Die Serum- und Glukokortikoid-induzierbare Kinase in der Regulation von natriumgekoppelten Aminosäuretransportern [Elektronische Ressource] = The serum and glucocorticoid inducible kinase in the regulation of sodium coupled amino acid transporters / von Jeyaganesh Rajamanickam

Physio

Sujets

• Biologie

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2007
• Nombre de lectures: 27
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Die Serum- und Glukokortikoid – induzierbare kinase in
der Regulation von natriumgekoppelten
Aminosäuretransportern

The Serum and Glucocorticoid inducible Kinase in the
regulation of sodium coupled amino acid transporters
















der Fakultät für Biologie

der Eberhard Karls Universität Tübingen

zur Erlangung des Grades eines Doktors der Naturwissenschaften




von
Jeyaganesh Rajamanickam
aus Madurai, Indien
vorgelegte
Dissertation
2007
































Tag der mündlichen Prüfung : 07.03.2007
Dekan : Prof. Dr. Friedrich Schöffl
1. Berichterstatter : Prof. Dr. Florian Lang
2. Berichterstatter : Prof. Dr. Fritz Götz



Contents
CONTENTS
ABBREVIATIONS ............................................................................................................................................... 1
1 ZUSAMMENFASSUNG............................................................................................................................. 3
2 SUMMARY.................................................................................................................................................. 6
3 INTRODUCTION ....................................................................................................................................... 9
3.1 THE SERUM AND GLUCOCORTICOID INDUCIBLE KINASE SGK1............................................................ 9
3.2 THE PROTEIN KINASE B...................................................................................................................... 13
3.3 THE UBIQUITIN LIGASE NEDD4-2 ....................................................................................................... 16
3.4 GLUTAMATERGIC NEUROTRANSMISSION............................................................................................ 20
3.4.1 Excitatory Amino Acid Transporters (EAATs) .............................................................................. 23
3.4.2 The Excitatory Amino Acid Transporter 2 (EAAT2) ..................................................................... 25
3.4.2.1 The Excitatory Amino Acid Transporter 4 (EAAT4).......................................................................... 26
4 AIM OF THE STUDY .............................................................................................................................. 28
5 MATERIALS AND METHODS.............................................................................................................. 30
5.1 SITE DIRECTED MUTAGENESIS ........................................................................................................... 30
5.1.1 Transformation of E.coli XL1-Blue Supercompetent Cells ........................................................... 34
5.2 PREPARATION OF CRNA..................................................................................................................... 34
5.2.1 Plasmid DNA linearization ........................................................................................................... 35
5.2.2 cRNA synthesis .............................................................................................................................. 36
5.3 XENOPUS LAEVIS OOCYTE PREPARATION ............................................................................................. 37
5.4 PROTEIN EXPRESSION IN XENOPUS LAEVIS OOCYTES............................................................................ 39
5.5 NEDD4-2 RNA SILENCING BY SIRNA ................................................................................................ 40
5.6 TRACER FLUX MEASUREMENTS ......................................................................................................... 41
5.7 DETECTION OF CELL SURFACE EXPRESSION BY CHEMILUMINESCENCE.............................................. 42
5.8 WESTERN BLOTTING .......................................................................................................................... 44
5.9 STATISTICAL ANALYSIS...................................................................................................................... 45
6 RESULTS................................................................................................................................................... 46
6.1 MODULATION OF EAAT2 BY SGK1-3 AND PKB................................................................................ 46
6.1.1 SGK1 stimulates EAAT2 activity and plasma membrane abundance ........................................... 46
6.1.2 SGK1 isoforms SGK2-3 and PKB similarly stimulate Nedd4-2 plasma membrane abundance ... 47
6.1.3 SGK1 enhances EAAT2 by inhibiting Nedd4-2 effects without altering Nedd4-2 expression....... 49
6.2 MODULATION OF EAAT4 BY SGK1 ................................................................................................... 53
6.2.1 SGK1 enhances EAAT4 mediated glutamate transport................................................................. 53
6.2.2 Disruption of the putative SGK1 phosphorylation sites (Thr40 and Thr504) on EAAT4 abrogates
transporter stimulation ................................................................................................................................ 54
6.2.3 Phosphorylation at Thr40 on EAAT4 is essential for transporter stimulation.............................. 56
6.2.4 Phosphorylation at Thr40 on EAAT4 is required for enhanced EAAT4 plasma membrane
abundance ................................................................................................................................................... 58
6.2.5 Nedd4-2 coexpression inhibits the activity of EAAT4 ................................................................... 59
6.2.6 Silencing of intrinsic xNedd4-2 upregulates EAAT4 function....................................................... 60
6.2.6.1 xNedd4-2 siRNA downregulates xNedd4-2 expression ...................................................................... 62
6.2.6.2 xNedd4-2 siRNA does not alter GLUT1 expression ........................................................................... 62
7 DISCUSSION............................................................................................................................................. 64
8 REFERENCES .......................................................................................................................................... 68
ACKNOWLEDGEMENT.................................................................................................................................. 89
CURRICULUM VITAE..................................................................................................................................... 90





Abbreviations
ABBREVIATIONS
2-DOG 2-deoxy-D-glucose
AMPA α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
ALS Amyotrophic lateral sclerosis
ATP Adenosine triphosphate
BSA Bovine serum albumin
cAMP Cyclic adenosine monophosphate
CNS Central nervous system
cRNA Complementary ribonucleic acid
EAATs Excitatory amino acid transporters
EGFR Epidermal growth factor receptor
ENaC Epithelial sodium channel
FSH Follicle stimulating hormone
Gln Glutamine
Glu Glutamate
GLUT1 Glucose transporter1
HA Haemagglutinin
HECT Homologous to E6AP-carboxyl-terminus
HEK293 Human embryonic kidney cells 293
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
IGF-1 Insulin-like growth factor-1
Nedd4-2 Neuronal cell expressed developmentally downregulated 4-2
NMDA N-methyl-D-aspartate
PBS Phosphate buffered saline
PCR Polymerase chain reaction
PDGF Platelet derived growth factor
PDK 3-phosphoinositide-dependent protein kinase
PI3K Phosphatidylinositol 3-kinase
PKB Protein kinase B
RISC RNA induced silencing complex
RLU Relative light units
ROMK1 Renal outer medullary potassium channel
Ser Serine
SGK Serum and glucocorticoid inducible protein kinase
1 Abbreviations
siRNA Short interference ribonucleic acid
TGF- β Transforming growth factor-β
Thr Threonine
TM Transmembrane domain
VGLUT Vesicular glutamate transporter






























2 Zusammenfassung
1 Zusammenfassung

Die Serum- und Glukokortikoid-induzierbare Kinase 1 (SGK1) ist eine
Proteinkinase welche die Funktion und Expression verschiedener Ionenkanäle und
Membrantransporter reguliert. Das Gen wird in Tumor- und Fibroblastenzellinien
unter dem Einfluss von Serum und Glukokortikoiden aufreguliert. Die Kinase wird
durch Phosphorylierung aktiviert durch Signale die weiter oberhalb in der
Signalkaskade die PI3 -Kinase aktivieren. Die Phosporylierung erfolgt durch die
Phosphoinositid-abhängige Kinasen 1 (PDK