor axon guidance. Neuronal overexpression of either Ckn-C or Ckn-Y drives ISNb bypass

(Table 2.1). Ckn-C is particularly potent, as overexpression gives 61% ISNb bypass (Fig. 2.3E), suggesting that Ckn-C blocks the function of protein(s) essential for ISNb pathfinding. In contrast, neither of the N-terminal SAM domain mutants, Ckn-A or Ckn-K, interferes with ISNb guidance when overexpressed, suggesting that an intact SAM domain is required for the Ckn overexpression

ISNb stall phenotypes. Furthermore, this analysis demonstrates that Ckn-K, the

LOF allele giving the highest frequency of ISNb bypass, does not have dominant- negative like activity and is a functional null. caskin and Lar interact genetically

Lar LOF mutants display ISNb bypass phenotypes similar to those displayed by

ckn LOF alleles (Table 2.2, Fig. 2.4B, and C) (Desai et al., 1997; Krueger et al.,

1996). We tested for dose-dependent genetic interactions between Lar and ckn

as genes acting in common molecular pathways often interact genetically (Fan et

al., 2003; Wills et al., 1999a). For this analysis, the Lar5.5 and Lar13.2 alleles were

used, both of which contain nonsense mutations in the extracellular domain and

are presumed nulls (Fig. 2.4C) (Krueger et al., 1996). Neither ckn nor Lar heterozygous embryos have ISNb guidance defects, whereas Lar5.5 cknK double

heterozygotes display bypass phenotypes with 39% penetrance (Fig. 2.4D, Table

2.2), which is similar to the severity observed when either gene alone is

homozygous mutant (Table 2.2, Fig. 2.4B, C). We next asked whether embryos

homozygous mutant for both cknK and Lar5.5 have an increased frequency of

pathfinding errors. Previous genetic analysis indicates that maternal Lar product

contributes to ISNb pathfinding (Fox and Zinn, 2005). Thus, if ckn and Lar

function in a common pathway, loss of ckn might compromise residual Lar

activity present in Lar zygotic mutants. Consistently, we observe increased

expressivity and penetrance of ISNb bypass when we remove ckn function in Lar

zygotic LOF embryos. Whereas Lar5.5 zygotic mutants have an ISNb bypass

frequency of 31%, in Lar5.5 cknK embryos the frequency of ISNb bypass rises to

49% (Table 2.2, Fig. 2.4E). This interaction is not allele specific as it is also observed in Lar13.2 cknA double homozygotes (Table 2.2). The enhanced bypass

phenotypes observed in Lar ckn double-homozygous embryos are consistent with genetic dependence of maternal Lar product on ckn function. However, ckn

may also participate in a parallel genetic pathway(s) that contributes to the

increased bypass frequency observed in Lar ckn embryos. Together, the

phenotypic similarity between ckn and Lar LOF alleles, as well as dominant

genetic interactions, suggest ckn and Lar may operate in a common signaling

pathway.

The N-terminal SAM domain of Caskin binds Lar

To test for a physical interaction between Ckn and Lar, we assayed whether they

interact in a yeast two-hybrid assay. Both full-length Ckn (Ckn-full) and an N- terminal fragment including both SAM domains (Ckn-SAM) interact with the Lar cytoplasmic domain (Lar-cyto) containing both D1 and D2 phosphatase domains

(Fig. 2.5A). The Drosophila genome contains two type IIA RPTPs—Lar and

PTP69D (Johnson and Van Vactor, 2003). Whereas PTP69D single mutant embryos have subtle ISNb phenotypes, the role of PTP69D in ISNb guidance is uncovered unambiguously in Lar; PTP69D double-mutant embryos, which display levels of ISNb bypass significantly elevated over that seen in Lar single mutants (Desai et al., 1996; Desai et al., 1997). The intracellular domains of Lar and PTP69D share extensive sequence similarities, raising the possibility that

Ckn binds PTP69D. However, ckn and PTP69D do not interact in the yeast two- hybrid assay (Fig. 2.5A), and we do not observe dominant genetic interactions between ckn and PTP69D alleles (Table 2.2). Interestingly, the penetrance of

ISNb bypass in ckn; PTP69D double homozygotes is not elevated over that observed in ckn homozygotes (Table 2.2). These data raise the possibility that ckn plays a specific role in the Lar signaling pathway. The physical interaction

between Ckn and Lar suggests that the pathfinding phenotype in ckn LOF mutant

embryos results from defective Lar signal transduction. Thus, we asked whether

Ckn mutant proteins corresponding to four alleles: Ckn-A, Ckn-C, Ckn-K, and

Ckn-Y, disrupt the Ckn/Lar association. Ckn-A and Ckn-K, the two variants that

alter the N-terminal SAM domain, abrogate binding to Lar, whereas Ckn-C and

Ckn-Y, which have intact SAM domains, retain Lar association (Fig. 2.5B). These

results indicate that the N-terminal SAM domain of Ckn is necessary for Lar

interaction and are in agreement with our in vivo data as cknA and cknK are the

two alleles that do not promote ISNb bypass when overexpressed (Table 2.1),

suggesting that the Ckn-A and Ckn-K mutant proteins are innocuous when

overexpressed in vivo as they lack Lar-binding activity and cannot compete with

wild-type Ckn for Lar binding. Together, these studies implicate deficits in Lar

signal transduction in ckn axon pathfinding defects. We next asked whether the

molecular relationship between Lar and Ckn is conserved. In support of this

hypothesis, both full-length mouse Caskin2 and a Caskin2 fragment containing

both SAM domains (mCaskin2-SAM) interact with two of three murine LAR

receptor family members, LAR and PTPσ, in a yeast interaction assay (Fig.

2.5C). These data suggest that Caskins contribute to Lar signaling in vertebrate systems and raise the possibility that murine Caskins interact preferentially with distinct RPTP type IIA family members—similar to the preference of Drosophila

Ckn for Lar over RPTP69D. Mouse Caskin2 also interacts with Drosophila Lar in yeast two-hybrid experiments (data not shown), supporting the model that the pathway is well conserved. The in vitro interaction led us to ask whether the

proteins interact in vivo. To this end, we tested whether vertebrate Caskin alters motor axon pathfinding when overexpressed in Drosophila neurons. We generated a UASline to drive overexpression of the Lar-binding region of mouse

Caskin2. elavGAL4/UAS-mCaskin2-SAM embryos display ISNb bypass phenotypes with a frequency of 24% (Table 2.1, Fig. 2.5D, E). Hence, mCaskin2 overexpression impairs Drosophila motor axon pathfinding, presumably by binding to the Lar receptor and blocking downstream signal transduction.

To advance the hypothesis that Ckn and Lar associate biochemically, we tested whether GST-Lar fusion proteins precipitate HA-Ckn from embryonic extracts.

GST fusions to the full length Lar cytoplasmic domain or to the membrane-distal inactive D2 phosphatase domain pull down HA-Ckn from embryo extracts (Fig.

2.5F), indicating that Lar and Ckn can form a complex. If Ckn and Lar interact directly, the two purified proteins would be expected to maintain the ability to form a complex. Consistent with a direct physical interaction, bacterially expressed and purified Lar and Ckn associate with each other (Fig. 2.5G). In sum, these data demonstrate that Ckn binds the Lar RPTP D2 phosphatase domain via its N-terminal SAM domain.

Ckn is sufficient to redistribute the cytoplasmic domain of Lar in S2R+ cells

We turned to adherent S2R+ cells to visualize the intracellular distribution of epitope-tagged Ckn protein. We first compared the subcellular localization of Ckn and Liprin-α. Like Ckn, Liprin-α is a SAM-domain protein that binds the D2 domain of the cytoplasmic tail of Lar via a SAM domain interaction (Serra-Pages

et al., 1995; Serra-Pages et al., 1998). Liprin-α and Lar have been shown to

colocalize with focal adhesion markers in Drosophila and vertebrate cell lines

(Hofmeyer et al., 2006; Serra-Pages et al., 1995). In agreement with these

studies, we find that Flag-Liprin-α exhibits punctate localization (Fig. 2.6A).

Notably, HA-Ckn displays a similar pattern of subcellular localization (Fig. 2.6B),

and Ckn and Liprin-α colocalize when cotransfected (Fig. 2.6D–F)—consistent

with the presence of Ckn, Liprin-α, and Lar in focal adhesions. Whereas full-

length Lar colocalizes with Liprin-α in S2R+ cells, we found that a tagged Lar

cytoplasmic domain construct (GFP-Larcyto) exhibits a diffuse cytosolic

distribution (Fig. 2.6C). The cytoplasmic localization of GFP-Larcyto presented us

with an opportunity to test whether Ckn can recruit Larcyto to Ckn-positive

punctae. In fact, when HA-Ckn is cotransfected with GFP-Larcyto, its distribution

shifts to Ckn positive punctae (Fig. 6G–I), consistent with a Ckn/Lar complex. We

next asked whether the ability of Ckn to relocalize Lar in cotransfection

experiments relies on its N-terminal SAM domain. In support of this hypothesis,

Ckn-C shifts the distribution of Larcyto to Ckn positive punctae, whereas Ckn-A and Ckn-K do not (Fig. 6J–O) (data not shown). These localization studies argue

that Ckn and Lar interact via the N-terminal SAM domain of Ckn and raise

questions about the the physical relationship between Lar, Ckn, and Liprin-α.

Ckn and Liprin-α do not simultaneously bind Lar

The molecular similarities between Ckn and Liprin-α and the evidence that they

function at distinct developmental stages (Ckn in guidance and Liprin-α in

synaptogenesis) raise the possibility that Liprin-α and Ckn occupy a shared/overlapping binding site(s) on the Lar receptor. Hence, we assayed for concurrent binding of Ckn and Liprin-α to Lar in a yeast three-hybrid assay (Fig.

