Identification and characterization of

Casein Kinase 2 as MuSK binding partner

Den Naturwissenschaftlichen Fakultäten der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades

vorgelegt von Tatiana Cheusova

aus Nowosibirsk, Russland

2006

Als Dissertation genehmigt von den Naturwissen- schaftlichen Fakultäten der Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 7. Juni 2006

Vorsitzender der Promotionskommission: Prof. Dr. D.-P. Häder Erstberichterstatter: PD Dr. F. Titgemeyer Zweitberichterstatter: Prof. Dr. M. Wegner

To my mother

Table of contents ______

Table of contents

Zusammenfassung...... 1 Summary...... 2 1. Introduction ...... 3 1.1. Structure and function of the NMJ...... 3 1.1.1. The presynaptic part is formed by motoneuron...... 4 1.1.2. The postsynaptic part is generated by myotubes...... 4 1.1.3. The role of Schwann cells at the NMJ...... 5 1.1.4. The basal lamina at the NMJ ...... 6 1.1.5. Physiology of the NMJ...... 6 1.2. Development of the NMJ...... 7 1.2.1. Origin of cells ...... 7 1.2.2. Establishment of nerve-muscle contact ...... 8 1.2.3. Postsynaptic differentiation ...... 9 1.3. Molecules and signaling cascades involved in the postsynaptic differentiation...... 10 1.3.1. -MuSK-rapsyn signaling cascade...... 10 1.3.2. Synapse specific transcription ...... 17 1.3.3. MuSK binding partners ...... 19 2. Aim of the study...... 23 3. Material and methods ...... 25 3.1. Materials ...... 25 3.1.1. Reagents...... 25 3.1.2. Devices ...... 26 3.1.3. Oligonucleotides...... 27 3.1.3. siRNAs...... 31 3.1.4. ...... 34 3.1.5. Kits and Columns ...... 34 3.1.6. Antibodies...... 35 3.1.7. Frequently used solutions ...... 36 3.1.8. Cell culture ...... 37 3.1.9. Animals...... 37 3.2. Methods...... 38 3.2.1. Molecular Biology Methods...... 38

I Table of contents ______

3.2.1.1. Isolation of plasmid DNA ...... 38 3.2.1.2. Determination of DNA/RNA concentration ...... 38 3.2.1.3. Electrophoretic separation of DNA fragments in agarose gel...... 39 3.2.1.4. PCR amplification of DNA...... 39 3.2.1.5. Cloning techniques...... 39 3.2.1.6. Plasmid constructs...... 40 3.2.1.7. Transformation of E. coli competent cells...... 42 3.2.1.8. Site directed mutagenesis...... 43 3.2.1.9. Lightcycler PCR...... 44 3.2.1.10. Total RNA isolation ...... 45 3.2.1.11. Complementary DNA-synthesis (Reverse Transcription) ...... 45 3.2.1.12. Yeast two hybrid (Y2H) techniques...... 46 3.2.2. Protein Biochemistry Methods ...... 49 3.2.2.1. Preparation of protein extract from cells and tissues...... 49 3.2.2.2. Immunoprecipitation ...... 49 3.2.2.3. Protein expression and extraction from bacteria...... 50 3.2.2.4. GST-pulldown...... 51 3.2.2.5. Determination of protein concentration ...... 51 3.2.2.6. Electrophoresis of proteins...... 51 3.2.2.7. Staining of protein gels ...... 52 3.2.2.8. Western blot ...... 53 3.2.2.9. In vitro kinase assay ...... 53 3.2.3. Cell culture methods...... 54 3.2.3.1. Cultivation of HEK293, Cos7, C2C12, MuSK-deficient myoblasts...... 54 3.2.3.2. Transient transfection of cells ...... 55 3.2.3.3. Luciferase reporter test...... 56 3.2.3.4. Agrin treatment ...... 57 3.2.3.5. Application of CK2 inhibitors...... 57 3.2.3.6. Immunocytochemistry...... 57 3.2.3.7. AChR cluster stability assay ...... 58 3.2.3.8. Quantification analysis of AChR clusters ...... 58 3.2.4.Animal care and immunohistochemistry methods ...... 58 3.2.4.1. Generation of muscle specific CK2β knockout animals...... 58 3.2.4.2. Genotyping...... 59

II Table of contents ______

3.2.4.3. Surgical Procedures...... 60 3.2.4.4. Immunohistochemistry...... 60 3.2.4.5. Mycroscopy, imaging and quantification of endplates...... 61 4. Results ...... 62 4.1. Searching for MuSK binding proteins ...... 62 4.1.1. Generation and characterization of MuSK baits for yeast two hybrid screens ...... 62 4.1.2. Outcome of the yeast two hybrid screens with MuSK baits...... 63 4.2. Detailed investigation of MuSK – CK2 interaction...... 65 4.2.1. Quantitative determination of CK2 transcript level in different tissues...... 65 4.2.2. Biochemical verification of the interaction of CK2 subunits with MuSK ...... 67 4.2.3. Mapping of interacting domains between MuSK and CK2β ...... 69 4.2.4. Localization of CK2 at the NMJ...... 72 4.2.5. Biological role of CK2 at the NMJ...... 76 4.2.5.1. Inhibition of CK2 activity ...... 76 4.2.5.2. Knockdown of CK2 subunits by using siRNA ...... 78 4.2.5.3. Phosphorylation of MuSK by CK2 ...... 80 4.2.5.4. Role of CK2 dependent serine phosphorylation of MuSK for AChR clustering ...... 82 4.2.5.5. Role of kinase insert domain of MuSK in AChR clustering...... 84 4.2.5.6. Mechanism of CK2 action...... 87 4.2.5.7. Generation and characterization of muscle-specific CK2β knockout mice...... 88 5. Discussion...... 94 5.1. Potential MuSK binding partners...... 94 5.2. CK2 – newly characterized MuSK binding partner...... 96 5.3. Phosphorylation of MuSK by CK2 is required for appropriate AChR clustering...... 97 5.4. KI domain of MuSK is involved in modulation of postsynaptic specialization...... 99 5.5. Role of CK2 in development of postsynaptic apparatus in vivo...... 100 6. Abbreviations...... 103 7. References ...... 105 Curriculum vitae ...... 114 Publications...... 115 Acknowledgments...... 116

III Zusammenfassung ______

Zusammenfassung

Die Synaptogenese an der neuromuskulären Verbindung erfordert u.a. die Bildung eines postsynaptischen Apparats, welcher duch die Sezernierung von Agrin an Nervenendigungen eingeleitet wird. Agrin stimuliert die muskel-spezifische Rezeptortyrosinkinase MuSK, dass seinerseits die Aggregation nikotinischer Acetylcholin-Rezeptoren herbeiführt. Signalwege, welche von MuSK aktiviert werden sind bisher nur unzureichend verstanden. Das Ziel der vorliegenden Arbeit war die Identifikation von Bindepartnern des MuSK. Dazu wurde das Verfahren des Hefe-2-Hybrid angewandt. Einer der Proteine, welcher mit der intrazellulären Region des MuSK interagiert, war die regulatorische β Untereinheit der (CK2β). Es konnte gezeigt werden, dass sowohl die katalytische α-, als auch die regulatorische β Untereinheit des CK2 in vivo mit MuSK interagieren. Zudem sind die Transkripte der CK2 Untereinheiten in der postsynaptischen Region der Myotuben adulter Mäuse angereichert. Inhibitor-, oder siRNA-vermittelte Reduktion der CK2 Aktivität beeinrächtigte die Aggregation nikotinischer Acetylcholin-Rezeptoren in Zellkulturmodell. Es konnte gezeigt werden, dass in vitro CK2 bestimmte Serine im ‚kinase insert’, einem bisher funktionell nicht charakterisiertem Epitop von MuSK phosphorylieren kann. Der Ausfall dieser Phosphorylierung geht mit einer fehlerhaften Aggregation der nikotinischen Acetylcholin-Rezeptoren einher. Weitere Experimente zeigten, dass diese Beeinträchtigung der Aggregation der Acetylcholin-Rezeptoren auch zu beobachten ist, wenn das ‚kinase insert’ von MuSK mit dem ‚kinase insert’ anderer Rezeptortyrosinkinasen ausgetauscht wird, welche keine CK2-phosphorylierbaren Aminosäuren enthalten. Die Behandlung von Myotuben-Kulturen mit einem CK2-Inhibitor zeigte, dass nicht die Kinase-Aktivität von MuSK abhängig von der Phosphorylierung genannter Serine ist, sondern die Stabilität der Acetylcholin-Rezeptoren. Schliesslich wurde die Bedeutung dieser Interaktion zwischen MuSK und CK2β in vivo untermauert. Die Deletion des CK2β in Myotuben von Mäusen führte zu einem myasthenischen Phänotyp. In dieser Studie wurde erstmals sowohl eine funktionelle Bedeutung für das ‚kinase insert’ Epitop von MuSK nachgewiesen, als auch die Abhängigkeit der Synaptogenese des postsynaptischen Apparates von Phosphorylierungen von Serinresten demonstriert.

1 Summary ______

Summary

The formation of the postsynaptic apparatus at the is initiated by the release of agrin from the nerve terminal and subsequent activation of the muscle-specific receptor MuSK which leads to the aggregation of nicotinic acetylcholine receptors. Signaling pathways downstream of MuSK are poorly understood. The goal of this study was to investigate MuSK downstream pathways by identification of MuSK interactors using a yeast two hybrid system. One of the identified proteins interacting with the intracellular domain of MuSK was the regulatory β subunit of the Casein Kinase 2 (CK2β). Our study has shown that both the catalytic α and the regulatory β subunits of CK2 interact with MuSK in vivo and that their mRNAs as well as proteins are concentrated at postsynaptic specializations of adult mice. Inhibition of CK2 activity either by chemical compounds or by siRNA in muscle cell culture resulted in impairment of AChR cluster morphology. Further investigations have revealed that CK2-mediated phosphorylation of MuSK occurs at serines 680 and 697 which belong to a domain of unknown function separating the kinase domain in two part and called ‘kinase insert’. The phosphorylation of these serine residues is required for appropriate AChR clustering. Consistently, the replacement of the MuSK kinase insert domain by kinase insert domains of other receptor tyrosine kinases containing potential CK2-phosphorylatable serines correlated with their ability to mediate proper AChR clustering. MuSK kinase activity was not changed, but AChR cluster stability dramatically decreased upon blockage of CK2. Muscle-specific ablation of CK2β in mice resulted in the change of CK2 activity and fragmentation of muscle endplates accompanied by a myasthenic phenotype. This study demonstrates that CK2-mediated phosphorylation of serine residues inside of the MuSK kinase insert domain plays an important role for the development of postsynaptic specializations at the neuromuscular junctions.

2 Introduction ______

1. Introduction

According to the definition of Sherrington, synapses are points of contact between two neurons (Pearce 2004). Nowadays, this term describes a sophisticated machinery which is required to ensure proper transmission of information between neurons (at the central nervous system; CNS) or neurons and muscle cells (at the peripheral nervous system; PNS). More precisely, synapses are composed of a presynaptic and a postsynaptic part. Neurotransmitter molecules are released from the presynaptic nerve terminal and interact with neurotransmitter receptors thereby activating them in the membrane of postsynaptic cell. To ensure a rapid and reliable transmission, first, the presynaptic terminal has to be organized in a way to maximize the efficacy of neurotransmitter secretion and, second, receptors at the postsynaptic membrane must be present in high density (a hallmark of postsynaptic specialization) directly opposite of the sites of neurotransmitter release. Despite good knowledge of synapse architecture, little is known about the processes, which lead to the presynaptic and postsynaptic differentiation. Much of current data originates from studies on the vertebrate neuromuscular junction (NMJ), a peripheral cholinergic synapse between motoneuron and skeletal muscle. This prototypical synapse offers a number of advantages, like large size, simplicity, accessibility, and availability of tools for its analysis (Sanes and Lichtman 2001).

1.1. Structure and function of the NMJ

The NMJ comprises portions of three cells – motoneuron, muscle fiber and Schwann cell. Basal lamina surrounds all three cells passing through the synaptic cleft and extending into the junctional folds formed by muscle fiber (Fig. 1).

3 Introduction ______

Fig. 1: Structure of the NMJ. The motor nerve terminal occupies a shallow gutter in the muscle fiber. The terminal Schwann cell caps the entire synaptic structure. Basal lamina passes through the synaptic cleft and extends into the junctional folds (from (Liyanage et al. 2002)).

1.1.1. The presynaptic part is formed by motoneuron

The motoneuron terminal is specialized for neurotransmitter release. It has a large number of synaptic vesicles containing the neurotransmitter acetylcholine (ACh), as well as numerous mitochondria, which provide the energy for its synthesis and release. Most of the vesicles cluster in the half-terminal that is opposed to the muscle fiber, whereas most of the mitochondria in the half-terminal beneath the Schwann cell (Fig. 1). Many of the vesicles are further focused at dense patches on the presynaptic membrane, called active zones, where they fuse with the presynaptic membrane thereby releasing their content into the synaptic cleft (Fig. 1) (Yee 1988). The best-studied molecules of the nerve terminal are proteins of the synaptic vesicles. Mostly these are the neurotransmitter ACh, the responsible for its synthesis choline acetyltransferase, and ACh transporter, which carries out vesicular storage of ACh by exchanging intravesicular protons for cytoplasmic ACh (Bravo and Parsons 2002; Calakos and Scheller 1996). Other components of synaptic vesicles are SNARE proteins, such as synaptobrevin and synaptotagmin that act as mediators of the vesicle fusion (Atwood and Karunanithi 2002).

1.1.2. The postsynaptic part is generated by myotube

The postsynaptic muscle membrane is specialized to respond effectively to released neurotransmitter. In the region which faces the motor nerve terminal it has a very high concentration of nicotinic acetylcholine receptors (AChRs) (>10000/µm2) (Salpeter and Loring 1985). Several actin binding proteins associate with the cytoplasmic portion of the

4 Introduction ______

AChRs thereby linking them to the muscle cytoskeleton, which is important for generation and maintenance of the high synaptic density of the receptors (Grady et al. 2000). The postsynaptic membrane of the muscle fibers is depressed into shallow gutters beneath the nerve terminals, and then further invaginated to form so-called junctional folds, which are about 0.1 µm wide, 1µm deep and spaced at 1-3µm intervals (Fig. 1). The throats of the folds open directly opposite of the active zones. AChRs are located at the crests and pathway down the sides of the folds, whereas voltage gated Na+-channels and the neural cell adhesion molecule (N-CAM) are found preferentially at the troughs of folds (Covault and Sanes 1986; Flucher and Daniels 1989). Such arrangements serve, first, to increase the contact area between nerve and muscle (the gutters), second, to have high AChRs density at ACh release sites (folds) and, at last, to facilitate the depolarization of the membrane leading to the generation of action potential and contraction of the muscle (distribution of AChRs and Na+- channels). Altogether, this organization enhances the efficacy of the synaptic transmission from nerve to muscle (Sanes and Lichtman 1999; Wood and Slater 1997). The cytoskeletal- binding protein composition of the folds is also heterogeneous: utrophin and α-dystrobrevin-1 are located together with rapsyn and AChRs at the tops of folds, while ankyrin, α- dystrobrevin-2 and dystrophin are concentrated at their bottoms (Flucher and Daniels 1989; Peters et al. 1998; Wood and Slater 1997). Cytoskeletal elements are most likely involved not only in generating the folds but also in maintaining the different domains with different proteins from which they are composed.

1.1.3. The role of Schwann cells at the NMJ

At the NMJ the entire synaptic structure is covered with specialized glial cells (Fig. 1). These cells are termed perisynaptic or terminal Schwann cells. In contrast to Schwann cells, which contact preterminal portions of the axon forming myelin sheaths, terminal Schwann cells are nonmyelinating. These two types of Schwann cells, though derived from the same progenitors, later, according to their functional specialization, differ structurally and express different genes. For example, myelin-forming Schwann cells highly express myelin basic protein, myelin-associated glycoprotein, and P0, whereas terminal Schwann cells are able to synthesize high levels of N-CAM and S-100 (Mirsky et al. 1996). Among functions of terminal Schwann cells are insulation of the nerve terminal from the environment and its supply with trophic sustenance. Terminal Schwann cells also play a role in recovery of synaptic integrity following injury (Son et al. 1996) as well as in modulation of synaptic

5 Introduction ______activity through short-term plasticity, which can contribute to the reliability of synaptic transmission (Colomar and Robitaille 2004).

1.1.4. The basal lamina at the NMJ

The structured form of extracellular matrix also known as basal lamina ensheaths the whole muscle fiber and passes at the site of nerve contact through the synaptic cleft, extending into the junctional folds (Fig. 1). The basal lamina of the NMJ contains members of large protein families like collagen IV, laminin, entactin, fibronectin, and heparan sulfate proteoglycans. Subsequently, synaptic and extrasynaptic portions of the basal lamina contain different isoforms of these proteins. The best known example is laminin, the β1 chain containing isoform of which is part of the extrasynaptic basal lamina, while β2 chain laminins are found exclusively in synaptic portion and originally were named s-laminins (Hunter et al. 1989). Other components strongly enriched in mature basal lamina include the collagen-tailed form of acetylcholinesterase (AChE), a set of glycoconjugates, and two signaling molecules, agrin and neuregulin (NRG) (Goodearl et al. 1995; Krejci et al. 1997; McMahan 1990; Scott et al. 1988). Synapse-specific components of basal lamina can be considered as candidate cues for regulation of synapse formation and function.

1.1.5. Physiology of the NMJ

Muscle contraction in response to a nerve impulse requires the sequential activation of at least five different sets of ion channels. First, the arrival of an action potential at the nerve terminal induces the opening of Ca2+ channels, which are located at active zones. As the [Ca2+] outside of cells is >1000 fold higher then inside, the Ca2+ ions flow into the nerve terminal. The increased [Ca2+] in the nerve terminal induces the fusion of synaptic vesicles at active zones and subsequent release of the neurotransmitter ACh into the synaptic cleft. In turn, ACh binds to postsynaptic muscular AChRs. The opening of AChRs leads to Na+ influx and local membrane depolarization, which induces opening of closely located voltage-gated Na+- channels. This results in further influx of Na+ and a self-propagating depolarization (action potential) that spreads through the entire muscle membrane. The generalized depolarization of the muscle cell membrane activates voltage-gated Ca2+-channels in specialized intracellular regions of muscle fiber, called transverse T-tubules. This induces the opening of Ca2+-release channels in the adjacent region of the sarcoplasmic reticulum and an efflux of Ca2+ ions in the cytoplasm from this intracellular reservoir. Sudden increase of the cytosolic Ca2+

6 Introduction ______concentration activates the muscle contraction (Alberts Fourth Edition; Sanes and Lichtman 1999). Unlike most synapses of the CNS the motor nerve of the NMJ releases more quanta of ACh than is required to induce an action potential and the following contraction of the muscle fiber. This excess of released ACh quanta establishes the safety factor of the NMJ, making the system extremely reliable (Slater 2003).

1.2. Development of the NMJ

1.2.1. Origin of cells

All three cell types composing the NMJ migrate a long distances to meet and establish a synapse (Fig. 2). The muscle fibers originate from mesodermal cells that acquire their myogenic identity in the dermatomyotomal portion of the somites. The myogenic cells migrate to sites of muscle formation, where they divide and differentiate into myoblasts. Beginning at embryonic day 11 (E11) in mouse, the myoblasts fuse in order to form multinucleated myotubes. At this time, genes that encode contractile and synaptic proteins are activated. Per definition, a myotube becomes a myofiber when the myonuclei move from the core of the tube to its periphery. Motoneurons arise in the ventral portion of the neural tube from multipotential progenitors. Motor axons exit the central nervous system through ventral roots or cranial nerves. They run long distances through peripheral nerves to the developing muscles. At E12-E13 the motor axons approach the muscle fibers and synapse formation starts.

7 Introduction ______

Fig. 2: Early steps of the NMJ formation. (a) Origin of cells comprising the NMJ. Myoblasts arise from somites, motor axons from somata in the neural tube, and Schwann cells from the neural crest. (b) Myotubes formed by fusion of myoblasts are approached by motor axons, closely followed by Schwann cells. (c) Initial contacts between growth cones of motor axons and myofibers are not specialized. (d) As development proceeds, presynaptic and postsynaptic specialization occurs, resulting at birth in (e) fully functional and multiply innervated NMJs (from (Sanes and Lichtman 1999)).

Schwann cells, the glial cells of the peripheral nervous system, derive from the neural crest, the dorsal part of the neural tube. Schwann cells and motor axons traverse the rostral halves of the somites, from which they acquire their segmental arrangement. Schwann cells become associated with motor axons somewhere within or near the somites and then follow them through the periphery into muscles. The motor axons supply Schwann cells with mitogenic stimuli and migratory guidance (Sanes and Lichtman 1999). It is assumed that prior to their contact the three cells types of the synapse already acquire their identity and express most of the proteins, which are subsequently concentrated at the NMJ. Even more interesting is that muscle cells are capable of formation of postsynaptic specialization (AChR clustering) already on early stages of embryogenesis and do not require nerve contact for this (Lin et al. 2001).

1.2.2. Establishment of nerve-muscle contact

During development, initial contact between motor nerve terminal and muscle fiber is established directly after the formation of the muscle fiber (Fig. 2) (Burden 2002). At this time the motor neuron is not specialized yet, but already capable of neurotransmission. Because initially both nerve terminal and muscle fiber lack their specialization, the efficacy of

8 Introduction ______the transmission is low. At E14 first signs of postsynaptic specialization indicated by the aggregation of AChR clusters appear on the surface of muscle fibers. These first clusters are aneural because they do not correspond to the geometry of the intercellular contacts. Innervation causes redistribution of these clusters and by E16-E18 all AChR clusters are matching to the nerve terminals. At birth, both nerve terminal and muscle fiber are greatly transformed and have accomplished their pre- and postsynaptic specialization. At this stage the NMJ is functional, but still immature (Luo et al. 2003b). In the first two or three postnatal weeks the NMJ undergoes a number of morphological changes which result in formation of pretzel-like shaped endplate innervated by a single-motor neuron. In mammals the main intramuscular nerve extends through the central region of the muscle, perpendicular to the long axis of the myotube. Individual motor axons branch and terminate forming synapses at the central end-plate band, which give the impression that the midpoints of muscle fibers are especially susceptible to innervation. An earlier hypothesis was that axons form synapses just at the sites of their entry into the muscle (Sanes and Lichtman 1999). Some observations of aneural AChR aggregates at the end-plate zone, which were supported further by genetic perturbation of motor neurons in animals throw into doubt this idea. In mice, lacking motoneurons AChR clusters (the sign of postsynaptic specialization) appeared in the muscle central end-plate zone, which has never seen a nerve. On the other hand, direct observation of synapse formation in nerve-muscle co-cultures has shown that neurites do not seek spontaneously formed high-density clusters of AChRs in uninnervated myotubes, but rather organize new clusters at initially unspecialized sites (Anderson and Cohen 1977; Frank and Fischbach 1979). One possible explanation is that a motor neuron randomly contacts aneurally formed AChR clusters, stabilizing them in case of matching and dispersing them in the case of mismatch. In this case, aneural clusters can serve as a back up system to ensure that every myotube will finally receive its synapse (Sanes and Lichtman 2001).

1.2.3. Postsynaptic differentiation

As soon as myoblasts fuse to form myotubes, they start to express AChR subunits and to assemble them into functional pentamers (α2βγδ), which will then be inserted in the plasma membrane. Originally, the distribution of the receptors is uniform with the density of ~1000/µm2. In the mature muscle the density runs up to >10000/µm2 at synaptic sites and

9 Introduction ______falls down to ~10/µm2 within a few micrometers from the synapse (Salpeter and Loring 1985; Salpeter et al. 1988). At least four distinct processes regulate this transition: 1. Redistribution of AChRs from the extrasynaptic to synaptic sites (Dai et al. 2000). 2. Increased stability of membranous AChRs from ~1 day (at embryonic stages) to 14 days (in adult) (Fambrough 1979). 3. Enhanced transcription of AChR subunit genes by synaptic nuclei (Schaeffer et al. 2001). 4. Repression of the AChR subunit gene expression in the extrasynaptic nuclei by electrical activity (Goldman et al. 1988).

1.3. Molecules and signaling cascades involved in the postsynaptic differentiation

1.3.1. Agrin-MuSK-rapsyn signaling cascade

During development of the NMJ postsynaptic specializations are induced by agrin, a heparan sulphate proteoglycan which is released by motoneurons. Originally agrin was isolated by McMahan and colleagues as factor of the basal lamina, which was sufficient to instruct the synapse to reform after denervation (Sanes et al. 1978). Expression studies in vivo and experiments on AChR aggregation in vitro allowed McMahan to postulate the “agrin hypothesis”, which reads that agrin is a critical nerve derived organizer of postsynaptic differentiation (McMahan 1990). Later this hypothesis has been confirmed. Synaptic differentiation was severely impaired in agrin knockout mice and led to premature death due to breathing failure (Gautam et al. 1996). Conversely, ectopic injection of expression plasmids expressing agrin into extrasynaptic region of innervated rodent muscle caused the formation of a fully functional postsynaptic apparatus in vivo (Jones et al. 1997). Agrin consists of more then 2000 amino acids and has a predicted molecular weight of 225 kDa. The actual mass of agrin is ~600 kDa due to the extensive N- and O- glycosylation of the protein N-terminus. The N-terminal region contains nine cysteine rich follistatin-like domains and laminin EGF-like region (Fig. 3). There are two serine/threonine-rich regions in the central part. The C-terminal region is characterized by four EGF-like repeats and three laminin G-like domains (Burden 2002).

10 Introduction ______

There are several agrin isoforms known, which are generated by alternative RNA splicing and have different tissue distribution and biological functions. Alternative splicing at the N- terminus causes the generation of two agrin isoforms. The first encodes a cleavable signaling sequence (SS) followed by the N-terminal agrin (NtA) domain responsible for laminin binding. The second isoform contains a non-cleavable signal-anchor, which converts agrin to a type II transmembrane protein.

Fig. 3: Schematic representation of the structure of agrin. GAG - glycosaminoglycan (from (Willmann and Fuhrer 2002)).

