REGULATION OF DBL FAMILY GUANINE NUCLEOTIDE EXCHANGE FACTORS By: MEGHANA BALARAM GUPTA Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Danny Manor Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY January 2014 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Meghana Balaram Gupta candidate for the Doctor of Philosophy degree* Dissertation Advisor: Danny Manor Committee Chair: Amy Wilson-Delfosse Committee Member: Bingcheng Wang Committee Member: Vera Moiseenkova-Bell Committee Member: Cathy Carlin Date of Defense: December 2, 2013 * We also certify that written approval has been obtained for any proprietary material contained therein. ii Dedication I dedicate this thesis to my parents. Without them, I would not be the person that I am today. Their unconditional love, support, and encouragement has helped me achieve something that at times I did not think was possible. I share the success of completing this thesis and earning my Ph.D. with my parents and my entire family. Without them, I am sure it would have been impossible. Mom and Dad, I hope that one day, I will be as loving and supportive towards my children as you have been to me. Thank you for everything you have given me and for all that you continue to do for me. I love you both. iii Table of Contents CHAPTER PAGE Table of Contents ............................................................................................................. iv List of Tables ................................................................................................................... vii List of Figures ................................................................................................................. viii Acknowledgements .......................................................................................................... ix Dbl Family GEFs................................................................................................................1 Abstract ...............................................................................................................................1 Chapter 1 ............................................................................................................................2 Literature Review ..............................................................................................................2 I. Mitogenic Pathways ......................................................................................................2 II. The Ras Superfamily GTPases ....................................................................................3 II.1 Ras Subfamily of GTPases ............................................................................................4 II.2 Ran Subfamily of GTPases ...........................................................................................4 II.3 Rab Subfamily of GTPases ...........................................................................................4 II.4 Arf Subfamily of GTPases ............................................................................................5 III. Rho Family GTPases: Activation and regulation of their cycle .............................7 III.1 Rho GTPase structure: requirements for nucleotide binding, GTP hydrolysis and effector recognition ..............................................................................................................7 III.2 Regulation of Rho GTPase cycling .............................................................................9 IV. The Dbl Family GEFs ..............................................................................................13 IV.1 The Dbl-Homology (DH) Domain: Structure & Function .......................................14 IV.2 The Pleckstrin Homology (PH) Domain ..................................................................16 V. Regulation of Dbl Family GEFs ................................................................................18 V.1 Coupling to Receptor Tyrosine Kinases and G-Protein Coupled Receptors .............18 V.2 Regulation of Dbl GEFs: Intra- and Inter- Molecular Modes of Regulation .............21 V.2.1 Examples of intra-molecular modes of regulation ......................................22 iv V.2.2 Release of inhibitory intra-molecular interactions ......................................22 V.2.3 Inter-molecular modes of regulation ...........................................................24 V.3 Other Modes of Dbl Regulation .................................................................................25 V.3.1 GEFs in signaling pathways .........................................................................25 V.3.2 Dbl GEFs mediate cross talk between pathways ........................................27 VI. GEF Dysfunction and Diseases ...............................................................................29 VI.1 Cancers .........................................................................................................29 VI.2 Developmental/Neurological Disorders .......................................................30 VI.3 Viral Infections .............................................................................................31 Statement of Purpose ......................................................................................................32 Chapter 2 .........................................................................................................................33 Plekhg4 is a novel Dbl-family GEF for Rho-family GTPases ......................................33 Capsule ...............................................................................................................................34 Abstract ..............................................................................................................................34 Introduction ........................................................................................................................35 Materials and Methods .......................................................................................................37 Results ................................................................................................................................40 Discussion ..........................................................................................................................47 Chapter 2 Figures ...............................................................................................................50 Chapter 3 .........................................................................................................................57 Tyrosine phosphorylation of Dbl regulates GTPase signaling ....................................57 Capsule ...............................................................................................................................58 Abstract ..............................................................................................................................58 Introduction ........................................................................................................................59 Materials and Methods .......................................................................................................61 Results ................................................................................................................................64 Discussion ..........................................................................................................................67 v Chapter 3 Figures ...............................................................................................................71 Chapter 4 ..........................................................................................................................80 Overall Summary and Future Directions ......................................................................80 4.1 Overall Summary .........................................................................................................80 4.1.1 Plekhg4 .........................................................................................................80 4.1.2 Dbl.................................................................................................................81 4.2 Future Directions .........................................................................................................82 4.2.1 Plekhg4 .........................................................................................................82 4.2.2 Dbl.................................................................................................................82 4.2.3 510Tyr is highly conserved in other Dbl-family GEFs ..................................83 4.3 Concluding Remarks .................................................................................................92 Literature Cited ...............................................................................................................93 vi List of Tables Table 1 List depicting coupling between cell surface receptors and GEFS ......................20 vii List of Figures Figure 1.1 Activation of cell surface receptor leads to Rho GTPase activitation ................3 Figure 1.2 General features of the G-Domain .....................................................................8 Figure 1.3 Cycling of Rho GTPases is tightly regulated ...................................................10