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Analysis of the effects of the Rap1 and Rasg genes on dictyostelium discoideum cell morphology Rebstein, Patrick James 1996

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ANALYSIS OF T H E EFFECTS OF T H E RAP1 A N D RASG GENES O N DICTYOSTELIUM DISCOIDEUM CELL MORPHOLOGY PATRICK JAMES REBSTEIN B.Sc, The University of Toronto, 1987 M.Sc, The University of Toronto, 1989 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR T H E DEGREE OF DOCTOR OF PHILOSOPHY in T H E FACULTY OF GRADUATE STUDIES (Department of Microbiology and Immunology) We accept this thesis as conforming Jtp^Jjie^required standard T H E UNIVERSITY OF BRITISH COLUMBIA January 19% © Patrick James Rebstein, 1996 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. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) A B S T R A C T The rapl gene of Dictyostelium discoideum is a member of the ras-gene superfamily of low molecular weight GTPase proteins. The D. discoideum rapl gene is expressed both during growth and development. To further characterize rapl, the sequence and organization of the genomic DNA encoding the rapl gene and 1 kbp of the 5' flanking region was determined. The 5' flanking region could activate expression of the (3-galactosidase reporter gene upon transformation into D. discoideum. To examine the action of the Rapl protein in D. discoideum, the rapl cDNA was expressed under the control of the inducible discoidin promoter. Overexpression of the Rapl protein correlated with the appearance of morphologically aberrant vegetative amebae: cells were extensively spread and flattened with an increase in F-actin staining around the cell periphery. Expression of the rapl cDNA also prevented cell detachment from the substratum upon treatment with azide. When starved D. discoideum amebae are stimulated with HL5 medium, the cells rapidly respond by rounding up. By contrast, the Rapl transformant cells showed a pronounced delay in rounding. Site directed mutagenesis was used to determine the requirements for specific conserved amino acids for these effects caused by overexpression of Rapl. The substitution G10V, predicted to prevent nucleotide binding, and the substitution S17N, predicted to restrict the protein to the GDP-bound state, both abolished the ability of Rapl to affect cell morphology, suggesting that GTP binding is required for Rapl activity. Moreover, the substitution G12V which is predicted to increase the proportion of protein binding GTP, modestly enhanced the ability of Rapl to inhibit the rounding of starved cells after nutrient stimulation. By contrast, substitutions at amino acid 38 in the presumptive effector domain reduced but did not totally abolish the ability of ii Rapl to affect cell morphology. The substitution T61Q, which impairs the ability of mammalian Rapl to revert the phenotype of Ras transformed mammalian cells, did not alter the effect of Rapl on D. discoideum cell morphology. Thus, although the effects of Rapl on D. discoideum cell morphology appear to be regulated by GTP, the unexpected effects of substitutions at amino acid positions 38 and 61 suggests that the Rapl induced effects may involve different effector and regulatory molecules from that required to revert the phenotype of Ras transformed cells. A D. discoideum transformant expressing activated RasG-G12T protein (RasG-G12T) had an altered cell morphology similar to that of Rapl overexpression: the cells became flattened and spread with a concomitant distribution of F-actin around the cell periphery. RasG-G12T cells also failed to round up and detach upon exposure to azide. However, expression of activated RasG-G12T resulted in the formation of multinucleate cells whereas Rapl expressing transformants remained mononucleate. iii TABLE OF CONTENTS Abstract ii Table of contents iv List of Tables viii List of Figures ix List of Abbreviations xi Acknowlegements xii Dedication xiii Introduction 1 Prologue 1 The Ras gene superfamily 1 Domains of Ras superfamily proteins 3 Regulators and effectors of the Ras superfamily 7 Biological roles of Ras 8 Rapl proteins 10 Mutational analysis of Rapl proteins 12 Regulation of the Rapl proteins 13 Summary : 14 Action of Ras superfamily proteins on cell morphology 15 The effects of the Ras superfamily on the cytoskeleton 17 Dictyostelium discoideum 22 Life cycle 22 Signal transduction events during the lifecycle of D. discoideum 24 Vegetative growth, starvation and aggregation 24 Cell differentiation 25 iv The ras gene superfamily in D. discoideum 27 Cell morphology of D. discoideum 29 Rationale and research objective 34 Materials and Methods 35 Materials 35 D. discoideum growth and differentiation 37 Growth of D. discoideum 37 Development of D. discoideum .. 37 Determination of cell viability 38 Induction of the discoidin promoter 38 Analysis of cell morphology 39 Analysis of cell-cell adhesion 40 Flow cytometric analysis 40 Transformation of D. discoideum 40 Dark held, Nomarski and fluorescence microscopy 41 Molecular Biology 41 Plasmid DNA preparation 41 Southern blot analysis 42 Preparation of cDNA probes 43 Sequencing 43 Electrophoresis and immunoblotting 43 p-galactosidase assay 44 Isolation of the rapl genomic region 45 Sequence analysis of the rapl genomic region 46 Vector constructions 47 Polymerase chain reactions 52 v Results 54 The Dictyostelium discoideum rapl gene: isolation of the genomic sequence and characterization of the promoter region 54 Introduction 54 Isolation of rapl genomic DNA 54 Nucleotide sequence of the rapl genomic DNA including the 5' region 55 Analysis of the rapl promoter 59 Altered morphology of vegetative amebae induced by increased expression of the Dictyostelium discoideum rapl gene 61 Introduction 61 Effect of Rapl overexpression on cell morphology 61 Time course analysis of Rapl protein levels and cell morphology after induction of the discoidin promoter 66 Localization of F-actin 70 Effects of Rapl expression on growth 70 The response of cells to azide treatment 74 Determination of the number of nuclei in transformed cells 74 Cell motility analysis '. 74 Analysis of morphology after HL5 stimulation 75 Localization of F-actin in cells treated with HL5 after starvation 79 Pattern of tyrosine phosphorylation of actin after HL5 stimulation 79 Analysis of the erasure response of Rapl cells 82 vi Identification of conserved residues of the Dictyostelium discoideum Rapl protein required to alter cell morphology . 84 Introduction 84 The effect of mutated rapl genes on cell morphology 84 F-actin distribution in transformed cells .92 Analysis of morphology after HL5 stimulation 92 The response of cells to azide treatment 95 Growth and development 95 Activation of the rasG gene alters cell morphology in D. discoideum 99 Introduction 99 The effect of RasG protein on D. discoideum cell morphology 99 The localization of F-actin in D. discoideum cells expressing RasG 100 The response of cells to azide treatment 106 Determination of the number of nuclei in transformed cells 106 Analysis of morphology after HL5 stimulation Ill Expression of Rapl protein in RasG-G12T cells and expression of RasG in Rapl cells 114 General Discussion 116 References 137 vii LIST OF TABLES Table 1. Oligonucleotides used for site directed mutagenesis of the rapl gene 51 Table 2. Erasure of the capacity for rapid developmental recapitulation 83 Table 3. The effect of site directed mutations in the rapl gene on cell morphology 87 Table 4. The proportion of vegetative cells with a flat spread morphology . 90 Table 5. The percentage of cells remaining adherent after treatment with azide 97 Table 6. Generation times of transformants expressing mutated Rapl proteins 98 , Table 7. The proportion of cells with a flat spread morphology 104 Table 8. The proportion of cells remaining adherent after treatment with azide 108 Table 9. The number of nuclei per cell..... 110 Table 10. The effects of conserved amino acid substitutions in Ras and Rapl proteins ,.• 122 viii LIST OF FIGURES Figure 1. Alignment and structure assignment for RaplA and Ras 4 Figure 2. GTP cascades controlling cell morphology and bud formation 20 Figure 3. The life cycle of D. discoideum 23 Figure 4. PCR mutagenesis of the rapl gene 50 Figure 5. Southern blot analysis of D. discoideum rapl genomic DNA 56 Figure 6. Genomic organization of the D. discoideum rapl gene 57 Figure 7. Nucleotide sequence encoding the rapl gene 58 Figure 8. f3-galactosidase expression under the control of the 1 kb 5' untranslated region of the rapl gene 60 Figure 9. The pVEII Rapl expression vector 63 Figure 10. Expression of Rapl protein in cells transformed with the pVEII Rapl vector 64 Figure 11. Morphology of vegetative Rapl cells 65 Figure 12. Forward and side light scatter analysis of vegetative Rapl cells 67 Figure 13. Time course analysis of cell morphology after induction of the discoidin promoter 68 Figure 14. Time course analysis of Rapl protein upon induction of the discoidin promoter 69 Figure 15. Localization of F-actin in Rapl cells. 