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Elucidating the Ras activation pathways in response to cyclic AMP in the aggregation of Dictyostelium… Kae, Helmut 2007

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E L U C I D A T I N G T H E R A S A C T I V A T I O N P A T H W A Y S IN R E S P O N S E T O C Y C L I C A M P I N T H E A G G R E G A T I O N O F DICTYOSTELIUM DISCOIDEUM by H E L M U T K A E B . S c , The University of British Columbia, 2000 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Microbiology and Immunology) T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A August 2007 © Helmut Kae, 2007 ABSTRACT Ras proteins are highly conserved molecular switches that regulate cellular responses to external stimuli. Dictyostelium discoideum contains an extensive family of Ras proteins that function in growth, cell type differentiation, and more. While there is much genetic evidence for the involvement of Ras proteins in these behaviours, little is known about the events that lead to their activation. The primary objective of this thesis project was to develop an efficient and rapid assay that allowed the measurement of activated Dictyostelium Ras proteins. Modification of an assay used successfully to measure activated Ras in mammalian cells demonstrated that the Ras Binding Domain (RBD) of the Schizosaccharomyces pombe Ras effector protein, Byr2, was able to bind to RasB, RasC, arid RasG. Furthermore, the use of dominant negative Ras mutants and nucleotide loading of cellular lysates showed that Byr2 (RBD) bound preferentially to Ras-GTP (activated Ras) over Ras-GDP (quiescent Ras). The ' R B D binding assay' was applied to elucidate the Ras activation pathways stimulated by c A M P during Dictyostelium aggregation. Previous experiments have suggested that RasC is involved in c A M P stimulated signalling events during Dictyostelium aggregation, and this was confirmed using the R B D binding assay, as levels of RasC-GTP. displayed a rapid and transient increase upon c A M P stimulation. Similarly, activated RasG also showed an increase in activation in response to c A M P . Through the use of various mutant strains, it was demonstrated that RasC and RasG activation is downstream of the c A M P receptor and its associated heterotrimeric G-protein. This thesis also provides evidence that the Dictyostelium Ras guanine nucleotide exchange factor (RasGEF) proteins can each act on specific Ras proteins. RasC activation was 11 abolished in a gefA mutant, while RasG activation was normal, indicating that R a s G E F A activates RasC, but not RasG. Conversely, RasC activation was normal in geJM and gefR~ mutants, whereas RasG activation was reduced in each strain, indicating that R a s G E F M and RasGEFR activate RasG. Analysis of P K B activation suggests that R a s G E F M is necessary for the activation of RasG in the pathways leading to P K B activation. Observing the streaming behaviour of aggregating gefR cells has suggested that RasGEFR may play a role in regulating chemotaxis. i i i "If we could just tap into the collective unconscious of the amoeba — then we *d know how it all began," Printed with permission from Sidney Harris © S. Harris T A B L E O F C O N T E N T S A B S T R A C T i i T A B L E O F C O N T E N T S v L I S T O F T A B L E S v i i i L I S T O F F I G U R E S ix A C K N O W L E D G E M E N T S x i i 1 I N T R O D U C T I O N 1 1.1 Ras Superfamily 1 1.2 Ras as a molecular switch 2 1.2.1 Monomeric GTPases 2 1.2.2 Heterotrimeric GTPases 3 1.3 Activators of Ras proteins 6 1.4 Inhibitors of Ras activation 8 1.5 Sub-cellular localization of Ras 9 1.6 Ras activation by extracellular ligands 10 1.7 Ras effector signalling pathways 12 1.8 Dictyostelium as a model organism 14 1.9 The life cycle of Dictyostelium discoideum 15 1.10 Dictyostelium Ras proteins 18 1.11 c A M P signalling during Dictyostelium aggregation 23 1.12 Involvement of Ras in aggregation 29 1.13 Thesis Objective 30 v 2 M A T E R I A L S A N D M E T H O D S 32 2.1 Materials 32 2.1.1 Reagents 32 2.1.2 Services 32 2.1.3 Dictyostelium strains 34 2.1.4 Plasmids and D N A 34 2.2 Methods 35 2.2.1 Cell culture and development of Dictyostelium discoideum 35 2.2.2 Inducible expression of activated Ras proteins 36 2.2.3 Preparation of pulsed cells 36 2.2.4 Cloning of GST-Byr2 (RBD) 37 2.2.5 Expression of G S T - R B D fusion proteins 39 2.2.6 Purification of G S T - R B D fusion proteins 39 2.2.7 R B D Binding Assay 40 2.2.8 Guanine nucleotide loading of Ras proteins 41 2.2.9 P K B phosphorylation assay 42 2.2.10 Immunoblot Analysis 42 3 R E S U L T S 44 3.1 Selection, cloning, and induction of G S T - R B D bacterial constructs 44 3.2 R B D interaction with endogenous levels of Ras 53 3.3 Optimizing protein load for a R B D pull down assay 55 3.4 Chemoattractant induced activation of RasC and RasG 57 vi 3.5 Ras activation is dependent on the c A M P receptor complex 59 3.6 Ras activation is independent of A C A , C R A C , and PiaA 61 3.7 R a s G E F A mediates activation of RasC 64 3.8 RasGEFR is required for maximal c A M P stimulated activation of RasG 67 3.9 R a s G E F M is required for maximal c A M P stimulated activation of RasG 71 3.10 R a s G E F C and R a s G E F D are not involved in c A M P stimulated activation of Ras 74 3.11 Over-expression of GbpD leads to an increase in activated R a p l , RasC 76 4 D I S C U S S I O N 78 4.1 Implications of the R B D Binding Assay as a tool for studying Ras activation 78 4.2 c A M P and Ras signalling 82 4.3 The involvement of G P C R in Ras activation 84 4.4 Role of RasGEFs in Ras activation 86 4.5 Ras activation and the adaptation response 95 4.6 Regulation of the cytoskeleton during aggregation by Ras 98 4.7 A n emerging model of Ras signalling during aggregation 101 4.8 Thesis synopsis and future directions 104 B I B L I O G R A P H Y 108 LIST OF TABLES Table 1. Summary of Dictyostelium Ras proteins and their proposed function 19 Table 2. Dictyostelium strain descriptions 33 Table 3. Predicted mass of G S T fusion proteins 46 Table 4. Observed rate constant, k o b s , of R a s G E F stimulated nucleotide exchange 66 v i i i LIST OF FIGURES F i g u r e 1. T h e G T P a s e as a c e l l u l a r s w i t c h 4 F i g u r e 2 . A l i g n m e n t o f R a s p r o t e i n s e q u e n c e s 5 F i g u r e 3 . T h e l i f e c y c l e o f Dictyostelium disco'uleum 17 F i g u r e 4 . T h e c A M P s t i m u l a t e d p a t h w a y s d u r i n g Dictyostelium a g g r e g a t i o n 2 3 F i g u r e 5 . T h e R B D B i n d i n g A s s a y 4 5 F i g u r e 6 . E x p r e s s i o n a n d p u r i f i c a t i o n o f G S T - R B D f u s i o n p r o t e i n s 4 7 F i g u r e 7 . B i n d i n g o f R B D s t o a c t i v a t e d R a s m u t a n t s 4 9 F i g u r e 8 . B i n d i n g o f G S T - R a f l a n d G S T - B y r 2 t o a c t i v a t i n g a n d d o m i n a n t n e g a t i v e R a s G m u t a n t s 5 2 F i g u r e 9 . G T P d e p e n d e n c e o f R B D - R a s i n t e r a c t i o n f r o m e n d o g e n o u s l e v e l s o f R a s 5 4 F i g u r e 1 0 . E f f e c t o f i n p u t p r o t e i n o n R B D b i n d i n g a s s a y 5 6 F i g u r e 1 1 . c A M P s t i m u l a t e d R a s a c t i v a t i o n 5 8 F i g u r e 1 2 . T h e c A M P r e c e p t o r c o m p l e x is r e q u i r e d f o r R a s a c t i v a t i o n 6 0 F i g u r e 1 3 . R a s a c t i v a t i o n i s u p s t r e a m o f A C A a n d i ts c y t o s o l i c r e g u l a t o r s 6 3 F i g u r e 1 4 . gef'A' m u t a n t s a r e d e f i c i e n t i n R a s C a c t i v a t i o n 6 5 F i g u r e 1 5 . L o s s o f R a s G E F R r e d u c e s a c t i v a t i o n o f R a s G d u r i n g d e v e l o p m e n t 6 8 F i g u r e 1 6 . S t r e a m i n g b e h a v i o u r o f A x 3 a n d gefK c e l l s d u r i n g a g g r e g a t i o n 6 9 F i g u r e 17 . c A M P i n d u c e d s t i m u l a t i o n o f P K B p h o s p h o r y l a t i o n 7 1 F i g u r e 1 8 . L o s s o f R a s G E F M r e d u c e s a c t i v a t i o n o f R a s G d u r i n g d e v e l o p m e n t 7 3 F i g u r e 1 9 . L o s s o f R a s G E F C o r R a s G E F D d o e s n o t a f f e c t R a s a c t i v a t i o n d u r i n g a g g r e g a t i o n 7 5 F i g u r e 2 0 . A c t i v a t i o n o f R a s p r o t e i n s b y G b p D in vivo 7 7 F i g u r e 2 1 . A l i g n m e n t o f R a s G E F p r o t e i n s e q u e n c e s 9 2 F i g u r e 2 2 . P r o p o s e d m o d e l f o r t h e i n v o l v e m e n t o f R a s C a n d R a s G i n c A M P s t i m u l a t e d e v e n t s d u r i n g Dictyostelium a g g r e g a t i o n 1 0 3 I X L I S T OF A B B R E V I A T I O N S A ampere or amp, unit measure for electric current A C A adenylyl cyclase A M P adenosine monophosphate ATP adenosine triphosphate b, bp base, base pair cAMP adenosine 3 ' ,5 ' -cycl ic monophosphate cAR cAMP receptor CFP cyan fluorescent protein cGMP guanosine 3',5'-cyclic monophosphate C R A C cytosolic regulator of adenylyl cyclase D A G diacylglycerol dNTP deoxynucleoside triphosphate EDTA ethylene diamine tetraacetic acid EGF epidermal growth factor EGFR epidermal growth factor receptor E R K extracellular regulated protein kinase g gravity, unit measure for centrifugal force GAP GTPase activating protein GC guanylyl cyclase GDP guanosine 5'-diphosphate GEF guanine nucleotide exchange factor GFP green fluorescent protein GPCR G-protein coupled receptor GST glutathione-S-transferase GTP guanosine 5'-triphosphate GTPyS non-hydrolysable GTP analogue h hour(s) HRP horseradish peroxidase H V R hypervariable region kDa kilo Dalton KK2 potassium based non-nutrient buffer L P A lysophosphatidic acid M A P mitogen activated protein M E K M A P K / E R K kinase mGDP fluorescent GDP analogue min minute(s) PAGE polyacrylamide gel electrophoresis PH pleckstrin homology PI(4,5)P2 phosphatidylinositol 4,5-bisphosphate PI3,4,5)P3 phosphatidylinositol 3,4,5-trisphosphate PI3K phosphoinositide 3-kinase P K A protein kinase A P K B protein kinase B PKC protein kinase C PLC phospholipase C PTEN phosphatase and tensin homolog PTK protein tyrosine kinase RBD Ras binding domain RGS regulator of G-protein signalling rpm revolutions per minute R T K receptor tyrosine kinase s second(s) SDS sodium dodecyl sulphate SH2 src homology 2 SH3 src homology 3 SOS Son of sevenless, a RasGEF TLC thin layer chromatography V volt(s), unit measure for electric potential YFP yellow fluorescent protein x i ACKNOWLEDGEMENTS I can clearly remember sitting in my third year industrial microbiology class, realizing that much of what is known about yeast genetics came about because we wanted to make better tasting beers. And that was the moment that my appetite for scientific research was first whetted. Many years, many beers, and many tears later, the long haul of my post-graduate degree is finally coming to a conclusion. And of course, there are many people who helped me along the way. I'm not sure if I am writing this if my lovely and caring wife, Amy, wasn't with me every step of the way. Always there with heartfelt encouragement and thoughtful motivation, she is the driving force behind all of the positive things that have happened to me since I have met her. I can only hope to provide her with the same type of support that she has shown me through the years. And to my L i l ' Man, for helping me keep my life in perspective, and for teaching me more than I could possibly teach him. To my parents, for always believing in me no matter the circumstances, but also for providing the tough words that are necessary to motivate the young and aimless. Thank you, because without your guidance I would not be here. To my brothers, for their camaraderie, and for keeping me grounded in a way that only my brothers can do. To Pops and Wah Wah, thank you for treating me like your son, and asking the "strange" questions that only make sense after a few days of thinking about them. To our "Lab Mom," Meenal, for keeping the Weeks lab running in a smooth and orderly fashion. Thanks Parvin, for all the support and encouragement, and the wonderful discussions about Dicty Ras research, sugary treats, and life in general that I will never forget. To my mentor, James, who taught me most of what I know, and whose fingerprints can be seen on everything that I do in the lab. Thanks to Michelle and Secko for being great friends, not just great colleagues. To Brent, who only wants to tell the truth, no matter if you want to hear it or not. To Rujun, Hermia, Zahara, Hanne, and even Megan, whose time with me was short, but appreciated. And to the unheralded work of our work study students, Minnette, Fariba, Tina, and Gigi, I think our lab would grind to a halt without them. To the members of my supervisory committee, Cal and Bob, for your words of encouragement, and offerings of constructive criticism. Big thanks to my co-supervisor, George, for your endless supply of questions, as they have left a mark on how I conduct myself today, and for being the best undergrad teacher that I have had. Big thanks to my supervisor, Gerry, for taking a chance on a slightly older than average 4 t h year student with average marks. And while I appreciate the kind words when things were going well, I will always remember the encouraging words when things were going not-so-well. Mahalo. xii 1 INTRODUCTION 1.1 Ras Superfamily The Ras Superfamily of proteins comprises a family of small (20-40 kDa) G T P binding proteins. Members of this superfamily are highly conserved, and orthologs are found in all i metazoans, yeast, slime mould, and plants (Takai et al., 2001). The Ras superfamily can be subdivided into 5 subfamilies on the basis of structural and functional similarities: Ras (gene expression, proliferation, differentiation); Rho (cytoskeletal rearrangement); Rab and A r f (vesicular transport, membrane trafficking); R a n (nuclear trafficking) (reviewed in Takai et al., 2001). The genes encoding Ras superfamily proteins are often mutated in cancers (Bourne et al., 1991), and thus Ras proteins have become a target for potential therapeutic agents (Fritz and Kaina, 2006; Shirai et al., 2007). Proteins are grouped in the Ras superfamily i f they share -30% amino acid identity, specific conserved domains for G T P binding, and a small domain that is necessary for binding to downstream effector proteins known as the 'effector domain' (Bourne et al., 1991). Ras, Rho, and Rab members have a G-terminal motif that undergoes post-translational prenylation, serving as a membrane anchor (Section 1.5). A r f proteins have an N-terminal glycine residue that is modified with myristic acid and serves the same purpose as C-terminal prenylation, while Ran proteins are not subject to such post-translational modifications (Takai et al., 2001). The Ras subfamily proteins were the first characterized members of the Ras superfamily, and have been the subject of intensive research due to their prominent involvement in oncogenesis. For the most part, Ras subfamily proteins serve to transduce extracellular signals into a wide array of intracellular signalling pathways by interacting with a diverse and catalytically distinct group of effector proteins. Members of the subfamily share -50% amino 1 acid identity, and include H-Ras, N-Ras, K-Ras, R-Ras, Rap, Ral , and Rheb (Colicelli , 2004). The remainder of this thesis wi l l deal with the topic of the Ras subfamily proteins, and hence the term 'Ras' w i l l be used in reference to the Ras subfamily, unless otherwise noted. 1.2 R a s a s a m o l e c u l a r s w i t c h The function of Ras GTPases is to couple extracellular signals into intracellular responses. This function is carried out by adopting two conformations, one of which allows for transmission of the extracellular signal, whereas the other conformation does not. Thus, Ras GTPases are often referred to as molecular switches in that they function in a way that is analogous to a household light switch. 1.2.1 M o n o m e r i c G T P a s e s The signalling function of Ras is carried out by cycling between a G D P bound and a G T P bound conformation (Figure 1A). G D P release from Ras results in the binding of G T P due to the fact that G T P is more abundant in the cell (Van Dyke et al., 1977), and binding of G T P results in a conformational change to two regions of Ras referred to as Switch 1 (residues 21-40 relative to H-Ras) and Switch 2 (residues 57-75) (Figure 2) (Quilliam et al., 2002). As this conformational change facilitates binding of Ras with its various downstream effector proteins, Ras-GTP is typically viewed as being turned 'On ' or activated. Hydrolysis of G T P to G D P by Ras returns Ras-GTP to Ras-GDP, effectively turning Ras 'Off. ' Interconversion between Ras-GDP and Ras-GTP can happen spontaneously, but the half-times of these reactions are typically too slow to be physiologically relevant (Neal et al., 1988). Thus, the Ras activation cycle is. catalyzed by two groups of proteins: guanine nucleotide exchange factors (GEFs) promote the release of G D P , and GTPase activating proteins (GAPs) enhance the GTPase activity of Ras-GTP. Therefore, the 2 concerted actions of these two groups of proteins are responsible for regulating the Ras activation cycle. 1.2.2 H e t e r o t r i m e r i c G T P a s e s The alpha subunit of the heterotrimeric G-protein family is related to the Ras protein superfamily, with a G T P binding domain that is structurally identical (Hamm, 1998). Given the importance of G-protein mediated signalling in Dictyostelium (Section 1.11), a brief description of the heterotrimeric GTPase cycle wi l l be presented (Figure IB) . Heterotrimeric G-proteins are trimers consisting of alpha, beta, and gamma subunits (abbreviated as G a , Gp\ and Gy, respectively) and are associated with G-protein coupled receptors (GPCR) . G P C R s are a large family of proteins with a canonical serpentine seven transmembrane domain, an intracellular cytoplasmic tail, and an extracellular ligand binding domain (reviewed in McCudden et al., 2005). Binding of a ligand induces activation of the G a subunit by guanine nucleotide exchange, which is the G T P binding protein that also functions to couple the heterotrimeric G -protein to its G P C R , and activation of G a leads to its dissociation from the G P y dimer. G a and G(3y can regulate signalling pathways independently, leading to the activation of enzymes such as adenylyl cyclases, phospholipases, and ion channels (Marinissen and Gutkind, 2001). The cycle is complete when G T P is hydrolysed to G D P , catalyzed by a group of proteins referred to as Regulators of G-protein signalling, or R G S . G a - G D P then reassociates with G(3y, terminating the signal. 3 Activating Signal B Activating Signal Guanine nucleotide Exchange Eactor (GEF) G-Protein Coupled Receptor (GPCR) "OFF" "ON" Downstream Downstream Downstream Signalling Signalling Signalling GTPase Activating Protein (GAP) Regulator of G-Protein Signalling (RGS) Figure 1. The GTPase as a cellular switch. (A) Model depicting the GTPase cycle of monomeric Ras superfamily proteins. Stimulation by an activating signal leads to displacement of G D P (depicted as two yellow circles) by G T P (depicted as three yellow circles), catalyzed by guanine nucleotide exchange factors (GEFs). G T P binding to Ras promotes a structural change facilitating the binding of Ras to various downstream effectors, propagating the signal. Ras is subsequently turned off through the action of GTPase activating proteins (GAPs) , which enhance the intrinsic GTPase activity of Ras proteins. (B) Model depicting the GTPase cycle of heterotrimeric G-proteins. Heterotrimeric G-proteins consist of G a , Gp\ and G y subunits. G a couples the G-protein to a serpentine G-protein coupled receptor (GPCR) , and G a - G D P is associated with G|3 and Gy, which functions as a dimer. Agonist binding to the G P C R , results in displacement of G D P by G T P to G a , and G a - G T P dissociates from the Gpy dimer. Both subunits can participate in signalling events. The cycle is completed by regulators of G-protein signalling, which catalyze the G a GTPase. 4 HRas RasD RasG RasB RasC RasS Rapi HRas RasD RasG • RasB .RasC RasS Rapl HRas Ml EYKLWV GAGGVGKS RasD — : — I - -G RasG . . __i> -G RasB - S V S N — — M - G - • RasC -S K L L - - - I - - D — — RasS - F . N F — L - - p — - - -Rapl '—PX R-F-I — L -S -VIAA " A A A A A A"AA-Af A A A A A V C W ALTIQLIQNHFVDEYDPTIEDSYRKQWIDGETCLLD ^: _ _ _ s — D — — - - T — Q - I A N — - — N — E - V Y M - - -C F-AQK— • — L : TTV E V-FV-GI—EK EV-SNQ-M-E A A A A A A A A A A A A A A A A A A A 51 OOOOOO EYSAMRDQYMRTGEGFLCVFAINOT ._ _ _ _ _ _ Q YS-TSRS-YDE-ASF — L - - — K - R — L - : - - Q - - YS-TSRS—DE-ASF- L—•—K-R D •—Q— YDVTSRT—E-NW- I——N--K--I . — I - S - R — - I - Y S - T S R A — - A V T T F — - L - - - - L S T Y - I OF--V— - : YS-TYLQ—KE—RLHNHLLK-™L-S--F QFT----L~-KN-Q~~V-~YS-ISNST:-NELPDL^^ L AA RTVESRQAQDLARSYGIP YI - DHE-Q-SVNEG-E-—KD-SLS FH - ESD-Q-TTGEG K-F-S-: FL - ENL-E-TEGEGSE--K-FSV- FL -PDKD-K-PPMEGKE—K-F-A- FL - NEY-E-STAEGBE—KKLNCK FL - HDQ-VISTE-GEE—-KF-DCYFL TRQGVEDAFYTLVREIR S.-IN—E—— S ~ — -I-VN--E s—-—. K-LN-DEC-FEV——K S-VN— E — F — K E - IN-SES—E VK NKVN—QI—N-I-Q-N HRas QHKLRKLNPPDESGP GCMSCK CVLS 18 9 RasD KE-KGDQSSGKAQKKKKQ - L I L • 186 RasG KD-KGDSK-EKGKXKRPLKA - T - L 189 Ra38 KS-KEPGRSKKDKK.-GI LKKFKGGD - L I L 197 RasC RWNQNPQ-EEMLPPKKRG - I I L 189 RasS KARQSNQHSNSGEQNTDQPIKKKKS -N-L 194 Rapl RKNPVGPP SKAK-K - A - L 186 54 54 54 .57 55 54 56 111 111 111 114 112 111 113 164 164 165 168 167: 165 168 Figure 2. Al ignment of Ras protein sequences. Gapped alignment of protein sequences of 6 Dictyostelium Ras subfamily proteins (RasB, RasC, RasD, RasG, RasS, Rapl ) with mammalian H-Ras. Numbers indicate position of amino acids in protein, dashes indicate amino acids that are identical to those in H-Ras, and boxes refer to the conserved GTP-binding domain. Residues marked with a (*) represent the Switch 1 region, residues marked with a (A) represent the Switch 2 region, and residues marked with a (o) represent the oc-helix 3 region. Figure modified from Daniel etal . (1995). 5 1.3 A c t i v a t o r s o f R a s p r o t e i n s The first RasGEF (CDC25p) was discovered in Saccharomyces cerevisiae in a genetic screen for components upstream of Ras (Broek et al., 1987). Biochemical confirmation of nucleotide exchange activity was shown a few years later (Jones et al., 1991). The first mammalian RasGEF cloned was SOS (Bowtell et al., 1992), discovered as a homologue of the Drosophila RasGEF, followed closely by the discoveries of the RasGEF, C D C 2 5 M m (also known as R a s G R F l ) (Martegani et al., 1992), the Ra lGEF , Ra lGDS (Albright et al., 1993), and the RapGEF, C 3 G (Knudsen et al., 1994). As mutations in Ras contribute to a large number of diseases, it should come as no surprise that several RasGEFs have been implicated as well . Renal carcinoma cell lines display increased Ras activation, and the amplification of SOS has been suggested to play a role in this process (Shinohara et al., 1997). Insertiortal mutagenesis has revealed that amplification of the RapGEF, GRP2 , induces acute myeloid leukemia in mice (Dupuy et al., 2001). Given that RasGEFs are often the limiting factor in Ras protein activation, their study has received much attention. While RasGEFs are typically multi-domain containing proteins, one common feature is the presence of a catalytic domain with significant homology to CDC25p , and this domain is often referred to as the C D C 2 5 domain. Sequence alignment of the -250 residue domain was found to contain five structurally conserved regions (scrl-5) (Boguski and McCormick , 1993), and solution of a crystal structure of a SOS-Ras complex revealed that scrl-3 form the Ras binding pocket (Boriack-Sjodin et al., 1998). RasGEFs preferentially bind to Ras-GDP over Ras-GTP, and three regions of Ras have been implicated in the RasGEF-Ras interaction: Switch 1, Switch 2, and a-helix 3 (residues 102-107) (Figure 2). Mutational analysis has revealed that 6 the Switch 2 region is important for tight binding to the RasGEF, and this binding results in the displacement of Switch 1, leading to the disruption of the nucleotide binding site and ultimately to G D P release (Hall et al., 2001; Wittinghofer, 1998). The family of RasGEFs is diverse and can be grouped into more than 10 different classes based on substrate specificity and structural similarities. SOS is ubiquitously expressed throughout mammalian tissues and cells, and believed to be the most commonly employed RasGEF, activating Ras in response to various growth factors via receptor tyrosine kinases (RTKs) , G P C R s , and immune cell receptor activation (Nimnual and Bar-Sagi, 2002). RasGEFs can also be regulated by intracellular secondary messengers, creating a network of intertwined signalling pathways. For example, R a s G R F l , expressed primarily in brain tissue, is regulated by the C a 2 + binding protein calmodulin through an IQ (ilimaquinone) domain at its N-terminus (Farnsworth et al., 1995). The RasGRP family is regulated by binding to diacylglycerol ( D A G ) , which serves to translocate RasGRP to the membrane. RasGRP is downstream of Phospholipase C y-1 activation, and has been implicated in the growth and development of immune cells (Dower et al., 2000; Ebinu et al., 2000). The Epac (also referred to as c A M P - G E F ) class of RasGEFs were isolated in a search of the human genome for sequences that contained both a RasGEF domain and a c A M P binding domain, and it has been shown in C H O cells that c A M P induces Rap l activation by Epac l (de Rooij et al., 1998). RasGEFs can also serve as dual specificity GEFs , catalyzing the activation of more than one GTPase. SOS, for example, contains a catalytically active RacGEF domain as well as its RasGEF domain, and expression of activated Ras (G12T) in COS-1 cells stimulates the RacGEF activity of SOS, suggesting that the R a c G E F activity of SOS is regulated by its RasGEF activity (Nimnual et al., 1998). The dual specificity of SOS provides a mechanism for the regulation of Rac mediated cytoskeletal 7 rearrangement by Ras, and in general, dual specificity GEFs can link GTPase pathways, creating a network o f GTPase signalling cascades. 