2.6P). Whereas Lar and Liprin-α interact in a yeast two-hybrid test, Ckn-SAM and

Liprin-α do not. We first asked whether a Lar/Gal4 DNA-binding domain fusion still interacts with a Ckn/Gal4 DNA activation domain fusion in the presence of

Liprin-α on a bridge vector. Liprin-α blocks the Ckn–Lar interaction (data not shown), consistent with a shared/overlapping binding site(s) for Ckn and Liprin-α.

As a more direct test, if Ckn and Liprin-α can co-occupy the Lar receptor, Lar is expected to serve as a bridge between Ckn and Liprin-α. The three proteins are not detected in a complex (Fig. 2.6P), suggesting that Lar associates either with

Ckn or with Liprin-α, but not both. These data are consistent with a common

Ckn/Liprin-α binding site on Lar and argue that the two proteins may compete for

Lar binding. Together, Ckn and Liprin-α are well situated to differentially regulate the interaction of Lar with downstream proteins that mediate the roles of Lar in axon outgrowth and synaptic development, respectively.

Caskin and Dock interact physically

Vertebrate Caskin proteins were recently identified in screens for Nck-binding proteins (Balazs et al., 2009; Fawcett et al., 2007). Nck/Dock proteins are

SH2/SH3-containing scaffolding proteins that participate in the signaling cascades of multiple guidance receptors (Fan et al., 2003; Garrity et al., 1996; Li et al., 2001; Schmucker et al., 2000; Song et al., 2003). Thus, we investigated

the possibility of Dock/Ckn interactions. Full-length Ckn and Dock interact in

yeast (Fig. 2.7A). Neither Ckn-SAM nor Ckn-C-terminal (Ckn-CT) constructs interact with Dock, implicating both N- and C-terminal Ckn sequences in the interaction. We further mapped the interacting domain(s) in Ckn using four mutant variants. Whereas Ckn-A and Ckn-Y retain Dock binding, neither Ckn-C nor Ckn-K interacts with Dock (Fig. 2.7B). Since Ckn-A abrogates Lar binding, these binding data allow us to separate the requirements for Ckn binding to Lar and Dock. Mapping the Ckn interaction domain within Dock using a series of deletion constructs (Fan et al., 2003) reveals that the second SH3 (SH3–2) domain of Dock is sufficient to bind Ckn (Fig. 2.7C).

Together, these mapping data argue that Ckn interacts with Lar and Dock via separable domains, as Ckn-A binds Dock but not Lar, and Ckn-C binds Lar but not Dock. To obtain additional support for this hypothesis, we asked whether Ckn simultaneously interacts with Dock and Lar in a yeast three-hybrid assay. Dock does not interact with the Lar cytoplasmic domain in a yeast two-hybrid assay

(Fig. 2.7D). However, with the addition of full length Ckn as a bridging protein in a yeast three-hybrid assay, Dock and Lar are found in a complex (Fig. 2.7D). The

Ckn mutant variants that do not bind either Lar or Dock individually cannot couple

Dock to Lar, demonstrating the specificity of the three-hybrid interaction. These results are consistent with our mapping data and indicate that Ckn interacts with

Lar and Dock via distinct domains. We further investigated the relationship between Ckn and Dock in S2 cells. In support of a physical interaction between

Ckn and Dock, Dock coimmunoprecipitates with Ckn in S2 cell lysates (Fig.

2.7E). Finally, we tested whether this interaction holds in embryos and find that

GST-Dock pulls down Ckn-HA from embryonic lysates (Fig. 2.7F). These results

argue that Ckn directly binds Dock and suggest that the two proteins interact in

axon development.

caskin and dock have redundant functions in axon outgrowth

To investigate whether these biochemical interactions have in vivo relevance, we

asked whether ckn and dock interact genetically. In agreement with published work (Desai et al., 1999), we find that dock mutants display a delay in neuromuscular junction (NMJ) formation by the RP3 motorneuron on the muscle

6/7 pair, and no defects are associated with loss of one copy of dock. The absence of pathfinding defects in zygotic dock LOF mutants is not the result of maternally derived Dock as motor axon pathfinding is also normal in embryos lacking both maternal and zygotic Dock (Desai et al., 1999). Given that Dock is strongly expressed in embryonic axons (Desai et al., 1999), normal motor axon pathfinding observed in dock LOF raises the possibility of redundancy. Therefore, we tested whether dock mutants are sensitive to ckn dosage. In support of the model that ckn and dock have shared functions in axon outgrowth, dose- sensitive interactions are observed between alleles of the two genes. In wild-type

embryos, ISNb pathfinding is complete by late stage 16, whereas in 23% of

dock3 cknk double heterozygotes, motor axons are still en route to their targets at

this stage. This dominant interaction suggests that dock and ckn contribute jointly

to the efficacy of motor axon outgrowth. To expand this analysis, we investigated

the effect of halving ckn dosage in a dock homozygous mutant background. Loss

of one copy of ckn has no effect on wild-type embryos but has a striking effect on

ISNb outgrowth in dock homozygotes. In dock3 cknK/dock3 + embryos, ISNb

outgrowth is incomplete in 43% (n = 187) of hemisegments, compared with 6% of dock3 homozygotes (Fig. 2.7H) (n = 110). In dock3 cknK double homozygotes,

the frequency of the “immature ISNb” phenotype rises to 65% (Fig. 2.7I) (n =

212). This interaction is not allele specific as 59% of dockP cknC hemisegments

exhibit delayed outgrowth (n = 178). Motor axons in the affected nerves are

loosely organized with multiple projections and resemble wild type axons at

earlier stages. Furthermore, the ISNd branch is frequently absent or reduced in

size. Examination of the ISNb/d choice point revealed defects in ISNb/d branch

segregation (Fig. 2.7J–L). Whereas in wild-type stage 16 embryos ISNb and

ISNd branches are tightly bundled and cleanly separated, a subset of ISNb/d

axons are entangled and remain clustered near the choice point in dock ckn

double mutants. Finally, we characterized the development of FasII-positive

longitudinal connectives. In late stage 16 wild-type embryos, the connectives are

tightly fasciculated and continuous (Fig. 2.7M). In contrast, the lateral two

longitudinals in dock ckn double mutants are poorly fasciculated and

discontinuous (Fig. 2.7N), demonstrating the activities of ckn and dock in axon

outgrowth are not limited to the periphery.

The pervasive defects in axonogenesis found in dock ckn mutants argue that ckn

activity is not limited to the Lar pathway, but rather that ckn likely participates in

signaling downstream of multiple receptors. Notably, we do not detect

appreciable ISNb bypass in dock ckn double homozygotes. However, the

absence of penetrant bypass phenotypes in dock ckn embryos may be

attributable to early pleiotropic defects in fasciculation and outgrowth and does

not exclude a role for dock in Lar-dependent signal transduction. Together, the data presented here identify caskin as a conserved adaptor protein required for

axon outgrowth and guidance.

Discussion

We demonstrate that Caskin mediates a novel Lar RPTP signaling cascade

during axonogenesis. Analysis of a panel of ckn LOF alleles indicates that ckn is

necessary for motor axon pathfinding as homozygous mutants display classic

bypass defects in the ISNb motor nerve. This phenotype is identical to that

displayed by Lar mutants, and genetic and biochemical interaction data

demonstrate that Ckn is a Lar-interacting protein. These studies position Caskin

to be a core member of a Lar-associated signaling complex that mediates its

function during axonogenesis.

Drosophila caskin encodes a conserved neuron-specific adaptor protein

Vertebrate Caskin was identified as a binding partner of the synaptic adaptor

protein CASK and competes for binding to the CaM kinase domain of CASK with

the PDZ (postsynaptic density-95/Discs large/zona occludens-1) protein Mint1

(Tabuchi et al., 2002). The CASK-binding site on Caskin maps to an N-terminal region not conserved in Drosophila, suggesting that fly Caskin does not bind

CASK. Consistent with this finding, Drosophila Caskin and CASK do not interact in a yeast interaction assay (data not shown). However, both mouse and fly

Caskin homologs bind LAR family members and Nck/Dock, in support of

considerable shared functions (Balazs et al., 2009; Fawcett et al., 2007).

Furthermore, overexpression of the Lar-binding domain of mouse Caskin in

Drosophila neurons yields a pathfinding phenotype like that of Lar and ckn LOF, suggesting that mouse Caskin competes with fly Ckn for binding to the Lar receptor to function as a dominant negative. These biochemical studies indicate that, whereas Caskins may have species-specific binding partners, Ckn function in Lar signal transduction is conserved.

Drosophila Lar also physically interacts with the Abl tyrosine kinase and its substrate the cytoskeletal regulator Ena (Wills et al., 1999). We are unable to detect physical interactions between Ckn and Abl or Ena, suggesting they bind

Lar independently (data not shown). This raises the possibility that Caskin and

Abl/Ena constitute parallel pathways downstream of the Lar receptor. Our allelic series enabled us to analyze the in vitro and in vivo activities of four Caskin mutant proteins: Ckn-A, Ckn-C, Ckn-K, and Ckn-Y. Ckn-A and Ckn-K contain alterations in the first SAM domain and block the interaction of Ckn with Lar, pointing to the importance of this domain for Lar/Ckn complex formation. The in vivo analysis of Ckn-A and Ckn-K is in strong agreement with the in vitro data as motor axon phenotypes are not associated with their overexpression, suggesting they do not interfere with Lar signaling in vivo. The behavior of Ckn-A and Ckn-K contrasts that of Ckn-C, which contains a C-terminal deletion. Ckn-C interacts with Lar, and its neuronal overexpression yields dominant-negative-like effects.

In fact, the penetrance of ISNb bypass associated with Ckn-C overexpression is

comparable with that observed in embryos lacking both maternal and zygotic Lar

(Fox and Zinn, 2005), suggesting that it effectively interferes with Lar activity.

