Proteomic identification of {C-EBPα [C-EBR-alpha] multiprotein complex reveals that JNK1, an activator of {C-EBPα [C-EBR-alpha] is downregulated in patients with acute myeloid leukemia (AML) [Elektronische Ressource] / vorgelegt von Arun Kumar Trivedi

Arun Kumar Trivedi

Description

Aus der Medizinischen Klinik und Poliklinik III, der Ludwig-Maximilians-Universitäts München, Direktor: Prof. Dr. med. Wolfgang Hiddemann Proteomic identification of C/EBPα multiprotein complex reveals that JNK1, an activator of C/EBPα is downregulated in patients with acute myeloid leukemia (AML) Dissertation zum Erwerb des Doktorgrades der Humanbiologie an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München Vorgelegt von Arun Kumar Trivedi, M.Sc. aus Obra Sonbhadra/Indien München 2006 From the Department of Internal Medicine III, Ludwig-Maximilians-University, Munich Chair: Prof. Dr. med. Wolfgang Hiddemann Proteomic identification of C/EBPα multiprotein complex reveals that JNK1, an activator of C/EBPα is downregulated in patients with acute myeloid leukemia (AML) Thesis Submitted for a Doctoral degree in Human Biology, at the Faculty of Medicine Ludwig-Maximilians-University, Munich Submitted by Arun Kumar Trivedi, M.Sc. From Obra Sonbhadra/India München 2006 2Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatter: Prof. Dr. med. W. Hiddemann Mitberichterstatter: Priv. Doz. Dr. P. Kahle Prof. Dr. W. Machleidt Mitbetreuung durch den promovierten Mitarbeiter: Priv. Doz. Dr. Gerhard Behre Dekan: Prof. Dr. med. D.

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 54
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait





Aus der Medizinischen Klinik und Poliklinik III, der Ludwig- Maximilians-Universitäts München, Direktor: Prof. Dr. med. Wolfgang Hiddemann

  Proteomic identification of C/EBPαmultiprotein complex reveals that JNK1, an activator of C/EBPα is downregulated in patients with acute myeloid leukemia (AML)        

      

 

 taerssDionti  zum Erwerb des Doktorgrades der Humanbiologie

an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München

Vorgelegt von Arun Kumar Trivedi, M.Sc. aus Obra Sonbhadra/Indien München 2006

From the Department of Internal Medicine III, Ludwig-Maximilians-University, Munich Chair: Prof. Dr. med. Wolfgang Hiddemann

   Proteomic identification of C/EBPαmultiprotein complex reveals that JNK1, an activator of C/EBPα is downregulated in patients with acute myeloid leukemia (AML)   

 













Thesis Submitted for a Doctoral degree in Human Biology, at the Faculty of Medicine Ludwig-Maximilians-University, Munich

Submitted by Arun Kumar Trivedi, M.Sc. From Obra Sonbhadra/India München 2006



2



Mit Genehmigung der Medizinischen Fakultät

Berichterstatter:



 Mitberictatsreth:ret

der Universität München

 Prof. Dr. med. W. Hiddemann

Priv. Doz. Dr. P. Kahle

 Prof. Dr. W. Machleidt





Mitbetreuung durch den

promovierten Mitarbeiter:



Dekan:







Priv. Doz. Dr. Gerhard Behre

 Prof. Dr. med. D. Reinhardt

Tag der mündlichen Prüfung: 15.02.2006











3



With permission from the Faculty of Medicine

 University of Munich

Supervisor/Examiner:



Prof. Dr. med. W. Hiddemann

 Co-Examiners: Priv. Doz. Dr. P. Kahle

 Prof. Dr. W. Machleidt





Co-Supervisor:



Dean:













Date of Submission:





Priv. Doz. Dr. Gerhard Behre

Prof. Dr. med. D. Reinhardt

28.09.2005

 Date of Oral Exam: 15.02.2006      





4

               

                        



Dedicated to my esteemed father Late Mr. Sita Ram Tiwari

5

Abbreviations:  AML ALL APL β-ME BR-LZ CLP CHAPS C/EBP

CML CK CHX 2 DE DTE DTT DHB DMEM DMSO EDTA EGTA

FAB GMPs

GST GCSF GCSFr HSC HA  IB



Acute Myeloid Leukemia Acute Lymphoid Leukemia Acute Promyelocytic Leukemia β-Mercaptoethanol Basic Region-Leucine Zipper Common Lymphoid Progenitor 3-[(3-Cholamidopropyl) Dimethylammonio]-1-Propanesulfonate CCAAT Enhancer Binding Protein Chronic Myeloid Leukemia Complex Karyotype Cycloheximide 2 Dimensional Gel Electrophoresis Dithioerythritol Dithiothreitol 2,5-Dihydroxy-Benzoicacid Dulbecco´s Modified Eagle Medium Dimethylsulfoxide Ethylenediamine Tetra-Acetic Acid Ethylene Glycol bis (2-aminoethyl ether)-N,N,N'N'-Tetra Acetic Acid French American British Classification

Granulocyte/Macrophage Progenitors Glutathione-S-Transferase Granulocyte Colony Stimulating Factor Granulocyte Colony Stimulating Factor Receptor Haematopoietic Stem Cell Hemeagglutinin  Immunoblot

