Proteomic analysis of leukaemogenic protein tyrosine kinase action

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Proteomic analysis of leukaemogenic protein tyrosine kinase action

François Griaud

Doctor of Philosophy (PhD)

2012-01

The University of Manchester

275

LIST OF CONTENTS ......................................................................................................................2LIST OF FIGURES ...........................................................................................................................6LIST OF TABLES .............................................................................................................................9LIST OF APPENDICES ...................................................................................................................9ABSTRACT ....................................................................................................................................10DECLARATION............................................................................................................................11COPYRIGHT STATEMENT ........................................................................................................11LIST OF ABBREVIATIONS ........................................................................................................12AMINO ACID CODE AND ISOTOPIC MASSES ..................................................................20ACKNOWLEDGEMENTS ...........................................................................................................21DEDICATION ...............................................................................................................................22CHAPTER 1. INTRODUCTION .................................................................................................231.1. GENERAL INTRODUCTION ............................................................................ 231.2. HAEMOPOIESIS ............................................................................................. 241.3. LEUKAEMOGENIC PROTEIN TYROSINE KINASES AND RELATED DISEASES ....... 271.3.1. BCR/ABL and chronic myeloid leukaemia (CML) ............................................... 291.3.2. JAK2 mutants and Ph-negative myeloproliferative diseases (MPDs)................... 321.3.3. FLT3 and acute myeloid/lymphoid leukaemia (AML/ALL) .................................. 331.3.4. KIT mutants and acute myeloid leukaemia (AML) .............................................. 331.3.5. TEL/PDGFRβ and chronic myelomonocytic leukaemia (CMML) ........................ 341.3.6. NPM/ALK and anaplastic large cell lymphoma (ALCL) ..................................... 341.3.7. Cell model for the study of leukaemogenic protein tyrosine kinases (PTKs) ......... 351.3.8. Conclusion ......................................................................................................... 351.4. EXCESSIVE PRODUCTION OF REACTIVE OXYGEN SPECIES (ROS) AND ITS CONSEQUENCES ON CELL REGULATION AND DNA INTEGRITY ................................. 361.4.1. Production and function of ROS in haemopoietic cells ........................................ 361.4.2. ROS are a source of DNA damage ...................................................................... 371.4.3. Antioxidant mechanisms in haemopoietic cells .................................................... 381.4.4. Oxidative stress by leukaemogenic protein tyrosine kinases (PTKs) .................... 411.4.5. Conclusion ......................................................................................................... 431.5. DNA DOUBLE STRAND BREAK SENSING/REPAIR SIGNALLING ........................ 451.5.1. DNA double strand breaks .................................................................................. 451.5.2. Response to DNA double strand breaks .............................................................. 461.5.3. DNA double strand break repair processes .......................................................... 551.5.4. Misregulation of the DNA damage response signalling and stimulation of unfaithful DNA double strand break repair processes by leukaemogenic PTKs ................ 581.6. APPROACHES AND TECHNOLOGIES IN PROTEOMICS ...................................... 6431.6.1. Approaches to the proteome definition ................................................................ 651.6.2. Principle of mass spectrometry ........................................................................... 671.6.3. Sources in mass spectrometry ............................................................................. 681.6.4. Analysers in mass spectrometry.......................................................................... 701.6.4.a. Quadrupole analyser ........................................................................................701.6.4.b. Ion trap analyser ...............................................................................................721.6.4.c. Time-of-flight (TOF) analyser .........................................................................751.6.4.d. Fourier transform ion cyclotron resonance (FTICR) analyser ....................761.6.4.e. Orbitrap analyser ..............................................................................................791.6.5. Detectors in mass spectrometry .......................................................................... 801.6.6. Tandem mass spectrometry ................................................................................. 811.6.7. Sequence databases and proteomic software ........................................................ 841.7. STRATEGIES FOR PHOSPHOPROTEOMICS ........................................................ 861.7.1. Function, control and identification of protein phosphorylation .......................... 861.7.2. Phosphoprotein and phosphopeptide immunoprecipitation .................................. 881.7.3. Ion metal affinity chromatography (IMAC) ........................................................ 891.7.4. TiO2 and ZrO2 affinity chromatography .............................................................. 891.7.5. Sequential elution from IMAC (SIMAC) ............................................................ 901.7.6. Strong cation exchange chromatography (SCX) .................................................. 901.7.7. Phosphosite tagging by chemical derivatization .................................................. 911.7.8. Experimental approaches for dissecting functional roles of protein phosphorylation . 911.8. STRATEGIES FOR QUANTITATIVE PROTEOMICS .............................................. 921.8.1. Strategies for label free quantification ................................................................. 931.8.2. Isotope-coded affinity tags (ICAT) ...................................................................... 931.8.3. Stable isotope labelling by amino acids in cell culture (SILAC) ........................... 941.8.4. Isobaric tags for relative and absolute quantification (iTRAQ™) ........................ 951.8.5. Absolute quantification in peptide mass spectrometry (AQUA™) ...................... 981.9. AIMS AND OBJECTIVES ................................................................................ 100CHAPTER 2. MATERIALS AND METHODS .......................................................................1012.1. CELL CULTURE ........................................................................................... 1012.1.1. Ba/F3 cell culture ............................................................................................. 