Although Ckn-C binds Lar, cknC homozygous LOF mutants display a “Lar-like”

ISNb phenotype, indicating that Lar signaling is blocked downstream of receptor

binding. Ckn-C does not interact with Dock, but this interaction is insufficient to

explain the cknC mutant phenotype since ISNb bypass is not associated with

dock LOF. The pathfinding phenotype observed in cknC embryos argues that the

allele also disrupts the interaction between Caskin and another unknown

downstream protein(s) essential for Lar signaling.

Ckn and Dock function redundantly in axon outgrowth

Dock/Nck are SH2/SH3-containing adaptor proteins that couple

phosphotyrosines on activated receptors to downstream signaling molecules via

SH2 and SH3 domain interactions, respectively (Buday et al., 2002). Dock also engages in a ligand-regulated SH3 domain interaction with the Robo receptor

(Fan et al., 2003), demonstrating that it is involved in diverse interactions downstream of guidance receptors. In this work, we demonstrate that Caskin

interacts with the second SH3 domain of Dock (SH3–2).

This domain has also been shown to interact with the cytoskeletal effector Pak

(Hing et al., 1999), raising questions about the relationship between Caskin and

Pak. It will be informative to determine whether Dock forms alternative

complexes with Caskin and Pak, or whether Dock binds Caskin and Pak

simultaneously.

The contrast between the ckn and dock single- and double- mutant phenotypes demonstrates that the adaptors have mostly redundant functions. Single-mutant analyses indicate that ckn plays a nonredundant role in Lar signaling, whereas dock has a unique role in synaptogenesis of the RP3 motorneuron. However, the outgrowth defects observed in dock ckn double mutants argue that these adaptors have overlapping roles in a number of signaling events. These data caution against drawing conclusions of cellular function based solely on single mutant analysis, as this obviously uncovers only the nonredundant functions of a protein.

The issue of genetic redundancy may be particularly acute in signaling systems involving multi-subunit complexes with many opportunities for parallel functions.

It will be important to identify additional binding partners of dock and ckn to determine whether they have a common set of interactors, or whether they impinge on the cytoskeleton via distinct, yet redundant, paths.

A Lar–Ckn pathway for axon pathfinding

The Lar receptor is a member of the type IIA subfamily of RPTPs, comprising Lar and PTP69D in flies (Johnson and Van Vactor, 2003). The single-mutant phenotypes of Lar and PTP69D indicate they have nonredundant functions in motor axon guidance, NMJ growth, and photoreceptor axon targeting (Clandinin et al., 2001; Desai et al., 1997; Hofmeyer and Treisman, 2009; Kaufmann et al.,

2002; Maurel-Zaffran et al., 2001). Several observations hint that the unique functions implied by the divergent phenotypes of Lar and PTP69D stem in part

from distinct ligand binding activities. Fox and Zinn (2005) observed that Lar and

PTP69D alkaline phosphatase fusion proteins have distinct embryonic staining patterns suggesting the presence of unique ligands. Furthermore, overexpression of a chimeric receptor composed of the Lar extracellular domain fused to the PTP69D intracellular domain rescues the LOF photoreceptor defect of Lar, whereas a PTP69D extracellular domain fusion to the Lar intracellular domain does not (Maurel-Zaffran et al., 2001)—arguing that Lar and PTP69D have overlapping intracellular partners and (at least partially) nonoverlapping extracellular ones. However, more recent data open the door for functional differences between the intracellular pathways activated by Lar and PTP69D. R7 photoreceptor axon targeting is independent of Lar phosphatase activity, but dependent on PTP69D phosphatase activity, suggesting that the receptors have distinct binding partners (Hofmeyer and Treisman, 2009; Prakash et al., 2009).

These findings are consistent with the work presented here. Both fly and vertebrate Caskins interact with subsets of LAR family receptors, raising the possibility that the intracellular signaling cascade(s) organized by Ckn contributes to the functional differences between Lar and PTP69D.

We investigated the physical relationship between Lar, Ckn, and Liprin-α and we find that these proteins do not form a ternary complex. These binding data support mapping studies indicating that Ckn and Liprin-α both interact with the

D2 phosphatase domain of Lar via SAM domain-mediated interactions. They further suggest sequential/competitive binding of Ckn and Liprin-α to the Lar receptor and raise the possibility of distinct neuronal functions. It is conceivable

that Ckn and Liprin-α both act downstream of Lar to mediate its activity during

axon outgrowth/pathfinding and synaptogenesis, respectively. To determine

whether Ckn function is specific for Lar signaling during axonogenesis, it will be

informative to test whether ckn LOF mutants exhibit defects in the assembly/localization of presynaptic components similar to that observed in Lar mutants (Kaufmann et al., 2002; Miller et al., 2005). Alternatively, the function of

Liprin-α in Lar signaling may be primarily to localize or maintain Lar at the

presynaptic terminal, whereas Ckn functions in downstream signal transduction.

This hypothesis is supported by evidence for a conserved function for Liprin-α in

synaptic protein targeting or anchoring (Ackley et al., 2005; Kaufmann et al.,

2002; Serra-Pages et al., 1998). A role for Liprin-α in trafficking is further

bolstered by conserved physical interactions between Liprin-α and Kinesin (Miller

et al., 2005; Shin et al., 2003), suggesting it is an adaptor protein for anterograde

transport of synaptic proteins. In this scenario, it is notable that Liprin-α function

is not required for pathfinding (Hofmeyer et al., 2006), arguing either that another

protein serves to localize Lar during guidance or that Lar activity in this process

does not require its tight localization to the axon terminal. This model is

consistent with the broad axonal localization of Lar during embryogenesis

(Krueger et al., 2003). Extracellularly, LAR family members interact with HSPGs

and CSPGs. In Drosophila, mutations in the HSPG syndecan (sdc) interact with

Lar in motor axon guidance, but homozygous LOF sdc embryos do not display

appreciable bypass phenotypes (Fox and Zinn, 2005), arguing that other ligands

are involved. Once these ligands are identified, it will be critical to determine

whether ligand binding influences the association of intracellular adaptors such as Liprin-α and Caskin with Lar. Recently, vertebrate LAR family members have moved into the spotlight in the field of axon regeneration, as PTPσ has been shown to be a receptor for CSPGs, which are dramatically upregulated at the lesion site and are strongly inhibitory to axon growth (Fry et al., 2010; Shen et al.,

2009). Strikingly, axons in PTPσ mutant mice have an enhanced ability for regeneration relative to wildtype mice. These studies suggest that blocking PTPσ signaling in injured axons might enhance recovery after spinal cord injury. Hence, the truncated forms of fly and vertebrate Caskins that interfere with Lar signaling are particularly interesting. The identification of such dominant-negative reagents allowing the blockade of Lar signal transduction in vivo may have clinical implications in axonal regeneration.

Figure 2.1

Figure 2.1: Neuronal overexpression of CG12424 disrupts axon guidance,

and CG12424 RNA localizes to the embryonic neuropil

(A,C) Stage 16 wild-type embryos, (B) stage 16 elavGAL4/UAS-CG12424 embryo, and (D) Hb9Gal4/UAS-CG12424 embryo labeled with mAb 1D4

recognizing all motor axons. (A, B) Pan-neuronal overexpression of CG12424

causes inappropriate midline crossing by ipsilateral axons (arrowheads in B). (C)

Two hemisegments of a wild-type embryo, and (D) an embryo with CG12424

overexpression in ventral and lateral motorneurons (Hb9>ckn). Hb9>ckn

embryos display ISNb stalls. Arrows in C, D indicate appropriate position of

dorsal-most ISNb synapse. This synapse is absent in Hb9>ckn embryos. (E)

Stage 16 wild-type embryo hybridized with anti-sense CG12424 RNA probe. The

embryo shown in (F-H) is double-labeled with CG12424 anti-sense RNA probe and BP102 to mark the CNS axon scaffold. CG12424 RNA is apparent in CNS axons. (A,B,E-H) Anterior up, (C,D) Anterior left. Scale bar: 15 µM

Figure 2.2

Figure 2.2: caskin gene structure and allele generation

(A) Alignment of Drosophila Caskin and murine Caskin2. Conserved domains are labeled. Percent amino acid similarity/identity in the two SAM domains and the C- terminal domain (CTD) are indicated. (B) Identity of eight caskin EMS alleles. (C) cknA and cknY are missense alleles, in which conserved amino acids in the first

SAM domain (cknA) or the CTD (cknY) are mutated. cknC and cknK contain small deletions. cknK returns to coding sequence in frame, while the cknC deletion results in a frame shift and truncated protein.

Figure 2.3

Figure 2.3: ckn LOF alleles disrupt ISNb motor axon pathfinding

(A-E) Two hemisegments of stage 16 embryos labeled with mAb 1D4 to mark motor axon projections. (A) ISNb-projecting axons of wild-type embryos innervate four ventrolateral muscles and adopt a characteristic branched morphology.

Black arrows indicate the ISNd branch (when present) in all panels. (B) In cknK/Df(2R)BSC330 null embryos, ISNb axons extend in a distinct fascicle adjacent to the primary ISN branch (parallel bypass; white arrow) and do not enter their muscle field. (C) cknK/Df(2R)BSC330 embryo in the plane of the VLM field. ISNb axons that bypassed the normal entry point into their muscle domain

“reach back” to contact their targets (white arrows). (D) In the cknD/cknK embryo,

ISNb axons fail to innervate their muscle targets and extend with ISN axons in a common fascicle (fusion bypass; white arrow). (E) In elavGAL4/UAS-cknC embryos, ISNb axons fail to separate from the ISN at the ventral choicepoint

(arrow) and exhibit bypass. (F) Schematic of wild-type and mutant ISNb/d nerve morphology. Exit Junction (EJ) is the choicepoint where ISNb axons defasciculate from the ISN. The asterisk indicates the point of ISNb/d branch segregation. Anterior is to the left in all panels. Scale bar: 15 µM

Figure 2.4

Figure 2.4: ckn and Lar interact genetically

(A-E) Two hemisegments of stage 16 embryos of the indicated genotypes labeled with mAb 1D4 to mark motor axons. (A) ISNb nerve of a wild-type embryo. (B) cknK mutant embryo displaying an intermediate level of ISNb bypass

(arrow). (C) Lar5.5 mutant embryo with parallel bypass phenotype in one hemisegment (arrow). (D) Lar5.5 cknK/+ + embryo with moderate frequency of

ISNb bypass (arrow). (E) Lar5.5 cknK double homozygous embryo exhibiting increased penetrance of ISNb bypass (arrows). Anterior is to the left in all panels.