1.4 I n h i b i t o r s o f R a s a c t i v a t i o n While much is known about GEFs and their role as regulators of Ras function, less is known about the G A P s that inhibit Ras. The reason for this is that G A P s were often thought to play a secondary role in Ras signalling, with their function being considered less important than that of the GEFs . For the most part, G A P s for the different Ras subfamilies don't share much sequence similarities, whereas G A P s for Ras proteins within each subfamily are highly related (Boguski and McCormick, 1993). As a result of this similarity, G A P s are predicted to exhibit a high substrate preference for members of one Ras subfamily. Analysis of a crystal structure of a RasGAP-Ras-GTP complex revealed that RasGAPs bind to Ras-GTP at the Switch 1 and Switch 2 regions via a -220 amino acid catalytic domain (Scheffzek et al., 1997). A n 'arginine finger' (R789 in mammalian p l 2 0 G A P ) that is conserved in all RasGAPs (Bernards, 2003) inserts itself into the active site of Ras to help stabilize the transition state formed during G T P hydrolysis (Scheffzek et al., 1997). Not much is known about how R a s G A P activity is regulated in vivo, but like RasGEFs, R a s G A P regulation is diversified (reviewed in Bernards, 2003). The most well studied mammalian R a s G A P is p i20 RasGAP, also referred to simply as RasGAP, a multidomain protein that includes SH2 (src homology 2), SH3 (src homology 3), and P H (pleckstrin homology) domains outside of the G A P catalytic domain. One study demonstrated that the SH2 domain mediates the recruitment of p i20 R a s G A P to activated R T K s in C H O cell lines (Kaplan 8 et al., 1990), although how this affects p l 2 0 R a s G A P activity is unknown. However, i f this translocation does facilitate G A P activation, this mechanism implies that the activation of Ras is coupled to the activation of its negative regulator since R T K s are often upstream of Ras activation. The R h o G A P , p l 9 0 B , is regulated by phosphorylation, and it has been shown in mouse embryo-derived fibroblasts that p l 9 0 B can be directly phosphorylated by the insulin receptor IGF-1 (Sordella et al., 2003). Phosphorylation of p l 9 0 B induces the translocation of p l90B to l ipid raft containing plasma membrane, where activated Rho has been shown to accumulate. Again, this mechanism joins activation of a GTPase with the activation of its G A P . 1.5 S u b - c e l l u l a r l o c a l i z a t i o n o f R a s The C-terminal sequences of Ras proteins are highly divergent, and hence the C-terminal -25 amino acids of Ras is often called the hypervariable region ( H V R ) (Takai et al., 2001). This is also the domain that is responsible for membrane anchoring of Ras. The 4 C-terminal amino acid residues are referred to as the CaaX motif, where ' C is a cysteine, 'a ' refers to an aliphatic amino acid, arid ' X ' is any amino acid residue. Isoprenoids, such as farnesyl or geranylgeranyl, are incorporated at the cysteine residue following cleavage of the last three amino acids, and the nature of the prenylation motif can influence the localization of Ras (Pechlivanis and Kuhlmann, 2006). Fatty acids, especially palmitate, can also be added to Ras proteins at cysteine residues other than the terminal cysteine, also contributing to sub-cellular localization (Schroeder et al., 1997). The fact that the H V R is so divergent suggests that Ras proteins exhibit different sub-cellular localization, and this has been shown to be true. H-Ras is associated equally with lipid raft and non-raft plasma membrane, and activation of H-Ras results in a shift to non-raft 9 membrane (Chen and Resh, 2001; Prior et al., 2001). K-Ras, on the other hand, is localized exclusively to non-raft plasma membrane (Prior et al., 2001), providing two separate platforms for the function of H-Ras and K-Ras. Ras activation has also been observed in non-plasma membrane domains! While Ras proteins have been visualized on the Golgi and E R membranes (Choy et al., 1999), it was not initially clear whether they contributed to signalling or exist there temporarily while being shuttled to the plasma membrane. The advent of fluorescent probes that can monitor Ras activation in live cells has demonstrated that the former is correct. E G F stimulation of COS-1 cells expressing wi ld type H-Ras demonstrated that Ras activation initially occurs at the plasma membrane, followed by a subsequent activation of Ras at the Golgi membrane (Chiu et al., 2002), demonstrating that Ras protein activation is not1 limited to the plasma membrane. 1.6 R a s a c t i v a t i o n b y e x t r a c e l l u l a r l i g a n d s The canonical mammalian Ras activation pathway is that of the epidermal growth factor receptor (EGFR) , belonging to the R T K family of proteins (Tan and K i m , 1999). R T K s consist of an extracellular domain required for ligand binding, one transmembrane domain that anchors the receptor to the membrane, and a cytoplasmic catalytic domain. The general mechanism for R T K activation begins with ligand binding, which induces homo-dimerization of the R T K and activation of the cytoplasmic kinase domain (reviewed in Olson and Marais, 2000). Activation of the kinase results in tyrosine phosphorylation of the cytoplasmic tails, and these phosphorylated tyrosines serve as docking sites for SH2 domain containing proteins, in particular Grb2, translocating these proteins from the cytosol to the activated receptor at the membrane. Grb2 is an adaptor protein consisting of a SH2 domain flanked by two SH3 domains, one of 10 which is constitutively associated with the RasGEF, SOS, through an interaction with a proline rich domain found at the C-terminus of SOS. If Ras is co-localized with the E G F R , translocation of Grb2 to the E G F R effectively serves to bring SOS in close proximity to Ras, allowing SOS to catalyze nucleotide exchange, thereby activating Ras. Ras activation can also be mediated by antigen receptors such as those found on B-cel l and T-cell lymphocytes (Downward et al., 1990). These receptors are hetero-dimers consisting of a large extracellular fragment, one transmembrane domain, and a short cytoplasmic tail. Ligand binding to the receptor recruits cellular protein tyrosine kinases (PTKs), which phosphorylate the cytoplasmic tails on tyrosine residues referred to as I T A M s (immunoreceptor tyrosine-based activation motifs). These phosphorylated tyrosine residues serve as docking sites for various SH2 domain containing proteins, ultimately leading to the recruitment of the Grb2-SOS complex to the membrane. A third Ras activation pathway begins with G P C R s . One of the first G P C R agonists connected to Ras activation was the mitogen Lysophosphatidic acid ( L P A ) (Luttrell et al., 1997). The mechanism by which L P A binding to its receptor activates Ras is unclear, but a number of the signalling components have been elucidated. One model that has been proposed is referred to as 'transactivation' (Marinissen and Gutkind, 2001). This involves the activation of cellular PTKs , such as Src or Pyk2, which phosphorylate the E G F receptor, leading to recruitment of the Grb2-SOS complex, and activating Ras. Another pathway involves PI3K signalling. Yart et al. (2002) demonstrated that blocking PI3Ky activity in Vero kidney epithelial cell lines through the use of dominant negative mutants results in a significant reduction in L P A stimulated Ras activation. Based on the reduction it was postulated that p l l O y activity generates phosphatidylinositol (3,4,5) trisphosphate (PI(3,4,5)P3) at the membrane, which serve as docking 11 sites for the adaptor protein G a b l , and in turn leads to the recruitment of the Grb2-SOS complex to the site of Ras. 1.7 Ras effector signalling pathways A large number of proteins have been characterized as Ras effector proteins, regulating many cellular processes. The first mammalian Ras effector, Raff, was isolated in a yeast two-hybrid screen (Vojtek et al., 1993). Ra f l is a serine/threonine kinase, and it was demonstrated that Ra f l interacted with H-Ras (G12T), but not H-Ras (S17N) (Vojtek et al., 1993). R a f l is recruited to the plasma membrane upon receptor stimulated Ras activation, and activated Raf l initiates a phosphorylation cascade that proceeds to M E K , and then leads to activation of the E R K proteins (Arbabi and Maier, 2002). Activated E R K s then translocate to the nucleus to activate various transcription factors such as E l k l and J U N , coupling Ras activation at the membrane to gene expression. Another well characterized Ras effector is Phosphoinositide 3-Kinase (PI3K) (reviewed in Katso et al., 2001). A lipid kinase, activated PI3K phosphorylates P I (4 ,5 )P2 at the 3' position to generate PI(3,4,5)P3. PI(3,4,5)P3 serves as a binding site for pleckstrin homology (PH) domain containing proteins, leading to the translocation of these proteins, usually from the cytosol, to the site of PI3K activity (Blomberg et al., 1999). A n important effector of PI3K is the protein kinase P K B , also referred to as Akt (Vanhaesebroeck and Waterfield, 1999). While P H domain dependent translocation is required for P K B to function (Bellacosa et al., 1998), activation of P K B is stimulated by phosphorylation on two sites, T308 and S473. Phosphorylation on T308 is mediated by the P H domain containing kinase, P D K 1 (Alessi et al., 1997), and phosphorylation on S473 is mediated by a complex of proteins, T O R Complex 2 or 12 T 0 R C 2 (Sarbassov et al., 2005). Activated P K B then phosphorylates downstream target proteins, including the pro-apoptotic group of transcription factors of the Forkhead family, inhibiting their function (Dijkers et al., 2002). PI3K products have also been shown to regulate RacGEF activity both in vitro (Han et al., 1998) and in vivo in COS-7 cell lines (Michiels et al., 1997), providing a mechanism for the role of PI3K in cell motility by inducing cytoskeletal rearrangement. The R a l G D S family, which includes R a l G D S , Rgl , Rlf, and Rgr, functions as G E F s for the Ral family of GTPases, and it has been demonstrated that Ra lGDS promotes the activation of Ral in COS-7 cells expressing activated H-Ras, but not activated R-Ras or Rap l (Urano et al., 1996), thereby stimulating mitogenesis. Phospholipase Ce (PLCe) is another important Ras effector. Expressing activated H-Ras in COS-7 cells stimulates P L C e activity (Kelley et al., 2001). The P L C family of proteins, of which only P L C e harbours a R B D , cleaves PI(4,5)P 2 at the plasma membrane to generate two important intracellular second messengers, diacylglycerol ( D A G ) and inositol (1,4,5) trisphosphate (IP3). D A G subsequently leads to the activation of the protein kinase P K C , whereas JP3 stimulates the increase of intracellular C a 2 + . Thus, Ras can integrate D A G and IP3 stimulated pathways through the activation of PLCe . Other examples of Ras effectors are A F 6 (inhibitor of cell proliferation in H E K 2 9 3 cell lines;(Radziwill et al., 2003), Nore l (tumor suppressor in A549 human lung tumor cell lines; (Vos et al., 2003), and p l 2 0 R a s G A P (Ridley et al., 1993), which has been implicated in cytoskeletal rearrangement via the activation of the Rho family GTPases in Swiss 3T3 fibroblast cell lines. Thus, the cellular role of Ras involves a complex balance of a number of signalling pathways. 13 1.8 Dictyostelium as a model organism Dictyostelium discoideum is a soil l iving amoeba where it feeds on bacteria in the wild, and is affectionately referred to as the 'social amoeba' because starvation initiates a developmental process that unites the normally solitary amoeba into a multicellular fruiting body containing -100,000 cells. Belonging to the class Mycetozoa, r R N A analysis suggests that early Dictyostelids diverged before the evolution of plant, animals, and fungi (Embley and Martin, 2006), while protein based analyses place the divergence closer to the point of divergence of animals and plants (Baldauf, 2003). While the evolutionary origin of Dictyostelium is somewhat cloudy, it does not deter from its role as a model organism. Dictyostelium is easily grown in the lab on both solid surfaces and in shaken culture with bacteria as a food source, and the generation of axenic strains has allowed for cultivation in liquid media, bypassing the need for providing bacteria as a food source. The Dictyostelium genome is haploid for most of its life cycle, and coupled with the tractability of its genome, allows for the targeted disruption of genes of study. The nature of the life cycle (Section 1.9) permits for growth and differentiation to be treated as independent processes, allowing for the characterization of growth specific or developmental specific genes. The Dictyostelium genome, which has been fully sequenced (Eichinger et al., 2005), encodes for many of the same components that are involved in human signalling, and it has proven to be useful for studying processes such as cytoskeletal organization, cell motility and chemotaxis, phagocytosis, and signal transduction (Affolter and Weijer, 2005; Aubry and Firtel, 1999; Franca-Koh et al., 2006; Lee et al., 2001; Manahan et al., 2004; Rupper and Cardelli,. 2001). Finally, given the similarity of its feeding mechanism to that of human phagocytes, Dictyostelium has proven useful for studying human diseases caused by intracellular pathogens 14 such as Legionella pneumophila, Mycobacterium marinum, and Salmonella typhimurium (Williams et al., 2006). 1.9 The life cycle of Dictyostelium discoideum The following section provides a brief overview of the Dictyostelium discoideum life cycle (Figure 3) (reviewed in Kessin, 2001). Dictyostelium lives as a single celled amoeba during the vegetative growth stage, feeding on bacteria, or in the case of the laboratory axenic strains, taking up nutrients from liquid media via pinocytosis. When the food source is depleted, cells within the population initiate the starvation response. Cells begin to emit pulses of c A M P -6-7 minutes apart, which elicits a variety of responses, including the activation of the aggregation stage adenylyl cyclase ( A C A ) , stimulating the1 synthesis of c A M P that functions as both an intracellular second messenger and as an extracellular signal to neighbouring cells, and the induction of the expression of genes required for aggregation and development. c A M P also functions as a chemoattractant. The relay of c A M P pulses allows for cell to cell communication such that up to 100,000 cells from as far away as 1 cm can coalesce together to form a 'loose aggregate.' Upon forming the aggregate, the mound tightens up and develops a tip (the 'tipped aggregate'). The tip elongates to form a vertical finger like structure, sometimes referred to as a 'standing slug,' which proceeds to fall over onto the surface and becomes a phototactic and thermotactic motile slug. In the slug, cells undergo a differentiation process that results in two general populations of cells, those destined to become spore cells (prespore cells), and those destined to become stalk cells (prestalk cells), with the tip of the slug consisting primarily of prestalk cells, and the rear % of the slug consisting primarily of prespore cells. Development finalizes with the 'culmination' stage, a process that describes the transition from the slug stage 15 to the final fruiting body. This involves a rounding up of the slug, invagination of the stalk cells through the middle of the round disc, and a resulting lifting of the prespore cells off the substratum by the elongating stalk tube. During this time the cells undergo the final differentiation process; the prestalk cells make a cellulose sheath, vacuolate, and eventually die, whereas the prespore cells are encapsulated in a wall of polysaccharides and cellulose. The final fruiting body consists of a sorus of spores held up by a long stalk tube, supported at the bottom by a basal disc. The spores can be spread through the action of passing animals or insects, for example, and in response to favourable growth conditions, the spores germinate into vegetative amoebae. The full process of development from amoeba to fruiting body requires about 24 hours to complete. • 16 Figure 3. The life cycle of Dictyostelium discoideum. Scanning electron micrograph depicting multicellular developmental structures Dictyostelium discoideum. Life cycle begins with 'vegetative cells' at the lower right corner of the figure, and proceeds clockwise. Electron micrograph is by Mark J. Grimson and R. Lawrence Blanton of the Biological Sciences Electron Microscopy Laboratory, Texas Tech University. Printed with permission from R. Lawrence Blanton. © M . J . Grimson and R . L . Blanton. 17 1.10 Dictyostelium Ras proteins The Dictyostelium discoideum genome contains 6 genes encoding for Ras subfamily proteins (Figure 2) that have been characterized to some extent: rasB, rasC, rasD, rasG, rasS, and rap A (summarized in Table 1), with at least 9 other putative ras genes present in the genomic database, including two more rap genes and one rheb gene (Weeks et al., 2005). This large number of ras genes is quite peculiar in that this number is almost half as many ras genes found in human (35) (Colicell i , 2004) despite the relatively small size of the Dictyostelium genome (-35 Mb) versus that of the human genome (-3.2 Gb). The Dictyostelium Ras proteins exhibit > 50% amino acid identity with mammalian H-Ras, with RasD and RasG sharing the highest similarity, 65% and 68%, respectively (Daniel et al., 1995). While the effector domains (residues 32-40 relative to H-Ras) of RasB, RasD, RasG and R a p l are identical to H-Ras (Daniel et al., 1995), a number of variations exist when including the residues that account for the Switch 1 region (Figure 2). A s residues from Switch 1 contribute to effector binding specificity, these differences would suggest that each Ras protein displays differential affinities for the same effector protein. The temporal regulation of expression is different for each of the characterized genes (Daniel et al., 1995), suggesting that each gene is required for mediating a specific set of functions during specific stages of the life cycle. Genetic analyses by targeted gene disruption have supported the notion that the Dictyostelium Ras proteins are not completely functionally redundant (reviewed in Weeks and Spiegelman, 2003; reviewed in Wilkins and Insall, 2001). 18 T a b l e 1. S u m m a r y o f Dictyostelium R a s p r o t e i n s a n d t h e i r p r o p o s e d f u n c t i o n . Protein Gene Function RasB rasB Inability to generate knockout suggests gene is essential for viability. Reduced expression of rasB coincides with reduced growth. Expression of activated RasB leads to increased rate of nuclear division. RasC rasC rasC cells fail to aggregate due to inability to activate A C A during early development. RasD rasD Expression of activated RasD suggests a role in cell type differentiation in late development. Phenotype of rasD' slug also suggests a role in photo- and thermotaxis. RasG rasG Expressed primarily during growth, rasG' cells display defects in cytokinesis, cytoskeletal regulation, and growth in shaken suspension. RasS rasS Loss of rasS leads to growth defects associated with a reduction in fluid phase endocytosis. Rap l rap A Inability to generate knockout suggests gene is essential for viability. Knockdown of rapA expression coincides with reduced growth rate and cell viability. The first Dictyostelium ras gene to be cloned in Dictyostelium was rasD (Reymond et al., 1984). The rasD m R N A is not expressed during vegetative growth, and only expressed at low levels during the aggregation stage (~8 hours from the onset of development), with maximal expression during culmination (-18-24 hours). When cells expressing activated RasD (G12T) are induced to undergo multicellular development, the aggregates exhibit an abnormal 19 morphology, with multiple tips protruding from the tipped aggregate (versus one tip for wi ld type cells), and development'proceeding no further (Louis et al., 1997). Another consequence of expressing RasD (G12T) is an enhancement of stalk cell specific genes, and a concomitant reduction in spore cell specific genes (Louis et al., 1997). These observations suggested that RasD is intimately involved in the developmental process, and in particular responsible for regulating spore and stalk cell differentiation (Table 1). Thus, it was somewhat of a surprise that a population of rasD knockout cells developed normally with a wild type stalk cell to spore cell distribution, and the only obvious phenotype was a motile slug that was not thermo- or phototactic (Wilkins et al., 2000). The role of RasD in cell type differentiation may have been masked by partial compensation by the highly homologous RasG (87 % amino acid identity to RasD) as there is an increase in RasG protein level in strains lacking rasD (Khosla et al., 2000). RasG is almost identical to RasD over the first 111 amino acids with only 4 differences (Figure 2) (Daniel et al., 1995). rasG m R N A is expressed predominantly during growth, with transcript levels coming down during aggregation, and not detectable by the 'standing slug' stage (16 hours) (Daniel et al., 1995). Consistent with the m R N A expression pattern that suggests RasG is important during vegetative growth, gene ablation of rasG results in cells that exhibit severe growth defects when grown axenically in suspension (Tuxworth et al., 1997). When grown in axenic medium on a solid surface, cells grow large and become multinucleate, indicating that rasG' cells have defects in cytokinesis (Table 1). rasG' cells also have defects in actin cytoskeleton regulation, in particular formation Of lamellipodia, filopodia, and reduced cell motility (Tuxworth et al., 1997). RasD can compensate for some RasG functions, as expressing rasD from the rasG promoter in rasG' cells results in rescue of the vegetative growth and cytokinesis defects (Khosla et al., 2000). However, RasD cannot fully compensate for the loss of 20 RasG as the expression of rasD from the rasG promoter in rasG' cells does not rescue the defects in cell motility. RasB is a unique protein in that it has been linked to the regulation of mitosis and immunofluorescence studies have revealed that RasB localizes to the nucleus (Sutherland et al., 2001), both novel attributes for Ras subfamily proteins. For most of the cell cycle, RasB is found in the nucleus; however, from metaphase to telophase of mitosis, RasB exits the nucleus to the cytosol, returning only once mitosis is complete. Expression of activated RasB (G15T), the equivalent of the activating H-Ras (G12T) mutation, results in cells that display an increased rate of nuclear division relative to the rate of cell division. Lending more evidence to the importance of RasB in regulating the cell cycle is the apparent inability to disrupt the rasB gene (Sutherland et al., 2001). A n initial population of what appeared to be rasB' cells grew extremely slowly, and displayed undetectable levels of RasB protein. Eventually, a subpopulation of cells was able to outgrow the slow growing cells, and this reversion to normal growth coincided with the restoration of RasB protein levels. Subsequent genetic studies demonstrated that this population of cells was able to undergo a recombination event that restored the expression of the rasB gene while still maintaining antibiotic resistance. These data suggest that RasB is essential and required for maintenance of normal cell division (Table 1). RasC is required for aggregation of Dictyostelium, as evidenced by the phenotype of a strain with a disruption of the rasC gene (L im et al., 2001). Further analysis demonstrated that the aggregation defect is due to an inability to produce c A M P upon receptor stimulation (Table 1). Supplying rasC cells with an exogenous supply of c A M P rescued the aggregation defect, suggesting that the primary function for RasC is to regulate A C A activity. rasC cells that have been starved without c A M P pulses chemotax to c A M P poorly. Supplying c A M P exogenously to 21 starving rasC cells results in a hyper-polar cell, relative to wild type (Lim et al., 2001), suggesting that RasC may also regulate aspects of cell polarity or cytoskeletal organization during chemotaxis. The expression pattern of rasS m R N A is similar to that of rasG, and disruption of rasS leads to cells that have defects in growth, endocytosis, and motility (Chubb et al., 2000). Unlike rasG cells, whose growth defects are primarily due to defects in cytokinesis, the growth defect of rasS' cells is a dramatically reduced rate of fluid phase endocytosis. rasS' cells are also elongated and exhibit a faster rate of motility, which coincides with an abundance of F-actin localized to the pseudopod. Chubb et al. (2000) proposed a model in which RasS regulates F-actin re-arrangement, and the need for actin in both feeding and motility means that one behaviour w i l l be compromised by the function of the other. Thus, the high motility of rasS' cells is proposed to inhibit endocytosis for feeding (Table 1). The gene encoding R a p l , rapA, exhibits a biphasic expression pattern with one peak during aggregation (~8 hours) and a second peak during culmination (18 hours) (Robbins et al., 1990). Like rasB, attempts at disrupting rapA have been unsuccessful, suggesting that rapA is essential for cell survival (Kang et al., 2002). Studies using inducible antisense constructs showed that a decrease in Rap l protein levels coincides with a reduction in growth rate and cell viability, supporting a role for R a p l in cell viability (Table 1). 