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Identification of the domains of RasG-G12T that are required to produce defects in aggregation and cytoskeletal… Zhang, Taiqi 1998

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IDENTIFICATION O F THE DOMAINS O F RASG-G12T THAT A R E R E Q U I R E D TO P R O D U C E D E F E C T S IN AGGREGATION AND CYTOSKELETAL FUNCTION IN DICTYOSTELIUM DISCOIDEUM by TAIQI Z H A N G B . S c , S ichuan University, 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT O F THE REQUIREMENTS FOR THE D E G R E E OF MASTER O F SCIENCE i n THE FACULTY O F G R A D U A T E STUDIES (Department of Microbiology and Immunology) W e accept this thesis as conforming to the required standard T H E UNIVERSITY O F BRITISH COLUMBIA February 1998 © Taiqi Zhang, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada Date 1WK 6 . 1^7 DE-6 (2/88) A B S T R A C T Dictyostelium transformants expressing an activated rasG gene, r a s G - G 1 2 T , do not aggregate when starved for nutrients. In addit ion, express ion of the activated R a s G protein results in vegetat ive cell populat ions with heterogeneous morphology. Some cel ls are extensively f lattened and spread and exhibit lateral or dorsal membrane ruffling while others which are less f lattened, exhibit prominent dorsal membrane ruffling. The expression of R a s G - G 1 2 T also causes a slight increase in the average number of nuclei and results in a redistribution of F-actin to the cell periphery. These results suggest that R a s G has roles in both cytoskeletal and developmental regulation in Dictyostelium. To identify the functional residues required for the downstream effects of activated R a s G , amino acid substitutions have been introduced into R a s G - G 1 2 T within the effector domain (Tyr32-Tyr40) or within the effector distal f lanking domain. Ce l ls expressing R a s G - G 1 2 T with amino acid substitutions in the effector domain (T35S or Y 4 0 C ) showed normal morphology. These cel ls also aggregated and differentiated normally, suggest ing that the defects cause by R a s G - G 1 2 T required interaction of the effector domain with a downstream protein(s). In contrast an amino acid substitution in the effector distal f lanking domain (T45Q) prevented the R a s G - G 1 2 T induced block in aggregation, but was not able to prevent the cytoskeletal defect. This result suggests that the cytoskeletal and developmental defects induced by R a s G - G 1 2 T result from the interaction of the protein with different downstream effector molecu les . i i T A B L E OF CONTENTS Abstract Table of Contents List of F igures v List of Tab les v i List of Abbreviations v i i Acknowledgement v i i i INTRODUCTION 1 Pro logue 1 The Ras gene superfamily 1 Biological roles of Ras 3 Domains of Ras proteins 4 Regulators and effector of Ras 8 Dictyostelium discoideum Life cycle 1 3 Signal transduction events during the life cycle of D. discoideum A 4 Vegetat ive growth and aggregat ion 1 4 C e l l d i f ferent iat ion 1 7 ras and ras related genes in D. discoideum 1 8 MATERIALS AND METHODS 2 1 M a t e r i a l s 21 i i i D. discoideum growth and differentiation 2 2 Induction of the discoidin promoter 2 3 Transformation of D. discoideum 2 3 Nuclear staining and analysis of cell morphology 2 4 Scann ing electron microscopy 2 5 P lasmid D N A preparation 2 5 Sequenc ing 2 6 Site directed mutagenesis and vector construct ions 2 6 E lec t rophores is and immunoblott ing 2 7 Chemotaxis and motility assays 2 9 RESULTS 3 0 Identification of the effector residues of R a s G - G 1 2 T that are required for phenotypes caused by activated R a s G In Dictyostelium discoideum. 3 0 In t roduc t ion 3 0 Overexpression of the mutated rasG-G12T genes 31 The effect of mutated r a s G - G 1 2 T genes on cell morphology 3 3 Determination of the number of nuclei in transformed cel ls 3 8 Developmental phenotypes of the transformants express ing r a s G -G 1 2 T / T 3 5 S , r a s G - G 1 2 T / Y 4 0 C or r a s G - G 1 2 T / T 4 5 Q 41 G E N E R A L DISCUSSION 4 4 BIBLIOGRAPHY 5 2 i v LIST OF FIGURES Figure 1. Three dimentional structure of the nuclotide-binding domain of human c -Ha- ras p21 5 Figure 2. R a s upstream signaling events in R7 development 9 Figure 3. Vertebrate Ras downstream signaling 1 2 Figure 4. The life cycle of D. discoideum 1 5 Figure 5. Amino acid substitutions of R a s G - G 1 2 T and vector c o n s t r u c t i o n s 2 8 Figure 6. Al ignment of the human H-Ras extended effector sequence with the Dictyostel ium R a s subfamily protein sequences 3 2 Figure 7. Express ion of R a s G proteins containing G12T /T35S , G 1 2 T / Y 4 0 C and G12T/T45Q substitutions 3 4 Figure 8. Morphology of vegetative cells 3 6 Figure 9. Scann ing Electron Microscopy of vegetative cel ls 3 7 Figure 10. Nuclear staining of Ras transformed cells 3 9 Figure 11. Chemotax is of pVEI I - rasG-G12T, pVE I I - r asG-G12T /T35S , p V E I I - r a s G - G 1 2 T / Y 4 0 C and pVE I I - r asG-G12T /T45Q transformants 4 3 v LIST OF T A B L E Table 1. The number of nuclei per cell LIST OF ABBREVIATIONS BS Bonner 's Sal ts buffer c A M P cycl ic adenosine 3', 5' -monophosphate c A R s c A M P receptors C R A C cytosol ic regulator of adenylyl cyc lase DIF di f ferent iat ion induc ing factor ECL enhanced chemi luminscence EGF epidermal growth factor G A P G T P a s e activating protein GEF guanine exchange factor MAP mi togen-ac t i va ted protein P B S phosphate buffered sal ine PDGF p la te le t -der ived growth factor PEG polyethy lene glycol P S F p re-s ta rva t ion factor P K A cAMP-dependen t protein kinase A S D S - P A G E sod ium dodecylsul fate polyacry lamide gel e lec t rophores is T B S Tris buffered sal ine T r i s t r i s (hyd roxyme thy l )am inomethane Tween 20 po lyoxyethy lene -20 -sorb i tan mono laura te Single letter code for amino acids: A , Alanine; R, Arginine; N, Asparag ine; D, Aspart ic acid; C , Cysteine; Q, Glutamine; E, Glutamic acid; G , Glyc ine; H, Histidine; I, lsoleucine;L, Leucine; K, Lysine; F, Phenyla lanine; P, Prol ine; S , Ser ine; T, Threonine; Y , Tyrosine; V, Val ine. v i i ACKNOWLEDGMENTS I thank Meenal Khos la for ass is tance in the vector construction and RasG-G12T strains. I thank Elaine Humphery for ass is tance with microscopy. I thank my committee members: Dr. Mike Gold and Dr. Tony Warren for their ass is tance. I thank past and present members of Dr. Week 's lab : Meenal Khosla, Patrick Rebstein, Sharon Louis, Kathy Luo, Zahara Jaffer, Kathryn Schubert, Shiv Sharma, Sam Abraham, Megan Delehanty, Brent Sutherland, James Lim and Loverne Duncan for their advice and support. Finally I thank my supervisors Drs. Gerry Weeks and George Sp iege lman for their guidance and efforts to make me think critically and also speak and write Engl ish correctly. v i i i INTRODUCTION 1 Pro logue The primary objective of this thesis was to identify the residues of R a s G - G 1 2 T that are required to produce defects in aggregation and cytoskeleta l function in Dictyostelium discoideum. The introduction will focus on a review of the ras gene superfamily emphas iz ing primarily on the relationship between the structure and function of the R a s proteins. I will a lso review the literature on R a s signal transduct ion pathways, placing a specia l emphasis on the downstream effectors of R a s . I will then d iscuss Dictyostelium discoideum, and its signal transduction pathways, before summariz ing what is known about the ras genes of Dictyostelium. The Ras gene superfamily The ras superfamily of genes encodes small monomeric G T P a s e s that functional ly resemble the heterotrimeric G proteins (reviewed in Zerial and Huber, 1995). The superfamily can be subdivided into three major groups (the ras, rho and rab sub-famil ies), based on the degree of shared amino acid conservation and the protein function (Kahn et al., 1992). More than 50 members of the mammalian ras superfamily have been identified to date. The ras sub-family includes the three human ras p ro to-oncogenes, Ki-ras, H-ras and N-ras , whose encoded products share 8 5 % amino acid identity (Bol lag and McCormick , 1991); the genes encoding the c losely related proteins R -Ras , TC21 and Ra l , that share - 5 5 - 5 0 % amino acid 2 identity with H-Ras (Cox et al., 1994; Graham et al., 1994), and the genes encoding the Rap proteins that share approximately 5 0 % amino acid identity with H-Ras (Bokoch, 1993; Noda, 1993). The ras sub-family of genes have been highly conserved throughout evolution, s ince Ras proteins from organisms as diverse as human and yeast share high levels of i den t i t y . The rab gene sub-family members encode proteins which share approximately 30% amino acid identity with the R a s protein (Rothman and Orc i , 1992). They play a role in the regulation of vesic le trafficking between intracellular organel les (Pfeffer, 1994), a process necessary for the b iogenesis of organel les and important in maintaining integrity of endomembrane compartments (Rothman, 1996). The rho gene sub-family encodes Rho, Rac and Cdc42 proteins which all share approximately 3 0 % amino acid identity with Ras protein (Nobes and Hal l , 1994). Rho-subfami ly members regulate signal transduction from receptors in the p lasma membrane, controll ing cellular events related to cel l shape , polarity, motility and cytoskeletal dynamics. Rho, Rac and C d c 4 2 induce distinct changes in act in-based cel l morphology when microinjected into mammal ian cel ls (Ridley et al., 1992; Paterson et al., 1990; Nobes and Hal l , 1995). Specif ical ly, Rho induces focal adhesion assembly and stress fiber formation, R a c induces lamell ipodia and membrane ruffling, and C d c 4 2 induces formation of f i lopodia. 