Figure 2.5

Figure 2.5 (Cont'd)

Figure 2.5: Ckn and Lar interact physically

(A) Yeast two-hybrid interactions indicate a direct physical interaction between

Ckn and Lar, but not Ckn and PTP69D. The cytoplasmic domain of Lar was

fused to the Gal4-DNA binding domain (bait). Schematics of the Ckn constructs

that were fused to the Gal4-activation domain (prey) are shown. Both full-length

Ckn and Ckn-SAM interact with Lar-cyto. In contrast, none of the Ckn constructs

interact with PTP69D. (B) Schematics of four caskin mutant variants are shown.

These mutant cDNAs were fused to the Gal4-activation domain (prey) and tested

for binding with the Lar-cyto domain fused to the Gal4-DNA binding domain

(bait). Both of the SAM domain mutations, Ckn-A and Ckn-K, abrogate Lar

binding, while both Ckn-C and Ckn-Y maintain Lar interactions. (C) The

cytoplasmic domains of the three vertebrate type IIA RPTPs were fused to the

Gal4-DNA binding domain (bait) and tested against full-length mCaskin2 and a

fragment of mCaskin2 containing both SAM domains fused to the Gal4-

activiation domain (prey). Lar and PTPs interact with mCaskin-SAM. Two hemisegments of stage 16 (D) wild-type and (E) elavGAL4/UAS-mCaskin2-SAM embryos labeled with mAb 1D4. Neuronal overexpression of mCaskin2-SAM yields ISNb bypass (arrow) suggesting that it interferes with Lar signal transduction in a dominant-negative fashion. (F) A GST-Lar-cyto fusion protein precipitates HA-Caskin from elavGAL4/UAS-HA-Caskin embryonic lysates, while

GST alone does not. GST-Lar D2 pulls HA-Caskin out of embryonic lysates arguing that Ckn binds the D2 domain. (G) A bacterially-expressed and purified

HA-Caskin fusion protein precipitates purified GST-Lar, while Ni-NTA beads alone cannot.

Figure 2.6

Figure 2.6: Ckn redistributes Larcyto in S2R+ cells, and Ckn and Liprin-α do not simultaneously bind Lar

S2R+ cells transfected singly with (A) Flag-Liprin-α, (B) HA-Ckn, or (C) GFP-

Larcyto. Flag-Liprin-α and HA-Ckn have punctate localization pattern, while GFP-

Larcyto has an even cytosolic distribution. (D-F) S2R+ cell cotransfected with

HA-Ckn and Flag-Liprin-α. The proteins colocalize. (G-I) S2R+ cell cotransfected with HA-Ckn and GFP-Larcyto. HA-Ckn recruits GFP-Larcyto to Ckn-positive punctae. (J-L) S2R+ cell cotransfected with HA-Ckn-K and GFP-Larcyto. HA-

Ckn-K does not relocalize Larcyto. (M-O) S2R+ cell cotransfected with Ckn-C and GFP-Larcyto. HA-Ckn-C relocalizes Lar-cyto to Ckn-positive punctae. (P)

Co-transformation of yeast with a Larcyto/Gal4-DNA binding domain fusion (bait) and either Ckn-SAM or Liprin-α Gal4-DNA activation domain fusions (prey) yield yeast two-hybrid interactions. Full-length Ckn could not be used in this assay as it activates transcription in the absence of the Gal4-activation domain. Ckn-

SAM/Gal4-DNA binding domain fusion (bait) and Liprin-α/Gal4-DNA activation domain fusions do not interact. If Ckn and Liprin-α bind Lar simultaneously, Lar is predicted to serve as a bridge in a yeast three-hybrid assay. An NLS was added to the N terminus of Lar to promote nuclear localization. No interaction between

Ckn and Liprin-α is detected in the presence of Lar, arguing that Ckn and Liprin-

α cannot bind Lar simultaneously.

Figure 2.7

Figure 2.7 (Cont'd)

Figure 2.7: Ckn exhibits physical and genetic interactions with Dock

(A) Full-length Ckn interacts with Dock in yeast two-hybrid. Ckn-Full, Ckn-SAM,

and Ckn-CT constructs were fused to Gal4-DNA activation domain (prey). Full-

length Dock was fused to the Gal4-DNA binding domain (bait). Only full-length

Ckn interacts with Dock. (B) Mutant cDNAs for four ckn alleles were fused to the

GAL4-DNA activation domain and tested against a Dock-Gal4-DNA binding domain bait plasmid. Neither Ckn-C nor Ckn-K binds Dock, while both Ckn-A and

Ckn-Y maintain the interaction. (C) A series of LexA-Dock fusions were tested for interaction with full-length Ckn-activation domain fusions. + +, Medium blue; -, white (no interaction) after one hour. (D) Schematic outlining the yeast three- hybrid interactions between Caskin, Lar, and Dock. Yeast were co-transformed with a Larcyto/Gal4-DNA binding domain fusion (bait) and a full-length

Dock/Gal4-activation domain fusion (prey). Dock and Lar do not interact. After two days, the yeast were mated to yeast transformed with Ckn-WT, Ckn-A, Ckn-

C, and Ckn-K. A nuclear localization signal (NLS) was added to the N terminus of

Ckn. Only wild-type Ckn couples Lar and Dock. Ckn-Y could not be tested in this assay as it activates transcription in the absence of the Gal4-activation domain.

(E) Flag-tagged Dock co-immunoprecipitates Caskin-HA from S2 cells. (F) A

Dock-GST fusion protein precipitates Ckn-HA from elavGal4/UAS-Caskin-HA embryos, while GST alone does not. (G-K) Two hemisegments of stage 16 embryos labeled with mAb 1D4 to mark motor axon projections. Anterior is to the left. Scale bar: 15 µM (G) ISNb nerve exhibits characteristic branched morphology in wild type. Black arrow indicates ISNd. (H) Axons in dock3

cknK/dock3 + embryos are still en route to their targets at late stage 16. (I) ISNb axons in dock3 cknK double homozygotes are loosely fasciculated and extend multiple projections. (J-L) ISNb/d axons do not separate in ckn dock double mutants, whereas in wild type embryos, ISNb and ISNd axons are tightly bundled and cleanly separated (J). ISNb/d axons remain tangled in the double mutant (K).

Schematic of the ISNb/d branch segregation defect is shown (L). The CNS midline of wild-type (M) and dock ckn (N) embryos labeled with ID4. Axons in the lateral two connectives are poorly fasciculated and stall in the double mutant.

Table 2.1: ISNb phenotype in ckn LOF and GOF embryos

a Hb9Gal4>cknWt 55% of ISNb axons displays stopshort phenotype b elav>cknWt 86% of ISNb axons displays stopshort phenotype

All embryos were scored at late stage 16/early stage 17. Partial parallel bypass was scored if a subset of ISNb axons entered the VLM field correctly. Parallel bypass was scored if ISNb axons extended next to ISN axons in a distinct fascicle. Fusion bypass was scored if ISN and ISNb axons could not be differentiated at the light microscope level. n= number of hemisegments scored.

Table 2.2: Genetic interactions between LOF mutations in Lar, ckn and ptp69D

CHAPTER 3

IDENTIFICATION OF CASKIN-INTERACTING PROTEINS AND THE REGULATORY MECHANISMS IN LAR-MEDIATED AXON PATHFINDING

Abstract

Caskin is a neuronal adaptor protein and its C terminus region is critical for transduction of LAR signaling into growth cone cytoskeleton remodeling. In order to elucidate how this signaling mechanism acts, I performed a yeast two-hybrid screen to isolate proteins that specifically interact with C-terminus Caskin.

Several novel interacting proteins were uncovered, most of which are expressed in the developing nervous system at the embryonic stage and have vertebrate homologues known to control axon guidance and neurite growth. Because

Caskin is capable of binding in a yeast two-hybrid interaction system with several different intracellular effector molecules, the goal of future studies will be to assess which Caskin interacting proteins mediate LAR signal transduction.

Additionally, we performed genetic studies that reveal abl GOF and LOF phenotypes are reciprocally identical to those of ckn (see the summary of phenotypes in Table 3.2). This suggests Abl kinase is a functional antagonist of

Caskin, presumably acting in a phosphorylation-dependent manner. In support of this notion, we found that Caskin can be tyrosine phosphorylated in the presence of Abl kinase in Drosophila S2R+ cells. We hypothesize that Abl-dependent tyrosine phosphorylation disrupts interactions between Caskin and its downstream binding partners, and in turn leads to defective motor axon guidance.

Introduction

LAR signaling is important for diverse cellular functions, including axon pathfinding, nerve regeneration, synapse formation, metabolic regulation, and tumor suppression (Chagnon et al., 2004; Johnson and Van Vactor, 2003).

Portions of LAR signaling cascades have been elucidated by identifying a variety of downstream substrates and interacting partners. However, very little is known about the mechanisms by which LAR converts extracellular stimuli into cytoskeletal reorganization and growth cone motility.

Several lines of evidence indicate that Rac GTPases are important for regulating axon pathfinding and neurite growth. Examination of single, double, and triple mutant Drosophila rac1, rac2 and mtl reveal that Rac GTPases have overlapping roles in establishing axonal repulsive signaling in the CNS (Hakeda-Suzuki et al.,

2002). In addition, Rac GTPases are required for proper motor axon targeting.