22 1.11 c A M P s i g n a l l i n g d u r i n g Dictyostelium a g g r e g a t i o n A s this thesis is primarily concerned with the events that occur during aggregation (the term 'early development' w i l l be used interchangeably with the aggregation stage), this section wi l l provide an overview of the c A M P stimulated signalling events during this time (summarized in Figure 4). c A M P signal transduction is required for the c A M P relay ( A C A activation) and for chemotaxis and motility. The latter employs components orthologous to that of mammalian signalling (reviewed in Manahan et al., 2004). A B F i g u r e 4. T h e c A M P s t i m u l a t e d p a t h w a y s d u r i n g Dictyostelium a g g r e g a t i o n . (A) Schematic of the c A M P stimulated signalling pathway leading to the activation of A C A . Both the PI3K and T O R C 2 pathways are required for A C A activation downstream of the c A M P receptor complex. PI3K activity results in the translocation of the P H domain containing protein C R A C from the cytosol to the membrane. RasC is also required for A C A activation, but it is not clear how RasC interacts with these pathways as both PI3K and T O R C 2 appear to be downstream of RasG (Section 1.12). A s A C A activation occurs at the rear of cells, it is possible that a signal is transmitted by RasC to A C A at the rear. (B) Schematic of the c A M P simulated pathways regulating chemotaxis, cell motility and cell polarity. A s with the pathways to A C A activation, both the PI3K and T O R C 2 are involved in cell motility and establishing cell polarity. PI3K activity results in the translocation of the P H domain containing protein P K B from the cytosol to the membrane. A separate pathway leading to the activation of G C is also required for the chemotaxis behaviour. 23 The primary receptor for c A M P at the aggregation stage, c A R l , is a typical G P C R , and is thought to mediate all c A M P stimulated events during aggregation (Pupillo et al., 1992). c A R l is predominantly associated with the heterotrimeric G-protein involving the G a 2 and G P y subunits (Parent and Devreotes, 1996). Binding of c A M P stimulates the exchange of G D P for G T P on G a 2 , which in turn leads to dissociation of G a 2 from G P y , and each of these are thought to initiate a separate signalling pathway; G p y is thought to activate PI3K and A C A (Parent and Devreotes, 1996; W u et al., 1995), whereas G a 2 is thought to stimulate the pathway leading to the activation of the aggregation stage guanylyl cyclases, sGC and G C A (Roelofs et al., 2001; Roelofs and Van Haastert, 2002). A C A , like the mammalian counterpart, consists of six membrane spanning domains, two large cytoplasmic domains, and functions as a homodimer. However, unlike the mammalian adenylyl cyclase, the Dictyostelium A C A does not contain a consensus G(3y binding site, and how G P y activates A C A is not Certain. What is known is that two cytosolic components are required, the Cytosolic Regulator of Adenylyl Cyclase ( C R A C ) and Pianissimo (PiaA) (Chen et al., 1997; L i l l y and Devreotes, 1994), and both of these proteins are necessary for A C A activation. C R A C is a P H domain containing protein and translocates to the front of migrating cells in response to c A M P , and this translocation is dependent on PI3K activity (Huang et al., 2003). P iaA is an orthologue of A V 0 3 , and is a part of the T O R C 2 complex, along with T O R , L S T 8 and the Dictyostelium homologue of mammalian S i n l , RIP3 (Lee et al., 2005). The T O R C 2 complex is required for A C A activation, cell motility, and cell polarity, and there is evidence that demonstrates that RasG is involved in the regulation of this complex (Section 1.12). c A M P stimulation of aggregation stage cells leads to a rapid production of c G M P , peaking -10 s after stimulation. Dictyostelium expresses 2 guanylyl cyclases during aggregation, 24 G C A and sGC (Roelofs and Van Haastert, 2002), homologous to 12 transmembrane domain and soluble guanylyl cyclases, respectively, and collectively referred to as G C . The evidence suggests that sGC is the primary G C during aggregation as gcaA cells produce normal levels of c G M P whereas sgcA' cells have a more dramatic reduction in c G M P production when stimulated with c A M P ; a sgcA'/gcd strain produces no measurable level of c G M P (Roelofs and Van Haastert, 2002). The role of c G M P as a second messenger is less clear. What is known is that c G M P is required for mobilizing myosin II to the rear of chemotaxing cells, and cells unable to produce c G M P have severe defects in chemotaxis efficiency (see below) (Bosgraaf et al., 2005; Roelofs and Van Haastert, 2002). A bioinformatic screen looking for c G M P binding proteins found only one protein, GbpC, with a demonstrated c G M P binding ability (Goldberg et al., 2002), and gbpC cells have a phenotype similar to cells that are unable to produce c G M P (Bosgraaf et al., 2005), suggesting that GbpC is needed to transduce the c G M P signal. Mutational analysis indicates that a small G-protein is upstream of G C and downstream of Goc2 (Roelofs et al., 2001), and analysis of c A M P stimulated c G M P production in a rasG' strain suggests that RasG mediates G C activity (Bolourani et al., 2006). A n important mediator of the intracellular c A M P signal is the c A M P dependent protein kinase, P K A . P K A is a heterodimer consisting of a catalytic kinase subunit, P K A - C , and a regulatory subunit, P K A - R (Saran et al., 2002). In the absence of a signal, P K A - C and P K A - R are associated, inhibiting P K A activity. Binding of c A M P to P K A - R forces dissociation of P K A - R from P K A - C , leading to the activation of P K A , and deletion of pkaR removes the c A M P dependent regulation of P K A activity, resulting in constitutive P K A activity (Zhang et al., 2003). P K A is required for all stages of development, including the transition from growth to development (Schulkes and Schaap, 1995), regulating intracellular c A M P degradation (Shaulsky 25 and Loomis, 1993), and terminal maturation of spore and stalk cells (Harwood et al., 1992; Mann et al., 1994). One role of P K A during aggregation is as a regulator of RegA, an intracellular c A M P specific phosphodiesterase that breaks down the c A M P generated by A C A (Shaulsky and Loomis, 1993). P K A is also involved in chemotaxis and cell motility, as constitutive P K A activity results in cells that display reduced cell speed and a loss of cell polarity (Zhang et al., 2003). However, the lack of known downstream substrates has prevented elucidating the role of P K A in chemotaxis. c A M P stimulation of the PI3K pathway has been suggested to be downstream of Gpy (Sasaki and Firtel, 2006). The pathway is complicated because Dictyostelium encodes at least five proteins that are similar to mammalian type I PI3Ks (Hoeller and Kay, 2007; Zhou et al., 1995). Dictyostelium PI3K1 and PI3K2 (encoded by pikA and pikB, respectively) are closely related orthologues of mammalian p i 10 PI3Ks (Zhou et al., 1995), and c A M P stimulation of aggregation stage cells leads to an increase in PI(3,4,5)P3 at the front of the cell, and a concomitant translocation of P H domain containing proteins such as P K B and C R A C to the site of PI3K activity (Huang et al., 2003; Loovers et al., 2006; M e i l i et al., 1999). A pikA'/pikB' strain exhibits dramatically reduced PI(3,4,5)P3 accumulation, and a defect in the c A M P stimulated activation of A C A (Funamoto et al., 2002), presumably due to the loss of C R A C function in this mutant. There has been significant controversy surrounding the role of PI3K as a mediator of chemotaxis (reviewed in Sasaki and Firtel, 2006). PI3K1 and PI3K2 both translocate to the front of migrating cells when placed in a gradient of c A M P , and pikA'/pikB' cells are poorly polarized and chemotax very slowly to the source of chemoattractant (Funamoto et al., 2002). Additionally, constitutive localization of PI3K1 to the entire cell periphery results in cells that 26 extend pseudopods in all directions (Funamoto et al., 2002). P T E N (encoded by the ptenA gene) is a lipid phosphatase that converts PI(3,4,5)P3 to PI(4,5)P2, effectively reversing the action of PI3K. Dictyostelium P T E N shows an intracellular translocation that is reciprocal to PI3K, i.e. towards the rear and sides of migrating cells. Cells that over-express ptenA have suppressed pseudopod formation at the lateral sides of the cell while chemotaxing, appearing hyper-polar relative to wild type cells (Funamoto et al., 2002). This led to a model of 'Local Excitation, Global Inhibition' (LEGI) in which PI3K plays the central role in chemotaxis (Ma et al., 2004). In this model, PI3K orients the cell by generating PI(3,4,5)P3 at the front of a chemotaxing cell (local excitation). This small gradient is then amplified through the action of P T E N (global inhibition), and the resulting polarity places the appropriate machinery into proper position to allow chemotaxis to occur. 1 Recent studies have refuted the role of PI3K as a mediator of chemotaxis. Chemotactic index is a quantitative measure of chemotaxis efficiency, and can be simply defined as the net displacement of a cell towards the source of the chemoattractant divided by the total displacement of the cell (Wessels et al., 2004), i.e. a cell that travels only in the direction of the chemoattractant w i l l have a C.I. of one, whereas a cell that moves back and forth but makes no net movement towards the chemoattractant source wi l l have a C.I. of zero. Thus, C.I. can be used to separate defects in motility from defects in chemotaxis. Quantitative analysis of chemotaxing cells revealed that there was no difference in C.I. between pikA'/pikB' cells and wi ld type cells, but there was a significant loss in cell speed and cell polarity, indicating that the defects in chemotaxis were actually defects in motility and polarity (Chen et al., 2007). Hoeller and Kay corroborate this report by observing that a Dictyostelium strain in which all five type-1 27 PI3K homologues have been knocked out shows no defect in C.I. when chemotaxing to c A M P , but suffers a significant decrease in the rate of motility (Hoeller and Kay, 2007). So what might be the controlling element for chemotaxis? A gcaA'/sgcA' mutant that is defective in the production of c G M P , and a gbpC mutant that is defective in transmitting signalling events downstream of c G M P production, both have a C.I. close to zero while chemotaxing to c A M P (Bosgraaf et al., 2002; Bosgraaf et al., 2005). These mutants demonstrate that c G M P is important for the chemotactic response. c G M P is required for mobilizing myosin II to the cell cortex, but cells lacking myosin II are still able to chemotax, albeit slower relative to wi ld type cells (Shelden and Knecht, 1995). This observation suggests that the role of c G M P in mobilizing the myosin II cytoskeleton is distinct from its role in chemotaxis1. Based on a quantitative analysis of pseudopod generation, a new model of chemotaxis has been proposed (Andrew and Insall, 2007). The 'Informed Choice' model was based on the observation that cells protrude pseudopods randomly, and these pseudopods often split, or bifurcate, with the pseudopod that orients itself spatially closer to the chemoattractant source becoming the dominant pseudopod, and a subsequent retraction of the spatially further pseudopod. Thus, small improvements in the direction of the leading pseudopod over time lead to efficient chemotaxis. While treatment of wild type cells with the PI3K inhibitor LY294002 did not affect the C.I. of chemotaxing cells, it did dramatically decrease the rate of pseudopod generation (Andrew and Insall, 2007). The same chemotactic behaviour was observed in human neutrophils and mouse embryonic fibroblasts treated with LY294002 (Andrew and Insall, 2007), suggesting a conserved mechanism regulating chemotaxis. Taken together, these data would suggest that PI3K is important in regulating the machinery involved in cell motility, but not in selecting the direction of cells during chemotaxis. 28 1.12 I n v o l v e m e n t o f R a s i n a g g r e g a t i o n Aside from the Dictyostelium P D K s , orthologues of well established Ras effectors in mammalian signalling pathways, a number of Ras signalling components have been implicated in A C A activation and chemotaxis during early development. The first was the RasGEF, R a s G E F A , formerly known as Aimless or A l e A , as gefA cells do not activate A C A in response to c A M P (Insall et al., 1996). Subsequent isolation of a rasC cell line with a similar phenotype suggested that R a s G E F A was, the activator of RasC (Lim et al., 2001); however, no further genetic or biochemical data was able to corroborate this. Another protein that was found to be necessary for A C A activation is Ras Interacting Protein 3, RIP3 (encoded by the ripA gene) (Lee et al., 1999). A s the name implies, RIP3 harbours a Ras Binding Domain (RBD), and its involvement in A C A activation provided a possible signalling pathway consisting of R a s G E F A to RasC to RIP3. However, RIP3 was shown to interact specifically with RasG, and not RasB, RasC, RasD, or RasS (Lee et al., 1999). This was somewhat surprising as there had been no prior indication that RasG was involved in A C A activation, with any evidence being tenuous at best, prior to this finding. RIP3 is part of the T O R C 2 complex of proteins, and along with the defects in A C A activation, ripA' cells don't aggregate and chemotax slower than wild type cells (Lee et al., 2005). A RIP3 protein that bears two point mutations in the R B D , K680E and R681E, does not bind to RasG (Lee et al., 2005). While over-expression of RIP3 rescues the phenotypes of a ripA' strain, over-expression of RTP3 (K680E, R681E) does not rescue the phenotypes of a ripA' strain (Lee et al., 2005), demonstrating that binding of RasG to RTP3 is necessary for the function of T O R C 2 . A s both RasG and T O R C 2 have been shown to be involved in the c A M P stimulated activation of P K B (Bolourani et al., 2006; Lee et al., 2005), it 29 may be that binding of RasG to RIP3 functions to localize T O R C 2 to the membrane, facilitating P K B activation. Two RasGEFs have been identified recently, and both demonstrate a role in early development. R a s G E F M was identified in a genomic search for RasGEF sequences, and is required for A C A activation, but not G C activation (Arigoni et al., 2005). gefM~ cells chemotax to c A M P poorly and do not polarize well . The observation that G C activity is unaffected in geJM cells suggests that the poor chemotaxis is due to defects in cell motility and/or polarity. Another RasGEF implicated in early development is GbpD, isolated in a search for c G M P binding proteins (Goldberg et al., 2002). While disruption of gbpD does not affect C.I. dramatically relative to wi ld type cells, gbpD' cells are hyper-polarized when chemotaxing to c A M P (Bosgraaf et al., 2005), suggesting that GbpD regulates aspects of determining cell polarity, perhaps as a negative regulator. Given the morphologic similarities of chemotaxing gbpD' and rasC cells (Bosgraaf et al., 2005; L i m et al., 2001), it is tempting to speculate that GbpD functions in the RasC pathway, perhaps as an activator of RasC. 1.13 T h e s i s O b j e c t i v e At the time I began my thesis project, much genetic and biochemical data was emerging that shed light on Ras signalling in Dictyostelium; however, not much was known about activation of the Ras proteins themselves. Many of the connections to pathways were speculative, for example the link between R a s G E F A and RasC, and the number of Ras pathway proteins identified began to create some confusion as to what the role of Ras really was. The objective of this thesis was to characterize the Ras activation pathway during Dictyostelium early development. This stage of the life cycle was chosen because the c A M P 30 signalling pathway was the best described, and given the implication of Ras in c A M P stimulated signalling pathways (Section 1.12), it was hypothesized that Ras was activated during this process. In order to confirm the role of Ras in the signalling events during aggregation, it must first be demonstrated that Ras is activated upon stimulation by c A M P . To begin such an undertaking, an assay was needed that was able to measure activated Dictyostelium Ras proteins. The first part of this thesis project was to modify an existing assay that was developed for monitoring mammalian Ras activation, and had been already modified for measuring not only Ras, but other small GTPases in both mammalian and other model systems. The second part of this project was to employ this assay to characterize the upstream factors that are involved in Ras protein activation in order to elucidate the Ras signalling pathways during early development. Based on the available data, it would be expected that RasC is activated downstream of c A R l and the Ga2(3y heterotrimeric G-protein, and activated RasC mediates the pathway leading to A C A activation as rasC cells are unable to activate A C A in response to c A M P . Furthermore, the striking similarities of the phenotypes of the gefA' and rasC cells suggest that R a s G E F A activates RasC. While there is not enough evidence to strongly implicate another Ras protein in the c A M P signalling pathway, the fact that rasC cells are able to chemotax efficiently to c A M P suggests that RasC is not the sole Ras responsible for mediating the downstream effects of c A M P . 31 2 MATERIALS AND METHODS 2.1 M a t e r i a l s 2.1.1 R e a g e n t s Restriction endonucleases, D N A polymerases and other D N A modifying enzymes were obtained from Invitrogen (Burlington, ON) . Growth media reagents were from Oxoid (Nepean, ON) , B B L (Cockeysville, M D ) , or B D Sciences (Mississauga, ON) . A l l chemicals were obtained from Fisher (Ottawa, ON) , Invitrogen (Burlington, ON) , or Sigma (Oakville, ON) , unless otherwise stated. A l l cloning and sub-cloning steps employed the use of chemical or electro-competent Escherichia coli strain X L 1-Blue M R F ' (Stratagene, LaJolla, C A ) . A l l protein expression vectors were transformed in Escherichia coli B L 2 1 DE3 (Invitrogen). Hybond P polyvinylidene difluoride (PVDF) membranes, horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody, enhanced chemiluminescence ( E C L ) , and E C L Plus reagents for immunoblot analysis were obtained from G E Healthcare (Baie d'Urfe, PQ). 2.1.2 S e r v i c e s A l l D N A sequencing was performed by the Nucleic A c i d and Protein Services (NAPS) unit (University of British Columbia, Vancouver, B C ) . A l l oligonucleotide synthesis was performed by Alpha D N A (Montreal, PQ). 32 Table 2. Dictyostelium strain descriptions. Strain Description Selection Reference Ax2. Parental axenic strain for following strains. Ax2::pVEH-rajB(G15T) Ax2 transformed with pVEII encoding activated rasB (G15T) expressed from a folate repressible discoidin promoter. 10 pg/ml G418 (Sutherland et al., 2001) (Blusch et al., 1992) A\2.:rasC-rasC (G13T) Ax2 transformed with pJLW25 encoding activated rasC (G13T) constitutively expressed from a rasC promoter. 10 pg/ml G418 (Lim, 2002) Ax2: :MB-rasG (G12T) Ax2 transformed with pMB38 encoding activated rasG (G12T) expressed from a tetracycline repressible TRE-Pmin_promoter. 10 pg/ml G418 5 pg/ml Blasticidin S (Secko et al., 2004) (Blaauw et al., 2000) Ax2::pVEII-rasG (G10V) Ax2 transformed with pVEII encoding dominant negative rasG (G10V) expressed from a folate repressible discoidin promoter. 10 pg/ml G418 Ax2::pVEII-rasG (S17N) Ax2 transformed with pVEII encoding dominant negative rasG (S17N) expressed from a folate repressible discoidin promoter. 10 pg/ml G418 (Khosla et al., 1996) gefM Chromosomal K O of gefM gene in Ax2. 5 pg/ml Blasticidin S (Arigoni et al., 2005) Ax3 Parental axenic strain for following strains. A\3:.actl5-gbpD Ax3 transformed with pMB74 encoding gbpD expressed from a constitutive high expression act 15 promoter. 10 pg/ml G418 (Kortholt et al., 2006) acciA' Chromosomal K O of acaA gene in Ax3. (Pitt etal., 1992) carA-/carC Chromosomal K O of car A and carC genes in Ax3. (Soedeetal., 1994) dag A Chromosomal K O of dag A gene in Ax3. (Lilly and Devreotes, 1994) gbpU Chromosomal K O of gbpD gene in Ax3. (Kortholt et al., 2006) gefA Chromosomal K O of gefA gene in Ax3. (Insall etal., 1996) gefC Chromosomal K O of ge/Cgene in Ax3. 5 pg/ml Blasticidin S (Wilkins et al., 2005) gefD- Chromosomal K O of gefD gene in Ax3. 5 pg/ml Blasticidin S (Wilkinset al., 2005) gefR- Chromosomal K O of gefR gene in Ax3. 5 pg/ml Blasticidin S (Secko et al., 2004) gpaB Chromosomal K O of gpaB gene in Ax3. (Wu etal., 1995) gpbA' Chromosomal K O of gpbA gene in Ax3 (Kumagai etal., 1991) piaA' Chromosomal K O of piaA gene in Ax3. (Chen etal., 1997) 33 2.1.3 Dictyostelium strains , Various Dictyostelium strains were sourced for use in this thesis. gefA', gefC, gefD', gefR' and their wildtype parental strains (Table 2) were from Dr. Robert Insall (University of Birmingham, Birmingham, U.K.). gefM~ and its parental strain (Table 2) were from Dr. Salvatore Bozzaro (University of Torino, Orbassano, Italy). gbpD' and Ax3::actl5-gbpD and their parental strains (Table 2) were obtained from Dr. Peter Van Haastert (University of Groningen, Haren, The Netherlands). acaA', carA'/carC, dagA', gpaB', gpbA, piaA' and their respective parental strains (Table 2) were obtained from the Dicty Stock Center (Columbia University, New York, NY). 2.1.4 Plasmids and DNA Various plasmids and D N A were generously provided by the following investigators: pGEX-Raf encoding GST-Rafl (RBD) from Dr. David Shalloway (Cornell University, Ithaca, NY) ; pSL333 and pSL335, encoding GST-PI3K1 (RBD) and GST-PI3K2 (RBD), respectively, from Dr. Richard A. Firtel (University of California at San Diego, La Jolla, CA); pGEX-RalGDS encoding GST-RalGDS (RBD) from Dr. Michael R. Gold (University of British Columbia, Vancouver, BC); Genomic D N A from Schizosaccharomyces pombe from Dr. Ivan Sadowski (University of British Columbia, Vancouver, BC). 34 2.2 Methods 2.2.1 Cell culture and development of Dictyostelium discoideum D. discoideum cells were grown axenically in HL5 medium (14.3 g peptone, 7.15 g yeast extract, 15.4 g glucose, 0.96 g Na 2 HP0 4 .7H 2 0 and 0.486 g K H 2 P 0 4 per litre of water) supplemented with 50 |lg/ml streptomycin at 22°C, either in Nunclon tissue culture dishes (Nunc, Rochester, NY), or in suspension in flasks shaken at 160 rpm (Watts and Ashworth, 1970). Transformed strains were selected and maintained in HL5 media supplemented with the following antibiotics as appropriate: 5 u.g/ml blasticidin S (Calbiochem, Darmstadt, Germany) or 10 u.g/ml G418 (Table 2). Cells were counted using a haemocytometer. Cells were maintained on tissue culture dishes, and passaged by scraping confluent plates and diluting 10-40 times into fresh HL5 on tissue culture plates. For growing cells in shake suspension, confluent tissue culture plates were scraped and resuspended in 60 ml HL5, and shaken at 160 rpm. Experiments conducted on cells growing exponentially used cells at a density of 2-4 x 106 cells/ml. For cryo-preservation, ~1 x 108 cells were frozen in 1.0 ml aliquots of HL5 supplemented with 10% DMSO. All cells were maintained for no more than 8 passages before recovering fresh stocks from cryo-preserved stocks. To observe aggregation streams, exponentially growing vegetative cells were washed three times with Bonner's Salts (10 mM NaCl, 10 raM KC1, 2 mM CaCl 2), and seeded at 5 x 105 cells/cm2 in tissue culture dishes submerged under Bonner's salts. Microscopic observations of streaming amoeba were performed using a Leica DM IL inverted microscope (Leica, Wetzler, Germany) fitted with a 4X objective lens. All images were captured using a Leica DFC350 F digital monochrome camera (Leica) in conjunction with Open Lab 4.0.4 software (Improvision, Lexington, MA). 35 2 . 2 . 