«DISSERTATION submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany ...»
ATYPICAL GTPASE RHOH
IN INTERLEUKIN-3 DEPENDENT
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
DISSERTATIONsubmitted 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 TRANSDUCTIONOF THE
ATYPICAL GTPASE RHOH
IN INTERLEUKIN-3 DEPENDENT
Prof. Dr. Michael Lanzer Prof. Dr. med. Alexander Dalpke Meiner Familie Table of contents Table of contents 1. Summary
2.1 Interleukin-3 (IL-3) and the IL-3 receptor
2.1.1 The JAK-STAT pathway
2.1.2 IL-3 mediated JAK-STAT signalling
2.1.3 The physiologic relevance of IL-3 and the IL-3 receptor
2.2 Rho GTPases member of the Ras superfamily of small GTPases
2.2.1 Structural features of Rho GTPases
2.2.2 Important regulation mechanisms of Rho GTPases
2.2.3 Function and signalling of Rho GTPases
2.3 The atypical GTPase RhoH
2.3.1 Structural features of RhoH
2.3.2 RhoH in signal transduction
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
3. Materials and Methods
3.1.1 Lab equipment
3.1.2 Expandable material
3.1.3 Purchaseable kits and buffers
3.1.4 General chemicals
3.1.5 DNA modifying enzymes
3.1.6 Stimuli for cell culture
3.1.7 Antibodies and isotypes
3.1.8 Size markers
3.1.11 Eukaryotic cells
3.1.12 Special software & databases
3.1.13 Buffers and working solutions
3.2.1 Eukaryotic cells
3.2.2 Prokaryotic cells
3.2.3 Molecular biological techniques
3.2.4 Biochemical and immunological methods
Table of contents4. Results
4.1 RhoH modulates IL-3 signalling through regulation of STAT activity and IL-3 receptor expression
4.1.1 Regulation of RhoH expression in the IL-3 dependent system
4.1.2 RhoH, an inhibitor of IL-3 induced proliferation
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
4.1.5 RhoH expression modulates IL-3 induced STAT5 activity
4.1.6 RhoH- a modulator of CD123 surface expression
4.1.7 RhoH reconstitution correlated with a downregulation of prognostic AML markers in THP-1 cells
4.1.8 RhoH expression pattern in lymphoma derived cell lines
4.1.9 Proteomics of RhoH in IL-3 dependent cell lines
4.2 RhoH in IL-3 derived bone marrow MCs
4.2.1 RhoH mRNA expression in IL-3 derived murine MCs
4.2.2 RhoH null HPCs show elevated IL-3 induced proliferation
RhoH null HPCs show lower p21waf/cip1 and p27kip1 mRNA expression only in early 4.2.3.
phase of IL-3 cultivation
4.2.4. RhoH null progenitors show no differences in the differentiation to MCs
4.2.5. RhoH is important for FcεRI mediated degranulation of MCs
RhoH-/- MCs show impaired release of IL-6 and TNF-α
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
5.3 RhoH expression in THP-1 cells, correlates with increased IRF-1 and decreased CD123 expression
5.3.1 RhoH is underexpressed in lymphoma derived cell lines with abberant signalling patterns and immunophenotype
5.4 RhoH interaction partner binding is IL-3 dependent
5.4.1 Similar proteins can bind to RhoH and to its effector domain mutant variant.............. 87 5.4.2 IL-3 treatment determines the subcellular localisation of RhoH interacting proteins... 89 5.4.3 RhoH interacts with Cofilin-1 in HEK cells
5.4.4 RhoH interacts with PTP1B in HEK cells
5.5 Summary and outlook (I): RhoH modulates IL-3 induced proliferation by finetuning IL-3 dependent JAK-STAT signalling
5.6 The function of RhoH in IL-3 mediated MC differentiation and function................ 95 5.6.1 RhoH mRNA expression is regulated during MC differentiation
5.6.2 RhoH null MC progenitors show enhanced proliferation in early phases of cultivation in IL-3 containing medium
5.6.3 No morphologic differences between RhoH null and wt MC
5.6.4 The lack of RhoH causes functionial defects in IL-3 derived MCs
Table of contents
5.7 Summary and outlook (II): The consequences of RhoH deficiency for IL-3 derived MCs
6.1 Supplementary tables
6.1.1 Thesis relevant protein hits
6.1.2 Gene ontology
6.2 RhoH protein sequence (mouse)
8. Publications and Presentations
Ran Ras related nuclear protein Ras Rat sarcoma rE Relative expression Rho Ras homologue RhoH Ras homologe H RLU Relative Light Units RNA Ribonucleic acid rpm Rotations per minute RPMI Roswell Park Memorial Institute RT Room Temperature RTK Receptor Tyrosine Kinase SCF-1 Stem cell factor-1 SCX Strong cation exchange SDS Sodium dodecyl sulfate Sec Second SHP Src homology region 2 domain-containing phosphatase SOCS Suppressor Of Cytokine Signalling Src Sarcoma SRF Serum Response Factor STAT Signal Transducers and Activators of Transcription Syk Spleen Tyrosine Kinase TBS Tris Buffered Saline TBST Tris Buffered Saline and Tween20 TCR T-Cell Receptor Tumour Necrotizing Factor alpha TNF-α TNP 2,4,6-Trinitrophenol TTF Translocation Three Four Tyk2 Tyrosine kinase 2 U Units V Volt WB Western-blot wt Wildtype Zap70 Zeta chain associated protein kinase 70 VI Summary
1. Summary The hematopoietic, atypical GTPase RhoH is reported to function as a negative regulator of progenitor cell differentiation, proliferation and survival. IL-3 is an important cytokine, signalling mainly via the JAKSTAT pathway and by this promoting hematopoiesis, progenitor cell survival and differentiation of myeloid cells, such as mast cells. This study aimed to investigate the role of RhoH and important components in the signalling network of RhoH in IL-3 mediated signal transduction. It was attempted to link published, as well as the gained, information of this study to profile hematopoietic malignancies with aberrant RhoH expression. Finally, complementary studies were performed with in vitro IL-3 derived mast cells to address the role of RhoH in IL-3 mediated cell differentiation and its function in mast cell mediated processes, such as anaphylaxis.