71 Figure 16. Growth of the Rapl transformant 72 Figure 17. Cell-cell adhesion properties of the Rapl cells 73 Figure 18. The effect of treating cells with azide 76 Figure 19. The motility of vegetative cells 77 Figure 20 Effect of HL5 stimulation on Rapl and Ax2 cells 78 Figure 21. Localization of F-actin in starved and HL5 stimulated cells. 80 ix Figure 22. Protein tyrosine phosphorylation after HL5 stimulation 81 Figure 23. Site directed mutations of conserved amino acids of Rapl... 86 Figure 24. Expression of Rapl protein containing G10V and S17N mutations 88 Figure 25. Cell morphology of transformants expressing mutated Rapl protein 89 Figure 26. Rapl protein expression under inducing conditions 91 Figure 27. Cell morphology and F-actin distribution 93 Figure 28. The response of starved cells to the reintroduction of HL5 medium '. 94 Figure 29. The percentage of cells responding to the reintroduction of HL5 medium after 5 and 10 minutes 96 Figure 30. Morphology of vegetative cells 101 Figure. 31. Induction of RasG protein expression 102 Figure 32. Forward and side light scatter analysis of RasG-G12T cells. 103 Figure 33. Localization of F-actin in Ras transformed cells 105 Figure 34. The effect of treating cells with azide 107 Figure 35. Nuclear staining of Ras transformed cells 109 Figure 36. The response of starved cells to FIL5 medium 112 Figure 37. The percentage of starved cells that respond to HL5 stimulation 113 Figure 38. Expression of Rapl protein in RasG-G12T cells and expression of RasG in Rapl cells 115 Figure 39. Comparison of azide treatment with the HL5 stimulation assay • • ••— 128 x LIST OF ABBREVIATIONS BSA bovine serum albumin CTAB cetyl trimethyl ammonium bromide ECL enhanced chemiluminscence EGF epidermal growth factor FITC-phalloidin fluorescein isothiocyanate-phalloidin GAP GTPase activating protein GTPyS guanosine 5'-(3-0-thio) triphosphate NGF nerve growth factor PBS phosphate buffered saline PEG polyethylene glycol PDGF platelet-derived growth factor RCF relative centrifugal force SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis TBS Tris buffered saline Single letter code for amino acids: A, Alanine; R, Arginine; D, Aspartic acid; C, Cysteine; Q, Glutamine; E, Glutamic acid; G, Glycine; H, Histidine; I, Isoleucine; M, Methionine; F, Phenylalanine; P, Proline; S, Serine; T, Threonine; W, Tryptophan; Y, Tyrosine; V, Valine. xi ACKNOWLEDGMENTS I thank Wolfgang Nellen for the pVEII vector; Meenal Khosla for assistance in the vector construction and the RasG and RasG-Gl2T strains; Jasmine Cohen for assistance with sequencing and the p-galactosidase assays; and Sharon Louis for the G10V and G12V mutated rapl cDNAs and helpful discussion. I thank the Gerish laboratory for performing the cell-cell adhesion analysis and the cell motility experiments. I thank Michael Schleicher, Matt Springer and Bruce Patterson for their helpful discussions on the effects of azide on D. discoideum. I thank Dennis Dixon for helpful discussions and assistance with the microscopy. I thank Dr. Frank Tufaro and Mr. Mike Weiss for letting me use their microscopes. I thank my committee members: Drs. Beatty, Jefferies, and Snutch for their assistance. I thank all members of Dr. Week's lab past and present who gave their advice and support. Finally I thank my supervisors Drs. Gerry Weeks and George Spiegelman for their guidance and efforts to make me think critically. I was a recipient of a Steve Fonyo Award from the National Cancer Institute of Canada and a University Graduate Fellowship. The research was supported by grants from the National Cancer Institute of Canada, the Medical Research Council and the BC Health Research Foundation. xii DEDICATION To Karen and Rachael. INTRODUCTION Prologue The primary objective of this thesis was to analyze the effects of expressing the ras-related rapl gene on cell morphology in Dictyostelium discoideum. In addition, the effects of expression of the rasG gene on cell morphology were also analyzed. In the Introduction, I shall initially review the literature on the ras gene superfamily focusing primarily on the well understood Ras proteins which have been extensively characterized both structurally and functionally. I will also review the literature on the rapl genes and other relevant ras-related genes. I will place a special emphasis on the roles that members of the ras gene superfamily play in modulating and regulating cell morphology. I will then discuss D. discoideum, focusing on the regulation of cell morphology during both growth and development before summarizing what is known about the ras and ras-related genes. The Ras gene superfamily The ras superfamily can be subdivided into three major groups that consist of the ras, rho and rab sub-families, based on the degree of shared amino acid conservation and in some cases on the protein function (Valencia et al, 1991; Kahn et al, 1992). More than 50 members of the mammalian ras superfamily have been identified to date. The ras sub-family includes the proto-oncogenes Ki-ras, H-ras and N-ras and the genes encoding the closely related proteins R-Ras and TC21 which share 55% identity with Ras (Cox et al., 1994; Graham et al, 1994) and the rap genes which encode proteins that share approximately 50% amino acid identity with Ras (Bokoch, 1993; Noda, 1993). The roles of the Rapl proteins is not fully understood but Rapl is capable of antagonizing transformation by Ras in some mammalian cells (Kitayama et al, 1989). The functions of the Rapl proteins will be discussed in more detail below. The ras sub-family of genes encodes proteins which are highly conserved throughout evolution. For example, the D. discoideum RasG protein has 69% conserved amino acid identity relative to H-ras (Robbins et al, 1989), while the D. discoideum Rapl protein is 73% identical to its human counterpart RaplA (Robbins et al., 1990), and the D. discoideum Ras and Rapl proteins are more highly related to the human Rapl and Rapl proteins than they are to the D. discoideum Rab and Rho proteins (Daniel et al., 1993a; Bush et al, 1993a). The identification of the presence of the same sub-families of the ras superfamily in divergent species likely represents some degree of conservation of function (reviewed by Valencia et al, 1991). In some cases, the ability to function equivalently has been demonstrated experimentally. For example, it has been shown that the mammalian H-ras gene can compliment ras deficient S. pombe and S. cerevisiae (Nadin-Davis etal, 1986; Kataoka et al, 1985; DeFeo-Jones et al, 1985). The rab gene sub-family members encode proteins which share approximately 30% amino acid homology with the Ras proteins (Rothman and Orci, 1992). Proteins in this sub-family have been shown to be required for vesicle transport between organelles in cells and for the production of synaptic vesicles (Rothman and Orci, 1992; Sudhof, 1995). The rho gene sub-family encodes Rho, Rac and Cdc42 proteins which all share approximately 30% amino acid identity with Ras proteins (Nobes and Hall, 1994). Rho, Rac and Cdc42Hs are capable of inducing the formation of actin stress fibers, membrane ruffles and filopodia, respectively (Ridley and Hall, 1992; Ridley et al, 1992; Kozma et al, 1995; Nobes and Hall, 1995). Domains of Ras superfamily proteins The Ras superfamily of proteins bind and hydrolyze GTP to GDP and it has been proposed that they act as molecular switches regulated by their nucleotide binding state (Bourne et al, 1990). The structures of the Ras and Rapl proteins have been determined by X-ray crystallography The structures, as determined by X-ray crystallography, of the two proteins are very similar (see Fig. 1 for an alignment of the structures of Ras and Rapl), suggesting a conserved mechanism for binding and hydrolysis of GTP (De Vos et al., 1988; Pai et al., 1990; Nassar et al., 1995). The amino acids required for binding and hydrolyzing GTP are located in 4 domains that are well conserved throughout the Ras superfamily and in additional flanking amino acids conserved within the Ras sub-family (Fig. 1). These domains have been identified by sequence comparison, analysis of mutated proteins and from the crystal structure of Ras and Ras related proteins (Pai et al, 1990; De Vos et al, 1988; Nassar et al, 1995). The a subunits of the heterotrimeric signal transducing G proteins and the bacterial elongation factor Tu also share limited amino acid homology in these regions reflecting a conservation of GTP binding domains across a broad range of proteins (Bourne et al, 1990). In the Ras sub-family these domains consist of residues 10-17; 53-62; 112-119 and 144-146, respectively (Bourne et al, 1991). (See Figure 1 for conservation of these domains between Ras and Rapl). The first domain (10-17) forms bonds with the a-and (3-phosphates of GTP or GDP; the second domain (53-62) forms a hydrogen bond with the y-phosphate of GTP; and the third domain (112-119) forms hydrogen bonds Res Rop M l E Y K L V V V G A G G V G K S A L T l O l l O N H F V O E M R E Y K L V V l G S G G V G K S A L T V O f V O G I F V [ K Y D P T I E O S Y Y O P T I E O S Y H K O V V I O G E T C L L 0 1 R K O V r v o C O O C M L E I i. x > i ).