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Proquest Dissertations The Role of Phosphatidylinositol Transfer Proteins in Phosphoinositide Signalling submitted by Siow Khoon Tan University College London as part of the requirements for the degree of Doctor of Philosophy University of London ProQuest Number: U644336 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U644336 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract The classical model of the phosphoinositide (PI) signalling pathway postulates that the substrate lipid, phosphatidylinositol (Ptdlns), is sequentially phosphorylated by PI kinases to form Ptdlns 4,5-bisphosphate (Ptdlns? 2), before being cleaved by phospholipase C (PLC) to generate two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol (DAG). Ptdlns is thought to be embedded in the membrane lipid bilayer throughout this process. However, several reports have strongly suggested that a Ptdlns transfer protein (PtdlnsTP) is not only essential but may also be a co-factor in this pathway. The experiments described in this thesis address a key aspect of this suggestion. The specific hypothesis to be tested is that the mammalian PtdlnsTPs PITPa and PITPp bind one or more Ptdlns kinases in order to present bound Ptdlns for phosphorylation. The results presented in this thesis provide evidence that Ptdlns bound to PITP is a direct substrate for PI kinases. Recombinant PITP was found to co-purify with PI kinases, which recognised and phosphorylated PITP-bound Ptdlns to produce Ptdlns 4-phosphate and PtdInsP 2 - Studies of a mutant PITP provided additional evidence for the above conclusion and also revealed that the co-purification of PITP and PI kinases was not dependent on the ability of PITP to bind Ptdlns. Liquid chromatographic analyses showed that PITP and PI kinases behaved as a complex. Furthermore, PLC and DAG kinase activities were also detected in the PITP-PI kinase complex. These studies provide strong support for the hypothesis that PITP is not only a component of a multi-enzyme signalling complex but that it also presents bound Ptdlns sequentially to the enzymes in a signalling cascade. Comparative studies were carried out on the two (alpha and beta) isoforms of PITP. Despite their different subcellular distribution, both isoforms were able to (i) transfer Ptdlns in vitro, (ii) reconstitute PLC signalling in permeabilised cells, and (iii) bind and present substrate to PI kinases in vitro. The biological significance of these results is discussed in the light of apparently complementary and conflicting studies by other groups. Acknowledgement Firstly, I would like to express my gratitude and sincere thanks to my thesis supervisor, Dr. Justin Hsuan, for the opportunity to work on this exciting project in his laboratory. He is a supportive and understanding mentor, who readily shares his knowledge and know­ how and is generous with his ideas, time and resources. I had a truly memorable time working with him. I would like to thank present and former members of the Hsuan laboratory, especially Shane Minogue, Damian Holliman, Maria dos Santos, Yvonne Fullwood, Mark Waugh, Nick Totty and Oanh Truong, for their help, advice, encouragement and generosity in sharing research materials. I also thank the staff and members of the Ludwig Institute for Cancer Research (LICR) for their help, support, advice, criticism, encouragement, patience, generosity and friendship during my stay at the institute. Many thanks to Prof. Shamshad Cockcroft (Dept, of Physiology, UCL) and her laboratory members (particularly, Geraint Thomas, Emer Cunningham and Andrew Ball) for their time, help, advice and generosity. I would also like to thank Drs. Peter Parker and Frank Cooke of the former Imperial Cancer Research Fund for access to his laboratory and for performing the lipid HPLC analyses, respectively. Last but not least, I would like to thank the LICR for its generous financial support. Table of Contents Abstract Acknowledgement 3 List of Tables 9 Abbreviations 10 Chapter 1 Introduction 1.1 Signal Transduction 12 1.2 Receptors 12 1.2.1 Epidermal Growth Factor Receptor 13 1.3 Signalling Pathways 18 1.4 Homology Domains 19 1.4.1 SH2 and Phosphotyrosine-binding Domains 19 1.4.2 SH3 and WW Domains 20 1.4.3 Pleckstrin Homology Domain 21 1.4.4 C2 Domain 22 1.4.5 PDZ Domain 22 1.4.6 FYVE Domain 22 1.4.7 ENTH Domain 23 1.4.8 PX Domain 23 1.5 Phosphoinositide Signalling Pathways 23 1.5.1 Phospholipase C 25 1.5.1.1 PLCÔ 27 1.5.1.2 PLCP 28 1.5.1.3 PLCy 29 1.5.1.4 PECs 33 1.5.2 Functional Roles of