The role of oncogenic Cbl mutants in Kit signaling and myeloid transformation [Elektronische Ressource] / by Srinivasa Rao Bandi

Bandi

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 51
• Langue: English
• Poids de l'ouvrage: 9 Mo

Extrait

The role of oncogenic Cbl mutants in Kit signaling
and myeloid transformation


Dissertation
to obtain the Degree of Doctor of Philosophy
at the Faculty of Natural Sciences



Submitted to the Faculty of Biochemistry, Chemistry and Pharmacy
of the Goethe University
in Frankfurt am Main



By
Srinivasa Rao Bandi
from India

Frankfurt am Main, 2009


(D30) Submitted to the Faculty of Biochemistry, Chemistry and Pharmacy of
The Goethe University in Frankfurt am Main



































Dean: Prof. Dr. Dieter Steinhilber
Examiners:
1. Examiner: Prof. Dr. Rolf Marschalek
2. Examiner: Prof. Dr. Hubert Serve
Date:




Contents


Contents
Summary ............................................................................................................................... 4
1 Introduction .................................................................................... 6
1.1 Hematopoiesis ........................................................................................................ 6
1.2 Leukemia ......................... 6
1.2.1 Classification of leukemia ................................................................. 7
1.2.2 Acute myeloid leukemia (AML) ......................................... 8
1.3 Receptor tyrosine kinases (RTKs) ........................................................................... 8
1.3.1 Activation of RTKs ...........................................................................................10
1.4 FMS-like tyrosine kinase 3 (Flt3) ............................................................................11
1.4.1 Flt3 receptor expression and activation ...........................................................11
1.4.2 Flt3 mutations..................................................................................................12
1.5 Kit ...........................................................................................................................13
1.5.1 Kit structure .....................................................................................................13
1.5.2 Stem cell factor ........................................14
1.5.3 Kit expression..................................................................14
1.5.4 Kit mutations ...................................................................14
1.6 Kit signaling ....................................................................................15
1.7 Activation of signal transduction pathways .............................................................15
1.7.1 MAP kinase pathway .......................................................................................15
1.7.2 PI-3-Kinase pathway .16
1.7.3 JAK/STAT pathway ..................................16
1.8 Src family tyrosine kinases (SFKs) .........................................................................17
1.9 Regulation of Flt3 and Kit .......................................................19
1.10 Ubiquitination ..............................................................................19
1.11 Cbl (Casitas B-lineage lymphoma) ......................................................................20
1.11.1 Cbl homologues and structure .........................................20
1.11.2 RTK regulation by Cbl .....................................................................................21
1.11.3 Phosphorylation of Cbl .....................22
1.11.4 Cbl as a multivalent adaptor protein ................................................................23
1.11.5 Cbl mutations in cancer ...................................................................................23
2 Aims of the study ..................................................................................25
3 Materials and methods ..................................26
3.1 Reagents and antibodies ........................................................................................26
3.2 Methods .................................................................................................................26
3.2.1 Generation of plasmid constructs ....................................................................26
3.2.2 Site-directed mutagenesis ...............................................................................26
3.2.3 Cell lines .........................................................................................................27
3.2.4 Retrovirus preparation ......................27
1 Contents


3.2.5 Transduction and transplantation of murine bone marrow ...............................27
3.2.6 Flow cytometric immunophenotyping (FACS analysis) ....................................28
3.2.7 Histopathology ................................................................................................28
3.2.8 Generation of stable cell lines .........................................................................28
3.2.9 Analysis of cell growth .....................................................................................29
33.2.10 [H]-thymidine incorporation .............29
3.2.11 Ubiquitination assays ......................................................................................29
3.2.12 Immunoprecipitation and Western blot analyses .............................................30
3.2.13 Internalization assays .......................30
3.2.14 Clonal growth in methylcellulose .....................................................................31
4 Results .........................................................................................................................32
4.1 Cbl mutants (Cbl-R420Q and Cbl-70Z) confer ligand-independent growth in
cooperation with Kit ..........................................................................................................32
4.1.1 Surface expression of Kit receptor ...................................................................32
4.1.2 Cbl mutants confer ligand-independent growth in association with Kit .............32
34.1.3 Enhanced [H]-thymidine incorporation in the presence of Cbl mutants and Kit
WT 33
4.1.4 Cbl mutants confer clonogenic growth in cooperation with Kit WT ...................34
4.2 Cbl mutants induce a generalized mastocytosis and a myeloproliferative disease in
a murine bone marrow transplantation model ...................................................................35
4.2.1 Cbl mutants induce hematologic disorder ........................35
4.2.2 Cbl-R420Q mutant induce myeloid leukemia ...................................................36
4.2.3 Cbl mutants induce a generalized mastocytosis ..............37
4.2.4 Granules in mast cells .....................................................................................39
4.3 Cbl mutants inhibit ubiquitination of Kit ...................................................................41
4.4 Cbl mutants inhibit internalization of ligand-activated Kit ........................................42
4.5 Cbl mutants associate with Kit ................................................................................42
4.6 Akt and Erk activation in the presence of Cbl mutants and Kit WT .........................43
4.7 Cbl mutants prolonged Kit mediated signaling ........................................................44
4.8 Kinase activity of Kit and Flt3 is dispensable for Cbl-70Z mediated transformation 45
4.9 Role of Akt in Cbl-70Z transformed Kit kinase dead cells .......................................46
4.9.1 Akt inhibition abolished transformation ............................................................47
4.9.2 Cbl-70Z do not confer autophosphorylation to kinase-dead (KD) receptors .....47
4.9.3 Kinase-dead Kit receptor does not undergo ubiquitination ...............................48
4.10 Src family tyrosine kinases (SFKs) are necessary for Cbl-70Z-mediated
transformation ..................................................................................................................49
4.10.1 Enhanced SFKs activity in the presence of Cbl mutants ..................................49
4.10.2 Constitutive association of Kit-KD, Cbl and SFKs ............49
4.10.3 SFK inhibitors inhibit SFK phosphorylation ......................................................50
4.10.4 Role of Fyn ......................................................................................................52
4.10.5 SFKs inhibition abolished Cbl-70Z transformation ...........52
2 Contents


4.10.6 Src inhibitors inhibit Akt pathway .....................................................................53
4.10.7 Kinase activity is required, but not essential ....................................................54
5 Discussion ................................................................56
6 Zusammenfassung .......................................................................67
7 References .....