Overexpression of dominant-negative Rac1 prohibits subsets of motor axons

(ISNb) from entering the ventral muscle field, inducing an ISNb bypass phenotype reminiscent of Dlar LOF mutants (Kaufmann et al., 1998). This evidence, together with evidence indicating rac1 and Dlar interact genetically, suggests that LAR signaling may modulate Rac1 activity via an unknown mechanism.

Activation of Rac GTPases is positively regulated by guanine nucleotide exchange factors (GEFs), which promote the exchange of GDP for GTP. In the

Drosophila visual system, Rac1 is activated by the dual Rho-GEF Trio and in turn

stimulates Pak, a cytoskeletal regulator, to direct photoreceptor cell (R cell) axon targeting (Newsome et al., 2000). Trio has been found to interact with LAR and

Netrin receptor DCC (deleted in colorectal cancer), suggesting it may serve as a molecular link between guidance receptors and cytoskeleton (Briancon-Marjollet et al., 2008; Debant et al., 1996). Although Drosophila Trio cannot directly interact with LAR because it lacks the conserved C terminus binding region, genetic interaction assays indicate that Trio functions downstream of LAR to mediate motor axon defasciculation and targeting (Bateman et al., 2000).

However, we did not observe a motor axon guidance phenotype associated with pak LOF mutants (data not shown). These observations suggest alternative mechanisms downstream of Trio that act to transduce LAR signaling.

In our previous studies, we identified Caskin as a neuronal adaptor protein that interacts with the LAR receptor and mediates LAR signaling. However, the downstream effectors that initiate each step of the subsequent signaling cascade remain elusive. Caskin is composed of two N-terminus SAM domains, an EVH1 binding motif, and one conserved C-terminus domain. These domains are involved in protein-protein interactions, and are thought to interact with the repertoire of proteins necessary for conveying diverse functions (Ball et al., 2002;

Qiao and Bowie, 2005). Sequence analysis of different ckn LOF mutants indicates that the first SAM domain and the conserved C terminus domain are required for LAR-mediated motor axon guidance. Our study demonstrated that the first SAM domain of Caskin is responsible for its interaction with LAR and the conserved C-terminus domain of Caskin is required for its interaction with

downstream effectors. We hypothesize that mutations abrogating LAR-Caskin or

Caskin-effector(s) interactions block signaling complex formation and thus result

in axon guidance defects.

Protein phosphorylation has been shown to be a key regulatory step in the

recruitment of signaling complexes to guidance receptors. For example, ligand- engaged Eph receptors are phosphorylated on multiple tyrosine residues, and in

turn Eph receptors increase their association with downstream interacting

proteins (Boyd and Lackmann, 2001; Yu et al., 2001). Similarly, protein

phosphorylation seems to be critical in LAR signal transduction. abl encodes a

tyrosine kinase that regulates cell adhesion and cytoskeletal dynamics, and has

been found to interact genetically with Dlar (Colicelli, 2010; Wills et al., 1999a). In

Drosophila, ectopic expression of Abl in neurons results in ISNb bypass

phenotype similar to that observed in Dlar LOF mutants. The induction of ISNb

bypass phenotype is dependent on the kinase activity of Abl, as overexpression

of kinase-dead Abl does not affect motor axon targeting. These data support the

idea that phosphorylation of substrates is determined by the enzymatic activities

of DLAR and Abl, and differential phosphorylation modifications on downstream

targets contribute to differential growth cone response (Wills et al., 1999a).

The Ena/VASP (Ena, vertebrate Mena, VASP, and EVL) proteins are a

conserved family of actin regulators, playing a crucial role in actin polymerization

and cell motility (Krause et al., 2003). In Drosophila, loss of Ena leads to a higher

penetrance of the ISNb bypass phenotype, with more than 90% of ISNb axons

failing to branch and enter the ventral muscle field. Thus, Ena may act as a

convergence point for multiple signal inputs (Wills et al., 1999a). Several lines of

evidence demonstrate Ena is a common substrate of both DLAR and Abl; its

phosphorylation is reciprocally regulated by DLAR and Abl activities. To date, it is unclear whether the phosphorylation of Ena is critical for cytoskeletal reorganization during axon guidance. Mutation of the six major Abl-dependent tyrosine phosphorylation sites in Ena does not alter Ena function in motor axon targeting (Lanier and Gertler, 2000). These data suggest that phosphorylation-

dependent regulation by DLAR and Abl may not occur on the substrate Ena.

We propose that Caskin phosphorylation, mediated by Abl kinase and DLAR

phosphatase, regulates growth cone behavior. Previous studies demonstrate abl

GOF embryos and ckn LOF mutants exhibit the same motor axon bypass

phenotype, raising a possibility that overexpression of Abl functionally

antagonizes Caskin (known interactions summarized in Table 3.2). Here we

demonstrate that ckn and abl interact genetically, and that Caskin is tyrosine

phosphorylated in an Abl kinase-dependent manner. This Abl-dependent tyrosine

phosphorylation does not abolish the interaction between DLAR and Caskin.

Thus, we hypothesize that tyrosine phosphorylation may inhibit Caskin’s

interaction with downstream effector(s). In order to test this idea, we conducted a

yeast two-hybrid assay to identify downstream binding partners of Caskin. We

found five interactors with potential roles in axon guidance: Mys (Myospheroid),

PTEN (phosphatase and tensin homolog deleted on chromosome ten), Graf

(GTPase Regulator Associated with FAK), GammaCop and Liprin-gamma.

These findings not only represent an important step toward understanding how

Abl-dependent phosphorylation regulates LAR-mediated motor axon guidance but also provide an insight into Caskin’s role in signal relays. Future work is

required to assess whether these Caskin binding partners have functional

importance.

Materials and Methods

Yeast strains and growth condition

Yeast two-hybrid experiments were performed with Saccharomyces cerevisiae

strains pJ694a (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4 gal80

LYS2:: GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ ) and SL3004 (MAT trp1-901

leu2 ura3 his3 gal4 gal80 lys2-801 ade2-101). Yeast strains were grown in YPD medium (yeast extract, peptone, and dextrose) or selected on proper synthetic dropout (SD) medium supplemented with dextrose (2%) at 30 °C.

DNA constructs cDNA clones encoding full-length Liprin-alpha (LD33094), and Liprin-beta

(RE16685) and Liprin-gamma (RE30524) were purchased from the Drosophila

Genomics Resource Center. To facilitate the introduction of in-frame DNA sequences into the yeast two-hybrid plasmids as well as other expression constructs, the corresponding coding sequences were first PCR-amplified from individual cDNA clones, and subcloned into the pCR8/GW/TOPO vector. The following yeast two-hybrid and expression constructs were made by Gateway recombination cloning (Invitrogen): pDEST-GBKT7 Liprin-alpha, pDEST-GBKT7

Liprin-beta, pDEST-GBKT7 Liprin-gamma, pDEST-GBKT CknCTD (600-927 aa) and pTHW CknFull (1-927 aa)

Yeast two-hybrid assay

The pJ694a yeast strain was transformed with 1 µg of pDEST-GBKT7 CknCTD

(600-927 aa) using the lithium acetate method and subsequently mated with the

SL3004 yeast strain pretransformed with a Drosophila embryonic cDNA Library.

Transformants were selected on –Leu/-Trp/-Ade/-His/SD plates by incubating plates at 30°C for 3 to 4 days. Positive colonies were then picked for DNA isolation and sequencing.

To test for protein interactions between Caskin and Liprins, we cotransformed the pJ694a yeast strain with CaskinFull AD and Liprin alpha/beta/gamma BD constructs (pDEST-GBKT7 Liprin-alpha, pDEST-GBKT7 Liprin-beta and pDEST-

GBKT7 Liprin-gamma) in a pairwise manner. Colonies were picked from the –

Trp/-Leu/SD plates and used to assay protein interactions by plating on -Trp/-

Leu/-His/-Ade/ SD plates. To further confirm protein–protein interactions, beta- galactosidase filter assay (colony filter lift assay) was performed as described previously.

Immunoblot

Drosophila S2R+ cells were transiently transfected with the indicated expression plasmids by FuGENE HD (Roche) and harvested after 48 hr. Cells were lysed in

IP buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1x protease

inhibitor (Roche) and 1x phosphatase inhibitor (Roche)) on ice for 15 min and

cleared by centrifugation. Caskin was immunoprecipitated using anti-FLAG M2

mAb (Sigma) prebound to protein G beads for 2 hr at 4°C. Samples were

separated by SDS-polyacrylamide gel electrophoresis, transferred to PVDF, and

immunoblotted using an anti-phosphotyrosine antibody (4G10, Millipore) and anti-FLAG antibody (Sigma).

Results and Discussion

Abl-dependent tyrosine phosphorylation occurs on Caskin

Tyrosine kinases play a vital role in regulating growth cone behavior. In

Drosophila abl GOF embryos, ISNb axons fail to defasciculate and instead follow

the ISN axon pathway toward the dorsal muscle region (ISNb bypass phenotype)

(Wills et al., 1999a). This phenotype is identical to that seen in ckn LOF embryos,

suggesting a model where Abl and Caskin function antagonistically in their

regulation of motor axon guidance (Table 3.2). In support of this notion, we found

that abl LOF and ckn GOF embryos exhibit a similar phenotype in which ISNb

motor axons stall at the proximal target muscles and fail to enter either the dorsal

or ventral muscle fields (Table 3.2). Interestingly, the penetrance of the ISNb stall phenotype is higher in ckn GOF embryos (80% by elavGAL4, Table 2.1) than in abl LOF mutants (24-63%)(Wills et al., 1999b), suggesting that overexpression of

Caskin may also antagonize other targets.

