ATYPICAL GUANINE NUCLEOTIDE EXCHANGE FACTORS FOR RHO FAMILY GTPASES: DOCK9 ACTIVATION OF CDC42 AND SMGGDS ACTIVATION OF RHOA Brant L. Hamel A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics. Chapel Hill 2010 Approved by: Henrik Dohlman, Ph.D. Brian Kuhlman, Ph.D. Matthew Redinbo, Ph.D. David Siderovski, Ph.D. John Sondek, Ph.D. ABSTRACT BRANT L. HAMEL: Atypical Guanine Nucleotide Exchange Factors for Rho Family GTPases: DOCK9 Activation of Cdc42 and SmgGDS activation of RhoA (Under the direction of John Sondek) Rho GTPases regulate diverse cellular processes ranging from cell morphology and motility to mitosis. The activation of Rho GTPases is tightly controlled by the actions of guanine nucleotide exchange factors (GEFs). While the mechanism of canonical Dbl family exchange factors is established, both DOCK proteins and SmgGDS catalyze nucleotide exchange by distinct mechanisms. The structure of the DOCK9 GEF domain bound to Cdc42 was recently described, while no structural information on SmgGDS is available. Here, we describe a C- terminal DOCK9 fragment, soluble in bacteria, that is sufficient to catalyze nucleotide exchange on Cdc42. We also provide evidence that full-length DOCK9 is significantly more active than the minimal GEF domain, implicating the ability of other domains to contribute to the DOCK9 exchange mechanism. In contrast to the reported ability of SmgGDS to activate both Rho and Ras family GTPases, we find exclusive activation of RhoA and RhoC both in vitro and in vivo. The mechanism of SmgGDS nucleotide exchange is shown to be distinct from Dbl family GEFs and to require the presence of an intact C-terminal polybasic region on the GTPase. Using a homology model of SmgGDS, an electronegative surface patch and a highly conserved binding groove are identified that are required for the ability of SmgGDS to interact with RhoA. Our results illustrate that further structural characterization is necessary for a fuller understanding of DOCK9 exchange and that SmgGDS is able to function as a bona fide GEF solely for RhoA and RhoC and does so through a unique interface distinct from other known Rho family exchange factors. ii This work is dedicated to my wife Sarah and my daughter Jillian, who have given me the strength and hope necessary for its completion. iii ACKNOWLEGEMENTS My sincere thanks go out to the many people who have made this work possible. The current and past members of the Sondek lab have created an exceptional work environment, not only providing technical assistance and advice, but providing genuine camaraderie and support throughout my tenure. I would like to particularly thank Cynthia Holley for many useful discussions on the DOCK family proteins and Rafael Rojas for his insights into SmgGDS function. Aurelie Gresset and Stephanie Hicks were invaluable for both their scientific enthusiasm and their scientific insights, as well as for their emotional support. I am also indebted to a number of collaborators whose work has enhanced this project. Elizabeth Monaghan-Benson from the Burridge laboratory at UNC-Chapel Hill was indispensible in performing assays for activated RhoA in cells. Tracy Berg and her mentor Carol Williams from the Medical University of Wisconsin gave us a number of insights into SmgGDS function and helped examine the in vivo effects of SmgGDS mutations. I would thank the members of my committee for their support, encouragement, and scientific advice. I am grateful to the staff of the Department of Biochemistry and Biophysics, the Biophysics program, and the Department of Pharmacology for all the behind the scenes work they do that allowed me to focus on science. I would also like to acknowledge the National Science Foundation for awarding me a graduate research fellowship. iv The support, advice, training, and mentorship of John Sondek has been very influential in shaping me as a scientist, so I thank him for his unwavering commitment to conducting excellent science. Finally I wish to acknowledge the love and support of my family: my parents, grandparents, and in-laws, but most importantly that of my wife Sarah who has stood by me through all the ups and downs of life and has given me more joy then I ever thought possible. v TABLE OF CONTENTS LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix LIST OF ABBREVIATIONS ............................................................................................ xi CHAPTER 1: INTRODUCTION ....................................................................................... 1 Part 1: G proteins and their activation by GEFs ............................................................ 1 G proteins have diverse biological roles ..................................................................... 1 The structure of a GTPase allows it to act as a molecular switch during a nucleotide exchange cycle ............................................................................................................ 8 The mechanisms of GTPase activation by GEFs ...................................................... 10 Part 2: The DOCK family of atypical exchange factors. ............................................. 14 Identification of DOCK family proteins as Rho GEFs ............................................. 14 Biological functions of DOCKs 1-5 ......................................................................... 17 The role of ELMO in regulating DOCKs 1-4 function ............................................. 21 The biological roles of DOCKs 6-11 ........................................................................ 23 DOCK family GEF activity and GTPase specificity ................................................ 27 Part 3: SmgGDS is a unique and atypical GEF ........................................................... 29 Discovery of SmgGDS as a GEF with unusually broad specificity ......................... 29 The GTPase polybasic region and isoprenoid modification and SmgGDS function 34 SmgGDS in biology and in cancer ............................................................................ 37 vi CHAPTER 2: PURIFICATION AND ANALYSIS OF DOCK9 CONSTRUCTS ABLE TO CATALYZE NUCLEOTIDE EXCHANGE ON CDC42 .......................................... 47 Background ................................................................................................................... 47 Experimental procedures .............................................................................................. 51 Results ........................................................................................................................... 54 Discussion ..................................................................................................................... 62 CHAPTER 3: SMGGDS IS A GUANINE NUCLEOTIDE EXCHANGE FACTOR THAT SPECIFICALLY ACTIVATES RHOA AND RHOC .......................................... 74 Background ................................................................................................................... 74 Experimental procedures .............................................................................................. 76 Results ........................................................................................................................... 80 Discussion ..................................................................................................................... 88 CHAPTER 4: CONCLUSIONS AND FUTURE DIRECTIONS ................................. 105 Conclusions ................................................................................................................. 105 Future directions ......................................................................................................... 107 REFERENCES ............................................................................................................... 110 vii LIST OF TABLES Table 1: Multiple mutations in RhoA have no effect on SmgGDS catalyzed exchange. ……..104 viii LIST OF FIGURES Figure 1: The Ras superfamily of small GTPases. ........................................................... 42 Figure 2: The ability of a GTPase to bind nucleotides and alter switch region conformation derives from its structure. ........................................................................... 43 Figure 3: GTPases alter their nucleotide state through a dynamic cycle with the help of regulatory proteins. ........................................................................................................... 44 Figure 4: The DOCK family of Rho GEFs. ...................................................................... 45 Figure 5: DOCK family members share a conserved core domain structure. .................. 46 Figure 6: Cloning and expression of DOCK9 yields soluble C-terminal constructs. ....... 66 Figure 7: DOCK9 CT forms a complex with nucleotide-depleted Cdc42. ....................... 67 Figure 8: DOCK9 CT specifically activates Cdc42 in a concentration dependent manner. ........................................................................................................................................... 68 Figure 9: Purification scheme for crystal trial