Studies on a novel poly(ADP-ribosyl)ation polymerase PARP-10 and its functional interaction with c-Myc [Elektronische Ressource] / vorgelegt von Mei Yu

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• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2005
• Nombre de lectures: 42
• Langue: English
• Poids de l'ouvrage: 3 Mo

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Studies on a novel poly(ADP-ribosyl)ation polymerase
PARP-10 and its functional interaction with c-Myc

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften
der Rheinisch-Westfälischen Technischen Hochschule Aachen
zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften
genehmigte Dissertation

vorgelegt von Diplom-Biologin
Mei Yu
aus
Jilin (China)

Berichter: Prof. Dr. Bernhard Lüscher
P. D. Dr. Harald Luksch



Tag der mündlichen Prüfung: 22. 06. 2005

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online
verfügbar.
Publication from this work:
Mei Yu, Sabine Schreek, Christa Cerni, Chantal Schamberger, Krzysztof Lesniewicz,
Elzbieta Poreba, Jörg Vervoorts, Gesa Walsemann, Joachim Grötzinger, Elisabeth
Kremmer, Yasmin Mehraein, Jürgen Mertsching, Regine Kraft, Matthias Austen,
Juliane Lüscher-Firzlaff and Bernhard Lüscher
PARP-10, a novel Myc-interacting protein with poly(ADP-ribose) polymerase
activity, inhibits transformation
Oncogene, 2005, 24, 1982-1993
Contents I
Contents
ABSTRACT V
ABBREVIATIONS VII
1 INTRODUCTION 1
1.1 Proto-oncoprotein c-Myc 1
1.1.1 Proto-oncogene c-myc 1
1.1.2 Expression, function and structure of c-Myc 1
1.1.3 The Myc/Max/Mad network 3
1.1.4 Target genes of c-Myc 4
1.1.5 Effect of c-Myc on cell cycle progression 5
1.1.6 c-Myc and transformation 5
1.1.7 c-Myc-dependent transactivation and repression 7
1.2 Modification of chromatin 9
1.3 Poly(ADP-ribosyl)ation 11
1.3.1 PARP-1 11
1.3.2 PARG 16
1.3.3 PARP and chromatin remodeling 17
1.3.4 Other PARP members 19
1.4 Aims of this study 21
2 RESULTS 23
2.1 Characterization of PARP-10 23
2.1.1 Consensus mapping of PARP-10 23
2.1.2 Subcellular distribution of PARP-10 24
2.1.3 PARP-10 inhibits cell proliferation 28
2.1.4 PARP-10 does not have an effect on colony formation 31
2.1.5 PARP-10 possesses PARP activity 32 Contents II
2.1.5.1 Glycine 888 is the catalytic center of PARP-10 32
2.1.5.2 PARP-10 derived from PARP-10-inducible cell line shows PARP
activity 34
2.1.5.3 Substrates of PARP-10 35
2.2 Biochemical and functional interaction of PARP-10 and c-Myc 40
2.2.1 In vivo interaction of PARP-10 and c-Myc 40
2.2.2 PARP-10 interacts with c-Myc in a stable cell line 41
2.2.3 Colocalization of PARP-10 and c-Myc in the nucleus 42
2.2.4 c-Myc interacts with PARP-10 in the nucleus 44
2.2.5 c-Myc binds to the C-terminus of PARP-10 45
2.2.6 PARP-10 represses the c-Myc-dependent transactivation 46
2.2.7 PARP-10 inhibits c-Myc/Ha-Ras- and E1A/Ha-Ras-dependent
transformation 48
3 DISCUSSION 52
3.1 The biochemical and biological function of PARP-10 52
3.1.1 Subcellular localization of PARP-10 52
3.1.2 Transport of PARP-10 54
3.1.3 Biological function of PARP-10 56
3.1.4 Poly(ADP-ribosyl)ation by PARP-10 60
3.2 The functional interaction of PARP-10 with c-Myc 63
3.2.1 PARP-10 interacts with c-Myc 63
3.2.2 PARP-10 represses the c-Myc-dependent transactivation 64
3.2.3 PARP-10 inhibits c-Myc/Ha-Ras- and E1A/Ha-Ras-dependent
transformation 66
4 MATERIALS AND METHODS 68
4.1 Materials 68
4.1.1 Chemicals 68
4.1.2 Synthetic oligonucleotides 68
4.1.3 Plasmids 69 Contents III
4.1.4 Antibodies 73
4.1.5 Bacterial strains 73
4.1.6 Materials for cell culture 74
4.1.7 Eukaryotic cells 74
4.1.8 Culture conditions 75
4.2 Methods 75
4.2.1 DNA methods 75
4.2.1.1 Transformation in competent E. coli cells 75
4.2.1.2 Isolation of plasmid DNA from E. coli 76
4.2.1.3 Quantitative analysis of nucleic acids 76
4.2.1.4 Modification of DNA 76
4.2.1.5 Molecular cloning by polymerase chain reaction (PCR) 77
4.2.1.6 PCR mutagenesis (site-directed mutagenesis) 77
4.2.1.7 DNA agarose gel electrophoresis and extraction 78
4.2.2 Methods for cell culture 79
4.2.2.1 Cryopreservation 79
4.2.2.2 Transient transfection 79
4.2.2.3 Generation of PARP-10 inducible cell lines 81
4.2.2.4 Single selection of inducible cell line 81
4.2.2.5 Colony formation assay 82
4.2.2.6 Bimolecular fluorescence complementation (BIFC) analysis 82
4.2.3 Biochemical methods 83
4.2.3.1 Prepare cell lysates 83
4.2.3.2 Immunoprecipitation (IP) 83