6



µg



µl

MEPs

MAPK



IEF

µCi



MS





NP40

NK

ng

IvtIn-vitro Translated

JNK c-Jun NH 2 -Terminal Kinase JNK* Kinase dead c-Jun NH 2 -Terminal Kinase (mutant JNK) MALDI Matrix Assisted Laser Desorption Ionisation- Time of Flight

Megakaryocyte/Erythroid Progenitors

Micro Curie

Nonidet P-40

Mass Spectrometry

TOF

UVB

IPTG

Ip

Micrograms

Microlitres

Mitogen Activated Protein Kinase

Isoelectric Focussing

Isopropyl-beta-D-Thiogalactopyranoside

Immunoprecipitation

7

Room Temperature

Ultra Violet Radiation B

Sodium Dodecyl Sulphate

Revolutions per Minute

World Health Organization

Nanogram

Time of Flight

Tris-Borate EDTA

Transactivation Elements

Transactivation Domain

Normal Bone Marrow

Peptide Mass Fingerprinting Isoelectric Point Radioimmunoprecipitation

PMF



pI

RIPA





WHO

nBM

TE

TBE

TAD

Normal Karyotype



RT

Rpm

SDS

Table of Contents:  1. Introduction:....................................................................................................10 1.1 Haematopoiesis: .................................................................................................10 1.2 Transcription factors involved in normal myelopoiesis ............................................. 11  1.3 Acute Myeloid Leukemia .....................................................................................12 1.4 Role of C/EBPαin myelopoiesis ...........................................................................15 1.5 C/EBPαinactivation in acute myeloid leukemia: .....................................................20 1.6 c-Jun NH 2 -terminal kinase1 (JNK1) ......................................................................23 1.6.1 JNK signalling in human leukemia and tumour development ..................................24 1.6.2 Stress activated kinases regulate protein stability ..................................................26 1.7 Proteomics ........................................................................................................27 1.8 Aim of the study .................................................................................................29 2. Materials and Methods....................................................................................30 2.1 Materials .........................................................................................................30 2.1.1 Mammalian Cell Lines ......................................................................................30 2.1.2 Cell Culture ....................................................................................................30 2.1.3 Immunoblots ...................................................................................................30 2.1.4 Antibodies ......................................................................................................31 2.1.5 2D-gel Electrophoresis ......................................................................................31 2.1.6 Peptides Extraction ..........................................................................................31 2.1.7 Mass Spectrometry: ..........................................................................................32 2.1.8 Chemicals .......................................................................................................32 2.2 Methods: ..........................................................................................................33 2.2.1 GST-Purification .............................................................................................33 2.2.2 GST-Pull Down ...............................................................................................34 2.2.3 2D-Gel Electrophoresis .....................................................................................34 2.2.4 MALDI-TOF Mass Spectrometry .......................................................................35 2.2.5 Plasmid Constructs ..........................................................................................35 2.2.6 Cell Culture ....................................................................................................36 2.2.7 Co-Immunoprecipitation ...................................................................................36 2.2.8In-vitroKinase Assay .......................................................................................36 2.2.9 Identification of Phosphopeptides by Mass Spectrometry .......................................37 2.2.10 Electrophoretic Mobility Shift Assay (EMSA) ....................................................38 2.2.11 Transient Transfections using LipofectAMINE Plus and Reporter Assays Firefly and Renilla Luciferase ....................................................................................................38 2.2.12In-vivoHA Ubiquitination Assay ......................................................................39 2.2.13 Pulse Chase Labelling .....................................................................................39 2.2.14 Western Blotting ............................................................................................40 3. Results:............................................................................................................40 3.1 Purification of GST fusion constructs ....................................................................40 3.2 2D Gel Electrophoresis of differentially interacting proteins .....................................42 3.3 JNK1 physically interacts with C/EBPαin-vitro .....................................................43 3.4 JNK1 physically interacts with C/EBPαin-vivo ......................................................44



8

3.5 JNK1 phosphorylates C/EBPαin-vitro ..................................................................45 3.6 Phosphopeptide analysis of immunoprecipitated C/EBPαfrom transiently cotransfected 293T with MKK7 confirms presence of one phophopeptide ...........................................47 3.7 JNK targets ubiquitination of C/EBPα: ..................................................................50 3.8 Anisomycin induced activation of JNK1 enhances C/EBPαprotein half life as compared to uninduced cells in pulse chase labelling assay ...........................................................51 3.9 Induced activation of JNK enhances C/EBPαprotein expression in Western blot analysis .............................................................................................................................52 3.10 MEKK1 cotransfection with C/EBPαleads to accumulation of C/EBPα . ..................54 3.11 Activated JNK1 enhances ability of C/EBPαto transactivate the minimal TK promoter driven by C/EBP DNA binding sites p(C/EBP) 2TK .....................................................55 3.12 JNK1 enhances C/EBPαDNA binding activity in Electrophoretic Mobility Shift Assay (EMSA): ................................................................................................................56 3.13 JNK1 mRNA expression is downregulated in AML subtypes ..................................57 3.14 Phospho JNK1 expression and its kinase activity is reduced in AML subtypes ...........59 4. Discussion.......................................................................................................60 5. Summary.........................................................................................................67 6.Zusammenfassung...70 7. References...72 8. Acknowledgement...........................................................................................83 9. Lebenslauf.......................................................................................................86 10.Appendix....90                         



9