Agrin RNA can be spliced in at least two other positions which are known as A/y and B/z sites (A and B – for chicken and y and z – for mammals) and are present in the C-terminal laminin G-like domains. Splicing at these sites gives rise to proteins that can contain 0 or 4 amino acids at the A/y site and 0, 8, 11 or, 19 (8+11) amino acids at the B/z site. The amino acid insertion at the B/z site is crucial for the AChR clustering ability of agrin. Although agrin is expressed not only by motoneurons, but also by the muscle and Schwann cell, its “active” B/z+ isoform is synthesized only by neurons (Bezakova and Ruegg 2003). After agrin is released by motoneuron it activates at the muscle cell membrane the MuSK. MuSK, which was discovered because of its abundance in the synapse-rich Torpedo electric organ is specifically expressed by skeletal muscle (Jennings et al. 1993) and is now considered as the most critical component of the agrin receptor complex. The extracellular part of MuSK has four immunoglobulin (Ig)-like domains and a cystein rich domain (named also C6-box) (Fig. 4). The first Ig-like domain is required for the activation of

11 Introduction ______

MuSK by agrin. The fourth Ig-like domain and C6-box are important for the co-clustering with rapsyn (Zhou et al. 1999). In avians, fish and amphibians, the extracellular region of MuSK also contains a kringle domain of unknown function (Fu et al. 1999; Ip et al. 2000; Jennings et al. 1993). The intracellular region of MuSK contains a ~50 amino acid juxtamembrane domain, a kinase domain, and a short 8- aa C-terminal domain. The kinase domain is responsible for the kinase activity of MuSK, which is “a must” for agrin-induced AChR clustering. The kinase domain is divided into two subdomains by a region of disordered and exposed conformation, named kinase insert (KI). Nothing is known about the function of MuSK KI up to now except that this region contains a serine residue, which is probably phosphorylated by some serine/threonine kinase (Till et al. 2002). The C-terminal domain of MuSK has a sequence corresponding to the consensus PDZ domain-binding motif, but the significance of this domain for AChR clustering has not been discovered yet (Zhou et al. 1999).

Fig. 4: Schematic structure of MuSK. In the MuSK cytoplasmic domain, the amino acids critical for agrin-induced AChR clustering are highlighted by gray circles (from (Willmann and Fuhrer 2002)).

Several facts argue in favor of MuSK being a component of the agrin receptor complex. First, MuSK knockout mice show at birth a similar phenotype to that of agrin null mice. Both mutants are immobile, cannot breath and die at birth. Muscle derived proteins, including AChRs, AChE and ErbB receptors, which are normally concentrated at the postsynapse, are uniformly distributed in myofibers of MuSK knockouts (DeChiara et al. 1996). Second, myotube culture derived from MuSK deficient mice fails to form AChR clusters in response to agrin. This defect can be rescued by reintroduction of MuSK. Third, the application of agrin to muscle cells induces a dimerization and a rapid phosphorylation of MuSK intracellular domain (Watty et al. 2000). Forth, the expression of a dominant negative MuSK

12 Introduction ______mutant in C2 cells prevents the AChR clustering in response to agrin. And finally, MuSK and agrin can be found crosslinked after the application of chemical cross-linkers to muscle cells (Glass et al. 1996; Hopf and Hoch 1998). Though it was not possible to demonstrate direct binding of purified agrin to MuSK, the abundance of evidence strengthen MuSK in its status of the major component of agrin receptor complex. Interestingly, earlier in development, the phenotype of MuSK knockout mice is different from that of agrin deficient mice. In agrin knockout mice at E14 AChR clusters are present and concentrated in the central region of the muscle. In contrast, the AChR clusters are absent from MuSK ablated mice at all stages of development (Lin et al. 2001). These data indicate that MuSK plays a central role during the first steps of postsynaptic differentiation, whereas agrin is required during later stages for the synaptic growth and maintenance. The next important player in the process of the AChR clustering, which acts in agrin signaling downstream of MuSK is rapsyn (receptor-associated protein at the synapse). Rapsyn is a peripheral membrane protein that is present at the postsynaptic membrane of the NMJ and associates with AChRs in a 1:1 stoichiometry (LaRochelle and Froehner 1986, 1987). In transfected non-muscle cells rapsyn is able to cluster itself and, upon co-transfection, AChRs and some other postsynaptic components (Huh and Fuhrer 2002). Rapsyn has an unique protein structure in which the N-terminal myristoylation sequence, which mediates the targeting of rapsyn to the plasma membrane is followed by eight tetratricopeptide repeats that are necessary for rapsyn self association (Fig. 5). The eighth TPR overlaps with a coiled-coil motif that is required for the binding of rapsyn to AChRs and their clustering. The ring zinc finger motif and a serine phosphorylation site at the C-terminus were suggested to be necessary for linking rapsyn with postsynaptic cytoskeleton proteins and implicated in AChR stability (Banks et al. 2003; Huh and Fuhrer 2002). Rapsyn null mice lack aggregates of AChRs and some other postsynaptic proteins such as dystroglycan, utrophin, syntrophin and ErbB receptors (Gautam et al. 1995). Interestingly, MuSK is still localized at the NMJ of rapsyn knockout mice and responds to agrin by tyrosine phosphorylation in rapsyn -/- myotubes (Apel et al. 1997). This shows that MuSK is a critical component of the primary synaptic scaffold, whereas rapsyn clearly acts downstream of MuSK.

13 Introduction ______

Fig. 5: Schematic structure of rapsyn (from (Willmann and Fuhrer 2002)).

Whereas the main players of synaptic differentiation, agrin, MuSK and rapsyn are already known, the complete picture of the signaling processes that lead to AChR clustering has still to be puzzled out. 1. What is the ligand of MuSK? Although agrin can activate MuSK in myotubes, it is not able to activate MuSK, which is over-expressed in fibroblasts and even myoblasts (Glass et al. 1996). These data strongly suggest that MuSK is complexed with another molecule that is selectively expressed in skeletal muscle and required for agrin binding. This hypothetical muscle accessory component (MASC) can be a second agrin-binding subunit of the heteromeric MuSK-receptor complex (Fig. 6). Particular carbohydrates, which are present on the myotube surface, the MuSK molecule or the amino-terminal part of agrin might also play the role of MASC (Parkhomovskiy et al. 2000). In this case, the first Ig-like domain of the MuSK extracellular part, which is necessary for agrin responsiveness, should serve as a for MASC or as a place for carbohydrate modification (Zhou et al. 1999). 2. How is MuSK activated? It has been shown that in response to agrin MuSK undergoes dimerization and tyrosine phosphorylation that occurs within minutes. Six of the nineteen intracellular MuSK tyrosine residues become phosphorylated upon activation (Watty et al. 2000). Besides tyrosines within the activation loop of the kinase domain, which are necessary for MuSK kinase activity, the juxtamembrane domain tyrosine Y553 (Fig. 4) appears to be required for agrin-induced AChR clustering (Herbst et al. 2002). Furthermore, the specificity of MuSK signaling is determined in particular by the juxtamembrane domain, since this region of MuSK even in the context of a different kinase (TrkA) domain sufficient to activate the signaling cascade leading to postsynaptic differentiation in vivo (Herbst et al. 2002). The amino acid sequence

14 Introduction ______surrounding Y553 represents the typical NPXY motif, which can interact with a PTB domain- containing protein, which in turn can recruit other components of downstream signaling. 3. How aggregation and stabilization of AChRs occurs? In myotube culture agrin induces the tyrosine phosphorylation of AChR β- and δ- subunits (Ferns et al. 1996). It is believed that tyrosine phosphorylation facilitates the interaction of AChRs with the cytoskeleton and contributes mainly to the stability of AChR clusters, but not to initial steps of their formation (Huh and Fuhrer 2002). The kinase, which phosphorylates AChRs appears to be different from MuSK and is a member of the Src kinase family (Src, Fyn, Yes). These kinases have been found in association with AChRs in myotubes and agrin was able to cause their rapid activation and tyrosine phosphorylation, which was dependent on rapsyn. Additionally, Src kinases have been shown to form complexes with and phosphorylate MuSK in myotube culture (Fuhrer and Yang 1996; Mittaud et al. 2001; Mohamed et al. 2001). Interestingly, Src kinases interact with both MuSK and AChRs to some extent even before the addition of agrin, thus implying the existence of some preassembled protein complexes. The available evidence suggests that a MuSK-Src kinase complex acts as the primary synaptic scaffold and clustered at first by agrin (Fig. 6). The AChR-Src kinase complex is recruited later due to a link between MuSK and rapsyn. The fourth Ig-like domain together with the C6-box within the extracellular portion of MuSK is required for the formation of the link with rapsyn, suggesting that this domain can be involved in interaction with rapsyn through hypothetical protein RATL (rapsyn-associated transmembrane linking molecule) (Fig. 6), which is still not found (Zhou et al. 1999). Dystrophin/utrophin glycoprotein complex (D/UGC) is involved in the stabilization of muscle sarcolemma by linking the cytoskeleton of the muscle fiber to the extracellular matrix. Present at both synaptic and extrasynaptic sites, D/UGC differs in its protein composition (Huh and Fuhrer 2002). Interactions with components of D/UGC have been shown for several proteins of postsynaptic specialization. α-dystroglycan binds to agrin (Fig. 6) and was originally proposed as a candidate for MASC. However, subsequent studies have shown that an agrin fragment incapable of dystroglycan binding still activates MuSK and induces AChR clustering (Hopf and Hoch 1996; Jacobson et al. 1998). Rapsyn interacts with utrophin mediating link between AChRs and cytoskeleton components (Fig. 6) (Huh and Fuhrer 2002). Studies on knockouts of components of the D/UGC have shown that this complex is largely dispensable for the initial formation of AChR clusters, but, instead, is mainly required for their postnatal maturation and stabilization (Deconinck et al. 1997; Grady et al. 2000).

15 Introduction ______

4. What else regulates postsynaptic aggregations? Agrin induced postsynaptic specializations are likely to be regulated by additional signaling intermediates. It has been shown that calcium influxes are required for the AChR clustering (Megeath and Fallon 1998). Moreover, activities of several intracellular enzymes in muscle, such as Rho-family GTPases and nitric oxide synthetases (NOS), are increased in response to agrin (Jones and Werle 2000). Rac and Cdc42 small GTPases are known to control the actin polymerization, for example by inducing focal reorganization of actin cytoskeleton in response to extracellular cues. In turn, it has been shown that AChR clusters are tightly associated with several cytoskeletal proteins, including actin and agrin increases this association. Using dominant negative forms of Rac and Cdc42 it has been proven that the activity of the small GTPases is necessary for agrin-induced AChR clustering. P21-activated kinase (PAK), a well-known cytoplasmic kinase involved in cytoskeleton regulation, can act downstream of small GTPases. PAK is activated by agrin in muscle cells in a Rac and Cdc42 dependent manner and its inhibition leads to attenuation of AChR cluster formation. These data suggest the existence of a signaling pathway involving small GTPases and PAK that regulates the stabilization of AChR clustering by promoting their linkage with the cytoskeleton (Luo et al. 2002; Weston et al. 2000).

16 Introduction ______

Fig. 6: Agrin induced signaling cascades leading to assembly of the postsynaptic membrane at the NMJ. In the absence of nerve-derived agrin, at least two pre-assembled signaling complexes (AChR complex and a MuSK complex) exist in the muscle membrane. Agrin causes rapid activation of MuSK, which triggers downstream signaling cascades with the involvement of calcium, Rac, Cdc42, NO, and actin. Upon agrin stimulation Src/Fyn kinases phosphorylate MuSK and AChRs. Preassembled AChR complexes bind to the MuSK complex through rapsyn. Dystrophin/utrophin glycoprotein complex (D/UGC) is additionally recruited, stabilizing the entire postsynaptic apparatus. α -DG, α -dystroglycan; p, tyrosine phosphorylation (Modified from (Huh and Fuhrer 2002)).

1.3.2. Synapse specific transcription

The postsynaptic specialization is not only the result of clustering of synaptic proteins, but also of selective transcription of their genes. This process is carried out by myofiber nuclei near the synaptic site. Synapse specific transcription ensures that AChRs and other postsynaptic components are available in the required density in the postsynaptic membrane. Defects in synapse targeted gene expression of the AChR ε subunit gene have been shown to be the cause of congenital myasthenia (Nichols et al. 1999). In search for nerve-derived inducers of the synapse-specific transcription ARIA (AChR-inducing activity), an isoform of the secreted growth factor neuregulin (NRG)-1 has been isolated (Falls et al. 1993). NRG, like agrin, is synthesized by motoneurons and secreted into the synaptic cleft. NRG receptors are transmembrane tyrosine kinases ErbB, which are concentrated at the postsynaptic site of the NMJ (Rimer et al. 1998). It has been shown that in vitro NRG stimulation of ErbBs leads

17 Introduction ______to the activation of MAP/ERK and the Phosphatidyl-Inositol 3 (PI3)-kinase pathways (Tansey et al. 1996). ERK in turn activates the Ets family transcription factor GABP, which binds to regulatory element in the AChR ε and δ promoters, termed N-box, and stimulates the transcription of respective genes (Koike et al. 1995; Schaeffer et al. 1998). For a long time it was believed that NRG and agrin work in parallel: agrin promoting AChR clustering and NRG – activating local transcription. Later a pile of evidence changed this belief, awarding to agrin the main role in both processes. It has been shown that even in the absence of neuronal NRG synapse specific transcription of AChR occurs in a normal way (Yang et al. 2001). It raised the possibility that agrin or MuSK can cluster muscle-derived NRG and ErbBs, thus stimulating the transcription of AChR genes in an autocrine way (Meier et al. 1998). The group of H.R. Brenner demonstrated that agrin can induce MuSK transcription and, possibly, the transcription of other synaptic genes not only by the autocrine NRG/ErbB pathway, but also by a novel “shunt” pathway in which agrin-MuSK signaling stimulates the activation of Rac and JNK independent of NRG/ErbB (Fig. 7) (Lacazette et al. 2003).

Fig. 7: Model of synaptic gene expression. Nerve-derived agrin activates preexisting MuSK to induce the expression of MuSK gene by (1) organizing an NRG/ErbB pathway, involving MuSK-induced recruitment of ErbB receptors and of muscle-derived NRG, and by (2) MuSK induced activation of JNK via Rac/Cdc42. With MuSK expression stabilized, the same pathways are used for AChR and ErbB genes expression. Expression can be strengthened by NRG-1 secreted from nerve terminal. P, tyrosine phosphorylation (from (Lacazette et al. 2003).

Recent data from the same group about conditional inactivation of ErbB2 and ErbB4 receptors in muscle demonstrated that development and maintenance of NMJ are only marginally affected in the absence of all NRG signaling (Escher et al. 2005). These data

18 Introduction ______argues in favor of a new scenario, where agrin, in vivo, regulates, may be together with NRG, synapse specific transcription and NRG signaling is redundant with that of agrin.

1.3.3. MuSK binding partners

In an attempt to find a link from agrin-activated MuSK to rapsyn that clusters AChRs, several groups started to identify proteins that interact with different domains of MuSK. By a proteomic approach Strochlic et al. have identified MAGI-1c as a MuSK binding partner. MAGI-1c belongs to the MAGUK family, which includes scaffolding proteins possessing multiple protein-protein interaction domains and is involved in cell polarity and the organization of signal transduction within cellular junctions. Among other domains MAGI-1c contains six PDZ domains, the fourth and fifth were shown to interact with the consensus C-terminal PDZ binding site of MuSK (Strochlic et al. 2001; Strochlic et al. 2005a). MAGI-1c was colocalized with adult rat NMJs, but was not concentrated at agrin- induces AChR clusters in C2 myotubes, suggesting that the protein is not involved in the clustering mechanism (Strochlic et al. 2001). These data correlate with the fact that the PDZ binding site of MuSK is dispensable for its clustering activity in the muscle cell culture (Zhou et al. 1999). As the other scaffolding proteins important for the organization of central excitatory synapses, e.g. PSD-95/SAP90, MAGI-1c might be involved in the formation of specialized signaling proteins complexes at the NMJ (Strochlic et al. 2001). Consistent with a number of data about the participation of actin in the process of AChR aggregation, several MuSK binding partners involved in actin cytoskeleton reorganization have been identified. Searching by yeast two hybrid (Y2H) technique for MuSK interacting proteins the group of L. Mei has revealed Dishevelled (Dvl) (Luo et al. 2002). Dvl was originally discovered in Drosophila, where it is implicated in the development of coherent arrays of polarized cells via the Wnt signaling pathway. The authors demonstrated the co- localization of Dvl with AChR clusters at the NMJ. The interaction of Dvl with MuSK was mapped to both the juxtamembrane and the kinase domains of MuSK, however this association was not facilitated by MuSK phosphorylation. The disruption of the interaction between MuSK and Dvl or inhibition of Dvl function attenuated agrin-induced AChR clustering in C2 cells and formation of neuromuscular synapses in culture. It has been shown that Dvl interacts with PAK1, an effector of Rac and Cdc42 in actin reorganization. PAK1 is activated in muscle cells upon agrin stimulation and this activation is required for AChR clustering. Consistently, induced PAK1 activation and subsequent AChR clustering was

19 Introduction ______attenuated in cells expressing Dvl mutants, suggesting the importance of Dvl for this event. Thus, the possible function of Dvl could be the recruitment of PAK to the MuSK signaling complex for AChR clustering (Luo et al. 2002). Another MuSK binding partner, potentially important in downstream signaling with involvement of actin reorganizers – geranylgeranyltransferase I (GGT I) has been identified by the same group in the Y2H screen with MuSK intracellular domain. It has been demonstrated that the α subunit of GGT I interacts with the kinase domain of MuSK. Agrin caused a rapid increase of GGT activity. The blockage of GGT I activity resulted in the abolishment of Rac1, Cdc42 and PAK1 activation in response to agrin and attenuated AChR cluster formation in muscle cells. Moreover, transgenic mice, expressing an inactive mutant of GGT I reveal defects in NMJ formation (Luo et al. 2003a). These data strongly indicate that the prenylation of agrin-induced signaling components and their following membrane targeting is important for AChR clustering. However, the role of MuSK-GGT I interaction in this model is still unclear. The tumor suppressor and actin binding protein Adenomatous Poliposis Coli (APC) has been shown to interact with the AChR β subunit (Wang et al. 2003). APC might help in localizing AChR to actin in the cytoskeleton, though its importance for AChR clustering in vivo has not been explored yet. Abelson tyrosine kinases Abl1 and Abl2 comprise a family of nonreceptor tyrosine kinases that regulate actin structure and presynaptic axon guidance. A.M. Pendergast and collaborators have hypothesized and successfully proven the role of Abl kinases in postsynaptic assembly. In the muscle Abl was localized to the postsynaptic site of the NMJ. In C2 culture Abl activity was required for the enhancement of agrin-induced MuSK tyrosine phosphorylation and AChR clustering. Moreover, Abl and MuSK interacted physically and effected reciprocal tyrosine phosphorylation. These findings suggest that Abl kinases might act by amplification of initial agrin-induced signaling through the tyrosine phosphorylation and also participate in cluster assembly by cytoskeleton remodelling (Finn et al. 2003).

20 Introduction ______

Fig. 8: MuSK binding partners and downstream signaling pathways. Cytoplasmic effectors of MuSK – Abl, GGT, Dvl – activate Rac/Cdc42-PAK1 leading to actin cytoskeleton reorganization and AChR aggregation, likely through APC. The scaffolding protein MAGI-1c, binding to the C-terminal consensus PDZ binding site of MuSK, potentially recruits multiple, not identified, signaling molecules. Adaptor protein 14-3-3-γ represses synaptic gene expression via inhibition MAPK-PI3K signaling pathways. MuSK - Syne-1 interaction mediates clustering of synaptic nuclei at the synaptic sites (modified from (Strochlic et al. 2005a)).

Another protein, which has been shown to interact with MuSK is Synaptic nuclear envelope-1 (Syne-1). This protein is enriched at the envelope of the synaptic nuclei (Apel et al. 2000). Syne-1 has two calponin-homology (CH) domains in the N-terminal region that binds to actin (Korenbaum and Rivero 2002). It is believed that Syne-1 can cluster synaptic nuclei by tethering their envelope to the actin cytoskeleton. MuSK in this case would operate through Syne-1 clustering myonuclei to the NMJ. By mass spectrometry analysis of MuSK crosslinked products from the postsynaptic membrane of Torpedo electrocytes. A.Cartaud and collaborators identified the adaptor protein 14-3-3-γ. The 14-3-3-γ was co-localized with AChRs at the NMJ and co-immunoprecipitated with MuSK. The over-expression of 14-3-3-γ in C2 myotubes specifically repressed transcription of several synaptic genes, pointing out the role of the protein in gene regulation. Consistently, MuSK expression potentiated 14-3-3-γ repression of transcription (Strochlic et al. 2004). It is suggested, that the repressive function of 14-3-3-γ targets the downstream effectors of the NRG/ErbB pathway, namely Raf-1 (Strochlic et al. 2005b). MuSK-14-3-3-γ

21 Introduction ______interaction then would be important for localization of 14-3-3-γ at the synaptic site, where it could modulate NRG/ErbB pathway. These data provide another piece of evidence, which proves the involvement of MuSK in the regulation of synaptic gene expression at the NMJ.

22 Aim of the study ______

2. Aim of the study

In the light of available data, MuSK plays a pleiotropic role during NMJ development. In the last few years, a number of MuSK binding partners have been reported, shedding some light onto the mechanism of its action. Despite of many efforts, the steps in agrin-MuSK signaling between MuSK activation and AChR cytoskeleton anchoring and clustering via rapsyn still are poorly characterized. Among objectives of this study were: (1) identification of linker protein between MuSK and rapsyn and (2) elucidation of MuSK downstream signaling events by identification of proteins associated with MuSK intracellular domain. The search for MuSK effectors was performed using a Y2H system that allows an in vivo detection of protein-protein interactions. In this system cDNA encoding a protein of interest is cloned into a bait vector, creating a fusion of a GAL4 DNA binding domain (DNA-BD) and a protein of interest. A second cDNA encoding an interacting protein (or a library of cDNAs encoding an entire collection of different potential interactors) is cloned into a prey vector, creating a fusion of an activation domain (AD) of GAL4 and the interacting protein. When bait interact with library fusion protein the DNA-BD and AD of GAL4 are brought into proximity, reconstitute transcription factor and mediate transcription of reporter genes in yeast cell, which results in their ability to grow on selective growth conditions. Since extracellular Ig-like IV domain together with C6-box of MuSK have been suggested to be involved in co-clustering of MuSK with rapsyn trough the hypothetical protein RATL (Zhou et al. 1999), we have chosen these domains for generation of the bait vectors used in Y2H for identification of RATL-like interactors. The bait bearing only the Ig-like IV domain should have helped to evaluate the impact of C6-box. For the identification of proteins involved in MuSK-downstream signaling cascade, the baits were constructed on the basis of MuSK intracellular domains. The juxtamembrane (JM) domain of MuSK contains a tyrosine embedded into a NPXY motif, phosphorylation of which is believed to generate a potential docking site for downstream signaling proteins (Herbst et al. 2002). In order to identify such proteins, a bait containing JM domain of MuSK was generated. In order to facilitate identification of MuSK downstream signaling components we have used bait mimicking the dimerized and phosphorylated form of MuSK intracellular domain, which

23 Aim of the study ______it acquires upon agrin stimulation (named MuSK2xwt). Another kinase-defective variant of the same bait was used to address the question about tyrosine-phosphorylation dependent binding of interactors. Identified by the Y2H MuSK binding partners were further characterized and their biological role for NMJ development was assessed.

24 Material and methods ______

3. Material and methods

3.1. Materials

3.1.1. Reagents

Basic solutions and reagents used have been purchased from Roche Molecular Biochemicals (Mannheim), Carl Roth (Karlsruhe), Promega (Mannheim) or Sigma (Deisenhofen). Specific reagents used were:

Yeast Two-Hybrid: 5-bromo-4-chloro-3-indoxyl-beta-D- Sigma galactopyranoside (X-Gal) β-Mercaptoethanol Sigma 3-amino-1,2,4-triazole (3-AT) Sigma DMSO Roth Minimal SD Base media Clontech Amino acids for drop out (DO) supplement Sigma Yeast cells AH109, HF7c Clontech Carrier DNA Clontech YPD Medium Clontech Agar Bacto E. coli: BL21(Rosetta) Competent cells Novagen Escherichia coli XL1-Blue Competent cells Novagen Isopropyl-beta-D-thiogalactoside (IPTG) Sigma LB Broth Sigma Molecular Biology and Biochemistry Standard Techniques: Agarose electrophoresis grade Gibco/BRL Ethidiumbromid Gibco/BRL DEPC-treated water Roth Oligo-dT Invitrogen 10x M MulV Reverse Transcriptase Buffer New England Biolabs dNTPs Fermentas DNA polymerase 10xBuffer, Mg free Fermentas MgCl2 Fermentas Ampicillin Sigma Luciferin Promega TRIzol Invitrogen Rotiphorese gel 40 (40% w/v acrylamide solution with Roth

25 Material and methods ______

0.8% bisacrylamide in ratio 29:1) TEMED Roth Dithiothreitol (DTT) Roth Protease inhibitor cocktail tablets-Complete and Applichem EDTA free Phenylenmethylsulfonylfluorid (PMSF) Roche Lysozym Roth Coomassie Brilliant Blau Roche Protein A Sepharose CL4-B Amersham Ni-NTA agarose Qiagen Glutathione SepharoseTM 48 Amersham Ammonium Persulphate (APS) Roth Nitrocellulose membrane PROTRAN BA85 Schleicher & Schuell Bioscience P81 phosphocellulose paper Whatman Immunocytochemistry: Sodium azide Sigma Bovine Serum Albumin (BSA) Sigma Fetal Calf Serum (FCS) Serva Mowiol Invitrogen Proteinase K Roth Radiochemicals γ-32P ATP Amersham Table 3.1. Specific reagents

3.1.2. Devices

Horizontal gel electrophoresis apparatus Gibco/BRL (33 cm x 42 cm) Horizontal gel electrophoresis apparatus OWI Electrophoresis Power Supply PS 304 Life Technologies Vertical mini-gel system Sigma Videodocumentation system for DNA- Biometra Gels Gel-Blotting apparatus Multiphor II Pharmacia Film developer X-Omat 1000 Prozessor Kodak Gel dryer SE1160 Hoefer Scientific Instruments Phosphoimager Molecular Dynamics Water Bath GFL Centrifuge 5415D Eppendorf Centrifuge 5417R Eppendorf Centrifuge 5810R Eppendorf Centrifuge RC 5B Plus Sorvall SpectroPhotometer Ultrospec 3000 Pharmacia Vortexer REAX2000 Heidolph Thermomixer compact Eppendorf Luminescence reader Lumat LB9501 Berthold

26 Material and methods ______

PCR-Thermocycler PTC-2000 Peletier MJ Research Thermal Cycler Lightcycler Roche Glass Teflon homogenizer Wheaton Homogenizer Kinematica AG (Dispersing and Mixing Technology) Ultrasonic Disintegrator Sonifier Branson Confocal laser-scanning microscope Leica (LSM 5 Pascal) Microscope MZ75 Leica Microscope DMIL Leica Inverted microscope (DMIRB) equipped Leica/ Princeton Instruments with a cooled MicroMax CCD camera Cryotome CM3050S Leica Microsystems, Nussloch Liquid-Scintillation machine Wallac Pharmacia 1410 Table 3.2. Devices

3.1.3. Oligonucleotides

Oligonucleotides were synthesized by MWG-Biotech AG or Invitrogen. Their positions on the relative transcripts are indicated and correspond to the beginning of the coding sequence for forward (bp-) and the end of complementary sequence for reverse (-bp) primer. In primers used for subcloning restriction sites sequences are included.