4.2.3.3 Measuring of transactivation 84
4.2.3.4 MTT assay 85
4.2.3.5 SDS-PAGE 85
4.2.3.6 Coomassie staining 86
4.2.3.7 Western blot 87
4.2.3.8 Purification of Glutathion-S-Transferase fusion proteins 88
4.2.3.9 In vitro translation/transcription 89
4.2.3.10 GST-pulldown 89
4.2.3.11 Immunofluorescence 89 Contents IV
4.2.3.12 PARP assay 90
5 REFERENCES 91
6 LEBENSLAUF 111
7 DANKSAGUNGEN 112 Abstract V
Abstract
The c-Myc oncoprotein regulates different aspects of cell behavior by
modulating gene expression. It was demonstrated that c-Myc stimulates
cell proliferation, inhibits differentiation, and induces apoptosis. At the
molecular level, c-Myc acts as a transcription factor by activating or
repressing target genes. It is suggested that c-Myc exerts many
functions by binding to other proteins, and several c-Myc-interaction
partners play roles in the c-Myc-dependent regulation of gene
expression and cell behavior. Thus, the characterization of novel c-Myc-
binding proteins could give an insight into known as well as novel roles
of c-Myc.
To unterstand the function of c-Myc, a screen for novel interaction
partners was performed by affinity column purifications, and PARP-10
was found to interact with c-Myc. PARP-10 is a member of the protein
family of poly(ADP-ribose) polymerases (PARPs) that are protein-
modifying and nucleotide-polymerizing enzymes able to catalyze the
+transfer of multiple ADP-ribose units from NAD to substrate proteins.
Poly(ADP-ribosyl)ation has been reported to regulate many cellular
processes such as DNA repair, genomic stability, cell cycle progression,
cell death, and gene transcription.
The aims of this study were to characterize PARP-10 biochemically and
biologically, and to analyze the biochemical and functional interaction
between c-Myc and PARP-10. This study shows that the novel PARP
family member, PARP-10, is localized to the cytoplasm and the nucleus,
PARP-10 contains a functional NES that mediates nuclear export which
is CRM1-dependent. PARP-10 poly(ADP-ribosyl)ates itself and core Abstract VI
histones, suggesting that it could play a potential role in the remodeling
of chromatin. PARP-10 also reduces the proliferation rate of a PARP-10-
inducible cell line, but shows no growth inhibitory effect on colony
formation. The interaction of PARP-10 with the proto-oncoprotein c-Myc
was shown in vivo and in vitro, furthermore the interaction occurs in the
nucleus. c-Myc interacts with the C-terminus of PARP-10. PARP-10 can
repress the c-Myc-dependent transactivation and inhibit c-Myc/Ha-Ras-
and E1A/Ha-Ras-dependent transformation indicating that PARP-10
might repress c-Myc function in target gene regulation and oncogenesis.
In summary, this study suggests that PARP-10 is a novel PARP enzyme
that interacts with the proto-oncoprotein c-Myc. PARP-10 inhibits the c-
Myc-dependent transactivation and transformation of rat embryo
fibroblasts.
Abbreviations VII
Abbreviations
Ab antibody
Amp ampicillin
APS ammonium persulfate
ATCC american type culture collection
ATP adenosintriphosphate
b basic region
BIFC bimolecular fluorescence complementation
bp base pair
BPB bromphenolblue
BSA bovine serum albumin
CB coomassie blue
CBP CREB binding protein
CDK cyclin-dependent kinase
cDNA complementary DNA
ChIP chromatin-immunoprecipitation
Ci curie
CMV Cytomegalie-virus
ddH Or double destilled water 2
DMEM Dulbecco´s modified Eagle medium
DMSO dimethylsulfoxid
DNA desoxyribonucleic acid
dNTP desoxynucleosidtriphosphate
DOC deoxycholate
DTT dithiothreitol
E. coli Escherichia coli
ECL enhanced chemiluminescence
EDTA ethlendiamintetraacetate
EGFP enhanced green fluorescence protein
EST expressed sequence tags
FCS fetal calf serum
GFP green fluorescence protein
GST glutathion-S-transferase
HA hemagglutenin
HAT histone-acetyltransferase
HDAC histone-deacetylase
Hebs hepes buffered saline
HEPES N-2-hydroxyethyl-piperazin-N`-2-ethansulfone acid
HLH helix-loop-helix
HMT histone-methyltransferase
HS horse serum
IE immediate early
Ig immunoglobulin
INR initiator-element
IPTG b-D-isopropyl-thiogalactopyranoside
kb kilobase
kDa kilodalton
LZ leucine zipper Abbreviations VIII
MB Myc box
MBP maltose-binding protein
MEM modified eagle medium
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
mRNA messenger-RNA
NAD nicotinamid-adenin-dinucleotide
NCBI National center of biotechnology information
NES nucleus export sequence
NLS nuclear localization signal
OD op