The Role of Phosphorylation in the Regulation of the Mammalian Target of Rapamycin A dissertation submitted to the University of London in candidature for the degree of Doctor of Philosophy By Susan Wai Yan Cheng Department of Biochemistry and Molecular Biology University College London 2004 1 UMI Number: U602837 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. Dissertation Publishing UMI U602837 Published by ProQuest LLC 2014. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract A key regulator of translation is the mammalian target of rapamycin (mTOR), a protein kinase member of the family of phosphatidylinositol kinase (PIK)-related kinases. mTOR is dually regulated by growth factors and nutrient availability, though the precise mechanisms by which mTOR is regulated are not well understood. The C-terminal of the mTOR catalytic domain has been of regulatory interest by the identification of the insulin stimulated and nutrient sensitive S2448 phosphorylation site. The functional significance of S2448 phosphorylation on the mTOR downstream targets p70 S6 kinase (S6K1) and eIF4E-binding protein 1 (4E- BP1) are unclear. A novel nutrient responsive mTOR phosphorylation site has been identified at T2446. In contrast to S2448 phosphorylation, T2446 is dephosphorylated when CHO-IR cells are insulin stimulated and phosphorylated when cells are nutrient deprived. Studies show that activation of AMP-activated kinase (AMPK) is concomitant with an increase in mTOR T2446 phosphorylation, paralleled by a decrease in S6K1 phosphorylation. Regulation of T2446 phosphorylation may involve AMPK. Phosphorylation at T2446 and S2448 is mutually exclusive. The functional significance of phosphorylation at T2446 and S2448 on the downstream target S6K1 was investigated by a mutational strategy where each site was substituted with non-phosphorylatable alanine or phospho-mimic glutamic acid. Evidence indicates that although phosphorylation of T2446 and S2448 is mutually exclusive in response to growth factors and nutrients, their individual phosphorylation may not be enough to have a direct effect on downstream S6K1 activity. Additionally, the tuberous sclerosis complex (TSC) may have positive regulatory effects on insulin signalling. Loss of TSC2 impairs insulin signalling by down- regulating the turnover of insulin receptor substrate-1 (IRS-1) protein, affecting associated class la phosphoinositide 3-kinase (PI3K) activity and downstream signalling; including suppression of PKB activation and mTOR S2448 phosphorylation. 2 Statement This thesis is an account of research conducted at the Department of Biochemistry and Molecular Biology at University College London, between September 1999 and September 2003. Except where references are given, this thesis contains my own original work, does not exceed the word limit stipulated by the University and is not substantially the same as any I have submitted for any other degree, diploma or examination. Some of the work presented in this thesis has been published elsewhere: Cheng, S. W. Y., Fryer, L., Carling, D. and Shepherd P. R. (2004) T2446 is a novel mTOR phosphorylation site regulated by nutrient status. The Journal o f Biological Chemistry, 279, 15719-15722 Harrington, L. S., Findlay, G. M., Gray, A., Tolkacheva, T., Wigfield, S., Barnett, J., r Leslie, N. R., Cheng, S., Shepherd, P. R., Gout, I., Downes, C. P. and Lamb, R. F. (2004) The TSC1-2 tumor suppressor controls insulin-PI3K signalling via regulation of IRS proteins. Journal o f Cell Biology. 166,213-223. 3 Acknowledgments I would like to acknowledge the support of Diabetes UK for their research studentship and the Elliot-Blake Studentship for additional funding. Thanks to Professor Peter Shepherd for his supervision. Well it definitely took longer than I thought and I’m just extremely glad to finish. I’d like to express thanks to people in the lab without whom I could not have completed my PhD. Firstly, to Dr Richard Brown and Dr Nathalie Daniele for their patience and guidance and most especially to Dr Lazaros Foukas for his time and advice both in the lab and during my write-up, and who acted as my substitute mentor. I would also like to thank the members of the Shepherd lab - past and present - sorry I can’t name you all! Everyone contributed to a humorous and harmonious lab and I greatly valued their friendship and encouragement during my time at UCL. Also, I’m grateful to Dr Ivan Gout, Dr Richard Lamb, Dr David Carling, Dr Guy Rutter and their lab members for their collaboration and for providing valuable reagents. I would also like to thank Vic and Simon the ‘Grammar Cops’ for critical reading of my thesis and Fei for referencing and printing. Finally, I’d like to thank my family and friends for their support and encouragement. March 2004 4 Contents Abstract....................................................................................................................... 2 Statement .................................................................................................................... 3 Acknowledgements .................................................................................................... 4 Abbreviations ............................................................................................................13 1 Introduction .......................................................................................................... 18 1.1 Initiation of the insulin signalling cascade ...................................................18 1.2 Overview of Translation ............................................................................. 20 1.3 The TOR kinase family............................................................................... 25 1.4 The Mammalian Target of Rapamycin ....................................................... 26 1.4.1 The modular domains of mTOR ............................................................ 26 1.4.1.1 mTOR HEAT domain ..................................................................... 26 1.4.1.2 mTOR FKBP/rapamycinbinding domain (FRB) ............................ 28 1.4.1.3 mTOR catalytic domain .................................................................. 28 1.4.1.3.1 mTOR autokinase activity and regulation by phosphorylation. 29 1.4.1.4 mTOR FAT and FATC domain ...................................................... 30 1.5 Targets of mTOR kinase activity .................................................................31 1.5.1 eIF4E binding protein (4E-BP1) ............................................................ 31 1.5.1.1 Phosphorylation of 4E-BP1 .............................................................32 1.5.2 S6 Kinases.............................................................................................. 35 1.5.2.1 Regulation of S6K1 by phosphorylation ......................................... 37 1.6 Additional targets of mTOR ...................................................................... 39 1.6.1 mTOR and Protein Phosphatase 2A (PP2A) ......................................... 39 1.6.1.1 Model for mTOR control of PP2A .................................................40 1.6.2 mTOR regulation of eEF2 ..................................................................... 43 1.6.3 mTOR regulation of other translation initiation factors ........................ 44 1.6.4 mTOR regulation of IRS-1 .................................................................... 44 1.6.5 mTOR regulation of PKC ...................................................................... 46 1.6.6 mTOR regulation of STATs .................................................................. 47 1.7 Regulation of mTOR .................................................................................. 48 5 1.7.1 The role of Raptor association with mTOR .......................................... 48 1.7.2 Amino acid regulation of mTOR and its downstream effectors............50 1.7.2.1 Branched chain amino acids and mTOR signalling .........................51 1.7.2.2 mTOR detection of amino acid levels .............................................53 1.7.2.3 Leucine regulation of mTOR ...........................................................54 1.7.3 Cytoplasmic nuclear shuttling of mTOR ...............................................55 1.7.4 Phosphatidic acid mediated mTOR signalling .......................................56 1.7.5 mTOR regulation by ATP levels ...........................................................56 1.7.5.1 Interplay between mTOR and AMPK .............................................57 1.7.6 TSC ....................................................................................................... 58 1.7.6.1 TSC and the mTOR signalling pathway ..........................................59 1.7.6.2 TSC2 GAP activity may regulate mTOR ........................................61 1.8 Role for mTOR in oncogenesis ................................................................... 63 1.8.1 mTOR and regulation of cell growth .....................................................63