3 Biological roles of Ras The ras genes were first identified as the transforming agents of the Harvey and Kirsten murine sarcoma viruses (reviewed by Barbac id , 1987; Lowy and Wi l lunsen, 1993). Identification of the mammal ian cellular ras homologues (H-ras, K-ras and N-ras) and the presence of mutated al leles in human tumors suggested a role for these genes in the control of cel lular proliferation. High incidence of ras oncogenes in cancers: adenocarc inomas of the pancreas (90%), the colon (50%) and the lung (30%), thyroid tumors (50%), and myeloid leukemia (30%) indicate the importance of the genes in human(reviewed by Bos, 1989). In vitro, microinjection of activated Ras proteins in NIH 3T3 cel ls caused cell proliferation, D N A synthesis and produced a malignantly t ransformed cel l appearance (Stacey and Kung, 1984) and the injection of antibody against R a s protein inhibited serum-st imulated growth of NIH 3T3 cel ls (Mulcahy et al., 1985). These studies further indicated a role for R a s in cel l proliferation and suggested a possible role in cytoskeletal regulat ion. R a s protein a lso functions in differentiation and development in some cel ls . Act ivated Ras protein induce the differentiation of P C 1 2 pheochromocytoma cells into neuronal cel ls (Hagag, 1986). The most clearly defined role for Ras in development, however, has been provided by a ser ies of genetic analysis of neuronal differentiation in the R 7 photoreceptor cel ls of the Drosophila eye (Wassarman etal., 1995) and vulval differentiation in C. elegans (Kayne and Sternberg, 1995). During Drosophila eye development, activation of Ras disrupts normal cel l fate specif icat ion in the compound eye and when microinjected into embryos, disrupts the terminal cell fates of posterior cel ls (Simon et al., 1991; Lu et al., 1993). In C. elegans, the let-60 ras gene is required for multiple 4 aspects of development. The vulval differentiation pathway has been the most intensively studied. Activation of R a s results in a multivulval phenotype while loss of Ras activity leads to a vulvaless phenotypes (Beitel et al., 1990; Han et al., 1990). Domains of Ras proteins The 21 kD Ras protein belongs to the highly conserved G T P a s e superfamily which also includes the a subunits of heterotrimeric G protein and G T P a s e s used in ribosomal protein synthesis such as bacterial elongation factor E F T u (Bourne et al., 1991). These proteins all cycle between an inactive GDP-bound form and an active GTP-bound form which al lows them to act as molecular switches for s ignal transduct ion pathways. The conformational state of Ras is regulated by two kinds of proteins, guanine nucleotide exchange factors (GEFs ) and G T P a s e -activating proteins (GAPs ) (Boguski and McCormick , 1993; Witt inghofer et al., 1997). The structures of the different forms of Ras have been determined by X-ray crystal lography (Fig. 1) and the functions have been studied by site-directed and random mutagenesis (De vos et al., 1988, Pai et al., 1989; Milburn et al., 1990; Nassar et al., 1995). The amino acid domains required for binding and hydrolyzing G T P are located in 4 conserved sequences consist ing of residues (10-17, 53-62, 112-119 and 144-146) respect ively (Bourne et al., 1991). The first domain (10-17) forms bonds with the a- and (3-phosphates of G T P or G D P (Lowy and Wi l lumsen, 1993). The second domain (53-62) forms a hydrogen bond with the y-phosphate of G T P ; and the third domain (112-119) forms hydrogen bonds with both the guanine ring and the first domain. The fourth domain (114-146) is 5 Figure"!. Three dimentional structure of the nuclot ide-binding domain of human c - H a - r a s p 2 1 . This figure is copied from (Pai et al., 1989). All loops and a few important amino-ac id res idues are label led for or ientat ion. 6 somewhat var iable and indirectly interacts with the guanine nucleot ide by stabi l iz ing the third domain. In the R a s protein, single amino acid substitutions within the first domain (amino acid 12 or 13), significantly decrease G T P a s e activity. These mutations are termed activating mutations, s ince they confer resistance to the action of the G A P , rendering the protein constitutively G T P - b o u n d . The activating effects of position 12 (or 13) mutations in R a s proteins have been demonstrated in mammalian cel ls (Barbacid, 1987; Lowy and Wi l lumsen, 1993), Drosophila (Fortini et al., 1992), C. elegans (Han and Sternbeerg, 1990), S . cerevisiae (Crechet et al., 1990) and Dictyostelium (Reymond et al., 1986; Khos la et al., 1996). In mammal ian cel ls these substitutions have often been identif ied in naturally occurring human tumors (Barbacid, 1987). Single amino acid substitut ion S 1 7 N strongly inhibits proliferation of NIH 3T3 cel ls (Feig and Cooper , 1988) and neuronal differentiation of P C 1 2 cel ls (Szeberenyi et al., 1990). This mutant protein is thought to bind exchange protein normally, thus promoting G D P release, but has a reduced affinity for G T P , and, as a result, remains bound to the exchange protein. This prevents activation of normal ras proteins, and thus inhibits s ignal t ransmiss ion (McCormick , 1994). The three-dimensional structure of the Ras protein changes upon G D P / G T P exchange. In particular, the conformations of the A s p 3 0 - A s p 3 8 and G l y 6 0 - G l u 7 6 regions change significantly, and these regions are cal led "switch I" and "switch II" respectively (Milburn et al., 1990). The switch I region is located in the second loop and essential ly over laps with the conserved effector region (Tyr32-Tyr40; S iga l et al., 1986; Wi l l umsen et al., 1986). Mutations that alter amino acid within this effector domain (32-40; Satoh et al., 1987) do not influence the guanine 7 nucleot ide-binding activity or the G T P a s e activity, but the biological effects such as morphological transformation of cultured f ibroblasts or stimulation of the yeast adenylate cyc lase are impaired. The effector domain is therefore bel ieved to interact with the downstream target effector (Marshal l , 1993). However, R a p l A is a nontransforming protein (K i tayama etal., 1989), although R a p l A residues 32-44 are identical to R a s , suggest ing that the transforming activity of R a s requires addit ional domains. Extensive genetic analysis suggested that region 26-48 should be considered to be an extended effector region. The amino acids most crit ical to biological function are G lu31 , Pro34, Thr35, A s p 3 8 , Tyr40, Va l45 and Gly48 (reviewed by Marshal l , 1993). The X-ray crystal structure of the complex between the Ras-re lated protein R a p l A in the G T P form and the Ras effector C-Raf1 structurally conf i rmed the interaction is mediated by main-chain and s ide-chain interaction of the effector residues in the switch I region of R a p l A (Nassar et al., 1995). An addit ional smal l domain is present at the carboxyl terminal of all Ras- re la ted proteins. This domain is subject to post translat ional process ing in R a s , resulting in addition of a farnesyl isoprenyl group, fol lowed by removal of the terminal 3 amino acids and then carboxymethylat ion of the resulting terminal cysteine (Hancock et al., 1989; C la rke , 1992). This domain with its subsequent modif ication is required for the attachment of Ras to the inner leaflet of the p lasma membrane s ince replacement of the cysteine with other amino ac ids prevents the associat ion of the protein with the membrane and blocks ability of R a s to transform cel ls (Guierrez et al., 1989; Wi l lumsen et al., 1984) . Regulators and effector of Ras 8 The binding and hydrolysis of G T P by Ras is regulated by other protein components. The G E F s function immediately upstream of Ras to stimulate the dissociat ion of G D P from Ras (reviewed in Boguski and McCormick , 1993). Several G E F s that interact with the R a s proteins have been identified (Feig, 1994) including the Drosophila S O S (son-of sevenless) (Simon et al., 1991), the mammalian S O S homolog (Bowtell et al., 1992) and S . cerevisiae C D C 2 5 (Broek et al., 1987). During R7 photoreceptor differentiation, the Drosophila S O S is linked to Drosophila homolog of the EGF-receptor sevenless via the adaptor protein DrK (downstream of receptor kinase). The Sev /Drk /Sos complex act ivates Ras1 (reviewed in Wassarman et al., 1995). Ras signaling terminates when G T P is hydrolyzed to G D P . G A P s inactivate Ras by accelerat ing the slow intrinsic rate of G T P hydrolysis by several orders of magnitude (Fig. 2). Five mammalian G A P s for Ras have been described (Boguski and McCormick , 1993; Wittinghofer et al., 1995), including p 1 2 0 G A P - t h e prototype for this c lass of proteins (Trahey and McCormick , 1987; Trahey et al., 1988 and Vogel et al., 1988), and neurof ibroma, the product of the type I neurofibromatosis (NF1) gene (Xu et al., 1990; Martin et al., 1990; and Bal lester et al., 1990). The molecules that interact with GTP-bound R a s and transmit the signal are general ly referred to as 'Ras effectors'. At present, there are several proteins have been shown to interact with R a s in a G T P -dependent manner (reviewed by Katz and McCormick, 1997). The most studied Ras effector is serine/threonine kinase Raf. B iochemica l and genetic studies identified the protein k inase Raf as involved in Ras-dependent signaling in mammalian cel ls, C. elegans and D. 9 Signals Plasma membrane Cytosol Sev Drk R7 cell GDP GTP Downstream events Figure 2. R a s upstream signaling events in R7 development. This figure is modified from (Alberts et al., 1994). Act ivated receptor tyrosine kinase Sev activates the guanine nucleotide releasing protein S o s by the small S H adaptor protein Drk. Sos stimulates the inactive R a s - G D P to active R a s - G T P , which activates Ras to relay the signal downstream. (Although not shown here, Ras is bound to the cytosol ic face of the p lasma membrane.) G A P counteracts the function of S o s by stimulating Ras to hydrolyze its bound G T P and become inactive. 