Phosphatidylinositol 4,5-bisphosphate 34 1.5.3 Phosphatidylinositol 4-Kinase 37 1.5.3.1 Structures and Characteristics 37 1.5.3.2 Regulations and Functions 42 1.5.4 Phosphatidylinositol Phosphate Kinase 45 1.5.4.1 Structures and Characteristics 45 1.5.4.2 Regulations and Functions 49 1.5.5 Phosphatidylinositol Transfer Protein 54 1.5.5.1 PITP 55 1.5.5.2 RdgB 59 1.5.5.3 Sec 14 61 1.5.5.4 Functions 62 1.6 Compartmentation of Signalling 68 Chapter 2 Materials and Methods 2.1 Molecular Biology 74 2.1.1 DNA Biochemistry 74 2.1.1.1 Polymerase Chain Reaction 74 2.1.1.1.1 Polymerase Chain Reaction Primers 75 2.1.1.2 Bacterial Transformation 77 2.1.1.3 Plasmid Preparation 78 2.1.1.4 DNA Digestion with Restriction Enzymes 79 2.1.1.5 Agarose Gel Electrophoresis of DNA 79 2.1.1.6 DNA Ligation 79 2.1.1.7 Automated DNA Sequencing 80 2.1.2 Protein Biochemistry 80 2.1.2.1 Polyacrylamide Gel Electrophoresis 80 2.1.2.1.1 Sodium Dodecyl Sulphate-PAGE 80 2.1.2.1.2 Native PAGE 80 2.1.2.1.3 Iso-Electric Focusing 81 2.1.2.2 Staining of Protein Gels 81 2.1.2.2.1 Coomassie Staining 81 2.1.2.2.2 Silver Staining 81 2.1.2.3 Semi-dry Electrophoretic Transfer of Protein 82 2.1.2.4 Western Blotting 82 2.1.2.5 Bradford Assay 82 2.1.3 Lipid Biochemistry 83 2.1.3.1 Separation of Inositol Phosphates 83 2.1.3.2 Identification of Inositol Phospholipids 83 2.1.3.2.1 Thin-layer Chromatography 83 2.1.3.2.2 Lipid Déacylation 84 2.2 Cell Culture 84 2.2.1 Insect Cell Culture 84 2.2.1.1 Routine Maintenance 84 2.2.1.2 Generation of Baculovirus Stock 85 2.2.1.2.1 Transfection of Sf9 cells 85 2.2.1.2.2 Plaque Assay 85 2.2.1.2.3 Virus Amplification 86 2.2.2 Mammalian Cell Culture 86 2.2.2.1 Routine Maintenance 86 2.2.2.2 In situ Immunofluorescence and Confocal Microscopy 87 2.3 Protein Expression and Purification 87 2.3.1 Expression System in Bacteria 88 2.3.1.1 Expression of Recombinant Proteins in co/z 88 2.3.1.2 Purification of Recombinant PITP 88 2.3.2 Expression System in Insect Cells 88 2.3.2.1 Expression of Recombinant Proteins in Sf9 Cells 88 2.3.2.2 Purification of PITP 89 2.3.3 Expression System in Mammalian Cells 89 2.3.3.1 Transfection of Cells 89 2.3.3.2 Immuno-affmity Purification of PITP 89 2.4 Assays for PITP, PLC and Lipid Kinases 90 2.4.1 PITP Assays 90 2.4.1.1 /« vzYro Ptdlns Transfer 90 2.4.1.1.1 Preparations of Reagents 90 2.4.1.1.2 Ptdlns Transfer Assay 91 2.4.1.2 Reconstitution of G-protein-regulated PLC Signalling 91 2.4.1.3 Reconstitution of EGF-dependent Phosphoinositide Kinases 92 2.4.2 Ptdlns 4-Kinase Assay 92 2.4.3 PLC Assay 92 2.4.3.1 PLC Assay using Exogenous Ptdlns ? 2 93 2.4.3.2 PLC Assay using Endogenous, Labelled Ptdlns ? 2 93 2.4.4 DAG Kinase Assay 93 Chapter 3 Reconstitution of Polyphosphoinositide Signalling by Both Isoforms of PITP 3.1 Introduction 94 3.2 Cloning and Expression of PITP 95 3.2.1 Cloning 96 3.2.1.1 Cloning Human PITPa 96 6 3.2.1.2 Cloning Rat PITPp 97 3.2.2 Expression and Purification of PITP 98 3.3 Functional Comparison of PITPa and PITPp 104 3.3.1 In vitro Ptdlns Transfer 104 3.3.2 Reconstitution of PI Signalling 106 3.3.2.1 Reconstitution in A431 Cells 106 3.3.2.2 Reconstitution in HL-60 Cells 109 3.4 Discussion 109 ipter 4 Presentation of Phosphatidylinositol to Lipid Kinases am Lipases by Phosphatidylinositol Transfer Proteins 4.1 Introduction 117 4.2 In vitro Interactions between PITP and Ptdlns 4-kinase 117 4.2.1 Interaction with Unpurified Ptdlns 4-kinase 117 4.2.2 Interaction with a Partially Purified Ptdlns 4-kinase 119 4.3 Co-purification of PI Kinases with PITP 120 4.3.1 Co-purification of Ptdlns 4-kinase Activity 120 4.3.2 Co-purification of PtdlnsP 5-kinase Activity 123 4.3.3 Confirmation of Lipid Identity 123 4.3.4 PITP-PI Kinase Interactions are Specific 126 4.4 PITP-bound Ptdlns as a Substrate 126 4.4.1 Phosphorylation by PI Kinases 126 4.4.2 Interactions are Independent of Lipid Type 131 4.5 Co-purification of DAG Kinase 131 4.6 Detection of Phospholipase Activity 133 4.7 Sf9 Cells as a Model of PITP Function 137 4.8 Expression of Recombinant PITPs in Cultured Mammalian Cells 137 4.8.1 Generation of New cDNAs and Preparations of Pure Plasmids 138 4.8.2 Mammalian Cell Transfection 138 4.8.2.1 COS-7 and A431 Cells 138 4.S.2.2 Human Embryonic Kidney Cells 139 4.8.3 Immunoprécipitation and PI Kinase Assays 141 4.8.4 Immunofluorescence Microscopy 144 4.8.4.1 Wildtype PITPa 144 4.8.4.2 Mutant PITPa 146 4.S.4.3 PITPp 146 4.8A 4 A33 148 4.9 Discussion 148 Chapter 5 Chromatographic and Electrophoretic Analyses of PITP and Co-purifying
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