Name of Sequence (5’-3’) Restriction Acc.No. Position the primer digestion site (bp) MuSK-17 CCGGAATTCATAGCTACCAATAAG EcoRI NM_010944 847- CAC MuSK-18 CCGGAATTCTGCCTGGCGGTAAAG EcoRI NM_010944 1201- GAG MuSK-21 ACGCGTCGACTTAGGAGTACGCAG SalI NM_010944 -1624 GCGAGAC MuSK-22 CCGGAATTCGAGTCGACCGCGGTG EcoRI NM_010944 1594- ACC MuSK-23 CGCGGATCCTTACAGGCTGAGCAA BamHI NM_010944 -1711 CTTAGG MuSK-38 GGAATTCTATTGCTGCCGAAGGAG EcoRI NM_010944 1552- GAAA MuSK-61 ACGCGTCGACTTACTTGGGATTCA SalI RNU34985 -1809 GAAGGA MuSK-62 ACGCGTCGACTTAGCGCTGCAGGA SalI RNU34985 -2164 TCCGGT MuSK-63 ACGCGTCGACTTACAGGTCACTGT SalI RNU34985 -2688 GGCTGA MuSK-64 ACGCGTCGACTTAGACGCCTACCG SalI RNU34985 -2724 TTCCCA Table 3.3. Oligonucleotides, which were used for the generation of MuSK bait plasmids for a yeast two hybrid (Y2H) screening and mapping experiments. Sequences of restriction digestion sites are underlined.

27 Material and methods ______

Name of Sequence (5’-3’) Restriction Acc.No. Position the primer digestion site (bp) CK2β-24 GGGGATCCGTATGAGCAGCTCAGA BamHI NM_001320 147- GGAG CK2β-25 GAAGATCTTTATCGGTAGTGAGGG BglII NM_001320 -481 ACCT CK2β-26 GAAGATCTTTAGTCCAAGATCATG BglII NM_001320 -505 TCTAG CK2β-27 GAAGATCTTTAGTCTTCCAGTTCTT BglII NM_001320 -532 CATC CK2β-28 GAAGATCTTTAGCACTTGGGGCAG BglII NM_001320 -760 TAGAG Table 3.4. Oligonucleotides used for subcloning of CK2β epitopes into the Y2H prey vector. Sequences of restriction digestion sites are underlined.

Name of Sequence (5’-3’) Restriction Acc.No. Position the primer digestion site (bp) CK2β-3 ATCAAGCTTCATGAGCAGCTCAGA HindIII NM_001320 341- GGAG CK2β-4 ATACTCGAGTCAGCGAATCGTCTT XhoI NM_001320 -988 GAC CK2β-7 ATAGGTACCATGAGCAGCTCAGAG KpnI NM_001320 341- GAG CK2β-12 ATAAAGCTTTCAGCGAATCGTCTT HindIII NM_001320 -988 GACTGG CK2β-29 CCGCTCGAGGATGAGTAGCTCTGA XhoI BC003775 147- GGAG CK2β-23 GGGGTACCTCAGCGAATAGTCTTG KpnI BC003775 -794 AC CK2α-3 CCGCTCGAGGATGTCGGGACCCGT XhoI BC060742 217- GCCA CK2α-4 GGGGTACCTTACTGCTGAGCGCCA KpnI BC060742 -1392 GC CK2α’-5 CCCAAGCTTGATGCCCGGCCCGGC HindIII BC057862 34- CGCG CK2α’-4 GGGGTACCTCATCGTGCTGCGGTG KpnI BC057862 -1086 AGAC Table 3.5. Oligonucleotides used for subcloning of CMV-driven plasmids expressing full length CK2β, CK2α or CK2α’. Sequences of restriction digestion sites are underlined.

Name of Sequence (5’-3’) Restriction Acc.No. Position the primer digestion site (bp) MuSK-39 GGAATTCGTATTGCTGCCGAAGGA EcoRI NM_010944 1552- GGAAA MuSK-40 CCGCTCGAGTTACTTAGGATTCAG XhoI NM_010944 -1698 AAGGAG MuSK-41 CCGCTCGAGTTAGCGCTGCAGGAT XhoI NM_010944 -2580 CCTGTG MuSK-42 CCGCTCGAGTTACAGGTCACTGTG XhoI NM_010944 -2055 GCTGAG MuSK-44 CCGCTCGAGTTAGACACCCACCGT XhoI MMU37709 -2755 TCCCTC Table 3.6. Oligonucleotides used for the PCR amplification of different MuSK intracellular domains for the generation of CMV – expression plasmids.

28 Material and methods ______

Name of Sequence (5’-3’) Restriction Acc.No. Position the primer digestion site (bp) CK2β-8 ATAAAGCTTTTATCGGTAGTGAGG HindIII NM_001320 -481 GACCGT CK2β-9 ATAAAGCTTTTAGTCCAAGATCAT HindIII NM_001320 -505 GTCTAG CK2β-10 ATAAAGCTTTTAGTCTTCCAGTTCT HindIII NM_001320 -532 TCATC CK2β-11 ATAAAGCTTTTAGCACTTGGGGCA HindIII NM_001320 -760 GTAGAG CK2β-12 ATAAAGCTTTCAGCGAATCGTCTT HindIII NM_001320 -988 GACTGG CK2β-16 CCGCTCGAGATGAGCAGCTCAGAG XhoI NM_001320 341- GAG CK2α-5 CCGCTCGAGATGTCAGGACCTGTG XhoI BC072167 172- CCAAG CK2α-6 CCCAAGCTTCTACTGAGTGGCTCC HindIII BC072167 -1377 AGCTG MuSK-47 CCCAAGCTTACCATGTATTGCTGC HindIII RNU 34985 1443- CGAAGGAGGAGAGAG MuSK-48 GGCCTCGAGTTGCTCTAGCTCAAG XhoI RNU 34985 -2727 AAATTCC Table 3.7. Oligonucleotides used for the generation of pGEXKG constructs, expressing the GST- fusions of MuSK intracellular epitope, CK2β, CK2α or CK2β epitopes. Sequences of restriction digestion sites are underlined.

Name of Sequence (5’-3’) Acc.No. Position the primer (bp) MuSK-67 CCTGGTCCTCCACCACTGGCCTGTGCAGAACAGCT NM_010944 2077- CTGCATTGCC MuSK-68 GGACCAGGAGGTGGTGACCGGACACGTCTTGCTG NM_010944 -2112 AGACGTAACGG MuSK-71 CGCACACTGTTTGCAGCCTCAGCCACGCTGACCTG NM_010944 2021- TCCACGAGGGCTCGGGTG MuSK-72 CACCCGAGCCCTCGTGGACAGGTCAGCGTGGCTG NM_010944 -2074 AGGCTGCAAACAGTGTGCG MuSK-73 CGCACACTGTTTGCAGCCTCAGCCACGATGACCT NM_010944 2021- GTCCACGAGGGCTCGGGTG MuSK-74 CACCCGAGCCCTCGTGGACAGGTCATCGTGGCTG NM_010944 -2074 AGGCTGCAAACAGTGTGCG MuSK-75 CGCACACTGTTTGCAGCCTCAGCCACGAGGACCT NM_010944 2021- GTCCACGAGGGCTCGGGTG MuSK-76 CACCCGAGCCCTCGTGGACAGGTCCTCGTGGCTG NM_010944 -2074 AGGCTGCAAACAGTGTGCG MuSK-77 GTCTAGCCCTGGTCCTCCACCACTGGACTGTGCAG NM_010944 2077- AACAGCTCTGCATTGCC MuSK-78 GGCAATGCAGAGCTGTTCTGCACAGTCCAGTGGT NM_010944 -2129 GGAGGACCAGGGCTAGAC MuSK-79 GCCCTGGTCCTCCACCACTGGAGTGTGCAGAACA NM_010944 2077- GCTCTGCATTGCC MuSK-80 GGCAATGCAGAGCTGTTCTGCACACTCCAGTGGT NM_010944 -2122 GGAGGACCAGGGC Table 3.8. Oligonucleotides used for the generation of MuSK point mutants. Sites of mutation are underlined.

29 Material and methods ______

Name of the Sequence (5’-3’) Restriction Acc.No. Position primer digestion site (bp) Musk.KIdel3 CGAATTCATGAGAGAGCTTGT EcoRI MMU37709 149- CAACATTC Musk.KIdel4 CCCTAGGAACTCATTGAGGTC AvrII MMU37709 2142- ACCATAG Musk.KIdel5 GCCTAGGGTGTGCAGAACAG AvrII MMU37709 2140- CTCTGC Musk.KIdel6 CTCTAGATTAGTATTGGTGAG XbaI MMU37709 -2646 GCCA IGF1R3 CTAGCGTCTCTGAGGCCAGAA Compatible with NM_010513 3279- GTGGAGCAGAATAATCTAGTC AvrII CTCATTCCTCCGAGCTTC IGF1R4 CTAGGAAGCTCGGAGGAATG Compatible with NM_010513 -3336 AGGACTAGATTATTCTGCTCC AvrII ACTTCTGGCCTCAGAGACG IR3 CTAGCGTCTCTGAGGCCAGAT Compatible with NM_010568 3367- GCTGAGAATAACCCAGGCCG AvrII CCCTCCCCCTACCTTGCAA IR4 CTAGTTGCAAGGTAGGGGGA Compatible with NM_010568 -3420 GGGCGGCCTGGGTTATTCTCA AvrII GCATCTGGCCTCAGAGACG PDGFbR3 AGCTAGCGCGACACTCCAACA NheI NM_008809 2240- AGCATTG PDGFbR4 GGCTAGCACTGGTGAGTCGTT NheI NM_008809 -2528 GATTAAG TrkC14.1 CTAGCGCTCTTTAATCCATCT Fit to AvrII S62933 2250- GGAAATGAT TTTTGTATATGGTGTGAG TrkC14.2 CTAGCTCACACCATATACAAA Fit to AvrII S62933 -2133 AATCATTTCCAGATGGATT AAA GAG CG Table 3.9. Oligonucleotides used for subcloning of MuSK kinase insert (KI) mutant constructs. Sequences of restriction digestion sites are underlined.

Name of the Sequence (5’-3’) Acc.No. Position Product primer (bp) size (bp) AChRα-1 ACGCTGAGCATCTCTGTCTT NM_007389 810- 448 AChRα-2 TTGGACTCCTGGTCTGACTT NM_007389 -1258 MuSK-24 GCCTTGGTTGAAGAAGTAGC NM_010944 115- 353 MuSK-25 CTTGATCCAGGACACAGATG NM_010944 -488 CK2β-1 AATGAGCAGGTGCCTCACTA BC003775 291- 376 CK2β-2 ACTCTGGATGCACCATGAAG BC003775 -667 CK2β-31 TCTGTGAGGTGGATGAAGAC BC003775 211- 453 CK2β-32 TGTGGATGCACCATGAAGAG BC003775 -664 CK2α-1 TGAGGATAGCCAAGGTTCTG BCO60742 944- 293 CK2α-2 TGCCATGCTAGTGGAACTCA BCO60742 -1256 CK2α’-2 CTGGCAGAGTTCTATCATCC BC057862 568- 489 CK2α’-2 CACGGTGTTCTCAGCACAAG BC057862 -1056 mbact111 TGGAATCCTGTGGCATCCATG NM_007393 885- 350 AAA mbact112 TAAAACGCAGCTCAGTAACA NM_007393 -1235 GTCCG Table 3.10. Oligonucleotides pairs used for quantitative PCR reactions. Size of resulting PCR product is given in bp.

30 Material and methods ______

Name of the primer Sequence (5’-3’) CK2β-17 GAGGGCATAGTAGATATGAATCTG CK2β-18 ATTTCTGAGATCGAGGCCAGTCTG CK2β-19 ATGAGTAGCTCTGAGGAGGTG CK2β-20 GGATAGCAAACTCTCTGAG HSA-CreF GACATGTTCAGGGATCGCCAGGCG HSA-CreR GACGGAAATCCATCGCTCGACCAG Table 3.11. Oligonucleotides used for genotyping PCR

3.1.3. siRNAs

For silencing of CK2 subunits two types of siRNA were used: pSUPERneoEGFP (Oligoengine) based and stealth siRNA. Targeting sequences were designed by Sfold program (Ding et al. 2004). Targeting oligonucleotides and stealth siRNAs were synthesized by Invitrogen.

Name Target Efficiency Primers Sequence of primers Acc.No. Position of used for or stealth siRNA inhibition subcloni ng pSuperNeo CK2ß No CK2ß- GATCCCCGCTCTG BC003775 203- EGFP (E979)- inhibition siRNA-5 GACATGATCTTAG CKIIβ- TTCAAGAGACTA siRNA-203 AGATCATGTCCA GAGCTTTTTGGAA A CK2ß- AGCTTTTCCAAAA siRNA-6 AGCTCTGGACAT GATCTTAGTCTCT TGAACTAAGATC ATGTCCAGAGCG GG pSuperNeo CK2ß No CK2ß- GATCCCCGCTCTG BC003775 154- EGFP (E979)- inhibition siRNA-3 GACATGATCTTAG CKIIβ- TTCAAGAGACTA siRNA-154 AGATCATGTCCA GAGCTTTTTGGAA A CK2ß- AGCTTTTCCAAAA siRNA-4 AGCTCTGGACAT GATCTTAGTCTCT TGAA CTAAGATCATGTC CAGAGCGGG pSuperNeo CK2ß 51% CK2ß- GATCCCCTCTTAC NM_0099 437- EGFP (E979)- siRNA-7 TGGACTCAATGA 75 CKIIβ- GTTCAAGAGACT siRNA-437 CATTGAGTCCAGT AAGATTTTTGG

31 Material and methods ______

CK2ß- AGCTTTTCCAAAA siRNA-8 ATCTTACTGGACT CAATGAGTCTCTT GAACTCATTGAGT CCAGTAAGAGGG pSuperNeo CK2ß 88% CK2ß- GATCCCCTGAGC NM_0099 453- EGFP (E979)- siRNA-9 AGGTGCCTCACTA 75 CKIIβ- TTTCAAGAGAAT siRNA-453 AGTGAGGCACCT GCTCATTTTTGGA AA CK2ß- AGCTTTTCCAAAA siRNA- A 10 TGAGCAGGTGCC TCACTATTCTCTT GAATAGTGAGGC ACCTGCTCA GGG pSuperNeo CK2ß 51% CK2ß- GATCCCCCCTGAT NM_0099 505 EGFP (E979)- siRNA- GAAGAGCTGGAA 75 CKIIβ- 11 GTTCAAGAGACTT siRNA-505 CCAGCTCTTCATC AGG TTTTTGGAAA CK2ß- AGCTTTTCCAAAA siRNA- A 12 CCTGATGAAGAG CTGGAAGTCTCTT GAACTTCCAGCTC TTCATCAGG GGG CKIIβ siRNA CK2ß 85% AGAAGAAUUCAU BC00375 189 -189 (stealth) UACCACGGAGCC C pSuperNeo CK2α 63% CK2α- GATCCCCGATGA BC060742 325 EGFP (E979)- siRNA-1 CTATCAGCTTGTT CKIIα- CTTCAAGAGAGA siRNA-325 ACAAGCTGATAG TCATCTTTTTGGA AA CK2α- AGCTTTTCCAAAA siRNA-2 AGATGACTATCA GCTTGTTCTCTCT TGAAGAACAAGC TGATAGTCATCGG G pSuperNeo CK2α 88% CK2α- GATCCCCGTGTTT BC060742 373 EGFP (E979)- siRNA-3 GAAGCCATCAAC CKIIα- ATTCAAGAGATG siRNA-373 TTGATGGCTTCAA ACACTTTTTGGAA A CK2α- AGCTTTTCCAAAA siRNA-4 AGTGTTTGAAGCC ATCAACATCTCTT GAATGTTGATGG CTTCAAACACGG G CKIIα siRNA CK2α No ACAAAGUCUUAC BC060742 129 - (stealth) inhibition CAACGUCUGCUU U

32 Material and methods ______pSuperNeo CK2α’ No CK2α’- ATCCCCCAATGA BC057862 220 EGFP (E979)- inhibition siRNA-1 GAGGGTGGTTGT CKIIα’- ATTCAAGAGATA siRNA-220 CAACCACCCTCTC ATTGTTTTTGGAA A CK2α’- AGCTTTTCCAAAA siRNA-2 ACAATGAGAGGG TGGTTGTATCTCT TGAATACAACCA CCCTCTCATTG GGG pSuperNeo CK2α’ No CK2α’- GATCCCCGATTCT BC057862 286 EGFP (E979)- inhibition siRNA-3 GGAGAACCTTCG CKIIα’- TTTCAAGAGAAC siRNA-220 GAAGGTTCTCCA GAATCTTTTTGGA AA CK2α’- AGCTTTTCCAAAA siRNA-4 AGATTCTGGAGA ACCTTCGTTCTCT TGAAACGAAGGT TCTCCAGAATCGG G pSuperNeo CK2α’ No CK2α’- GATCCCCCCTTCG BC057862 298 EGFP (E979)- inhibition siRNA-5 TGGTGGAACAAA CKIIα’- TTTCAAGAGAATT siRNA-298 TGTTCCACCACGA AGGTTTTTGGAAA CK2α’- AGCTTTTCCAAAA siRNA-6 ACCTTCGTGGTGG AACAAATTCTCTT GAAATTTGTTCCA CCACGAAGGGGG pSuperNeo CK2α’ No CK2α’- GATCCCCCTTGGT BC057862 173 EGFP (E979)- inhibition siRNA-7 CGGGGCAAGTAT CKIIα’- ATTCAAGAGATA siRNA-173 TACTTGCCCCGAC CAAGTTTTTGGAA A CK2α’- AGCTTTTCCAAAA siRNA-8 ACTTGGTCGGGG CAAGTATATCTCT TGAATATACTTGC CCCGACCAAG GGG pSuperNeo CK2α’ No CK2α’- GATCCCCAGGAC BC057862 342 EGFP (E979)- inhibition siRNA-9 CCTGTGTCAAAG CKIIα’- ACTTCAAGAGAG siRNA-342 TCTTTGACACAGG GTCCTTTTTTGGA AA CK2α’- AGCTTTTCCAAAA siRNA- AAGGACCCTGTG 10 TCAAAGACTCTTG AAGTCTTTGACAC AGGGTCCTGGG

33 Material and methods ______

pSuperNeo CK2α’ No CK2α’- GATCCCCAGAGG BC057862 275 EGFP (E979)- inhibition siRNA- TTAAGATTCTGGT CKIIα’- 11 TCAAGAGACCAG siRNA-275 AATCTTAACCTCT CGTTTTTGGAAA CK2α’- AGCTTTTCCAAAA siRNA- ACGAGAGGTTAA 12 GATTCTGGTCTCT TGAACCAGAATC TTAACCTCTCG GGG CKIIα’ CK2α’ 72% UAUCAUGCUCGC BC057862 690 siRNA -690 (inhibits UAACAUGCAGCC (stealth) CK2α by C 65%) CKIIα’ CK2α’ No UGAGCAGGAUGA BC057862 569 siRNA -569 inhibition UAGAACUCUGCC (stealth) A CKIIα’ CK2α’ 44% UCCUGCUCAGGA BC057862 585 siRNA -585 GUACAAUGUUCG (stealth) A CKIIα’ CK2α’ 73% AUUCGAACAAGC BC057862 746 siRNA -746 (inhibits UGGUCAUAGUUG (stealth) CK2α by U 27%) Table 3.12. siRNAs.

3.1.4. Enzymes

Restriction enzymes New England BioLabs, Gibco/BRL, Roche, Fermentas RNase A Roth M MulV Reverse Transcriptase New England Biolabs APex TM Heat-Labile alkaline Epicenter phosphatase Taq DNA polymerase Fermentas T4 DNA Roche Table 3.13. Enzymes used

3.1.5. Kits and Columns

Plasmid DNA Purification Kit Macherey-Nagel (Nucleobond AX) DNA Purification Kit (Nucleospin Macherey-Nagel Extract) High Pure Plasmid Isolation Kit Roche Quikchange XL Site-directed Stratagene Mutagenesis Kit Lightcycler–FastStart DNA Master Roche

34 Material and methods ______

SYBR Green Kit Centricon Plus-20 Centrifugal Filter Amicon bioseparations Device Table 3.14. Kits and Columns used

3.1.6. Antibodies

A. Primary antibodies

Antigen Use and Dilution Company/Suppliers (species) T7 WB 1:10000 Novagen (mouse monoclonal) IP 1:1000 myc WB 1:10000 Cell Signaling (mouse monoclonal) IP 1:1000 α−ΗΑ WB 1:10000 Cell Signaling (mouse monoclonal) IP 1:1000 α-CK2β S.269 IHC 1:500 Dr. Mathias Montenarh (rabbit polyclonal) α-CK2β IHC 1:200 Dr. Claude Cochet (rabbit polyclonal) α-CK2β WB 1:250 Drs. Olaf-Georg Issinger and 123-GLSDI-127 Brigitte Boldyreff (mouse monoclonal) α-CK2β WB 1:250 BD (Transduction Laboratories) (mouse monoclonal) α-CK2β WB 1: 10000 Calbiochem (mouse monoclonal) α-CK2α WB 1:1000 Upstate (rabbit polyclonal) IHC 1:100 α-MuSK Rb194T WB 1:1000 Dr. Markus Ruegg (rabbit polyclonal) IHC 1:1000 IP 1:10 α-MuSK PA1-1740 WB 1:100 ABR (rabbit polyclonal) IHC 1:30 α-MuSK ab5618 WB 1:100 Abcam (rabbit polyclonal) IHC 1:30 α-MuSK 20kD WB 1:3000 Amir Khan (rabbit polyclonal) α−Synaptophysin IHC 1:200 DAKO (rabbit polyclonal) α−NF 200 IHC 1:5000 Chemicon (rabbit polyclonal) α−NF 165 IHC 1:1000 Developmental Studies (mouse monoclonal) Hybridoma Bank, Iowa Table 3.15. Primary antibodies used in WB, IHC and IP

B. Secondary antibodies

Antigen Use and Dilution Company/ Suppliers (species) Goat anti-mouse IgG HRP- WB 1:3000 Amersham Pharmacia Biotech

35 Material and methods ______conjugated Protein A HRP-conjugated WB 1:3000 Amersham Pharmacia Biotech Cy2-cojugated goat anti-mouse IHC 1:100 Dianova IgG Cy2-cojugated goat anti-rabbit IHC 1:100 Dianova IgG Cy3-cojugated goat anti-mouse IHC 1:200 Dianova IgG Cy2-cojugated goat anti-mouse IHC 1:200 Dianova IgG Alexa 488 (green IHC 1:500 Molecular Probes fluorescence) conjugated goat anti-rabbit IgG Table 3.16. Secondary antibodies used in WB, IHC and IP

3.1.7. Frequently used solutions

Solution name Composition 10xDNA loading buffer 50% Glycerin, 0.1% Xylene cyanol FF, 0.1% Bromophenol blue 1xTBE buffer 88 mM Tris, 88 mM Boracid, 2 mM EDTA pH 8.3 3x Lammli buffer 187 mM Tris, 6% SDS, 30% Glycerin, 0.02% Bromophenol blue, 15% β- Mercaptoethanol 1xPBS 140 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 x 2 H2O, 1.5 mM KH2PO4 PBST PBS, 0.1% Tween-20 Mowiol 6 ml water was added to 6.0 g Glycerol and 2.4 g Mowiol and left for 2 h at RT. Afterwards, 12 ml 0.2 M Tris pH 8.5 was added and the solution was rotated for 24 h at 53˚C, followed by centrifugation at 3220 rcf and aliquoting. Mowiol solution was stored at -20˚C for up to 12 months. 4% PFA 20 g Paraformaldehyde (PFA) was dissolved in 300 ml water (65ºC). After pH was adjusted to 7.4 water was added up to 500 ml, and the solution was sterile filtered, aliquoted and stored at -20ºC. Tail lysis buffer 50 mM Tris-HCl pH 8.0, 100 mM EDTA, 0.1 M NaCl, 1% SDS Table 3.17. Frequently used solutions

36 Material and methods ______

3.1.8. Cell culture

Human embryonic kidney 293 cells American Type Culture Collection (HEK293) Cos7 American Type Culture Collection C2C12 Gift from Prof. Hans-Rudolf Brenner MuSK-deficient myoblasts (MuSK-/-) Gift from Drs. Ruth Herbst and Steven C3.16 Burden HEK293 cells expressing continuously Gift from Dr. Stefan Kröger secretable active (4.8.) or inactive (0.0) agrin Dulbecco’s MEM (DMEM) with Gibco/BRL GlutamaxTM-1 with Sodium Pyruvate and 4500 mg/L Glucose Fetal Calf Serum (FCS) Invitrogen Heat-inactivated Horse Serum (HS) Invitrogen Chick Embryo Extract (CEE) SLI Mouse recombinant interferon-γ Sigma Matrigel Becton Dickinson Apigenin Sigma, HCLP grade 2-Dimethylamino-4,5,6,7-tetrabromo-1H- Gift from Drs. Flavio Meggio and benzimidazole (DMAT) Lorenzo A. Pinna Rhodamine-α-bungarotoxin (Rh-α-BTX) Molecular Probes, Eugene or Alexa-α-bungarotoxin (Alexa-α-BTX) SuperFect Qiagen LipofectamineTM 2000 Invitrogen DEAE-Dextran Pharmacia, Sigma Chloroquin Sigma Table 3.18. Cell culture materials.