>. x >, y i. J i x n 56 L D T A G O E E Y S A M R D O Y M R T G C G F L C V r A I N N T K S F L D I H O Y B E Q I K R V K O S D D V P M V , 56 L D T A G T E O r T A M R O L Y M K N G O G F A L V Y S I T A O S T F N O L O D L R E O I L R V K O T C O V P M I P5 c4 J6 , , 3 L V G N K C O L - A A R T V E S R Q A Q D L A R S Y G - I P Y 1 E T S A K T ' R C G V f O A F Y T L V R c I R O H • 113 L V G N K C 0 L C D E R V V G K C Q G G" N L A R 0 w C N C A f L E S S A K S K I N V N L I F Y O L V R O 1 N R -B RAP1_DICDI MPLREFKIWLGSGGVGKSALTVQFVQGIFVEKYDPTIEDSYRKQVEVDSNQCML 55 RAPA_HUMAN M - - REYKLVVLGSGGVGKSALTVQFVQGIFVEKYDPTIEDSYRKQVEVDCQQCML 53 RASK HUMAN MT- -EYKLVWGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLL 53 — * * _ * _ * * _ * _ * * * * * * * * * _ * _ _ * _ * * _ * * * * * * * * * * * * * _ * ' _ * . * RAP1_DICDI EILDTAGTEQFTAMRDLYMKNGQGFVLVYSIISNSTFNELrPDLREQILRVKDCED 110 RAPA_HUMAN EILDTAGTEQFTAMRDLYMKNGQGFALVYSITAQSTFNDLQDLREQILRVKDTED 108 RASK HUMAN DILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKS FEDIHHYREQIKRVKDSED 108 ****** *__**** * * _ * * * * * * * * * **** _ * RAP1_DICDI VPMVLVGNKCDLHDQRVISTEQGEEIiARKFGDCYFLEASAKWKVNVEQIFYNLIR 165 RAPA_HUMAN VPMILVGNKCDLEDERVVGKEQGQNLARQWCNCAFLESS AKSKINVNEIFYDLVR 163 RASK HUMAN VPMVX.VGNKCDLPS-RTVDTKQAQDLARSYGI -PFIETSAKTRQRVEDAFYTLVR 161 * * * * * * * * * * * _ * . . . . * . * * . . * . * * * . . * . . * * .r**" . * RAP1_DICDI QIN--RKNPVGPPSKAK SKCALL 186 RAPA_HUMAN QIN—RKTPVEKKKPKK KSCLLL 184 RASK HUMAN EIRQYRMKKLNSSDDGTQGCMGLP-CVVM 189 Figure 1. Ahgnment and structure assignment for Rapl A and Ras A) This figure is modified from (Nassar et al, 1995). p sheets (open arrows)\ and a helixes (spiral bars) for H Ras (Ras) and human Rapl A (Rap). Residues found in all GTP-binding proteins are underlined; effector residues 32-40 are bracketed. B) Alignment of D. discoideum Rapl (RAP1 DICDI) with human Rapl A (RAPA HUMAN) and K-Ras (RASK HUMAN) with both the guanine ring and the first domain. The fourth domain (144-146) is somewhat variable and indirectly interacts with the guanine nucleotide by stabilizing the third domain. A n additional domain, the core effector domain (extending from amino acids 32-40), is conserved in the Ras sub-family but is not conserved between members of the Ras superfamily with the exception of threonine 35 which is required to coordinate a Mg2+ ion. Mutations which disrupt this domain abolish the transforming ability of Ras (McCormick, 1994; Marshall, 1993). It has been proposed that this domain interacts with downstream effector molecules such as Raf (Avruch et al, 1994). Comparison of the crystal structure of Ras-GDP with that of Ras-GTP has revealed differences in the effector region amino acids 32-40 and as well as in another external loop region from amino acids 60-76. It has been hypothesized that changes in the structure of these two loops are involved in the transduction of a signal to the effector protein (Brunger et al, 1990; Milburn et al, 1990). Although the effector domain is conserved in all the Ras sub-family proteins, they do not function equivalently (Bourne et al, 1991). Ral cannot transform cells (Feig and Emkey, 1993); TC21 can transform cells in a manner equivalent to Ras; R-Ras can transform cells but does not cause a characteristic transformed cell morphology while Rapl can antagonize transformation by Ras (Graham et al, 1994; Bokoch, 1993; Cox et al, 1994; Noda, 1993). These results suggest that additional effector specificity lies outside this region. Mutagenesis studies have revealed additional requirements within the extended effector domain of Ras, which encompasses amino acids 25-46 (Nur-E-Kamal et al., 1992). Rapl and R-Ras diverge from Ras within this region, which possibly explains their differing effects in transforming cells. In the Rho and Rab sub-families there are amino acid substitutions within the core effector domain and there is evidence that these proteins interact with quite different effector proteins (reviewed in Nobes and Ha l l , 1994; Chant and Stowers, 1995) A n additional small domain is present at the carboxyl terminal of all Ras-related proteins. This domain is subject to post translational processing i n Ras, resulting i n removal of the terminal 3 amino acids, followed by addit ion of a farnesyl isoprenyl group and carboxymethylation of the processed terminal cysteine (Hancock et al., 1989; Clarke, 1992). This domain with its subsequent modification is required for the attachment of Ras to the inner leaflet of the plasma membrane and disruption of this modification blocks the ability of Ras to transform cells (Guierrez et al., 1989; Williamsen et al., 1984). Additional features in each Ras protein also contribute to specify its intracellular location, for example, a sequence rich in lysine near the carboxyl terminus i n K-Ras and a palmitoylation modification near the C terminus in H-Ras and N-Ras are also required for localization to the inner leaflet of the plasma membrane (Clarke, 1992). The post translational processing of R a p l proteins is similar to of Ras, but a geranylgeranyl moiety is added rather than a farnesyl group (Buss et ai, 1991). However, the location of Rap l in the cell, presumably specified in part by this modification, has not been conclusively established. In platelets, R a p l has been reported to be localized i n the cytoplasmic fraction and to be translocated to a cytoskeletal fraction upon thrombin activation of platelets, suggesting that its location i n the cell is subject to addit ional regulation (Fischer et al., 1990). In contrast, i n neutrophils, Rapl A has been reported to bind to cytochrome b558 of N A D P H oxidase and this binding is inhibited by Rapl A phosphorylation (Bokoch et ah, 1991). Two conflicting reports have localized Rap l to the Golgi and the late endocytic compartments in fibroblasts (Beranger et al, 1991; Pizon et al, 1994) . In addition, chimeric Ras proteins containing Rapl amino acids from position 66 or position 111 to the carboxyl terminal are still capable of transforming cells (Buss et al., 1991; Zhang et al., 1991), implying that some Rapl protein might also be located at the inner leaflet of the plasma membrane. Regulators and effectors of the Ras superfamily The binding and hydrolysis of GTP by Ras is regulated by other protein components, which in some cases also serve to transmit information. The switch from the GDP bound state to the GTP bound state is mediated by guanine nucleotide exchange factors which stimulate the dissociation of GDP from Ras (reviewed in Boguski and McCormick, 1993). The exchange factors function immediately upstream of Ras and are linked to receptors via adapter proteins such as GRB2 and She (reviewed in Schlessinger, 1994; Pawson, 1995) . Several Ras exchange factors have been identified, possibly linking Ras to a different receptors (Boguski and McCormick, 1993). Elucidation of the effector molecule downstream of Ras had been an elusive goal, but recently three effectors which interact directly with Ras have been identified in mammalian cells: Raf, phosphatidylinositol-3-OH kinase (Pl-kinase) and mitogen-activated protein kinase kinase kinase (MEKK1) (Warne et al; 1993; Moodie et al, 1993; Koide et al, 1993; Zhang et al., 1993; Viciana et al, 1994; Russell et al, 1995). Raf and MEKK1 transduce a signal by activating the MAP kinase cascade while Pl-kinase phosphorylates phosphoinositides at the 3' position (reviewed in Blumer and Johnson, 1994; Cantley etal, 1991). Ras signaling terminates when GTP is hydrolyzed to GDP. The proteins GAP and NFI enhance the rate of GTP hydrolysis (Trahey and McCormick, 1987; Martin et al, 1990). It is possible that GAP, in addition to downregulating Ras, may also function as an effector molecule. GAP is required for Ras dependent inhibition of atrial muscarinic K+ channels and GAP is capable of altering cytoskeletal structure and cell adhesion in a manner independent of Ras (Yatani et al., 1990; Martin er: al, 1992; McGlade et al, 1993). However the mechanism whereby GAP causes these effects is not clear. Numerous exchange factors and GAPs which regulate the guanine nucleotide binding and hydrolysis of other Ras superfamily proteins have been identified (Boguski and McCormick, 1993), suggesting that the regulation of these proteins is similar to that for Ras. Biochemical analysis has revealed an additional level of complexity as some regulatory proteins appear to be able to interact with more than one Ras superfamily member. For example, the exchange factor, smgGDS, interacts functionally in vitro with Ras, RhoA and Rapl (Mizuno et al, 1991) while RalGDS interacts with both Ral and Ras in vitro (Hofer etal, 1994). Furthermore, R-Ras interacts with Ras-GAP, NFI and also Raf (Spaargaren et al, 1994; Rey et al, 1994). Although, the significance of these interactions remains to be demonstrated in vivo, the potential clearly exists for complex interacting networks of Ras related proteins. Biological roles of Ras Activated ras genes were first identified in viral or tumor cell D N A based on their ability to transform NIH3T3 cells (reviewed by Barbacid, 1987; Der, 1989; Lowy and Willumsen, 1993). Mutations at codons 12,13 and 61 were identified in the three ras genes, H-ras, Ki-ras and N-ras , in many human tumors (reviewed by Bos, 1989). Mutated ras genes were identified in 80% of pancreatic tumors, 50% of colon carcinomas, 30% of non-small cell lung carcinoma and 20% of melanomas. Different tumor types were usually found to contain mutations in specific ras genes. For example, pancreatic and non-small cell lung tumors had mutations in Ki-ras, while melanomas contained mutations in N-ras. Microinjection of activated Ras proteins in NIH 3T3 cells caused cell proliferation and resulted in a transformed cell appearance (Feramisco et al, 1984; Stacey and Kung, 1984; Mulcahy et ah, 1985). Taken together, these observations strongly implicated mutated ras genes in the process of human neoplasia. This hypothesis was confirmed experimentally by the observation that transgenic mice expressing an activated N-ras gene from the MMTV promoter elicited tumors in the mammary and salivary glands (Mangues et al, 1990; Mangues et al, 1992). In vitro, activated Ras protein transforms cells and bypasses the requirements for serum and anchorage dependent growth (Hall et al, 1983; Spandidos and Wilkie, 1984; Paterson et al, 1987) while microinjection of Ras-GTP stimulates DNA synthesis in fibroblast cells (Stacey and Kung, 1984; Feramisco et al, 1984). These studies suggest a role for Ras in cell proliferation. Conversely, microinjection of Ras specific antibody blocks the mitogenic activity of serum and growth factors (Mulcahy et al., 1985). Furthermore, the proportion of Ras-GTP increases after treatment of cells with serum or growth factors such as PDGF or EGF, suggesting that Ras mediates the response to these factors, and is consistent with the idea that binding of GTP results in an active form of the protein (Satoh et al, 1990b; SaXoh etal., 1990a). A role for Ras proteins in differentiation and development has also been well documented. PC12 pheochromocytoma cells develop into neural type cells upon treatments with NGF in a process which is dependent on Ras (Bar-Sagi and Feramisco, 1985; Satoh et al, 1987). In addition, transfection of Ras can induce differentiation of 3T3-L1 fibroblasts into adipocytes (Benito et al, 1991). A role for Ras in development has also been identified by genetic analysis in C. elegans and D. melanogaster (Beitel et al., 1990; Simon et al., 1991; Lu etal., 1993). In C. elegans, activation of Ras produces a multivulval phenotype while loss of Ras activity leads to a vulvaless phenotype, again consistent with a switching function for Ras in a signaling pathway. In D. melanogaster, activation of Ras disrupts normal cell fate specification in the compound eye and when microinjected into embryos, disrupts the terminal cell fates of posterior cells (Simon et al, 1991; Lu et al, 1993). Although Ras proteins have been implicated in both proliferation and diverse developmental processes, it appears that in many cases that Ras transduces a signal via a similar pathway that is conserved through evolution. The Ras effector, Raf, is required for the stimulation of proliferation by Ras and also for the developmental events mediated by Ras in C. elegans and D. melanogaster (Moodie and Wolfman, 1994). Furthermore, the subsequent activation of a MAP kinase cascade by Raf was also found to be an evolutionarily conserved feature. Rapl proteins The rapl genes (Pizon et al, 1988a) (also called Krev-1 (Kitayama et al, 1989) and smg p21 (Kawata et al, 1988)) encode low molecular weight GTPase proteins and have been identified in organisms as diverse as S. cerevisiae and mammals (Schejter and Shilo, 1985; Pizon et al, 1988a; Pizon et al, 1988b; Kawata et al, 1988; Bender and Pringle, 1989; Robbins et al, 1990; Hong et al, 1990; Hariharan et al, 1991). A defining characteristic of Rapl proteins is a conserved threonine at position 61 which is a conserved glutamine in Ras genes (Bourne et al, 1991; Noda, 1993). In humans there are two rapl genes, rapl A and raplB, which encode proteins that are 95% identical to each other (Pizon et al, 1988a; Pizon et al, 1988b). In addition, a related pair of genes encode the Rap2A and Rap2B proteins, which are approximately 60% identical to Rapl A and RaplB (Pizon et al, 1988a; Farrel et al, 1990; Ohmstede et al, 1990). The Rap2A protein cannot antagonize Ras-induced transformation (Jimenez et al, 1991). A raplA cDNA, Krev-l, was isolated by its ability to suppress the transformed phenotype of Ki-ras transformed NIH 3T3 cells (Kitayama et al, 1989). Flattened, more adherent cells with reduced tumorigenicity were isolated following transfection with the rapl A cDNA. Rapl proteins antagonize Ras function in a variety of in vitro assays. They inhibit Ras-dependent activation of MAP kinases, ERK1 and ERK2 (Cook et al, 1993); N-Ras induced inhibition of M2-muscarinic receptor coupled K + channels in heart (Yatani et al, 1991) and H-Ras induced activation of germinal vesicle breakdown in Xenopus laevis oocytes (Yatani et al, 1991; Campa et al, 1991). In view of the conservation of core effector domain between Rapl and Ras proteins (Pizon et al, 1988a; Kitayama et al, 1989) (Fig. 1), it has been proposed that Rapl directly competes with Ras for a downstream effector proteins such as Raf or possibly Ras-GAP, both of which have been shown to interact directly with Rapl (Freeh et al, 1990; Zhang et al, 1993). The Rapl proteins do not appear to act solely as antagonists of Ras since they appear to have different roles in various cell types, and they may signal through as yet unknown effector molecules (Kitayama et al, 1989; Yoshida et al., 1992). In platelets, RaplB is activated by agents that elevate intracellular cAMP (Altschuler et al., 1995). Rapl has also been implicated in the oxidative burst in B lymphocytes (Maly et ah, 1994). In S. cerevisiae, the rapl gene homologue RSR1/BUD1 is required for bud site localization, a process proposed to involve the orientation and localization of the actin cytoskeleton in the cell (Bender and Pringle, 1989; Chant and Herskowitz, 1991). Finally, Rapl can elicit some effects that are similar to those of Ras. In Swiss 3T3 cells, microinjection of RaplB in the presence of insulin partially mimics the effect of Ras, causing DNA synthesis and membrane ruffling (Yoshida et al, 1992) and in S. pombe, both the human RaplA and RaplB cDNAs were isolated as genes capable of suppressing the morphological and sporulation defects in a rasl mutant strain (Xu et al., 1990). Mutational analysis of Rapl proteins Mutagenesis and domain exchanges have been used to compare the Rapl and Ras proteins (Zhang et al, 1990; Kitayama et al, 1990; Zhang et al, 1991). Using cell transformation as a criteria for Ras activity and transformation suppression as a criteria for Rapl activity, domain exchange experiments located the amino acids which specified Ras or Rapl activity to a limited region flanking the core effector domain (Zhang et al, 1990). Further analysis showed that simply substituting Rapl amino acids 26, 27, 30, 31 and 45 with the corresponding residues from Ras was sufficient to generate a protein with Ras-like properties in both mammalian and yeast biological assays (Marshall et al., 1991). These results suggests that effector specificity is encoded in a small number of amino acids in extended effector region flanking the core effector. Mutational analysis of Rapl amino acids that are conserved between Ras and Rapl suggested a similar relationship between structure and function for the two proteins. Mutations that activate Ras cause enhanced tumor suppression by Rapl while mutations in the core effector region that block transformation by Ras cause attenuated tumor suppression by Rapl (Kitayama et ah, 1990). The requirement for GTP binding for Rapl activity was also evaluated by microinjecting RaplB into Swiss 3T3 cells in the presence of insulin (Yoshida et al, 1992). When bound to the non hydrolysable GTP analog GTPyS, RaplB induced DNA synthesis and membrane ruffling, whereas GDP-RaplB did not. By contrast, analysis of RaplA proteins in Epstein-Barr virus transformed human B lymphocytes found that both activating and dominant negative mutant proteins but not wild type Rap blocked the phorbol ester stimulated oxidative burst (Maly et al., 1994). This result may mean that in some cases the cycling of Rapl from a GTP to GDP bound form is required for activity. Regulation of the Rapl proteins Like Ras, Rapl proteins are regulated by exchange factors and GAPs. A Rapl specific GAP (Rapl-GAP) that shows no sequence homology with Ras-GAP has been isolated and cloned from bovine brain (Rubinfeld et al., 1991). Rapl-Gap is phosphorylated in vivo and can be phosphorylated in vitro by cAMP dependent protein kinase and also by p34 cdc2 (Polakis et al., 1992). The effect of phosphorylation on Rapl-GAP is not known. An exchange factor, smgGDS, which stimulates the rate of Rapl GDP/GTP exchange has been cloned (Kaibuchi et al., 1991). As mentioned previously, smgGDS is also active on other Ras related proteins (Kaibuchi et ah, 1991). To date, no specific downstream effectors of the Rapl proteins have been identified, although RaplA has been shown to enhance protein kinase C activity in an in vitro assay (Labadia et al, 1993). Rapl also interacts with