1 0 melanogaster (reviewed in Dickson et al., 1994). Yeast two hybrid and GST- fus ion protein binding studies indicate Raf is a direct target of R a s (Vojtek et al., 1993; Aelst et al., 1993; Zhang et al., 1993; Warne et al., 1993). Raf plays an essential role downstream of Ras in tyrosine kinase-st imulated s ignal ing pathways promoting cel l growth and differentiation (Marshal l , 1996). The stimulation of a receptor tyrosine k inase (RTK) results in the recruitment of the guanine nucleot ide-releasing factor S o s (Schlessinger, 1994) to the p lasma membrane through the S H 2 domain of an adaptor protein Grb2 (Clark etal., 1992; Lowenstein etal., 1992). S o s activates R a s and promotes its direct binding to Raf and translocates Raf to the p lasma membrane (Moodie etal., 1993; Warne etal., 1993; Zhang et al., 1993). Activated Raf is the member of a M A P kinase cascade that results in the activation of ERK1 and /or E R K 2 which, in turn, phosphory late and activate transcript ional factors that induce transcription of genes required for entry into S phase of the cel l cycle (reviewed in Marshal l , 1995; McCormck, 1995; Marshal l , 1996; Katz and McCormick , 1997). R a l - G D S (Ral guanine nucleotide dissociat ion stimulator), an exchange factor for the Ras-re lated protein Ral was also identified to interact with R a s through the effector loop in a GTP-dependent manner in vitro in insect cel ls and in the two hybrid systems (Hofer et al., 1994; Spaargaren and Bischof, 1994; Kikuchi et al., 1994), making it a candidate for a biological effector of Ras act ion. It was d iscovered recently (Urano et al., 1996) that Ras can stimulate exchange activity of R a l G D S in cel ls. R a l G D S may mediate its effects via Ra l , although this issue is still controversial (Katz and McCormick , 1997). At present, the function of Ral is unknown. 11 Identification the proteins in cell extracts that bind to p 2 1 R a s in a GTP-dependen t manner has provided direct biochemical ev idence for the interaction between phosphoinosit ide 3 -OH kinase (PI 3-kinase) and ras (Rod r i guez -V i c i ana et al., 1994). PI 3-kinase interacts with R a s - G T P but not with R a s - G D P and is activated both in vitro and in vivo as a result of this interaction (Kodaki et al., 1994; Rodr iguez-V ic iana et al., 1996). PI 3-k inase has been implicated in the regulation of the actin cytoskeleton by growth factors such as P D G F and insulin (Kotani et al., 1994; Wennst rom et al., 1994; Nobes et al., 1995). Furthermore, PI 3-kinase may provide a link between Ras and the Rho G T P a s e s and appears to function upstream of Rac , s ince generating 3' phosphorylated phosphoinosi t ides activate Rac guanine nucleotide exchange factors (Hawkins et al., 1995), mediating Ras control of the cytoskeleton (Fig. 3). Other putative mammal ian Ras effectors (Katz and McCormick , 1997) include the family of Ras GTPase-ac t iva t ing proteins, among them p 1 2 0 G A P , neurofibromin, and Gap1 (Marshal l , 1996), but it is still open question whether G A P s and neurofibromin are both regulators and effectors of R a s . Two serine/threonine k inases other than Raf: M E K kinase 1 (MEKK1) and protein kniase Ct, ( P K C Q bind Ras , although it is not known whether M E K K 1 or PKCt ; are activated directly by Ras or not. Two structurally related proteins A F - 6 and Canoe have been found recently to bind G T P - R a s (Kur iyama et al., 1996), but the function of these proteins are not known. Finally, kinase suppressor of Ras (KSR-1) has been shown to be downstream of ras genetically, but there is no b iochemical ev idence for interaction between Ras and K S R - 1 . Recent evidence has indicated that Ras proteins can regulate more than one signal transduction pathway. Raf-dependent pathway is 1 2 1 Vesicular transport? 1 (^RaT) MEK 1 ERK 1 PI-3K Actin cytoskeleton Cell division Differentiation Figure 3. Vertebrate Ras downstream signal ing. This figure is modified from (Marshal l , 1996). The strongest candidates for physiological effectors of vertebrate Ras are shown along with the pathways they are proposed to control. 1 3 essent ia l for f ibroblast proliferation but not needed for R a s to induce membrane ruffling in f ibroblasts (Joneson et al., 1996). Further analys is indicates PI 3-kinase is the effector by which Ras induces membrane ruffling, act ing through Rac (Ridley, 1994; Rodr iguez-V ic iana et al., 1997). Efficient neoplast ic transformation by R a s requires activation of PI 3-kinase in addition to Raf. (Rodr iguez-Vic iana et al., 1994; Kl inghofer et al., 1996; Marsha l l , 1996; Rodr iguez-V ic iana et al., 1997). In fact, in f ission yeast S . pombe, Ras has been shown to directly regulate two effectors. One is a M A P Kinase kinase kinase, Byr2, and the other is S c d 1 , a guanine nucleotide exchange factor for the Rho family protein C d c 42, that is involved in regulation of cell morphology (Chang et al., 1994). Dictyostelium discoideum Life cycle D. discoideum is a simple eukaryote that has a life cycle that renders it attractive for studies on both proliferation and differentiation s ince the p rocesses are largely distinct (Loomis, 1982; Firtel et al., 1989; Mann et al., 1991). Vegetative D. discoideum cells exist as individual amoebae and use phagocytosis to ingest bacteria and pinocytosis ingest liquid media. Upon nutrient deprivat ion, the cel ls initiate an interactive developmental program. Approximately 3 h after starvat ion, cel ls within the population initiate a c A M P - m e d i a t e d response/re lay cascade by secret ing pulses of c A M P into its surroundings. Nearby cel ls respond by moving towards the st imulus and by producing c A M P pulses themselves. This chemotaxis and signal relay results in the formation of an aggregate of cel ls. The aggregation process begins at 5 h and typically requires 2 h for completion. By 12 h after starvation, a single tip, which functions as an organiz ing center for 1 4 morphogenesis, can clearly be distinguished on the mound. At the same t ime, the initial spat ial pattern of two functional ly distinct cel l types (prestalk and prespore) is establ ished. The tipped mound gradually extends vertically and then falls to the substratum forming a migrating slug by 16 h of development. The anterior 2 0 % of the slug is composed of prestalk cel ls and the posterior 80% is composed of predominantly prespore cel ls . Under appropriate condit ions, the migrating slug culminates with the formation of a mature fruiting body containing 8 0 % spore cel ls and 2 0 % vacuolated dead stalk cel ls rising from a basal disk. The process is complete by 25 h after starvation (Fig. 4) Signal transduction events during the life cycle of D. discoideum Vegetative growth and aggregation Vegetat ive D. discoideum cel ls prey on bacteria by detecting and chemotax ing toward folate and pterin, two bacterial metabol i tes (Pan et al., 1972; Pan et al., 1975), The transition from single cel ls to multicellularity is mediated by a variety of s ignal ing molecu les . Growing amoebae secrete an autocrine factor, prestarvation factor or P S F , which accumulates in proportion to cell density, serving as a sensor for the availabil i ty of nutrients (Clarke et al., 1987, Rathi et al., 1991; Rathi and Clarke, 1992). At a high PSF/bac te r ia ratios, a prestarvation response is initiated and the expression of several genes involved in early aggregation is increased. (Burdine and Clarke, 1995). Another factor, Spores C D C D 1 5 Amoebae Prespore cells Prestalk cells Aggregation Migrating slug r o 12 Spore head Stalk Fruiting body 16 ~ T ~ 20 24 Development (h) Figure 4. The life cycle of D. discoideum Black areas represent prestalk and stalk cel ls while c lear areas represent prespore and spore cel ls. The time in development is shown below in hours. 1 6 condit ioned medium factor (CMF) provides a similar density sens ing function which is involved in regulating c A M P signal ing after starvation has initiated (Gomer etal., 1991; Yuen etal., 1991). Aggregat ion in Dictyostelium is mediated by chemotact ic responses to pulsati le extracellular c A M P that binds to cel l surface receptors and act ivates intracellular s ignal ing pathways including the activation of adenylyl cyc lase, leading to the secret ion of c A M P and the relay of the chemotact ic s ignal ; the activation of guanylyl cyc lase , which is important for chemotaxis; and the expression of genes (including the c A M P receptor cAR1 and the coupled G a 2 subunit), whose encoded products are essent ial for the aggregation process. These pathways are regulated by oscil latory pulses of c A M P , which control the sequent ia l activation and adaptation of these pathways (Van Haastert 1991; Devereotes 1994; Firtel 1995; Firtel 1996). During aggregat ion, c A M P , acting through c A M P receptor (cAR1), st imulates adenylyl cyc lase producing c A M P , which is then relayed outward from aggregation center to st imulate addit ional cel ls . Fol lowing st imulation, there is a subsequent adaptation response leading to an inactivation of adenylate cyc lase . Extracellular phosphodiesterase (PDE) hydrolyzes the excess extracellular c A M P , thus preparing the cell for response to the next pulse. The roles of many of the components in aggregation have been defined by b iochemica l , physiological and genetic studies (reviewed in Firtel, 1995; Firtel, 1996; Parent and Devreotes, 1996). Activation of adenylyl cyc lases requires the G protein containing the G a 2 subunit coupl ing to cAR1 and c A R 3 (Kumagai etal., 1991; Kesbeke etal., 1988; Insall etal., 1994a) and Gp subunit regulates adenylyl cyc lase directly. Cytosol ic regulator of adenylyl cyc lase ( C R A C ) is also required