3.1.9. Animals.

Wild type C57/BL6 mice were purchased from Charles River Laboratories. CK2βloxP/loxP conditional knockout mice were generated by Thierry Buchou, University of Southern Denmark (Buchou et al. 2003). HSA-Cre transgenic mice were a kind gift from Prof. Hans- Rudolf Brenner.

37 Material and methods ______

3.2. Methods

Standard methods were performed according to the following book: Sambrook, J., Russell D.W. Molecular Cloning: A Laboratory Manual, 3rd edition (Volume 1-2-3) Cold Spring Harbor Laboratory Press, 2001

3.2.1. Molecular Biology Methods

3.2.1.1. Isolation of plasmid DNA

Up to 10 µg of plasmid were isolated using the following protocol. Bacterial cells were pelleted by centrifugation. Cell pellet was resuspended in 100 µl of buffer S1. Then 200 µl of S2 buffer was added and mixed gently by inverting to avoid shearing of the genomic DNA. After 5 min of incubation 150 µl of S3 buffer was added and mixed gently. The mixture was centrifuged at 4°C (20000 rcf for 10min). To precipitate the plasmid DNA 1 ml of absolute Ethanol was added to the supernatant, mixed and centrifuged at 4°C (20000 rcf for 10 min). The pellet was centrifuged once more with 300µl of 70% Ethanol at 4ºC (14000 rpm for 5 min), air dried, and resuspended in 50 µl water.

S1 Buffer 50 mM Tris, 10 mM EDTA, 100 µg/ml RNase A pH 8.0

S2 Buffer 200 mM NaOH, 1% SDS

S3 Buffer 2.8 M KAc pH 5.1 Table 3.19. Solutions for isolation of plasmid DNA

Larger amounts of plasmid (20-2500 µg) were prepared with the use of Macherey-Nagel Plasmid DNA Purification Kit (Nucleobond AX) according the manufactures protocol, which is based on a modified alkaline lysis procedure followed by binding of plasmid DNA to an anion-exchange resin under appropriate low-salt and pH conditions. RNA, proteins and low- molecular-weight impurities are removed by a medium-salt wash; DNA is eluted in a high- salt buffer and then concentrated and desalted by isopropanol precipitation.

3.2.1.2. Determination of DNA/RNA concentration

38 Material and methods ______

The concentration of DNA/RNA in solution was determined in a spectrophotometer, measuring the absorption of the solution at 260 nm and using the following formulas:

1A260 =50 µg double stranded DNA

1A260 =33 µg single stranded DNA

1A260 =40 µg RNA

3.2.1.3. Electrophoretic separation of DNA fragments in agarose gel

DNA was loaded on 0.7-2 % agarose gels prepared in 1xTBE buffer with 0.5 µg/ml ethidium bromide. The electrophoresis was performed for ~1 h at 120 V (Horizontal gel electrophoresis apparatus – Gibco/BRL). The DNA fragments were visualized under UV light.

3.2.1.4. PCR amplification of DNA

The Polymerase-Chain-Reaction (PCR) was performed according to the method of Saiki (Saiki et al., 1986). A standard PCR reaction to amplify DNA from plasmid template contained about 50 ng plasmid DNA, forward and reverse primers (10 pmol each), 200 µM dNTPs, 1xTaq-polymerase buffer + (NH4)2SO4, 2 mM MgCl2 and 1U Taq DNA polymerase (Fermentas), in a total volume of 50 µl. The amplification was carried out in a PCR-Thermocycler PTC-2000 Peletier Thermal Cycler (MJ Research). The amplification conditions were as follows: (1) Initial denaturation for 2 min at 94°C; (2) 25-30 cycles of 10-30 sec at 94°C, 10-30 sec at the annealing temperature of the primer pair and extension of 1 min/kbp at 72°C; (3) Incubation for 10 min at the extension temperature to allow for the complete amplification of all products. The annealing temperature, the time of denaturation, annealing and the extension were optimized for each experiment. The PCR products were then analyzed in an agarose gel by separation according to their size (see 3.2.1.3.).

3.2.1.5. Cloning techniques

The DNA fragment to be subcloned was amplified by PCR (see 3.2.1.4.) using specific primers containing unique restriction sites, analyzed on an agarose gel (see 3.2.1.3.) and purified either by using DNA Purification Kit - Nucleospin Extract (Macherey Machinery) or by the Tombstone procedure. For that the gel area directly above (towards the cathode) and under (towards to anode) the band of the PCR product was cut and pieces of DE81 cellulose

39 Material and methods ______paper were inserted into the nicks. During further migration of the DNA the desired PCR product was transferred and bound to the lower DE81 paper, while the upper DE81 paper prevented contamination of the PCR product with other DNA fragments. The DE81 paper with the PCR product was removed from the gel and placed into a 0.5 ml PCR tube with a hole in the bottom and inserted into the 1.5 ml Eppendorf tube. Residual TBE buffer was removed from the DE81 paper by centrifugation (16000 rcf for 30 sec.). DNA of the PCR fragment was eluted three times from the DE81 paper with high salt Tombstone buffer (1 M LiCl, 20% Ethanol, 10 mM Tris pH 7.5, 1 mM EDTA) and precipitated with three volumes of absolute Ethanol, washed with 80% Ethanol, air dried and dissolved in 20 µl of water. The purified PCR product and the subcloning vector were digested in a total volume of 20 µl comprising of about 1 U of restriction enzyme per 1 µg of DNA, 2 µl of 10x corresponding restriction buffer and sterile water. When the vector ends were blunt or compatible with each other, the vector was dephosphorylated prior to ligation to prevent self-ligation. To remove 5’ phosphates from the vector, 1 U of APex TM Heat-Labile alkaline phosphatase (Epicenter) was added directly to the digestion reaction. The reaction was incubated for 20 min at 37°C. Phosphatase was heat inactivated at 70°C for 5 min. After enzymatic digestion or dephosphorylation the vector or PCR product were purified by agarose gel electrophoresis. Before ligation the concentrations of PCR-fragment and vector were roughly estimated on an agarose gel. A typical ligation reaction contained vector and insert at a molar ratio of about 1:2 (600 ng total DNA), 1x ligase buffer (New England BioLabs), and 1 U of T4 DNA Ligase (New England BioLabs) in a volume of 10 µl. The incubation was carried out at RT for 5 h or at 16°C for 12-16 h. After that, 1 µl of the ligation reaction was transformed in Escherichia coli competent cells.

3.2.1.6. Plasmid constructs

Plasmid constructs generated according to the basic cloning techniques (see 3.2.1.5.) are listed in the Table 3.12. For primers sequences and Acc.No. position see 3.1.3.

Plasmid name Primers used for Restriction digestion sites Template used for subcloning used for subcloning PCR pCMX.PL1-T7-CK2β CK2β-3+CK2β-4 HindIII/XhoI pGADGH-CK2β-full length (fished by pCMV5-myc-CK2β CK2β-7+CK2β-12 KpnI/HindIII Y2H) pCMX.PL1-T7-CK2β−m CK2β-29+CK2β-23 XhoI/KpnI 1st cDNA of C2C12 pCMX.PL1-T7- CK2α−3+CK2α-4 XhoI/KpnI CK2α−m

40 Material and methods ______pCMX.PL1-T7- CK2α’−5+CK2α’-4 HindIII/KpnI CK2α’−m pGEXKG-CK2β-47 CK2β-16+CK2β-8 XhoI/HindIII pGADGH-CK2β-full length (fished by pGEXKG-CK2β-55 CK2β-16+CK2β-9 XhoI/HindIII Y2H) pGEXKG-CK2β-64 CK2β-16+CK2β-10 XhoI/HindIII pGEXKG-CK2β-140 CK2β-16+CK2β-11 XhoI/HindIII pGEXKG-CK2β-full CK2β-16+CK2β-12 XhoI/HindIII length pGEXKG-CK2α-full CK2α−5+CK2α-6 XhoI/HindIII pTX-HX-CK2α length pCMX.PL1-T7-MuSK- MuSK-39+MuSK-40 EcoRI/XhoI pMT-MuSK-full- 563 length pCMX.PL1-T7-MuSK- MuSK-39+MuSK-41 EcoRI/XhoI 682 pCMX.PL1-T7-MuSK- MuSK-39+MuSK-42 EcoRI/XhoI 857 pCMX.PL1-T7-MuSK- MuSK-39+MuSK-44 EcoRI/XhoI 868 pET28b-MuSK-868 MuSK-47+MuSK-48 HindIII/XhoI pCDNA3-MuSK2xwt pET28b-MuSK-868-A- MuSK-47+MuSK-44 HindIII/XhoI pMT-MuSK-A- 683/699 683/699 Table 3.20. Plasmid constructs. pcDNA-MuSK2xwt-myc and pcDNA-MuSK2xkd-myc constructs have been generated and kindly provided by the group of Prof. Hans-Rudolf Brenner. For their generation two intracellular domains of MuSK or its kinase defective mutant (aa 467-868; Acc.No. U34985) were linked together by five E/G (E-Glutamic Acid, G-Glycin) modules. The first intracellular MuSK domain was subcloned into HindIII/NheI of myc-tagged pcDNA3 (Invitrogen). The second intracellular MuSK domain was joint by ligation into the NheI and EcoRI sites of the plasmid. For the generation of pSUPERneoGFP based siRNAs (see 3.1.3.) used for silencing of mouse CK2β/CK2α/CK2α’ a pair of oligonucleotides according to the following design was synthesized:

compatible with BglII 5’ – GATCCCCC ( sense ) TTCAAGAGA (anti sense) TTTTTGGAAA – 3’ 3’ – GGG (anti sense) AAGTTCTCT ( sense ) AAAAACCTTTTCGA – 5’ hairpin loop compatible with HindIII

Sense and anti sense specific sequences targeting the mRNA of corresponding genes were designed with the software Sfold (Ding et al. 2004). The oligos were hybridized and subcloned into restriction digestion sites HindIII/BglII of pSUPERneoEGFP(E979) (Oligoengine) destroying at the same time the BglII site.

41 Material and methods ______

CMV expression plasmids pCMX.PL1, carrying the luciferase gene and CK2β/CK2α/ CK2α’ cDNA as bicistronic message were constructed as follows. Luciferase gene was amplified by PCR from the plasmid pGL2-basic (using primers Luc-1: 5’- CGGGATCCATGGAAGACGCCAAAAAC-3’ and Luc-2: 5'- CGGGATCCTTACAATTTGGACTTTCC-3' and subcloned into BamHI site of pCMX.PL1 (then named pCMX.PL1-Luc). cDNAs of CK2β/CK2α/CK2α’ were cut out from pCMX.PL1-T7-CK2β−m/CK2α−m/CK2α’-m respectively and ligated into the XhoI/KpnI (for CK2β and CK2α) or HindIII/KpnI (CK2α’) of pCMX.PL1-Luc. For generation of pcDNA3-MuSK∆KI, lacking kinase insert (KI) domain (aa 667-697), the 3’ portion of mouse MuSK cDNA downstream of the KI was amplified by PCR using primers Musk.KIdel5 (containing AvrII site) and Musk.KIdel6 (containing XbaI site) and subcloned into pCR2.1 vector (Invitrogen). The 5’ portion of MuSK upstream of the KI was amplified using primers Musk.KIdel3 (containing EcoRI) and Musk.KIdel4 (containing AvrII) and ligated into pCR2.1-MuSK-3’, opened by EcoRI/AvrII digestion. MuSK chimeras, containing KIs of other receptor tyrosine kinases were generated on the basis of pCR2.1- MuSK∆KI construct. KIs of Insulin-Like 1 (ILGFR-1), (IR) and TrkC were generated by hybridization of the following primers, containing overhangs compatible with AvrII site: IGF1R3 with IGF1R4, IR3 with IR4 and TrkC14.1 with TrkC14.2 respectively. Platelet-Derived Growth Factor-β Receptor (PDGFβR) ΚΙ was amplified by PCR from mouse muscle 1st strand cDNA, using primers PDGFbR3 and PDGFbR4, containing NheI site compatible with AvrII. All kinase inserts were ligated into pCR2.1-MuSK∆KI vector, opened with AvrII digestion. The MuSK KI chimera cassettes were excised from pCR2.1 by EcoRI/XbaI digestion and transferred into pcDNA3. For the construction of pcDNA3-MuSK-full-length vector full length cDNA of mouse MuSK (Acc.No. MMU37709) was cut out from pMT-MuSK-full-length (gift from Dr. Christian Fuhrer) and ligated into EcoRI/XbaI sites of pcDNA3. The generation of pCEFL-HA-CK2α is described elsewhere (Korn et al. 2001).

3.2.1.7. Transformation of E. coli competent cells.

While XL1-blue cells were used for subcloning of plasmid vectors, BL21 cells were used for protein expression. 1-10 ng of plasmid DNA or an aliquot from a ligation reaction (1 µl) were added to 50 µl of E. coli XL1-Blue or BL21 (Rosetta) electrocompetent cells (Novagen). The cell-DNA

42 Material and methods ______mixture was transferred into a prechilled electroporation cuvette (EquiBio). DNA was transformed into the bacteria cells by means of an electric impulse (1800 V, 7.5 ms). After electroporation the bacteria were shaken for 30 min at 37°C in 200 µl of LB medium, then plated on LB plates containing antibiotic (100 µg/ml ampicillin or kanamicin) and incubated at 37°C for about 16 h, until colonies appeared.

3.2.1.8. Site directed mutagenesis.

Substitutions of S (Serine) residues within the KI of MuSK by A (Alanine), E (Aspartic Acid) or D (Glutamic Acid) were introduced into the plasmid pMT-MuSK-full-length, using the Quikchange XL Site-directed Mutagenesis Kit (Stratagene). Primers for the site directed mutagenesis should fulfill the following criteria: have a length 25-50 b, Tm>78°C and carry a mutation of nucleotide(s), which would lead to the exchange of the desired amino acid without a shift in the open reading frame of the targeted gene. A plasmid carrying the target gene serves as a template for the mutagenesis PCR. Usage of PfuTurbo DNA-Polymerase ensures high fidelity of the PCR reaction. Following DpnI digestion results in the elimination of the methylated form of the template plasmid. Remaining unmethylated plasmid produced by PCR and carrying the mutated gene is subsequently transformed into E. coli competent cells. A 20 µl PCR reaction contains: 2 µl of 10x reaction buffer, 1.2 µl Quick solution, 0.4 µl dNTP Mix, 125 ng of two primers and 10 ng of template plasmid. Combinations of the primers and templates used al well as the resulting mutations are indicated in the Table 3.21.

Primer combination Template Mutation Resulted plasmid MuSK-71+MuSK-72 pMT-MuSK- full-length S-683 -> A pMT-MuSK-A-683 MuSK-67+MuSK-68 pMT-MuSK- full-length S-699 -> A pMT-MuSK-A-699 MuSK-67+MuSK-68 pMT-MuSK-A-699 S-683 -> A pMT-MuSK-A-683/699 MuSK-73+MuSK-74 pMT-MuSK- full-length S-683 -> D pMT-MuSK-D-683 MuSK-77+MuSK-78 pMT-MuSK- full-length S-699 -> D pMT-MuSK-D-699 MuSK-73+MuSK-74 pMT-MuSK-D-699 S-683 -> D pMT-MuSK-D-683/699 MuSK-75+MuSK-76 pMT-MuSK-D-683 D-683->E pMT-MuSK-E-683 MuSK-79+MuSK-80 pMT-MuSK-D-699 D-699->E pMT-MuSK-E-699 MuSK-79+MuSK-80 pMT-MuSK-D-683 D-699->E pMT-MuSK-E-683/699 Table 3.21. Combination of primers (see Table 3.8.) and templates used for site directed mutagenesis of MuSK.

Mutagenesis PCR was carried out according to the program indicated in the Table 3.22.

43 Material and methods ______

Step Number of cycles Temperature Time

Denaturation 1 95°C 1 min 95°C 50 sec Amplification 18 60°C 50 sec 68°C 7 min Elongation 1 68°C 7 min Table 3.22. The program of the site directed mutagenesis PCR.

The PCR product was digested with DpnI at 37°C for 1 h. XL1-Blue electrocompetent bacteria were transformed with 1 µl of digested PCR product. Plasmid DNA was isolated from bacteria clones and verified by sequencing.

3.2.1.9. Lightcycler PCR

Lightcycler PCR allows to quantitatively estimate the amounts of gene transcripts and to compare them for different tissues, cells or developmental stages. The Lightcycler–FastStart DNA Master SYBR Green Kit allows to quantify the amount of PCR products by means of incorporated SYBR Green fluorescence, which is proportional to the amount of double stranded DNA. Total RNA was extracted from cells (C2C12 myoblasts, C2C12 myotubes treated with the inactive 0.0. or active 4.8. isoform of agrin) or tissues (muscle, brain, synaptic and extrasynaptic regions of diaphragm) and then transcribed into complementary DNA (cDNA). cDNA was used as a template for Lightcycler PCR reaction. A 10 µl reaction contained 1 µl of cDNA (in dilution 1:10), 1.2 µl MgCl2 (25 mM), 1 µl of two primers (10 pmol each) and 1

µl MasterSG mix (Taq DNA–Polymerase, SYBR Green, dNTPs and 10 mM MgCl2). Lightcycler PCR was performed, using the Lightcycler Thermal Cycle System (Roche). The PCR program is given in the Table 3.23.

44 Material and methods ______

Step Number of cycles Temperature Time Denaturation 1 95°C 8 min

35 95°C 0 sec

Amplification 62°C 7 sec

72°C 1 min

1 95°C 30 sec

Elongation 1 67°C 30 sec 1 95°C 0 sec Table 3.23. The program of the Lightcycler PCR.

The amount of gene transcripts was defined for MuSK, AChR α subunit, CK2β, CK2α and

CK2α’ and normalized to the amount of transcripts of a housekeeping gene (β-actin). Respective primers used for PCR reactions and sizes of resulting PCR products are listed in the Table 3.10. (see 3.1.3.).

3.2.1.10. Total RNA isolation

Total RNA was extracted from mouse brain, leg muscle, extrasynaptic and synaptic regions of diaphragm, C2C12 myoblasts, C2C12 myotubes treated with inactive 0.0. or active 4.8. isoforms of agrin using the TRIzol Reagent (Invitrogen). 1 ml of TRIzol reagent was used per 50-100 mg of tissue or per 10 cm cell plate area. Tissues were homogenized using an automatic homogenizer (Kinematica), insoluble material was removed by centrifugation (12000 g for 10 min at 2-8°C). After 5 min at RT 0.2 ml chloroform was added per 1 ml TRIzol Reagent, vortexed for 15 sec, incubated additional 3 min at RT and centrifuged (12000 g for 15 min at 2-8°C). The aqueous phase was transferred to a fresh tube and RNA was precipitated with 0.5 ml isopropanol per 1 ml TRIzol Reagent for 10 min at RT. After centrifugation (12000 g for 15 min at 2-8°C) the RNA pellet was washed with 75% Ethanol, briefly dried and dissolved in RNAse free water. RNA was aliquoted and stored at -80°C.

3.2.1.11. Complementary DNA-synthesis (Reverse Transcription)

The reverse transcription (RT) allows the transcription of RNA in complementary DNA (cDNA) that can be subsequently used as template for PCR. For cDNA-synthesis, 2 µg of total RNA were incubated in a total volume of 15.5 µl, including 1 µl of oligo-dT (0.5 µg/µl) (Invitrogen) and DEPC water at 70°C for 10 min and then quickly chilled on ice to open secondary structures. The following mix was then added to each tube: 2.5 µl of 10x First-

45 Material and methods ______

Strand Buffer (New England BioLabs), 4 µl dNTP mix (10 mM) and 2 µl of DEPC water and incubated 1 min at 37°C. After that 200 U of M MulV Reverse Transcriptase (New England BioLabs) were added and the reaction was incubated for 50 min at 37°C. Finally the enzyme was inactivated at 70°C for 15 min and the cDNA was aliquoted and stored at –80°C.

3.2.1.12. Yeast two hybrid (Y2H) techniques

The MATCHMAKER Two-Hybrid System 3 (Clontech), was used to identify protein-protein interactions in yeast cells. It comprises a bait sequence expressed as a fusion to the GAL4 DNA-binding domain (DNA-BD) from the pGBKT7 plasmid (Table 3.24.), while prey sequences are expressed as a fusion to the GAL4-activation domain (AD) from pGADT7 or from pGAD424 (Table 3.24.). When bait and prey fusion proteins interact, the DNA-BD and AD of GAL4 are brought into proximity, thus activating transcription of reporter genes. Yeast strains HF7c and AH109 have been used for the Y2H screens. The elimination of false positives have been performed by using the reporter genes - HIS3, lacZ (for HF7c) and ADE2, HIS3, lacZ (for AH109), which are under the control of distinct GAL4 upstream activating sequences (UASs) and TATA boxes. For generation of bait constructs for the Y2H screens, different domains of muscle receptor tyrosine kinase (MuSK) were amplified by PCR using pMT-MuSK-full-length as a template and subcloned in frame into the EcoRI/SalI restriction sites of pGBKT7 (Table 3.25.). MuSK2xwt and MuSK2xkd baits were generated and kindly provided by the group of Prof. Hans-Rudolf Brenner. In brief, two intracellular domains of rat MuSK or its kinase defective mutant (Acc.No. U34985) were linked together by five E/G modules. As restriction sites for the first intracellular MuSK domain NcoI and EcoRI and for the second intracellular MuSK domain EcoRI and SalI of pGBKT7 were used.

Cloning vectors Epitope Yeast selection Bacterial selection pGBKT7 (bait) myc TRP1 kanamicin pGADT7 (prey) LEU2 ampicillin pGAD424 (prey) LEU2 ampicillin Control vectors Epitope Yeast selection Bacterial selection pGADT7-T HA LEU ampicillin pGBKT7-53 myc TRP1 kanamicin Table 3.24. Yeast vectors used.

46 Material and methods ______

Bait MuSK domains Aa Primers used for subcloning (see Table position 3.3.) pGBKT7-K3 C6 box + Ig-like IV 232-491 MuSK-17 + MuSK-21 pGBKT7-K4 Ig-like IV 350-491 MuSK-18 + MuSK-21 pGBKT7-JM Juxtamembrane (JM) 481-520 MuSK-22 + MuSK-23 pGBKT7- Two times whole 2x (467- Gift from Prof. Hans-Rudolf Brenner MuSK2xwt intracellular domain 868) Table 3.25. Bait plasmids generated for Y2H screens.

Each cloned bait was first sequenced and then tested for transactivation of the yeast reporter genes by transforming HF7c and AH109 yeast cells with the bait plasmid together with empty prey plasmid pGADGH. The transformed cells were plated on yeast selection media with different concentration of 3-amino-1,2,4-triazole (3-AT, Sigma; 0/5/10 mM), a competitive inhibitor of the yeast HIS3 protein (His3p), used to suppress background growth of yeast cells. To screen for MuSK interacting proteins, yeast HF7c and AH109 cells were sequentially transformed with each bait vector and 1 µg of a human HeLa cDNA MATCHMAKER library (Clontech), using the lithium acetate method (Schiestl and Gietz 1989) (see Table 3.26.).

1x Tris EDTA (TE) pH 7.5 From 10x TE: 100 mM Tris-Cl, 10 mM EDTA 1x TE/LiAc pH 7.5 From: 10x TE, 10x LiAc 40% PEG 3350 PEG/LiAc/TE pH 7.5 1x TE buffer 1x LiAc DNA carrier Yeastmaker carrier DNA DMSO 10% final concentration Table 3.26. Solutions for yeast transformation.

The yeasts were plated on media selecting transformants (SD -Leu, -Trp) to estimate the efficiency of transformation and on the media selective for the reporter gene activations (SD - Leu, -Trp, -His or SD -Leu, -Trp, -His, -Ade) containing 5 or 10mM 3-AT. Yeast colonies growth was evaluated after 5 days of incubation at 30°C. Selected positive clones were further confirmed by colony-lift filter assays for β-galactosidase activity according to the Clontech Manual. Transformation efficiency was calculated for every screen counting colonies (c.f.u. - 2 3 4 colony forming unit) growing on SD -Leu, -Trp dilution plates (1:10; 1:10 ; 1:10 ; 1:10 ) according to the formula:

47 Material and methods ______

(counted c.f.u.) x total suspension volume (µl) = total c.f.u. dilution factor

Prey plasmids were isolated from about 700 positive yeast colonies for all screens together, according to the Clontech Manual protocol for preparation of plasmids from yeasts and then shuttled into E. coli XL1-Blue electrocompetent cells. Prey plasmids were re-transformed back into yeast cells together with respective baits or empty pGBKT7 vector to ensure that positive clones do interact with the bait but not with the GAL4-DNA-BD. Library inserts of positive, re-tested interactors were sequenced and analyzed with protein and nucleotide databases of the National Center for Biotechnology Information (NCBI, Bethesda, MD) using the Basic Local Alignment Search Tool (BLAST). For binary epitope-mapping Y2H studies PCR-amplified intracellular epitopes of mouse MuSK (Acc.No. MMU37709) were subcloned into pGBKT7 using restriction digestion sites EcoRI and SalI (see Table 3.27.).

Bait MuSK epitope aa position Primers used for subcloning (see Table 3.3.) pGBKT7-MuSK-563 JM 467-563 MuSK-38 + MuSK-61 pGBKT7-MuSK-682 JM + half of kinase domain 467-682 MuSK-38 + MuSK-62 pGBKT7-MuSK-857 JM + kinase domain 467-857 MuSK-38 + MuSK-63 pGBKT7-MuSK-868 Whole intracellular domain 467-868 MuSK-38 + MuSK-64

Table 3.27. MuSK bait constructs used in epitope-mapping experiments.

Different epitopes of human CK2β (Acc.No. NM_001320) were PCR amplified and subcloned into the restriction sites BamHI and BglII of the prey vector pGAD424 as GAL4 AD fusions (see Table 3.28.).

Bait Aa position Primers used for subcloning (see Table 3.4.) pGAD424-CK2β-47 1-47 CK2β24 + CK2β-25 pGAD424-CK2β-55 1-55 CK2β24 + CK2β-26 pGAD424-CK2β-64 1-64 CK2β24 + CK2β-27 pGAD424-CK2β-140 1-140 CK2β24 + CK2β-28

Table 3.28. CK2β prey constructs used in epitope-mapping experiments

For binary Y2H interaction assays transactivation of reporter genes was analyzed in the HF7c yeast strain on selective plates SD –Leu, -Trp, -His + 10 mM 3-AT. Functionality of Y2H assays was always tested using the positive control plasmids pGBKT7-53 or pGADT7-T of

48 Material and methods ______the MATCHMAKER GAL4 Two-Hybrid System 3 (Clontech), encoding for the murine tumor suppressor protein p53 and the SV40 large T-antigen, respectively (Table 3.24.).