the Ras regulator Ras-GAP but Ras-GAP and NFI do not stimulate the GTPase activity of Rapl (Freeh et al, 1990; Zhang et al., 1991). In addition, Rapl interacts with the Ras effector, Raf, in a yeast two hybrid assay (Zhang et al, 1993). Whether such non-functional interactions are responsible for the antagonism of Ras transformation is not yet clear and it remains to be determined whether Rapl can interact with other recently identified Ras effectors or the Ras exchange factor. It is also plausible that Ras and Rapl act via the same effectors in cases where Rapl and Ras have similar effects. For example, human Rapl is capable of activating the Ras effector adenylate cyclase in S. cerevisiae (Ruggieri et al., 1992). Rapl is a substrate for protein kinase A and protein kinase G in vitro (Kawata et al, 1989; Miura et al, 1992). Phosphorylation of RaplB occurs in platelets upon treatment with agents such as prostaglandins which increase intracellular cAMP, suggesting that protein kinase A may phosphorylate Rapl in vivo (Kawata et al, 1989; Siess et al, 1990). The smg GDS induced activation of RaplB is enhanced upon phosphorylation of RaplB in vitro (Itoh et al, 1991), suggesting a mechanism whereby phosphorylation may regulate Rapl. Summary Given the diverse effects of Rapl proteins and their sometimes antagonistic, sometimes complementary relationship with Ras and the failure thus far to detect a definitive Rapl specific effector, it remains to be determined if Rapl proteins function in the Ras signal transduction pathway by using or sequestering some of the components of that pathway or if Rap acts in an independent pathway. It is unclear whether the diverse effects of Rapl proteins reflects a diversity of molecular interactions, or whether a single role for Rapl underlies these numerous effects. A c t i o n of Ras superfamily proteins on cell morphology The cytoskeleton of a cell is required for the structural integrity and shape of the cell and consists of a dense network made up of numerous components. These cytoskeletal elements form a matrix with a measurably rigid structure capable of resisting mechanical stress in a manner analogous to the rigid strength of geodesic domes designed by R. Buckminister Fuller (Wang et al, 1993; Heidemann, 1993). This matrix must also by necessity be capable of rapid disassembly and reassembly as a cell undergoes processes such as cellular division, developmentally regulated changes in shape, cell movement, and chemotaxis. Dramatic cytoskeletal changes also accompany the process of cellular transformation and occur during the treatment of cells with growth factors. Electron microscopy analysis has revealed that the cytoskeleton consists of 3 primary structures: microtubules, intermediate filaments and microfilaments. Biochemical analysis has shown that microtubules consist predominantly of tubulin; intermediate filaments are made up of a mixture of proteins; and microfilaments are made up of actin. I will restrict my focus to the actin cytoskeleton because it is subject to modification by Ras-related proteins. The actin cytoskeleton consists of monomeric actin subunits assembled into fibers which are in turn linked to create a matrix by actin crosslinking proteins (reviewed in Matsudaira, 1991). Many other actin associated proteins regulate the degree of actin polymerization: by preventing the addition of further subunits to the fiber; by sequestering free actin subunits so as to prevent their assembly; and by severing actin fibers (Luna and Condeelis, 1990; Button et al, 1995). Actin filaments are present in numerous distinct forms in different subcellular structures in mammalian cells (reviewed in Matsudaira, 1991). Stress fibers consist of long bundles of actin filaments that traverse the cell and are linked to the extracellular matrix via integrins and focal adhesion complexes. Focal adhesions are involved in cell-substratum adhesion and consist of protein complexes linked to stress fibers. Lamellipodia are broad but thinly spread actin containing regions observed at the periphery of adherent cells. Membrane ruffles are similar to lamellipodia and are made up of actin containing wavy curtain-like structures on the cell surface and cell periphery. Filopodia are narrow peripheral structures containing 'microspikes' of polymerized actin and are proposed to have a sensory function. As well as regulation by the assembly, disassembly and crosshnking of actin, the actin cytoskeleton is acted upon by force-generating myosin molecules. The conventional force-generating myosin, myosin II, forms filaments which bind to actin fibers and generate contractile forces (reviewed in Spudich, 1994; Ruppel and Spudich, 1995). Myosin molecules are involved in processes including muscle-driven movement, maintaining cell morphology and cell motility. In D. discoideum, myosin II is required for a normal cell morphology (Knecht and Loomis, 1987; De Lozanne and Spudich, 1987). Many unconventional or nonfilamentous myosins have also been isolated (classes I, and III-VIII) (Titus et al, 1994; Bement et al, 1994). The roles of such unconventional myosins have not been determined to date. It has been estimated that a typical mammalian cell has at least 11 myosins consisting of 1 or 2 members from each myosin class and it has been speculated that each myosin family member may mediate a different actin-based process (Bement et al, 1994). The effects of the Ras superfamily on the cytoskeleton Structures such as membrane ruffles, stress fibers and focal adhesions are produced in response to growth factors and appear to involve members of both the Ras and Rho sub-families. Microinjection of activated H-Ras into fibroblasts results in a refractile transformed morphology and increased membrane ruffling and blebbing (Feramisco et al., 1984; Bar-Sagi and Feramisco, 1986). Microinjection of activated K-Ras into Swiss 3T3 cells also causes an increase in membrane ruffling and a decrease in stress fiber formation (Yoshida et al., 1992). Interestingly, microinjection of RaplB bound to GTPyS also caused membrane ruffling in Swiss 3T3 cells but did not have a significant effect on stress fiber formation. The relationship between Rapl and Ras in causing these partially similar phenotypes is not known. Microinjection studies have also identified roles for a number of Rho sub-family members in regulation of the cytoskeleton. Microinjection of an activated RhoA protein into serum starved Swiss 3T3 cells causes rapid stress fiber and focal adhesion formation (Ridley and Hall, 1992). Addition of either serum or lysophosphatidic acid caused a similar effect, suggesting that RhoA may mediate some of the effects of growth factors on the cytoskeleton. Specifically blocking RhoA function in vivo by microinjection of a dominant negative form of RhoA, generated by ribosylation with the exoenzyme C3 transferase from Clostridium botulinum, or C3 transferase alone blocked the effects of serum or lysophosphatidic acid, suggesting that RhoA is essential for the assembly of focal adhesions and stress fibers induced by growth factors (Ridley and Hall, 1992). Microinjection of an activated Racl into Swiss 3T3 cells stimulates actin filament accumulation at the plasma membrane, forming lamellipodia and membrane ruffles (Ridley et al, 1992). Induction of membrane ruffling by growth factors or an activated H-ras protein could be blocked by a dominant inhibitory mutant Racl protein, suggesting that endogenous Racl is required for growth factor induced membrane ruffling. In addition, a delayed response to Racl is the formation of actin stress fibers. The results of blocking Rho activity with C3 transferase suggested that growth factors act through Racl to stimulate a Rho dependent response (stress fiber formation) as well as a Rho independent response (ruffling). Analysis of the human Cdc42 protein revealed that it too can regulate actin structures. Microinjection of human Cdc42 protein into Swiss 3T3 cells promoted the formation of peripheral actin microspikes and filopodia (Kozma et al, 1995; Nobes and Hall, 1995). Treatment of cells with the growth factor bradykinin had a similar effect which was blocked by microinjection of a dominant negative form of Cdc42. This result suggested that the endogenous Cdc42 protein is required for the formation of these peripheral actin structures. Analysis of the cells microinjected with Cdc42 also caused the formation of ruffles as well as stress fiber formation suggesting that perhaps Rac and RhoA were also being activated. Inactivation of the endogenous Rac and Rho proteins with a dominant negative Racl protein and C3 toxin blocked the formation of these structures, suggesting that Cdc42 acts in part via Rac and Rho. A model summarizing the relationship between the three Ras-related proteins is shown in Figure 2 (Chant and Stowers, 1995). The model is based on the two lines of evidence discussed above. Firstly, the pathway appears to be branched because upstream components such as Cdc42 cause multiple effects while downstream components such as Rho cause a single effect. Secondly, the proteins appear to function in a linked manner rather than independently, because