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May 1 1 2011 Libraries The Identification of the mTOR-Regulated Phosphoproteome and a Mediator of Feedback Inhibition to P13K-Akt MASSACHUSET~s INSTT FE by OF TECHNOLOGY Peggy Ping Hsu MAY 11 2011 A.B. Molecular Biology LIBRARIES Princeton University, 2003 SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFULLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOLOGY AT THE ARCH NES MASSACHUSETTS INSTITUTE OF TECHNOLOGY JUNE 2011 @ 2011 Peggy Ping Hsu. All rights reserved. The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium now known or hereafter created. Signature of Author: ___ Department of Biology April 27, 2011 Certified by: David M. Sabatini Associate Professor of Biology Member, Whitehead Institute for Biomedical Research Thesis Supervisor Accepted by: Stephen P. Bell Professor of Biology Co-Chair, Committee for Graduate Students The Identification of the mTOR-Regulated Phosphoproteome and a Mediator of Feedback Inhibition to P13K-Akt by Peggy Ping Hsu Submitted to the Department of Biology on April 27, 2011 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology ABSTRACT The mTOR protein kinase nucleates two complexes, mTORC1 and mTORC2. Collectively, the two complexes regulate processes important for cell growth and proliferation, including protein synthesis, autophagy, metabolism, and cytoskeletal maintenance. Despite this diverse array of cellular functions, few mTOR substrates are known. To address this deficit, we defined the mTOR-regulated phosphoproteome by quantitative mass spectrometry and characterized the primary sequence motif specificity of mTOR using positional scanning peptide libraries. We found that the phosphorylation response to insulin is largely mTOR-dependent and that mTOR regulates the phosphorylation of many proteins not presently appreciated to be linked to mTOR signaling. The mTOR kinase, moreover, exhibits a preference for proline, hydrophobic, and aromatic residues at the +1 position which is unique among all kinases previously profiled. Grbl 0 is an adaptor protein and negative regulator of growth factor signaling identified as an mTORC1 substrate that mediates the inhibition of P13K typical of cells lacking TSC2, a tumor suppressor and negative regulator of mTORC1. Phosphorylation of Grbl 0 is important for its inhibitory function as well as for its stability. While acute mTORC1 inhibition results in changes in Grb1O and IRS1 phosphorylation which partially reactivates Akt in TSC2-null cells, chronic mTORC1 inhibition causes Grbl 0 destabilization, IRS protein stabilization, and a complete resensitization of Akt to insulin and IGF-1. These changes in Grbl 0 and IRS protein abundance are likely to be the most important effects of mTOR inhibitors to consider in their clinical use. Finally, the discovery of Grbl 0 as an mTORC1 substrate validates our phosphoproteomic approach and suggests that the other potential downstream effectors we identified may also serve as starting points for new areas of investigation in mTOR biology. Thesis Supervisor: David M. Sabatini Title: Associate Professor of Biology, MIT; Member, Whitehead Institute for Biomedical Research For my parents Acknowledgments I cannot sufficiently express my gratitude and respect for my advisor, David Sabatini. He is the scientist that I could only wish to become: insightful, creative, intelligent, fearless, and efficient. I especially appreciate that David gave me the freedom to follow my own path while focusing me when I had wandered astray. I am also grateful to have had Mike Yaffe and Mike Hemann on my thesis committee for the past few years. I have learned quite a bit from them in both my formal meetings as well as in casual conversations. I must also thank Brendan Manning, whose work I have long admired, and Piyush Gupta for being on my defense committee. David has built a lab of bright, fun, and ambitious people, and I have enjoyed working with every single one of them. Siraj first introduced me to the lab, and Dos showed me how to properly Western blot. The graduate students who came before me - Shomit, Tim, and Yasemin - inspired me with their hard work and independence. Tony has been a fantastic collaborator, and from him I learned how to be a more efficient experimentalist. I have enjoyed conversations with Dudley over coffee, and Do and Yoav in tissue culture. Carson has also become a close friend in the lab. He is a person whose scientific advice I value enormously and also someone who I have enjoyed getting to know over the past few years. I respect him not only for his intellect but also the manner in which he lives his life. I especially want to acknowledge Kathleen and Heather. I do not know what I did to deserve Kathleen's help, but I have immensely enjoyed working with her. Her thirst for learning and self-improvement are inspiring. She has given me great life advice and constant encouragement. And Heather has been a great friend in the lab. I too admire her for her independence and assertiveness, but also for her loyalty to friends and excitement about both the little and small things in life. I have really enjoyed coming to work knowing that I have a friend as a baymate. I would like to thank my PhD classmates, MD classmates, the many amazing people working at the Whitehead, and friends I have met along the way for their Acknowledgments support. Lauren and Derek have been amazing friends, and in Lauren I am so thankful to have found a friend with whom I can share all aspects of the MD PhD journey. I also have to thank Renuka, Jasper, Vincent, and Michelle for their help, friendship, and commiseration. Through it all, Dan has been my best friend, and I have enjoyed sharing life and science with him. I admire the ways he thinks about scientific problems and his generous, patient, and caring nature. I do not know what I would do without him, or how I would have finished this thesis! Finally, I would like to thank my family. While they were not part of the day-to-day aspects of the PhD, knowing that they believed in me and were there for me no matter what was a great source of strength and comfort. I see myself in them, and know that I would not be where I am without them. Table of Contents Ab s tra ct ............................................................................................................................................................................... 2 Ac kn ow le dg m e nts4.......................................................................................................................................................4 Chapter 1 Introduction Su m m a ry ......................................................................................................................................................................... 12 Rapamycin and its molecular target.........................................................................................................13 Upstream signaling: mTOR senses the nutrient and metabolic state of the cell..19 Downstream functions: mTOR is the master regulator of cell growth................... 23 The pathway is complex: interconnected feedback loops.............................................. 34 T he role of mT O R in cance r...........................................................................................................................3 7 mT OR inh ib ito rs......................................................................................................................................................... 40 Introduction to the work presented in this thesis..................................................................... 43 Fig u re s ............................................................................................................................................................................. 44 Re fe re nc e s ................................................................................................................................................................... 4 9 Chapter 2 Characterization of the mTOR-regulated phosphoproteome S um m a ry ........................................................................................................................................................................ 7 2 Intro du c tio n................................................................................................................................................................... 73 Re sults and Discussio n......................................................................................................................................7 5 Identification of AGC kinase substrates and proline-directed phosphorylations downstream of mTOR by immunoaffinity phosphopeptide isolation Identification of the mTOR-regulated phosphoproteome by quantitative mass spectrometry Acknowledgments Torin1 mimics serum deprivation and is a more complete inhibitor than rapamycin Definition of a consensus mTOR phospho-acceptor motif Classification of the mTOR-regulated phosphoproteome Fig u re s .............................................................................................................................................................................. 8 5 Mate rials a nd Metho ds......................................................................................................................................10 0 A ckno w le dg e me nts..............................................................................................................................................10
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