3.2.2. Protein Biochemistry Methods

3.2.2.1. Preparation of protein extract from cells and tissues.

For preparation of protein extract HEK293 cells were transfected with SuperFect (Qiagen) in 10 cm plates, Cos7 cells were transfected in 10 cm plates using DEAE-dextran technique followed by chloroquin treatment (see 3.2.3.2.). Whole cell protein extracts were prepared from transfected HEK293 or Cos7 cells or nontransfected C2C12 cells. For that, cells were lysed in the presence of 2 µg/µl leupeptin and aprotinin in ice-cold buffer containing 10 mM Hepes pH 7.9, 0.2 mM EDTA, 2 mM DTT, and 1% Nonidet P-40. Immediately after the lysis, NaCl was added to a final concentration of 400 mM. After incubation for 15 min (for HEK293 cells) or 30 min (for C2C12 cells) under constant rotation, cell debris was removed from the extract by centrifugation. For preparation of muscle tissue extract, mouse hind limb muscles were frozen in liquid nitrogen, mashed and further homogenized in ice cold lysis buffer, containing 10 mM Hepes pH 7.9, 0.2 mM EDTA, 2 mM DTT, and 1% Nonidet P-40, 2 µg/µl leupeptin and aprotinin for 10 min. Cell debris was removed from the extract by centrifugation (14000 rpm for 10 min).

3.2.2.2. Immunoprecipitation

For immunoprecipitation experiments HEK293 or Cos7 cells were transiently transfected with one of the following CMV-expression plasmids encoding for: T7- or myc-tagged MuSK2xwt and MuSK2xkd constructs; HA-tagged full length CK2α; T7- or myc-tagged full length CK2β; T7-tagged MuSK C-terminal truncations (see 3.2.1.6.). Protein extract was prepared as described above (see 3.2.2.1.). For each extract the protein concentration was determined (see 3.2.2.5.) or expression level of proteins was analyzed by Western blot (see 3.2.2.8.) and adjusted. Lysates containing expressed proteins were mixed and the final volume set to 500 µl with the buffer containing 10 mM Hepes pH 7.9, 0.2 mM EDTA, 10 mM PMSF, 1 mM leupeptin and aprotinin. 1 µl of a monoclonal antibody against the T7- (Novagen), myc- or HA- (Cell Signaling) was added and the reaction was incubated under constant rotation at 4°C

49 Material and methods ______for 30 min. Next, 20 µl of in PBS equilibrated Protein A Sepharose CL-4B beads (Amersham) were added and incubation continued overnight. After washing the beads three times with buffer containing 50 mM Hepes pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100, 10 mM PMSF, 1 mM leupeptin and aprotinin the precipitated proteins were analyzed by SDS-PAGE and Western blot. For immunoprecipitation of endogenous CK2β and MuSK proteins from C2C12 cells or muscle tissue, a polyclonal rabbit serum against MuSK (Abcam) was pre-conjugated with Protein A Sepharose. 20 µl of antibody was incubated with 40 µl of Protein A Sepharose in 1 ml of PBS overnight at 4°C under constant rotation. Then Sepharose beads containing MuSK antibody on their surface were washed two times with 500 µl of PBS and equilibrated with PBS in a 1:1 ratio. A 10 µl aliquot of MuSK- antibody-Sepharose conjugate was added to the extracts. Samples were incubated under the constant rotation overnight. After washing three times with a buffer containing 50 mM Hepes pH 7.5, 50 mM NaCl, 1 mM EDTA, 10% glycerol, 10 mM PMSF, 1 mM leupeptin and aprotinin proteins bound to the Sepharose beads were resolved by SDS gel and analyzed by Western blot.

3.2.2.3. Protein expression and extraction from bacteria

Full length or 3’-terminal truncations of CK2β cDNA were ligated in-frame to the coding sequence of glutathione-S- (GST) in pGEXKG (see 3.2.1.6.). cDNA encoding for the intracellular domain of rat MuSK or its alanine mutant (S683/699A) were fused in frame to the His-tag of pET28b (see 3.2.1.6.). Plasmids were transformed in E. coli BL21 (Rosetta).

Bacteria expressing GST-fusions of CK2β were grown in 50 ml cultures until OD600 0.4 and protein expression was induced by 1 mM isopropyl-beta-D-thiogalactoside (IPTG; Sigma) for 4 h. Afterwards bacteria were collected by centrifugation, incubated in sonification buffer (50 mM NaH2PO4, 300 mM NaCl, 25 U/ml Benzonase, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µl/ml Triton X-100, 10 µg/ml DNAseI, 15 U/µl Lysozym) at 4°C for 30 min, lysed by sonification and centrifuged (14000 rpm for 10 min). The supernatants containing the GST- fusion proteins were collected and used freshly for the further GST-pulldown experiments (see 3.2.2.4.). Bacteria expressing His-tag fusions of MuSK intracellular domain or its mutant were grown in 4 l culture until OD600 1.0 and protein expression was induced by 1 mM IPTG for 4 h. Bacteria were pelleted by centrifugation. The pellet was dissolved in 270 ml buffer A, pH 8.0

(6 M guanidine hydrochloride, 0.1 M NaH2PO4, 0.01 M Tris pH 8.5) and shook overnight at 250 rpm. The lysate was centrifuged to remove cell debris, the supernatant was collected and

50 Material and methods ______incubated with 6 ml Ni-NTA agarose (Qiagen) under constant rotation during 3 h at RT. Ni-

NTA beads were washed five times in buffer B pH 8.0 (8 M Urea, 0.1 M NaH2PO4, 0.01 M Tris), three times with buffer B pH 6.6 and the protein was eluted by washing the beads five times with 7.5 ml buffer B pH 4.5. The eluted protein was concentrated to a volume of 2.5 ml using Centricon Plus-20 column (Amicon Bioseparations). The protein was additionally purified by SDS gel electrophoresis. The band of the expected size was identified by Coomassie staining (see 3.2.2.7.) and isolated. The gel piece was placed into a dialysis tube and the protein was eluted from the gel and purified from SDS by horizontal electrophoresis (50 mA for 5 h in SDS-running buffer (see 3.2.2.6.) and then 50 mA for 2 h in running buffer without SDS). Resulting protein was concentrated by Centricon Plus-20 column to a final concentration of 200-500 ng/µl.

3.2.2.4. GST-pulldown

The supernatants containing the GST-fusion proteins were supplemented by 30 µl equilibrated Glutathione-Sepharose beads and incubated under constant rotation at 4°C for 2 h. After washing three times with washing buffer containing 4.3 mM Na2HPO4, 1.47 mM

KH2PO4, 1.37 mM NaCl, 2.7 mM KCl an aliquot of the beads was loaded on a SDS gel for estimation of the concentration by a Coomassie Brilliant Blue (Roche) staining. Then the beads carrying the GST-fusion protein were incubated together with 25 µl of extract from Cos7 cells expressing the desired protein after transient transfection. After washing three times with the washing buffer, proteins bound to the beads were analyzed by SDS-PAGE and Western blot.

3.2.2.5. Determination of protein concentration

Protein concentration was estimated according to the slightly modified method of Bradford (Bradford 1976). A calibration curve from the concentration measurement of BSA samples (1 mg/ml; A280 = 0.66) in different dilutions was used as a standard for the protein sample measurements. 0.5, 1, 5 and 10 µl of protein extract dissolved in 800 µl of PBS were mixed with 200 µl of Bradford reagent (BIO-RAD). The absorbance of the samples at 595 nm was then measured. All samples were prepared and analyzed in duplicate. Protein concentration was calculated per 1 µl of protein extract.

3.2.2.6. Electrophoresis of proteins

51 Material and methods ______

Proteins were resolved on denaturing SDS polyacrylamide gels, using the Vertical Mini-gel system (Sigma). The separating gel contained 8, 10 or 12.5% polyacrylamide depending on the molecular weight of the protein and the stacking gel was 5% (see Table 3.29, 3.30.). The proteins were mixed with sample loading Lammli buffer, denatured at 100°C for 5 min and loaded on the gel. Electrophoresis was started at 100 V until the probes entered the separating gel and was then carried out constantly at 120 V for 1-2 h in SDS gel running buffer depending on the protein dimensions.

Separating gel buffer 1.5 M Tris-Base pH 8.8, 0.4% (w/v) SDS Stacking gel buffer 500 mM Tris-HCl pH 6.8, 0.4% (w/v) SDS 10 x Running buffer 250 mM Tris-Base pH 8.3, 1.92 M Glycin 1% (w/v) SDS Table 3.29.: Solutions for SDS-PAGE.

Separating gel 8% 10% 12.5% Stacking gel 5% H2O 2.9 ml 2.5 ml 2 ml H2O 1.5 ml Separating gel 1.5 ml 1.5 ml 1.5 ml Stacking gel buffer 625 µl buffer Acrylamide/Bisac Acrylamide/Bisacryl 1.625 ml 2 ml 2.5 ml 425 µl rylamide (30%) amide (30%) APS (20% w/v) 22.5 µl 22.5 µl 22.5 µl APS (20% w/v) 5 µl TEMED 5 µl 5 µl 5 µl TEMED 2 µl Table 3.30. Separating gels and stacking gel for SDS-PAGE (calculated for one gel of 11 cm x 8 cm x 0.7 mm size).

3.2.2.7. Staining of protein gels

Coomassie staining was used to detect proteins in SDS polyacrylamide gels. After electrophoresis, the gel was placed in the staining solution (30% Methanol, 10% acetic acid, 0.05% Coomassie Brilliant Blue) for 15 min at RT and then destained overnight in a solution containing 25% Methanol and 15% acetic acid. The gel was dried on Whatman paper covered with a cellophane sheet on a Gel dryer SE1160 (Hoefer Scientific Instruments).

52 Material and methods ______

3.2.2.8. Western blot

Proteins resolved on SDS polyacrylamide gels were transferred to nitrocellulose membrane in blotting buffer (see Table 3.29.) for ~1.5 h at 150 mA. The membrane was then blocked in blocking buffer (see Table 3.31.) for 1 h at RT and further incubated for a minimum of 1 h up to overnight with the first antibody diluted in washing buffer (see Table 3.31.). A monoclonal antibody directed against the T7-tag (Novagen), myc-tag, HA-tag (Cell Signaling) and against CK2β (gift from Drs. Olaf-Georg Issinger and Brigitte Boldyreff) or polyclonal sera against either CK2α (Upstate) or MuSK (ABR, Abcam, 194T or 20kD) served as primary antibodies. 1:10000 dilution was used for anti-T7- and anti-myc-tag antibodies; 1:250 dilution - for monoclonal anti-CK2β antibody and 1:100 for all polyclonal antibodies (except 1:3000 for α- MuSK 20kD). The membrane was washed three times for 5 min with the washing buffer, incubated for 1 h with secondary antibody (Horseradish-peroxidase-coupled-Protein A or anti-mouse-Ig-coupled-horseradish-peroxidase in 1:3000 dilution), and subsequently washed three times for 5 min with the washing buffer. The bound antibodies were detected by the ECL system (Amersham) according to the manufacturer’s protocol, and the membrane was exposed to X-ray film super RX (FUJI Medical) that was developed in a film developer X- Omat 1000 Prozessor (Kodak).

Blotting buffer 480 mM Tris, 380 mM Glycin, 0.1% SDS, 20% Methanol Blocking buffer PBS, 0.1% Tween-20, 5% dry milk powder Washing buffer PBS, 0.1% Tween-20 Table 3.31. Western blot buffers

3.2.2.9. In vitro kinase assay

Kinase activity of protein kinases (MuSK and CK2) was determined as amount of radioactive phosphate incorporation into their substrates. A known substrate of MuSK is enolase (Feder and Bishop 1990; Mohamed et al. 2001). MuSK protein was isolated by immunoprecipitation from protein extracts of C2C12 myotubes, which were treated for 16 h with agrin 0.0. or agrin 4.8. and in the presence or absence of 40 µM 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT; gift from Drs Flavio Meggio and Lorenzo A. Pinna). 1/3 of the extract from one 10 cm plate (200 µl) was incubated overnight with 15 µl of MuSK-antibody-protein A Sepharose conjugate (see

53 Material and methods ______

3.2.2.2.) in a total volume of 1 ml adjusted by PBS. Immunoprecipitates were washed three times in buffer containing 50 mM Hepes pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100, 10 mM PMSF, 1 mM leupeptin and aprotinin and then two times in kinase buffer (10 mM MnCl2, 50 mM Tris, pH 7.4). Beads with precipitated MuSK were resuspended in 40 µl of the kinase buffer and 1µg of acid denatured enolase was added. The reaction was started by the addition of 0.8 µl γ-32P ATP (specific activity 60000 cpm/pmol) at RT and stopped by addition of 20 µl 3x Lammli after 10 min. After boiling for 5 min, the samples were resolved by 10% SDS-gel for radiography. For acid denaturation of enolase 1 vol of enolase (1.5 mg/ml, Sigma) was mixed with 1 vol of 50 mM acetic acid and incubated for 5 min at 30°C. Denaturation was stopped by the addition of 1 vol of 1 M Hepes pH 7.4. Recombinant intracellular domain of MuSK from rat or its alanine mutant were expressed and purified from bacteria (see 3.2.2.3.). 2.5 µg of the recombinant proteins were phosphorylated in vitro by 150 ng of CK2α (Biaffin) alone or together with 20 pmol CK2β (gift from Dr.

Olaf-Georg Issinger) in a 20 µl volume containing 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 1 mM DTT and 100 µM γ-32P ATP with specific activity 3.0 Ci/mmol. After 30 min of incubation at 30°C the reaction was stopped by the addition of 10 µl of 3xLammli and the samples were resolved by 10% SDS-gel for radiography. CK2 activity was measured in 10µg mouse muscle lysates as described above using 10 µM of 800 µM synthetic peptide substrate RRRDDDSDDD.

3.2.3. Cell culture methods

3.2.3.1. Cultivation of HEK293, Cos7, C2C12, MuSK-deficient myoblasts.

HEK293 or Cos7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM;

Gibco/BRL) containing 10% (v/v) fetal calf serum (FCS; Invitrogen) at 37°C and 5% CO2. C2C12 cells were maintained for proliferation in DMEM containing 20% (v/v) FCS, for differentiation the medium was replaced by DMEM with 5% (v/v) heat-inactivated horse serum (HS, Invitrogen) at the same growth conditions. Myotubes were formed after 4-6 days. MuSK-deficient myoblasts (gift from Drs. Ruth Herbst and Steve Burden) were proliferated in DMEM containing 10% (v/v) FCS, 10% (v/v) HS, 0.5% chick embryo extract (CEE; SLI), 0 mouse recombinant interferon-γ (Sigma) at 33 C, 10% CO2. For differentiation, MuSK- deficient myoblasts were transferred to the proliferation medium lacking CEE and interferon-

54 Material and methods ______

γ at 39°C, 10% CO2. For MuSK-deficient cells dishes were coated with Matrigel (Becton Dickinson). Myotubes were usually observed after 2-3 days.

3.2.3.2. Transient transfection of cells 24 h before transfection exponentially growing cells were replated in growth medium and transfected at 50-60% (HEK293, Cos7) or 80-90% (C2C12, MuSK-deficient myoblasts) confluence.

A. SuperFect transfection

For preparation of protein extracts HEK293 cells were transiently transfected with CMV expression plasmids (see 3.2.2.2.) in 10 cm dishes with in total 10 µg of expression vectors using SuperFect (Qiagen). In order to test the efficiency of siRNAs against different CK2 subunits HEK293 cells were transfected in 6 cm plates with 1 µg of CMV-expression vectors encoding for full length CK2 subunits together with 3 µg of the respective pSUPERneoEGFP(E797)-siRNA constructs or with 100 pmol of stealth siRNAs. For 10 cm plate transfection DNA was dissolved in 300 µl of DMEM medium lacking FCS. Then 30 µl of SuperFect reagent was added to the DNA solution and mixed by vortexing for 10 sec. The samples were incubated for 5-10 min at RT to allow transfection complex formation. In the meantime the growth medium was aspirated from the dishes and the cells were washed once with 4 ml of PBS. Then 3 ml of cell growth medium (containing FCS) were added to the reaction tube containing the transfection complex, mixed by pipetting and immediately transferred to the cells. Cells were incubated with the transfection complexes for 3-12 h under their normal growth conditions. After that the medium containing the remaining complexes was removed from the cells, cells were washed once with 4 ml of PBS and fresh growth medium was added. Transfection efficiency was controlled by transfection of one cell plate with the plasmid expressing Green Fluorescence Protein (GFP) with nuclear localization signal – pnlsGFP (Hashemolhosseini et al. 2000). At 48 h post-transfection, cells were harvested for extract preparation as described (see 3.2.2.1.).

B. DEAE-Dextran transfection

In some cases Cos7 cells were transfected using the DEAE-Dextran technique. The transfection reaction for 10 cm dish was made as follows: 10 µg of expression plasmid DNA were mixed with 187.5 µl of PBS and 375 µl of DEAE-Dextran. The mix was added to the growth medium of the cells. After incubation for 30 min under standard growth conditions,

55 Material and methods ______the medium was replaced by 8 ml of fresh medium and 80 µl of Chloroquin was added. Cells were incubated for 3 h, then the growth medium was changed once again. At 48 h post- transfection, cells were harvested for extract preparation as described (see 3.2.2.1.).

C. LipofectamineTM 2000 transfection

For immunocytochemistry C2C12 and MuSK-deficient cells were transiently transfected in 3.5 cm plates with 1 µg of plasmid expressing nuclear localized GFP – pnlsGFP together with either 3 µg pSUPERneoEGFP(E797)-siRNA constructs or 3 µg expression plasmid containing one of the MuSK serine or KI mutants. 24 h before transfection cells were replated in growth medium without antibiotic. 4 µg of DNA were dissolved in 250 µl of DMEM medium lacking FCS. 10 µl of Lipofectamine were diluted separately in 250 µl DMEM medium lacking FCS. After incubation for 5 min at RT the mixtures were combined and incubated for 20 min at RT. Then 500 µl of DNA-Lipofectamine complex was added to each plate. The medium was replaced by differentiation medium after one day. In order to check silencing of CK2α, CK2α’ and CK2β by luciferase activity tests HEK293 cells were transfected in 24 well dishes with 0.125 µg of luciferase- CK2α/CK2α’/CK2β cDNA fusion constructs (see 3.2.1.6.) and 0.375 µg of pSUPERneoGFP(E797)-siRNA constructs or 20 pmol of stealth siRNA. In this case 1µl of Lipofectamine was used.

3.2.3.3. Luciferase reporter test.

The ATP dependent oxidation of luciferin by luciferase is accompanied by the light emission, which can be measured. The luciferase activity test was performed to check the ability of different siRNAs to knockdown the mRNAs of CK2 subunits (α, α’, β). The cDNAs of the respective genes were subcloned together with the luciferase gene to be transcribed as a bicistronic message (see 3.3.1.6.). An effective siRNA would target the chimeric luciferase- CK2subunit mRNA which would result in its degradation and lead to a decrease of luciferase activity. HEK293 cells transfected with the constructs described above were lysed 48 h posttransfection. The lysis of the cells was performed in 300 µl per well of Luciferase Harvest/Assay Buffer, which contained 88 mM Tris/MES pH 7.8, 12.5 mM MgAc, 2.5 mM ATP, 1 mM DTT and 0.1% Triton X-100. Measuring of the chemiluminescence reaction was performed for 200 µl of the cell lysate in a luminometer (Berthold–Lumat LB9501), after injection of 100 µl of 0.5 mM luciferin dissolved in 5 mM KHPO4. The luciferase activity was calculated in Relative Light Units (RLU).

56 Material and methods ______

3.2.3.4. Agrin treatment

The production of agrin-conditioned media was performed as described before (Kröger 1997). In brief, stably transfected HEK293 cells (gift from Dr. Stephan Kröger) expressing continuously secreted active agrin 4.8. or inactive agrin 0.0. were grown in DMEM containing 10% FCS until 80-90% confluence. The terms inactive and active reflect agrin originating either from isoform agrinA0B0 or agrinA4B8 respectively (Gesemann et al. 1995). After another 4 days of proliferation in serum-free DMEM, agrin-conditioned medium was collected, aliquoted and frozen. Agrin-conditioned medium was added at 1:8 dilution (125 µl per 3.5 cm dish) to C2C12 myotubes. AChR aggregates were detected or quantified 16 h later as described below (see 3.2.3.8.).

3.2.3.5. Application of CK2 inhibitors

Inhibition of endogenous CK2 activity was performed using either apigenin (Sigma, HCLP grade) or 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT; gift from Drs Flavio Meggio and Lorenzo A. Pinna) (Pagano et al. 2004). Stock solutions for both inhibitors were generated by dissolving them in DMSO at 100 mM or 10 mM, respectively. 4 h before adding agrin-conditioned media to C2C12 or MuSK-deficient cells apigenin was added to the cell cultivation medium at different concentrations: 0/10/20/40/60/80µM. 12 h later another 25% of apigenin was added to compensate for the degradational loss of inhibitor activity. DMAT was added to the cell cultivation medium at the same time as agrin-conditioned medium at indicated concentrations: 0/10/20/40/60/80µM. In both cases cells were incubated in total 16 h with agrin-conditioned medium. The same amounts of DMSO added to C2C12 or MuSK-deficient cells served as controls.

3.2.3.6. Immunocytochemistry

AChR clusters on the surface of C2C12 or MuSK-deficient cells were visualized by α- Bungarotoxin staining. Cultivation medium was aspirated from the dishes and cells were washed three times with PBS. Then cells were fixed with 2% PFA solution in PBS for 20 min at RT. After washing three times with PBS cells were stained for 1 h with Rhodamine- conjugated-α-Bungarotoxin (Rh-α-BTX), which was applied in 1:2500 dilution in PBS. After washing three times with PBS the bottoms of the dishes with cells on their surface were cut out and mounted on cover slides with Mowiol. Slides were analyzed and documented using

57 Material and methods ______the Cy3 filter of a Leica inverted microscope (DMIRB) equipped with a cooled MicroMax CCD camera (Princeton Instruments, Stanford, CA).

3.2.3.7. AChR cluster stability assay

For assessing AChR cluster stability C2C12 cells were treated for 16 h with agrin 4.8. alone or together with 60 µM apigenin, washed and then maintained in fresh cultivation medium with or without apigenin at the same concentration. Cells were fixed with 2% PFA at 0/4/ 8h and stained with Rh-α-BTX (see 3.2.3.6.).

3.2.3.8.Quantification analysis of AChR clusters

The numbers of AChR aggregates were quantified as follows: using the Cy3 filter pictures were taken from 8 areas exhibiting similar myotube density by phase contrast microscopy at 100x magnification. AChR clusters were counted for each area, normalized to mock treatment set to 100%, and the standard deviation was calculated for the 8 areas. Quantification analysis of AChR clusters was performed for three independent experiments. Quantification of the AChR cluster density and length was done using Scion Image program as describe before (Jacobson et al. 2001). In brief, pictures of MuSK-deficient myotubes or C2C12 cells expressing clusters on their surface were taken by fluorescence microscopy at 400x and 200x magnification respectively. Images were imported into the Scion Image program in grey scale mode. Size scale calibration was set to 0.58 pixel/µm or to 1.16 pixel/µm respectively. The total cluster area was determined by circumscribing clusters using free hand tool and then Measure command. The clusters were highlighted then using Density slice tool and the area occupied by clusters was measured using Analyze particle command. The resulting values were summed to give the particle area. The AChR density, given as a percentage, was calculated as particle area divided by total area of the cluster. The length of AChR clusters was arbitrary assigned as number of pixels. Statistical analysis was performed using unpaired two-tailed t-test.

3.2.4.Animal care and immunohistochemistry methods

3.2.4.1. Generation of muscle specific CK2β knockout animals

For the generation of muscle specific CK2β knockout, mice of the CK2βloxP/loxP genotype were crossed with HSA-Cre transgenic mice to get CK2βloxP/+, HSA-Cre offspring. Then

58 Material and methods ______

CK2βloxP/+, HSA-Cre mice were crossed with CK2βloxP/loxP mice to get animals with CK2βloxP/loxP, HSA-Cre genotype.

3.2.4.2. Genotyping

DNA for genotyping was obtained from tail tips. The samples were lysed in 185 µl of Tail lysis buffer with 15 µl proteinase K (20 µg/µl; Roth) for 1-2 h at 55˚C. The DNA was precipitated with 150 µl of isopropanol. After centrifugation for 20 min at 16000 rcf the DNA was washed with 1 ml of 70% ethanol, dried and dissolved in 500 µl of water. Genotyping PCRs for identification of homozygous CK2βloxP/loxP and HSA-CRE animals were performed according to the schemes given in the Table 3.32 and 3.33 respectively.

PCR mix Number of Step Temperature Time cycles

H20 6.1 µl Denaturation 1 94°C 4 min

10xTaq-polymerase 1.2 µl 35 94°C 30 sec buffer+(NH4)2SO4 Amplification

MgCl2 (25 mM) 1 µl 55°C 30 sec dNTP (10 mM) 1.2 µl 72°C 30 sec

CK2β-17/19 0.125 µl Elongation 1 72°C 8 min

CK2β-18/20 0.125 µl

Taq DNA polymerase 0.25 µl 1 10°C 10 min Genomic DNA 2 µl

Table 3.32. Scheme for CK2βloxP/loxP genotyping. For the sequences of the primers see 3.1.3.

PCR mix Number of Step Temperature Time cycles

H20 11.2 µl Denaturation 1 94°C 3 min

10xTaq-polymerase 2 µl 35 94°C 30 sec buffer+(NH4)2SO4 Amplification

MgCl2 (25 mM) 2.4 µl 60°C 30 sec dNTP (10 mM) 2 µl 72°C 1 min

HSA-Cre-F 0.125 µl Elongation 1 72°C 2 min HSA-Cre-R 0.5 µl

Taq DNA polymerase 0.5 µl 1 10°C 10 min Genomic DNA 1 µl

59 Material and methods ______

Table 3.34. Scheme for HSA-Cre genotyping. For the sequences of the primers see 3.1.3.