Given that Caskin is a LAR binding partner and its function is negatively

regulated by Abl kinase, we hypothesized that Caskin is a common substrate of

both LAR and Abl and its state of phosphorylation contributes distinct aspects of

axon targeting. To test this idea, we first examined genetic interactions between

ckn and abl. We found that reduction of abl by half genetic dosage dramatically

rescues axon targeting deficits in ckn LOF phenotype; homozygous cknk mutants

exhibit 41% bypass penetrance (n=218, Table 2.1, Figure 3.1B) whereas cknk /

cknk; abl2/+ mutants only display a 4% penetrance in motor axon guidance

defects (n=90, Figure 3.1C). This data suggests that Caskin is a potential substrate of Abl and that reduction of Abl-dependent tyrosine phosphorylation on

Caskin relieves motor axon guidance defects in ckn LOF embryos.

To examine whether Caskin can be tyrosine phosphorylated in an Abl kinase- dependent manner, we cotransfected both Caskin and Abl protein expression constructs in Drosophila S2R+ cells. We found that coexpression of both Caskin and Abl leads to a significant increase in Caskin tyrosine phosphorylation relative to Caskin phosphorylation in the absence of Abl kinase (Figure 3.2A). We used

Scansite, a prediction software, to find potential tyrosine phosphorylation sites in

Caskin. We found that there are three putative Abl phosphorylation sites located at the N-terminus (Y33) and C-terminus (Y517 and Y724) portions of Caskin.

Future experiments will be necessary to determine whether these predicted tyrosine residues are indeed subject to the kinase activity of Abl.

How does Abl-dependent phosphorylation of Caskin inhibit LAR signaling and lead to motor axon guidance defects? One possibility is that phosphorylation of

Caskin disrupts its interaction with LAR. However, our genetic and biochemical analysis does not fit with this model. cknk allele encodes a truncated Caskin protein that cannot bind to LAR. The genetic rescue of cknk bypass phenotype by decreasing abl genetic dosage is not likely due to Ckn-K regaining its capacity to bind with LAR. Furthermore, we observe that Caskin is capable of binding with

LAR in the presence of Abl (Figure 3.2B). In a new model, we propose that

Caskin may become hyperphosphorylated as a result of defective dephosphorylation reactions in Dlar null mutants or ckn mutants (cknA and cknK).

Reduction in Abl kinase activity may return phosphorylation modifications to

normal levels, subsequently restoring interactions between Caskin and its

downstream interactors (Figure 3.3).

Taken together, we hypothesize that phosphorylation modifications on Caskin

prevent its interaction with downstream targets and impede signaling complex

assembly. In abl GOF, Dlar LOF, or ckn LOF embryos (cknA and cknK),

hyperphosphorylated Caskin causes signaling complex assembly to fail, and this

failure contributes to axon targeting defects. In contrast, a relatively high level of

dephosphorylated Caskin exists in abl LOF or ckn GOF embryos, generating an

overabundance of signaling complexes that act to stop axon growth.

Isolation of Caskin-interacting proteins

To begin to explore how Abl-dependent phosophorylation regulates LAR-

mediated axon guidance, we first sought to indentify downstream Caskin

interactors. We used the C-terminal portion of Caskin (594-924 aa) to search for

interacting proteins in a Drosophila embryonic yeast-two hybrid cDNA library.

Protein-protein interaction occurring in the yeast two-hybrid assay leads to activation of the reporter genes ADE2, HIS3 and LacZ, and allows yeast to survive on the appropriate selection plates. We screened approximately 106 clones and isolated 80 clones with the strongest protein interactions. We discarded redundant clones with identical size inserts to eliminate potential duplicates among these 80 clones. The remaining clones were sequenced and

the results of our screen are listed in Table 3.1. Four of these interacting proteins

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have potential roles in axon guidance: Graf, Mys, GammaCop and PTEN. Here I briefly introduce their functions.

Drosophila Graf (GTPase regulator associated with FAK) contains a central

RhoGAP domain and a C-terminus SH3 domain which binds to FAK (Hildebrand et al., 1996). Study of the vertebrate homologue of Graf suggests that RhoGAP activity is essential for Graf function in modulating actin polymerization. Ectopic expression of Graf in fibroblast cells induces filopodia extension, whereas expression of a mutated Graf variant lacking RhoGAP activity decreases the number of stable filopodia (Taylor et al., 1999). Rho GTPases, which are known to play a crucial role in axon guidance, are positively regulated by guanine nucleotide exchange factors (GEFs) and negatively regulated by GTPase activating proteins (GAPs). Genetic studies in Drosophila provide compelling evidence that Trio and Rac1 might function together in LAR signaling. Given that

Graf has an antagonistic function on Rho GTPases activities, it may be interesting to assess whether Graf represses Trio function.

Drosophila Myospheroid (Mys) encodes a common beta integrin subunit that assembles with other alpha integrin subunits to form heterodimeric receptors.

Emerging evidence reveals the involvement of integrin-mediated signaling in axon guidance and outgrowth. For example, Sema7A is found to bind to the beta1 integrin subunit, which in turn leads to the activation of FAK. Abrogating receptor engagement using a function blocking anti-beta1 integrin antibody neutralizes Sema7a mediated axon growth (Pasterkamp et al., 2003). In addition, inactivation of Drosophila intergrin receptors or their downstream mediators

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causes ISNb motor axon defasciculation defects (Hoang and Chiba, 1998;

Huang et al., 2007). Interestingly, Dlar and mys display genetic interaction in their organization of actin filaments (Bateman et al., 2001). Thus, whether Caskin functions in integrin-mediated signaling and allows crosstalk between LAR and integrin receptors requires further investigation.

Drosophila GammaCop is a component of COPI coatomer which is involved in the vesicular trafficking (Grieder et al., 2008). Several studies in yeast and other model systems indicate that inhibition of GammaCop causes deficits in both anterograde and retrograde transport (Gaynor and Emr, 1997; Lee et al., 2004).

Interestingly, an RNAi screen in Drosophila primary neuronal cultures identified

GammaCop as a regulator of axon growth (Sepp et al., 2008). It is likely

GammaCop functions in the sorting and transport of “growth-associated proteins” between the soma and the axon terminus to stimulate neurite growth. A growing body of evidence suggests that endocytosis of ligand-receptor complexes may act as a mechanism for guidance receptor activation and signaling (Bashaw and

Klein, 2010; Cowan et al., 2005; Hines et al., 2010). For example, bidirectional endocytosis of Eph/Ephrin has been shown to regulate cell detachment and induce axonal repulsive signaling. Thus, whether GammaCop triggers

LAR/Caskin internalization into signaling endosomes or recycles endosomes to the surface of the growth cones requires further investigation.

PTEN counteracts PI3K/Akt signaling and has been shown to regulate several aspects of neuronal development, including neurite growth, neuronal arborization, and myelination (Cotter et al., 2010; Kwon et al., 2006). Recently,

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PTEN was found to couple Sema signaling in the induction of growth cone collapse (Oinuma et al., 2010). Sema stimulation leads to the activation of PTEN via PTEN dephosphorylation. Although PTEN has many known target substrates, which substrate molecules are responsible for altering actin dynamics remains elusive. Because Caskin serves as a LAR adaptor protein, it is possible that LAR localizes PTEN via Caskin to effect changes in growth cone motility.

In summary, our yeast two-hybrid screen identified several potential axon guidance regulators interacting with C-terminus. Functional characterization of these proteins will provide an important step in elucidating Caskin’s function in different cellular contexts, and particularly in LAR-mediated motor axon guidance. It will be exciting to assess whether Caskin-interacting proteins identified in our screen have genetic interactions with LAR and Caskin in their

LOF ISNb bypass phenotype. Interestingly, although protein sequences in each domain of Caskin are highly conserved between Drosophila and mammalian homologues, the flanking regions vary widely between species. Therefore, it is still unclear whether the interaction networks surrounding Caskin are similar from

Drosophila to higher vertebrates. For example, mammalian Abi-2 and CASK were found to interact with Caskin1 in vitro (Balazs et al., 2009; Tabuchi et al.,

2002). However we did not observe interaction between their Drosophila homologues and Drosophila Caskin in the yeast two-hybrid interaction assay

(Table 3.3). This suggests that mammalian Caskin may have novel protein functions evolved through a different set of interactions.

Interactions between Caskin and Liprin family proteins

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The Drosophila Liprin family consists of Liprin-alpha, Liprin-beta and Liprin- gamma (Astigarraga et al., 2010). Each member of the Liprin family has three

SAM domains which have known function in mediating protein-protein interactions. A number of studies have established a function for Liprin-alpha in synaptogenesis. However, the roles of Liprin-beta and Liprin-gamma in neural development are largely unexplored. Recently a vertebrate homolog of Liprin- gamma, KazrinE, has been shown to associate with acetylated microtubules in human keratinocytes (Nachat et al., 2009). Overexpression of KazrinE leads to profound changes in cell shape, suggesting the possibility that KazrinE may regulate the cytoskeleton. Given that growth cones are enriched with acetylated

α-tubulin, and that Caskin and Liprins are capable of binding with LAR, we assessed whether Caskin interacts with Liprin family proteins to induce cytoskeleton rearrangement.

We first used yeast two-hybridization to assess pairwise interactions between

Caskin and Liprins. We found that neither Liprin-alpha nor Liprin-beta is able to interact with Caskin. We did however observe physical interaction between

Liprin-gamma and Caskin. Interactions were further confirmed by analysis of β- galactosidase activity, indicating that Caskin and Liprin-gamma form a complex that activates the LacZ reporter gene. Interestingly, the cknY allele, which bears with a point mutation in the conserved C-terminal domain (CTD), is defective in binding of Liprin-gamma (Table 3.3). Because our genetic and biochemical studies indicate the CTD of Caskin is critical for relaying signaling events downstream of LAR, the specific interaction observed between Liprin-gamma

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and Caskin’s CTD suggests that Liprin-gamma may function downstream of

Caskin.

In contrast to KazrinE’s ubiquitous expression, Liprin-gamma is exclusively

expressed in the developing Drosophila nervous system at an embryonic stage

(Astigarraga et al., 2010; Nachat et al., 2009). Biochemical studies have shown

that Liprin-gamma interacts with LAR and Liprin-alpha via different regions.

