Analysis of signaling mechanisms essential to mature B cell viability [Elektronische Ressource] / von Alina Patke

Alina Patke

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2007
• Nombre de lectures: 26
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Analysis of Signaling Mechanisms Essential to
Mature B Cell Viability
D i s s e r t a t i o n
zur Erlangung des akademischen Grades
d o c t o r r e r u m n a t u r a l i u m
(Dr. rer. nat.)
im Fach Biologie
eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin
von
Diplom-Biologin Alina Patke
geboren am 29. März 1976 in Berlin
Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Christoph Markschies

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Christian Limberg
Gutachter: 1. Prof. Dr. Peter-Michael Kloetzel
Gutachter: 2. Prof. Dr. Wolfgang Lockau
Gutachter: 3. Prof. Dr. Alexander Tarakhovsky
Tag der mündlichen Prüfung: 17.9.2007
Abstract
The maintenance of mature peripheral B cells depends on at least two
survival cues, tonic signaling from the B cell receptor (BCR) complex and the
extracellular cytokine B cell activating factor of the TNF family (BAFF). In
addition to enhancing viability, BAFF controls the functional efficiency of the
peripheral B cell pool by regulating complex physiological processes including
cell growth, metabolism, energy homeostasis and entry into the cell cycle.
BAFF-mediated induction of two molecular mechanisms, namely activation of
the Akt signal transduction pathway and upregulation of the oncogenic kinase
Pim-2 results in the modification of effector proteins including transcription
factors and regulators of protein synthesis which are capable of executing the
observed cellular physiological changes. The classic protein kinase C β is
instrumental in BAFF-induced Akt-activation and PKC β-deficient B cells and
mice show signs of partial refractiveness to BAFF. The protein tyrosine kinase
Syk plays a role in early B cell development and is activated in mature B cells
by immunogenic BCR-stimulation. Inducible ablation of Syk in mice results in
the loss mature B cells from the peripheral lymphoid organs and reveals an
indispensable function for Syk in tonic BCR survival signaling.

Kurze Zusammenfassung
Die Langlebigkeit reifer periphärer B Zellen ist abhängig von mindestens zwei
Überlebenssignalen, einem tonischen Signal, welches vom B Zellrezeptor
ausgeht und dem Zytokin B Zell aktivierender Faktor der TNF-Familie (BAFF).
BAFF fördert nicht nur das Überleben von reifen B Zellen, sondern kontrolliert
auch deren Funktionstüchtigkeit, indem es vielschichtige physiologische
Prozesse wie Zellwachstum und –metabolismus, Energiehaushalt und Eintritt
in den Zellzyklus reguliert. Zwei BAFF-induzierte molekulare Mechanismen,
zum einen die Aktivierung des Akt Signaltransduktionsweges sowie die
erhöhte Expression der onkogenen kinase Pim-2 zum anderen, führen zu
Veränderungen in Effektorproteinen welche in der Lage sind diese
physiologischen Zellveränderungen auszulösen. Die BAFF-induzierte
Aktivierung von Akt hängt von der klassischen Proteinkinase C (PKC) β ab
und sowohl PKC β-defiziente B Zellen als auch Mäuse zeigen Anzeichen von
Unsensitivität gegenüber BAFF-Stimulation. Die Proteintyrosinkinase Syk
spielt eine Rolle während der frühen B Zellentwicklung und wird in reifen B
Zellen durch Stimulation des B Zellrezeptors aktiviert. Induzierbare
Inaktivierung von Syk in Mäusen führt zum Verschwinden reifer B Zellen aus
den periphären lymphoiden Organen, was auf eine unverzichtbare Funktion
von Syk in der Vermittlung des tonischen B Zellrezeptorsignals schliessen
läßt.