3.2.4.3. Surgical Procedures

Adult C57/BL6 wild type mice were anesthetized by intraperitoneal administration of a Ketamine–Rompune mixture (100 mg/per kg body weight Ketanest (Pfizer); 5 mg/per kg body weight Xylacin (Bayer)) for surgery using standard aseptic technique. A skin incision was made on the lateral thigh to expose the left biceps femoris muscle, and a longitudinal incision was made to expose and transect the sciatic nerve at the level of its trifurcation. After sciatic nerve transaction the mice were stitched. 5 days postoperatively mice were sacrificed and soleus and gastrocnemius muscles were dissected. Frozen sections of rat soleus muscle, which was ectopically injected with plasmid encoding for agrin 4.8. (Hashemolhosseini et al. 2000) were kindly provided by Prof. Hans-Rudolf Brenner.

3.2.4.4. Immunohistochemistry

For the preparation of frozen sections for immunohistochemical analysis, all muscles were quick-frozen in prechilled isopentane and embedded in Tissue-Tec (Leica Instruments). 12 µm slices were prepared using a cryotome (Leica Microsystems, Nussloch) and placed on glass slides (HistoBond, Adhesion Micro Slides, Jung HistoService). The sections were air- dried for 1 h at RT and stored at -80˚C or directly subjected to the immunohistochemical stainings. Unfixed sections were rinsed with PBS and permeabilized for 5-10 min in PBS supplemented with 0.1% Triton-X100. Further, sections were blocked in blocking solution (10% FCS and 1% BSA) for 1-2 h and stained for AChR with Rh-α-BTX (1:2500 dilution) or incubated with rabbit anti-CK2β (at 1:500 or 1:200 dilution; gift from Drs. Mathias Montenarh or Claude Cochet respectively) or anti-CK2α antibody (at 1:1000 dilution; Upstate Biotechnology) dissolved in the blocking solution at 4˚C in a humid environment overnight. After washing the sections six times for 5 min in PBS, secondary antibodies conjugated to Cy2, Cy3 or Alexa 488 immunofluorescent dyes (Dianova, Molecular probes) were applied in 1:100, 1:200 or 1:500 dilutions respectively for 1 h at RT. Subsequently, sections were washed six times for 5 min in PBS and covered with Mowiol. For whole mount preparations the hind limb muscles were isolated from the adult CK2βloxP/loxP, HSA-Cre mice and their wild type littermates. The hind limb muscles (soleus,

60 Material and methods ______gastrocnemius and extensor digitorum longus (EDL)) were quickly fixed in 4% PFA for 10 min and then teased in thin bundles of 5-10 myotubes. The tissue was blocked in 100 mM glycin in PBS for 15 min, permeabilized in 0.5% Triton X-100, 5% BSA, 1%FCS for 1 h and incubated with rabbit anti-Neurofilament antibody (at 1:5000 dilution; Chemicon) overnight. Then the tissues were washed six times for 10 min in PBS and incubated for 1-2 h with secondary antibodies conjugated to Alexa 488 (at 1:500 dilution; Molecular Probes) together with Rh-α-BTX. After washing six times for 10 min in PBS the tissues were covered with Mowiol.

3.2.4.5. Microscopy, imaging and quantification of endplates.

Sections were analyzed and documented using a Leica inverted microscope (DMIRB) equipped with a cooled MicroMax CCD camera (Princeton Instruments, Stanford, CA) or a Leica confocal microscope TCS SL equipped with Leica confocal software TCS SL version 2.5.1347a. The confocal stacks were shown as average projections comprising several stacks taken with an interval of about 0.5 µm. For quantification of AChR cluster disassembly, endplates were divided according to their morphology in four categories: A-intact; B-slightly impaired (with several brakes); C-fragmented; D-disassembled in micro aggregates of AChRs. Statistics were performed by averaging three sets of independent quantifications of minimum 50 endplates for each muscle.

61 Results ______

4. Results

4.1. Searching for MuSK binding proteins

4.1.1. Generation and characterization of MuSK baits for yeast two hybrid screens cDNA representing extracellular (Ig-like IV and C6-box: aa 232-491; or IgIV-like alone: aa 350-491) or intracellular (JM: aa 481-520) domains of MuSK have been subcloned in frame with GAL4-DNA-BD in pGBKT7 and named K3, K4 and JM bait respectively (Fig. 9).

Fig. 9: Different MuSK domains were used as baits for yeast two hybrid (Y2H) screens. K3 bait composed of Ig-like IV and C6-box and K4 bait comprising Ig-like IV domain only were used in order to identify RATL. Intracellular baits: JM representing juxtamembrane domain and MuSK2xwt mimicking active MuSK intracellular domain conformation were used for identification of MuSK downstream effectors. Candidates from Y2H screen with MuSK2xwt were checked for interaction with kinase defective MuSK2xkd bait.

All expression cassettes were verified by sequencing. The expression of the K3, K4 and JM baits was verified using Western blot of protein extracts prepared from transformed yeast cells (Fig. 10).

62 Results ______

Fig. 10: Expression of the Y2H baits K3, K4 and JM in yeast. Expression of myc-tagged K3, K4 (A) and JM (B) Y2H baits was verified in two different yeast strains HF7c and AH109 by Western blot with α-myc antibody. JM bait expression was under detection limit in HF7c strain. Position of SeeBlue protein ladder (Invitrogen) is indicated on the left side of each blot.

A bait comprising two complete intracellular domains of MuSK fused by a flexible G/E linker (named MuSK2xwt) and its kinase defective mutant bearing substitution of lysine in the ATP- binding site (Zhou et al. 1999) of the kinase domain (named MuSK2xkd) (Fig. 9) have been generated and tested regarding expression and auto-tyrosine phosphorylation in the group of Prof. Hans-Rudolf Brenner (Biocenter, Basel, Switzerland). Autophosphorylation has been observed in yeast and mammalian cells for MuSK2xwt protein but not for MuSK2xkd mutant.

4.1.2. Outcome of the yeast two hybrid screens with MuSK baits

Bait constructs were tested for autonomous trans-activation of yeast reporter genes. With some of the baits 3-AT was used to avoid trans-activation. For the screen, a commercial HeLa cDNA library (Clontech) was used. In all screens, the number of examined colonies was higher than the number of independent clones of the library to ensure that statistically every clone of the library was tested at least one time (1x106; Table 4.1.). The isolated yeast clones after screening by selective growth conditions on auxotrophic markers were further analyzed for β-galactosidase gene activation. Afterwards, prey plasmids were isolated from yeast clones and transformed into E. coli. To avoid further work with identical clones, prey plasmids were compared by their restriction patterns after EcoRI/XhoI or ApaI digestion. Prey plasmids were then re-transformed into yeast cells together with the specific bait to confirm specificity of protein-protein interaction or with an empty bait plasmid pGBKT7 to check whether the candidate interacts with GAL4 DNA-BD alone. All candidates identified by screening with the JM bait interacted with the GAL4 DNA-BD and could not be further considered as specific. The inserts of isolated and evaluated prey plasmid were analyzed by

63 Results ______sequencing. Coding sequences of proteins (or part of the proteins) corresponding to the yeast candidates were identified by the BLAST program (Basic Local Alignment Search Tool at the National Center for Biotechnology Information) using sequences of prey plasmids. Only candidates, whose coding sequences were in frame with the GAL4 AD have been selected for further investigation. Only clones representing proteins, which spatially and subcellularly co-localize with the respective MuSK domains used as baits for their identification, were considered as potential MuSK binding partners (Table 4.1.).

Bait Conditions N° of Candidates Confirmed Potential MuSK N° of independent binding partners identification clones (aa) screened MuSK2xwt (HF7c) 4x106 493 44 Nedd9 2 -T-L-H+ (aa 500-762) 10mM3AT Casein 167 kinase2β (CK2β) (aa 1-215) Hypothetical 1 protein (aa 110-376) DKFZp434A1319 LIM domain only 2 7 (aa 945-1350) K3 (AH109) 2.4x106 66 21 EFEMP-1/ S1-5 4 -T-L-H-A (aa 87-494)/ (aa 4-388) K4 (AH109) 0.6x106 3 1 - -T-L-H-A

(AH109) 1.9x106 89 22 WISP2 5 -T-L-H (aa 1-251) +5mM3AT Laminin receptor 1 1 (aa 127-898) JM (AH109) 3x106 30 - -T-L-H-A

(HF7c) 3.6x106 14 - -T-L-H + 10mM3AT

Table 4.1. Results of the Y2H screens with the MuSK domains. Only relevant candidates, e.g. with the corresponding tissue expression and sub-cellular localization to that of MuSK, are shown in the column “potential MuSK binding partners”. The number of independent sequences and amino acid region of candidate obtained from the Y2H screen are indicated in the last column.

One of the candidates, which have been identified many times in the Y2H screen with the MuSK intracellular domain is regulatory β subunit of Casein Kinase 2 (CK2β). CK2 is highly conserved and ubiquitously expressed serine/threonine kinase, which is involved in many

64 Results ______biological processes such as gene expression, protein synthesis and signal transduction (Meggio and Pinna 2003; Olsten and Litchfield 2004). Interaction of this protein with MuSK has been further studied in details.

4.2. Detailed investigation of MuSK – CK2 interaction

To evaluate the significance of the interaction between MuSK and CK2 following experiments have been performed: - Investigation of the temporal and spatial expression profile of CK2 at the NMJ - Biochemical verification of the interaction between MuSK and CK2 - Mapping of the binding epitopes for both proteins - Co-localization of MuSK and CK2 proteins at the NMJ - Investigation of the biological significance of the interaction between MuSK and CK2

4.2.1. Quantitative determination of CK2 transcript level in different tissues

After identification of CK2β interacting with MuSK in yeast, we wanted to know if CK2 is expressed in the same tissues and cells as MuSK. Moreover, considering the fact that transcripts of MuSK as well as other proteins of postsynaptic specialization, for example AChR subunits, are upregulated at the synapse (Moore et al. 2001; Witzemann et al. 1991), we intended to investigate whether the same is true for CK2. Taking into account that the CK2 holoenzyme is a tetramer, consisting of two catalytic α or α’ subunits and two regulatory β subunits we decided to investigate the expression profile of all subunits. It seemed to be even more interesting to investigate the distribution of all CK2 subunits, because CK2 can act in some processes without regulatory subunit and there is a functional specialization between α and α’ subunits in different tissues (Boldyreff and Issinger 1997; Chen et al. 1997; Escalier et al. 2003; Xu et al. 1999). The quantity of transcripts was estimated by quantitative real time PCR for all CK2 subunits as well as for some proteins concentrated at the NMJ, e.g. MuSK and AChR α subunit. For 1st strand cDNA synthesis RNA was prepared from synaptic and extrasynaptic regions of mouse diaphragm, C2C12 myoblasts, and C2C12 myotubes treated with conditioned media containing either nerve- derived agrin 4.8., which is able to induce clustering of AChRs or an inactive muscle-derived st isoform agrin 0.0. As a negative control for MuSK and AChRα expression 1 strand cDNA from brain has been used.

65 Results ______

As expected, MuSK and AChRα transcripts accumulate in the synaptic part of the diaphragm (Fig. 11 A, B). Their quantity is higher in myotubes than in myoblasts and subsequent treatment of myotubes with agrin 4.8. induces further increase of the transcripts level.

Fig. 11: Distribution of MuSK and AChRα transcripts. The level of MuSK (A) and AChRα (B) mRNA was defined by real time PCR in brain, synaptic and extrasynaptic regions of diaphragm, C2C12 myoblasts and myotubes treated with muscle-derived agrin 0.0. or nerve-derived agrin 4.8. Quantification data were normalized to β-actin for each sample. Gene expression level in C2C12 myotubes was always set to 1.

Next, we investigated the expression profile of the CK2 subunits. As CK2 is an ubiquitous protein, the expression of all CK2 subunits is detected in both brain and muscle tissues, whereas muscle contains a higher amount of CK2 transcripts (Fig. 12). CK2β and CK2α’ transcripts are enriched in the synaptic region of the diaphragm (Fig 12 B, C). Although expression level of all CK2 subunits is higher in C2C12 myotubes than in myoblasts, only CK2 α’ subunit transcription is slightly elevated in myotubes in response to agrin 4.8. treatment (Fig. 12 B).

66 Results ______

Fig. 12: Distribution of CK2 subunits transcripts. Relative amounts of CK2 α (Α), α’ (B) and β (C) subunits transcripts were measured for brain, extrasynaptic and synaptic areas of diaphragm, C2C12 myoblasts and myotubes treated with either agrin 0.0. or agrin 4.8. by quantitative real time PCR. All quantification data were normalized to β- actin and calibrated for each CK2 subunit to the level of their transcripts in C2C12 myoblasts, which was set to 1.

4.2.2. Biochemical verification of the interaction of CK2 subunits with MuSK

In order to confirm the interaction between CK2β and the intracellular domain of MuSK by co-immunoprecipitation experiments both proteins were over-expressed in mammalian cells. For that, full length human CK2β identified by Y2H screen was subcloned into a CMV- expression vector in frame with the T7-tag and transiently transfected together with an expression plasmid encoding a myc-tagged MuSK2xwt (pcDNA3-MuSK2xwt-myc) into HEK293 cells. The cells were harvested after 48 h, protein extracts were prepared and used for co-immunoprecipitation. As a negative control protein extract containing only MuSK2xwt-myc was used. Immunoprecipitation of CK2 β subunit with anti-T7-tag antibodies resulted in co-immunoprecipitation of MuSK2xwt, which was detected by Western blot with anti-myc antibodies (Fig. 13). The CK2β preferentially interacted with the tyrosine- phosphorylated form of MuSK intracellular domain, which corresponds to the upper band detected for MuSK2xwt (Fig. 13, Fig. 14 A). Since CK2 in most cases acts as a tetramer

67 Results ______composed of β and α subunits, interaction between HA-tagged catalytic α subunit and MuSK2xwt has also been studied. α subunit of CK2 was also able to bind to the intracellular domain of MuSK, but the observed interaction was very weak (Fig. 13).

Fig. 13: Co-immunoprecipitation of MuSK2xwt by CK2α and CK2β. Protein extracts of HEK293 cells containing over-expressed either HA-tagged CK2α or T7-tagged CK2β together with MuSK2xwt-myc were used for immunoprecipitation with anti-HA-tag or anti-T7- tag antibodies respectively. Western blot with α-myc antibodies has shown that MuSK2xwt is co- precipitated by CK2α and CK2β. Phosphorylated form of MuSK2xwt interacts strongly with CK2β. Position of SeeBlue protein ladder is indicated on the left (the figure is kindly provided by Amir Khan).

To reveal if the α and β subunits of CK2 bind to the same epitope of MuSK, the interaction between MuSK2xwt and CK2β has been studied under the excessive amounts of CK2α and vice versa. It has to be noted, that due to the ubiquitous expression of CK2 protein, α and β subunits are always present at least in minor amounts in every cell system. Precipitation of T7-tagged CK2β in the presence of high amounts of over-expressed CK2α resulted in the same efficient co-precipitation of MuSK2xwt (Fig. 14 A). The interaction was also observed between MuSK2xwt and CK2α when CK2β subunit was over-expressed (Fig. 14 B). At last, a strong binding between CK2α and CK2β was not affected by the presence of high amounts of MuSK2xwt, which most likely reflects existence of additional CK2β – CK2α complexes independent of MuSK (Fig. 14 C).

68 Results ______

Fig. 14: Co-immunoprecipitation of CK2β, CK2α and MuSK2xwt. Co-immunoprecipitation experiments were performed with protein extracts of HEK293 cells transiently transfected with T7-tagged CK2β, HA-tagged CK2α and myc-tagged MuSK2xwt. (A) Interaction between MuSK2xwt and CK2β was not affected by over-expression of CK2α. (B) Immunoprecipitation of MuSK2xwt in the presence of over-expressed CK2β resulted in co- precipitation of CK2α. (C) Strong binding between CK2β and CK2α was not disturbed by over- expression of MuSK2xwt. Position of protein ladder is indicated on the left of each blot.

As a next step, the interaction between MuSK and CK2β was analyzed in vivo. Precipitation of endogenous MuSK protein from C2C12 myotubes or mouse hind limb muscle tissue lysates resulted in co-immunoprecipitation of endogenous CK2β (Fig. 15 A, B).

Fig. 15: Co-immunoprecipitation of endogenous MuSK and CK2β. Interaction between endogenous MuSK and CK2β has been shown by co-immunoprecipitation experiments using (A) protein extract of C2C12 myotubes and (B) of mouse hind limb muscle. MuSK protein was precipitated by a mixture of three α-MuSK antibodies (ABR, Abcam and 194T) conjugated to protein A Sepharose beads, CK2β was detected by Western blot with α-CK2β specific antibodies (123-GLSDI-127). Position of protein marker is indicated on the left of each Western blot.

4.2.3. Mapping of interacting domains between MuSK and CK2β

To identify the epitopes of MuSK interacting with CK2β a series of C-terminal deletions of MuSK intracellular domain were generated (Fig. 16). First, deletion mutants were subcloned

69 Results ______as a fusion to GAL4 DNA-BD in the Y2H bait vector and used in binary Y2H interaction assays with the full length CK2β identified by the screen.

Fig. 16: MuSK deletion constructs. C-terminal deletions of MuSK intracellular domain are shown. The numbers in the name of the constructs correspond to the C-terminal aa positions of truncation. All truncations were subcloned into the Y2H bait vector pGBKT7 and into CMV-expression vector.

CK2β interacted with MuSK2xwt, MuSK2xkd and with whole MuSK intracellular domain (MuSK-868) (Fig. 17 A).

Fig. 17: Mapping the epitope of MuSK interacting with CK2β. Epitope of MuSK interacting with CK2β was mapped by binary Y2H assay (A) and by co- immunoprecipitation (B) to the kinase domain. (A) C-terminal truncations of MuSK were expressed as GAL4 DNA-BD fusions, whereas full length CK2β was fused to GAL4 AD. Interaction between MuSK constructs and CK2β is indicated by the yeast growth. Positive control implies interaction between murine tumor suppressor protein p53 and the SV40 large T-antigen encoded by pGBKT7-53 and pGADT7-T (Clontech) respectively. (B) Co-immunoprecipitation of myc-tagged CK2β by different T7-tagged truncations of the MuSK intracellular domain over-expressed in HEK293 cells.

70 Results ______

While deletion of MuSK C-terminal domain (MuSK-857) enclosing PDZ-binding motif did not affect interaction with CK2β, the truncation of kinase domain to the half (MuSK-682) completely abolished the binding (Fig. 17 A). To confirm the results of Y2H experiments the same MuSK intracellular domain C-terminal truncations were expressed as T7-tagged proteins and used in immunoprecipitation experiments together with myc-tagged full length CK2β. From MuSK deletion mutants only MuSK-868 and MuSK-857 were able to interact with CK2β that corresponded to the Y2H data (Fig. 17 B). To identify the epitope of CK2β which interacts with MuSK we followed the same strategy generating a series of deletion constructs (Fig. 18). For CK2β the following epitopes are known: (1) a ‘destruction box’ which is responsible for CK2β turnover; (2) an ‘acidic loop’ which interacts with basic residues present on CK2α and mediates the association of the holoenzyme with the plasma membrane; (3) a ‘zinc finger domain’ which is responsible for the dimerization of CK2 β subunits; (4) a ‘positive regulatory domain’ at the C-terminus which appears to play a role in the oligomerization of the kinase. Starting from the C- terminus, we subsequently chopped of CK2β domains, subcloned the remaining parts as GAL4 AD- or as GST-fusions and used them with MuSK2xwt in Y2H assays and GST- pulldown experiments, respectively.

Fig. 18: CK2β constructs used for epitope mapping. Domain structure of CK2β protein is shown. C-terminal truncations enclosing different domains were generated and subcloned either into the Y2H prey vector or as GST-fusions. The numbers in the name of the constructs correspond to C-terminal aa position of truncation.

In contrast to the full length protein all deletion mutants of CK2β failed to interact with MuSK2xwt in both assays (Fig. 19 A, B).

71 Results ______

Fig. 19: Mapping of epitope of CK2β interacting with MuSK. Epitope of CK2β interacting with MuSK was identified in binary Y2H experiments (A) or by GST- pulldown (B) with MuSK2xwt. (A) CK2β truncations were expressed as preys and MuSK2xwt as a bait. Interaction was revealed by the yeast growth only for full length CK2β. (B) GST-fusions of CK2β epitopes were immobilized on Glutathione Sepharose beads and incubated with the extract of Cos7 cells transiently transfected with myc-tagged MuSK2xwt. Interaction was detected by Western blot with α-myc antibodies.

4.2.4. Localization of CK2 at the NMJ

MuSK is specifically expressed in muscle and concentrates at the postsynaptic sites. Therefore, the next step was to study the distribution of CK2 subunits at the NMJ. Frozen cross-sections of the soleus mouse muscles were stained with antibodies specifically recognizing either the β or α subunits of CK2. The same sections were incubated with Rhodamine-α-Bungarotoxin (Rh-α-BTX), which labels AChRs clustered at the postsynaptic sites of the NMJ. While immunoreactivity of both CK2α and CK2β proteins was revealed in the cytoplasm of muscle fibers (reflecting ubiquitous expression of CK2), the signal was significantly concentrated at the NMJ and co-localized there with Rh-α-BTX-stained AChR clusters (Fig. 20).

72 Results ______

Fig. 20: Co-localization of CK2 subunits with the NMJ. Immunofluorescence micrographs of cross-sections of mouse soleus muscle stained with CK2α (upper panel) or CK2β (lower panel) antibodies shown in green together with Rh-α-BTX shown in red. Both subunits of CK2 are concentrated at the sites of AChR clustering. Scale bar 20 µm.

To rule out a presynaptic localization of CK2 subunits in motor nerve terminals or terminal Schwann cells, we followed two different strategies. Firstly, transection of the sciatic nerve, which leads to degeneration of nerve terminals which form synapses on the soleus muscle was performed for one of the hind limbs of a mouse. The contra-lateral hind limb served as a control. After 5 days post denervation immunohistochemistry of the muscle crossections revealed that CK2β and CK2α proteins are still maintained co-localized with AChR clusters, suggesting that CK2 subunits are at least in part located at the postsynapse (Fig. 21).

Fig. 21: Localization of CK2 subunits at the postsynaptic specializations. Immunohistochemical labeling of denervated mouse soleus muscle with antibodies specific for CK2α (upper panel) or CK2β (lower panel) shown in green together with Rh-α-BTX shown in red. CK2 subunits are co-localized with AChR clusters in the absence of the nerve terminal. Scale bar 20 µm.

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Secondly, ectopic endplates were generated in vivo by injection plasmids expressing an active isoform of agrin (agrin 4.8.) together with nlsGFP into anesthesized rats (Fig. 22 A). The ectopic injections where performed by Prof. Hans-Rudolf Brenner (Biocenter, Basel, Switzerland). Such ectopic endplates are known to be free of nerve terminals and Schwann cells, but contain all proteins, which are normally concentrated at postsynaptic specializations (Jones et al. 1997). We observed by immunostaining that CK2 β and α subunits are localized at such ectopic endplates (Fig. 22 B).

Fig. 22: Co-localization of CK2 subunits at the ectopic endplates with AChR clusters. (A) Ectopic endplates, which are free of nerve terminal and Schwann cells were generated by injection of plasmids encoding active isoform of agrin pcAgrin 748 and nuclear localized GFP (pnlsGFP) into the NMJ free site of muscle (Hashemolhosseini et al. 2000). (B) Immunofluorescence confocal images of the region of rat soleus muscle expressing ectopic postsynaptic specialization stained with Rh-α- BTX (red) and antibodies against CK2α (upper panel) or CK2β (lower panel, green). Overlaid single Z-projections are shown. Scale bar 80 µm – upper image, 40 µm – lower image.

MuSK, as a key regulator of postsynaptic specialization, mainly plays its role during the late phase of embryogenesis and early postnatal stage when the establishment of NMJs takes place. At these stages, the MuSK protein is highly expressed, though later during development it becomes downregulated (Valenzuela et al. 1995). To study whether the CK2 expression profile correlates with that of MuSK, mice hind limb muscles were examined at different developmental stages (E18.5, P0 and P7) for the presence of CK2 subunits at the NMJ. Staining with CK2β-specific antibodies has shown that accumulation of the protein at postsynaptic sites detected by Rh-α-BTX staining of AChRs occurs starting from the first postnatal week, while at the embryonic stage CK2β immunoreactivity is uniformly distributed in the cytoplasm of muscle cells (Fig. 23).

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Fig. 23: Accumulation of CK2β at the NMJ during development. Immunofluorescence micrographs of crossections of mouse hind limb muscles at different developmental stages (E18.5, P0 and P7) stained with α-CK2β antibody (shown in green) together with Rh-α-BTX (shown in red). Immunoreactivity for CK2β starts to be concentrated at the NMJ sites at P7 stage. Scale bar 10 µm.

Similarly, concentration of the signal for α subunit of CK2 became detectable at postsynaptic membranes only at P7 and later (Fig. 24), suggesting that α and β subunits act together at the late stages of the NMJ development.

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Fig. 24: Accumulation of CK2α at the NMJ during development. Immunofluorescence micrographs of crossections of mouse hind limb muscles at different developmental stages (E18.5, P0 and P7) stained with α-CK2α antibody (shown in green) together with Rh-α-BTX (shown in red). Immunoreactivity for CK2α starts to become concentrated at the NMJ sites at P7 stage. Scale bar 5 µm.

4.2.5. Biological role of CK2 at the NMJ

Given that CK2 interacts with MuSK and that the subunits of CK2 are concentrated at the NMJ on mRNA and protein level, we decided to examine the biological significance of CK2 for postsynaptic specializations.

4.2.5.1. Inhibition of CK2 activity

The physiological role of CK2 for postsynaptic assembly was studied using murine C2C12 myoblasts. C2C12 myoblasts were differentiated into myotubes under the low-serum conditions. Upon treatment with agrin 4.8., the myotubes formed on their surface postsynaptic specializations characterized by clustering of AChRs (Fig. 25 A). In order to investigate the role of CK2 for AChR clustering, its activity was blocked by pharmacological inhibitors either apigenin (Sigma, HCLP grade) or DMAT (2-Dimethylamino-4,5,6,7-tetrabromo-1H- benzimidazole; gift from Drs Flavio Meggio and Lorenzo A. Pinna). DMAT was recently published to be highly specific for CK2 (Critchfield et al. 1997; Pagano et al. 2004). To cover the time of agrin-MuSK signaling, inhibitors were added to the myotubes before application of agrin and treatment was continued for 16 h. The aggregation of AChRs was not abolished

76 Results ______in the presence of both inhibitors, but the number of clusters was significantly increased (Fig. 25 A).

Fig. 25: Inhibition of CK2 activity affects AChR clustering. (A) Immunofluorescence images of C2C12 cells treated with agrin 4.8. and either mock (DMSO) or CK2 inhibitors – 60 µM apigenin or 40 µM DMAT as indicated. Rh-α-BTX staining (depicted in red) shows that amount of AChR clusters is increased upon CK2 blockage. Scale bar 100 µm. (B) MuSK protein was immune-precipitated from either mock or 40 µM DMAT treated C2C12 cells after application of inactive agrin 0.0. or active agrin 4.8. Subsequently, the MuSK kinase activity was measured in in vitro by [32 P] γ –ATP incorporation assay against enolase (an in vitro MuSK substrate). As it is shown in autoradiography enolase phosphorylation reflecting MuSK kinase activity is induced by application of agrin 4.8., but not affected by DMAT.