1012.1.2. Ba/F3 cell stable gene transfection .................................................................... 1012.1.3. Ba/F3 cell transient gene transfection ............................................................... 1022.1.4. Preparation of Lin- cells and mast cells (CD45 WT/null).................................. 1032.1.5. Human primary cell culture ............................................................................. 1042.1.6. K562 cell culture .............................................................................................. 1042.1.7. Chemicals and inhibitors .................................................................................. 1042.1.8. γH2AX assay ................................................................................................... 1052.1.9. Apoptosis assay ................................................................................................ 1062.1.10. Flow cytometry analysis of Thoc5/Fmip tyrosine phosphorylation .................... 1062.1.11. Immunofluorescence of Thoc5/Fmip tyrosine phosphorylation .......................... 1072.2. SAMPLE PREPARATION FOR PROTEIN AND PROTEOMIC ANALYSIS ............... 1072.2.1. Whole cell lysis ................................................................................................. 1072.2.2. Nuclear preparation .......................................................................................... 10842.2.3. Protein assay .................................................................................................... 1082.2.4. Western blot analysis ....................................................................................... 1092.2.5. Study of the etoposide-induced genotoxic stress with iTRAQ™ peptide labelling ... 1102.2.6. Phosphopeptide enrichment with TiO2 affinity chromatography ........................ 1112.2.7. Phosphopeptide fractionation with high-pH reversed phase (RP) chromatography.. 1112.3. MASS SPECTROMETRY ................................................................................. 1122.3.1. Study of the etoposide-induced genotoxic stress ................................................ 1122.3.2. Data analysis .................................................................................................... 1132.3.3. Validation with selected reaction monitoring (SRM) ........................................ 1152.4. STATISTICAL METHODS ............................................................................... 1162.4.1. Parametric tests ................................................................................................ 1162.4.2. Non-parametric tests ........................................................................................ 1172.4.3. Standard error of the mean ............................................................................... 118CHAPTER 3. SITE-SPECIFIC QUANTITATIVE PHOSPHOPROTEOMIC ANALYSIS OF BCR/ABL PTK CROSSTALK ON ETOPOSIDE-INDUCED DNA DAMAGE RESPONSE IDENTIFIES A SYNERGISTIC PHOSPHORYLATION CHANGE ON DEVELOPMENTAL PROTEIN HEMOGEN..........................................................................1193.1. INTRODUCTION .......................................................................................... 1193.2. RESULTS ..................................................................................................... 1203.2.1. Induction of DNA damage and DNA repair with etoposide .............................. 1203.2.2. Phosphoproteomic analysis of etoposide-induced events in the presence and absence of BCR/ABL PTK ............................................................................................. 1273.2.3. γH2AX, ATM and 53BP1 respond to etoposide ................................................ 1333.2.4. BCR/ABL PTK signalling acts on proteins within multiple pathways .............. 1373.2.5. Phosphosite-specific overlap of etoposide and BCR/ABL PTK signalling ........... 1413.2.6. Validation of Hemogen phosphorylation on serine 380 with AQUA™ .............. 1583.2.7. Pathway analysis pinpoints the B-WICH and NF-κB complexes as potential mediators of BCR/ABL PTK-mediated genomic instability............................................ 1663.3. DISCUSSION ................................................................................................ 171CHAPTER 4. A SIGNALLING PATHWAY FROM RECEPTOR ACTIVATION AND OXIDATIVE STRESS TO THO COMPLEX PHOSPHORYLATION POTENTIATED BY LEUKAEMOGENIC PROTEIN TYROSINE KINASES AND STEM CELL HOMING CHEMOKINES .............................................................................................................................1824.1. INTRODUCTION .......................................................................................... 1824.2. RESULTS ..................................................................................................... 1854.2.1. Structure/function analysis on NPM/ALK reveals the key residues for activation of Thoc5/Fmip tyrosine phosphorylation ....................................................................... 1854.2.2. Thoc5/Fmip phosphorylation on tyrosine 225 is increased by oxidative stress ... 1874.2.3. Nucleocytoplasmic distribution of Thoc5/Fmip tyrosine phosphorylation .......... 1904.2.4. Thoc5/Fmip tyrosine phosphorylation relies on proto-oncogene Src .................. 1944.2.5. CD45/Src signalling pathway is upstream of Thoc5/Fmip tyrosine phosphorylation ............................................................................................................ 2004.2.6. THOC5/FMIP tyrosine phosphorylation is elevated in primary CML stem cells .... 20354.2.7. Thoc5/Fmip phosphorylation on tyrosine 225 in mediation of apoptosis in response to oxidative stress ......................................................................................................... 2074.2.8. Thoc5/Fmip tyrosine phosphorylation does not mediate protein levels of Hsp40 and Hsp70 in response to etoposide and H2O2 ............................................................... 2094.3. DISCUSSION ................................................................................................ 213CHAPTER 5. GENERAL DISCUSSION, FUTURE WORK AND CONCLUSION .........2205.1. GENERAL DISCUSSION ................................................................................ 2205.2. FUTURE WORK ............................................................................................ 2245.3. CONCLUSION .............................................................................................. 226CHAPTER 6. REFERENCES .......................................................................................................227CHAPTER 7. APPENDICES ......................................................................................................2627.1. γH2AX ASSAY WITH FLOW CYTOMETRY ..................................................... 2627.2. APOPTOSIS ASSAY WITH FLOW CYTOMETRY ................................................ 2637.3. COMPARISON BETWEEN QSTAR® XL AND QSTAR® ELITE ....................... 2657.4. PHOSPHOENTITIES POTENTIALLY REGULATED BY ETOPOSIDE AND/OR BCR/ABL PTK ...................................................................................................... 2667.5. PHOSPHOENTITIES OF THO COMPLEX MEMBERS IN THE PRESENCE OF BCR/ABL PTK AND ETOPOSIDE ............................................................................ 2747.6. CONTROLS FOR ABSOLUTE QUANTIFICATION (AQUA™) OF HEMOGEN PHOSPHORYLATION ON SERINE 380 ....................................................................... 275