for A C activation (Lilly and Deverotes 1994) possibly by interacting with Gp subunit (Insall 1 7 et al., 1994b; Touhara et al., 1994). cAMP-dependent protein k inase (PKA) , presumably activated by increased intracellular c A M P , has a lso been shown to be required for A C activation. A possible role for Ras in aggregation has been indicated by the finding that E R K 2 and a G E F are essential for aggregation (Segal l et al., 1995; Insall et al., 1996). However, cel ls expressing an activated form of rasG fail to aggregate, suggesting that R a s G - G T P could be negative regulator of the signal transduction pathway that leads from c A M P receptor to adenylate cyc lase activat ion. Cel l di f ferentiat ion Upon the formation of the mound, rising levels of c A M P activate a second signal ing pathway that results in the repression of aggregat ion-stage gene expression and the induction of postaggregative gene expression and the regulatory cascade that leads to the subsequent express ion of prestalk- and prespore-speci f ic genes , cel l- type differentiation, morphogenesis (Abe and Yanag i sawa 1983; Schni tz ler et al., 1994, 1995; Firtel 1995). In contrast to the cAMP-med ia ted responses during aggregat ion, post-aggregate gene express ion and cel l -type differentiation require a high, continuous level of c A M P . The express ion and activation of the transcription factor G B F is essent ia l for prestalk and prespore gene expression. The differentiation of cel ls into prestalk and prespore cel ls within the multicellular aggregate is regulated by a number of factors including c A M P (reviewed in Gross , 1994; Firtel, 1995; Firtel, 1996). c A M P initially promotes the development of both prestalk and prespore cel ls but at later s tages acts to promote spore development and inhibit stalk 1 8 development. Although both prestalk and prespore gene express ion requires G B F function, they are regulated by addit ional distinct mechan isms. Protein kinase A (PKA) , is required for normal development and has been shown to be a positive regulator of prespore gene expression and spore maturation, presumably functioning to mediate the effects of intracellular c A M P (Simon etal., 1989; Firtel and Ahapman, 1990; Mann et al., 1994; Hopper et al., 1995). Protein kinase A also appears to inhibit prestalk development but plays a role in subsequent stalk formation (Mann and Firtel, 1993; Harwood etal., 1992b). Glycogen synthase kinase 3 (GSK-3) and E R K 2 are also essential for prespore gene expression (Harwood etal., 1995, Gask ins etal., 1996), but it is unknown whether G S K 3 , P K A and E R K 2 function in common or parallel pathways. The morphogen DIF (differentiation-inducing factor) is required for the prestalk pathway and inhibits the prespore pathway (Kopachik et al., 1983; Wi l l iams etal., 1987). DIF activates a S T A T (Signal Transducers and Act ivators of Transcript ion) protein which induces prestalk cel l differentiation and represses stalk cel l differentiation (Kawata et al., 1997). The low affinity c A M P receptor c A R 4 is also required for prestalk cel l differentiation (Louis et al., 1994). Ras and ras related genes in D. discoideum In Dictyostelium, five ras genes (rasD, rasG, rasB, rasS, and rasC) and one rap gene (rap7) have been reported, each having a specif ic express ion pattern (Reymond etal., 1984; Robbins etal., 1989; Daniel et al., 1993; Daniel etal., 1994) and recently two additional genes have been identified (Wilkins and Insall, unpublished). The first five ras genes share the four wel l -conserved GTP-b ind ing domains as well as the C -1 9 terminal C A A X box. rasG encodes a protein which shares 6 9 % overall amino acid identity with the human H-ras gene product, and the gene is expressed during growth and early development with express ion decl ining markedly during aggregation (Robbins et al., 1989). rasD, whose gene product share 6 5 % identity with H-ras, is expressed only during late aggregation and slug formation (12-16 hours) (Reymond et al., 1984). The R a s G and R a s D proteins are 82% identical to each other and they have identical effector and effector-proximal domains. The binding site for the Ras-spec i f i c monoclonal antibody, Y13-259 , is a lso totally conserved in R a s D and R a s G . Four other ras sub-family genes {rasB, rasC, rasS, and rapl) encode products more distantly related to Ha- ras . The R a s B , R a s C and R a s S gene products share 59%, 56% and 54% amino acid identity respect ively with H-ras. r a s B is maximally expressed during vegetat ive growth and early development but expression remains relatively high during the remainder of development (Daniel et al., 1994). r a s C is expressed maximally during aggregation and slug migration but signif icant express ion is detected during vegetat ive growth throughout the remainder of development (Daniel et al., 1993). r a s S is expressed only during aggregation (4-8 hours). As ide from their patterns of express ion, little is known about the functions of Dictyostelium Ras protein. Overexpress ion of an activated form of R a s D (G12T) gene during development resulted in formation of aggregates with multitips, instead of the normal single tips, and a block in further development (Reymond et al., 1986). Further analysis has indicated that this development defect is accompanied by a profound change in the fate decis ion; prestalk cell gene expression is markedly enhanced and prespore gene expression is almost totally inhibited (Louis et al., 1997), suggest ing that R a s D has a role in regulating cell type 20 determinat ion during differentiation. Surpr is ingly, rasD null mutant show temporal ly and morphological ly normal development although there is a signif icant increase in spore cell formation. (Khosla and Wi lk ins, unpubl ished data). Overexpress ion of rasG containing an activating mutation ( R a s G -G12T) during growth caused a block in aggregation (Khosla etal., 1996). The defect in aggregation was rescued by pulsing cel ls with c A M P and by mixing with wild type cel ls , suggest ing that the mutant cel ls are able to receive c A M P signals but not able to generate them. In contrast, cel ls overexpressing wild type ras G gene or a presumptive dominant negative r a s G - S 1 7 N differentiated normally, suggest ing that the inhibition of the initiation of aggregation is due to enhanced levels of R a s G - G T P . r a s G -G 1 2 T expression also caused marked cytoskeletal changes and wild type rasG produced less pronounced morphological changes. r a s G - S 1 7 N produced a different cell shape change, and the r a s G null cel ls have a defect in the cytoskeleton, which results in abnormal cytokinesis when cel ls are grown in suspension (Tuxworth et al., 1997). These results are all consistent with R a s G having a role in regulating the cytoskeleton. Final ly transformants overexpress ing r a s G or r a s G - G 1 2 T also exhibit a slight defect in cytokinesis, they have on average 1.7 nuclei per cell under condit ions where wild type cel ls have a single nucleus. 2 1 MATERIALS AND METHODS M a t e r i a l s The following reagents were purchased from suppl iers indicated in brackets. Cyc l ic adenosine monophosphate (cAMP) , streptomycin, ampici l l in, and folate (Sigma, St. Louis, U S A ) ; X-ray film (Kodak, Canada) ; R a d i o l a b e l e d [ a 3 5 S ] d A T P (Dupont N E N C a n a d a Inc.); filters (Mill ipore, U S A ) ; the enhanced chemi luminescence kit for western blot analys is (Amersham, Canada) ; NuclebondR Ax-20 kit for isolating plasmid D N A was (Macherey-Nage l , Germany) ; Bacter iological peptone and yeast extract for the D. discoideum growth media (Oxoid, UK) and for bacterial growth media (Canadian Life Technologies G I B C O BRL) ; G418 (Geneticin), restriction endonuc leases and modifying enzymes (G IBCO B R L ) ; Sequenase (United States Biochemical) . All other chemicals were purchased from Fisher Scientif ic C o . (Vancouver, Canada) or B D H (Vancouver, Canada) . Hoechst 33258 dye was a gift from Dr. R. E. W. Hancock's laboratory (UBC) . Ol igonucleot ides were synthesized by the N A P S facility (UBC) . The preparation and use of the R a s G - G S T - f u s i o n protein antibody for western blot analysis has been descr ibed previously (Khosla et al., 1994) Goat anti-rabbit antibody, conjugated to horseradish perox idase was purchased from Amersham (USA) . The E. coll strains DH5aF ' , XL-1 and RZ1032 were used for the various cloning manipulations. The genotype of DH5ocF' is: F'/endA1 hsdR17{rk-mk+) supE44 thi-1 recA1gyrA(Na\r) relA1 A(lacZYA-argF)U169 deoR (08OdlacA(lacZ)M15) (Raleigh et al., 1989). The genotype of X L 1 M R F ' is: endA1, hsdR17 (rK-, ITIK+), supE44, thi-1 , I-, recA1, gyrA96, relA1, (lac-), F'[proAB, / ac /9ZAM15 , Tn10 (tetR)] (Stratagene). The genotype of 22 RZ1032 is: HfrKL 16PO/45[]ysA (61-62)7 dut 1, ung1, thil, relA1Zbd-279:: Tn10supE44 (Kunkel et al., 1987). The pVEII vector was donated by Wolfgang Nel len and modified to remove the discoidin A T G translation site by Meenal Khosla (Rebstein etal., 1993). t h e p T Z 1 9 R - r a s G - G 1 2 T vector was constructed by Meenal Khos la (Khosla etal., 1996). D. discoideum growth and differentiation The parental axenic line Ax-2 strain of D. discoideum that was used in all experiments was grown axenical ly in HL5 medium (Watts and Ashworth, 1970) (14.3 g neutral ized bacteriological peptone, 7.15 g yeast extract, 0.96 g N a 2 H P 0 4 and 0.486 g KH2PO4 per liter of water) with gyratory shaking at 175 rpm at 22°C or on S M nutrient agar (10 g glucose, 10 g neutral ized bacteriological peptone, 1g yeast extract, 1 g M g S 0 4 - 7 H 2 0 , 1.55 g NH2PO4.H2O, 1 g KHPO4 and 20 g bacto-agar per liter of water) plates in associat ion with E. aerogenes. Cel l numbers were determined in duplicate with a