dominant negative forms of the presumptive downstream proteins such as Rho block some but not all of the effects of upstream proteins such as Cdc42. An additional level of complexity to the model is that Rac and Rho can also be activated independently by growth factors, but this can be explained by the branched nature of the pathway. A precedent for such a complex model linking multiple Ras-related proteins comes from the genetic analysis of bud site selection in S. cerevisiae (Fig. 2) (reviewed in Chant and Stowers, 1995). In S. cei'evisiae, the site of the daughter bud is not randomly selected but depends on the GTPase cycle of the Rapl homologue, RSR1/BUD1, which is linked in turn to the GTPase cycle of Cdc42. A series of Rho proteins are then proposed to function downstream of Cdc42 and perhaps act on the cytoskeleton itself. The protein Cdc24 potentially links RSR1/BUD1 and Cdc42, because it binds to RSR1/BUD1 and also functions as an exchange factor for Cdc42. Figure 2. GTP cascades controlling cell morphology and bud formation This figure is modified from (Chant and Stowers, 1995). (A) The pathway that controls cytoskeletal polarization during vegetative division or mating. (B) The proposed pathway that controls the formation of filopodia (microspikes), lamellipodia (ruffles) and stress fibers with focal adhesions. Continuous arrows indicate physical contact. Broken arrows indicate indirect interactions. ^ Intrinsic spatial signals for budding Mating pheromone t BUD5 ( BUD1 BUD2 V CDC24 GEF CCDC42) RH01 RH02 RH03 RH04 B PDGF or i n s u l i n LPA RAS (CDC42) Rlopodia XT C RAC Lamellipodia C RHCy Cytoskeletal polarization Stress fibers Adhesion Dictuostelium discoideum Life cycle D . discoideum is a simple eukaryote which serves as an experimentally tractable model system for addressing many biological questions. D . discoideum cells exist as solitary amebae that feed on bacteria but, when starved, aggregate and form a developmental structure (reviewed in Gross, 1994; Kay, 1994; Firtel, 1995) (Fig. 3). The cells cease growing when deprived of nutrients and signal to each other via pulses of cAMP. Cells respond by moving towards the stimulus and by producing cAMP themselves. This chemotaxis and signal relay results in the formation of an aggregate of cells. The aggregate becomes compact and tipped and the tip eventually elongates to form a finger-like structure. This structure falls over and continues to elongate producing a pseudoplasmodium or slug. The slug is capable of migrating towards conditions of optimal temperature and light. The slug tip consists of prestalk cells which make up approximately 20% of the cell total and the rear 80% of the slug consists of prespore cells. The ratio of prestalk to prespore cells is fixed. If the front or back of the slug is removed experimentally, the remaining cells will again assume the proportions of 20% prestalk 80% prespore cells. The maturation of the structure occurs as the prestalk cells at the tip of the structure migrate down through the center of the prespore cell mass in a motion which resembles that of an inverse fountain. The consequences of this prestalk cell movement is the formation of a stalk which raises the prespore mass away from the substratum. During this process the prespore cells develop into mature spores. Figure 3. The life cycle of D. discoideum Black areas represent prestalk and stalk cells while clear areas represent prespore and spore cells. The time in development is shown below in hours. Signal transduction events during the lifecvcle of D. discoideum Vegetative growth, starvation and aggregation Vegetative D. discoideum cells prey on bacteria by detecting and chemotaxing toward folate and pterin, two bacterial metabolites (Pan et al, 1972; Pan et al, 1975). The mechanism that D. discoideum cells use to respond to folate and pterin is not well understood, although it is known to require the G(3 and Ga4 members of the heterotrimeric G protein family (Wu et al, 1995; Hadwiger et al, 1994; Burdine and Clarke, 1995). Folate binding sites have also been detected on the surface of D. discoideum cells (De Wit, 1982; De Wit etal, 1985). To determine when to stop growing and initiate collective development, cells evaluate both their nutritional status and also their density. This latter process is achieved by secreting and responding to a prestarvation factor (PSF) (Clarke et al, 1987; Clarke et al, 1988). Another factor, conditioned medium factor (CMF) provides a similar density sensing function which regulates signal transduction after starvation has initiated (Yuen et al., 1995). After cells starve and enter into development, the reintroduction of nutrients will cause the cells to dedifferentiate although such cells retain the capacity to rapidly reenter the developmental program if nutrients are removed for a second time (Waddell and Soil, 1977; Soli and Waddell, 1975; Kraft et al, 1989). This 'memory' exists for approximately 90 minutes and is then lost by a process that has been termed erasure. Presumably, such flexibility reflects selection pressures of living in an environment where the food supply is variable and intermittent. The cAMP signal relay response mediating the process of aggregation and early gene expression has been extensively analyzed (reviewed in Gross, 1994; Firtel, 1995). The signal relay machinery consists of a serpentine cAMP receptor, cARl, linked to a heterotrimeric G protein made up of Ga2, Gp and presumably a hitherto unidentified Gy subunit. Signal transduction also appears to involve the protein kinase ERK2. Starving cells emit and respond to pulses of cAMP. The binding of cAMP to receptors activates phospholipase C, guanylate cyclase and Ca2+ influx. Protein kinase A, presumably activated by increased intracellular cAMP, has also been shown to be required for aggregation. Cells chemotax toward cAMP and in addition respond by activating adenylate cyclase, which produces further cAMP allowing a 'relay' of the signal. Cells also secrete a cAMP phosphodiesterase which degrades extracellular cAMP, preventing its over-accumulation. Cells also respond to cAMP signals by expressing early developmental genes. Cell differentiation The differentiation of cells into prestalk and prespore cells within the multicellular aggregate is regulated by a number of factors including cAMP (reviewed in Gross, 1994; Firtel, 1995). Levels of cAMP increase during development and the presence of continuously high levels of cAMP represses the expression of genes involved in early development and stimulates expression of genes involved in later processes. cAMP initially promotes the development of both prestalk and prespore cells but at later stages acts to promote spore development and inhibit stalk development. Four developmentally regulated cAMP receptors and 8 heterotrimeric Ga protein subunits have been identified, suggesting the potential for several distinct cAMP pathways. Protein kinase A 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 cAMP (Simon et al, 1989; Firtel and Chapman, 1990; Harwood et al, 1992a; Harwood et al, 1992b; Simon et al, 1992; Anjard et al, 1992; Mann et al, 1994; Hopper et al, 1995). Protein kinase A also appears to inhibit prestalk development and to play a role in stalk formation (Mann and Firtel, 1993; Harwood et al, 1992b). An additional factor, stalk cell differentiation inducing factor (DIF) promotes prestalk cell differentiation and suppresses prespore cell differentiation (Kopachik et al., 1983; Williams et al., 1987). Finally, ammonia, which accumulates as cells catabolize protein and RNA by endogenous respiration during development, serves as a negative regulator for the process of culmination - the formation of a mature fruiting body from a slug (reviewed in Gross, 1994). Although the factors which promote the development prestalk and prespore cells have been identified, the question of how the proportions of the two cell populations is determined remains unanswered. Prestalk and prespore cells have been observed to start differentiating in an interspersed manner within the early aggregate, suggesting an initially cell-autonomous process rather than a response to an external gradient (Williams et al, 1989). Subsequently there is a migration of prestalk cells to the tip of the aggregate and this is believed to be in response to cAMP (Traynor et al, 1992). Within the aggregate and slug, exposure to morphogens promotes the continued development of prestalk and prespore cells. At least three prestalk cell types, as defined by differential patterns of gene expression, have been identified (Williams et al., 1989; Jermyn et al., 1989). There are also interactions between the prestalk and prespore cells which result in the regulation of the final proportion of the two cell populations (Raper, 1940; Shaulsky and Loomis, 1993). Factors such as position within the cell cycle during vegetative growth prior to the initiation of development also influence cell fate (Maeda et al., 1989; Gomer and Firtel, 1987). A number of models have been proposed to explain the proportioning of the stalk and spore populations, utilizing some or all of the morphogens