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Phosphoproteomic Investigation of Differential Signalling Downstream of Class IA PI3K Isoforms

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Phosphoproteomic Investigation of Differential Signalling Downstream of Class IA PI3K Isoforms Phosphoproteomic Investigation of Differential Signalling Downstream of Class IA PI3K Isoforms PhD Thesis of Michael Hartley Walsh Principal Supervisor: Professor Bart Vanhaesebroeck Second Supervisor: Dr Pedro Cutillas Submitted in fulfilment of the requirements of the Degree of Doctor of Philosophy 1 Statement of Originality I, Michael Hartley Walsh, confirm that the research included within this thesis is my own work or that where it has been carried out in collaboration with, or supported by others, that this is duly acknowledged below and my contribution indicated. Previously published material is also acknowledged below. I attest that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge break any UK law, infringe any third party’s copyright or other Intellectual Property Right, or contain any confidential material. I accept that the College has the right to use plagiarism detection software to check the electronic version of the thesis. I confirm that this thesis has not been previously submitted for the award of a degree by this or any other university. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. Signature: M. H. Walsh Date: 22/11/2014 2 Acknowledgements I must first thank Bart Vanhaesebroeck for giving me the opportunity to work in an environment which engenders personal growth and independence, and on projects which hold such interest and importance. The knowledge and guidance of my second supervisor Pedro Cutillas have been supremely useful, and I wish to thank him for always being there when called upon. The Centre for Cell Signalling has been a wonderful place to work, and everyone in it past and present has played a part in getting me to where I am. Ardent thanks go to Larissa for doing much more than she ever needed to, and to Claire for her support in some of the hardest times. I thank Salma for her work from which much of this project sprang, and for her help throughout. Ed, Michalina, Aine and Alex P have provided much needed material and moral support which has been indispensable. Thanks also to Alex M, Alex S, Benoit, Daniele, Ellie, Ezra, Inma, J.C., Khaled, Luisa, Maria, Pedro Casado, Regine, Roberto, Samira, Sandra, Veronica, Vinni, Wayne and York for their help along the way. I would like to thank the Biotechnology and Biological Sciences Research Council and AstraZeneca for the funding which allowed the project and my PhD to happen. I am grateful to Kevin Hudson for his discussions, and to the graduate tutors who have helped my progression. I am especially grateful to the BBSRC and the Society of Biology for providing the means and opportunity for me to explore a non-academic path, and to Bart for recognising my need for this, and for his support in granting me the freedom to pursue it. Final, great, and sincere thanks go to my parents and family, whose limited knowledge of my project has never precluded concern, enquiry, or encouragement. 3 Abstract The PI3K family is central to numerous cellular processes in both health and disease. The class IA isoforms of PI3K control such outputs as proliferation, metabolism and survival through their well-characterised function as lipid kinases, with their signalling thought to predominantly mediated by the Akt/PKB protein kinase. However there exist other signalling routes, including from the lipid kinase activity through other effectors, but also through a protein kinase function of the class IA isoforms themselves. Mass spectrometry is a powerful tool which has been central to the recent advances in phosphoproteomic techniques. It is now possible to use mass- spectrometry to probe the phosphoproteome of any number of systems in an unbiased and global manner. In this project, we aimed to advance our understanding of two aspects of class IA PI3K signalling which are relatively poorly understood. We used phosphoproteomic techniques which allowed us to provide answers to some old questions which have up to now proved elusive. First, we investigated the protein kinase activity of p110α. We used an in vitro protein kinase assay and coupled this to mass spectrometry techniques to identify direct substrates of p110α. We proposed two novel protein substrates and attempted to characterise them further, although we were hampered by lack of available biochemical tools. Second, we investigated the differential phosphoproteomes of the ubiquitously expressed class IA PI3K isoforms p110α and p110β in a panel of breast cancer cell lines. We used mass spectrometry-based phosphoproteomics and found significant differences in signalling between p110α and p110β in 4T1 cells, including differential regulation of previously described PI3K effectors, amongst them the Akt substrate PRAS40, and potential novel targets. Additionally, we found that some of these effects were conserved between cell lines. 4 Table of Contents Statement of Originality……………………………………………………………..2 Acknowledgements………………………………………………………………….3 Abstract………………………………………………………………………………..4 Table of Contents…………………………………………………………………….5 Abbreviations…………………………………………………………………………9 1. Introduction .............................................................................................. 12 1.1. Phosphoinositide 3-kinases (PI3Ks) ............................................................. 12 1.1.1. PI3K classification ................................................................................. 12 1.1.2. Class I PI3K structure ............................................................................ 14 1.1.3. Signalling upstream of class I PI3K........................................................ 16 1.1.4. Signalling downstream of class I PI3K ................................................... 18 1.1.5. Tools for studying PI3K function ............................................................ 35 1.1.6. Isoform-specific functions of class IA PI3K ............................................ 36 1.1.7. Disease ................................................................................................. 39 1.1.8. Pharmacological Intervention ................................................................. 41 1.2. Proteomics ................................................................................................... 45 1.2.1. Post-translational modifications ............................................................. 46 1.2.2. Instrumentation ...................................................................................... 51 1.2.3. Quantification ........................................................................................ 53 1.2.4. Validation and characterisation of phosphosites .................................... 57 1.3. Aims ............................................................................................................. 59 2. Materials and Methods ............................................................................. 61 2.1. Antibodies ..................................................................................................... 61 2.2. Other materials and reagents ....................................................................... 61 5 2.3. Cell culture ................................................................................................... 63 2.3.1. Cell lines ................................................................................................ 63 2.3.2. Growth maintenance ............................................................................. 64 2.3.3. Inhibitor treatments ................................................................................ 64 2.4. Cell lysate preparation .................................................................................. 65 2.4.1. Cell lysis ................................................................................................ 65 2.4.2. Determination of protein concentration .................................................. 65 2.5. Sodium dodecyl sulphate polyacrylamide gel electrophophoresis (SDS-PAGE) 66 2.5.1. Western Blotting .................................................................................... 66 2.5.2. Staining ................................................................................................. 67 2.6. In vitro protein kinase assay ......................................................................... 67 2.6.1. With recombinant proteins ..................................................................... 67 2.6.2. With peptides ......................................................................................... 67 2.7. Preparation of samples for MS ..................................................................... 68 2.7.1. In vitro protein kinase assay for MS ....................................................... 68 2.7.2. In-solution digestion ............................................................................... 68 2.7.3. Desalting ............................................................................................... 69 2.7.4. Phosphopeptide enrichment .................................................................. 69 2.8. Liquid chromatography mass spectrometry .................................................. 69 2.9. Bio-informatic analysis .................................................................................. 71 2.9.1. Peptide production ................................................................................
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