FOR MY PARENTS







ABSTRACT II
KURZE ZUSAMMENFASSUNG III
ABBREVIATIONS IV
1 INTRODUCTION 1
1.1 Brief overview of B cell development
1.2 Mature B cell survival through tonic BCR signaling 3
1.2.1Composition of the BCR-complex and its signaling mechanisms 3
1.2.2The role of Syk in B cell biology8
1.3 Mature B cell survival through BAFF signaling 13
1.3.1 The BAFF-related protein network
1.3.2 The functions of BAFF and BAFF-R in B cell survival and beyond 15
1.3.3 The functions of TACI, BCMA and APRIL 18
1.3.4 The molecular consequences of BAFF-signaling 19
1.4 Lymphocyte survival through growth factor signaling 22
1.4.1 The association between the trophic state of a cell and its viability 22
1.4.2 Growth factor signaling is transduced by the PI3K-Akt pathway 23
1.4.3 Association between the PI3K pathway and cell transformation 28
1.5 The aims of this study 29
2 MATERIALS AND METHODS 30
2.1 Materials 30
2.1.1 Equipment and consumables
2.1.2 Standard buffers 31
2.1.3 Oligonucleotides 32
2.1.4 Antibodies 34
2.2 Methods 37
2.2.1 Molecular Biology
2.2.2 Cellular Methods 39
2.2.3 Biochemical Methods 45
2.2.4 Other Methods 50
2.2.5 Mice 52
I 3 RESULTS 53
3.1 Part 1: BAFF acts as a survival and growth factor on B cells 53
3.1.1 BAFF promotes B cell growth and metabolic fitness
3.1.2 BAFF activates the PI3K-Akt signaling pathway 59
3.1.3 PKC β controls BAFF-mediated Akt-activation 64
3.1.4 Other BAFF-induced signaling events are PKC β-independent 68
3.1.5 PKC β-deficiency impairs BAFF-induced cellular responses and
causes a defect in peripheral B cell maturation 70
3.2 Part 2: The role of Syk in B cell survival 74
3.2.1 Arrested B cell development in Syk-deficient mice
3.2.2 Aberrant B cell development in the absence of Syk is a B cell intrinsic
phenomenon 76
3.2.3 Syk is indispensable for mature B cell survival 79
3.2.4 Development of an experimental system for inducible Syk-ablation in
vitro 82
4 DISCUSSION AND FUTURE PERSPECTIVE 93
4.1 BAFF-mediated effects on B cell physiology 94
4.2 A novel BAFF-induced signaling mechanism 97
4.3 The role of Syk in mature B cell survival 104
4.4 Development of a novel protein knockout system for Syk inactivation in
B cells 106
4.5 Other potential methods for rapid inducible Syk inactivation 110
4.6 B cell survival mechanisms which could represent targets of the Syk-
mediated tonic BCR signal 112
5 SUMMARY 116
6 ZUSAMMENFASSUNG 117
7 REFERENCES 119
8 ACKNOWLEDGEMENTS 160
9 ERKLÄRUNG 161
II 10 PUBLICATIONS 162
11 APPENDIX 163

III Abbreviations
Standard abbreviations used throughout this work include chemical symbols,
SI units as well as one and three letter amino acid codes.
Non-standard abbreviations are explained in the text upon first citation and
are listed in the following:

4E-BP1 4E binding protein 1
A1 BCL2-related protein A1
Act1 NF- κB activator 1
AMP Adenosine monophosphate
AMPK AMP-activated protein kinase
APRIL a proliferation-inducing ligand
ATM ataxia telangiectasia mutated
ATP Adenosine triphosphate
Bad Bcl-2-associated death promoter
BAFF B cell activating factor of the TNF family
BAFF-R BAFF receptor
Bak BCL2-antagonist/killer
Bax Bcl2-associated X protein
Bcl-2 B-cell lymphoma 2
Bcl-xL Bcl2-like 1
Bcl10 B-cell lymphoma 10
BCMA B-Cell Maturation Antigen
BCR B cell receptor
Bid BH3 interacting domain death agonist
Bim Bcl-2 interacting mediator of cell death
BLAST basic logical alignment search tool
BLNK B cell linker protein
BrdU bromodeoxyuridine
BSS Hanks Balanced Salt Solution
Btk Bruton’s tyrosine kinase
Carma1 caspase-recruitment domain membrane-associated guanylate
kinase protein 1
Cbl Casitas B-lineage lymphoma
CCCP carbonyl cyanide m-chlorophenyl hydrazone
IV CD Cluster of Differentiation
Cdc cell division cycle
Cdk cyclin-dependent kinase
CFDA-SE carboxyfluorescein diacetate succinimidyl ester
CFSE carboxyfluorescein succinimidyl ester
CHX cycloheximide
CLL chronic lymphocytic leukemia
CRD Cystein-Rich Domain
Cre Cyclization recombination
Csk C-terminal src-kinase
DAG diacylglycerol
DNA deoxyribonucleic acid
DNA-PK DNA-dependent protein kinase
EDTA ethylenediamine tetraacetic acid
EGTA ethylene glycol tetraacetic acid
eIF4E eukaryotic translation initiation factor 4E
Erk extracellular signal-regulated kinase
ES embryonic stem
FACS fluorescence activated cell sorting
FBS fetal bovine serum
Fc fragment crystallizable
FITC fluorescein isothiocyanate
FoxO Forkhead box O
G1 Gap1
GAP GTPase activating protein
GC germinal center
GFP green fluorescent protein
Gsk-3 gycogen synthase kinase 3
Gst Glutathione S-transferase
HA hemagglutinin
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HK hexokinase
Ig immunoglobuline
IFN interferon
ILK integrin-linked kinase 1
V I κB inhibitor of nuclear factor- κB
IKK I κB kinase
IP inositol-1,4,5-trisphosphate 3
IPTG isopropyl- β-D-thiogalactopyranoside
ITAM immunoreceptor tyrosine-based activation motif
ITIM ne-based inhibitory motif
Jnk Jun N-terminal kinase
LB Luria-Bertani
loxP locus of X-over of P1
MACS magnetic cell sorting
MALT1 (mucosa-associated-lymphoid-tissue lymphoma-translocation
ge