AN INVESTIGATION OF PHOSPHO-REGULATION AND THE ROLE CTP:PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE α IN PROMOTING RAS-INDUCED MALIGNANT TRANSFORMATION OF INTESTINAL EPITHELIAL CELLS by Michael J. McPhee Submitted in partial fulfilment of the requirements for the degree of Master of Science at Dalhousie University Halifax, Nova Scotia August 2018 © Copyright by Michael J. McPhee, 2018 DEDICATION PAGE I want to dedicate this thesis to my mother Beverly, my sister Rachel, and to my brother Chris for all their love and encouragement over the years. I cannot imagine completing this work without their support. I also want to dedicate this thesis to my loved ones who have suffered from cancer: my amazing grandmother Myrna McPhee, Roy Fletcher, Everett Lavender, and to my late father, Clyde, who passed away from melanoma in July of 2014. This work is also dedicated to my many talented friends who have created a wonderful community here in Halifax and Dartmouth for artists and musicians. ii TABLE OF CONTENTS LIST OF FIGURES ............................................................................................................................... v ABSTRACT .......................................................................................................................................... viii LIST OF ABBREVIATIONS USED ............................................................................................ ix ACKNOWLEDGEMENTS ............................................................................................................ xii CHAPTER 1: INTRODUCTION ................................................................................................... 1 1.1 Project overview .......................................................................................................................... 1 1.2 Phosphatidylcholine ................................................................................................................... 2 1.2.1 Overview of phosphatidylcholine structural properties and biosynthesis .................................. 2 1.2.2 Phosphatidylcholine function in membrane biogenesis ............................................................. 5 1.2.3 Phosphatidylcholine as a substrate for lipid second messengers and inflammatory mediators .. 7 1.3 The CDP-choline pathway ................................................................................................... 13 1.3.1 Choline transporters ................................................................................................................ 13 1.3.2 Choline kinase ......................................................................................................................... 14 1.3.3 CTP:phosphocholine cytidylyltransferase ............................................................................... 17 1.3.4 CCTα regulation is correlated with dephosphorylation ............................................................ 19 1.3.5 Transcriptional regulation of CCTα ........................................................................................ 22 1.3.6 Choline/choline-ethanolamine phosphotransferase ................................................................. 24 1.3.7 The role of the CDP-choline pathway in cancer ..................................................................... 24 1.3 Autophagy ................................................................................................................................... 27 1.3.1 Overview of the autophagy pathway ....................................................................................... 27 1.3.2 The role of phospholipids in the autophagy pathway .............................................................. 29 1.4 ER Stress Pathways ................................................................................................................ 33 1.4.1 The unfolded protein response ................................................................................................ 33 1.4.2 The Keap1-Nrf2 pathway ........................................................................................................ 34 1.5 Research objectives and rationale for investigation .................................................. 35 CHAPTER 2: Materials and Methods ......................................................................................... 37 2.1 Materials ..................................................................................................................................... 37 2.2 Cell culture .................................................................................................................................. 38 2.3 Plasmid transfection of HeLa cells ................................................................................... 38 2.4 Retroviral and lentiviral production and cell transduction .................................... 39 iii 2.5 SDS-PAGE and immunoblotting of cell lysates ........................................................... 40 2.6 Immunofluorescence ............................................................................................................... 41 2.7 Measuring autophagic flux in IEC-ras34 ....................................................................... 42 2.8 Measuring ER stress in IEC-ras cultured on agarose ............................................... 42 2.9 Oleate treatment of cultured cells ..................................................................................... 43 2.10 Statistical analysis ................................................................................................................. 43 CHAPTER 3: Results ......................................................................................................................... 45 3.1 Using phospho-specific antibodies to monitor CCTα phosphorylation ............. 45 3.1.1 Oleate induces dephosphorylation of CCTα at Ser315/Ser319 but not Tyr359/Ser362 ........... 45 3.1.2 Choline depletion induces dephosphorylation of CCTα at Ser315/Ser319 in IEC-ras ............. 58 3.1.2 3 CCTα is phosphorylated at Ser315/Ser319 in IEC-ras grown detached from the ECM ...... 64 3.2 The role of PC biosynthesis in promoting cancer cell survival ............................. 68 3.2.1 Choline depletion does not cause p62 or LC3b-II accumulation in HCT116 cells ................. 68 3.2.2 Choline depletion does not affect autophagic flux in IEC-ras ................................................. 68 3.2.3 Choline depletion and CCTα knockdown does not induce the UPR through PERK- eIF2α- ATF4-CHOP in IEC-ras ........................................................................................................... 71 3.2.4 Choline depletion does not induce the Keap1-Nrf2 pathway in IEC-ras ................................. 76 CHAPTER 4: Discussion ................................................................................................................... 79 4.1 CCTα activity is correlated with phosphorylation at Ser315/Ser319 .................. 79 4.2 Choline depletion and CCTα knockdown induce p62 accumulation by an unknown mechanism .............................................................................................................. 86 4.3 Conclusions ................................................................................................................................. 87 REFERENCES ...................................................................................................................................... 88 iv LIST OF FIGURES Figure 1.1. PC structure and placement in lipid bilayers. ..............................................3 Figure 1.2 The CDP-choline pathway. ..........................................................................4 Figure 1.3 Phospholipase cleavage sites on PC. ...........................................................8 Figure 1.4 Representation of CCTα structure, membrane binding capability, and P region residues. .......................................................................................18 Figure 1.5 The autophagy pathway .............................................................................28 Figure 3.1 Target residues of anti-pCCT antibodies raised against p-Ser315/Ser319 and p-Tyr359/Ser362. ....................................................46 Figure 3.2 Anti-p-Ser315/Ser319 but not anti-p-Tyr359/Ser362 detects both overexpressed CCT isoforms in HeLa cells. .............................................47 Figure 3.3 Anti-p-Ser315/Ser319 and anti-p-Tyr359/Ser362 detect endogenous CCT isoforms in Caco2 and Caco2 CCTαKO cells. .................................48 Figure 3.4 Oleate treatments induce dephosphorylation of Ser315/Ser319 but not Tyr359/Ser362 in HeLa cells. ...................................................................50 Figure 3.5 Oleate treatment induces dephosphorylation of Ser315/Ser319 but not Tyr359/Ser362 in F8 human fibroblasts. ................................................51 Figure 3.6. Oleate treatment induces dephosphorylation of Ser315/Ser319 but not Tyr359/Ser362 in Caco2 cells ...................................................................52 Figure 3.7 Oleate induces translocation of CCTα in HeLa cells. ................................53 Figure 3.9 Oleate-induced translocated CCTα is dephosphorylated at Ser315/Ser319 in HeLa cells and F8 human fibroblasts. ........................55 Figure 3.10 Oleate treatment induces dephosphorylation of Ser315/Ser319 but not Tyr359/Ser362