Given that both LAR and Liprin-alpha function in retinal axon targeting as well as

synaptic morphogenesis, phenotypic characterization of Liprin-gamma mutants

were previously analyzed in these two developmental contexts. Surprisingly,

Liprin-gamma mutants do not exhibit any overt defects in either their retinal axon

projections, or their synaptic morphology at larval NMJs (Astigarraga et al.,

2010). Given their physical interaction, it will be important to assess whether

Liprin-gamma is involved in LAR signaling within other contexts. Future work will

assess Liprin-gamma’s involvement in motor axon guidance and potential

genetic interactions with LAR and Caskin in this context.

Conclusion

In this study, we demonstrate Caskin becomes tyrosine phosphorylated in the presence of Abl kinase and its Abl-dependent phosphorylation does not abrogate interaction with LAR phosphatase. An attractive hypothesis is that LAR dephosphorylates Caskin allowing it to interact with downstream effectors.

Although validation of our model awaits the identification of Caskin’s downstream

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targets and demonstration of phosphorylation-dependent interactions, genetic interactions between ckn and abl are suggestive.

Our previous work demonstrated that C-terminus Caskin is responsible for relaying LAR signaling events; overexpression of truncated Caskin lacking the C- terminal region blocks LAR signal transduction and induces motor axon bypass phenotype. Our identification of interacting proteins that bind to C-terminus

Caskin provides an important step in dissecting the molecular basis of LAR- mediated motor axon guidance. Although most of these proteins have potential roles in axon guidance, future experiments will be necessary to determine their function in the context of LAR signaling.

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Figure 3.1

k k wild type ckn /ckn

A B

k k 2 ckn /ckn ;abl /+

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Figure 3.1: Reduction in abl genetic dosage rescues ckn LOF phenotype

Abdominal segments of late 16/17 stage embryos were stained with mAb 1D4 to

label motor axon projections. Anterior, left; dorsal up.

(A) Wild type ISNb-projecting axons defasciculate from ISN and display a

stereotypic innervation pattern in the ventrolateral muscle field (arrows). (B) In

cknK/ cknK LOF mutants, ISNb axons follow the ISN axon pathway (arrows) and do not enter their target muscle field. (C) cknK/ cknK ; abl2/+ embryos. ISNb axons

separate from ISN and re-enter the ventrolateral muscle field at the normal entry

point. Axons are able to recognize distal synaptic targets (arrows). Nonetheless,

innervation defects are still evident.

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Figure 3.2

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Figure 3.2: Abl-dependent tyrosine phosphorylation occurs on Caskin

(A) Full length Caskin-HA is transfected alone or co-expressed with Abl kinase in

the S2R+ cells. Caskin was immunoprecipitated using anti-HA antibody and subsequently western blotted using anti-phosphotyrosine antibody (4G10). The blot was stripped and reprobed using anti-HA antibody (12CA5) to confirm equal

loading of Caskin. In the presence of Abl kinase, Caskin can become tyrosine

phosphorylated.

(B) Full length Caskin-Flag together with different DLAR variants were co-

expressed in S2R+ cells in the absence or presence of Abl kinase. DlarD1CS

encodes a catalytic dead version of DLAR whereas DlarPPLL is a monomeric

form of DLAR. Equal amount of DLAR was used to immunoprecipitate Caskin-

Flag. Interactions between Caskin and these DLAR variants remain even in the

presence of Abl kinase.

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Figure 3.3

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Figure 3.3: Model of the interplay between Abl and Caskin during motor axon guidance

(A) In wild type, the phosphorylation state of Caskin is regulated by the enzymatic activities of both Abl kinase and DLAR phosphatase. Caskin with no

Abl-dependent tyrosine phosphorylation can assemble downstream binding partners, leading to target recognition events. (B) In ckn LOF mutants (cknA and cknK), disruption of DLAR-Caskin interactions prevents DLAR dephosphorylation of Caskin. In this circumstance, Caskin accumulates tyrosine phosphorylation over time and in turn dissociates from the signaling complex. (C) In cknK/cknK; abl2/+ embryos, reduction in abl expression returns the phosphorylation state of

Caskin to normal levels, allowing Caskin to interact with downstream binding partners.

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Table 3.1: Caskin-interacting proteins indentified by a yeast two-hybrid screen

Gene Function Numbers CG8948(Graf) RhoGAP 2 CG1528 (GammaCop) Vesicle transport 4 CG5671(PTEN) Protein tyrosine phosphatase 1 CG1560 (Mys) Cell adhesion molecule 1 CG4145 (Collagen type IV) Cell adhesion molecule 1 CG1651 (ANK) Cytoskeletal linker protein 3 CG11963 (Skap) MT-associated protein 1 CG2198 (Ama) Ig superfamily protein 2 CG8863 (DnaJ-like-2) Molecular chaperone 1 CG10528 Unknown 1 CG7052 Unknown 1 CG3348 Unknown 1 CG6783 Unknown 1 CG7533 Unknown 1 The C-terminal portion of Drosophila Caskin (amino acids 594–924) was used as bait to screen a Drosophila embryonic yeast two-hybrid cDNA library. Strong potential interactors were isolated under high stringency conditions. The

‘Numbers’ column represents redundancies in clone identification.

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Table 3.2: GOF and LOF phenotypes in motor axon guidance genes

Gene GOF phenotypea LOF phenotype Dlar wt Bypass ckn Stall Bypass ena wt Bypass rac 1 n.d Bypass abl Bypass Stall trio n.d Stall a Neuronal GOF phenotype was examined in late stage 16 embryos.

Bypass phenotype was defined as the failure of ISNb axons to defasciculate from

ISN; axons remain in the common ISN pathway and project to the dorsal muscle field. Stall phenotype was defined as normal ISNb entry into the ventrolateral muscle field, but failure of axons to reach more distal synaptic targets.

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Table 3.3: Pairwise interaction between Caskin and candidate proteins

Bait Prey Interaction Ckn-SAM Ckn-Full - Caki Ckn-Full - Ena Ckn-Full - Pak Ckn-Full - Abi Ckn-Full - Homer Ckn-Full - Cdk5 Ckn-Full - Trio GEF domain Ckn-Full - Liprin-alpha Ckn-Full - Liprin-beta Ckn-Full - Liprin-gamma Ckn-Full + Liprin-gamma Ckn-Y -

A yeast two-hybrid assay was used to examine the interactions between indicated proteins.

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CHAPTER 4

General Discussion

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Redundancy and complexity of LAR signaling

Vertebrate LAR family proteins share high sequence homology in both their extracellular and intracellular regions, suggesting they may interact with the same binding partners and confer functional redundancy. In mammals, LAR family proteins have overlapping expression patterns in the developing nervous system. Single knockout of LAR, PTPsigma, or PTPdelta may therefore yield an underestimate of each receptor’s role neural development. Further characterization and comparison of double and triple knockout mice may unravel physiological functions conveyed by the LAR family.

Notably, LAR family proteins exhibit overlapping but not identical functions. For example, PTPsigma and PTPdelta function redundantly in regulating motor axon targeting (Uetani et al., 2006). Loss of PTPsigma causes impaired proprioception and abnormal brain development whereas deficiency of PTPdelta contributes to a retarded spatial learning ability. It was thought that differential binding of ligands or differential regulation of downstream substrates contributes to LAR family protein heterogeneity. For example, TrkC interacts with PTPsigma, but not with LAR or PTPdelta, to promote glutamatergic synapse formation (Takahashi et al., 2011). My work identified Drosophila Caskin as a novel LAR-interacting protein and showed that this interaction is evolutionarily conserved. Interestingly, mouse Caskin2 associates with LAR and PTPsigma, but not PTPdelta. The related mouse Caskin2 homologue, Caskin1, is also expressed in the developing nervous system (Tabuchi et al., 2002). Caskin1 and Caskin2 have small sequence differences in the N terminus region, SAM domains, and the conserved

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C-terminal domain, suggesting they may not have the same binding partners.

Consistent with this idea, we found preliminary yeast two-hybrid evidence

indicating that Caskin1 interacts with LAR and PTPdelta, but does not interact

with PTPsigma. This suggests a model where Caskin1 and Caskin2 act in the

divergence of LAR signaling based on their differential binding to the three LAR

family receptors.

LAR family receptors are capable of forming homodimers and heterodimers, with

substrate availability a likely factor determining these interactions. LAR family

proteins confer functional redundancy by binding the same partners or delineate

their signaling by assembling different intracellular complexes. Therefore

together, a complete picture of LAR family signaling cascades is likely to be

highly complex.

LAR signaling cascades in axon pathfinding

My thesis work provided both genetic and biochemical evidence demonstrating

the involvement of Caskin in LAR mediated axon pathfinding. Phenotypic

analysis in ckn GOF and LOF embryos reveals Caskin has an important role in

growth cone decision-making. Changes in gene expression correlate with

discrete axon guidance behaviors; augmenting caskin expression causes axons

to stall at early choice points whereas reduction of caskin results in a bypass

phenotype. It is worth noting that Caskin lacks intrinsic enzymatic activity and

thus cannot directly act to regulate cytoskeleton. Therefore it is likely that Caskin

regulates the assembly of signaling complexes, and the genetic dosage of caskin

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correlates with the number of active signaling complexes. We propose that when axons reach their targets, ligand-engaged LAR causes Caskin to assemble downstream mediators, generating a “stop signal” that arrests growth cone advance.

