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The Role of Ras and Its Downstream Targets on Cell Cycle Control

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The Role of Ras and Its Downstream Targets on Cell Cycle Control The Role of Ras and its Downstream Targets on Cell Cycle Control Bryony S. Wiseman A thesis submitted in partial fulfillment of the degree of Doctor of Philosophy, University of London. March 1999 Growth Control and Development Laboratory Imperial Cancer Research Fund 44, Lincoln’s Inn Fields London ProQuest Number: U641977 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. uest. ProQuest U641977 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract The oncogene Ras can both stimulate and inhibit cell proliferation depending on the cellular context in which it is activated. Ras is believed to mediate its effects through activation of downstream effector proteins. The best characterised of these is the serine/threonine kinase, Raf. Through conditional activation of Ras or Raf we have shown that depending on the intensity of signal activation, these proteins are able to both stimulate and inhibit proliferation in murine fibroblasts. Strong signals are inhibitory to growth which is shown to be dependent upon an induction of the cell cycle inhibitor p2l‘^‘P ’ which prevents upregulated G1 cyclins from forming active complexes. Weaker signals can induce DNA synthesis in low mitogen conditions since they can upregulate cyclin D1 expression, yet are unable to stimulate p21^^' expression. Thus we demonstrate that Raf controls opposing cellular responses through differential activation of cell cycle regulatory proteins. Ras however demonstrates a greater propensity to stimulate growth than Raf which correlates with an ability to downregulate Raf induction of p21^'P '. The Ras effectors PI 3-kinase and Rif are unable to reproduce this effect and furthermore, RhoA, which has been proposed to downregulate is also unable to attenuate induction of p21^'P ’ by Raf. Indeed we show that RhoA and PI 3-kinase co­ operate with Raf to induce p21^^\ Furthermore Raf and PI 3-kinase co-operate to induce DNA synthesis in the presence of high levels of p21^^\ In addition we investigate the ability of activated Rac, which has been implicated as a downstream target of Ras, to control the cell cycle. It is demonstrated that Rac^'^ is able to stimulate anchorage-independent growth and upregulate cell cycle regulatory proteins, yet is unable to stimulate mitogen-independent growth. Thus cell growth control by Ras depends both on signal strength and a panel of downstream targets. Acknowledgements Firstly, I would like to thank Hucky for giving me the opportunity to work in his laboratory and for his guidance during my time there. I would like to acknowledge the work of Andreas Sewing which is included in this thesis. Furthermore I would like to thank J.Bos, P.Brennan, D.Cantrell, C. Dickson, J.Downward, M.Fried, M.Gossen, T.Jacks, C.Marshall, G.Peters, S.Reeves, A.Ridley, P.Rodriguez-Viciana, R.Treisman and E.Sahai for reagents and/or helpful discussions. The FACS laboratory and the photography department at ICRF have also given invaluable assistance. I would especially like to thank Alan Hall and Fiona Watt for interesting conversations, support and perspective. In the lab I would like to thank Alison whose irrepressible and unrelenting questioning taught me scientific method but also for support, proof-reading and friendship. Thanks also to Sharon whose sharp wit and words of comfort kept my feet on the ground; Susan, the Bolton boys still remember; David P., for calmness in chaos and for knowing who Little Jimmy was; to Andreas for all his help and much silliness; David S. for his interesting points of view; Liz whose presence continuously reassures me that life can be fun and who has it all to come; Soo for, amongst other things, pointing out that this sentence is too long; Sally for head-banging reminiscences; Betina for vibrancy in a dull place; Laurent for showing me that after it all you can still be chilled; Beatrice who is just wonderful; Nacho, with whom I have been through so many tears, smiles and the odd scowl -thank you for everything and finally Frank-I understand! Outside of the lab, thank you everyone who has made this time enjoyable, but especially to Nicky for being there through it all ; Jo for trying to make me look good and for being such an understanding friend these past few months; Shugs for supporting me even when my ambitions seemed to come first; to Matt-man for the dog with pants, the dropped pints and for endlessly supporting me down Marlboro Road; Robby for friendship that I can't pin down to anything, but may have something to do with an appreciation of the virtues of shallowness; Ann for helping me split up sentences over the past few months; the Boys for making our house a real-life soap opera and I would like to give a special thank you to Romana and Fiona without whose advice and honesty I may not have got this far. Finally I would like to thank my family for their help, support and reassurance through out the past few years. This thesis is dedicated to C.H.Peters and T.G.Dupe without whom none of this would have been possible. 'L o n g IS THE WAY AND HARD, THAT OUT OF HELL LEADS UP TO LIGHT.' -John M iltonP a r a d is e L o s t Table Of Contents Title Page..........................................................................................................................................................1 Abstract...............................................................................................................................................................2 Acknowledgements..........................................................................................................................................3 Table of Contents.......................................................................................................................................... 4 List of Figures................................................................................................................................................7 List of Tables................................................................................................................................................. 8 Abbreviations....................................................................................................................................................9 1. - INTRODUCTION.............................................................................................................................. 12 1.1 Cell C ycle Co n t r o l ....................................................................................................................................................... 14 1.1.2 Cell cycle regulatory proteins and tumorigenesis........................................................................................ 19 1.2 R a s ..............................................................................................................................................................................................22 1.2.1 Background.................................................................................................................................................................... 22 1.2.2 The Ras Superfamily.................................................................................................................................................22 1.2.3 Ras in human tumorigenesis..................................................................................................................................23 1.2.4 Activation of Ras........................................................................................................................................................ 24 1.2.5 Cellular Effects of Ras.................................................................................................. 27 1.2.6 Ras signal transduction through ...................................................................................................................Raf. 29 1.2.7 Activation of MAP kinase cascades...................................................................................................................... 33 1.2.8 Alternative Ras effectors...........................................................................................................................................37 1.2.9 Ras Effectors................................................................................................................................................................. 39 1.2.10 PI 3-kinase and its role as a Ras effector..........................................................................................................41 1.2.11 Ral-GEFs and their roles as Ras effectors....................................................................................................... 45 1.3 R a c a n d R ho-fa m ily pr o t e in s ................................................................................................................................... 49 1.3.1 Background...................................................................................................................................................................
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