2 I n d u c i b l e e x p r e s s i o n o f a c t i v a t e d R a s p r o t e i n s Ax2::MB-rasG (G12T) (Table 2) expresses activated RasG from a tetracycline repressible promoter (Blaauw et al., 2000). Growth in HL5 supplemented with 5 |4g/ml tetracycline represses expression of the protein. To induce expression, exponentially growing cells were harvested by centrifugation (500 x g, 5 min) and washed three times with HL5 without tetracycline. Cells were resuspended in HL5 without tetracycline at 2-4 x 106 cells/ml, and shaken in flasks at 160 rpm. Maximal expression is in 4 hours (Secko et al., 2004). Ax2::pVEII-rasfl (G15T), Ax2::pVEII-rasG (G10V), Ax2::pVEII-rasG (S17N) (Table 2) express RasB and RasG mutants from a folate repressible discoidin promoter (Blusch et al., 1992). Growth in HL5 supplemented with 1 mM folate represses expression of the proteins. To induce expression, exponentially growing cells were harvested by centrifugation (500 x g, 5 min) and washed three times with HL5 without folate. Cells were resuspended in HL5 without folate at 5 x 105 cells/ml, and shaken in flasks at 160 rpm. Maximal expression is within 48-60 hours (Khosla et al., 1996; Sutherland et al., 2001). As high cell density represses expression from the discoidin promoter, cells were diluted such that the.cell density did not exceed 2 x 106 cells/ml. 2 . 2 . 3 P r e p a r a t i o n o f p u l s e d c e l l s To prepare pulsed cells, exponentially growing cells were harvested by centrifugation (500 x g, 5 min), and washed three times with KK2 (20 mM potassium phosphate, pH 6.1). Cells were resuspended at 5 x 106 cells/ml in KKo, and shaken in a flask at 160 rpm for 30 min. Cells were pulsed with 100 pi pulses of cAMP, as applied by using a Polystaltic pump (Buchler, Fort Lee, NJ), to a final concentration of 50 nM. Timing of the pulses was every 6 min, as controlled 36 by a Lab Controller timer ( V W R , Mississauga, ON). After pulsing, cells were harvested by centrifugation (500 x g, 5 min), washed two times with K K 2 , and resuspended at 5 x 10 7 cells/ml in K K 2 . 2.2.4 C loning of GST-Byr2 (RBD) The fragment encoding the R B D of the byr2 gene product (encoding for amino acid residues 1-237) was amplified from S. pombe genomic D N A by P C R such that an £ c o R I restriction site was engineered at the 5' end and a Sail restriction site was engineered at the 3' end. The oligonucleotide primers used were as follows: Byr2 Forward 5' G A A T T C A T G G A A T A T T A T A C C T C G A A 3' Byr2 Reverse 5' G T C G A C A A C G C C G A G A G T T T G A T A T T G T 3' Standard P C R reactions were conducted in 10 ul mixtures in glass capillary tubes containing 5.58 ul H 2 0 , 1.0 | i l 10X Idaho P C R buffer (500 m M Tris, p H 8.3, 2.5 mg/ml B S A , 20% (w/v) sucrose, 1 m M cresol red, 30 m M M g C l 2 ) , 0.5 p i dNTPs (2.5 m M ) (GE Healthcare), 1.0 ul of each oligonucleotide (2.5 mM) , 1.0 p i S. pombe genomic D N A (250 ng/ul) and 0.12 pi Taq D N A polymerase (5 U/ul) . The capillary tubes were set up in an Idaho Rapidcycler (Idaho Technologies, Idaho Falls, ID) and the following cycling parameters were employed: • 2cyc le so f D = 9 2 ° C f o r 6 0 s , A=56°C for 7 s, E=72°C for 60 s • 36 cycles of D=92°C for 1 s, A=56°C for 7 s, E=72°C for 60 s 37 • 1 cycle of E=72°C for 120 s D is the denaturation temperature, A is the annealing temperature, E is the extension temperature. The temperature ramping rate, S, for all cycling was set at 6.0. The P C R samples were resolved on 1.0 % agarose gel electrophoresis in T B E (89 m M Boric A c i d , 89 m M Tris, 2 m M E D T A ) buffer. The resulting ~700bp fragment was excised from the gel and extracted using a QIAquick Gel Extraction K i t (Qiagen, Mississauga, ON) , as per manufacturer's instructions. The extracted D N A fragment was ligated into a p G E M - T Easy (Promega, Madison, WI) cloning vector and transformed into electrocompetent E. coli X L l - B l u e M R F \ The p G E M - T Easy-byr2 (RBD) vector was purified from ampicillin resistant colonies using a QIAprep Spin Miniprep K i t (Qiagen), as per manufacturer's instructions,1 and sequenced to confirm the identity of the fragment. Plasmid D N A from one clone with an exact copy of the genomic byr2 (RBD) was digested with EcoRl and Sail to excise the D N A fragment. The p G E X 4T-1 expression vector (GE Healthcare) was digested with EcoRl and Sail to allow for the directional cloning of thei>yr2 (RBD) D N A fragment. The resulting digested D N A was resolved on 1 % agarose gel electrophoresis and the desired D N A fragments were extracted from the gel using a QIAquick Gel Extraction Ki t . 100 ng of the digested p G E X 4T-1 were mixed with 42 ng of the byr2 (RBD) D N A fragment (a 3;1 molar ratio) in a 10 | i l ligation reaction using T4 D N A ligase and incubated overnight at 15°C, as per manufacturer's instructions. 4 | i l of the ligation mixture were used to transform chemically competent E. coli B L 2 1 D E 3 , and transformants were confirmed by sequencing. 38 2 . 2 . 5 E x p r e s s i o n o f G S T - R B D f u s i o n p r o t e i n s G S T - R B D fusion proteins were prepared by the following procedure. One colony was picked and used to inoculate 5 ml of L B media supplemented with 50 (ig/ml ampicillin. 2.5 ml of the overnight culture was used to inoculate 500 ml of L B media supplemented with 50 u.g/ml ampicillin, and grown in shake flasks at 37°C until the ODgoo was 0.6-0.8. The cells were induced with 0.1 m M IPTG and grown for 18-20 hours at 22°C. Cells were harvested by centrifuging at 5,000 x g for 10 min. The bacterial pellet was resuspended in 10 ml S T E buffer (10 m M Tr i s -CL (pH 8.0), 150 m M N a C l , 1 m M E D T A ) and frozen overnight. The cell suspension was thawed and treated with lysozyme (0.05 mg/ml, final concentration) and incubated on ice for 30 min. 100 ul D T T (1 M ) and 1.4 ml Sarkosyl (10 %) were added just prior to lysis. Cells were lysed on ice by sonication using an Ultrasonic processor X L (Misonix Inc., Farmingdale, N Y ) in 6, 10 s pulses at power setting 4, and then centrifuged at 17,000 x g for 30 min at 4°C. 2 ml Triton X-100 (20 %)' was added to the supernatant and the total volume was brought up to 20 ml such that the final concentrations of Triton X-100 and Sarkosyl were 2 % and 0.7 %, respectively. The lysates were separated into 10 ml aliquots and snap frozen in a dry ice/ethanol bath. 2 . 2 . 6 P u r i f i c a t i o n o f G S T - R B D f u s i o n p r o t e i n s Glutathione-sepharose beads ( G E Healthcare) are supplied in a 70 % bead, 30 % ethanol mixture. 665 u.1 of glutathione-sepharose beads (equivalent to 500 ul dry volume of beads) were washed three times with PBS (2.7 m M KC1, 1.4 m M K H 2 P 0 4 , 4.3 m M N a 2 H P 0 4 • 12H 2 0 , 137 39 m M N a C l , p H 7.5), and subsequently topped with 500 pi PBS to create a 50:50 bead to buffer slurry. 1 ml of a 50:50 glutathione-sepharose bead slurry was added to 10 ml bacterial lysates containing G S T - R B D fusion protein, and tumbled end over end for 2 hours at 4°C. Beads were harvested by centrifugation (500 x g, 5 min, 4°G), washed 3 times with ice cold S T E buffer (10 m M Tr is -Cl , p H 8.0; 1 m M E D T A ; 150 m M NaCl) , and resuspended in 500 p i S T E resulting in a 50:50 bead to buffer slurry. The quality of each preparation of the G S T - R B D proteins was determined by testing the ability of each G S T - R B D to bind to activated RasC and RasG over a range of increasing input cellular protein similar to that shown in Figure 10. 2 . 2 . 7 R B D B i n d i n g A s s a y Exponentially growing cells were harvested by centrifugation (500 x g, 5 min), washed three times with KK2, and resuspended at 5 x 10 7 cells/ml in K K 2 . Cells were lysed in an equal volume of cell suspension and ice cold 2 X R B D - L B (20 m M Sodium Phosphate, p H 7.2, 2 % Triton X-100, 20 % Glycerol, 300 m M N a C l , 20 m M M g C l 2 , 2 m M E D T A , 2 m M N a 3 V 0 4 , 10 m M NaF, with two tablets of 'Complete protease inhibitor cocktail ' (Roche, Basel, Switzerland) added per 50 ml buffer). For c A M P stimulated cells, 6 h pulsed cells were stimulated with 200 n M (final concentration) c A M P , and 400 | i l aliquots were taken at the selected times, lysed in an equal volume of ice cold 2 X R B D - L B , and incubated on ice for 5 min. Efficiency of cell lysis was estimated visually by using a haemocytometer. The lysates were cleared by centrifugation (13,000 x g, 10 min, 4°C). Protein concentrations were determined using the DC Protein Assay (Bio-Rad, Hercules, C A ) according to manufacturer's instructions, and lysates were diluted to a concentration of 1 mg/ml. 400 pig of protein lysates were incubated with 50 pi of 50:50 G S T -40 R B D bead slurry, and the mixture was tumbled end over end at 4°C for 1 h. Beads were harvested by centrifugation and washed 3 times in ice cold I X R B D - L B . Beads were suctioned dry using a 22V2G syringe needle (Beckton Dickinson, Franklin Lakes, NJ). 50 p L of I X SDS-P A G E loading buffer (50 m M Tris-Cl , p H 6.8, 1.6 % (w/v) SDS, 100 m M D T T , 0.002 % (w/v) bromophenol blue, 5 % (v/v) glycerol) were added to the pelleted beads, and the mixture was heated at 100°C 5 min. A 10 JLLI sample of the lysates was added to 50 p.1 of 6 X S D S - P A G E loading buffer. Approximately 8.3 [ig of protein lysate were added to one lane of the SDS-P A G E gel and included as a load control to assess the level of total Ras protein. The samples from the R B D binding assay were loaded on the gel representing the pool of activated Ras from approximately 160 p,g of total protein. Samples were analyzed for bound Ras proteins by immunoblot analysis. 2 . 2 . 8 G u a n i n e n u c l e o t i d e l o a d i n g o f R a s p r o t e i n s Guanine nucleotide loading of cell lysates was accomplished by modification of the protocol described by Taylor and Shalloway (1996). Exponentially growing cells were harvested by centrifugation (500 x g, 5 min), washed three times with K K 2 , and resuspended at 5 x 107 cells/ml in K K 2 . Cells were lysed in an equal volume of cell suspension and ice cold 2 X G T P -L B (20 m M Sodium Phosphate, pH 7.2, 2 % Triton X-100, 20 % Glycerol, 300 m M N a C l , 10 m M E D T A , 2 m M N a 3 V 0 4 , 10 m M NaF, with two tablets of 'Complete protease inhibitor cocktail ' (Roche) added per 50 ml buffer). Either 1 m M (final concentration) G D P or GTPyS, a non-hydrolysable G T P analogue, was added and incubated at room temperature for 10 min. M g C l 2 was added to a final concentration of 50 m M and incubated at room temperature for 30 41 min. Protein concentrations were determined using the DC Protein Assay, according to manufacturer's instructions, and subjected to the R B D binding assay. 2 . 2 . 9 P K B p h o s p h o r y l a t i o n a s s a y 7 ' 6 h pulsed cells were resuspended at 5 x 10 cells/ml in K K 2 supplemented with 1 m M caffeine, and shaken at 200 rpm for 30 min (Jeon et al., 2007). Caffeine treatment has been shown to block the c A M P relay (Brenner and Thorns, 1984), and is used to prevent auto stimulation by inhibiting endogenous c A M P pulsing, as a means to synchronize cells prior to stimulation. Cells were stimulated with 15 u M (final concentration) c A M P , and 100 ul samples were taken at the indicated time points and lysed in 20 ul 6 X S D S - P A G E loading buffer. Samples were heated to 100°C for 5 min and phosphorylated P K B was detected by immunoblot analysis using a polyclonal anti-phospho-threonine specific antibody (Cell Signaling Technology, Danvers, M A ) . Phosphorylated P K B appears as a -51 kDa band, and the identity of this band has been previously confirmed as P K B with the use of an anli-Dictyostelium P K B antibody (L im et al., 2001). 2 . 2 . 1 0 I m m u n o b l o t A n a l y s i s Protein samples were prepared by collecting cells by centrifugation (500 x g, 5 min) and lysing directly in I X S D S - P A G E loading buffer. Other times, cells in suspension in K K 2 buffer were directly lysed by mixing with 6 X S D S - P A G E loading buffer in a volume ratio of 5:1. Soluble lysate fractions were collected by mixing with 6 X S D S - P A G E loading buffer in a 42 volume ratio of 5:1. Samples were boiled at 100°C for 5 min and used for discontinuous SDS-P A G E or stored a t -86°C. Discontinuous S D S - P A G E gels consists of an upper protein stacking gel (3.9 % bis-acrylamide (1:19), 125 m M Tris-Cl , p H 6.8, 0.1 % SDS, 0.03 % A P S , 0.1 % T E M E D ) and a lower protein resolving gel (10 % bis-acrylamide (1:19), 375 m M Tris -Cl , p H 8.8, 0.1 % SDS, 0.03 % A P S , 0.07 % T E M E D ) . Samples were electrophoresed at 100 V for 100 min. The electrophoresis buffer was 25 m M Tris, 192 m M glycine, 0.1 % SDS, p H 8.3. Separated proteins were blotted onto Hybond P P V D F membranes using a M i n i Protean II electroblot apparatus (Bio-Rad) at 0.2 A for 1 h submerged in ice-cold transfer buffer (25 m M Tris, 192 m M glycine, 20 % methanol). The gels were stained with Coomassie blue solution (0.025 % w/v Coomassie G-250, 10 % v/v acetic acid) and examined visually for equal loading and even transfer. Membranes were incubated for 1 h at 22°C with gentle agitation, in blocking buffer consisting of 5 % w/v milk (non-fat dried powder) in T B S - T (50 m M Tris -Cl , pH 7.4, 154 m M N a C l , 0.1 % Tween20) when using Ras isoform specific antibodies, or in 5 % w/v bovine serum albumin (BSA) in T B S - T when probing for phosphorylated P K B , and then incubated with anti-Ras isoform specific primary antibodies in 1 % w/v non-fat milk powder/TBS-T overnight at 22°C or with anti-phospho-threonine antibodies in 5 % w/v B S A / T B S - T overnight at 4°C on a rotary shaker. Membranes were washed 3 times for 5 min each with T B S - T and incubated with H R P conjugated goat anti-rabbit secondary antibody (GE Healthcare) at 1:5000 dilution in 1 % w/v mi lk /TBS-T for 1 h at 22°C on a rotary shaker. Membranes were washed 3 times for 5 min each with T B S - T , developed with E C L or E C L Plus, as indicated, according to manufacturer's instructions and exposed to X - O M A T X K - 1 X-ray fi lm (Eastman Kodak Company, Rochester, N Y ) for the appropriate amount of time. 43 3 RESULTS 3.1 Selection, cloning, and induction of GST-RBD bacterial constructs Taylor and Shalloway developed an assay that is able to measure the level 'of activated Ras from cellular lysates (Taylor and Shalloway, 1996). This assay is not too labour intensive, requiring as much work as an ordinary immunoprecipitation, and it does not require the use of radioactivity like previous methods used to study Ras activation (Satoh et al., 1988). The assay, referred to here as the ' R B D binding assay' (Figure 5), exploits the specific nature of the interaction between Ras and its effectors. The region of the effector proteins that binds to Ras is called the Ras binding Domain (RBD) , and the R B D binds to Ras-GTP with an affinity that is three orders of magnitude greater than its affinity for Ras-GDP (Herrmann et al., 1995). Taylor and Shalloway fused the R B D of mammalian Raf l to a Glutathione-S-transferase (GST) affinity tag and incubated this fusion protein with cellular lysates. B y virtue of the G S T affinity tag, the G S T - R B D - R a s - G T P complex can be purified from cell extracts using glutathione-sepharose beads, and the bound Ras protein can be detected via immunoblot analysis using anti-Ras antibodies. 44 asBindingDomain Binding Assay 1) Add RBD to cell lysate GST • • T ? i S Ras-GDP Ras-GTP < ^ 0 fph. GST 2) RBD binds specifically to Ras-GTP rob r~c& GST GST L2r 3) Immunoblot Ras (S17N) Ras (G12T) Figure 5. The R B D Bind ing Assay. The R B D Binding assay is based on the observation that the R B D of Ras effector proteins binds with a much higher affinity to Ras-GTP than Ras-GDP. The R B D is fused to a G S T affinity tag, incubated with cellular lysates, and the GST-RBD-Ras -G T P complex can be purified using glutathione-sepharose beads. The affinity purified Ras can be detected by immunoblot analysis with anti-Ras antibodies. 45 The most important consideration in developing an assay to measure activated Ras was to find a R B D that was able' to bind to the different Dictyostelium Ras proteins. In the t Dictyostelium Ras proteins, the effector domains are almost identical to the effector domain of H-Ras, with only one amino acid difference in RasC (D38N, relative to H-Ras) and RasS (I36L) (Figure 2). Thus it seemed plausible that Dictyostelium Ras proteins would be able to bind to the R B D of mammalian effector proteins. A s the R B D s of Ra f l and Ra lGDS had been successfully employed for Ras subfamily activation assays (Kang et al., 2002; Taylor and Shalloway, 1996), these two R B D s were chosen for initial study. E. coli strains expressing GST-Raf 1 (RBD) and G S T - R a l G D S (RBD) (referred to as G S T - R a f l and G S T - R a l G D S , respectively, unless otherwise noted) were obtained as gifts from D . Shalloway and M . Gold, respectively, and protein was expressed and purified as described in Materials and Methods. Both G S T - R B D proteins were expressed at high levels and the predominant protein band in the purified fraction corresponded to the predicted molecular weight of the fusion protein, based on amino acid sequence (Figure 6, Table 3). T a b l e 3 . P r e d i c t e d m a s s o f G S T f u s i o n p r o t e i n s . G S T a l o n e G S T - B y r 2 G S T -R a l G D S G S T - R a f l G S T -P I 3 K 1 G S T -P I 3 K 2 P r e d i c t e d s i z e o f f u s i o n p r o t e i n , k D a 29.0 56.2 45.8 41.9 58.1 54.5 Note: Predicted molecular weight was calculated by amino acid sequence using ProtParam tool (http://us.expasy.org/tools/protparam.html). 46 Figure 6. Expression and purification of GST-RBD fusion proteins. 500 m L of L B media were inoculated with 5 m L of an overnight culture of E. coli BL21 DE3 strains carrying plasmids that encoded GST-Byr2 , G S T - R a l G D S , G S T - R a f l , GST-PI3K1, and GST-PI3K2 fusion proteins as labelled on figure. Cells were grown at 37°C until OD600 -0.8, induced with 1 m M JPTG, and incubated for 18 h at 22°C. Samples were collected before adding JPTG (labelled 'Uninduced') and after induction (labelled 'Induced'). Cells were harvested by centrifugation (5000 x g, 10 min, 4°C), and lysed by sonication. Lysates were cleared by centrifugation (12,000 x g, 30 min, 4°C). 50 ul of a 50:50 glutathione-sepharose bead slurry were added to 1 m L bacterial cell lysates and incubated at 4°C for 2 hours. Beads were pelleted by centrifugation and washed three times with ice cold PBS . 50 ul of I X SDS loading buffer were added to the pelleted beads, and protein was eluted by boiling for 5 min (labelled 'Pul l Down') . 10 ul were loaded onto a S D S - P A G E gel (10% acrylamide). The gel was stained with Coomassie Blue. Protein size standards are shown, in kDa. 47 The ability of G S T - R a f l and G S T - R a l G D S to bind to activated Ras was tested in lysates of Ax2::pVEJJ-rasfl (G15T)j Ax2::rasC-rasC (G13T), and A x 2 : : M B - r a s G (G12T), Dictyostelium strains that express the activated Ras mutants RasB (G15T), RasC (G13T), or RasG (G12T) (Table 2). These Ras mutants are the equivalent of the H-Ras (G12T) mutation that renders the GTPase activity non-functional, and locks the Ras protein in its G T P bound conformation. These three Ras proteins were chosen due to the availability of Ras isoform specific antibodies generated by our lab (Lim, 2002; Robbins et al., 1991; Sutherland, 2001). Cells were lysed in a phosphate buffer as described in Materials and Methods, as it has been shown that this type of buffer is efficient in immunoprecipitations of phosphorylated proteins (Yurchak et ali, 1996). Both RasB (G15T) and RasG (G12T) were able to bind t o G S T - R a f l and G S T - R a l G D S (Figure 7 A , 7C). There was no binding detected to a G S T alone control (Figure 7 A , 7C), demonstrating that the contaminating protein bands present in the purified G S T - R B D preparations recovered from E. coli (Figure 6) do not contribute to a non-specific binding of Ras. No detectable level of RasC (G13T) was able to bind to G S T - R a l G D S (Figure 7B), and binding of RasC (G13T) to G S T - R a f l could only be demonstrated i f blots were over exposed (Figure 7B, data not shown). 48 m Ui c CO o ± < CO o CO CD 1— CN CN ro co D_ a. m h- i— h-co CO CO CD o o CO Q CD CO rr • i -co CD CO DC h-CO o R a s B (G15T) B CO rr o c o CO CD co 1— CN GD T — ro co i _ >> CO H — CO CL 0_ m DC rr h- I— h- H co CO co CO co o CD CD CD O mm R a s C (G13T) co ro rr "ro o CO c o < I-CM Q CM CD T -CO CO co ro CL Q . CO rr rr 1 1- V- r— i — i — co co CO CO CO CD o o CD CD mm • F i g u r e 7. B i n d i n g o f R B D s t o a c t i v a t e d R a s m u t a n t s . Ax2::pVEII-ra*fi (G15T) and Ax2::MB-rasG (G12T) (Table 2) were induced to express activated Ras protein. Ax2::ra,sC-rasC (G13T) cells (Table 2) constitutively express activated RasC. Exponential stage (2-4 x 10 6 cells/ml) Ax2::pVEII-rasfl (G15T), Ax2::rasC-rasC (G13T), and A x 2 : : M B - r a s G (G12T) were harvested by centrifugation, and 2.5 x 107 cells were lysed in 1 ml R B D - L B , incubated on ice for 10 min, and lysates were cleared by centrifugation (13,000 x g, 10 min, 4°C). 50 pi of GST-R a f l , G S T - R a l G D S , GST-Byr2 , GST-PI3K1 , GST-PI3K2, or G S T only bound to glutathione-sepharose beads were added to 500 pg total cellular protein, and the resulting mixtures were subjected to the R B D Binding assay. After elution of the bound Ras protein in 50 \il of I X SDS loading buffer, 10 Lll of the samples were fractionated by S D S - P A G E (10 % acrylamide) and bound Ras protein was detected by immunoblot analysis. 10 p i of 6 X SDS loading buffer were added to 50 p.1 of Dictyostelium lysates (1 mg/ml), indicated as 'Total Ras,' and approximately 8.3 p:g total protein were loaded on the gel. (A) RasB (G15T), (B) RasC (G13T), (C) RasG (G12T). 49 The Dictyostelium genome contains a number of genes encoding PI3Ks (Zhou et al., 1995), and it has been well established that mammalian Ras-GTP binds to and activates mammalian PI3K (Pacold et a l , 2000; Rubio et al., 1997). In light of this link, we tested the ability of the R B D s of Dictyostelium PI3K1 and PI3K2 to bind to Dictyostelium Ras. These R B D s had previously been reported to bind to RasD and RasG, but not to any other Dictyostelium Ras proteins when tested in a yeast two-hybrid assay (Funamoto et al., 2002; Lee et al., 1999). E. coli strains expressing GST-PI3K1 (RBD) and GST-PI3K2 (RBD) (referred to as GST-PI3K1 and GST-PI3K2, respectively, unless otherwise noted) were obtained as a gift from R. Firtel, and protein was expressed and purified as described in Materials and Methods. Both G S T - R B D proteins were able to be purified from bacterial extracts, but at levels lower than that of G S T - R a f l and G S T - R a l G D S (Figure 6). While a band was present in the purified fraction that corresponded to the predicted size of the fusion protein (Table 3), the fusion protein was subject to degradation, as evidenced by the presence of a number of other bands (Figure 6). A s these bands were also present in the bacterial whole cell lysates, it would suggest that both GST-PI3K1 and GST-PI3K2 experienced degradation in vivo (Figure 6). GST-PI3K1 and G S T -PI3K2 did not to bind RasB (G15T) and RasC (G13T) (Figure 7 A , 7B), supporting the results of the yeast two-hybrid assay. While GST-PI3K1 and GST-PI3K2 were able to bind RasG (G12T), the relative amount of bound RasG was significantly lower when compared to that bound to G S T - R a f l , G S T - R a l G D S , and GST-Byr2 (Figure 7C). The low amount of bound RasG may reflect the in vivo affinity of Ras for the R B D s of PI3K1 and PI3K2, or the relative instability of the GST-PI3K1 and GST-PI3K2 fusion proteins, rendering these fusion proteins inadequate for use in an assay to measure activated Ras. 50 While both Ra f l and Ra lGDS were very efficient at binding to RasB and RasG, neither of these R B D s were able to interact very well with RasC. Although RasC shares very high overall sequence identity with H-Ras, the amino acid difference at position 39 (the equivalent of position 38 in H-Ras), asparagine instead of aspartate, had been shown through a mutational analysis of H-Ras to abrogate binding to its effector proteins (Akasaka et al., 1996). This change may explain the inability of G S T - R a f l , G S T - R a l G D S , GST-PI3K1 , and GST-PI3K2 to bind to activated RasC. Another Ras effector, the Schizosaccharomyces pombe kinase Byr2, had been shown to retain the ability to bind to H-Ras containing the D 3 8 N substitution (Akasaka et al., 1996), and thus it was postulated that the Byr2 R B D may bind activated RasC as well . In order to test this hypothesis, the R B D of Byr2 was cloned from S. pombe genomic D N A via P C R , as described in Materials and Methods. Protein frofn the E. coli strain expressing GST-Byr2 (RBD) (referred to as GST-Byr2 , unless otherwise noted) was expressed and purified as described in Materials and Methods, and the predominant protein band in the purified fraction corresponded to the predicted molecular weight of GST-Byr2 (Figure 6, Table 5). A s predicted, Byr2 was able to bind to activated RasC (G13T) (Figure 7B). Furthermore, Byr2 was able to bind to RasB (G15T) and RasG (G12T) as well (Figure 7 A , 7C). These results demonstrate that Byr2 can be used as a tool to measure the level of activated RasB, RasC, and RasG from Dictyostelium cell lysates. To investigate the G T P dependence of the RBD-Ras interaction, two strains, A x 2 : : p V E H -rasG (G10V) and Ax2: :pVEII-rasG (S17N) (Table 2), expressing dominant negative mutants were employed. These strains over-express RasG (G10V) and RasG (S17N), respectively. In mammalian Ras, these mutations result in an inability of the Ras protein to exchange G D P for G T P , even in the presence of a RasGEF (John et al., 1993), locking Ras in a G D P bound 51 conformation. A x 2 : : M B - r a s G (G12T) (Table 2) was used as a positive control. G S T - R a f l and GST-Byr2 were used in this experiment as both G S T - R B D s were effective at recovering RasG-G T P from cellular lysates (Figure IC). Figure 8 shows that although RasG (G10V) and RasG (S17N) were highly expressed in cell lysates, they did not bind to either GST-Raf l or GST-Byr2 , as evidenced by the lack of a signal corresponding to RasG in the immunoblot. RasG (G12T) was effectively recovered by both GST-Raf l and GST-Byr2 (Figure 8). A faint signal was seen when lysates of RasG (S17N) were incubated with G S T - R a f l or GST-Byr2 . The signal could be representative of the activated pool of endogenous RasG, but the relative amount of this band is negligible when compared to RasG (G12T) (Figure 8). Furthermore, the amount of endogenous RasG-GTP would be expected to be low, as dominant negative Ras proteins have been shown to interfere with activation of endogenous Ras, at least in higher eukaryotes (Feig, 1999). These results show that the interaction between the G S T - R B D s and Ras is dependent on Ras being in its G T P bound, or activated form. To ta l R a s GST-Raf1 GST -Byr2 P > z P > z P > z c N O r - . C\J o r - CM o 5 5 m 5 5 « 5 5 5 o o o o G o o o o ( / ) ( / ) ( / ) c o c a o ( o w e / ) (0 (0 (0 ( 0 ( 0 ( 0 ( 0 ( 0 ( 0 rr cr cr cr cr cr cr cr cr * 9 w Figure 8. Binding of GST-Rafl and GST-Byr2 to activating and dominant negative RasG mutants. Ax2: :MB-ra .vG (G12T), Ax2 : :pVEU-rasG (G10V), and Ax2::pVEII-rasG (S17N) (Table 2) were induced to express activated Ras protein. Lysates were prepared as described in Materials and Methods and activated Ras was measured as described in Figure 7. 