The Role of CCL5/RANTES in Regulating Cellular Metabolism in Activated T cells by Olivia Chan A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Immunology University of Toronto © Copyright by Olivia Chan, 2011 The Role of CCL5/RANTES in Regulating Cellular Metabolism in Activated T cells Olivia Chan Master of Science Graduate Department of Immunology University of Toronto, 2011 Recruitment of effector T cells to sites of infection is essential for an effective adaptive immune response. The inflammatory chemokine CCL5/RANTES activates its cognate receptor, CCR5, to initiate cellular functions including chemotaxis. This thesis describes the signaling events invoked by CCL5 and its ability to regulate the energy status of activated T cells. CCL5 treatment in ex vivo activated human T cells induced the activation of AMPK and downstream substrates ACC1, PFKFB2 and GSK-3. Evidence is provided that CCL5 treatment is able to induce glucose uptake in an mTOR-dependent manner. Using 2-deoxy-D-glucose, an inhibitor of glucose uptake, and Compound C, an inhibitor of AMPK, evidence is provided that demonstrate that CCL5-mediated chemotaxis is dependent on metabolic events, since these inhibitors perturb chemotaxis in a dose-dependent manner. Collectively, these studies suggest that CCL5 may also influence the metabolic status of activated T cells by simultaneously activating the AMPK and mTOR pathways. ii ACKNOWLEDGEMENTS I would like to dedicate this thesis to everyone who supported me throughout my graduate studies, and would like to express my gratitude to the individuals whom, without which, none of this could have been possible. Eleanor, your guidance, without fail, has always pointed me in the right direction. Whenever things looked grim, your encouragement and advice would always put me back on the right path. Thank you for your patience and mentorship throughout my studies – you have been an incredible driving-force that has helped me grow both as a scientist and as an individual. I am especially thankful for the opportunity to have gone to multiple international conferences, and the chance to present my work around the world. I look forward to working with you in the future, as we further embark on the „CCL5-era‟ in the lab! To my supervisory committee members, Dr. Pam Ohashi and Dr. Juan Carlos Zuniga-Pflucker, I am grateful for your helpful guidance and input into my work. Also, thank you Dr. Shannon Dun for taking the time to discuss my research plans and long term goals. To past and present Fishies, I offer my sincerest thanks for all your emotional and scientific support (especially your PBMCs). Beata, you‟ve given multi-tasking a whole new meaning, and I look to you as a constant source of inspiration and motivation. You‟ve been the shoulders I could always depend on and a friend I could always turn to. Thank you for holding my hand through some of the toughest times during my studies. Daniel, without a doubt, you‟ve been an invaluable „metabolism-and-protein-translation- buddy‟. Thank you for our many scientific discussions – which have always made the realm of metabolism a little less daunting. Thank you for all your encouragement and friendship throughout the years, and keep working on your chopstick skills! Carole, I‟ve always been determined to answer at least one of your unanswerable questions! Thank you for taking the time to guide and mentor me throughout my project and for letting me pick-your-brain about experimental design. I wish you, Kip and Kaycee all the very best. Leesa, I can‟t help but chuckle whenever I come across or hear about PKR (I think that video is still on my desktop…). Thank you for sharing your incredible energy with the lab, and although there is much work to be done to improve your Chinese accent, there is no doubt that you are well on your way towards obtaining your Ph.D. Ben, thank you for bringing Fail Blog into our daily lunch routine (or was that Craig?). I wish you all the best in your Ph.D. journey, and remember to take advantage of the lab hammock when you truly need it! Craig, I look forward to the day when you and Ben revolutionize IGSA (with karaoke nights!). Thank you for bringing your calming-influence to the lab and I wish you best of luck in your Ph.D. studies. Thomas, Danlin and Ramtin, thank you for your continuous support throughout the years and for always being an email away! You‟ve been brilliant mentors and role models, and I wish you all happiness in your new lives. iii To my Mom, Dad and Oscar, thank you for your constant vote of confidence. Mom and Dad, you give me strength and hope during my rough patches – thank you for believing, before I did, in my ability to achieve something substantial. Oscar, you never fail to cheer