Functional characterization of leucine-rich repeat kinase 2 (LRRK2) dimerization [Elektronische Ressource] / vorgelegt von Christian Klein

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Functional characterization of leucine-rich repeat kinase 2 (LRRK2) dimerization Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften der Fakultät für Biologie und der Medizinischen Fakultät der Eberhard-Karls-Universität Tübingen vorgelegt von Christian Klein aus Dachsbach, Deutschland September 2008 Tag der mündlichen Prüfung: 1. Dezember 2008 Dekan der Fakultät für Biologie: Prof. Dr. H. A. Mallot Dekan der Medizinischen Fakultät: Prof. Dr. I. B. Autenrieth 1. Berichterstatter: Prof. Dr. T. Gasser 2. Berichterstatter: Prof. Dr. T. Stehle Prüfungskommission: Prof. Dr. T. Gasser Prof. Dr. T. Stehle Prof. Dr. B. Schloßhauer Prof. Dr. P. J. Kahle Prof. Dr. O. Rieß M e i n e r F a m i l i e g e w i dm e t And if we are herejust to e ase God’s sorrowLord come to meand I ’ll e ase your mindPSYCHOTIC WALTZ Into The Everflow (1992) Contents 1 SUMMARY .................................................................................1 2 INTRODUCTION ........................................................................3 2.1 Parkinson’s disease................................................................................... 3 2.1.1 Clinical definition................................................................................ 3 2.1.2 Epidemiology and economic impact .................................................. 4 2.1.
Publié le : mardi 1 janvier 2008
Lecture(s) : 31
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Source : TOBIAS-LIB.UB.UNI-TUEBINGEN.DE/VOLLTEXTE/2008/3653/PDF/DISSERTATION_CLK_2008.PDF
Nombre de pages : 113
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Functional characterization of
leucine-rich repeat kinase 2 (LRRK2)
dimerization




Dissertation

zur Erlangung des Grades eines Doktors
der Naturwissenschaften


der Fakultät für Biologie
und
der Medizinischen Fakultät
der Eberhard-Karls-Universität Tübingen



vorgelegt
von

Christian Klein
aus Dachsbach, Deutschland

September 2008


















Tag der mündlichen Prüfung: 1. Dezember 2008

Dekan der Fakultät für Biologie: Prof. Dr. H. A. Mallot
Dekan der Medizinischen Fakultät: Prof. Dr. I. B. Autenrieth

1. Berichterstatter: Prof. Dr. T. Gasser
2. Berichterstatter: Prof. Dr. T. Stehle

Prüfungskommission: Prof. Dr. T. Gasser
Prof. Dr. T. Stehle
Prof. Dr. B. Schloßhauer
Prof. Dr. P. J. Kahle
Prof. Dr. O. Rieß M e i n e r F a m i l i e g e w i dm e t And if we are here
just to e ase God’s sorrow
Lord come to me
and I ’ll e ase your mind
PSYCHOTIC WALTZ Into The Everflow (1992) Contents


