Identification and characterization of casein kinase 2 as MuSK binding partner [Elektronische Ressource] / vorgelegt von Tatiana Cheusova

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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......................................................
Publié le : dimanche 1 janvier 2006
Lecture(s) : 58
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Source : WWW.OPUS.UB.UNI-ERLANGEN.DE/OPUS/VOLLTEXTE/2006/398/PDF/TATIANACHEUSOVADISSERTATION.PDF
Nombre de pages : 122
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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. Agrin-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. Enzymes.. 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 Casein
Kinase 2 (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 neuromuscular junction is initiated by the
release of agrin from the nerve terminal and subsequent activation of the muscle-specific
receptor tyrosine kinase 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).
3Introduction
___________________________________________________________________________

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 enzyme 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
2concentration of nicotinic acetylcholine receptors (AChRs) (>10000/µm ) (Salpeter and
Loring 1985). Several actin binding proteins associate with the cytoplasmic portion of the
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