me up following rather disappointing experiments. Thank you for your tremendous support and encouragement, and I can‟t wait to see what life has in store for you! Thanks to all my friends and labmates who have donated blood to „fuel‟ the studies undertaken in my project. None of this would have been possible without your generous donations; and I may owe royalties to many of you. Finally, a heartfelt thank you goes to my fellow students of the Immunology Department for their support and friendship. Nothing beats sharing a pint of beer together when experiments go awry, and I look forward to working with all of you again in the future. iv Table of Contents Title Page………………………………………………………………………………….. i Abstract…………………………………………………………………………………ii-iii Acknowledgments……………………………………………………………. ………..iv-v Table of Contents……………………………………………………………………….v-vi List of Figures………………………………………………………………………….... vii List of Tables…………………………………………………………………………….viii List of Abbreviations………………………………………………………………….. ix-xi CHAPTER 1: INTRODUCTION…………………………………………………... 1-52 1.1 Chemokine Superfamily……………………………………………………………….2 1.1.1. Classification……………………………………………………………………2 1.1.2. Chemokine Structure……………………………………………………………4 1.1.3. Chemokine-mediated Signaling………………………………………………... 7 1.1.3.1. Jak-Stat Pathway…………………………………………………………11 1.1.3.2. MAPK Signaling Cascade……………………………………………….12 1.2 Chemokine Receptors……………………………………………………………….. 15 1.2.1. Classification…………………………………………………………………. 15 1.2.2. Chemokine Receptor Structure and Ligand Binding…………………………. 18 1.2.3. Receptor Dimerization and Internalization…………………………………… 19 1.3. Chemokine/ Chemokine Receptor Functions………………………………………. 20 1.3.1. Chemotaxis…………………………………………………………………… 20 1.3.1.1. Cellular Polarization……………………………………………………. 20 1.3.1.2. The Rho Family GTPases in Cytoskeletal Rearrangement…………….. 21 1.3.1.3. Activation of the PI-3‟K Pathway……………………………………… 22 1.3.1.4. The mTOR/4E-BP1 Pathway and Chemotaxis………………………… 24 1.3.2. Role in determining Cellular Fate…………………………………………….. 32 1.3.2.1. T cell Differentiation and Activation…………………………………… 33 1.3.2.2. Role in Cell Death………………………………………………………. 35 1.4. mTOR Signaling and Metabolic Regulation……………………………………....... 36 1.4.1. Growth Factor and Nutrient-Sensing by mTOR……………………………… 36 1.4.1.1. AMPK-regulation of mTOR……………………………………………. 39 1.4.2. mTOR Signaling in Lymphocyte Trafficking………………………………… 40 1.4.3. mTOR-mediated Proliferation………………………………………………... 41 1.5. Energy Metabolism and the T cell Response……………………………………….. 43 1.5.1. Regulation of T lymphocyte Metabolism…………………………………….. 43 1.5.2. Quiescent Cells and Oxidative Phosphorylation……………………………… 44 1.5.3. Proliferating Lymphocytes and Glycolysis…………………………………... 45 1.5.3.1. PI-3‟K Signaling in Aerobic Glycolysis………………………………... 46 1.5.4. Energy Regulation during an Immune Response…………………………….. 48 v 1.6. Thesis Hypothesis and Objectives………………………………………………….. 52 CHAPTER 2: MATERIALS AND METHODS…………………………………. 53-57 2.1. Cells and Reagents ………………………………………………………………….54 2.2. Immunoblotting …………………………………………………………………….55 2.3. Flow Cytometric Analysis ………………………………………………………….55 2.4. Chemotaxis Assay …………………………………………………………………..56 2.5. Glucose Uptake Assay ………………………………………………………………56 2.6. AMPK Antibody Signaling Array ………………………………………………….57 2.7 Statistical Analysis……………………………………………………………………57 CHAPTER 3: RESULTS…………………………………………………………... 58-87 3.1. CCL5 induces phosphorylation of proteins in the AMPK signaling pathway……... 59 3.2. CCL5-mediated glucose uptake is mTOR-dependent……………………………… 69 3.3. CCL5-mediated glucose uptake is not accompanied by changes in the surface expression of nutrient receptors ………………………………….....................................74 3.4. Glucose uptake and AMPK signaling are required for efficient CCL5-mediated chemotaxis ………………………………………………………………………………80 3.5. CCL5-induced AMPK signaling phosphorylates the 4E-BP1 repressor of mRNA translation………………………………………………………………………………... 80 CHAPTER 4: DISCUSSION………………………………………………………. 88-97 CHAPTER 5: FUTURE DIRECTIONS………………………………..…………98-101 CHAPTER 6: REFERENCES…………………………………………………...102-118 vi LIST OF FIGURES CHAPTER 1 Figure 1.1. Chemokines share similar structure………………………………………….. 5 Figure 1.2. Chemokine-induced signaling pathways……………………………………... 9 Figure 1.3. The MAPK signaling cascade………………………………………………. 13 Figure 1.4. Two-dimension depiction of CCR5 and residues critical for ligand binding and signaling transduction………………………………………………………………. 16 Figure 1.5. mTOR signaling complexes………………………………………………….26 Figure 1.6. Regulation of cap-dependent mRNA translation ……………………………30 CHAPTER 3 Figure 3.1. CCR5 surface expression is induced upon T cell activation in the presence of cytokines………………………………………………………………………………….60 Figure