Introduction: Chronic myeloid leukaemia is a blood cancer which progresses from a chronic phase to an acute blast crisis if untreated. Disease progression and treatment resistance may be precipitated by the mutator action of BCR/ABL protein tyrosine kinase (PTK), but only few protein phosphosites involved in the DNA damage response have been investigated with respect to BCR/ABL action.Aim: The aim of this PhD project was to demonstrate that BCR/ABL PTK expression can affect the response to genotoxic stress signalling at the protein phosphorylation level.Methodology: Etoposide-induced DNA damage response has been studied in control and BCR/ABL PTK-expressing Ba/F3 cells using apoptosis and γH2AX assays. Quantitative phosphoproteomics was performed with iTRAQ™ peptide labelling to discover putative modulated phosphorylation sites. Absolute quantification (AQUA™) performed with selected reaction monitoring was used to validate discovery phosphoproteomics. The effect of genotoxic stress on the THO complex protein Thoc5/Fmip was studied using western blots.Results: The expression of BCR/ABL PTK induced γH2AX phosphorylation after etoposide exposure. This was associated with the modulation of H2AX tyrosine 142 phosphorylation, MDC1 (serines 595 and 1053) and Hemogen serine 380 phosphorylation among proteins regulated by both BCR/ABL PTK and etoposide. We identified that leukaemogenic PTKs mediate Thoc5/Fmip phosphorylation on tyrosine 225 via Src proto-oncogene and oxidative stress, while ATM and MEK1/2 may control its phosphorylation. Human CD34+ CD38- leukaemic stem cells showed pronounced level of THOC5/FMIP tyrosine phosphorylation. Expression of phosphomutant Thoc5/Fmip Y225F might reduce apoptosis mediated by etoposide and H2O2.Conclusion: BCR/ABL PTK can sustain, create, block and change the intensity of protein phosphorylation related to genotoxic stress. Modulation of H2AX, MDC1, Hemogen and Thoc5/Fmip post-translational modifications by BCR/ABL PTK might promote unfaithful DNA repair, genomic instability, anti-apoptotic signalling or abnormal cell differentiation, resulting in leukaemia progression.

• Chronic Myeloid Leukaemia
• BCR/ABL
• Quantitative Phosphoproteomics
• 8-plex iTRAQ
• TiO2
• Phosphosites
• H2ax
• Hemogen
• Mdc1
• Thoc5
• Src

• Griaud, Francois

uk-ac-man-scw:185184

Griaud, Francois

15th January, 2013, 19:28:34

Griaud, Francois

12th February, 2013, 19:28:59