described above and invoking gradients of the morphogens (reviewed in Gross, 1994). However, to date, evidence for the spatial distribution of the above morphogens is not convincing (Weeks and Gross, 1991). Ras and ras related genes i n D. discoideum D. discoideum expresses five ras genes (Reymond et al., 1984; Robbins et ah, 1989; Daniel et al., 1993b; Daniel et al, 1993a). rasG encodes a protein which shares 69% overall amino acid identity with the human H-ras gene product, and the gene is expressed in vegetative and early developing cells with expression declining markedly during aggregation (Robbins et al., 1989). The RasB and RasC gene products share 59% and 56% amino acid identity, respectively, with the human H-ras gene product (Daniel et al., 1993a; Daniel et al, 1993b). rasB is maximally expressed during vegetative growth and early development but expression remains relatively high during the remainder of development (Daniel et al, 1993b). rasC is expressed maximally during aggregation but significant expression is detected during vegetative growth and the remainder of development (Daniel et al, 1993a). Two additional Ras genes, rasS and rasD, whose gene products share 54% and 65% amino acid identity, respectively, with H-ras, are expressed in a more restricted manner (Daniel et al, 1993a; Reymond et al, 1984). RasS is expressed only during aggregation (4-8 hours) while rasD is highly expressed only during late aggregation and slug formation (12-16 hours). rasD gene expression is induced by cAMP and is expressed in at least 50% of cells during aggregation but is subsequently restricted to the prestalk cells by an unknown mechanism (Jermyn and Williams, 1995). The existence of 5 ras genes in D. discoideum each with a specific pattern of expression, raises the question of whether they each have distinct roles and this question remains to be answered. The presence of amino acid differences within the core effector domain of RasC and RasS may mean that they interact with different effectors than do the other three Ras gene products (Daniel et al, 1993a). The Ras proteins have not been definitively linked to any specific growth or developmental signaling pathway in D. discoideum. Expression of an activated RasG protein in vegetative cells under the control of the discoidin promoter blocks aggregation and northern blot analysis showed only low levels of induction of the cAMP receptor, cARl, which is normally expressed early after only 2-4 hours development, suggesting a role in early development (M. Khosla, personal communication). Expression of an activated rasD gene under the control of the rasD promoter resulted in cells which were arrested as multitipped aggregates (Reymond etal, 1986). Northern blot analysis showed a dramatic increase in prestalk specific ecmA mRNA levels and a decrease in prespore specific sp60 mRNA levels (S. Louis, personal communication), indicating that expression of activated rasD disrupts pattern formation. Eight rho and 5 rab genes have been isolated from D. discoideum (Bush et al, 1993a; Bush et al, 1993b; Vithalani et al, 1995). The rho related genes each exhibit unique patterns of mRNA expression during growth and development, again consistent with the idea that they may have different roles during the D. discoideum life cycle. In contrast, four of the five rab genes have similar expression profiles. Disruption of one rho gene interfered with cytokinesis (Vithalani et al, 1995) but the roles of the remaining rho and rab genes remain to be established. Only one rap gene, rapl, has been identified in D. discoideum (Robbins etal., 1990) despite extensive searching for additional rapl genes (Daniel, 1993). The Rapl gene product shares 76% amino acid identity with the human Rapl A protein and approximately 50% amino acid identity with the H-ras gene product (Robbins et al., 1990; Daniel, 1993). A complex pattern of rapl mRNA expression is observed (Robbins et al., 1990). A single 1.1 kb mRNA is present during vegetative growth and early development. As development continues, this message is replaced by two mRNAs of 1.0 and 1.3 kb. mRNA levels are maximal during aggregation, then decline during slug formation, but increase again slightly at culmination. Separation of prestalk and prespore cells by a percoll gradient followed by mRNA analysis did not show any enrichment in either prestalk or prespore cells (Robbins, 1991). However, in contrast to the complex developmental pattern of mRNA expression, western blot analysis showed that protein levels remained constant throughout growth and development (M. Khosla, personal communication). Cell morphology of D. discoideum D. discoideum has become an important experimental model for analysis of the function of actin associated proteins and myosin motors, using techniques for gene targeting and gene replacement (Patterson etal, 1991). The actin-associated proteins a-actinin, ABP-120, coronin, synexin, ponticulin and profilin have each been eliminated by gene disruption techniques (Witke et al, 1987; Cox et al, 1992; de Hostos et al, 1993; Hitt et al, 1994; Doring et al, 1991). Similarly, several myosin I genes have been disrupted (Jung et al, 1993; Jung and Hammer III, 1990; Peterson et ai, 1995). Many of these gene disruption mutants strains appear morphologically normal and exhibit only subtle changes in their motility. However, disruption of the myosin II light or heavy chain genes results in cells which are enlarged, flattened and multinucleate (Knecht and Loomis, 1987; De Lozanne and Spudich, 1987). Such cells are incapable of division in suspension but continue to grow until they lyse. These cells are no longer able to cap surface proteins, have reduced cortical tension and no longer respond to azide treatment by contracting and detaching from the substratum (Pasternak et al, 1989). Surprisingly, such cells are still motile and can stream, aggregate and develop to the mound stage, although development arrests at this stage (Knecht and Loomis, 1987; De Lozanne and Spudich, 1987; Springer et al, 1994). This range of defects suggests that myosin II is involved in cytokinesis, receptor capping, control of cell morphology and morphogenesis. Some of the components involved in regulating myosin II in D. discoideum have been identified. Myosin II is structurally and functionally similar to non muscle myosin II in other organisms (reviewed in Tan et al., 1992; Spudich, 1994). Myosin II consists of a hexamer of subunits (2 heavy chains, 2 essential light chains and 2 regulatory light chains) forming a structure with two globular heads and a helical coiled-coil tail. The two globular heads consist of the amino terminals of two heavy chains and the myosin essential light and regulatory light chains which the tail consists of the carboxyl terminals of heavy chains. The hexamers assemble into filaments which are capable of contracting, and which are analogous to those in muscle. Both myosin heavy chain and regulatory light chains are subject to phosphorylation (reviewed in Tan et al., 1992; Hammer III, 1994). Myosin heavy chain phosphorylation occurs on multiple residues in the tail and this prevents assembly of filaments capable of exerting force. Amino acid substitutions which mimic the phosphorylated state of the myosin II heavy chain tail prevent the assembly of myosin onto the actin cytoskeleton, while disruption of three sites of phosphorylation results in the overassembly of myosin II on the actin cytoskeleton in vivo (Egelhoff et al, 1993). To date, three myosin heavy chain kinases have been identified and one of these is a member of the protein kinase C family (Maruta et al., 1983; Cote and Bukiejko, 1987; Ravid and Spudich, 1989; Ravid and Spudich, 1992). Myosin light chain is phosphorylated by myosin light chain kinase which causes an increase in actin activated ATPase activity of myosin II in vitro (Griffith et al., 1987). Phosphorylation of myosin light chain does not regulate the assembly/disassembly of myosin II, a situation different from that in higher eukaryotes (Tan et al, 1992). As D. discoideum amebae start to chemotax toward cAMP, they become much more elongated, and while streaming together make head to tail contacts. Observation of single cells responding to a pulse of cAMP has revealed that cells initially cringe before extending a pseudopod in the direction of the cAMP stimulus (Futrelle et al., 1982). There is a rapid accumulation of F-actin into the cytoskeleton (peaking 5 s after the cAMP stimulus) and phosphorylation of the myosin II heavy chain accompanied by an association of myosin II with the cytoskeleton, occurs 25-30 s after the cAMP stimulus (McRobbie and Newell, 1984; McRobbie and Newell, 1983; Berlot et al., 1985; Berlot et al., 1987). The myosin light chain is also phosphorylated following cAMP treatment (Berlot et al., 1985). The transient increase in intracellular cGMP which occurs following cAMP stimulus has been shown to be important for the regulation of the myosin II responses (Liu and Newell, 1991; Liu etal., 1993; Liu and Newell, 1994). Strains with a defective cGMP-specific phosphodiesterase have persistent elevated levels of cGMP following a cAMP stimulus