RhoH overexpression in BaF3 cells resulted in inhibition of IL-3 induced proliferation correlating with increased STAT1 and decreased STAT5 activity. Downregulation of RhoH was followed by an increase in IL-3 proliferation associated with enhanced STAT5 phosphorylation. Altered STAT1 activation induced increased expression of the cell cycle inhibitors (CDKIs) p21waf/cip and p27kip. Enforced IL-3 induced STAT5 activation in cells with low RhoH expression led to enhanced sensitivity of cells to IL-3, as a result of an upregulation of the STAT5 dependent transcription factor IRF-1 which induced increased IL-3 alpha chain (CD123) surface expression. RhoH expression was tested in lymphoma cell lines which were characterised based on their immunophenotype, STAT and GTPase activity. RhoH was strongly underexpressed in almost every cell line, suggesting that RhoH plays an important role in the formation of hematopoietic cancer types. To gain further insight into the IL-3 dependent signalling networks of RhoH, cytokine treatment dependent proteomic experimebyents were performed with IL-3 dependent BaF3 cells.
A cytokine dependent pattern of protein interactions was identified. The interaction with RhoH was verified in HEK cells for two proteins: Cofilin-1 and PTP1B. Both of these proteins were found to have effects that can be related to the previous findings of this study in IL-3 mediated signalling. Finally, experiments in mast cells were performed to identify the role of RhoH for IL-3 dependent cell differentiation and function, indicating that RhoH deficiency does not affect the IL-3 induced differentiation. However, the loss of RhoH has functional consequences on mast cell biology, such as decreased cytokine production and mast cell mediator release.
These findings link RhoH expression and RhoH modulated IL-3 induced proliferation on the one hand to STAT1 dependent induction of CDKIs and on the other hand to the increased STAT5 dependent CD123 expression. It is suggested that RhoH is a tandem regulator of IL-3 dependent STAT5/STAT1 activation, most likely by newly discovered interactions with key signalling proteins such as PTP1B and Cofilin-1.
Altered RhoH expression pattern in different hematopoietic disorders also suggests that the identified role of RhoH in IL-3 dependent signalling is transferrable to aberrant signalling in cancerogenous diseases.
Furthermore, it was postulated that, although RhoH is dispensable for IL-3 mediated myeloid mast cell differentiation, the lack of RhoH has serious impact on mast cell mediated effects.
1. Zusammenfassung Die hämatopoetische GTPase RhoH ist ein negativer Regulator für die Vorläuferzelldifferenzierung, Proliferation sowie für das Überleben hämatopoetischer Stammzellen. Die Regulation der Differenzierung und des Überlebens dieser Zellen erfolgt hauptsächlich über den IL-3 abhängigen JAK-STAT Signalweg.
Ziel dieser Studie war es die Rolle von RhoH in der IL-3 abhängigen Signaltransduktion zu untersuchen und neue Komponenten der RhoH vermittelten Signaltransduktion zu identifizieren. Zusätzlich wurde anhand von Hinweisen aus der Literatur verschiedene Lypmhoma Zellinien hinsichtlich ihrer RhoH Expression im Zusammenhang mit der Aggressivität der jeweiligen Tumorzellen analysiert. Ausserdem sollte der Einfluss von RhoH auf die IL-3 abhängige Mastzelldifferenzierung und deren Funktion (z.B.
Anaphylaxie) mittels in vitro generierter Mastzellen geklärt werden.
Die Überexpression von RhoH in der murinen Pro-B-Zelllinie Baf3 korrelierte mit der Inhibition IL-3 vermittelter Proliferation einhergehend mit einer erhöhten STAT1 und verminderter STAT5 Phosphorylierung. Umgekehrt führte die Inhibition der RhoH Expression zu einer verstärkten IL-3 vermittelten Proliferation, verbunden mit einer erhöhten STAT5 Phosphorylierung. Die verstärkte STAT1 Aktivität induzierte eine erhöhte Expression der Zell Zyklus Inhibitoren (CDKIs) p21waf/cip1 und p27kip1 in RhoH überexprimierenden Zellen, während die erhöhte STAT5 Aktivierung in RhoH defizienten Zellen in einer gesteigerten Expression des STAT5 abhängigen Transkriptionsfaktors IRF-1 resultierte. Dies führt wiederum zur Überexpression der IL-3 α Kette CD123 an der Zelloberfläche.