Signal transduction of the atypical GTPase RhoH in Interleukin-3 dependent cell systems [Elektronische Ressource] / presented by Mehtap Gündogdu

Mehtap Gündogdu

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2011
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

SIGNAL TRANSDUCTION
OF THE
ATYPICAL GTPASE RHOH
IN INTERLEUKIN-3 DEPENDENT
CELL SYSTEMS



DISSERTATION
submitted to the Combined Faculties for the
Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences









presented by
Dipl.-Ing. (FH) Mehtap Gündogdu
born in Hof a.d. Saale




DISSERTATION








submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences












presented by
Dipl.-Ing. (FH) Mehtap Gündogdu
born in Hof a.d. Saale
Oral examination: ……………………….



SIGNAL TRANSDUCTION
OF THE
ATYPICAL GTPASE RHOH
IN INTERLEUKIN-3 DEPENDENT
CELL SYSTEMS









Referees:

Prof. Dr. Michael Lanzer

Prof. Dr. med. Alexander Dalpke




Meiner Familie
Table of contents

Table of contents
1. Summary .................................................................................................................... 1
1. Zusammenfassung ..................................................................................................... 2
2. Introduction ............................................................................................................... 3
2.1 Interleukin-3 (IL-3) and the IL-3 receptor ...................................................................... 3
2.1.1 The JAK-STAT pathway ................................................................................................. 4
2.1.2 IL-3 mediated JAK-STAT signalling .............................................................................. 5
2.1.3 The physiologic relevance of IL-3 and the IL-3 receptor ................................................ 6
2.2 Rho GTPases member of the Ras superfamily of small GTPases ................................. 7
2.2.1 Structural features of Rho GTPases................................................................................. 8
2.2.2 Important regulation mechanisms of Rho GTPases ........................................................ 9
2.2.3 Function and signalling of Rho GTPases ...................................................................... 10
2.3 The atypical GTPase RhoH ............................................................................................ 10
2.3.1 Structural features of RhoH ........................................................................................... 11
2.3.2 RhoH in signal transduction .......................................................................................... 11
2.3.3 RhoH, a negative modulator of hematopoetic proliferation and its function in cancer . 12
2.3.4 RhoH, inhibitor of IL-3 mediated proliferation and signalling: Preliminary data ......... 13
2.4 Aim of this study .............................................................................................................. 14
3. Materials and Methods ........................................................................................... 15
3.1 Material ............................................................................................................................ 15
3.1.1 Lab equipment ............................................................................................................... 15
3.1.2 Expandable material ...................................................................................................... 16
3.1.3 Purchaseable kits and buffers ........................................................................................ 17
3.1.4 General chemicals ......................................................................................................... 17
3.1.5 DNA modifying enzymes .............................................................................................. 19
3.1.6 Stimuli for cell culture ................................................................................................... 19
3.1.7 Antibodies and isotypes ................................................................................................. 20
3.1.8 Size markers .................................................................................................................. 21
3.1.9 Vectors ........................................................................................................................... 21
3.1.10 Oligonucleotides ............................................................................................................ 22
3.1.11 Eukaryotic cells ............................................................................................................. 23
3.1.12 Special software & databases ........................................................................................ 24
3.1.13 Buffers and working solutions ....................................................................................... 24
3.2 Methods ............................................................................................................................ 26
3.2.1 Eukaryotic cells ............................................................................................................. 26
3.2.2 Prokaryotic cells ............................................................................................................ 30
3.2.3 Molecular biological techniques .................................................................................... 32
3.2.4 Biochemical and immunological methods .................................................................... 37

I
Table of contents

4. Results ....................................................................................................................... 48
4.1 RhoH modulates IL-3 signalling through regulation of STAT activity and IL-3
receptor expression .......................................................................................................... 48
4.1.1 Regulation of RhoH expression in the IL-3 dependent system ..................................... 48
4.1.2 RhoH, an inhibitor of IL-3 induced proliferation .......................................................... 49
4.1.3 RhoH overexpression results in increased IL-3 dependent STAT1 activation .............. 51
4.1.4 RhoH modulated IL-3 dependent STAT1 activation correlates with expression of
CDKIs ............................................................................................................................ 52
4.1.5 RhoH expression modulates IL-3 induced STAT5 activity .......................................... 54
4.1.6 RhoH- a modulator of CD123 surface expression ......................................................... 56
4.1.7 RhoH reconstitution correlated with a downregulation of prognostic AML markers in
THP-1 cells .................................................................................................................... 58
4.1.8 RhoH expression pattern in lymphoma derived cell lines ............................................. 59
4.1.9 Proteomics of RhoH in IL-3 dependent cell lines ......................................................... 62
4.2 RhoH in IL-3 derived bone marrow MCs ..................................................................... 71
4.2.1 RhoH mRNA expression in IL-3 derived murine MCs ................................................. 71
4.2.2 RhoH null HPCs show elevated IL-3 induced proliferation .......................................... 72
waf/cip1 kip1
4.2.3. RhoH null HPCs show lower p21 and p27 mRNA expression only in early
phase of IL-3 cultivation ............................................................................................... 73
4.2.4. RhoH null progenitors show no differences in the differentiation to MCs ................... 74
4.2.5. RhoH is important for Fc RI mediated degranulation of MCs ..................................... 75
-/-
4.2.6. RhoH MCs show impaired release of IL-6 and TNF- ............................................. 76
5. Discussion ................................................................................................................. 78
5.1 RhoH expression is regulated by IL-3 and modulates IL-3 induced proliferation .... 78
5.2 RhoH: Fine-tuning of IL-3 dependent JAK-STAT signalling ..................................... 79
5.3 RhoH expression in THP-1 cells, correlates with increased IRF-1 and decreased
CD123 expression ............................................................................................................ 82
5.3.1 RhoH is underexpressed in lymphoma derived cell lines with abberant signalling
patterns and immunophenotype ..................................................................................... 83
5.4 RhoH interaction partner binding is IL-3 dependent .................................................. 85
5.4.1 Similar pr