hemacytometer. The transformed Ax-2 strains were maintained in HL5 medium in the presence of 10 | ig/ml G418 (Geneticin) and 1 mM folate in rotatory agitated suspens ion (175 rpm) at 22°C. D. discoideum development on filters was initiated as previously descr ibed (Khos la et al., 1990). Exponential ly growing vegetat ive cel ls at a density of between 1 x 1 0 ^ and 2 x 1 0 ^ cells per ml were harvested by centrifugation at 700 x g for 2 min and then washed twice in Bonner 's salt (10 mM NaCI, 10 mM KCI and 2 mM CaCl2 ) , (Bonner, 1947). For development, 2.5 x 1 0 7 washed cells were plated on a 4.0 cm diameter nitrocellulose filter (pore s ize = 0.45 | im), resting on a Bonner 's salt (BS) saturated pad in a 60 mm petri d ish. The filters were incubated at 22°C 23 in a moist chamber. To observe D. discoideum development fol lowing growth on bacter ia was accompl ished by pipetting 1- 5 JLLI of 2 x 10^ cel ls /ml Dictyostelium cel ls in HL5 medium onto a freshly inoculated lawn of Enterobacter aerogenes on an S M nutrient agar plate. Plates were incubated at 22°C and after the D. discoideum cel ls had consumed the bacteria (usually 4 days), development could be observed in the zone depleted of bacter ia. Induction of the discoidin promoter To maintain strains containing genes under the control of the d i sco id in dis I y gene promoter in a suppressed state, 1 mM folate was added to the HL5 medium. To maximally induce expression from the discoidin promoter, cel ls were incubated with condit ioned HL5 medium, s ince condi t ioned medium contains a pre-starvation factor ( P S F ) (Clarke et al., 1987; Clarke et al., 1988), that induces express ion from the discoidin promoter (Rathi et al., 1991). Condit ioned HL5 medium was prepared by growing Ax-2 cel ls to a density of approximately 2 x 1fj6 ce l ls /ml , removing the cel ls by centrifugation and filtering the medium through a 0.2 | im pore s ize nitrocellulose filter. In some exper iments, induction was ach ieved by growing cel ls to 1-2 x 10^ cel ls/ml in the absence of folate. Transformation of D. discoideum D. discoideum Ax-2 cel ls were transformed by the calc ium phosphate precipitation technique (Nellen et al., 1984) in Bis-Tr is HL5 (Egelhoff et al., 1989). A total of 8 x 1 0 6 exponent ia l -phase Ax -2 ce l ls 24 were incubated with 10 |xg of vector DNA in the form of calc ium phosphate D N A precipitate for 4 hours, the cel ls were given a 2 minute osmotic shock with 15% glycerol as previously descr ibed (Early and Wi l l iams, 1987). Transformants were selected in HL5 medium containing 30 i ig/ml G 4 1 8 , 50 ng/ml stretomycin, 50 Lig/ml ampici l l in, and 1mM folate and colonies were visible after approximately 14 days . Individual c lones were picked using a pipette and transferred initially to 24 well plates, and then to 100 mm plates. Once cell growth was well es tab l ished, stable transformants were maintained in shake suspens ion in HL5 media containing 10 ixg/ml G418 , 50 pig/ml st reptomycin, 50 u\g/ml ampici l l in, and 1 mM folate. Nuclear staining and analysis of cell morphology To examine cell morphology, cel ls were plated 3 x 1 0 ^ ce l l s / cm^ on a g lass coversl ip in a 60 mm petri dish and incubated for 24 hours either in the presence of 5 ml of conditioned HL5 medium or in HL5 medium containing 1mM folate to induce or repress the discoidin p romo te r , respectively. To determine nuclear number, the adherent cel ls were washed , fixed with 3.7% formaldehyde in B S for 10 minutes and then washed 3 times with P B S (8 g NaCI, 0.2 g KCI, 1.44 g N a H P 0 4 , 0.24 g KH2PO4 in one liter of distil led water). Cel ls were then permeal ibi l ized for 5 minutes in -20°C actone, rehydrated in P B S , overlaid with 0 .0005% Hoechst dye #33258 for 5minutes and then washed with P B S (Harlow and Lane, 1988). The g lass coversl ips were briefly dried before mounting on g lass s l ides using 5 0 % glycerol as mounting solution and v iewed with a Ze i ss Axiophot microscope equipped with epi f luorescence. Ce l l morphology of the same cells were then observed using phase contrast 25 microscopy. All photographs were taken using Kodak T M A X 400 black and white f i lm. Scanning electron microscopy A total 4 x 1 0 3 cel ls were plated on 1/2 inch g lass coversl ip in a 60mm petri dish and incubated for 24 hours in the presence of 5 ml of condit ioned HL5 medium to induce the discoidin promoter. The coversl ips were removed and the cel ls were fixed in 2 .5% gluteraldehyde buffered with 100mM sodium cacodylate, pH 7.4. The cel ls were then treated successful ly with 1% O s 0 4 , 2% Tannic acid and 1% O s 0 4 again. The attached cel ls were dehydrated in a graded ethanol ser ies and critical point dried with liquid carbon dioxide. The coversl ips with adhering dried cel ls were mounted on specimen stubs, sputter coated with gold and v iewed with an Cambr idge 250T Scanning Electron Microscope. Plasmid DNA preparation Plasmid D N A was isolated from DH5ocF' E. coli cel ls using alkaline lysis miniprep procedure (Maniatis et al., 1989) or a PEG-prec ip i ta t ion large sale procedure (Maniatis et al., 1989). Competent DH5ocF' E. coli cel ls were prepared using the rubidium chloride technique and transformation of competent cel ls was done as descr ibed (Maniatis et al., 1989). Transformed D H 5 a F ' E. coli cel ls were selected on LB ampicil l in plates (10 g Bacto-tryptone, 5 g Bacto yeast extract, 10 g NaCI, 15 g Bacto-agar pH 7.0, with 60 | ig/ml ampici l l in) . 26 Sequenc ing Single stranded D N A was generated following infection of D H 5 a F ' with K0 7M 13 helper phage, by P E G precipitation. The precipitated D N A was isolated on g lass filters and then eluted into T E (Maniatis et al., 1989). Double stranded DNA was isolated using the NucleobondR Ax-20 kit Macherey-Nagel (Duren, Germany). DNA sequence was determined by the dideoxy chain termination method (Sanger et al., 1977) with modified T7 D N A polymerase. Sequencing reactions were performed according to the manufacturer 's (United States Biochemical) protocol except that the Sequenase reaction buffer was added after D N A denaturation. Site directed mutagenesis and vector constructions Missense mutations of r a s G - G 1 2 T gene were created as fol lows. The p T Z 1 9 R - r a s G - G 1 2 T vector (Khosla etal., 1996) was transformed into Escherichia coli strain RZ1032 (Kunkel et al., 1987). Uraci l containing s ingle-st randed phagemid D N A was isolated after infection with the helper phage, M13K07 (Vireira et al., 1987) and was used as a template for o l igonucleot ide-directed mutagenesis reaction (Kunkel et al., 1987). To create the mutations, the mutagenic ol igonucleot ides had the fol lowing s e q u e n c e s : 5 ' - T A C G A T C C A T C T A T C G A A G - 3 ' (T35S) 5 ' - C T A T C G A A G A T T C A T G T A G A A A A C A A G T T A C - 3 ' (Y40C) 5 ' - C A T A C A G A A A A C A A G T T C A A A T T G A T G A A G A A A C T T G - 3 ' (T45Q) In each case the substituted bases are underl ined. The mutated genes were transformed into Escherichia coli strain D H 5 a F ' (Stratagene). To 2 7 obtain the pVEI I - rasG construct, the pVEII vector was digested by Kpn\, and treated with T4 DNA polymerase to generate blunt ends. The rasG inserts were isolated by an BglW EcoRl d igest ion, treated with K lenow and d N T P s to generate blunt ends and then ligated into blunt ended pVEII vector. The r a s G fragment was ligated into the pVEII vector in the sense orientation downstream of the discoidin promoter and created the 3' f lanking E c o R l site (Fig. 5). The junctions of the constructs were confirmed by E c o R l digestion and by double stranded DNA sequencing react ions. pVEII vector also contains the actin 15-Tn903 resistance casset te (Blusch et al., 1992) as a G418 selectable marker. The constructs were transformed into X L - 1 . Electrophoresis and immunoblotting S D S - P A G E and immunoblotting techniques were performed as descr ibed (Robbins, 1991). For western blot analysis of transformants express ing genes under the control of the discoidin promoter, cel ls were inoculated at a density of 5 x 1fj5 cel ls/ml and grown for 24 hours in shake suspens ion in either condit ioned medium or HL5 containing 1mM folate. Cel ls were lysed in 1% S D S . Protein concentration was estimated by UV Absorbance (Harlow and Lane, 1988). About 20 \ig of protein from each transformant was mixed with an equal volume of 2 x loading dye (20% glycerol, 10% p-mercaptoethanol, 4 .6% S D S , 125mM Tr is-HCI, pH 6.8), boiled for 5 min, then were electrophoresed on a 12% S D S -p loyacry lamide ge l . Figure 5. Amino acid substitutions of R a s G - G 1 2 T and vector c o n s t r u c t i o n s The posit ions of the substituted amino acid are indicated, the hatched box indicate the proposed effector domain. The mutated r a s G -G 1 2 T genes were cloned under the control of dicoidin promoter in the pVEII vector. 