10 fal of 6 X SDS loading buffer were added to 50 ul of Dictyostelium lysates (1 mg/ml), indicated as 'Total Ras,' and approximately 8.3 ug total protein were loaded on the gel. ' G S T - R a f l ' refers to Ras-G T P bound to G S T - R a f l , and 'GST-Byr2 ' refers to as Ras-GTP bound to GST-Byr2 . 52 3 . 2 R B D i n t e r a c t i o n w i t h e n d o g e n o u s l e v e l s o f R a s The experiments in Figure 7 and 8 were carried out with Dictyostelium strains that over-express activated Ras proteins in order to ensure that activated Ras was not a limiting factor. However, studies measuring Ras-GTP in N I H 3T3 cells have shown that physiological levels of activated endogenous Ras are much lower (1.3 fmol per mg of cellular protein) relative to N I H 3T3 cells that over-express H-Ras (G12T) (2049 fmol per mg of cellular protein) (Scheele et al., 1995). Thus, in order to determine whether or not the RBD-Ras interaction was strong enough to detect only endogenous Ras-GTP, protein lysates from wild type A x 3 cells were isolated and incubated with GTPyS or G D P to load Ras protein in the lysates with either nucleotide (Materials and Methods). Using G S T - R a f l , GST-Byr2 and G S T - R a l G D S , RasG was detected from GTPyS loaded lysates (Figure 9C), in support of the data from Figure 7C. Contrary to the data seen in Figure 7C, GST-PI3K1 and GST-PI3K2 did not pull down detectable levels of RasG (Figure 9C). One possible explanation for this observation is that the apparent instability of GST-PI3K1 and GST-PI3K2 (Figure 6) had an effect On the structure of the fusion proteins, affecting their ability to bind to RasG. When lysates were loaded with G D P prior to incubation with GST-Byr2, the amount of RasG recovered was either reduced from the levels seen with GTPyS, or completely abolished (Figure 9C), which showed that the interaction was selective for RasG-G T P over RasG-GDP. 53. CO CO in c to o E < o CO B CM Q * CM CD T— CO CO W— CO * ^ CO CL CL CO rr rr h- h- 1— CO CO CO CO CO CD o o o a — mm CO ra o co co co co rr < o. CL m cr rr <g H I— I— I— h- I— o co w w co co co H CD CD CD CD CD CD R a s B GTPyS RasB G D P R a s C GTPyS R a s C G D P co Q k ^ CN CD CD rr < CL CL m rr cr c i i I I co I— I— I— I— fc H 5 CO CO CO CO CO CO K CD CD CD CD CD CD R a s G G T P 7 S R a s G G D P Figure 9 . G T P dependence of R B D - R a s interaction from endogenous levels of Ras. Exponential stage (2-4 x 10 6 cells/ml) Ax3 cells (Table 2) were harvested by centrifugation, and 2.5 x 107 cells were lysed in 1 ml R B D - L B , incubated on ice for 10 min, and lysates were cleared by centrifugation (13,000 x g, 10 min, 4°C). Lysates were loaded with either 1 m M GTPyS or 1 m M G D P , and activated Ras was measured as described in Figure 7. Bound levels of (A) RasB, (B) RasC, and (C) RasG protein were detected by immunoblot analysis. 10 pi of 6 X SDS loading buffer were added to 50 pi of Dictyostelium lysates (1 mg/ml), indicated as 'Total Ras,' and approximately 8.3 pg total protein were loaded on the gel. 54 Figure 9 A shows the ability of the G S T - R B D proteins to bind to RasB. Consistent with the results from Figure 7 A employing cells over-expressing RasB (G15T), GST-Byr2 , G S T - R a f l and G S T - R a l G D S were able to bind to RasB-GTP in cellular lysates, while the GST-PI3K1 and GST-PI3K2 proteins were not. A s shown for RasG, G D P loading of lysates prior to incubation with the G S T - R B D s completely abolished the interaction with RasB (Figure 9A) . The pull down performed on the GTPyS loaded lysates shows that it was possible to detect RasC-GTP only when using GST-Byr2 , and the amount of RasC bound to GST-Byr2 was reduced when lysates were incubated with G D P prior to incubation with GST-Byr2 (Figure 9B). Consistent with the experiments using cells Over-expressing activating mutants (Figure 7B), none of the other R B D s permitted detection of RasC-GTP (Figure 9B). 3.3 O p t i m i z i n g p r o t e i n l o a d f o r a R B D p u l l d o w n a s s a y The data in Figures 7, 8, and 9 was from experiments that employed 500 | lg of total cellular protein and 100 ul of a 50:50 bead:buffer slurry (Materials and Methods). The amount of cellular protein and G S T - R B D bead volume was not an issue as the aim of the aforementioned experiments was to determine whether the R B D s were able to bind to activated Ras or not. However, i f the beads are saturated with Ras-GTP in the absence of any stimulus, an increase in activated Ras in response to a stimulus wi l l not be detected. To investigate the effect of input cellular protein, lysates from cells loaded with GTPyS were used in a pull down with 50 ul of a 50:50 GST-Byr2 bead slurry. The cell lysates were loaded with GTPyS in order to simulate maximum activation of RasC and RasG. The volume of GST-Byr2 bead slurry was chosen as an amount that would be practical to use in future experiments. The results of the experiment show that the amount of RasC-GTP binding to 50 ul GST-Byr2 bead slurry saturated when 400 ug of 55 Dictyostelium extract protein were added (Figure 10B). RasG-GTP binding to 50 ul GST-Byr2 bead slurry saturated between 400 to 500 (ig Dictyostelium extract protein, as evidenced by monitoring the unbound fraction of RasG (Figure 10A). Thus, the amount of Dictyostelium extract protein used in subsequent experiments was 400 (ig. GST-Byr2 Unbound Total jtein ( R a s G o o o o o o o o o o Prot i  (ua) o o o o o o © o o o o o v^ y; Gn i - CN co Tt io n ^ <v w -* w B GST-Byr2 Total o o o o o o Protein (ua) o o © o © LO R a s C — Figure 10 . Effect of input protein on R B D binding assay. Ax3 cells were loaded with GTPyS, and 2.5 x 10 7 cells were lysed in 1 ml R B D - L B , incubated on ice for 10 min, and lysates were cleared by centrifugation (13,000 x g, 10 min, 4°C). Total protein was quantified using Bio-Rad D C Protein Assay. Increasing quantities of total protein ranging from 50 to 500 | ig were incubated with 50 (il of GST-Byr2 bound to glutathione-sepharose beads, and the mixture was subjected to the R B D binding assay as described in Figure 7. After elution of the bound Ras protein in 50 ul of I X SDS loading buffer, 10 ul of the samples were fractionated by S D S - P A G E (10 % acrylamide) and bound (A) RasG and (B) RasC protein was detected by immunoblot analysis. 10 ul of 6 X SDS loading buffer were added to 50 ul of Dictyostelium lysates (1 mg/ml), indicated as 'Total Ras,' and approximately 8.3 | ig total protein was loaded on the gel. 10 ul of 6 X SDS loading buffer were added to 50 ul of the supernatant (1 mg/ml) from the first centrifugation before the first wash, indicated as 'Unbound,' and approximately 8.3 fig total protein were loaded on the gel. 'GST-Byr2 ' refers to Ras bound to GST-Byr2 , 'Unbound' refers to fraction of Ras not bound to GST-Byr2 . 56 3.4 Chemoattractant induced activation of R a s C and R a s G Previous evidence had suggested that RasC is involved in regulating signalling events during the early development of Dictyostelium: Ablation of the rasC gene results in cells that are unable to undergo development, largely due to an inability to activate A C A (Lim et al., 2001). As no other ras gene knockout strains characterized at the time this thesis was started showed any defects in early development, it was hypothesized that RasC was activated by c A M P in early developmental cells, and that RasC was the sole Ras protein involved in c A M P stimulated signalling. To test the first part of this hypothesis using the R B D binding assay, wi ld type Ax3 cells (Table 2) were suspended in non-nutrient buffer and induced to initiate early development by applying exogenous pulses of c A M P (final concentration 50 nM) for 6 h (referred to as pulsed cells henceforth), and then stimulated with 200 n M c A M P (Materials and Methods). These exogenous pulses mimic the developmental program of Dictyostelium, thus all strains, even those defective in endogenous signalling, undergo synchronized developmental changes. Aliquots were taken at the indicated time points after the 200 n M stimulation, lysed, and the level of activated Ras was measured from each aliquot using the R B D binding assay. RasC displayed a rapid and transient increase in G T P binding upon c A M P stimulation, with peak activation levels reached within 10 s of stimulation (Figure 11 A ) . A second, less intense peak was observed starting at 80 s and remained on until at least 150 s (Figure 11 A ) . However, the timing and intensity of the second peak was variable and thus its physiological relevance is unclear at this time. This data provided direct evidence that RasC activation is responsive to c A M P signalling events. 57 Total Ras-GTP R a s Time (s) o 10 20 30 40 60 70 so 90100110120150 RasC RasG ••^ '•W*: ^ ' •  B Ras-GTP Total Ras Time (s) 0 10 20 30 40 60 70 80 90 100 * RasB Figure 11 . c A M P stimulated Ras activation. Pulsed Ax3 cells were harvested by centrifugation (500 x g, 5 min), washed 2 times with KK/>, and resuspended in K K 2 at 5 x 10 7 cells/ml. Cells were stimulated with 200 n M c A M P , and 400 p.1 of cells were added to 400 pi 2 X R B D - L B before stimulation and at the time points indicated, and incubated on ice for 10 min. Lysates were quantified and 400 p.g of protein lysate were added to 50 p.1 of GST-Byr2 bound to glutathione-sepharose beads. After elution of the bound Ras protein in 50 pi of I X SDS loading buffer, 10 pi of the samples were fractionated by S D S - P A G E (10 % acrylamide) and bound Ras protein was detected by immunoblot analysis using specific antibodies against (A) RasC and RasG, (C) RasB. 10 p.1 of 6 X SDS loading buffer were added to 50 pi of Dictyostelium lysates (1 mg/ml), indicated as 'Total Ras,' and approximately 8.3 pg total protein were loaded on the gel. 'Ras-GTP' refers to Ras bound to GST-Byr2 . The experiment is representative of four separate experiments. 58 RasG also displayed a similar rapid and transient activation upon c A M P stimulation (Figure 11A). Up to this point, all the evidence had suggested that RasG functioned primarily during the growth stage of Dictyostelium cells. Thus it was not expected for RasG to be responsive to c A M P , so this observation was somewhat surprising. Subsequent generation of a rasC/rasG double knockout strain corroborated the involvement of RasG in early development (Bolourani et al., 2006; Section 4.2). Activation of RasB was measured as a control as the available evidence suggested that RasB is involved in the control of the cell cycle rather than development (Sutherland et al., 2001). No increase in activated RasB was observed in response to c A M P stimulation (Figure 1 IB) . 3.5 R a s a c t i v a t i o n i s d e p e n d e n t o n t h e c A M P r e c e p t o r c o m p l e x c A M P signalling during early development involves a G P C R , c A R l (encoded by the car A gene), that binds c A M P with high affinity, as well as a low affinity G P C R , c A R 3 (carC) (Soede et al., 1994); a carA'/carC double knockout is unable to initiate any c A M P induced signalling during aggregation (Insall et al., 1994; Soede et al., 1994). Thus, in order to determine whether the activation of RasC and RasG was dependent on the c A M P receptors, pulsed A x 3 and carA' /carC cells (Table 2) were stimulated with c A M P and assayed for activation of Ras. No increase in activated RasC or RasG was observed in pulsed carA'/carC cells, indicating that all RasC and RasG activation during early development is dependent on c A M P receptors c A R l and c A R 3 (Figure 12A). 59 Ras-GTP R a s G RasG Ras-GTP Time (s) o 10 60 o 10 60 RasC RasG Total Ras A x 3 carA/carC ft J J ^ V J 10 60 0 10 60 * Time (s) 0      " $ RasC B Ras -GTP I ° t a l Ras A x 3 gpaB- ft J> Time (s) 0 10 60 0 10 60 RasC Total Ras A x 3 gpbA ft Figure 12. The c A M P receptor complex is required for Ras activation. Pulsed A x 3 , carA' /carC, gpbA, gpaB' cells (Table 2), were harvested by centrifugation (500 x g, 5 min), washed 2 times with K K i , and resuspended in KK2 at 5 x 107 cells/ml. Cells were stimulated with 200 n M c A M P , and activated Ras was measured as described in Figure 11. 10 | i l of 6 X SDS loading buffer were added to 50 u.1 of Dictyostelium lysates (1 mg/ml), indicated as 'Total Ras,' and approximately 8.3 ug total protein were loaded on the gel. 'Ras-GTP' refers to Ras bound to GST-Byr2 from (A) carA/carC, (B) gpbA, and (C) gpaB strains. The experiments are representative of three separate experiments. 60 There are two general types of signalling pathways that transmit signals induced by c A M P : those that involve the heterotrimeric G-protein associated with the c A M P receptor, and those that do not (reviewed in Manahan et al., 2004). The Dictyostelium genome contains a single gene that encodes the Gj3 protein, gpbA, component of the heterotrimeric G-protein, so all G-protein mediated events are dependent on G ^ (Wu et al., 1995). To determine the involvement of G(3 in Ras activation, pulsed A x 3 and gpbA' cells (Table 2) were stimulated with c A M P and assayed for activation of Ras. The increase in RasC or RasG activation observed in A x 3 cells in response to c A M P stimulation was not seen in the gpbA' mutant (Figure 12C). This indicated that the G(3 protein is required for c A M P dependent RasC and RasG activation. There are at least 11 genes that encode for G alpha proteins, but only one, gpaB (Ga2 protein), is believed to mediate the stimulatory pathways associated with c A R l during aggregation (Kumagai et al., 1991). To test whether Gcc2 is necessary for c A M P stimulated activation of RasC and RasG, pulsed A x 3 and gpaB' cells (Table 2) were stimulated with c A M P and assayed for activated Ras. No increase in activation of RasC and RasG was observed in cells lacking the gpaB gene (Figure 12B). The results of these experiments demonstrate that during aggregation, RasC and RasG activation are dependent on the c A M P stimulus signalling through the c A M P receptor and its associated G protein complex Ga2Py. 3.6 R a s a c t i v a t i o n i s i n d e p e n d e n t o f A C A , C R A C , a n d P i a A Two cytosolic proteins have been shown to be required for the activation of A C A (encoded by the acaA gene) downstream of the c A M P receptor: C R A C , or the 'Cytosolic Regulator of Adenylyl Cyclase' {dagA) and Pianissimo (piaA) (Chen et al., 1997; L i l l y and Devreotes, 1994). Their involvement in Ras activation was tested by stimulating pulsed A x 3 , acaA', dagA', and 61 piaA' cells (Table 2), and assaying for activated RasC and RasG. None of the strains exhibited a defect in the activation of RasG and RasG (Figure 13). These data demonstrated that A C A , C R A C , and PiaA are not involved in c A M P simulated activation of RasC and RasG. Taken together with the previous experiments (Figure 11, 12), these results demonstrate that RasC and RasG activation lie downstream of the c A M P receptor and its associated G protein. Previous results have indicated that RasC is required for A C A activation (L im et al., 2001). The c A M P dependent activation of RasG clearly depends on c A R l (Figure 12A), and the apparent involvement of RasG in regulating the T O R C 2 complex (Lee et al., 2005), which is also required for A C A activation, also places RasG activation upstream of A C A and its regulators. It should be noted that the loss of C R A C and PiaA resulted in an increased level of activated RasC at 60 s relative to wi ld type (Figure 13A, 13B), and the loss of A C A resulted in an increased level of activated RasG at 60 s, relative to wild type (Figure 13C). The reason for this is not clear, but as c A M P is an important intracellular second messenger, it is possible that the defect in c A M P stimulated A C A activation demonstrated by these strains (Chen et al., 1997; L i l l y and Devreotes, 1994; Pitt et al., 1992) results in a deregulation of Ras activation (Section 4.5). 62 o o r n TOtal R a s - G T P R a s Ax3 dagA /> Time (s) o 10 60 0 10 60 v 6 R a s c m m ^ mm R a s G B R a s - G T P Tota l R a s Ax3 piaA 4> # ^ <?x T i m e (s) 0 10 60 0 10 60 R a s C R a s G C R a s - G T P To ta l R a s Ax3 a c a A T i m e (s) 0 10 60 0 10 60 R a s C R a s G H i Figure 13. Ras activation is upstream of A C A and its cytosolic regulators. Pulsed A x 3 , dagA, piaA, and acaA cells (Table 2), were harvested by centrifugation (500 x g, 5 min), washed 2 times with KK2, and resuspended in KK2 at 5 x 10 7 cells/ml. Cells were stimulated with 200 n M c A M P , and activated Ras was measured as described in Figure 11. 10 p.1 of 6 X SDS loading buffer were added to 50 pi of Dictyostelium lysates (1 mg/ml), indicated as 'Total Ras,' and approximately 8.3 pg total protein were loaded on the gel. 'Ras-GTP' refers to Ras bound to GST-Byr2 from (A) dagA, (B) piaA, (C) acaA strains. The experiments are representative of three separate experiments. 63 3.7 R a s G E F A mediates activation of R a s C Insall et al. identified R a s G E F A as a component involved in G-protein mediated c A M P stimulated signaling events (Insall et al., 1996). Strains with a disrupted rasC gene have similar phenotypes td those of the gefA' mutant, and hence it was speculated that R a s G E F A might mediate RasC activation (L im et al., 2G01). However, expression of activated RasC in gefA' cells fails to rescue the phenotypic defects of the gefA' strain (C.J. L i m , unpublished observations). This was not entirely unexpected as the expression of activated RasC in rasC cells does not fully rescue the defect i i i multicellular development, and appears to be detrimental to development when expressed in wild type cells (Lim, 2002). Thus, the relationship between RasC and R a s G E F A remained unclear. The R B D binding assay allowed for an alternative strategy to test this relationship. Pulsed Ax3 and gefA' cells (Table 2) were stimulated with c A M P , and the levels of activated Ras were measured at various time points post stimulation. In cells lacking R a s G E F A , no activation of RasC was observed, indicating that R a s G E F A is required for all of the c A M P stimulated RasC activation (Figure 14). In contrast, there was no reduction of RasG activation upon c A M P stimulation in gefA' cells compared to wi ld type cells, indicating that R a s G E F A is not involved in RasG activation (Figure 14). This result led to a collaborative effort with A . Kortholt from the Van Haastert lab, who had established an in vitro fluorescence nucleotide exchange assay to identify substrates for different Dictyostelium RasGEFs. This assay involves loading the Ras protein with a fluorescent analogue of G D P , 2'-, 3'-bis(O)-(N-methylanthraniloyl)-guan0sine diphosphate, m G D P , and incubating Ras-mGDP in the presence of a RasGEF (Lenzen et al., 1995). m G D P displays a higher level of fluorescence in the hydrophobic environment of the G T P binding pocket relative to the aqueous environment of the buffer solution, so RasGEF stimulated nucleotide release can be monitored as 64 a decay in fluorescence over time. Nucleotide exchange in the absence of a RasGEF was measured to determine the intrinsic nucleotide exchange activity of the Ras protein, and used as a background control. The rate of fluorescence decay can be fitted onto a single exponential curve to derive the observed rate constant, k 0 Ds- As summarized in Table 4, results from this assay confirmed that R a s G E F A mediates nucleotide release specifically from RasC, and not any of the other Ras proteins tested (Kae et al., 2007). It bears pointing out that the loss of gefA appeared to coincide with a prolonged RasG activation (Section 4.5). Ras-GTP Total Ras Ax3 g e f A ^ Of Time (s) 0 10 60 0 10 60 RasC iiiliHli iiHHii RasG Figure 14. gefA' mutants are deficient in RasC activation. Pulsed Ax3 and gefA' cells (Table 2), were harvested by centrifugation (500 x g, 5 min), washed 2 times with K K 2 , and resuspended in K K 2 at 5 x 107 cells/ml. Cells were stimulated with 200 n M c A M P , and activated Ras was measured as described in Figure 11. 10 ul of 6X SDS loading buffer were added to 50 ul of Dictyostelium lysates (1 mg/ml), indicated as 'Total Ras,' and approximately 8.3 [ig total protein were loaded on the gel. 'Ras-GTP' refers to Ras bound to GST-Byr2 . The experiment is representative of three separate experiments. 65 T a b l e 4. O b s e r v e d r a t e c o n s t a n t , k p b s , o f R a s G E F s t i m u l a t e d n u c l e o t i d e e x c h a n g e . Intrinsic / x lcrV +ve Control Ax lO^s 1 Fold Increase, +ve/ Intrinsic RasGEFA / x i o - V Fold Increase RasGEFA/ Intrinsic RasGEFR / x i o - V Fold Increase RasGEFR/ i Intrinsic GbpD / x l O V Fold Increase GbpD/ Intrinsic RasB 1.2 2.3 1.9 1.1 0.9 1.2 1.0 1.1 0.9 RasC 1.2 2.7 2.3 5.70 4.8 1.1 0.9 1.4 1.2 RasD 2.9 8.0 2.8 2.7 0.9 2.6 0.9 3.1 ,1.1 RasG 0.4 6.0 .15 0.5 1.2 2.7 6.8 0.3 0.8 RasS 1.1 2.4 2.2 1.1 1.0 1.2 1.1 11 1.0 Rapl 0.4 2.8 7.0 0.4 1.0 0.3 0.8 2.7 6.8 The in vitro nucleotide exchange assay employs a fluorescent analogue of G D P , m G D P . m G D P has a higher level of fluorescence in the hydrophobic G T P binding pocket of Ras, so nucleotide release can be observed as a decrease in fluorescence as m G D P is released into the aqueous environment of the surrounding buffer. The observed rate of decay can be fitted onto a single exponential curve to derive the observed rate constant, kobs- 'Intrinsic' refers to the k 0 b S in the absence of a RasGEF, and serves as a background control. '+ve Control ' refers to the k 0 b S when the Ras protein was incubated with the positive control: mammalian R a s G R F l for RasB, RasC, RasD, RasG, RasS, and mammalian C 3 G for R a p l . 'Fold increase' is the ratio of the kobS for the indicated RasGEF-Ras reaction over the kobS for the intrinsic value. The data from this table was summarized from Kortholt et al. (2006) and Kae et al. (2007), and the analysis was performed by A . Kortholt. 66 3.8 R a s G E F R i s r e q u i r e d f o r m a x i m a l c A M P s t i m u l a t e d a c t i v a t i o n o f R a s G Secko et al. (2004) used a proteomics based approach to search for proteins involved in the RasG signalling pathway. This screen involved looking for proteins that underwent changes in their state of phosphorylation, as assayed with antibodies specific for phosphorylated serine, threonine and tyrosine residues, in response to the expression of activated RasG (G12T). One signalling protein identified was RasGEFR, which displayed a decrease in threonine phosphorylation when RasG (G12T) was expressed. A s it has been shown in mammalian systems that RasGEF phosphorylation can lead to either positive or negative regulation of RasGEF activity (Kesavapany et al., 2004; Yang et al., 2003), it was speculated that RasGEFR either mediates RasG activation, and that this activation is itself regulated by RasG, or R a s G E F R functions downstream of RasG signalling, activating a different Ras GTPase. The availability of a gefR' strain allowed a test of the first possibility. Pulsed A x 3 and gefR' cells (Table 2) were stimulated with c A M P , and assayed for activated Ras. There was a partial reduction in RasG activation in gefR' cells, suggesting that R a s G E F R is partially responsible for the activation of RasG (Figure 15). Since the reduction in RasG activation was only partial, it would appear that there are at least two RasGEFs that mediate c A M P stimulated RasG activation. This conclusion is consistent with the observation that gefR' cells display no apparent defect in early development (Secko et al., 2004), although the phenotypic examination of the gefR' cells was limited to the ability of the mutant cells to complete multicellular development ( D . M . Secko, unpublished observations). There was no defect in RasC activation in gefR' cells (Figure 15); however, the level of RasC-GTP in gefR' cells at 60 s was elevated compared to wi ld type cells (Section 4.5). The in vitro nucleotide exchange assay performed by A . Kortholt demonstrated that RasGEFR mediates G D P release specifically from RasG (summarized in Table 4), confirming the in vivo 67 activation data (Figure 15) and indicating that the observed decrease in RasG activation is likely due to the specific loss of RasGEFR activity on RasG (Kae et al., 2007). Time (s) RasC RasG Ras-GTP Ax3 gefR-0 10 60 0 10 60 Total Ras 4> # l i U M Figure 15. Loss of R a s G E F R reduces activation of R a s G dur ing development. Pulsed A x 3 and gefR' cells (Table 2), were harvested by centrifugation (500 x g, 5 min), washed 2 times with K K 2 , and resuspended in K K 2 at 5 x 10 7 cells/ml. Cells were stimulated with 200 n M c A M P , and activated Ras was measured as described in Figure 11. 