How does LAR orient growth cones in response to different guidance cues? Our data suggests that Caskin serves as a molecular link between LAR and downstream cytoskeleton regulators. We hypothesize that local activation of LAR leads to asymmetric distribution of Caskin, which in turn regulates the spatial assembly of signaling complexes. Genetic interactions observed between LAR and Trio, Rac1, and Ena/VASP suggest that Caskin may interact with these molecules to relay guidance signals. Although Caskin was found to associate with LAR via its SAM domain, we did not observe any direct interaction in the yeast two-hybrid interaction assay between Caskin and Trio, and Ena/VASP

(Table 3.3), suggesting the existence of other unidentified interactors. Utilizing a yeast two-hybrid screening approach, we identified several proteins that interact with C-terminus Caskin. Functional characterization of these interacting proteins may provide insight into Caskin’s downstream signaling. Notably, although Dock was found to associate with Caskin, and Dock/Pak signaling is known to regulate actin dynamics and axon guidance in visual system (Hing et al., 1999; Newsome et al., 2000), we did not observe synergistic genetic interactions between Caskin and Dock/Pak. Thus, at least in context of motor axon guidance, Dock/Pak are unlikely to act downstream of Caskin in cytoskeleton regulation.

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The extracellular region of LAR contains three immunoglobulin-like (Ig) and nine fibronectin type III (FNIII) domains. These domains are responsible for recognizing extracellular ligands and initiating signaling cascades that underlie axon targeting and synaptogenesis. Phenotypic rescue experiments using different variants of LAR in the Drosophila visual system reveal that FNIII domains 7–9 are required for photoreceptor axon targeting (Hofmeyer and

Treisman, 2009). Importantly, a ligand responsible for LAR-mediated axon guidance has yet to be identified. The HSPGs Sdc and Dlp are high affinity ligands for LAR, and bind to Ig domains via heparan sulfate (HS) chains.

Surprisingly, mutants lacking either Sdc or Dlp exhibit synaptic abnormalities but do not exhibit axon guidance defects. Thus, Sdc and Dlp are unlikely to be relevant motor axon guidance ligands in Drosophila. We cannot exclude the possibility, however, that functional redundant ligands compensate to provide directional information. Further identification of extracellular ligands for LAR will elucidate how LAR discriminates between choice points along axon trajectories.

Engagement of RPTPs by their ligands is thought to alter phosphatase activity.

Crystallography of different RPTPs has demonstrated that receptor dimerization leads to occlusion of both D1 and D2 catalytic sites and subsequently inactivates phosphatase activity. The crystal structure of intracellular LAR indicates that LAR cannot undergo dimerization, however, biochemical studies suggest that full length LAR can form a dimer (Hofmeyer and Treisman, 2009). To date, little is known about how LAR activity changes upon binding of different guidance cues.

It has been hypothesized that ligand binding may either induce or disrupt LAR

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dimerization to alter phosphatase activity, ultimately changing the phosphorylation status of downstream effectors for forward transduction of LAR signaling. LAR appears to employ distinct mechanisms to control axon projections in different circumstances. For example, motor axon guidance defects in Dlar LOF embryos can be dramatically rescued by decreasing abl gene dosage, suggesting phosphorylation regulation is required in motor axon targeting (Wills et al., 1999a). In contrast, the phosphatase activity of LAR is dispensable for axon targeting in the Drosophila visual system. Introducing catalytically inactive LAR is still capable of restoring axon abnormalities in Dlar

LOF mutants (Hofmeyer and Treisman, 2009). Our work shows that Caskin functions downstream of LAR in regulating motor axon guidance defasciculation events. It may be interesting to assess Caskin function in the Drosophila visual system to address whether Caskin has a more general role in LAR-mediated axon guidance.

Receptor dimerization may also alter binding affinities between LAR and its downstream effectors. Although several LAR-interacting proteins have been found, their preference for association with monomeric or dimeric states of LAR remains to be studied. We proposed that Caskin is capable of binding with monomeric LAR because Caskin was found to co-immunoprecipitate with

DlarPPLL, a DLAR variant that cannot dimerize and exists in monomeric form.

Our study has shown that Caskin is capable of relocalizing LAR into Caskin positive puncta in Drosophila S2R+ cells. To further confirm that Caskin can

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interact with monomeric LAR, we can coexpress monomeric DlarPPLL and

Caskin and see if they are colocalized in the S2R+ cells.

Caskin in neural development

Caskin likely participates in multiple signaling pathways, regulating a vast number of physiological and developmental processes. Although Caskin exhibits a unique function in LAR-mediated motor axon guidance, our phenotypic analysis of ckn and dock double mutants argues that they have redundant function in promoting axon growth. Interestingly, whereas Dock binds to Pak to regulate cytoskeletal dynamics, we did not observe a physical interaction between Caskin and Pak, raising the possibility that Caskin may act through a distinct path to exert its action on cytoskeleton.

Our work and previous studies have together identified several binding partners of Caskin, providing a first step toward understanding Caskin’s molecular function (Balazs et al., 2009; Fawcett et al., 2007). In particular, some guidance receptors such as EphA2, L1CAM, LAR and PTPsigma were found to interact with Caskin in the yeast two-hybrid interaction assay. Whether these in vitro interactions are relevant to physiological binding and function requires further investigation. Because Caskin was thought to be a molecular scaffold protein, it is possible that Caskin acts via multiple mechanisms to direct axon targeting in different neuron types. Although our genetic interaction assays suggest that the physical association between Caskin and Dock does not mediate LAR signaling,

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we cannot exclude the possibility that they function together in other axon

pathways.

Caskin may function in contexts outside axon guidance. Caskin1 was originally

identified in rat brain extract as a CASK-interactor, and was shown to form a

complex with synaptic proteins (Tabuchi et al., 2002). RNA blotting analysis and

immunohistochemical studies have shown that Caskin has strict central and

peripheral nervous system expression, suggesting that Caskin may have an

essential role in neural function. In the adult rat brain, Caskin1 is primarily

localized to the neuropil and is enriched in synaptic areas. Given that CASK is

involved in synaptic assembly and function, the formation of a CASK-Caskin1

synaptic complex may point to function for Caskin1 in synaptic transmission.

Several studies have defined crucial functions for LAR in synapse formation and maturation (Dunah et al., 2005; Johnson et al., 2006; Kaufmann et al., 2002;

Kwon et al., 2010; Woo et al., 2009). For example, genetic ablation of Dlar in

Drosophila results in a reduction of synapse number and impairment of synaptic activity. The associations between Liprin-alpha and LAR were found to be required for LAR mediated synaptic maturation and plasticity. Our study demonstrated that the interaction of Liprin-alpha and Caskin with DLAR is mutually exclusive. These results are consistent with a model in which LAR couples different binding partners to exert distinct developmental processes; LAR regulates axon guidance via Caskin whereas LAR promotes synaptogenesis via

Liprin-alpha. However, we cannot exclude the possibility that Caskin participates in synapse morphogenesis by regulating the interaction between LAR and Liprin-

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alpha. Due to the early requirement of Caskin in axogenesis, employing

approaches that conditionally inactivate Caskin at a later developmental stage may allow examination of Caskin in synaptic development.

Axon regeneration

After injury to the CNS astrocytes become reactive, upregulating the intermediate filaments vimentin and glial fibrillary acidic protein and forming a scar around the lesion replete with chondroitin sulfate proteoglycan (CSPG), an inhibitor of axonal regeneration (Davies et al., 1997; Davies et al., 1999). Therefore astrocytic reactivity leads to both a physical and chemical barrier to axonal regeneration.

CSPGs comprise a core protein with multiple sulfated glycosaminoglycan (GAG) side chains (Silver and Miller, 2004). Extensive work has demonstrated that specific sulfation, predominately 4-sulfation, on sugar moieties is responsible for inhibition of axon growth (Gilbert et al., 2005; Wang et al., 2008). Degradation of sugar moieties from CSPG core proteins by chondroitinase ABC (Bradbury et al.,

2002), prevention of sugar addition to core proteins by decreasing xylosyltransferase expression (Hurtado et al., 2008) or reducing levels of 4- sulfated sugar moieties by inactivation of chondroitin sulfotransferase all represent potential strategies for neutralizing ligand-receptor binding and shaping a permissive growth environment for regenerating axons.

Several lines of evidences indicate that CSPGs act through signaling cascades to impede axon regeneration. For example, axons growing on CSPGs in culture have elevated levels of phosphorylated protein kinase C (PKC) and epidermal

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growth factor receptor (EGFR) (Koprivica et al., 2005; Sivasankaran et al., 2004).

Pharmaceutical inhibition of PKC or EGFR can help axons overcome CSPG inhibition, indicating that signaling crosstalk may exist between aPKC and activated EGFR. Recently, PTPsigma was uncovered as a receptor for CSPGs.

Biochemical assays indicate that a string of positively charged amino acids in the first Ig-like domain of PTPsigma is responsible for recognizing sugar moieties of

CSPGs. To establish the role of PTPsigma in mediating CSPG inhibition, functional studies were performed to show that DRG neurons lacking PTPsigma exhibited an increased ability to cross a CSPG gradient in vitro. Interestingly,

EGFR has been shown to be a substrate of PTPsigma in vitro (Suarez Pestana et al., 1999). Thus, it may be interesting to assess whether ligand engaged

PTPsigma activates EGFR and in turn inhibits axon regeneration.

Axon regeneration might be enhanced by targeting PTPsigma signaling.

Approaches that focus on preventing receptor-ligand interactions, such as function blocking PTPsigma antibodies or a soluble PTPsigma extracellular domain, might be developed to enhance axonal regeneration after injury. On the other hand, blockade of intracellular PTPsigma signaling is another way to neutralize CSPG inhibition. Given that truncated Caskin C terminus exhibited dominant negative activity in LAR-mediated axon guidance, it will be intriguing to test whether overexpression of this truncated Caskin is capable of interrupting

PTPsigma signaling and restoring axon growth in the presence of CSPGs. In addition, our sequence analysis of Caskin mutant alleles reveals a protein contact site for Caskin-LAR interaction. This contact site is localized in the first

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SAM domain and shares conserved amino acid sequences to vertebrate Caskin homologues. This information may yield the development of peptide inhibitors mimicking Caskin–LAR contact sites. Further studies will examine whether these strategies can disrupt the interaction between Caskin and LAR family members and attenuate CSPG inhibition of axon regeneration.

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