29 After e lect rophoresis , the protein were transferred to a nitrocel lulose filter for 1 hour at 90V (Towbin et al., 1979). Presta ined molecular weight markers (BioRad) were used to estimate protein s i zes . The nitrocellulose blots were stained with Ponceau S (Harlow and Lane, 1988) to confirm that equal amounts of protein had been loaded and transferred in all lanes. The membrane was blocked at room temperature with 1 x T B S (8 g NaCI, 0.2 g KCI, 3 g Tris-HCI in 1 liter H2O, pH 7.4) 5% skim milk and o .1% Tween-20 at least for 1 h.and were then washed twice for 5 min. with T B S and 1% Tween-20. The R a s G protein was detected with a specif ic a n t i - R a s G - G S T protein antibody at a 1: 500 dilution containing 0 .5% powered milk, 0 .1% Tween-20 and incubated with nitrocellulose membranes at room temperature for overnight. The membranes were then washed four times (for 5 min. each) in T B S - T w e e n . And the binding of the ant i -RasG antibody was detected using a secondary goat anti-rabbit antibody by E C L which was recorded on X-ray film. Chemotaxis and motility assays Chemotax is assays were performed as previously descr ibed (Khosla et al., 1996). Chemotax is to folate was assessed by plating approximately 1 0 6 cells in 1 JLII on 2 % agar plates containing P D F / M E S (20 mM KCI, 1.2 mM M g S 0 4 , 7.5 mM morpholinethanesulfonic acid [MES; pH 6.5]) and 40 i iM folate. The average distance migrated by the halo of cel ls that escape from the cell mass was determined after 24 h. Chemotax is to c A M P was determined in an identical fashion except that the 2 % agar plates contained P D F / M E S and 10 uM c A M P , and the cells were shaken at 150 rpm for 4 h in P D F / M E S at 5 x 1 0 6 cel ls per ml prior to plating. R E S U L T S 30 Identification of the effector residues of RasG-G12T that are required for phenotypes caused by activated RasG In Dictyostelium discoideum In t roduct ion Recent studies have provided overwhelming evidence that R a s regulates at least two distinct s ignal transduction pathways in mammal ian cel ls ; one involves the Raf-dependent activation of E R K 1 and E R K 2 , resulting in transcription of genes that regulate growth and differentiation; the other pathway is Raf- independent and involves activation of Rho family G T P a s e s which play a role in actin cytoskeletal reorganizat ion (Katz and McCormick , 1997). Partial loss of function mutants of Ras have been used help to define its downstream effector pathways (White et al., 1995; Joneson et al., 1996; Rodr iguez-V ic iana et al., 1997). For example in quiescent f ibroblasts, ectopic express ion of activated H - R A S (H-RASV12) induces membrane ruffling, M A P kinase activation and stimulation of D N A synthesis. When secondary mutations (T35S or Y 4 0 C ) were introduced into the activated H - R A S effector domain (32-40), either M A P kinase activity (Y40C) or the membrane ruffling (T35S) effects were not induced (Joneson et al., 1996). It has been suggested that the effector mutations S35 and C40 in a V12 background correlate with either of two distinct R a s effector pathways to induce transformation in mammal ian cel ls (Rodr iguez-V ic iana et al., 1997). Express ion of activated R a s G (G12T) protein in vegetative D. discoideum cel ls resulted in cytoskeletal and cytokinesis defects and 3 1 cel ls that did not aggregate when starved for nutrients (Khosla et al., 1996). To identify the effector resides of the R a s G that block the aggregat ion and/or produce the cytoskeletal and cytokinesis defects in Dictyostelium and to determine if there are multiple downstream effectors involved, secondary mutations (T35S, Y 4 0 C ) were introduced into r a s G - G 1 2 T within the effector domain (32-40) and a mutation (T45Q) was introduced within one of the effector f lanking regions (Fig. 6). Residue 45 is not conserved in most of the Dictyostelium R a s subfamily proteins and the amino acid in R a s G is different from that found in mammalian Ras (Fig. 6). In mammalian Ras , V45 has been found to be one of the most critical amino acids required for R a s function (reviewed by Marshal l , 1993). A V 4 5 E mutation prevented an activated ras gene from producing foci formation in NIH 3T3 fibroblast cell and from promoting neurite outgrowth of P C 1 2 cel ls (Marshal l et al., 1991; Fu j i ta -Yosh igak i ef al., 1991). Overexpression of the mutated rasG-G12T genes Mutated rasG genes T35S (ACT->TCT), Y 4 0 C (TAC->TGT), or T45Q ( A C C - > C A A ) were c loned downstream of the folate-repressible discoidin (disly) promoter in the vector pVEII (Fig. 5), and the vector was introduced into D. discoideum by transformation using the calc ium phosphate precipitate procedure. Transformants were selected in the presence of 30 j ig G418 to select for the uptake of the vector and 1mM folate to repress the discoidin promoter. For each transformation twelve G 4 1 8 resistant c lones were isolated and R a s G protein levels under inducing condit ions were determined by Western blot analysis using a RasG-spec i f i c antibody. The western blot 32 26 * * * 48 H-Ras N H F V D E Y D P T I E D S Y R K Q V V I D G RasG _ _ _ I _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ T - - E RasD _ _ _ I _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ S _ _ D RasB - - - I E - - - - - - - - - - - R - C Q V - E RasC - - - I A - - - - - - - N - - - - - - N - - E RasS _ _ _ _ _ _ _ _ _ _ T J - - - - - - - T T V - -Figure 6. Al ignment of the human H-Ras extended effector sequence with the Dictyostelium Ras subfamily protein sequences . Numbers indicate amino acid posit ions in each protein (aa 26-48). Stars indicates amino acid positions of mutants (35, 40 or 45). Dashes indicate amino ac ids identical to those in H-Ras . The effector domain residues (Sigal ef al., 1986) are underl ined. 33 data for six of the isolates are shown in Figure 7. Most of the levels of R a s G were considerably higher than that found in the parental Ax-2 cel ls (Fig. 7). For each double mutant construct, a transformant c lone that expressed a level of R a s G protein equal to or higher than that of original p V E I I - r a s G - G 1 2 T transformant was selected for further analys is (Fig. 7). The effect of mutated rasG-G12T genes on cell morphology To determine the effects of high levels of the mutated R a s G protein on cell morphology in vegetative Dictyostelium, t ransformants were plated on g lass coversl ips and incubated for 24 hours in condit ioned medium. Condi t ioned medium contains a pre-starvation factor (PSF) (Clarke et al., 1987; Clarke et al., 1988), which enhances express ion from the discoidin promoter (Rathi et al., 1991). The cel ls were fixed with formaldehyde and then observed by phase contrast microscopy. The transformant c lones express ing r a s G - G 1 2 T / T 3 5 S (Fig. 81) or r a s G -G 1 2 T / Y 4 0 C (Fig. 8J) had a morphology that resembled the parental Ax-2 cel ls (Fig. 8K). In contrast, many of the cel ls in a population expressing high levels of r a s G - G 1 2 T / T 4 5 Q exhibited a flattened and spread out morphology (Fig. 8 panel E, F, G , H) that was similar to that of cel ls expressing activated r a s G - G 1 2 T (Fig 8 panel A, B, C , D). To examine if there were subtle dif ferences in the morphology of the transformants express ing r a s G - G 1 2 T a n d transformants expressing r a s G - G 1 2 T / T 4 5 Q that were not readily apparent by phase contrast microscopy, vegetat ive cel ls were observed by scanning electron microscopy (SEM) and their appearance is shown in Figure 9. The transformant expressing r a s G -G12T /T45Q (Fig. 9C and 9D) resembled the r a s G - G 1 2 T transformant (Fig. 9A and 9B). Both populations exhibited heterogeneity in their morphology. 3 4 Figure 7. Expression of R a s G proteins containing G12T /T35S , G 1 2 T / Y 4 0 C and G 1 2 T / T 4 5 Q substi tut ions. Cel ls were incubated in condit ioned medium for 24 hours and then lysed in 1% S D S , 20 i ig of total protein was separated by S D S - P A G E , transferred to nitrocellulose and probed with an an t i -RasG antibody. Lane 1 is the pVEI I - rasG-G12T transformant. Lane 2 is the parental Ax-2 . Lane 3-8 represent independent transformants expressing the doubly mutated R a s G proteins RasG-G12T7T35S (panel A) , R a s G - G 1 2 T / Y 4 0 C (panel B) or R a s G - G 1 2 T / T 4 5 Q (panel C) . The arrows indicate the t ransformants se lec ted for further detai led ana lys is . 3 5 36 Figure 8. Morphology of vegetative cel ls pVEI I - rasG-G12T (A, B, C , D), pVEI I - rasG-G12T/T45Q (E, F, G , H), p V E I I - r a s G - G 1 2 T / T 3 5 S (I), pVEI I - / -asG-G12T/Y40C (J) and Ax-2 (K), cel ls were incubated with condit ioned media for 24 hours and fixed with formaldehyde and then photographed with phase contrast optics. The cell morphology of Ax-2 and r a s G - G 1 2 T cells shows no significant change before and after fixation with formaldehyde (data not shown). The bar is 10 j im . 3 7 Figure 9. Scann ing Electron Microscopy of vegetative cel ls pVEI I - rasG-G12T (A and B); pVEII- / -asG-G12T/T45Q (C and D) and Ax-2 (E) cel ls were grown in condit ioned medium for 24 hours, f ixed, dehydrated, critical point dried, coated with gold and then photographed using scanning electron microscope as descr ibed in the Materials and Methods. 3 8 S o m e cel ls were extensively flattened and spread and exhibited cons iderab le dorsal membrane ruffling with many fine elongated f i lopodia (Fig. 9B and 9D), whereas some cells were flattened and spread but exhibi ted lateral membrane ruffling with few elongated f i lopodia (Fig. 9A and 9C) . Other cells in the two populations had an appearance more character ist ic of wild type Ax-2 cel ls (Fig. 9E) . Determination of the number of nuclei in transformed cells There was an increase in the number of multinucleate cel ls in populat ions of cel ls expressing rasG-G12T, suggest ing a defect in cytokinesis (Rebstein, 1996). When cel ls were induced with condit ioned medium during growth on a surface, multinucleate cel ls were frequently observed in transformants expressing rasG-G12T/T45Q (Fig. 10C). In contrast, most of the cel ls expressing rasG -G12T/T35S (Fig. 10A) or /-asG-G12T/Y40C (Fig. 10B) had single nuclei like Ax-2 (Fig. 10E). The number of nuclei in the cel ls transformed with pVEI I -rasG -G12T/T35S, pVEII-rasG-G12T/Y40C and pVEII-rasG-G12T/T45Q was determined and compared to the number in Ax-2 and rasG-G12T cel ls (Table 1). Transformants expressing rasG-G12T/T45Q had an average of 2.0 nucle i /ce l l , which was signif icantly different from the value (1.2 nuclei /cel l ) for Ax -2 (P<.05), but was not signif icantly different from the value (1.7 nuclei/cell) for pVE I I - r asG-G12T transformant (P>.05). Folate repression of the discoidin promoter, which drives express ion of the introduced ras gene, abol ished the multinucleate phenotype of cel ls t ransformed with rasG-G12T or rasG-G12T/T45Q (Table 1). To test if growth condi t ions affected cytok ines is , the transformants express ing either rasG-G12T or rasG-G12T/T45Q were induced with condi t ioned 3 9 . • A • * • • • 1 • B * •* • * : :» c • "'At E • • • • • * • Figure 10. Nuclear staining of Ras transformed cel ls p V E I I - r a s G - G 1 2 T / T 3 5 S (A), pVE I I - r asG-G12T /Y40C (B), pVE I I - r asG-G 1 2 T / T 4 5 Q (C), pVEI I - rasG-G12T (D) and Ax-2 (E) cel ls were incubated in condit ioned media for 24 hours. The cel ls were fixed and stained with Hoechst dye as described in the Material and Methods. 