The increase of the number of AChRs after treatment with inhibitors was dose-dependent, with the number of clusters being 2.5-fold higher at the maximal inhibitor concentration used (Fig. 26 A). The use of higher concentrations of CK2 inhibitors turned out to be toxic for the cells. Interestingly, after detailed investigation it appeared that the high number of AChR clusters after the inhibition of CK2 runs on account of very small cluster. At the same time AChR aggregates of average and big size were still present as in the control situation (Fig. 26 B). The change in AChR cluster formation upon application of the inhibitors was not induced by a substantial change in MuSK kinase activity because the ability of MuSK to phosphorylate its in vitro substrate enolase was not affected in the presence of DMAT (Fig. 25 B).

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Fig. 26: Blockage of CK2 activity affects AChR clustering. (A) Pharmacological inhibitors of CK2 activity applied to C2C12 myotubes in increased concentrations (10 µM – 60 µM) together with agrin 4.8. induced a dose-dependent increase of number of AChR clusters. AChR clusters were counted in at least eight different fields expressing the same cell density at 100x magnification. The number of clusters for mock control was set to 100%. (B) Morphometrical analysis of AChR clusters formed after agrin 4.8. and either 60 µM apigenin or 40 µM DMAT treatment shows that inhibition of CK2 activity leads to appearance of big population of small clusters. The length of AChR clusters was measured by means of Scion Image program and presented in pixels.

4.2.5.2. Knockdown of CK2 subunits by using siRNA

In order to elucidate the requirement of different CK2 subunits for postsynaptic specialization an additional approach of RNA interference has been used. Specific siRNAs knocking down the genes encoding CK2 β, α and α’ subunits have been designed by Sfold software (Ding et al. 2004) and either synthesized as stealth siRNA or expressed from plasmid pSUPERneoGFP (Oligoengine). The knockdown efficiency was confirmed by luciferase reporter assay. For that luciferase gene was subcloned as a bicistronic message together with CK2 subunits cDNA in CMV expression vector. The vectors were co-transfected with siRNAs (or siRNA producing vectors) targeting CK2 subunits into HEK293 cells. Targeting of the bicistronic luciferase-CK2 subunit mRNA transcribed from the vector by specific siRNA led to its degradation resulting in reduced luciferase activity. According to this assay, the siRNAs CK2α-373 and CK2α’-746 specifically inhibited synthesis of the mRNA of the respective CK2 subunits by about 80% (Fig. 27 A, E). siRNA CK2α’-690 effectively knocked down mRNA of both CK2 α and α’ subunits (Fig. 27 A, E). Similarly, the efficiency of the CK2β- specific siRNA (CK2β-189) was confirmed (Fig. 27 C). Next, the knockdowns of CK2α, α’ and β were confirmed on the protein level. For that plasmids expressing T7-tagged CK2 subunits were transfected together with respective siRNAs into HEK293 cells and the expression of proteins was determined by Western blot using α-T7-tag antibodies (Fig. 27 B, D, F).

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Fig. 27: Knockdown of CK2 β, α or α’ subunit by different siRNAs. Efficiency of CK2 β, α or α’ knockdown by different siRNAs was assessed in luciferase-reporter assay (A, C, E) and on protein level by Western blot (B, D, F). (A, C, E) Luciferase activity was measured after co-transfection of HEK293 cells with plasmid encoding luciferase-CK2β/α/α’ bicistronic messages and defined siRNAs. Luciferase activity in control cells transfected with nonspecific siRNA was set to 100%. (B, D, F) Western blots of protein extracts of HEK293 cells transfected with vectors encoding T7-tagged CK2β/α/α’ subunits and indicated siRNAs. β-Tubulin amount in extracts was taken as a loading control. Note, that siRNAs CK2α-373 and CK2α’-746 specifically inhibit CK2α or CK2α’ subunit expression respectively. siRNA CK2α’-690 effectively inhibits both CK2 catalytic subunits. CK2β is knocked down by siRNA CK2β-189.

To investigate the biological consequences of knocking down the expression of CK2, we transfected the effective siRNAs into MuSK-deficient myoblasts. Unlike C2C12 myotubes, MuSK-deficient myotubes fail to cluster AChRs in response to agrin treatment, unless forced to express MuSK by transfection of a respective expression plasmid. Like this, these cells allow to avoid interference of endogenous MuSK and to study AChR clustering specifically in MuSK-transfected cells. Specific siRNAs were transfected together with pMT-MuSK-full- length plasmid into MuSK-deficient myoblasts, which were subsequently differentiated by special medium and incubation conditions into myotubes. Transfected cells were detected by fluorescence of co-transfected pnlsGFP. After application of agrin for the last 16 h, cells were fixed and stained for AChR clusters with Rh-α-BTX. Transfection of siRNAs specific against either CK2α or CK2α’ resulted as in the case of inhibitor application in appearance of high amount of undersized AChR clusters (Fig. 28 A).

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Fig. 28: Wile depletion of CK2 α or α’ subunits level results in formation of high amount of undersized AChR clusters, inhibition of either both catalytic subunits or regulatory subunit of CK2 leads to myoblasts death. (A) Images of MuSK-deficient cells transfected with full length MuSK and siRNAs (as indicated) are shown. AChR clusters are stained with Rh-α-BTX (red). Transfected cells detected by fluorescence of co-transfected nuclear localized GFP (green). Scale bar 50 µm. The amount of GFP-positive cells was quantified per 3.5 cm plate. Blockage of both CK2 α and α’ subunits by siRNA CK2α’-690 (B) or CK2 β subunit by siRNA CK2β-189 (C) disturbed cell survival.

While there was no apparent abnormality in differentiation and survival of MuSK-deficient myoblasts transfected with siRNAs active against only one of catalytic α subunits, the ablation of both α and α’ subunits by transfection of siRNA CK2α’-690 led to arrest of cell differentiation and survival. The effect was quantified by calculation of the number of the GFP-positive cells transfected with control or specific siRNA after 5 days of differentiation (Fig. 28 B). The same effect was observed when CK2β subunit expression was suppressed by siRNA CK2β-189 (Fig. 28 C).

4.2.5.3. Phosphorylation of MuSK by CK2

After we found out that MuSK interacts with CK2 it was obvious to speculate that CK2, as a serine/threonine kinase might phosphorylate MuSK. Indeed, a phosphorylated serine residue has been previously identified within the intracellular part of MuSK (Till et al. 2002). In silico study showed that four serines (S-678, S-680, S-690 and S-697) which are located within the kinase insert (KI) domain of MuSK might be phosphorylated by CK2. To identify which of four serines are indeed phosphorylated, we started a collaboration with group of Prof. Jorge Allende (Universidad de Chile, Santiago, Chile). They have checked a phosphorylation of synthetic peptides (named MuSK667 and MuSK687) representing the parts of MuSK KI and

80 Results ______carrying serines potentially phosphorylated by CK2 or their corresponding mutants (named MuSK667-S678A, MuSK667-S680A and MuSK687-S690A, MuSK687-S697A), carrying substitutions of serines to alanines, which can not be phosphorylated (Fig. 29 A). Both wild type peptides were highly phosphorylated by CK2 α subunit and addition of CK2 β subunit (resulting in holoenzyme formation) led to further enhancement of the phosphorylation rate (Fig. 29 B). While mutations of serine residues 678 and 690 did not have any prominent effect on phosphorylation, substitutions of serines 680 and 697 to alanines significantly affected phosphorylation of respective peptides either by CK2α or by holoenzyme, suggesting that serines 680 and 697 can be phosphorylated by CK2.

Fig. 29: Identification of MuSK serine residues potentially phosphorylated by CK2. (A) Amino acid sequences of synthetic peptides representing MuSK KI. Serines - potential targets of CK2 phosphorylation or their alanine substitutions are shown in bold. First number in peptide name corresponds to its starting aa position in MuSK protein sequence. Three first R residues are required for binding of peptides to the phosphocellulose membrane. (B) In vitro kinase assay for peptides shown in (A) performed with CK2α (black bars) and holoenzyme (open bars). The amount of incorporated isotope is given in pmol and indicated above or within the respective bars. The figure is kindly provided by Prof. Jorge Allende (Universidad de Chile).

We verified the in vitro phosphorylation data obtained with small peptides using the whole intracellular domain of MuSK or respective S680/S697A mutant. These MuSK proteins were expressed in E.coli, purified and subsequently subjected to phosphorylation by CK2α or CK2 holoenzyme.

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Fig. 30: In vitro phosphorylation of MuSK intracellular domain by CK2. Autoradiography after phosphorylation of MuSK intracellular domain or its alanine mutant S680/697A either by CK2α or holoenzyme.

Consistent with the phosphorylation of the peptides, the high degree of radioactive phosphate incorporation was observed for the wild type MuSK intracellular domain, but not for its alanine mutant S680/697A. However, in case of the MuSK intracellular domain the use of the holoenzyme compared with the use CK2α alone resulted in a lower phosphorylation rate of the protein (Fig. 30).

4.2.5.4. Role of CK2 dependent serine phosphorylation of MuSK for AChR clustering

To investigate if CK2-mediated phosphorylation is of any relevance in vivo, we mutated the respective serine residues. Either we replaced them by alanines (S680/697A) to abolish phosphorylation or by aspartic (S680/697D) or glutamic acid (S680/697E) which mimic constitutive phosphorylation. Mutations were introduced into the plasmid pMT-MuSK-full- length, encoding full-length mouse MuSK by use of a commercial Mutagenesis (Stratagene). The expression of MuSK mutants was confirmed in HEK293 cells (Fig. 31 A).

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Fig. 31: Ability of different MuSK mutants to restore AChR clustering in MuSK-deficient cells. (A) Expression of MuSK mutants, carrying substitutions of serines to alanines, aspartic or glutamic acids at positions 680 and 697 was confirmed in HEK293 cells by Western blot with α-MuSK antibodies (MuSK20kD). (B) Immunofluorescence images of MuSK-deficient myotubes transfected with wild type MuSK or its different mutants after agrin 4.8. application. AChR clusters stained with Rh-α-BTX are shown in red. Transfection of myotubes with MuSK plasmids is confirmed by co- transfected pnlsGFP (green). Treatment with CK2 inhibitor DMAT is indicated. DMAT treatment or mutation of serines 680/697 of MuSK to alanines (S680/697A) leads to formation of highly dispersed AChR clusters (arrowheads), whereas transfection of aspartic (S680/697D) or glutamic acid (S680/697E) MuSK mutants results in normal AChR clusters formation (arrows) even in the presence of DMAT. Scale bar 50 µm.

The ability of the MuSK mutants to restore the agrin-stimulated AChR clustering in MuSK- deficient myotubes was studied. Plasmids, encoding MuSK mutants were transfected together with the nlsGFP expression plasmid into MuSK-deficient myoblasts, which were subsequently differentiated into myotubes. As a control, plasmid encoding wild type MuSK was transfected. Upon agrin application the cells transfected with the wild type MuSK exhibited on their surface regular dense patches of AChR clusters (Fig. 31 B). When the cells

83 Results ______expressing wild type MuSK were treated with inhibitor of CK2 activity DMAT appearance of a bulk of very small AChR aggregates occupying large area of myotubes was detected (Fig. 31 B). Transfection of MuSK alanine mutant lacking potential CK2 phosphorylation sites (S680/697A) resulted in the same phenotype of AChR clusters. Additional treatment of the cells with DMAT did not further affect AChR clustering. Conversely, transfection of aspartic (S680/697D) or glutamic acid (S680/697E) MuSK mutants mimicking constant phosphorylation at aa positions 680 and 697 led to appearance of the regular size dense AChR patches. The normal morphology of the AChR aggregates in the cells expressing S680/697D or S680/697E mutants was preserved even in the presence of the DMAT, specifying the importance of MuSK phosphorylation at aa positions 680/697 for the normal AChR clustering. To confirm changes in visual appearance of the AChR clusters the morphometrical analysis of the respective cluster areas was performed. Indeed, mutation of serines 680/697 and/or CK2 inhibition lead to statistically significant decrease in density of AChRs, while mutations mimicking constitutive phosphorylation retain AChR at high density patches even in the presence of CK2 inhibitor (Fig. 32).

Fig. 32: Quantification analysis of AChR clusters restoration by different MuSK serine mutants in MuSK-deficient cells. Density of AChR clusters was calculated in Scion Image program (as described in Material and Methods). AChR clusters formed in the cells transfected with wild type MuSK and subsequently treated with DMAT or in the cells transfected with S680/697A MuSK mutant have significantly lower density than AChR clusters in control. * P-values <0.0001.

4.2.5.5. Role of kinase insert domain of MuSK in AChR clustering

Considering that we identified CK2-phosphorylatable serine residues within the KI of MuSK, we asked if their presence inside of KI might be required for modulation of AChR clustering. We decided to delete the KI of MuSK or to replace it by the KI of other receptor tyrosine

84 Results ______kinases and to study if these MuSK mutants are able to induce AChR clustering upon agrin 4.8. application. For our replacement studies, we chose the KIs of different receptor tyrosine kinases, such as Platelet-Derived Growth-Factor β Receptor (PDGFβR), Insulin-Like Growth Factor Receptor 1 (ILGFR-1), Insulin Receptor (IR) and receptor tyrosine kinase TrkC with KI of 14 aa (TrkC-14). Potential CK2-phosphorylatable serines are located within the KIs of two receptor tyrosine kinases of our choice: PDGFβR and TrkC-14 (Fig. 33).

Fig. 33: In silico identification of potential CK2 phosphorylation sites in KIs of different receptor tyrosine kinases. All serine residues found in KIs (aa sequences are indicated) of the receptor tyrosine kinases shown in red. Serines potentially phosphorylatable by CK2 were found in KIs of PDGFβR and TrkC-14 (underlined).

The MuSK KI mutants have been transiently transfected into HEK293 cells and their expression was verified by Western blot using α-MuSK antibody (MuSK 20kD) (Fig. 34 A). The ability of MuSK KI mutants to restore AChR clustering was analyzed upon their transfection into MuSK-deficient cells. MuSK-deficient myotubes expressing MuSK KI deletion mutant or MuSK in which KI was replaced by that of TrkC-14 were not able to form AChR clusters in response to agrin 4.8. application (Fig. 34 B). On the contrary, MuSK mutant carrying the KI of PDGFβR was able to mediate normal AChR clustering (Fig. 34 B). These data suggested that amino acid residues critical for AChR clustering are present in the KIs of MuSK and PDGFβR but absent from the KI of TrkC-14.

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Fig. 34: Restoration of AChR clustering by different MuSK KI mutants in MuSK-deficient cells. (A) Expression of MuSK mutants with deletion or substitution of KI domain by KIs of different receptor tyrosine kinases was confirmed in HEK293 cells by Western blot with α- MuSK antibody. (B) Images of MuSK-deficient cells transfected with different MuSK KI mutants. Transfected cells are identified by co-transfection of nlsGFP (green). AChR stained with Rh-α-BTX (red). Deletion of MuSK KI or its substitution with KI of TrkC-14 completely abolish AChR clustering. Substitution of MuSK KI by KI of PDGFβR doesn’t have any effect on AChR cluster morphology (arrows). Conversely, AChR clusters are affected in MuSK-deficient cells transfected with MuSK carrying KI of ILGFR-1 or IR (arrowheads). Scale bar 50 µm.

Interestingly, transfection of MuSK bearing ILGFR-1 or IR KIs resulted in the formation of undersized AChR aggregates similar to that observed upon transfection of MuSK alanine mutant S680/697A or CK2 inhibition, corroborating the importance of serine phosphorylation within the KI domain for regular AChR clustering. The observed changes in the AChR clusters morphology were quantified by morphometrical analysis of respective cluster areas (Fig. 35).

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Fig. 35: Quantification analysis of AChR clusters restoration by different MuSK KI mutants in MuSK-deficient cells. Density of AChR clusters was calculated in Scion Image program (as described in Material and Methods). AChR clusters formed in the cells transfected with MuSK bearing KI of ILGFR-1 or IR upon agrin stimulation have significantly lower density than regular AChR aggregates formed upon transfection with wild type MuSK or MuSK ∆KI (PDGFβR) mutant. * P-values <0.0001.

4.2.5.6. Mechanism of CK2 action

Up to now, our results demonstrated the requirement of CK2-dependent phosphorylation of specific serine residues within the KI of MuSK for AChR clustering. Inhibition of CK2 activity or knockdown of CK2 protein or mutagenesis of respective serines resulted in appearance of dispersed and undersized AChR clusters. We asked if the changed morphology of AChR clusters is caused by a change in AChR cluster stability. To determine stability of AChR clusters, C2C12 myotubes were treated with agrin 4.8. alone or together with CK2 inhibitor apigenin (60 µM), then agrin-containing medium was replaced by fresh medium and the number of AChR clusters was assayed after 0, 4 and 8 h. The number of AChR clusters at 4 and 8 h was calculated as percentage of the value at time point 0 h, which was set to 100%. As previously, the application of CK2 inhibitor led to the appearance of a high amount of small AChR aggregates, but the rate of their dispersal after removal of agrin together with the inhibitor was comparable with that for the mock control (Fig. 36, blue and red bars). However, when the treatment with apigenin was continued after agrin withdrawal, AChR clusters disappeared much faster than in the mock control. The significant difference between apigenin and mock treated cells was observed already within 4 h. These data suggest that CK2 activity is required for stabilization of AChR clusters.

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Fig. 36: AChR cluster stability. C2C12 myotubes were stimulated with agrin 4.8. in the presence of CK2 inhibitor apigenin (60 µM) or mock solution of DMSO (red and blue bars respectively). After agrin removal apigenin treatment was stopped (yellow bar) or continued (grey bar). Amount of AChR clusters left on myotube surface was quantified at 0, 4 and 8 h. AChR cluster stability was affected in the presence of CK2 inhibitor. * P-values <0.0003.

4.2.5.7. Generation and characterization of muscle-specific CK2β knockout mice

To explore the biological impact of CK2 for the postsynaptic specialization at the NMJ it is necessary to examine mice after knocking out the genes of CK2 subunits specifically in myonuclei. Considering interaction between CK2β and MuSK, we started by generating a muscle-specific CK2β knockout. Fortunately, mice with manipulated genomes where the first two exons of CK2β flanked by insertion of loxP sites (with the ATG located inside the second exon) were already available (Buchou et al. 2003). In order to get muscle specific CK2β knockout mice, first, we crossed CK2βloxP/loxP mice with transgenic mice expressing Cre recombinase under the human skeletal actin (HSA) promoter (Schwander et al. 2003). The resulting CK2βloxP/+,HSA-Cre mice were subsequently breaded with CK2βloxP/loxP mice to get CK2βloxP/loxP,HSA-Cre offspring. Expression of the HSA-Cre recombinase in muscle precursors from E9 stage (i.e. before NMJ formation at E13) should lead to deletion of first two exons of CK2 gene resulting in its disruption selectively in muscle tissue. CK2βloxP/loxP,HSA-Cre mice identified by genotyping PCR (see Methods) were found at all developmental stages (E10.5, E15.5, E18.5 and adult; Fig. 37).

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Fig. 37: Genotypes of mice obtained from CK2βloxP/+,HSA-Cre x CK2βloxP/loxP breadings. CK2βloxP/+,HSA-Cre mice were crossed with CK2βloxP/loxP mice to get animals with CK2βloxP/loxP,HSA- Cre genotype. At least 20 embryos were genotyped for each embryonic stage (E10.5, E 15.5 and E18.5) and 60 mice at the adult age (P30). CK2βloxP/loxP,HSA-Cre mice were found at all developmental stages in the ratio closed to Mendalian.

In order to prove the disruption of the CK2β gene in the muscle of these mice, we performed quantitative real-time PCR. One of the primers for this PCR was designed to anneal within the first exon in order to be able to detect its deletion. Indeed, the quantity of CK2β transcripts was significantly reduced in the muscles of mutant mice at both embryonic and adult stages (Fig. 38). Residual detected levels of CK2β mRNA in the muscles of the mutants reflect the presence of the large fraction of non-muscle cells (aprox. 60%) in adult muscle tissue (Escher et al. 2005).

Fig. 38: Levels of CK2β transcripts in leg muscle of wildtype and CK2β mutant mice. CK2β transcript levels, measured by real-time RT-PCR, are reduced in muscles of mutant CK2βloxP/loxP, HSA-Cre mice compared to wildtype litters at both embryonic and postnatal stages. The residual transcripts in the muscles of mutant mice originate from non-muscle cells which make up approx. 60% of all nuclei in adult leg muscle.

Consistent with the muscle-specific deletion of the CK2β gene no immunoreactivity for CK2β protein was detectable at the NMJs of the adult mutant mice (Fig. 39).

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Fig. 39: Immunoreactivity for CK2β protein is absent from the muscle of CK2βloxP/loxP,HSA-Cre mouse. Immunofluorescence micrographs of cross-sections of wildtype and CK2βloxP/loxP,HSA-Cre mice (P210) gastrocnemius muscles stained with α-CK2β antibody (shown in green) together with Rh-α- BTX (shown in red). Immunoreactivity for CK2β is present in the muscle and concentrates at the NMJ of wildtype, but not CK2βloxP/loxP,HSA-Cre mice. Scale bar 10 µm.

Phenotypically, the mutant mice did not show any abnormalities during the first two months of age. However, from P60 on the mutant mice started to display myasthenic characteristics. Their grip strength, which was reflected by the time for which the mice were able to cling up side down onto a wire grid began to decrease and at the age of six month went down to at average 5-10 sec compared to 2 min for the wildtype litters (Fig. 39).

Fig. 39: Grid test. The time for which CK2β mutant mice of different age or their wildtype littermates can cling upside- down onto a horizontal wire mesh was measured. The test was discontinued after 120 sec. The mutant mice of more then 60 days age hold on the mesh significantly shorter time than their wildtype littermates. Means ± S.E. (N=6 for each age).

One reason for the observed myasthenia of these mice might be affected AChR aggregates at the NMJ. Therefore whole mount preparations of different hind limb muscles (soleus,

90 Results ______gastrocnemius and extensor digitorum longus (EDL)) as well as diaphragmas from the mutant mice and their wildtype litters of 210-240 days of age were stained with Rh-α-BTX. Consistent with myasthenic appearance, the morphology of AChR aggregates was severely affected in the CK2β mutants. The endplates were mostly fragmented and continued to disintegrate further into small patches or spots of AChRs (Fig. 40, types C and D).

Fig. 40: AChR clustering is affected in CK2β mutant mice. Confocal micrographs of wildtype and CK2βloxP/loxP,HSA-Cre mice (P210) gastrocnemius endplates stained with Rh-α-BTX (shown in red). AChR clusters in CK2β mutant muscles display different degree of fragmentation. AChR aggregates were grouped according to the following criteria. Type A: AChR cluster represent uninterrupted pretzel shaped structure; Type B: endplate is broken up into several longitudinal and circular structures (arrowheads); Type C: AChR aggregates are made up of only circular structure; Type D: endplate consist of circular structures and spots of AChR (arrows).

We further quantified the phenotype of the affected endplates in mutant mice. According to their morphology, the AChR clusters were divided into four types: Type A. AChR cluster represent a typical uninterrupted pretzel shaped structure; Type B. pretzel-shaped structure is broken up into several longitudinal and circular structures; Type C. AChR aggregates are only made up of circular structure; Type D. endplates consist of circular structures and small spots of AChR aggregates. The quantification was performed for the soleus and gastrocnemius muscles of wildtype and mutant mice and showed that endplates of wildtype mice mainly fall into A and B categories (99%) with only 1% corresponding to the C type. The D type of endplates was not found in the muscles of wildtype mice. Conversely, aprox. 90% of the endplates of the mutant mice corresponded mainly to the Type C and D endplates (Fig. 41).

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Fig. 41: Quantification analysis of AChR cluster affection in CK2β mutant mice. At least 100 images from 4 CK2βloxP/loxP, HSA-Cre and wildtype (P210-P240) soleus and gastrocnemius muscles were grouped independently by 3 individuals according to the criteria described in text. Percentages of each type averaged from the individual assessments (mean ± S.E.) are given in column graphs. Types C and D of endplates are more prominent in muscles of mutant mice, while types A and B in the muscles of wildtypes.

It might be that the motoneurons terminals are also affected in the muscles of the mutant mice. To inspect carefully the NMJs of mutant mice for the morphology of the motoneuron terminals, their hind limb muscle fibers were teased and stained with Rh-α-BTX together with an antibody against neurofilament, detecting motoneurons. Interestingly, even in spite of severe disruption of AChR clusters, the nerve terminal arborization was not looking affected in the mutant mice. The nerve branches contacted in some cases, even very small spotty patches of AChRs (Fig. 42). In some cases, we failed to see a dendrite co-localizing with spots of AChR clusters. These data suggest that defects of the NMJ observed in CK2β mutants are muscle specific.

Fig. 42: Nerve morphology at the NMJ of CK2βloxP/loxP, HSA-Cre mice. Confocal image of extensor digitorum longus (EDL) endplate stained with Rh-α-BTX (red) for AChR and with α-neurofilament antibody for the motor nerve terminal (green). Morphologically normal nerve terminal contact spotty subsynaptic AChR aggregates (arrow). Rarely AChR aggregates, which are not accompanied by the nerve terminal are detectable (arrowhead).

92 Results ______

The deletion of the gene encoding CK2β in myotubes implies that the CK2 holoenzyme is disrupted and this in turn leads to the change of CK2 activity. To investigate the CK2 enzymatic activity in the muscle after ablation of the CK2β gene, protein extracts were prepared from a hind limb muscle of the mutant and wildtype mice and used in in vitro phosphorylation assays with a CK2-phosphorylatable peptide as substrate (see Methods). Surprisingly, the CK2 activity in the muscle of mutant mice was higher then in wildtypes. Concomitantly, a decrease of CK2 activity was observed during aging for both mutant and wildtype animals (Fig. 43). Note that because of the decline of CK2 activity, eight month old animals display roughly the same amount of activity like one month old wildtypes (Fig. 43).