10 p i of 6 X SDS loading buffer were added to 50 pi of Dictyostelium lysates (1 mg/ml), indicated as 'Total Ras,' and approximately 8.3 p.g total protein were loaded on the gel. 'Ras-GTP' refers to Ras bound to GST-Byr2 . The experiment is representative of four separate experiments. 68 In light of the above observations, the developmental phenotype of gefR' cells was scrutinized in more detail. When Dictyostelium cells are placed on plastic dishes under non-nutrient buffer, wild type cells aggregate via a process called streaming, and this behaviour initiates after ~10h in starvation buffer, as was the case for Ax3 cells (Figure 16). Similar to A x 3 cells, gefR' cells also formed aggregation stream in about 10 h (Figure 16), unlike rasG' cells, which exhibit a delay of about 4 h relative to wild type cells (Bolourani et al., 2006). It's been shown that A C A activity is required for cells to be able to form streams (Kriebel et al., 2003), so the observation that gefR' cells were able to form streams suggests that RasGEFR is not required for A C A activity. It appears that the streams formed by gefR' cells are less robust than the streams formed by wi ld type cells, (Figure 16), a phenotype similar to rasG' cells (Bolourani et al., 2006). As RasG is involved in regulating chemotaxis and cell motility (Bolourani et al., 2006), the difference in streaming pattern displayed by gefR' cells would suggest that RasGEFR may be involved in one of these processes. Figure 16. Streaming behaviour of A x 3 and gefR' cells dur ing aggregation. Cells were seeded as a monolayer at a density of 5 x 10" cells/cm"" and submerged under Bonner's salts solution. Images were photographed at 10 hours after the initiation of starvation. Ax3 gefR-69 c A M P stimulation of aggregation stage cells results in the rapid activation of P K B , and loss of P K B has been associated with defects in cell motility and polarity (Mei l i et al., 1999). It has been shown that P K B activation is dramatically reduced in rasG cells relative to wild type cells (Bolourani et al., 2006). c A M P stimulated activation of Dictyostelium P K B requires the phosphorylation of two threonine residues (T278 and T435) on P K B . A n immunoblot analysis using an anti-phospho-threonine antibody detects a number of threonine phosphorylated proteins in Dictyostelium cell lysates, including a -51 kDa band that corresponds to P K B . A n anti-Dictyostelium P K B specific antibody has confirmed that the band corresponds to P K B (Lim et al., 2001). Mutation of the threonine residues to non-phosphorylatable alanines results in loss of both in vivo phosphorylation of P K B and in vitro protein kinase activity, demonstrating that the phosphorylation state of P K B can be used as a direct measure of P K B activation (Lim, 2002). In order to determine the involvement of R a s G E F R in the RasG mediated activation of P K B , pulsed A x 3 and gefR' cells were incubated in the presence of 1 m M caffeine for 30 min and stimulated with 15 p M c A M P , as described in Materials and Methods. Samples were collected at the time points indicated, and activation of P K B was assayed for by immunoblot analysis using a phospho-threonine specific antibody. Phosphorylation of P K B was rapid and transient in both A x 3 and gefR' cells, peaking at 10 s post-stimulation and returning to basal levels by 30 s (Figure 17A). Thus, a loss of RasGEFR did not affect P K B phosphorylation. A s defects in either the PI3K or T O R C 2 signalling pathways results in a loss of P K B activation (Lee et al., 2005; M e i l i et al., 1999), the result from Figure 17A implies that RasGEFR is not involved in the regulation of either of these pathways. 70 A Ax3 gefR Time (s) 0 10 30 60 0 10 30 60 p P K B - ^ {MB B Ax2 gefM~ Time (s) 0 10 30 60 0 10 30 60 p P K B ^ F i g u r e 1 7 . c A M P i n d u c e d s t i m u l a t i o n o f P K B p h o s p h o r y l a t i o n . (A) Pulsed Ax3 and gefR', (B) A x 2 and gefM cells (Table 2) were harvested by centrifugation (500 x g, 5 min), washed two times with K K 2 , resuspended at 5 x 107 cells/ ml in K K 2 supplemented with 1 m M caffeine, and incubated with shaking for 30 min. 100 pi of cell suspension was added to 20 p.1 6 X SDS loading buffer before stimulating cells with 15 p M c A M P (final concentration) and at the time points indicated after stimulation. Samples were resolved by S D S - P A G E , and phosphorylated P K B was detected by immunoblot analysis. The band corresponding to phosphorylated P K B is indicated as ' p P K B . ' 3.9 R a s G E F M i s r e q u i r e d f o r m a x i m a l c A M P s t i m u l a t e d a c t i v a t i o n o f R a s G Using a bioinformatic approach to search the Dictyostelium genome for regulators of Ras, Arigoni et al. (2005) identified a RasGEF, R a s G E F M . When the gefM gene was ablated, the strain displayed defects in early development. gefM cells have chemotaxis and polarity defects, and development arrests after forming small, flat aggregates (Arigoni et al., 2005). Given the involvement of R a s G E F M in early developmental processes, it was postulated that R a s G E F M may regulate RasG activation. Pulsed wild type A x 2 and gefM cells (Table 2) were stimulated with c A M P and assayed for activated RasC and RasG. c A M P stimulation of A x 2 cells resulted 71 in a similar rapid increase in RasG activation when compared to A x 3 cells (compare Figures 11 and 18). However, unlike in 'Ax3 , RasG activation in A x 2 did not decrease to near basal levels at 60 s (compare Figures 11 and 18). This difference in the RasG activation profile observed may be attributed to differences between the wild type A x 2 and A x 3 strains. A x 3 was initially generated by mutagenesis of a non-axenic strain (Loomis, 1971), whereas A x 2 was generated from a non-axenic strain without using mutagenesis (Watts and Ashworth, 1970), and the different methods used in generating these strains is evidenced by the fact that A x 3 contains a -755 kb duplication of Chromosome 2 (Eichinger et al., 2005; Loomis et al., 1995). While it has been proposed that the duplication has no severe consequences on either growth or development (Loomis et al., 1995), it has been shown that A x 2 aggregates earlier than A x 3 , and that culmination proceeds directly from 'finger' to 'fruiting body' (Figure 3), bypassing the motile slug stage present in A x 3 (Kellerman and McNal ly , 1999). Whether or not these differences are related to the observed difference in RasG activation is unclear at this time. Compared to the activation in the parental A x 2 strain, there was a partial reduction in RasG activation when gefM cells were stimulated with c A M P (Figure 18), suggesting that R a s G E F M is involved in mediating RasG activation along with RasGEFR. However, the decrease in RasG activation in gefM' cells is modest relative to A x 2 , and since there is no in vitro data to confirm that RasG acts as a substrate for R a s G E F M , it is possible that the decrease in activated RasG is due to some unspecified secondary effect such as gene expression of a regulator of RasG. There was no dramatic difference in c A M P stimulated RasC activation between A x 2 and A x 3 strains (Figures 11 and 18), and while the activation of RasC was not affected in gefM cells, the level of RasC-. G T P at 60 s was higher in gefM cells than A x 2 (Figure 18, Section 4.5). 72 Time (s) RasC RasG Ras-GTP Ax2 gefM-0 10 60 0 10 60 Total Ras Figure 18. Loss of R a s G E F M reduces activation of R a s G dur ing development. Pulsed A x 3 and gefM cells were harvested by centrifugation (500 x g, 5 min), washed 2 times with K K 2 , and resuspended in K K 2 at 5 x 10 7 cells/ml. Cells were stimulated with 200 n M c A M P , and activated Ras was measured as described in Figure 11. 10 ul of 6 X SDS loading buffer were added to 50 ul of Dictyostelium lysates (1 mg/ml), indicated as 'Total Ras,' and approximately 8.3 ug total protein were loaded on the gel. 'Ras-GTP' refers to Ras bound to GST-Byr2 . The experiment is representative of three separate experiments. The polarity defect observed in chemotaxing gefM cells (Arigoni et al., 2005) suggested that there may be an effect on either the PI3K or T O R C 2 pathways. P K B phosphorylation is an effective way to determine the involvement of R a s G E F M in these pathways as both PI3K and T O R C 2 lead to P K B activation (Lee et al., 2005; M e i l i et al., 1999). Pulsed A x 3 and gefM cells were incubated in the presence of 1 m M caffeine for 30 min, stimulated with 15 u M c A M P , and samples were collected at the time points indicated. Samples were resolved using S D S - P A G E , transferred to a P V D F membrane, and probed for phosphorylated P K B using a polyclonal anti-phospho-threonine antibody. c A M P mediated P K B activation in gefM' cells was reduced 73 relative to A x 2 (Figure 17B), demonstrating that R a s G E F M is involved in P K B phosphorylation. While the amounts of protein loaded onto the gel were normalized by observing Coomassie blue staining of the gels following electrophoresis (data not shown), there was no way to measure the level of total P K B protein in these blots, so it is possible that the decrease in phosphorylated P K B could be a reflection of a decrease in total P K B . Assuming that the level of total P K B is not affected by the loss of R a s G E F M , these data support the idea that R a s G E F M regulates RasG mediation of the pathways leading to P K B phosphorylation. 3 . 1 0 R a s G E F G a n d R a s G E F D a r e n o t i n v o l v e d i n c A M P s t i m u l a t e d a c t i v a t i o n o f R a s The availability of other gef gene strains (Wilkins et al., 2005) provided an opportunity to search for other RasGEF proteins involved in the c A M P mediated RasC and RasG signalling pathways. c A M P stimulated RasC and RasG activation was normal in gefC and gefD' strains (Table 2, Figures 19A and 19B), indicating that RasGEFC and R a s G E F D are not involved in c A M P stimulated RasC or RasG activation during early development, however, the activation of RasC and RasG was prolonged in gefC cells (Section 4.5). It cannot be ruled out that R a s G E F C and R a s G E F D regulate the activation of either RasC or RasG in response to other stimuli. 74 A Ras-GTP Total Ras Ax3 gefC Time (s) 0 1 0 6 0 0 1 0 6 0 RasC RasG WkWk B Ras-GTP Total Ras Ax3 gefD Time (s) 0 1 0 6 0 0 1 0 6 0 RasC mm > — RasG «m mm mm mm F i g u r e 1 9 . L o s s o f R a s G E F C o r R a s G E F D d o e s n o t a f f e c t R a s a c t i v a t i o n d u r i n g a g g r e g a t i o n . Pulsed A x 3 , (A) gefC and (B) gefD' cells (Table 2), were harvested by centrifugation (500 x g, 5 min), washed 2 times with K K 2 , and resuspended in K K 2 at 5 x 10 7 cells/ml. Cells were stimulated with 200 n M c A M P , and activated Ras was measured as described in Figure 11. 10 pi of 6 X SDS loading buffer were added to 50 pi of Dictyostelium lysates (1 mg/ml), indicated as 'Total Ras,' and approximately 8.3 p,g total protein were loaded on the gel. 'Ras-GTP' refers to Ras bound to GST-Byr2 and 'Total Ras' refers to total Ras present in cell lysates. The experiments are representative of three separate experiments. 75 3.11 O v e r - e x p r e s s i o n o f G b p D l e a d s t o a n i n c r e a s e i n a c t i v a t e d R a p l , R a s C GbpD was isolated in a bioinformatic search for c G M P binding proteins, and along with a cyclic nucleotide binding domain, GbpD was found to contain a putative C D C 2 5 domain (Goldberg et al., 2002). Over-expression of GbpD protein results in cells that are flat, exhibit greatly increased adhesion, and extend many lateral pseudopodia (Bosgraaf et al., 2005), a phenotype that bears a striking resemblance to cells that over-express activated Rap l (Rebstein et al., 1997), suggesting a possible role for GbpD as a regulator of Rap l activation. To investigate the role of GbpD in mediating R a p l activation, the steady-state levels of activated R a p l was measured in vegetative A x 3 , gbpD' and GbpD over-expressing (Ax3::actl5-gbpDOE) cells (Table 2). While there was no difference in activated R a p l levels between A x 3 and gbpD' cells, Ax3: :ac t l5-gbpD 0 E cells displayed an increase in activated Rap l (Figure 20A). A s a control, activated RasC and RasG levels were also measured. The low level of activated RasC in A x 3 and gbpD' cells (Figure 20A) could be a reflection of the importance of RasC during early development over vegetative stage events. This is mirrored by the m R N A expression pattern of the rasC gene, which is lower in vegetative cells, increasing to maximal expression during early development (Lim et al., 2001). While only a trace amount of RasC-GTP was seen in A x 3 and gbpD' cells, there was a modest increase in RasC-GTP observed in Ax3::actl5-gbpDOE cells (Figure 20A). While these results suggest that GbpD affects both RasC and R a p l activation, the modest increase in activated RasC in Ax3: :ac t l5-gbpD 0 E cells could be an indirect effect of over-expressing gbpD. Pulsed gbpU cells stimulated with c A M P displayed no defects in RasC activation (Figure 20B), showing that GbpD is not necessary for c A M P stimulated activation of RasC during early development. The activated levels of RasG-GTP remained similar between the three strains tested (Figure 20A), suggesting that GbpD is not involved in mediating RasG 76 activation. Consistent with the evidence that suggests GbpD is a RapGEF, not a RasGEF, an in vitro nucleotide exchange assay demonstrated that GbpD exhibits specific activity for Rap l (Table 4), and not RasC or RasG (Kortholt et al., 2006). Rap1-GTP Total Rapl B Ras-GTP Total Ras Ax3 gbpCT 4> $ Time (s) o 10 60 o 10 60 RasC ~ <HP *•» • « • * • Figure 20. Act ivat ion of Ras proteins by G b p D in vivo. (A) Exponentially growing A x 3 , gbpD', and Ax3::actl5-gbpD cells (Table 2) were harvested by centrifugation (500 x g, 5 min). 2.5 x 10' cells were lysed in 1 ml R B D - L B , incubated on ice for 10 min, and lysates were cleared by centrifugation (13,000 x g, 10 min, 4°C). Activated Ras was measured as described in Figure 7. 10 ul of 6 X SDS loading buffer were added to 50 ul of Dictyostelium lysates (1 mg/ml), indicated as 'Total Ras,' and approximately 8.3 ug total protein were loaded on the gel. Representative of three separate experiments. (B) Pulsed A x 3 and gbpD' cells, were harvested by centrifugation (500 x g, 5 min), washed 2 times with K K 2 , and resuspended in K K 2 at 5 x 107 cells/ml. Cells were stimulated with 200 n M c A M P , and activated Ras was measured as described in Figure 11. 10 ul of 6 X SDS loading buffer were added to 50 ul of Dictyostelium lysates (1 mg/ml), indicated as 'Total Ras,' and approximately 8.3 ug total protein were loaded on the gel. 'Ras-GTP' refers to RasC bound to GST-Byr2 . The experiment is representative of three separate experiments. 77 4 DISCUSSION 4.1 I m p l i c a t i o n s o f t h e R B D B i n d i n g A s s a y a s a t o o l f o r s t u d y i n g R a s a c t i v a t i o n Previous data had implicated a role for RasC as a major regulator of the c A M P signalling pathway during Dictyostelium early development. Phenotypic characterization of a rasC knockout strain revealed that RasC was required for the optimal activation of A C A and P K B in response to c A M P (L im et al., 2001), and it was hypothesized that RasC was activated by c A M P and mediated at least some of the signalling pathways downstream of the c A M P signal. In order to test the hypothesis, an assay was needed that could be used to measure levels of activated Ras. Much of the early work done in studying the Ras G T P cycle was performed with the use of radioactivity (Satoh et al., 1988). This involved labelling cells with 3 2 P , immunoprecipitating Ras from cell lysates, and resolving G T P from G D P on thin layer chromatography (TLC) . However, there are a number of problems associated with using this method. First and foremost is the requirement of millicurie levels of 3 2 P . This requires extensive safety procedures and equipment, potentially excluding some labs from performing this assay. Another problem is the fact that the cells require incubation in phosphate free medium for several hours. This has been shown to lead to a decrease in intracellular A T P , potentially affecting normal physiological processes (Atkinson, 1968). Finally, incubating cells in the presence o f ' P can lead to cell cycle arrest, or even apoptosis (Yeargin and Haas, 1995), making this assay problematic when studying Ras activation in growing or developing cells. Another method described measured Ras-GTP enzymatically (Scheele et al., 1995). Much like the radionucleotide based assay, this method begins by immunoprecipitating total Ras from cell lysates. The bound nucleotides are released from Ras, and subsequent enzymatic reactions employing N D P Kinase and Luciferase are used to quantify G T P and G D P . This assay 78 is useful for its ability to measure absolute levels of G T P and G D P bound to Ras to femtomolar accuracy, and it has been used to measure Ras-GTP in human tissues in situ (Guha et al., 1996). However, this assay has not been widely used, as evidenced by the fact that it has been cited only 59 times since 1995 (Web of Science), perhaps due to the seemingly labour intensive nature of this assay. For example, measurement of G D P requires conversion of cellular G D P to [ P ]GTP using N D P kinase, and then resolving the samples by T L C . Measurement of G T P involves converting the cellular G T P to A T P using N D P kinase, and then converting A T P to A M P and light, which is measured in a photon counting luminometer, using Luciferase (Scheele et al., 1995). The method devised by Taylor and Shalloway (1996) is advantageous because it does not require the incorporation of any radioactivity, and it is not as labour intensive as the previous methods described (Figure 5). The G S T - R B D fusion can be easily expressed and purified from bacteria, and the glutathione-sepharose resin is commercially available from a large number of manufacturers. Furthermore, pan-Ras antibodies that w i l l recognize most Ras isoforms are also available commercially. The pan-Ras antibody from Calbiochem (Cat # OP40), for example, reacts with Dictyostelium RasB, - D , - G , and - S . On the other hand, i f the objective of the study involves the assay of a specific Ras protein, isoform specific antibodies are required. In specialized cell types that may express only one isoform of Ras, this is not usually a concern; however, Dictyostelium expresses a number of ras genes at any one time during the course of its life cycle. For example, vegetatively growing cells express at least RasB, RasC, RasG, RasS, and Rap l (Daniel et al., 1995). While the pan-Ras antibody can be used to determine whether any Ras proteins are activated, only isoform specific antibodies can be used to determine which specific Ras proteins are activated. 79 The use of a R B D has been extended to visualize Ras activation in living cells (reviewed in Walker and Lockyer, 2004). This has been accomplished by tagging the R B D to a fluorescent tag such as green fluorescent protein (GFP), and the resulting probe can be used to provide both temporal and spatial information about Ras activation. However, since many R B D s can bind to multiple Ras proteins, identifying the specific activated Ras species in vivo may be virtually impossible. For example, Sasaki et al. (2004) observed that GFP-Raf 1 (RBD) localizes to the membrane that is spatially closest to the c A M P gradient (the leading edge) in chemotaxing Dictyostelium cells. PI3K1 localizes to the same area in chemotaxing cells when placed in a spatial gradient of c A M P (Funamoto et al., 2002). Since RasG is involved in regulating PI3K1 activity (Bolourani et al., 2006), and Raf l (RBD) does not interact well with RasC (Kae et al., 2004), Funamoto et al. concluded that the GFP-Raf 1 (RBD) probe was detecting only activated RasG. What this analysis cannot rule out is the possibility that another Ras protein that can bind R a f l (RBD) is also activated by c A M P . One method of visualizing the activation of a specific Ras protein in vivo employs a F R E T based probe that incorporates both the Ras protein and the R a f l - R B D as one fusion protein, flanked by Y F P and C F P at the N - and C-termini, respectively (Mochizuki et al., 2001). This probe is capable of observing isoform specific Ras activation because only the intramolecular interaction elicits the F R E T response, while binding of endogenous Ras wi l l not. One problem with this probe is the possibility that the proximity of the R B D may competitively interfere with R a s G A P function (Mochizuki et al., 2001), thereby affecting the extent of the activation observed. Another possible way to monitor activation of a specific Ras is to identify a R B D that wi l l interact with only one Ras protein. The R B D from Dictyostelium Phg2 shows the most potential as it has been shown via a yeast two-hybrid assay that Phg2 binds only to Rap l (Gebbie et al., 2004; G . Weeks, unpublished observations). 