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Regulation of Growth Factor and Nutrient Sensing Pathways by Human Papillomavirus E6 Proteins

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Regulation of Growth Factor and Nutrient Sensing Pathways by Human Papillomavirus E6 Proteins Regulation of Growth Factor and Nutrient Sensing Pathways by Human Papillomavirus E6 Proteins The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Spangle, Jennifer Marie. 2012. Regulation of Growth Factor and Nutrient Sensing Pathways by Human Papillomavirus E6 Proteins. Doctoral dissertation, Harvard University. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:10345147 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA © 2012 Jennifer Marie Spangle All rights reserved Karl Münger Jennifer Marie Spangle Regulation of Growth Factor and Nutrient Sensing Pathways by Human Papillomavirus E6 Proteins High-risk human papillomaviruses (HPVs) are associated with nearly all cases of cervical cancer and also contribute to other types of anogenital and oropharyngeal cancers. The high-risk HPV E6 oncoprotein contributes to malignant progression in part by the targeted degradation of the tumor suppressor p53. The activation of growth factor and nutrient sensing pathways including receptor protein tyrosine kinases (RPTKs) and mTORC1 may also support cellular transformation. Moreover, previous studies suggested that HPV16 E6 activates mTORC1. We are particularly interested in understanding the mechanisms by which HPV E6 activates mTORC1 and the function of mTORC1 activation in HPV infection. Here we show that high-risk HPV16 E6 activates mTORC1 signaling and increases cap dependent translation through an increase in S6K signaling and an increase in 4E-BP1 phosphorylation. Mechanistically we found that HPV16 E6 activates AKT under conditions of nutrient deprivation. The combined approach of phospho-tyrosine immunoprecipitations and Western blot identified HPV16 E6 mediated activation of a subset of receptor protein tyrosine kinases. HPV16 E6 activates RPTKs at least in part by increasing the internalization of phosphorylated and activated receptor species. The signaling adaptor protein Grb2 associates with HPV16 E6, and Grb2 knockdown abrogated HPV16 E6 mediated mTORC1 activation. We hypothesize that Grb2 may be important in relaying E6 mediated RPTK activation to downstream signaling cascades. iii In this dissertation we also evaluate mTORC1 signaling and cap dependent translation in cells expressing HPV16 E6 mutants and E6 proteins from other HPV types. Binding to p53 and the association with proteins that contain an LXXLL motif are important for HPV16 E6 mediated mTORC1 activation. An increase in mTORC1 activation and cap dependent translation is shared between high-and low-risk mucosal, but not cutaneous HPV E6 proteins. Association with proteins through their LXXLL binding motif is also important for low-risk mucosal HPV E6 activation of mTORC1 and cap dependent translation. Shared mucosal E6 activation of mTORC1 indicates that mTORC1 may be important for the viral lifecycle in mucosal epithelia. However, it does not rule out the possibility that together with other properties of high-risk HPV E6 proteins, mTORC1 activation may promote transformation. iv ACKNOWLEDGEMENTS I would like to thank my PhD advisor and mentor Karl Munger. Karl has been my biggest academic champion and from him I have learned more than I can fit within the pages of this dissertation. As a supportive mentor, he has given me countless opportunities to test potentially ludicrous ideas in lab and try my hand at training and teaching others. His open mind and willingness to explore new areas has made my time as a student exciting, especially when we learned together. Beyond the bench I have learned and benefited from Karl’s quick and exacting eye in designing experiments and compiling presentations and manuscripts. This is a skill that will never fail to be useful, and I am forever grateful. Most importantly Karl continues to encourage me to pursue a career as an academic scientist and has made it clear that I can always come back to talk. Thank you for this time and training in your lab and I certainly look forward to future discussions. I would like to thank my dissertation advisory committee. Many thanks to Fred Wang for his critical view and concern over the bigger picture. He taught me how to see the forest through the trees - how to organize and prioritize experiments, and how to determine which questions are important, all with the model in mind. Sheila Thomas so generously shared reagents and protocols, and it was a pleasure having a signaling expert with whom I could discuss experimental nuances. I cannot thank my committee without thanking Nick Dyson for the support and