40 Table 1. The number of nuclei per cell Number of nuclei Suspension Growth Surface Growth S t r a i n Induced 1 3 Induced 1 5 R e p r e s s e d 0 p V E I I - r a s G - G 1 2 T 1.5 (1) 1.7 ± 0.30 (4) 1.1 (1) p V E I I - / - a s G - G 1 2 T / T 3 5 S N/D 1.2 ± 0.04 (4) 1.1 (1) p V E I I - r a s G - G 1 2 T / Y 4 0 C N/D 1.2 ± 0.08 (3) 1.0 (1) p V E I I - / - a s G - G 1 2 T / T 4 5 Q 2.2 (1) 2.0 ± 0.16 (3) 1.0 (1) A x - 2 1.4 (1) 1.2 ± 0.06 (4) 1.2 (1) Mean ± standard deviation of the number of experiments shown in brackets. An average of 200 cel ls were analyzed per experiment. b Ce l ls were grown in conditioned HL5 media for 24 h. c Ce l ls were grown in HL5 media with 1mM folate for 24 h. 4 1 media during growth in suspension culture and then were plated on a surface for 30 minutes before nuclear staining. The average number of nuclei for Ax-2 was slightly increased to 1.4 nuclei /cel l , but the number for r a s G - G 1 2 T / T 4 5 Q transformant was still higher (Table 1). Developmental phenotypes of the transformants expressing rasG-G12T /T35S. rasG-G12T/Y40C or r a s G - G 1 2 T / T 4 5 Q R a s G protein levels expressed during growth in the presence of folate (Khos la et al., 1996) or during growth on bacteria (Khosla , unpubl ished data) were found to be relatively low. However, these relatively low levels of act ivated R a s G - G 1 2 T were sufficient to block aggregat ion (Khos la et al., 1996). All the isolated transformants express ing r a s G - G 1 2 T / T 3 5 S , r a s G - G 1 2 T / Y 4 0 C or r a s G - G 1 2 T / T 4 5 Q (twelve independent c lones from each transformation) dif ferentiated normally after growth on bacteria (data not shown). To determine if the express ion of high levels of protein would produce the defect in aggregation, the r a s G - G 1 2 T / T 3 5 S , r a s G - G 1 2 T / Y 4 0 C and r a s G - G 1 2 T / T 4 5 Q transformants that had been selected for the previously descr ibed morphological and cytokinesis studies were grown in HL5 medium in the absence of folate to densit ies of between 1 x 1 0 ^ and 2 x 1 0 ^ cel ls per ml, plated on a Mill ipore filter and then incubated under starvation condit ion to induce differentiation. These transformants a lso formed aggregates and differentiated normally (data not shown). In addit ion, normal development occurred when the transformants were incubated on non-nutr ient agar. S ince cel ls transformed with pVEI I - / -asG-G12T exhibi ted reduced chemotaxis to c A M P and folate and also reduced random motility (Khosla 42 et al., 1996), the abilit ies of the various transformants to respond to a c A M P and folate concentration gradients were tested. The transformant express ing r a s G - G 1 2 T / T 3 5 S exhibited c A M P chemotaxis that was indist inguishable from that for Ax -2 . In contrast, the cel ls express ing r a s G - G 1 2 T , r a s G - G 1 2 T / Y 4 0 C and r a s G - G 1 2 T / T 4 5 Q exhibi ted signif icantly reduced chemotaxis to c A M P compared to cel ls expressing r a s G -G 1 2 T / T 3 5 S (P<.05) (Fig. 11) Simi lar ly p V E I I - r a s G - G 1 2 T / T 3 5 S transformant exhib i ted normal chemotaxis to folate whereas the cel ls expressing r a s G - G 1 2 T and rasG-G 1 2 T / T 4 5 Q exhibited significantly lower chemotaxis (Fig. 11). The r a s G -G 1 2 T / Y 4 0 C strain appeared to exhibit reduced levels of chemotaxis to folate, but the average level for three exper iments was not signif icantly lower than that of the r a s G - G 1 2 T / T 3 5 S strain (P>.05). 4 3 Q. s a E o 1.2' 1.0' 0.8 H 0.6' 0.4' 0.2' 0.0' x x x X X X x x x X X X x x x X X X x x x X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X II • i f i i • To folate E3 To cAMP 71. v ^ x x x x x T12 T12S35 T12C40 T12Q45 Strain Figure 11. Chemotax is of pVEI I - rasG-G12T, p V E I I - r a s G - G 1 2 T / T 3 5 S , p V E I I - r a s G - G 1 2 T / Y 4 0 C and p V E I I - r a s G - G 1 2 T / T 4 5 Q t ransformants. Chemotax is to folate and c A M P was measured as descr ibed by (Browning et al., 1995). For each of the transformants, the height of the bar represents the mean distance migrated in response to folate (cross bars) and to c A M P (hatched bars), relative to Ax-2 . The error bars indicate the standard error of the mean from three independent expe r imen ts . General D iscuss ion 4 4 The overexpression of an activated rasG gene in vegetative cel ls of Dictyostelium has multiple effects. r a s G - G 1 2 T transformants fail to aggregate upon starvation due to an impairment in cycl ic A M P signal relay (Khosla et al., 1996), suggest ing R a s G - G T P is a negative regulator of the signal transduction pathway involved in c A M P relay. In addition part of the act ivated R a s G transformant population exhibit morphological abnormal i t ies that are accompan ied by alterations in cytoskeleta l function (Rebste in, 1996). These morphological abnormali t ies range from cel ls that are flattened and spread with numerous f i lopodia, lateral and dorsal ruffles to cel ls that are less flattened but display large circular dorsal ruffles (Cardell i and Bush , unpublished data). The cel ls that have the flattened morphology have increased F-actin located around the cell periphery (Rebstein, 1996). The expression of R a s G - G 1 2 T also causes a slight increase in the average number of nuclei per cell (Rebstein, 1996). A r a s G null mutant in which expression of R a s G was completely abol ished was capable of undergoing normal development but exhibited a wide range of defects in the control of the actin cytoskeleton, including defective cell movement, a loss of cell polarity, absence of normal lamel l ipodia, formation of unusual smal l , punctuate polymer ized actin structure and a large number of abnormally long f i lopodia. However, the most dramatic defect is in cytokinesis. r a s G null cel ls become mult inucleate when grown in suspens ion (Tuxworth et al., 1997). S ince both the activated r a s G transformants and rasG null transformants have altered morphology and exhibit defective cytokinesis, R a s G clearly has a role in the regulation of cytoskeletal function in Dictyostelium. However , the r a s G null transformants differentiate normally whereas the 4 5 act ivated rasG transformants fail to aggregate, suggest ing the defect in aggregat ion and the defects in cytoskeletal function and cytok inesis involve different downstream pathways of R a s G . In mammal ian cel ls , the most thoroughly studied Ras-dependent pathway involves the activation of the M A P K cascade . Upon receptor-act ivat ion, G T P - b o u n d Ras binds cytoplasmic Raf-1 and translocates it to the p lasma membrane where Raf-1 kinase becomes activated. Act ivated Raf results in activation of a R a f - > M E K - > E R K , kinase cascade . This act ivat ion is c lear ly required for transformation of rodent f ibroblasts because expression of dominant-negative versions of M E K (Cowley et al., 1994), or the use of a synthetic inhibitor of M E K , blocks transformation by Ras (Dudley et al., 1995). R a s proteins have been associated with control of the cytoskeleton in mammal ian cel ls. Act ivated Ras proteins have been implicated in triggering actin fi lament accumulat ion at the p lasma membrane: the formation of membrane ruffles via a Rac-dependent process, and the formation of actin stress fiber and focal adhesion development v ia a Rho-dependent process (Ridley and Hal l , 1992; Ridley et al., 1992). Recent results a lso support the existence of a Ras effector-mediated R a c / R h o s ignal ing pathway which is distinct from R a f / M A P k inase pathway which is required for full R a s transformation (Rodr iguez-V ic iana et al., 1997). R a s proteins with relatively subtle mutations in the effector region (32-40) exhibit a partial loss of function in that interaction with some effectors is maintained but interaction with others is lost. These mutants have been used to correlate effector interaction with biological function, providing further ev idence of branch points in Ras signal ing (White et al., 1995; Joneson et al., 1996; Rodr iguez-V ic iana et al., 1997). For example, V 1 2 S 3 5 Ras binds to and activates Raf-1 and the M A P kinase 46 cascade but does not activate PI 3 kinase and does not cause membrane ruffling. In contrast, V 1 2 C 4 0 Ras binds and activates PI 3-kinase and induces membrane ruffling but does not activate Raf and does not activate M A P kinase. When introduced into NIH 3T3 cel ls, neither of the mutant genes alone were able to induce transformation, but express ion of both V 1 2 C 4 0 and V 1 2 S 3 5 Ras in the same cell resulted in transformation. (Rod r i guez -V i c i ana et al., 1997). This indicates that membrane ruffling and activation of M A P kinase represent distinct R a s effector pathways, through PI 3-kinase and Raf respectively, that both are required for cell t r a n s f o r m a t i o n . In f ission yeast S . pombe, the Ras protein has been shown to regulate two effectors: Byr2 which functions in an analogous manner to Raf as a M A P kinase kinase kinase, in the pheromone M A P K pathway (Wang etal., 1991), and