80 Whether Phg2 is able to interact with any of the other Ras proteins is not yet known, but the possibility of a R a p l specific probe remains promising. During the R B D binding assay, the protein content in the Dictyostelium cell lysates must be quantified to ensure equal input of total protein. As a result there wi l l be a period of time during which cell lysates are left on ice, and during this time Ras-GTP wi l l be susceptible to R a s G A P activity. This is only a problem until lysates are incubated with G S T - R B D , which acts as a competitive inhibitor to R a s G A P activity, since they both bind to Ras at the same site, provided that the amount of G S T - R B D is far greater than the amount of cellular RasGAPs. In order to avoid G A P activity, one must work quickly and leave cell lysates in ice at all times. The DC Protein assay used in this thesis is fast, and it can be carried out in less than 30 min. Another potential limitation is the question of the linearity of the R B D binding assay, i.e. does a band that is twice the intensity indicate that there is twice the amount of Ras-GTP? The data from Figure 10 suggests that this assay is not linear as increasing the total protein content from 200 to 400 ug results in bands representing RasC-GTP and RasG-GTP that appear to be more than 2 times in intensity. This is an issue when the results show a reduced activation of Ras, as is the case for RasG activation in gefM and gefR' cells (Figures 15 and 18). While it is easy to speculate that the approximately half reduction of RasG activation observed in both strains means that R a s G E F M and RasGEFR are the only two RasGEFs that mediate RasG activation in response to c A M P , given the non-linearity of the assay, this can be a dangerous assumption to make. Finally, when the signal of the activated Ras detected is low relative to the background control, interpretation of the results can be potentially difficult. A s small variations of Ras-GTP can occur due to experimental error while handling the samples, small differences observed in Ras-81 G T P may not be significant. Thus, in the cases when the differences in Ras-GTP are small, large number of repetitions must be performed in order to ensure the validity of the results. Structural determinants of the specificity of the RBD-Ras interaction were analyzed by screening H-Ras proteins harbouring mutations in the Switch 1 region (Akasaka et al., 1996). It was discovered that a H-Ras (D38N) mutant lost the ability to bind to Ra f l while still retaining the ability to bind to Byr2. A n alignment of R B D sequences does not provide much insight into how this specificity is determined. A structure of a Byr2-Ras complex has revealed that K101 of Byr2 interacts with D38 of H-Ras, most likely by forming a salt bridge between the two residues (Scheffzek et al., 2001). Thus, it is curious that substituting the negatively charged aspartate residue with a positively charged asparagine doesn't abolish the binding interaction. Furthermore, substituting D38 with the negatively charged but longer side chain of glutamate does abolish the Byr2-Ras interaction (Scheffzek et al., 2001), whereas H-Ras with a position 38 substitution to an alanine binds to Byr2 with similar affinity as H-Ras (D38N) (Ohnishi et al., 1998), demonstrating that the structure of Ras residue 38 is more important than its charge. The corresponding residue of Ra f l to Byr2 (K101) is a Lysine, suggesting that the factors that determine specificity are very subtle. A s expressing H-Ras (D38N) rescued the sporulation defect of a S. pombe strain bearing a rasl gene ablation (Akasaka et al., 1996), it appears that the mutation does not severely affect the overall structure of Ras. 4.2 c A M P and Ras signalling The main objective of this thesis project was to develop an assay that allowed for the measurement of activated Ras, and using this assay, determine the involvement and regulation of RasC in the c A M P signalling pathway. Figure 11A demonstrates that c A M P stimulation of 82 pulsed cells led to a rapid increase in activated RasC, peaking at 10 s after stimulation, substantiating the hypothesis that RasC is involved in regulating c A M P stimulated signalling events. The activation of RasC was transient, with a return of RasC-GTP to basal levels by 40 s post stimulation. A subsequent second burst of activation was seen at 80 s and remained on until at least 150 s, although the magnitude of this second peak was significantly lower than the initial peak at 10 s (Figure 11 A ) . Whether this observed second peak is physiologically relevant is not known at this time, but one intriguing consequence of this second peak is the regulation of the cytoskeleton during chemotaxis. Both actin and myosin II filaments rapidly localize to the cell cortex upon c A M P stimulation, followed by a depolymerization and return of actin and myosin II to the cytosol by about 90 to 100 s There is evidence that RasC may be involved in the depolymerization of actin and myosin II as chemotaxing rasC cells have defects in the depolymerization events (Wessels et al., 2004; Section 4.6). The observation that activated RasG levels increased upon c A M P stimulation was initially surprising, because there was limited evidence to suggest that RasG was involved in early development. For example, expression of activated RasG (G12T) results in cells that are unable to aggregate, and while this was initially believed to be an indicator of a role for RasG in the onset of development, over-expression of wi ld type RasG or a dominant negative RasG (S17N) results in no defects in development (Khosla et al., 1996). Therefore, the block in aggregation caused by expressing RasG (G12T) was explained as the result of RasG (G12T) interfering with other Ras signalling pathways. Another link between RasG and development was suggested when RIP3 was shown to bind specifically to RasG, and ablation of ripA produced cells that did not aggregate or develop (Lee et al., 1999). While RIP3 has been shown to bind specifically to RasG, the phenotypes of ripA' and rasG cells were so dissimilar it was 83 deemed unlikely that RasG signalled through RIP3 (Lee et a l , 1999; Tuxworth et al., 1997). The finding that RasG is activated by c A M P shed new light on the connection between RasG and RIP3. 4.3 T h e i n v o l v e m e n t o f G P C R i n R a s a c t i v a t i o n i Dictyostelium discoideum possesses four car genes that encode for c A M P receptors, named car A to carD, but only one, carA, is highly expressed during early development (6 h post starvation) (Chen et al., 1996). Another, carC, is expressed during mid-development (9 h post starvation), and can somewhat compensate for the loss of carA (Soede et al., 1994). carB and carD are not expressed until late development, and a carA'/carC double knockout is incapable of initiating c A M P stimulated signalling events (Soede et al., 1994). RasC and RasG activation was abolished in the car double knockout strain (Figure 12A), demonstrating that Ras activation during aggregation in response to c A M P requires c A R l and c A R 3 receptors. While the majority of the stimulatory pathways downstream of c A R l are regulated by the activation of the associated heterotrimeric G-protein, Goc2py, there are a number of responses that function in response to c A M P but independently of Goc2(3y. The c A M P mediated activation of the M A P kinase E R K 2 has been shown to be partly independent of Goc2py (Briscoe et al., 2001; Maeda et al., 1996), suggesting the possibility that a Ras protein may be involved in mediating some G-protein independent responses given that M A P kinase cascades are typically downstream of Ras proteins in mammals (Arbabi and Maier, 2002). However, the relationship between Ras and E R K 2 in Dictyostelium has been somewhat controversial. A s measured by an in vitro kinase assay, c A M P stimulated activation of E R K 2 peaks one min post-stimulation, and returns to basal levels by three min post-stimulation (Knetsch et al., 1996). Expression of an 84 activated RasD (G12T) in wild type cells results in an approximately 4-fold increase in the basal level of c A M P stimulated E R K 2 activity, with a subsequent decline in E R K 2 activation to levels comparable to wild type by 3 min post-stimulation. It was postulated that activated RasD (G12T) stimulates the activation of E R K 2 , hence the elevated basal level, but does not affect the subsequent adaptation of E R K 2 , leading to E R K 2 inactivation. Aubry et al. (1997) observed the opposite effect. Expression of activated RasD (Q61L) in wild type cells results in a significant reduction in c A M P stimulated E R K 2 activation, as measured by an in-gel kinase assay. Furthermore, the basal level of activated E R K 2 was the same as that seen in wi ld type cells, and the kinetics of E R K 2 activation mirrored that of wi ld type cells. The observation that c A M P stimulated RasC and RasG activation is ablated in gpaB' or gpbA' cells (Figure 12B and 12C) demonstrates that RasC and RasG activation in response to c A M P is linked to Ga2(3y, suggesting that RasC and RasG are not involved in the G-protein independent activation of E R K 2 . Upon agonist binding to the c A M P receptor, both G a 2 and the G p y dimer participate in distinct signalling pathways, and a combination of genetic and in vitro data has suggested that Goc2 is involved in the pathway leading to guanylyl cyclase activation, and G P y is responsible for PI3K and adenylyl cyclase activation (Okaichi et al., 1992; Roelofs et al., 2001; W u et al., 1995). Activated RasC and RasG are also thought to participate in discrete pathways, with RasG involved in regulating the chemotactic response and cell polarity, which includes the activation of guanylyl cyclase and PI3K, respectively, and RasC in adenylyl cyclase activation (Bolourani et al., 2006). Thus, it can be speculated that Goc2-GTP mediates the activation of RasG in the pathway leading to G C activation, whereas the G P y dimer mediates the activation of both RasC and RasG for the activation of A C A and PI3K, respectively. A model of dual Ras protein 85 activation from one G P C R has been proposed in HEK293 cells (Schmitt and Stork, 2002). Stimulation of the (32-adrenergic receptor with isoproterenol leads to the activation of Ras and R a p l . Furthermore, through the use of dominant negative mutants it was demonstrated that Ras activation was dependent on G(3y, and Rap l activation was dependent on Got (Schmitt and Stork, 2002). This model of G-protein dependent Ras activation provides a mechanism for how, two distinct signalling pathways originating from one stimulus can be activated and regulated. 4.4 R o l e o f R a s G E F s i n R a s a c t i v a t i o n The Dictyostelium genome encodes 15 Ras subfamily proteins (Weeks et al., 2005), a number that is considerably larger than that found in worms and yeast, which contain between four and ten (Weeks et al., 2005). In addition, there are more gef genes than ras genes encoded by the Dictyostelium genome (Wilkins et al., 2005), suggesting that at least some Ras proteins are activated by more than one RasGEF. Consistent with this idea is the fact that of the ten gef gene knockouts that have been described, four gef gene knockout strains have no distinct phenotypes (Wilkins et al., 2005). The isolation of a gefA' strain that failed to aggregate was the first indication of a Ras protein being involved in c A M P signalling during Dictyostelium development (Insall et al., 1996). The subsequent isolation of a rasC mutant with a similar phenotype suggested the possibility that RasC functioned downstream of R a s G E F A (Lim et al., 2001). The pattern of Ras activation in the gefA' mutant provided convincing evidence that R a s G E F A is necessary for the activation of RasC in response to c A M P , but not required for RasG activation (Figure 14). This has been verified by the use of an in vitro nucleotide exchange assay (Table 4). R a s G E F A 86 catalyzed the removal of G D P from RasC, but not from other Dictyostelium Ras proteins, confirming that R a s G E F A is specific for RasC. A proteomic study has shown that RasGEFR is one of the proteins that could be identified as exhibiting an alteration in tyrosine phosphorylation in response to the expression of activated RasG (Secko et al., 2004). This suggested a possibility that RasGEF could mediate activation of RasG as there are examples of RasGEF regulation by phosphorylation. Stimulation of rat forebrain slices with the vasodilator forskolin leads to phosphorylation of R a s G R F l , and this phosphorylation is required for maximal RasGEF activity (Yang et al., 2003). Conversely, it has been shown in C H O cells that RasGRF2 is phosphorylated by the cyclin dependent kinase, Cdk5, and this phosphorylation serves to downregulate the R a c G E F activity of RasGRF2 (Kesavapany et al., 2004). However, there wds no direct evidence to suggest that RasGEFR mediated RasG activation. The finding of a reduction in the magnitude of RasG activation in response to c A M P in a gefR' mutant (Figure 15) provided evidence that RasGEFR is responsible for mediating at least some of the RasG activation. This conclusion was confirmed by an in vitro nucleotide exchange assay which showed that RasGEFR was specific for RasG (Table 4; Kae et al., 2007). A closer examination of the gefR' phenotype revealed that gefR' cells do not have defects in the activation of P K B (Figure 17A), demonstrating that RasGEFR is not responsible for RasG mediated activation of either PI3K or the T O R C 2 complex. A n examination of the streaming characteristics of gefR' cells during aggregation revealed that, unlike rasG cells, there was no delay in the onset of aggregation (Figure 16). However, the streams formed by gefR' cells appeared to involve fewer cells and thus appeared less dense when compared to streams formed by wi ld type cells (Figure 16). It has been shown that A C A activity is required for stream formation (Kriebel et al., 2003), so the fact that gefR' cells are able to stream would 87 suggest that A C A activity is not abolished in this strain. Instead it can be speculated that the less robust streams formed by gefR' cells is due to a defect in either motility or chemotactic efficiency. Since RasG activation is not completely abolished in the gefR' strain, it would appear that there is at least one other RasGEF responsible for the activation of RasG upon c A M P stimulation. One candidate RasGEF is R a s G E F M . c A M P stimulation of gefM' cells resulted in a moderate reduction of RasG activation, relative to wild type (Figure 18), demonstrating that R a s G E F M , like RasGEFR, is required for maximal activation of RasG. It has been shown in splenic B cells that both RasGRP 1 and RasGRP3 contribute to maximal activation of Ras, detected using a pan-Ras antibody, and loss of either RasGEF results in a decrease in activation (Coughlin et al., 2005). Furthermore, R a s G R P l and RasGRP3 appear to mediate different pathways as rasgrpl'' and rasgrp3'A knockout mice exhibit different phenotypes. A model involving the regulation of RasG activation by two RasGEFs can provide a means to regulate two independent pathways mediated through RasG. However, without supporting in vitro evidence, it is impossible to conclude that R a s G E F M regulates RasG activation. RasG has been linked to the c A M P mediated activation of P K B (Section 1.13). The observed reduction in the activation of P K B in gefM cells suggests that R a s G E F M mediates the RasG dependent activation of P K B (Figure 17B), and the reduced P K B activation is consistent with the defects in cell polarity and motility seen in both gefM' and rasG' cells (Arigoni et al., 2005; Bolourani et al., 2006). However, at this point the evidence linking R a s G E F M and RasG is circumstantial, and it cannot be ruled out that the reduced activation of both RasG and P K B could be due to some secondary effect such as a change in gene expression, or the result of an indirect effect on a separate signalling pathway. We do know that RasGEFC and RasGEFD are 88 not involved in the activation of RasG in response to c A M P , as neither the gefC nor gefD' mutants were defective in RasG activation (Figure 19). GbpD was isolated in a search for c G M P binding proteins identified by bioinformatic analysis and was found to contain a domain with homology to CDC25p (Goldberg et al., 2002). A gbpD strain revealed an involvement in early developmental signalling, as gbpD' cells chemotax faster due to a hyper-polarized cell shape, a phenotype that is similar to rasC cells (L im et al., 2001), and to cells that over-express a dominant negative R a p l (S17N) (Jeon et al., 2007). Over-expression of GbpD, on the other hand, leads to cells that are more adherent when placed on a solid surface, and a loss of polarity when chemotaxing (Bosgraaf et al., 2005). Assaying for activated Ras revealed that over-expression of GbpD results in an increase in activated R a p l and RasC, but not RasG (Figure1 20A). However, an in vitro nucleotide release assay demonstrated that GbpD can catalyze activation of R a p l , and not RasC (Table 4), indicating that the in vivo increase in activated RasC-GTP is not likely a direct effect (Kortholt et al., 2006). In support of these observations, c A M P stimulated RasC activation in a gbpD' strain is largely unaffected (Figure 20B). It has been shown that c A M P stimulation of aggregation stage cells results in transient activation of R a p l (Jeon et al., 2007). Furthermore, expression of a dominant negative Rap l (S17N) results in cells that hyper-polarize during chemotaxis, and expression of an activated Rap l (G12T) abolishes cell polarity. However, use of dominant negative.and activating Rap l mutants could interfere with other Ras signalling pathways (Feig, 1999), including those of RasC and RasG, so the physiological effects of these Rapl mutants may be the result of perturbing the signalling of another Ras protein. How Rapi integrates with the Ras signalling pathway, i f at all, is unclear at this time, but there is evidence to suggest that 89 c A M P stimulated activation of Rap l lies downstream of either RasC or RasG (P. Bolourani, unpublished observations). Initial experiments have shown a high degree of RasGEF-Ras specificity in Dictyostelium. These findings seem to be in contrast to the situation in mammalian cells where overlapping specificities have been reported. For example, SOS exhibits in vitro exchange activity on H - , K - , N - , M - , and R-Ras2 (Nielsen et al., 2001; Ohba et al., 2000; Porfiri et al., 1994), and R a s G R F l has been shown to activate H - , K - , N - , M - , R-, and R-Ras2 (Nielsen et al., 2001; Ohba et al., 2000). In an organism like Dictyostelium, activation of Ras pathways by specific interactions with a RasGEF would be an efficient mechanism to ensure high fidelity as there are normally multiple Ras and RasGEF proteins present. During aggregation there are 18 gef genes and at least 7 ras genes expressed (Chisholm et al., 2006; Daniel et al., 1995; Wilkins et al., 2005). This mechanism may not be as important in metazoans, as many RasGEFs have tissue or cell specific expression, bypassing the need for tight substrate specificity. For example, R a s G R P l is the only RasGRP family member to be expressed in T cells and mediates all of the D A G stimulated Ras activation (Dower et al., 2000), while R a s G R F l is predominantly expressed in brain tissue (Guerrero et al., 1996). Little is known regarding the Ras structural determinants that might be involved in conferring RasGEF specificity. Analysis of a SOS-Ras crystal structure has revealed that 3 regions of Ras are involved in the RasGEF-Ras interaction; the Switch 1 region (residues 21-40), the Switch 2 region (57-75) and the helix oc3 region (95-111) (Boriack-Sjodin et al., 1998). RasC and RasG exhibit a number of differences in the Switch 1 (4 out of 19 residues), Switch 2 (2 out of 19), and helix a3 regions (6 out of 17) (Figure 2), providing a variety of possible determinants of specificity. However, RasGEFR is capable of activating RasG but is without 90 activity for RasD (Table 4), despite the fact that RasD and RasG are highly related (89% identity), including identical Switch 1, Switch 2, helix oc3 regions (Figure 2). The observation that the CDC25 domain of mammalian RasGRFl, used as a positive control, was able to activate all the Dictyostelium Ras proteins indiscriminately in an in vitro nucleotide exchange assay (Table 4) would indicate that the factors that determine the RasGEF-Ras interaction specificity are subtle, and involves other residues outside of the Switch 1, Switch 2, and helix a3 regions. The region of SOS neighbouring the Ras-SOS interface is surrounded by three residues that have been proposed to be involved in mediating RasGEF specificity. When residues R826, T935, and E1002 of SOS are mutated to alanines there is no effect on the binding and activation of Ras by SOS (Hall et al., 2001). However, a mutation of yeast CDC25p in the residue equivalent to R826 of SOS disrupts the ability of CDC25p to bind to Ras (Park et al., 1994), and a mutation in RasGRFl in the residue equivalent to T935 of SOS disrupts the catalytic activity on Ras (Vanoni et al., 1999). Alignment of the protein sequences of RasGEFA and RasGEFR (Figure 21) reveals that at the position corresponding to R826 of SOS, RasGEFA has a negatively charged aspartate residue, whereas RasGEFR has a positively charged asparagine residue, and at the position corresponding to E1002 of SOS, RasGEFA has a leucine residue, whereas RasGEFR has a gap in its alignment. RasGEFA has a threonine residue and RasGEFR has a serine residue at the position corresponding to T935 of SOS (Figure 21). If the structures of RasGEFA and RasGEFR are analogous to the structure of SOS, it's possible that the differences in the residues at position 826 and 1002, relative to SOS, contribute to the specificity of the RasGEF-Ras interaction observed. There are also a number of differences in other residues that have been implicated in the SOS-Ras interaction when comparing the alignment of the RasGEFA and RasGEFR sequences (Figure 21), providing a number of possible residues that may be involved 91 in mediating specificity. A detailed mutational analysis wi l l be needed to determine which of these residues, or any other residues, are the important determinants that confer specificity for the RasGEF-Ras interaction. GefA - NLSIYDIDEEEIARQLTLIEFEIYRNIKPPELLNQSwNMTK jKSRAPNl GefR TALFLLLNESEIARQLTLIDFNIFSKIQPTELLDQSraiDSJKFKSPNJ SOS TFDLLTLHPIEIARQLTLLESDLYRAVQPSELVGSVpl^ED |EINSPN| G R F 1 AEPFENHSALEIAEQLTLLDHLVFKKIPYEEFFGQG^gLEgNERTPYl IKE. JK C | | | E •' 4 0 5 . 1 4 9 9 8 3 2 1 0 9 0 GefA GefR SOS G R F 1 GefA GefR SOS G R F 1 GefA GefR SOS G R F 1 FJ T M I I Q T T K V K A R A R M M T R F I K I A D H L K N L N N Y M S S Q I L W Q E D I E E R V K V F E K F I L I S K Y L R E . M N N F 1 ' : L | . 'Fj K C I V E T E N L E E R V A W S R I I E I L Q V F Q E L N N F H | L I | S E I I R N E D I N A R V S A I E K W V A V A D I C R C L H N Y | .'pCYTREE KKTFAL DHTFEQ K K T W L K LSAQTMRTYSDL LSPNSLSIYNSL iPSRQKKILEE. VSKQTKALIDKLl AU. • . :-'<T •,.-u(?rv!:<N EBH¥KKY1 I R T R L Q N V P P - M L ^ ,v:.-:;vr-ip rmy.,;'-:,!. R S - V S K N P P - L L ; - - ,s L!L:OLU L MLO A.: L K L R S 1 N P P - C \ " IREALKNCDPPCV ' : " I ' : L T : - J- w r ™i —i !-• V G G K Q V S L I N F T K R T - D K I N N L I N F Q K R E L L K R H G K E L I N F S K R R | — Y T E D G L V N F S K M : JCRVLS E VQQCQQQT KY E F P W E P I H T L L T E L P A E | T G E I Q Q Y Q N Q P Y C L R V E S D I K R F F E N L N P M G - N S ] I I S H I I R E I R Q F Q Q T A Y K I E H Q A K V T Q Y L L D Q S F V M D -4 6 5 1 5 5 9 8 9 2 1 1 5 0 N F V T D 5 2 4 — 1 6 1 2 E - — V ' 9 4 7 N - 1 2 0 6 Q Y Q 5 8 4 STP 1 6 6 3 [ E F T 1 0 0 6 — E 1 2 5 7 F i g u r e 21. A l i g n m e n t o f R a s G E F p r o t e i n s e q u e n c e s . Gapped alignment of protein sequences of the C D C 2 5 catalytic domains of the Dictyostelium R a s G E F A and RasGEFR, and mammalian SOS and R a s G R F l (labelled GRF1) . Alignment was performed using C L U S T A L W multiple sequence alignment program, version 1.83. Numbers indicate position of amino acids in protein and dashes indicate gaps in alignment. Shaded residues represent those that are involved in the RasGEF-Ras interaction, as deduced from the SOS-Ras crystal structure (Boriack-Sjodin et al., 1998; Hal l et al., 2001). Residues marked on the bottom with (*) are conserved residues, those marked with (:) are 'strongly' conserved as defined by C L U S T A L W , and residues marked with (.) are 'weakly' conserved as defined by C L U S T A L W . Residues marked on top with a (•!)) represent the residues corresponding to R826, T935, and E1002 of SOS. 92 A n in vitro study employing purified Ras proteins demonstrated that Ras prenylation may play a role in contributing to RasGEF substrate specificity in mammalian cells (Porfiri et al., 1994). The proteins were expressed in Sf9 insect cells so that the Ras proteins underwent the correct post-translational modification that is absent when expressed in bacteria. The report demonstrates that SOS does not exhibit in vitro exchange activity on unprenylated H-Ras or K -Ras4B, and this specificity is limited to prenylation as changes to the palmitoylation sites of H -Ras have no effect. On the other hand, C D C 2 5 is able to mediate nucleotide exchange on either form of Ras, indicating that prenylation does not determine specificity for all RasGEFs. Both RasC and RasG are predicted to undergo the same post-translational modification, a geranylgeranylation (Moores et al., 1991), suggesting that prenylation does not account for the difference in specificity towards R a s G E F A and' RasGEFR. Furthermore, the results from an in vitro nucleotide exchange assay shown in Table 4 employed a C-terminally truncated form of RasC (Kae et al., 2007), supporting the notion that regions of Ras other than the C-terminus are important in determining substrate specificity for R a s G E F A and RasGEFR. However, it cannot be ruled out that post-translational modification is a determinant for substrate specificity for other Dictyostelium RasGEFs. It remains to be seen how Dictyostelium RasGEFs direct Ras activation in vivo. In mammalian Ras pathways, a large number of RasGEFs associate with adaptor proteins that mediate their cellular localization and facilitate Ras activation. A model has been proposed in H E K 2 9 3 epithelial kidney cell lines that involves a direct interaction of a G P C R and a c A M P regulated RasGEF, CNrasGEF. Pak et al. (2002) showed that CNrasGEF binds directly to the G P C R P l -AR, and in conjunction with binding to c A M P , CNrasGEF activates Ras directly upon agonist binding to p l -AR. This model provides the first evidence of Ras activation directly from 93 a G P C R . While a key feature of most RasGEFs is the inclusion of other signalling domains that provide clues in elucidating Ras pathways, many Dictyostelium RasGEFs contain very few signalling motifs outside of the catalytic domain (Wilkins et al., 2005). CNrasGEF, for example, contains a P D Z domain which is necessary for its interaction with p i - A R and a c N M P binding domain which binds c A M P and serves as a way to regulate its activity (Pak et al., 2002}. In contrast, R a s G E F M and RasGEFR contain no other signalling domains outside of the catalytic domain (Wilkins et al., 2005), and the only other signalling motif that can be found on R a s G E F A is a L i s H domain (Wilkins et al., 2005), a poorly characterized motif that has been postulated to be a possible protein oligomerization domain (K im et al., 2004). The L i s H domain has also been suggested to regulate microtubule dynamics (Ernes and Ponting, 2001). ' The function of the microtubule cytoskeleton in chemotaxis is unclear, but it has been observed that Dictyostelium cells exclude the microtubule cytoskeleton from the leading pseudopod during chemotaxis to c A M P (Rubino et al., 1984), and that the microtubule organizing centre localizes to the rear of cells when chemotaxing to folate (Sameshima et al., 1988). This type of distribution has also been observed in dHL-60 cells, a neutrophil-like cell line, where disruption of the microtubule cytoskeleton using the microtubule depolymerising drug, nocodazole, increases cell polarity (Xu et al., 2005). Furthermore, treating cells with nocodazole results in a decrease in C.I. of cells chemotaxing towards f M L P , while increasing the migration speed -50 % (Xu et al., 2005). R a s G R F l has been shown to associate with microtubules in vitro, and R a s G R F l transfected into COS-7 cells localizes to microtubules in vivo (Forlani et al., 2006). A s this interaction has no effect on microtubule dynamics, it has been suggested that the association serves as an anchor for R a s G R F l (Forlani et al., 2006). Even though the domain involved in R a s G R F l localization shows no homology to the L i s H domain 94 present in R a s G E F A , it could be possible that R a s G E F A demonstrates a similar localization to the microtubule network. 