encouragement he offered over the years, and for serving as the chair of my defense examination committee. The Munger lab, both past and present, has fostered an exceptional environment for a growing scientist. Karl, for all of his features described above, but also for the unique skill of v selecting, developing and maintaining a network of lab mates that are supportive and considerate. The Mungers can be a critical bunch, but as a whole the lab has always been full of constructive and helpful coworkers that are also friends. Molly McLaughlin-Drubin has been there from the beginning, and has been a knowledgeable source for experimental design and manuscript organization. She is also a fantastic role model for women in science. From her I have learned what to expect on the long and arduous path ahead and how also to make the most of it and I know these lessons will go on beyond the writing of this dissertation. It is nothing short of a joy to have common interests of cats and crafting and I look forward to many years of friendship ahead. Taking this path for my PhD has genuinely been the best decision that I have made – pushing boundaries and having those boundaries push right back. It was here that I met some of the most amazing people in my life, including life long friends and classmates, lab mates and role models, and my husband Andreas. I thank him for also committing to a PhD on the 8th floor, Channing labs. Thank you Andreas for eating all the baked goods I make, but also for listening to every practice talk and reading every manuscript including this one, multiple times. I cannot express enough gratitude for you challenging me to exceed my own personal expectations. Thank you for supporting me in times self-doubt and it is a pleasure to share the occasional small successes that come from unexpected experiments and places. This experience would not be complete with thanking my parents for their encouragement from the beginning. Thank you for encouraging me to look within myself for strength and to be self sufficient. As a child, after I took a test or received a report card, my parents would ask me, “Did you do your best? Did you try your hardest? If so, you did all you could and for that we are proud of you.” Thank you both for motivating me to do “my” best but vi still being proud when I wasn’t ”the” best –it turns out that has been excellent training for this career. I look forward to many more years of phone conversations on my way to and from work nearly every day, so we can experience the best of being a scientist together. vii TABLE OF CONTENTS ABSTRACT……………………..……………………..……………………..………………....iii ACKNOWLEDGEMENTS………………………………..……………………..……………..v CHAPTER ONE: Introduction 1.1 Human Papillomaviruses…………………………………………………..……...2 1.1.1 Classification……………………………………………………………....2 1.1.2 HPV lifecycle and encoded proteins………………………………………4 1.1.3 HPV associated disease: Prevalence, Diagnosis, Treatment and Prevention ……………………..……………………..……………………..………………...9 1.1.4 HPV associated carcinogenesis and oncogenic activities of the HPV E6 oncoprotein……………………..……………………..…………………………11 1.2 Activation of growth factor responsive receptor protein tyrosine kinases………18 1.2.1 ErbB/HER family – Epidermal Growth Factor Receptor…………..……18 1.2.2 IR/IGFR……………………………………....………………………….19 1.3 mTOR signaling: Regulation and the function of mTORC signaling in the initiation of cap dependent translation……………………..……………………23 1.3.1 Upstream mTORC1: AKT phosphorylation and inhibition of TSC2 relieves mTORC1 inhibition……………………..……………………..…….….26 1.3.2 Additional mechanisms of mTORC1 regulation………………………...28 1.3.3 mTORC1 effectors and translation initiation: S6K and 4E-BP1………...29 1.4 DNA viruses and the regulation of the RPTK/PI3K/AKT/mTORC1 signaling axis ……………………..……………………..……………………..…………..……35 SUMMARY AND SIGNIFIGANCE……………………..……………………..…….……….39 viii CHAPTER TWO: The human papillomavirus type 16 E6 oncoprotein activates mTORC1 signaling and increases protein synthesis Abstract…………………………………………………………………………………..43 Introduction………………………………………………………………………………44 Materials and Methods…………………………………………………………………...46 Results……………………………………………………………………………………49 Discussion…………………………………………………………………………..……61 CHAPTER THREE: The mechanism by which mucosal human papillomavirus E6 proteins activate mTORC1 and increase protein synthesis is dependent on an intact LXXLL binding motif Abstract…………………………………………………………………………………..71 Introduction………………………………………………………………………………72 Materials and Methods…………………………………………………………………...76 Results……………………………………………………………………………………81 Discussion………………………………………………………………………..………91 CHAPTER FOUR: The HPV16 E6 oncoprotein promotes the phosphorylation of receptor protein tyrosine kinases and increases internalization
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