1 SUMMARY .................................................................................1
2 INTRODUCTION ........................................................................3
2.1 Parkinson’s disease................................................................................... 3
2.1.1 Clinical definition................................................................................ 3
2.1.2 Epidemiology and economic impact .................................................. 4
2.1.3 Neuropathology ................................................................................. 5
2.1.4 Etiology.............................................................................................. 7
2.1.5 Pathogenesis................................................................................... 12
2.2 PARK8: Leucine-rich repeat kinase 2 (LRRK2) ..................................... 15
2.2.1 Phenotype and neuropathology....................................................... 15
2.2.2 Structure.......................................................................................... 16
2.2.3 Expression and localization ............................................................. 18
2.2.4 Mutations......................................................................................... 18
2.2.5 Putative functions and effects of mutations ..................................... 19
2.3 The ROCO protein family......................................................................... 23
2.4 Goals of this study................................................................................... 24
3 RESULTS.................................................................................27
3.1 Characterization of the αMid LRRK2 antibody ...................................... 27
3.2 LRRK2 homodimerizes via its ROCO domain ....................................... 29
3.3 LRRK1 homodimerizes via its ROCO domain ....................................... 33
3.4 LRRK2 and LRRK1 heterodimerize via their ROCO domains .............. 35
3.5 DAPK1 demonstrates homodimerization capability and shows
heterodimerization potential with LRRK ROCO kinases ...................... 37
LRRK23.6 The ROCO homodimerization is independent of GTP .................. 38
LRRK23.7 Familial PD mutations in the ROCO domain alter ROCO
homo- and heterodimerization................................................................ 41
LRRK2
3.8 The ROCO fragment exerts an inhibitory effect on
LRRK2 kinase activity.............................................................................. 43
3.9 Influence of familial and artificial mutations in the
LRRK2ROCO fragment on the LRRK2 kinase inhibiting effect................ 47
4 DISCUSSION ...........................................................................51
LRRK2
4.1 The ROCO domain directs LRRK2 dimerization............................ 51
LRRK2
4.2 Predicted model of the ROCO self-interaction............................... 53
i Contents
4.3 LRRK1 homodimerization and LRRK2/LRRK1 heterodimerization .... 55
4.4 Putative functions of the mammalian ROCO domain ........................... 56
4.5 Influence of artificial and familial PD mutations on
LRRK dimerization ................................................................................... 57
LRRK2
4.6 ROCO exerts an inhibitory effect on LRRK2 kinase activity ....... 59
LRRK24.7 Predicted model of the ROCO -mediated
LRRK2 kinase inhibition.......................................................................... 61
4.8 Outlook...................................................................................................... 63
5 MATERIALS AND METHODS..................................................67
5.1 Chemicals ................................................................................................. 67
5.2 Solutions and buffers .............................................................................. 69
5.2.1 Molecular biology............................................................................. 69
5.2.2 Yeast two-hybrid (Y2H).................................................................... 70
5.2.3 Protein biochemistry ........................................................................ 70
5.2.4 Cell biology...................................................................................... 72
5.3 Vectors and oligonucleotides ................................................................. 72
5.4 Antibodies................................................................................................. 73
5.5 Molecular cloning..................................................................................... 74
5.5.1 PCR, agarose gel electrophoresis and extraction of DNA ............... 74
5.5.2 Enzymatic digestion of DNA fragments and plasmid DNA .............. 74
5.5.3 Ligation of digested DNA fragments and transformation
of DNA by electroporation................................................................ 75
5.5.4 Production of electrocompetent cells............................................... 75
5.5.5 Amplification, purification and validation of plasmid DNA ................ 76
5.5.6 Cloned constructs............................................................................ 77
5.6 MBP-Mid fusion protein expression and purification ........................... 78
5.7 Y2H interaction study .............................................................................. 80
5.8 Cell culture, transfection, cell harvest and cell lysis ............................ 81
5.9 Immunoprecipitation, GTP-sepharose affinity purification and
nucleotide competition assays ............................................................... 81
5.10 SDS-PAGE, Coomassie staining, Western blotting
and densitometry ..................................................................................... 82
5.11 In vitro kinase assay ................................................................................ 82
6 REFERENCES .........................................................................83
ii List of Figures and Tables


Figure 2-1 Scheme of the nigrostriatal pathway in health and PD. 6
Figure 2-2 LB pathology. 7
Figure 2-3 Pathways leading to PD. 13
Figure 2-4 Domain architectures of mammalian ROCO kinases. 17
Figure 2-5 Mutations in LRRK2. 19
Figure 3-1 Characterization of the αMid LRRK2 antibody. 26
Figure 3-2 The Mid LRRK2 antibody immunoprecipitates LRRK2 27
protein.
Figure 3-3 LRRK2 homodimerizes via its ROCO domain. 29
Figure 3-4 LRRK2 does not interact with ArmAnk, MidLRR, LRR or 31
WD40 domains in co-IP assays.
Figure 3-5 LRRK1 homodimerizes via its ROCO domain. 32
Figure 3-6 LRRK2 and LRRK1 heterodimerize via their ROCO domain. 34
DAPK1 shows potential for homo- and heterodimerization. 36 Figure 3-7
LRRK2Figure 3-8 The ROCO homodimerization is independent of GTP. 37
LRRK2
Figure 3-9 Familial PD mutations in the ROCO domain alter ROCO 40
homo- and heterodimerization.
LRRK2
Figure 3-10 The ROCO fragment exerts an inhibitory effect on 43
LRRK2 kinase activity.
LRRK2Effects of familial and artificial mutations in the ROCO 46 Figure 3-11
fragment on LRRK2 kinase activity.
LRRK2
Figure 4-1 Predicted model of the ROCO interaction. 52
Figure 4-2 Homegenic dimerization is a prerequisite for LRRK2 kinase 58
activity.
LRRK2
Figure 4-3 Possible mechanisms of the ROCO mediated inhibitory 60
effect on LRRK2 kinase activity.



Table 2-1 Genetic loci associated to PD. 9

iii Abbreviations

°C degree celcius
μ micro
aa amino acid
AD Alzheimer’s disease
ANK ankyrin
ARM armadillo
ASK1 apoptosis signal-regulatng kinase 1
BCA Bicinchoninic Acid Assay
BSA bovine serum albumine
C. elegans Caenorhabditis elegans
COR C-terminal of ROC
D. melanogaster Drosophila melanogaster
DA dopaminergic
DAPK1 death-associated protein kinase 1
DD death domain
DMEM Dulbecco’s Minimal Essential Medium
E. coli Escherichia coli
ER endoplasmatic reticulum
ERK extracellular signal regulated kinase
ERM ezrin/radixin/moesin
g gravitation constant
g gram
h hour
HEK human embryonal kidney
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HRP horseradish-peroxidase
HSP heat shock protein
IP immunoprecipitation
IPTG isopropyl-ß-D-thiogalactopyranoside
kD kilodalton
KSR kinase suppressor of Ras
l liter
LB Luria-Bertani
LB Lewy Body
LRR leucine-rich repeat
LRRK2/1 leucine-rich repeat kinase 2/1
iv

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