4.5 R a s a c t i v a t i o n a n d t h e a d a p t a t i o n r e s p o n s e The stimulatory signalling events initiated by c A M P during early development in Dictyostelium have been studied for many years (reviewed in Manahan et al., 2004). To respond to cyclic pulses of a stimulant, however, there must be a period when the signalling components are deactivated, during which time the cells remain unresponsive to the signal and cells return to a basal state. In Dictyostelium, this process is referred to as the adaptation response, and the mechanism for how this proceeds remains largely unresolved (Brzostowski et al., 2004). The loss of A C A activity in acaA', dagA\ and piaA' strains appeared to have an effect on the deactivation of Ras. RasC activation was prolonged in dagA', and piaA' cells (Figure 13A, 13B), whereas RasG activation was prolonged in acaA' (Figure 13C). It is possible that the loss of A C A activity has an effect on the adaptation response, and one possible regulator of the adaptation response is P K A , an important transducer of the intracellular c A M P signal. c A M P stimulation of wild type cells results in a rapid activation of the M A P kinase, E R K 2 (Maeda et al., 1996), and it has been postulated that E R K 2 regulates the level of intracellular c A M P levels through the regulation of phophodiesterases (Maeda et al., 2004). Cells with an ablation of the gene encoding E R K 2 do not aggregate (Segall et al., 1995), presumably due to the elevated action of c A M P phosphodiesterases, and the aggregation defect can be rescued by expressing constitutively active P K A (Aubry et al., 1997), placing it genetically downstream of E R K 2 . However, E R K 2 activation is prolonged in pica' cells (Aubry et al., 1997), suggesting that 95 intracellular c A M P is important for the adaptation response, and the prolonged activation of RasC and RasG observed (Figure 13) may be due to a defect in this adaptation response. In some of the studies on Ras activation reported in this thesis, loss of a RasGEF was associated with prolonged activation of those Ras proteins that were still activated. In wild type cells, Ras activation was rapid, peaking by 10 s, and returning to near basal levels by 40 s (Figure 11). gefA' cells exhibited no decrease in RasG activation by 60 s (Figure 14), gefM' and gefR' cells exhibited a level of RasC activation at 60 s that was greater than basal levels (Figures 15 and 18), and gefC cells exhibited a greater level of both activated RasC and RasG by 60 s compared to basal levels (Figure 19A). Given the general model of the G T P dependent activation cycle of Ras proteins (Figure 1), this extended peak of Ras-GTP might reflect reduced activity of a negative regulator of Ras, such as a RasGAP. Nine genes have been annotated in the Dictyostelium genome that encode for proteins with putative R a s G A P domains (Chisholm et al., 2006), but none have been characterized thus far. Two proteins with R a s G A P domains, D G A P 1 and G A P A , have been identified as IQGAPs , and the evidence suggests that these two proteins act on Rho family GTPases, and not Ras (Adachi et al., 1997; Faix et al., 1998). Whether the 9 putative RasGAPs show specificity towards individual Ras proteins is unknown at this time. One candidate negative regulator of Ras activation in c A M P stimulated events is the G alpha protein, G a 9 (gpal)', which has been implicated in the negative regulation of events downstream of c A R l (Brzostowski et al., 2004). While the majority of c A R l is coupled to Gcc2p.Y (Kumagai et al., 1991), Brzostowski et al. (2004) have demonstrated that a small but significant population of c A R l has also been shown to be coupled to Ga9Py. Dissociation of / c A R l with its heterotrimeric G-protein reduces its affinity for c A M P , and c A M P binding is 96 inhibited by about 75 % in membranes from wild type cells when the heterotrimeric G-proteins are activated in vitro using GTPyS. In contrast, membranes from gpaB' cells display only -15 % inhibition when stimulated with GTPyS, indicating that the majority of c A R l is coupled to Ga2 . Ectopic expression of gpal in gpaB' cells is able to partially restore the GTPyS stimulated inhibition of c A M P binding to membranes from gpaB' cells to -35 %, indicating that Goc9 is also able to couple to c A R l (Brzostowski et al., 2004). Gene ablation studies have revealed that a gpaT strain exhibits extended peaks of c A M P stimulated c A M P , c G M P and PI(3,4,5)P3 accumulation (Brzostowski et al., 2004). It is striking that the initial activation kinetics are largely unaffected, with the gpaT cells often displaying an equal or a small increase in activation relative to wi ld type cells. However, the adaptation response, defined as the return to basal levels, is significantly delayed. Expression of a constitutively activated G a 9 (Q196L) results in a faster adaptation response. As the loss of Got9 affects multiple activation pathways, i.e. the PI3K pathways, A C A activation, and c G M P accumulation, it would suggest that G a 9 acts as a general negative regulator of c A M P signalling, functioning to turn off all pathways mediated by c A R l . How G a 9 mediates this regulation is unknown as there is a dearth of information regarding the components of the adaptation response, but it is conceivable that G a 9 functions as a negative regulator of both RasC and RasG activation given the involvement of G a 9 in multiple signalling pathways (Brzostowski et al., 2004). There are some examples of G A P s functioning downstream of G P C R in mammalian cells. G a l 2 - G T P interacts with G A P l m via a tandem P H / B H (Btk homology motif) domain, and this interaction has been shown to stimulate G A P activity in a cell free in vitro assay (Jiang et al., 1998). Additionally, while E G F stimulation of M E G - 0 1 human leukemia cells stimulates Ras activation, concomitant over-expression and activation of G a l 2 with the agonist U46619 97 reduces this activation, demonstrating that activated G a l 2 potentiates G A P l m activity in vivo as well . On the other hand, the Goo protein regulates G A P activity in a different manner. In H E K 2 9 3 cells, Rap l G A P co-immunoprecipitates with Gao-GDP, but not the G T P bound isoform (Jordan et al., 1999). Over-expression of Goto results in an increase in activated R a p l , whereas over-expression of a constitutively activate Gc<o (Q205L) diminishes activated R a p l , suggesting that Gao-GDP regulates Rap l activity by sequestering R a p l G A P . If the adaptation response in Dictyostelium is indeed mediated by G A P regulation, the available evidence suggests that G a 9 lies upstream of the putative RasGAPs for RasC and RasG. 4.6 R e g u l a t i o n o f t h e c y t o s k e l e t o n d u r i n g a g g r e g a t i o n b y R a s Chemotaxing Dictyostelium cells, like all motile cells, undergo orchestrated chemoattractant induced changes to the cytoskeleton (Franca-Koh et al., 2006). After an initial "cringe" response in which cells round up and cease motion, the cytoskeleton polarizes such that F-actin assembles at the leading edge, which is the site of new pseudopod formation, and myosin II assembles at the rear and sides of the cell to provide cortical tension and to retract the trailing uropod. These two responses have been reported to be regulated by PI3K activity and c G M P , respectively (Bosgraaf et al., 2002; Chen et al., 2003), and there is an increasing body of evidence to suggest that RasG is involved in both responses. It has been well documented that mammalian Ras-GTP binds to PI3K and stimulates PI(3,4,5)P3 formation (Kodaki et al., 1994; Rubio et al., 1997). Similarly, RasG-GTP has been shown to bind to Dictyostelium PI3K1 and PI3K2 in a yeast two hybrid assay (Funamoto et al., 2002), and a R B D binding assay (Figure 7C). Although it has yet to be determined whether this binding stimulates PI3K activity, a PI3K1 isoform that harbours a mutation in the R B D is unable to generate PI(3,4,5)P3 at the 98 leading edge in response to c A M P when expressed in a pi3kl~/pi3k2~ double knockout. Furthermore, constitutively localizing PI3K1 to the membrane via a myristoylation motif does not rescue this defect, suggesting that binding of Ras to PI3K does not function to merely anchor PI3K to the membrane (Funamoto et al., 2002). These data have been interpreted as indicating that RasG is required for PI3K stimulated PI(3,4,5)P3 formation, and consequently for actin polymerization. The evidence for RasG mediated regulation of myosin II polymerization is equally compelling. Chemotaxing cells rapidly polymerize myosin II to the cortex at the rear and sides of the cell when challenged with c A M P , and this response is defective in a sgcA'/gca double knockout strain, cells that are unable to synthesize c G M P (Bosgraaf et al., 2002). These cells are motile, but they display a loss in polarity during chemotaxis, and have a severe defect in chemotactic efficiency as measured by the C.I. rasG cells display a similar phenotype in that they are also unable to properly polarize during chemotaxis, yet still retaining the ability to locomote (Bolourani et al., 2006). While rasG cells display a significant reduction in C.I., the defect is not as severe as that of the sgcA'/gcd strain. The difference in C.I. between rasG and sgcA'/gca cells can be accounted for by the fact that while both receptor and GTPyS stimulated c G M P accumulation is significantly reduced in rasG cells, an appreciable level of c G M P is still synthesized (Bolourani et al., 2006, and P. Bolourani, unpublished observations). c G M P synthesis is completely abolished in rasC/rasG double null cells; and these cells have the same dramatic defect in C.I. as sgcA'/gca' cells. These data suggest that the inefficient chemotactic response of rasG cells may be in part a consequence of defects in mobilizing both the actin and the myosin cytoskeleton. 99 RasC, on the other hand, appears to antagonize the pathways leading to actin and myosin polymerization. In a study to examine the chemotactic properties of the rasC cells, it was found that both the actin and myosin II cytoskeletons polymerize and mobilize to the cell cortex in response to c A M P much like wild type cells (Wessels et al., 2004). While wild type cells subsequently depolymerise and reorganize actin and myosin II away from the cortex to the cytosol, both remain in the cortex of rasC cells, leading to a hyper-polarized cell morphology. The depolymerization of actin and myosin II occur at approximately 80 to 90 s after the c A M P stimulus, which coincides with the second, more gradual peak of activated RasC (Figure 11), so it is possible that RasC is involved in the depolymerization of both actin and myosin II filaments during chemotaxis. Vegetative rasC cells display polymerized actin staining at several sites around the membrane, whereas wild type cells typically show a single focus of polymerized actin (L im et al., 2005), supporting a negative regulator role for RasC. It has been shown in HCT116 human cancer cell lines that K-Ras regulates depolymerization of the cytoskeleton (Pollock et al., 2005). HCT116 cells have one mutant K-Ras ( G l 3 D ) allele, the equivalent to H-Ras (G12T), and these cells are devoid of stress fibres and focal adhesions when placed on glass coverslips. Disruption of the mutant K-Ras allele restores stress fibre and focal adhesions, suggesting that the absence of stress fibres and focal adhesions in HCT116 cells is a result of the presence of activated K-Ras. Furthermore, despite the absence of stress fibres, HCT116 cells display an elevated level of activated Rho, which is involved in the formation of stress fibres, and treatment of HCT116 cells with Rho inhibitors results in the reformation of stress fibres, suggesting that K-Ras mediates its effects on the cytoskeleton by negative regulation of Rho activity, but not by the activation of Rho (Pollock et al., 2005). Whether RasC functions through mediating the activity of a Rho family GTPase remains to be seen, but as Ras has been well 100 documented to mediate cytoskeletal rearrangement upstream of small GTPase cascades in other cells, this notion is not out of the realm of possibility. 4.7 A n e m e r g i n g m o d e l o f R a s s i g n a l l i n g d u r i n g a g g r e g a t i o n The work in this thesis project demonstrated that c A M P stimulation of Dictyostelium cells during the aggregation stage resulted in the activation of RasC and RasG (Figure 11), and that this activation is downstream of the c A M P receptors, c A R l and eAR3 , as well as its associated heterotrimeric G-protein, Ga2(3y (Figure 12). Furthermore, it appears that all c A M P mediated RasC activation is mediated by R a s G E F A (Figure 14), whereas RasG activation is mediated by at least 2 GEFs , RasGEFR and R a s G E F M (Figurel5, 18). Closer examination of the phenotypes of gefR' and gefM cells has revealed a possible role for R a s G E F R in chemotaxis (Figure 16) and R a s G E F M in regulating cell polarity and motility through the activation of the pathway leading to P K B phosphorylation (Figure 17B). How the Dictyostelium RasGEFs are regulated is unknown at this time, but some speculations can be made based on models of analogous systems. The most common way to regulate RasGEF mediated activation is to regulate the localization of the RasGEF, usually via the action of adaptor proteins. In the case of R a s G E F A , the L i s H domain may function to localize R a s G E F A in the proximity of RasC when stimulated with c A M P . Since no obvious motifs exist on R a s G E F M and RasGEFR, the model would depend on the existence of novel binding sites for protein-protein interaction domains that would be analogous of SH2 or SH3 domains. RasGEFR undergoes a decrease in threonine phosphorylation in response to the expression of activated RasG. Given that RasGEFR exhibits in vivo (Figure 15) and in vitro (Table 4) activity on RasG, the phosphorylation of RasGEFR could be a positive regulator of its 101 RasGEF activity, and the subsequent activation of RasG provides feedback inhibition on the activity of RasGEFR. In other cells, activation of signalling pathways downstream of Ras has been shown to induce negative feedback inhibition of Ras activation. Expression of activated Raf l in B J fibroblasts results in an increase in E R K activation when treated with tamoxifen, but in a decrease in Ras-GTP levels, demonstrating that activation of Ras pathways can lead to inhibition of Ras (Courtois-Cox et al., 2006). Whether this inhibition of Ras is through the activation of a RasGAP, or inhibition of a RasGEF is unknown. A picture of the c A M P stimulated Ras signalling pathways is beginning to emerge (Figure 22). The current evidence suggests that all Goc2Py dependent signalling is mediated by the Concerted functions of RasC and RasG. RasG appears to regulate at least three pathways: 1) activation of G C (Bolourani et al., 2006), 2) activation of PI3K (Bolourani et al., 2006; Funamoto et al., 2002), and 3) regulation of the T O R C 2 complex via an interaction with RIP3 (Lee et al., 2005). Taken together, the predominant role of RasG is in chemotaxis and in regulating cell motility and polarity. The observation that RasG may be activated at the leading edge of chemotaxing cells is significant as it had been shown that G C , PI3K, and RIP3 translocate to the leading edge when placed in a c A M P gradient (Funamoto et al., 2002; Lee et al., 2005; Sasaki et al., 2004; Veltman et al., 2005), reflecting RasG interaction with its downstream effectors. The streaming pattern of gefR' cells (Figure 16) suggests that there may be defects in cell motility or chemotaxis as the streams formed by gefR' cells appear less dense when compared to streams formed by wi ld type cells. A quantitative analysis of the chemotactic behaviour of gefR' cells could provide more insight into the role of RasGEFR in this process. Since R a s G E F M is required for maximal P K B phosphorylation (Figure 17B), it is possible that R a s G E F M activates RasG in a pathway that targets either PI3K or T O R C 2 . 102 CAR1 I I I I I I I h ^ _ — . * —• Front of Cell " R a s G E F R R a s G I Chemotaxis Actin/Myosin II depolymerization? Rear of Cell cAR1 F i g u r e 22. P r o p o s e d m o d e l f o r t h e i n v o l v e m e n t o f R a s C a n d R a s G i n c A M P s t i m u l a t e d e v e n t s d u r i n g Dictyostelium a g g r e g a t i o n . c A M P stimulation leads to a number of responses, but for simplicity have been separated into 3 functions: chemotaxis, cell polarity/motility, and activation of A C A . Activation of RasC and RasG are downstream of the G P C R , c A R 1, and its associated G-protein, Ga2Py. Activation of RasG occurs at the front of chemotaxing cells (Sasaki et al., 2004), and is mediated by at least two RasGEFs: The role of RasGEFR is not clear, but is speculated to mediate RasG activation in the pathway regulating chemotaxis, and R a s G E F M regulates RasG activation in the pathway regulating cell polarity and motility. On the other hand, RasC activation regulates the pathway leading to A C A activation, and speculated to be activated at the rear of chemotaxing cells based on the observation that A C A is localized at the rear of cells (Kriebel et al., 2003). Some unclear relationships exist that cannot be addressed with this model. For example, it is clear that RasG signals to PI3K, and that PI3K and its effector protein C R A C are required for A C A activation, so it is not clear why the loss of RasG results in only a modest decrease in A C A activation (Bolourani et al., 2006). The role of RasC in regulating depolymerization of the actin and myosin II cytoskeletons is unresolved at this time, and thus its role in these processes has been marked with a question mark. 103 The main role of RasC is in the activation of A C A . RasC in turn is activated by R a s G E F A , but how RasC activation leads to A C A activation is not clear at this point. In vivo localization studies indicate that A C A activation occurs at the rear of the cell (Kriebel et al., 2003), so the stimulatory c A M P signal must transduce from the front of the cell to the back. A n intriguing possibility is that RasC is activated at the rear of the cell, placing it in proximity to A C A , and thereby providing a model for RasC mediated activation. However, unlike yeast A C A , Dictyostelium A C A does not have a R B D , so there must be at least one other regulatory molecule that transduces the c A M P signal from activated RasC to A C A . It should be noted that there is no evidence of where in the cell RasC is activated. However, given that the microtubule organizing centre is localized towards the rear of chemotaxing Dictyostelium Cells (Rubino et al., 1984; Sameshima et al., 1988), and that there may be a possible association of R a s G E F A to the microtubule cytoskeleton via its L i s H domain (Ernes and Ponting, 2001; Wilkins et al., 2005), the idea that RasC is activated at the rear is not out of the realm of possibility. 4.8 T h e s i s s y n o p s i s a n d f u t u r e d i r e c t i o n s The work presented in this thesis has made an impact in the field of Ras signalling in general, and in Dictyostelium Ras research specifically. This work demonstrated for the first time that chemoattractant signalling in aggregating Dictyosteliumcells involves two Ras proteins, RasC and RasG. During this process RasC is activated by R a s G E F A in the pathway leading to A C A activation. RasG, on the other hand, appears to be regulated by two RasGEFs, R a s G E F M and RasGEFR, with R a s G E F M involved in the pathway leading to P K B phosphorylation, and RasGEFR potentially involved in chemotaxis. The initial published work (Kae et al., 2004) has stimulated an interest in the use and adaptation of the assay, leading to a 104 growing number of collaborative efforts, with the results of some of these partnerships summarized in this thesis. As the number of components involved in the Ras signalling pathways in Dictyostelium increases, so does the complexity in regards to how everything fits together. The R a s G - P B K pathway, for example, is particularly confusing. A l l the evidence suggests that RasG is involved in mediating the PI3K and T O R C 2 pathways. Both of these pathways are required for A C A activation, and rasG cells are only modestly diminished in their ability to activate A C A (Bolourani et al., 2006). However, activation of A C A is far more affected by a loss of RasC with rasC cells being unable to aggregate due to loss of A C A activation (L im et al., 2001). It is not known how A C A is activated, so it is not clear how the c A M P signal is transmitted from either RasC or RasG to A C A , but it is presumed that there are at least two pathways. A t this time, the exact nature of the involvement of R a s G E F M in RasG activation is not clear. The data presented in this thesis (Figure 17B and 18) provide preliminary evidence, but in vitro nucleotide release activity is needed to confirm that R a s G E F M is an activator of RasG. Furthermore, obtaining a Dictyostelium P K B specific antibody wi l l determine whether the levels of total P K B are equal between A x 2 and gefM cells (Section 3.9), and this wi l l assign a definitive role for R a s G E F M in the signalling pathway leading to P K B phosphorylation/activation. Identification of a RasC interacting protein would go a long way to shed some light on the RasC signalling pathway. Along with its role in A C A activation, RasC also appears to be involved in cytoskeletal rearrangement during chemotaxis. In particular, RasC appears to be required for the depolymerization of both the actin and myosin II cytoskeletOns. While RasC exhibits a second peak of activation when stimulated with c A M P (Figure 11) that coincides with 105 the timing of the depolymerization of actin and myosin II, it is possible that the improper depolymerisation of actin and myosin II in chemotaxing rasC cells could be a result of the inability to activate A C A , as c A M P is an important second messenger involved in mediating the activation of c A M P binding proteins such as P K A (Zhang et al., 2003). A yeast two-hybrid screen recently undertaken by the Weeks lab wi l l hopefully shed some light on this subject by finding which components transmit the c A M P stimulated signal downstream of RasC. The role of negative regulators of Ras is an interesting avenue of study. The genetic data suggests that Gcc9 is the key negative regulator for c A M P stimulated events (Section 4.5). Thus, one would expect that Ras activation in a gpal strain would be sustained for a longer period of time. Finding the specific RasGAPs for RasC and RasG, on the other hand, requires some more effort. A method involving brute force would be to knock out all of the gap genes and determine the effect of their loss on Ras activation. A more directed approach could involve screening for RasGAPs that interact with activated RasC and/or RasG using a yeast two-hybrid assay first. A n y gap genes that show a positive interaction can then be targeted for knockout, and its role in the negative regulation of Ras can be studied via the R B D binding assay, as well as phenotypic effects of the gene knockout. The use of G F P - R B D probes can provide important information about the in vivo localization of activated Ras. However, the relative promiscuity of the Ras -RBD interaction makes it difficult to be able to differentiate between the different Ras isoforms. One solution to this problem is to use isoform specific R B D s . Thus far, the R B D of the protein kinase Phg2 has proven to be specific for R a p l when tested for its ability to bind to 5 members of the Dictyostelium Ras subfamily (Gebbie et al., 2004; G . Weeks, unpublished observations)). While the Dictyostelium genome encodes for at least 10 more Ras proteins, it remains promising that 106 the R B D of Phg2 can be used as a R a p l specific probe. Another method of obtaining isoform specific R B D s is to generate them using mutagenesis! This would involve randomly mutagenizing R B D s and screen for those that bind to only one Ras protein via a yeast two-hybrid screen. A similar type of strategy has been used to generate more efficient G F P probes, and fluorescent probes of different colours (Zhang et al., 2002). The tool developed by Taylor and Shalloway just over 10 years ago has grown tremendously in its use and applications. As there is still much more to learn about the regulation and localization of Ras, I foresee no end to how this assay can be further modified, improved, and applied. 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