Fig. 43: CK2 activity in muscles of CK2β mutant and wildtype mice. CK2 activity was measured in muscle lysates of CK2β mutant and wildtype mice of different age. CK2 activity estimated as amount of isotope incorporated in synthetic CK2 substrate peptide was increased in CK2β mutant compare to wildtype mice, but decreased with age of animals of both genotypes.

93 Discussion ______

5. Discussion

5.1. Potential MuSK binding partners

Identification of protein interactions is a prerequisite for revealing cascades of signaling pathways. The state of knowledge at the NMJ regarding signal transduction is very limited. The identification of interactions at the NMJ would allow better understanding of synapse development and pathology. For example, recent data about interaction between MuSK and the C-terminus of ColQ, a collagenic tail recruiting acetylcholinesterase (AChE) to the NMJ, has approved that MuSK is responsible for synaptic localization of AChE and explained certain forms of congenital myasthenic syndromes associated with mutations in the C- terminal part of ColQ (Engel et al. 2003). Using the Y2H technique, we looked for proteins, which interact with different domains of MuSK. MuSK extracellular parts (C6-box and Ig-like IV domain) have been used as baits for identification of the hypothetical protein RATL which is believed to link MuSK to rapsyn (Zhou et al. 1999). The disadvantage of Y2H screening with extracellular parts of protein is that they can not be appropriately post-translationally modified, for example glycosylated, inside of the yeast cell that might result in a loss of desired interactions. Indeed, two glycosylation sites have been found within the extracellular domain of MuSK which we used in our screens (Watty and Burden 2002). Nevertheless, we have succeeded in screening with these MuSK baits arguing that extracellular modifications might not be relevant for some interactions. Among proteins obtained from these screens are an EGF-domain containing protein of the extracellular matrix, EFEMP-1/S1-5; a member of connective tissue growth factors family shown to be induced by Wnt signaling called WISP2 and Laminin Receptor-1 (LR-1). EFEMP-1/S1-5 is a novel extracellular protein containing six EGF-like repeats. The EFEMP- 1 gene product have been found to be up regulated in Werner syndrome of premature aging (Lecka-Czernik et al. 1995). Single-amino acid substitutions in the EFEMP-1 protein leads to another disease - Malattia Leventinese - an inherited macular degenerative syndrome, characterized by abnormal accumulation of EFEMP-1 protein inside of retinal pigment epithelium cells (Marmorstein et al. 2002). The multidomain protein structure may indicate

94 Discussion ______that EFEMP-1 protein interacts with extracellular matrix components and serves to connect and integrate the function of multiple partner molecules. Two more proteins found to interact with the extracellular part of MuSK are WISP2 and LR1. WISP2 belongs to the subfamily of connective tissue growth factors. WISP proteins are up-regulated downstream of the Wnt pathway (Pennica et al. 1998). On the other hand, several members of Wnt signaling pathway have been already shown to act at the NMJ (Luo et al. 2002; Luo et al. 2003a; Wang et al. 2003). Interestingly, out of all WISP proteins only WISP2 is expressed in skeletal muscle (Pennica et al. 1998). LR1 is a 67kD non-integrin expressed on different cell types including muscle cells. It recognizes several non-identical hydrophobic domains on elastin, laminin, and type IV collagen (Hinek 1996). These components of basement membranes are also present in the basal lamina of the NMJ and some of them play an important role during NMJ development (Patton et al. 1997). Interactions between MuSK and EFEMP-1, WISP2 and LR-1 have to be confirmed and require further investigation. Among proteins found to bind to the active form of the intracellular MuSK domain in our Y2H screens we have selected as the most interesting for our further studies neural precursor cell expressed developmentally down-regulated 9 (Nedd9), Lim Domain Only 7 (LMO7) and Casein Kinase 2 β (CK2β). Nedd9 is a member of small Cas (including Cas130, Nedd9 and Sin) which acts as “docking” molecules in intracellular signaling cascades. Cas proteins contain N- terminal SH3-domains, cluster of SH2 binding motives and a serine-rich region. These proteins have been shown to participate as docking molecules in JNK, ERK and Rac signaling pathways known to be activated by agrin at the NMJ (Jones and Werle 2000; Lacazette et al. 2003; Weston et al. 2003). Moreover, interactions with Nedd9 were demonstrated for other signaling molecules which are involved in the NMJ development, such as Src and Abl kinases (Finn et al. 2003; Mohamed et al. 2001). LMO7 is a novel protein, containing alternatively spliced C-terminal LIM domain and found in nuclear and cytosolic cell fractions. LMO7 being widely expressed is present in skeletal muscle in high amounts (Putilina et al. 1998). The deletion of the LMO7 gene in mice leads to their death between birth and weaning accompanied by retinal and muscular degeneration and growth retardation (Semenova et al. 2003). CK2β is a regulatory subunit of CK2 – a ubiquitously expressed and constitutive active serine/threonine kinase involved in many cell processes. Among CK2 substrates are many proteins involved in different processes inside of cell including signal transduction.

95 Discussion ______

5.2. CK2 – newly characterized MuSK binding partner

CK2β was identified numerous times in our Y2H-screen with the intracellular domain of MuSK. Nevertheless, CK2β has not been found in the screens with the extracellular domains of MuSK that suggests the specificity of the interaction. In all interaction assays CK2β bound stronger to the phosphorylated form of MuSK, suggesting that the MuSK - CK2β interaction is strengthened after the activation of MuSK followed by autophosphorylation of its intracellular domain. Although CK2 regulatory β subunits normally associate with CK2 catalytic α subunits building the heterotetrameric holoenzyme, the α subunit is constitutively active and can function independently (Heriche et al. 1997). Controversially, function of CK2β independent of α subunit has also been reported (Bosc et al. 2000; Heriche et al. 1997). Our interaction assays have shown a weak, most likely transient interaction between MuSK and CK2α. This interaction was not influenced by an excess of CK2β, suggesting that α and β subunits do not bind to the same MuSK epitope. Most likely, CK2β binds to MuSK at first and operates further as a docking platform recruiting α subunit. Epitope mapping studies have shown interaction between the positive regulatory domain at the C-terminal of CK2β with the kinase domain of MuSK. It is also possible that CK2β binds to the juxtamembrane region of MuSK and the kinase domain is involved in the proper conformation or exposure of the juxtamembrane domain (Luo et al. 2002). Other proteins involved in the development of the NMJ such as Dishevelled or Geranylgeranyltransferase I also bind to the kinase domain of MuSK (Luo et al. 2002; Luo et al. 2003a). Since processes of signal transduction are regulated by phosphorylation of serine, threonine or tyrosine residues, it is not surprising that kinase domains of transmembrane receptors carrying most of phosphorylation sites (Watty et al. 2000) become the target for upstream regulators or the place of binding for downstream signaling effectors. The C-terminal of CK2β comprising 155-196 aa has been shown to be responsible for dimerization of β subunits, association with the α subunit, and up-regulation of CK2 activity (Boldyreff et al. 1993; Krehan et al. 1996). In particular the area between 155-167 aa is required for dimerization of β subunits, whereas the 172-196 aa motif appears to be crucial for stabilizing the association of α and β subunits (Boldyreff et al. 1993; Boldyreff et al. 1996; Krehan et al. 1996; Marin et al. 1997). However, this facts do not exclude the involvement of the C-terminus of CK2β in interaction with MuSK as other proteins such as c-

96 Discussion ______

Mos and A-Raf have been shown to interact with the same region of CK2β (Boldyreff and Issinger 1997; Chen et al. 1997).

5.3. Phosphorylation of MuSK by CK2 is required for appropriate AChR clustering

Since CK2 subunits were found to bind to MuSK and concentrate at the postsynaptic site of the NMJ in vivo we asked a biological role of CK2 during the postsynaptic development. Two different approaches, fist, blockade of CK2 by chemical compounds and, second, knockdown of CK2 subunits by specific siRNA have been used. If first approach allowed to estimate an impact of CK2 activity on AChR clustering, the second could help to disclose a role of different CK2 subunits in this process. In both cases ablation of catalytic activity of CK2 led to appearance of high amount of undersized AChR clusters, suggesting that CK2 activity is not required for initiation of AChR aggregation per se but rather important for the appropriate condensation of micro-aggregates into the regular size endplates. In spite of high similarity between CK2α and α’ subunits (aprox. 90% within their catalytic domain) and considerable functional overlap, there is an evidence for their functional specialization in some physiological processes (Litchfield 2003). While siRNA knockdown of only one of CK2 subunits did not interfere with cell survival and differentiation into myoblasts, it did influence the normal AChR aggregation, implying that one subunit can compensate for the loss of the other during the cell proliferation, but presence of both subunits is prerequisite for the normal postsynaptic development. Previous studies hinted at serine phosphorylation inside the kinase insert (KI) domain of MuSK (Till et al. 2002). We discovered that CK2 (catalytic subunit and holoenzyme) was able to phosphorylate MuSK at serine residues 680 and 697 located in the KI region. If the phosphorylation of these two serines is of fundamental importance for the MuSK signaling, one would expect the residues to be conserved in different species. To see if serines 680 and 697 are conserved in KIs of MuSK from the other species we have performed an alignment of mouse, rat, human, zebrafish, xenopus, chick and torpedo MuSK (Fig. 44). Corroborating their functional importance serine residues 680 and 697 are indeed present in the KIs of MuSK of most of the species. Moreover, serine 697 shows a higher degree of conservation among species that correlates with our its higher phosphorylation rate (Fig. 29 B, Fig. 44).

97 Discussion ______

Fig. 44: Conservation of MuSK serine residues 680 and 697 in different species. Alignment of KI areas of MuSK protein from different species. Sequences corresponding to KI is underlined. Serine residues 680 and 697 are boxed. Serine 697 is conserved in all indicated species.

The fact that mutation of serines 680 and 697 in the MuSK KI to the non-phosphorylatable amino acids led to the similar phenotype of AChR cluster impairment as in experiments with CK2 inhibition further supported a view that CK2-mediated phosphorylation of MuSK at serines 680 and 697 is required for appropriate AChR clustering. CK2 has been shown to participate in Wnt/β-catenin signaling pathway, phosphorylating and by this way stabilizing cytosolic levels of Dishevelled and β-catenin proteins (Song et al. 2000; Song et al. 2003). On the other hand different members of this pathway, i.e. APC, Dishevelled, β-catenin have been reported to bind to the main molecules of the NMJ (AChR β subunit, MuSK, rapsyn, see introduction) and influence agrin induced AChR clustering. In this regard, blockage of CK2 activity, leading to increased number of AChR clusters, can exert also through the Wnt/β-catenin pathway by reducing the levels of cytosolic β-catenin. Consistent with this hypothesis increased levels of β-catenin were found to inhibit AChR clustering (Luo et al. 2003b; Sharma and Wallace 2003). Alternatively, CK2 can influence AChR clustering through both MuSK- and Wnt/β-catenin signaling pathways. The fact that substitution of CK2-phosphorylateble serines of MuSK by amino acids mimicking permanent phosphorylation led upon their transfection in MuSK-deficient cells to formation of the regular sized AChR clusters even in the presence of CK2 inhibitors corroborated the specific involvement of CK2 in regulation of AChR clustering by modulation of agrin-MuSK, but not the Wnt-signaling. Regarding appearance of high amount of undersized AChR cluster aggregates upon blockage of CK2-mediated serine phosphorylation of MuSK, different mechanisms of CK2 action can be proposed. The involvement of Rac/Rho small GTP-ases is likely as they act at early stages of AChR clustering, promoting initial aggregation of diffused AChRs into micro clusters (Rac) and subsequent condensation of these micro clusters into full-size AChR aggregates

98 Discussion ______

(Rho) (Weston et al. 2003). It is tempting to speculate that MuSK phosphorylation by CK2 in this case is important for coordination of Rac/Rho activities and blockage of CK2 leads either to upregulation of Rac or downregulation of Rho. Alternatively, AChR gene upregulation can be involved. Recently, a new shunt pathway involving agrin-induced activation of Rac and acting in synaptic gene regulation has been described by the group of H.-R. Brenner (Lacazette et al. 2003). It can be proposed that CK2, phosphorylating MuSK acts as a suppressor of Rac activity. As follows inhibition of CK2 results in upregulation of AChR gene expression through the Rac-JNK pathway on the one hand and Rac-dependent micro cluster generation on the other hand. Both hypotheses have to be further experimentally proven. Our data about the decreased stability of AChR clusters in the presence of CK2 inhibitors suggests another mechanistical aspect of CK2 action, where CK2-mediated phosphorylation of MuSK is required for AChR cluster stabilization. Further studies are required to understand which MuSK-downstream signaling processes involved in AChR clusters stabilization are regulated by its serine phosphorylation.

5.4. KI domain of MuSK is involved in modulation of postsynaptic specialization

Many receptor tyrosine kinases such as PDGFβ-, ILGF-, Insulin-Receptors or c-kit contain in their cytoplasmic portion a hydrophobic region of about 100 amino acids. These region named kinase insert (KI) divides tyrosine kinase domains in two parts (Hubbard and Till 2000; Ullrich and Schlessinger 1990). The function of KI is poorly understood, but it is speculated that along with juxtamembrane domain and C-terminus the KI domain contains amino acid residues that are autophosphorylated upon binding of the ligand to the receptor and serve as binding sites for the modular domains of other proteins (Hubbard and Till 2000). For example, KI of PDGFβ-Receptor contains an autophosphorylated upon ligand binding tyrosine, which recruits downstream effector - PI3-kinase (Kazlauskas and Cooper 1989). Therefore, KIs of receptor tyrosine kinases can play an important role in the processes of signal transduction. Our experiments with MuSK KI deletion mutant or MuSK KI chimeras show that the KI of MuSK receptor tyrosine kinase has an important function in agrin-induced AChR clustering. Thus, it can be hypothesized that serine phosphorylation within MuSK KI is

99 Discussion ______required for establishing of docking sites for certain intracellular proteins that participate in agrin-mediated signaling required for the proper AChR clustering. Recently, the adaptor protein 14-3-3 γ has been identified as a MuSK binding partner. (Strochlic et al. 2004; Strochlic et al. 2005a). Since 14-3-3 proteins interact with their various partners in a serine phosphorylation dependent manner a serine phosphorylation of MuSK KI domain by CK2 might be of biological significance. The fact that 14-3-3 γ is involved in the regulation of synaptic transcription can argue in favor of the hypothesis regarding involvement of CK2 in AChR subunits gene regulation. Further investigation will enlighten the potential implication of CK2 in regulation of MuSK-14-3-3 γ interaction. Interestingly, the KIs of other receptor tyrosine kinases like Trk seem to have completely different functional specialization rather disrupting then mediating biological signaling. Splice variants of TrkC carrying 14- or 39- aa insert within their tyrosine kinase domain are not able to mediate neurotrophin-3 induced signaling because of their inability to bind such downstream effectors as Sch and phospholipase Cγ (Guiton et al. 1995). Consistent study has shown that introduction of 14- aa KI of TrkC into TrkA receptor, which normally lacks the KI domain leads to repression of TrkA downstream signaling (Meakin et al. 1997). Accordingly, the introduction of 14- aa KI of TrkC into MuSK has also led to its inability to conduct agrin- initiated signaling. These findings suggest the completely different mechanisms of regulation of activity of Trk and PDGF-Receptors by KIs, assigning MuSK to be more similar to the group of PDGF-Receptors.

5.5. Role of CK2 in development of postsynaptic apparatus in vivo.

Our in vitro and cell culture studies demonstrated the importance of CK2-dependent MuSK phosphorylation for AChR clustering and their maintenance. Nonetheless, not all aspects of synaptic differentiation can be studied using a tissue culture system since requirements for synaptic proteins in tissue culture may differ from that in vivo. For example, rapsyn is required to cluster MuSK in cultured cells but not during the synapse development in vivo (Gillespie et al. 1996; Moscoso et al. 1995). Similarly, the extracellular domain of MuSK is indispensable for AChR clustering in cell culture but not essential in vivo (Apel et al. 1997; Sander et al. 2001). Thus, in vivo studies were required to prove the impact of CK2 during the NMJ development.

100 Discussion ______

Study of CK2α knockout in yeast has shown that this subunit can functionally replace CK2α’ and vise versa, but deletion of both subunits is lethal (Padmanabha et al. 1990). Conversely, ablation of regulatory β subunit did not affect yeast cell survival (Bidwai et al. 1995). In mice only CK2α’ subunit knockout has been studied so far, showing the inability of CK2α to compensate for α’ only during spermatogenesis (Xu et al. 1999). In contradiction to the yeast studies, the CK2β knockout mice died at embryonic stage shortly after implantation, suggesting that in mammals CK2β protein is important at early stages of development for the cell survival (Buchou et al. 2003). Consistently our cell culture data has shown that inhibition of CK2β subunit by siRNA in myoblast cells leads to arrest of their proliferation and differentiation. To reveal the biological importance of CK2β for NMJ development we have used a conditional knockout approach selectively ablating CK2β in muscle tissue. Mice carrying a deletion of CK2β in muscle displayed muscle weakness starting from the third month of age. This phenotype correlated with the time of onset of their postsynaptic membrane impairment. Phenotypically AChR clusters of CK2β muscle knockout mice are similarly affected as observed for other myasthenic mice lacking components of dystrophin/utrophin-glycoprotein complex (D/UGC). The endplates are fragmented and often contain small patches or granules of AChRs (Grady et al. 2000). This phenotype is reminiscent of AChR impairment in C2C12 cell culture after the blockade of CK2 or mutation of potential CK2 phosphorylation sites on MuSK. Similarly, myotubes lacking α-dystrobrevin or dystoglycan form in response to agrin plenty of small AChR aggregates (Grady et al. 2000). The late onset of phenotype in vivo shows that components of D/UGC are required not for initial stages of postsynaptic specialization but rather for subsequent maturation and stabilization of the muscle endplate. For CK2β the situation is similar. Firstly, blockage of CK2 in C2C12 cell culture does not result in the loss of agrin-induced AChR clusters, but rather affects their morphology. Secondly, stability of AChR aggregates is dramatically reduced upon CK2 inhibition. And, finally, in vivo CK2 subunits concentrate at the postsynaptic site of the NMJ only starting from the early postnatal age when AChR plaques are already formed. Recently Src and Abl class of kinases has been reported to act downstream of MuSK and regulate the stability of AChR clusters controlling AChR-cytoskeleton linkage (Finn et al. 2003; Mittaud et al. 2004; Smith et al. 2001). The fact that CK2 participates in stabilization of postsynaptic apparatus suggests the possible involvement these kinases in CK2-mediated MuSK signaling. Alternatively, since ablation of CK2β in muscle leads to the NMJ phenotype

101 Discussion ______similar to that of D/UGC components knockout mice, CK2-mediated phosphorylation of MuSK can be important for recruitment of these components to the AChR clusters during the endplate maturation. Interestingly that in cell culture in the presence of agrin and absence of CK2 activity we immediately observe an impairment of AChR cluster morphology, in contrast, in vivo this defect shows up only late postnatally. The existence of some other not yet identified factors, promoting the proper aggregation of the AChR receptors at the late embryonic and early postnatal stages might explain this contradiction. The other difference of our in vivo system from the cell culture experiments is that in CK2β muscle knockout mice we still have both CK2 catalytic subunits present. Moreover, surprisingly, our studies show that though CK2 activity decrease with the age, deletion of CK2 β in the muscle leads to upregulation of CK2 activity. Our in vitro data do not provide clear definition of how does CK2β regulates CK2α activity on MuSK. Considering the fact that CK2 activity is downregulated during the aging it can be proposed that decrease in the recruitment of CK2α to MuSK due to CK2β ablation can be compensated in young, but not in old animals by the increased CK2α activity. The study of mouse mutant having deletion of catalytic CK2 subunits or carrying substitution of MuSK genomic locus by MuSK S680/697A mutant would provide the final explanation of how CK2 subunits counteract during MuSK phosphorylation. Taken together, we our study has shown for the first time an interaction between CK2 and the main organizer of the postsynaptic specialization – MuSK. These data provides the first description of the role for the MuSK KI domain. CK2-mediated phosphorylation of serines inside of this region of MuSK appears to be critical for the downstream signaling events which lead to stabilization and maintenance of postsynaptic specialization at the NMJ. The data presented here can be of general importance for the understanding of processes of signaling transduction during synaptogenesis not only at the NMJ but also in the CNS, since CK2 enzyme has already been shown to be involved in the several neural functions (Blanquet 2000).

102 Abbreviations ______

6. Abbreviations

aa amino acid APS Ammonium Persulphate 3-AT 3-amino-1,2,4-triazole ATP adenosine triphosphate C6-box cysteine-rich domain (box) 1st cDNA first strand complementary deoxyribonucleic acid ColQ collagen Q Cy2, Cy3 carbocyanine 2 and 3 DMSO dimethylsulphoxide DNA deoxyribonucleic acid DNase deoxyribonuclease DO drop out DTT dithiothreitol dNTP deoxyribonucleotide triphosphate ECL enhanced chemiluminescence EDTA ethylenediamine tetra-acetic acid EFEMP-1 EGF-containing fibulin-like extracellular matrix protein 1 ERK extracellular-signal regulated kinase Ets domain E twenty-six domain EGF epidermal growth factor FGF fibroblast growth factor Fig. figure GABP GA-binding protein GST glutathione-S-transferase HB-GAB heparin-binding growth-associated molecule Ig immunoglobulin IP immunoprecipitation JNK c-Jun N-terminal kinase kd kinase defective ∆ΚΙ deletion of kinase insert lacZ β-galactosidase gene from E.coli MAPK mitogene activated MAGUC membrane-associated guanylate kinase mRNA messenger RNA NP-40 Nonidet P-40 OD optical density P0 myelin protein zero PBS phosphate-buffered saline PCR polymerase chain reaction PDZ PSD-95/Discs-large/ZO-1 PEG polyethylenglicol PFA paraformaldehyde PMSF phenylenmethylsulfonylfluorid

103 Abbreviations ______

PTB phospho-tyrosine binding motif RNA ribonucleic acid RNase ribonuclease RT reverse transcription RT-PCR reverse transcription followed by polymerase chain reaction S100 calcium-binding protein SDS sodium dodecyl sulphate siRNA small interfering RNA SNAP-25 synaptosomal-associated protein of 25kDa SNARE soluble N-ethyl maleimide sensitive factor attachment protein receptor TBE Tris/boric acid/EDTA TEMED N,N,N’,N’-Tetramethylethylenediamine TM transmembrane Tris Tris-(hydroxymethyl)-aminomethane Trk Tyrosine receptor kinase WB Western blot wt wildtype X-Gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside

Units bp base pair ˚C degree Celsius cm centimeter cpm counts per minute h hour kD kilodalton M, mM, µM molar, milimolar, micromolar min minute ng, mg, µg nanogram, milligram, microgram ml, µl milliliter, microliter p pico pH -log H+ concentration rpm revolutions per minute rcf=g relative centrifugal field RLU Relative Light Units RT room temperature s second U Unit V Volt

104 References ______

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Curriculum vitae

Name: Tatiana Cheusova Date of birth: 06.09.1978 Place of birth: Novosibirsk, Russia Nationality: Russian Marital status: Single

Education: 1985-1995 Secondary school, Novosibirsk, Russia 1995-2000 Novosibirsk State University, Russia 1998-2000 Master course at the State Research Center of Virology and Biotechnology ”Vector” Title of the thesis: “Ultrastructural Study of Marburg virus replication cycle”. Degree: M.Sc. in Biology Specialization – cytology and genetics (July 2000) 2000-2002 State Research Center of Virology and Biotechnology ”Vector”, Senior scientific employee 2002-2006 University of Erlangen-Nuremberg, Germany PhD course at the Faculty of Medicine, Institute of Biochemistry. Title of the thesis: “Identification and characterization of Casein Kinase 2 as MuSK binding partner.”

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Publications

Cheusova T., Khan M.A., Schubert S.W., Gavin A.-C., Buchou T., Jacob G., Sticht H., Allende J., Boldyreff B., Brenner H.-R., Hashemolhosseini S. (2006) Casein kinase 2 dependent serine phosphorylation of MuSK regulates acetylcholine receptor aggregation at the neuromuscular junction. Genes Dev; 20 (13)

Schubert S.W., Kardash E., Khan M.A., Cheusova T., Kilian K., Wegner M., Hashemolhosseini S. (2004) Interaction, cooperative promoter modulation, and renal colocalization of GCMa and Pitx2. J Biol Chem 26;279(48) 50358-65

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Acknowledgments

First, I would like to express my gratitude to my supervisor PD. Dr. Said Hashemolhosseini for giving me the opportunity to do my doctoral thesis at the Institute of Biochemistry, University of Erlangen-Nuremberg. I would like to thank him for his constant guidance, understanding and patience during the PhD course, and for his critical reading of this thesis. I am grateful to Prof. Dr. Has-Rudolf Brenner for the help in starting our scientific project. I would also like to thank him for the guidance through the whole our study and great assistance in the manuscript writing. I am indebted to PD Dr. Fritz Titgemeyer for undertaking the revision of this thesis. I would like to thank Prof. Dr. Michael Wegner for the help in difficult moments during my PhD course and all his group members for the helpful advices and constant support in my experimental work. I also thank Dr. Elisabeth Sock for correction of my thesis. Very special thanks to my friend Amir Khan for his daily support; help with experiments for the manuscript and for correction of my thesis. Many sincerer thanks to other lab members and my dear friends Steffen Schubert and Nicolas Lamoureux for their help in experimental and life difficulties and for creating friendly atmosphere in the lab. I would like to acknowledge the group of Prof. Jorge Allende for performance of in vitro phosphorylation experiments; Heinrich Sticht for in silico studies; Diana Hittmeyer for the help in kinase assays. I thank Christian Fuhrer for MuSK constructs; Claude Cochet, Olaf- Georg Issinger, Mathias Montenarh, and Markus Ruegg for providing us with antibodies; Ruth Herbst and Steve Burden and Stefan Kröger for cells; Lorenzo Pinna and Flavio Meggio for the CK2 inhibitor DMAT; Michael Sendtner for help in performing ischiadicus lesions. Moreover, I wish to thank my family and all my friends from Russia and from the Institute for their unfailing support and encouragement during my work in Germany. I am especially grateful to Vitaly Vatsko for his respect of my wishes, constant love and great patience. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) (HA 3309/1-1,2).

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