S c d 1 , a guanine presumptive nucleotide exchange factor for the Rho family protein Cdc42 and therefore involved in the regulation of the actin cytoskeleton (Chang etal., 1994). Thus the S c d 1 - R a s interaction may be analogous to the interaction between R a s and Rho signal ing pathways in mammal ian cell sys tems. The aim of the work presented here was to determine if Dictyostelium R a s G acts through more than one downstream effector. If the effects of R a s were transmitted through a single effector, then appropriately chosen secondary mutations of r a s G - G 1 2 T should inhibit all effects. Overexpress ion of R a s G - G 1 2 T with amino acid substitut ions at either posit ions 35, 40, or 45 did not induce the defect in aggregat ion, indicating that both the effector domain (amino acids 35 and 40) and the effector distal f lanking domain (amino acid 45) were required for activated R a s G to prevent aggregation. In contrast, overexpress ion of R a s G - G 1 2 T with a substitution at position 45 induced the cytoskeletal 47 changes whereas overexpression of R a s G - G 1 2 T with substitut ions at posit ions 35 and 40 did not. Cel ls expressing r a s G - G 1 2 T / T 4 5 Q exhib i ted similar morphology and cytokinesis defects to those seen in cel ls express ing r a s G - G 1 2 T . This result indicates that the effector domain is important for inducing the cytoskeletal defect whereas the effector distal f lanking domain is not. The fact that the position 45 change affects the disruption of aggregation by R a s G - G 1 2 T but not the disruption of the cytoskeleton, suggests that the change at position 45 does not induce a general deleter ious conformational change in the Ras protein. Consis tent with this conclus ion is the finding that replacing V45 with E in mammal ian R a s has little effect on the intrinsic G T P a s e activity of Ras and does not effect its interaction with G A P , but prevents an act ivated ras gene from producing foci in NIH 3T3 fibroblast cell or neurite outgrowth of P C 1 2 cel ls (Marshal l et al., 1991; Fuj i ta-Yoshigaki et al., 1991). Res idue 45 is different in most of the Dictyostelium R a s subfami ly proteins. The change from T to Q substitutes the amino acid present at position 45 in R a s B into R a s G . R a s G and R a s B share identical effector domain Cel ls expressing rasB -G12T aggregate normally and have abnormal morphology (Delehanty, Spiegelman and Weeks , unpubl ished observat ion), suggest ing that R a s G - G 1 2 T / T 4 5 Q might be reacting with the same effector as R a s B - G 1 2 T . However, the cel ls expressing R a s B -G 1 2 T and R a s G - G 1 2 T / T 4 5 Q have different morphological defects and it is therefore unlikely that the R a s G - G 1 2 T / T 4 5 Q protein interacts with R a s B e f f e c t o r . In mammal ian R a s , residue 35 is required for membrane ruffling and residue 40 is required for activation of M A P kinase activity (Joneson et al., 1996). Both of these residues are required for the abnormal 48 development and cell morphology of Dictyostelium, that is induced by activated R a s G . However, there was a slight difference between the effects of R a s G - G 1 2 T / Y 4 0 C and R a s G - G 1 2 T / T 3 5 S in Dictyostelium. The p V E I I - r a s G - G 1 2 T / Y 4 0 C transformant exhibited reduced chemotax is to c A M P whereas there was no reduction in c A M P chemotaxis for the pVEII-r a s G - G 1 2 T / T 3 5 S transformant. Further analysis will be needed to address the question as to whether residues 35 and 40 interact with ident ical or distinct s ignal ing pathways. Overexpress ion of an activated R a s G - G 1 2 T protein during growth inhibited the ability of cel ls to aggregate upon starvation while overexpression of wild type R a s G had no effect on aggregation (Khosla et al., 1996). These data suggested that G T P bound R a s G negatively regulates aggregat ion, and that the cell has sufficient regulatory capacity to compensate for the overexpression of wild type R a s G , but not activated R a s G . Consistent with this idea, the rasG null mutant has no aggregation defect. The available data suggests that r a s G - G 1 2 T interferes with the c A M P relay (Khosla et al., 1996). E R K 2 , which is the homologue of mammalian M A P kinase, is required for aggregation. E R K 2 activation is inhibited in cel ls overexpressing activated R a s D (Aubrey et al., 1997) or R a s G (Kosaka etal, 1998). Recent results suggest E R K 2 functions as a negative regulator of the cAMP-spec i f i c phosphodies terase RegA , and therefore down regulates intracellular c A M P levels and blocks the c A M P relay (Lu et al, unpublished observations). These data suggest that there might be a negatively regulated R a s signal transduction pathway to control initiation of aggregation in Dictystelium . It would be informative to determine if a regA null mutant could rescue the aggregation defect phenotype of cel ls expressing r a s G - G 1 2 T . Further more, protein-protein interaction studies could be performed to try to 4 9 show direct interactions between R a s G and E R K 2 or some other downstream effector that might couple R a s G to E R K 2 . It will a lso be important to determine if E R K 2 directly interacts with RegA . Whereas the aggregation defect appears to be specif ic for rasG-G 1 2 T transformants, both r a s G - G 1 2 T , gain of function, transformants and rasG null, loss of function, transformants exhibit abnormal morphologies. Although the morphological defects are not the same in the two cel ls , these results are consistent with the fact that the inhibition of aggregat ion involves a different effector than the defect in cytoskeleton. However, is not known how the Dictyostelium cytoskeleton is regulated by R a s G . The Dictyostelium strain expressing R a s G - G 1 2 T / Y 4 0 C didn't have an abnormal cell morphology, whereas a similarly mutated H-Ras protein produced an abnormal cell morphology when expressed in mammal ian cel ls. These results suggest that there might be novel R a s effectors that regulate cytoskeleton in Dictyostelium. Attempts should be made to identify direct R a s G downstream effector(s), using yeast two-hybr id, GST- fus ion proteins or Immunoprecipitation to help address the poss ib le pathway(s) that regulate cytoskeleton in Dictyostelium. Ras proteins have been associated with both control of the cytoskeleton and cel l proliferation in mammal ian cel ls but not with cytok inesis . However s ince cytokinesis defects are more difficult to determine in mammal ian cel ls than in Dictyostelium , it therefore is conceivable that Ras does play a role in mammalian cell cytokinesis. The most distinctive phenotype of the r a s G null cel ls is the defect in cytokinesis. Although the suspens ion culture growth rate of the r a s G null t ransformants was reduced, this was attributed to the defect in cytokinesis. However, more recent results have suggested that the 50 growth and cytokinesis defects may be distinct. When either rasG or rasD were transformed into the rasG null cel ls , a wild type phenotype was produced. However, when the rasB gene was transformed into rasG null ce l ls , it rescued the cytokinesis defect but not the growth defect, suggest ing that the defect in growth in the rasG null cel ls is independent from the defect in cytokinesis. (Khosla, unpubl ished data). None of the phenotypes of the three double rasG mutants strains obtained during this work demonstrated a separat ion of the cytokinesis and cytoskeletal defects and there is, therefore, no ev idence for an involvement of two different signal ing pathways. Thus the defect in cytokinesis may be a manifestation of general disruption of the actin cytoskeleton. However the possibil ity that R a s G controls a s ignal ing pathway that specif ical ly regulates cytokinesis can not be ruled out. An Dictyostelium homologue of mammalian Rac , R a c E , has been charac te r i zed (Larochel le et al., 1996). r a c E null cel ls exhibit defective cytok ines is , but the actin cytoskeleton rearrangements involved in phagocytos is , receptor capp ing, cort ical contract ion, and chemotax is appear normal. This suggests that R a c E might act on only one of the downstream R a s G effector pathways that is involved speci f ical ly in cytokinesis. However, when undergoing cytokinesis, the r a c E null mutant fails to complete contraction whereas the r a s G null mutant fails to complete cytokinesis occurs later during the process(rev iewed in Ch isho lm et al., 1997). These observations suggest R a c E is not directly regulated by R a s G . Genes that encode homologues of the mammalian p110 PI 3-kinase have also been cloned in Dictyostelium (Zhou et al., 1995). PI 3-kinase null cells do not grow in shake culture (Zhou et al., 1995), but exhibit nearly normal growth on a surface. These results suggest that the PI 3-kinase null cel ls have defects in cytokinesis that 5 1 are similar to those exhibited by the rasG null cel ls and this raises the possibil i ty that PI 3-kinase is downstream of R a s G . However, there is as yet no direct evidence for this hypothesis. The PI 3-kinase null mutant has an additional developmental defect that is not exhibited by the r a s G null ce l ls : aggregates produce multiple tips and abnormal fruiting bodies. However during development PI 3-kinase might act downstream of one of the other Ras proteins. Although both the loss of R a s G function and the gain of function produced cytokinesis defects in transformants, the changes were quite distinct. The abnormali t ies in the activated r a s G transformants were more obvious when cel ls were grown on a plastic surface while the abnormal i t ies in r a s G null cel ls were most clearly found when cel ls were grown in suspens ion . 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