and in these strains, there is prolonged association of myosin LI with the cytoskeleton and a delay in myosin II light and heavy chain phosphorylation. It has been proposed that guanylate cyclase is activated via Ca2+ influx (Newell and Liu, 1992). However, the pathways mediating the phosphorylation of myosin via myosin kinases in response to cAMP have not been fully elucidated. The response of starved cells to nutrients has some similarities to the cAMP cringe response; When nutrient medium is reintroduced to starved cells, the ameboid cells rapidly round up and become refractile in a process which has been compared to the response of mammalian cells to serum (Schweiger et al, 1992; Howard et al., 1993). The HL5 stimulation response is accompanied by a rapid tyrosine phosphorylation of a subset of cellular proteins including actin (Schweiger et al, 1992; Howard et al, 1993). Disruption of tyrosine phosphatase PTP1, but not PTP2, caused a more rapid and more prolonged phosphorylation of actin and an acceleration of cell rounding when starved cells were returned to growth medium (Howard et al., 1993; Howard et al., 1994). Overexpression of PTP1 decreased the amplitude and duration of actin phosphorylation and also diminished the cell rounding response (Howard et ai, 1993). In addition, treatment of vegetative cells with the phosphatase inhibitor, phenylarsine oxide, caused cell rounding accompanied by actin phosphorylation (Schweiger et al, 1992). Although some of the components involved in the cAMP signal transduction relay in D . discoideum have been identified, the details of the signal transduction to the cytoskeleton are not known. Similarly the response of starved cells to nutrients has not been well characterized. One strategy for studying the control of the cytoskeleton is to identify signal transduction molecules which, when disrupted or overexpressed, affect cell morphology and motility. Overexpression of Gal or the expression of a calmodulin antisense RNA both produce large multinucleate cells that are reminiscent of those caused by disruption of myosin II heavy chain and profilin (Kumagai et al, 1991; Liu et al, 1992). Rationale and research objective At the outset of this work, the function of the D. discoideum rapl gene was unknown. The objective of my research has been to further characterize the organization of the rapl gene and to evaluate its role in D. discoideum by overexpression of wild type and mutant Rapl proteins. Site-directed mutations were generated in rapl to target conserved amino acids previously shown to be important for activity in either mammalian Rapl or Ras proteins or both. During the course of this work, I noted that a transformant expressing an activated RasG-G12T protein (generated by M. Khosla) shared some of the characteristics of transformants overexpressing the Rapl protein, and therefore I characterized this transformant in more detail. MATERIALS AND METHODS Materials G418 (Geneticin) was purchased from Sigma (USA), FITC-phalloidin was purchased from Molecular Probes (USA) and X-ray film was purchased from Kodak (Canada). Radiolabeled [oc32p] dCTP was purchased from ICN How Labs (Canada) and [35s] dATP was purchased from Dupont NEN Canada Inc., filters were purchased from Millipore (USA) and the enhanced chemiluminescence kit for western blot analysis was purchased from Amersham (Canada). Bacteriological peptone and yeast extract were purchased from Oxoid (UK). All other chemicals were purchased from Fisher ScientiEc Co. (USA) or BDH (Canada). Hoechst 33258 dye was a gift from Dr. Hancock's laboratory. Oligonucleotides were synthesized by Dr. Sadowski's laboratory (UBC) or the NAPS unit (UBC). Restriction endonucleases or modifying enzymes were purchased from GIBCO BRL (Canada). Taq polymerase was purchased from Promega (USA); Vent polymerase was purchased from New England Biolabs (USA) and Sequenase was purchased from United States Biochemical. The anti-Rapl peptide antibody and the anti-RasG-GST-fusion protein antibody were generated by Steve Robbins (Robbins, 1991). The anti-phosphotyrosine antibody IgG2bk was initially purchased from Upstate Biotechnology Inc. (USA) but subsequently was a gift from Dr. Mike Gold. Goat anti-mouse IgG antibody conjugated to horseradish peroxidase and goat anti-rabbit IgG antibody conjugated to horseradish peroxidase, the secondary antibodies for ECL analysis, were purchased from Amersham (USA). The E. coli strain DH5ctF' was used for bacterial transformations. The genotype of DH5aF' is: F/endAl //sdRi7(rk"mk+) supE44 thi-1 iecAl gyrA(Nair) relAl A(lacZYA-argF)U169 deoR (08OdlacA(lacZ)Ml5) (Raleigh et al, 1989). The pVEH vector was donated by Wolfgang Nellen, the pDdGal 17 vector was donated by Adrian Harwood and the pVEIIGal vector was donated by Birgitte Wetterauer. D. discoideum growth and differentiation Growth of D. discoideum The Ax2 strain of D. discoideum that was used in all experiments was grown axenically in HL5 medium (Watts and Ashworth, 1970) (14.3 g neutralized bacteriological peptone, 7.15 g yeast extract, 0.96 g Na2HPC»4 and 0.486 g KH2PO4 per liter of water) with gyratory shaking at 150 rpm at 22°C or on plates in association with E. aerogenes. Cell numbers were determined in duplicate with a hemacytometer. The transformed Ax2 strains were maintained in HL5 medium in the presence of 10 /ig/ ml G418 (Geneticin) except the wild type Rapl transformant (containing the pVEII Rapl vector) (Rebstein et al, 1993) which was maintained in the presence of 50 ug/ ml G418. Development of D. discoideum D. discoideum development on filters was initiated as previously described (Khosla et al, 1990). Exponentially growing cells were washed twice in BS buffer (10 mM NaCl, 10 mM KC1 and 2 mM CaCl2) (Bonner, 1947) by centrifugation at 700g for 3 minutes and 2.5 X 10^  cells were plated on a 4.0 cm diameter filter (pore size = 0.45 um), resting on a BS buffer-saturated pad in a 60 mm petri dish. The filters were incubated at 22°C in a moist chamber. In some experiments, cells were developed in suspension in an Erlenmeyer flask at a density of 2 X10^ cells/ml with gyratory shaking at 150 rpm at 22°C. D. discoideum development following growth on bacteria was accomplished by pipetting 1-5/d of cells in HL5 medium onto a freshly inoculated lawn of Enterobacter aerogenes on an SM nutrient agar plate (10 g glucose, 10 g neutralized bacteriological peptone, 1 g yeast extract, 1 g MgS04-7H20,1.55 g NaH2P04 H20,1 g K H P O 4 and 20 g bactoagar per liter of water). Plates were incubated at 22°C and after the D. discoideum cells had consumed the bacteria (usually 4 days), development ensued in the zone depleted of bacteria. To analyze the erasure response, a modification of a previously described procedure was used (Kraft et al., 1989). Cells were starved in BS buffer in suspension, as described above, for 8 hours, incubated in HL5 medium for either 1 or 2 hours under shake condition, washed twice in BS buffer and then allowed to develop on filters as described above. Determination of cell viability Cells were grown to high density in the absence of folate, washed twice, and approximately 100 cells were plated in 100 mm tissue culture plates. After allowing the cells to adhere for 30 minutes, HL5 was added back either immediately or after 8 hours of starvation. Cells were counted after 6 days. The percentage of cells viable after starvation was determined by counting the number of CFU before and after starvation on duplicate plates. Induction of the discoidin promoter To maintain strains containing genes under the control of the discoidin dis I y gene promoter in a suppressed state, 1 mM folate was added to the HL5 medium. To induce expression from the discoidin promoter, cells were incubated with conditioned HL5 medium, since conditioned medium contains a pre-starvation factor (PSF) (Clarke et al, 1987; Clarke et al, 1988), that induces expression from the discoidin promoter (Rathi et al, 1991). Conditioned HL5 medium was prepared by growing Ax2 cells to a density of approximately 2 x 10^  cells/ml, removing the cells by centrifugation and filtering the medium though a 0.2 um pore size nitrocellulose filter. The discoidin promoter is also induced between 4 and 8 hours of development (Blusch et al, 1992), which allows the effects of genes expressed from this promoter to be analyzed during early development. To ensure continuous high expression of the Rapl and RasG proteins from the start of development, cells were grown under inducing conditions in conditioned medium for 24 hours prior to the initiation of development. In some experiments, induction was achieved by growing cells to 1-3 xlO^ cells/ml in the absence of folate. Development was then initiated as described above. Analysis of cell morphology To examine cell morphology, cells were plated at 3x10^  cells/ cm^ on a glass coverslip 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 1 mM folate to induce or repress the discoidin promoter, respectively. To evaluate the ability of the cells to undergo the nutrient stimulus induced cell rounding, the cells were then starved for 8 hours in 5 ml BS buffer and then exposed to 5 ml of HL5 medium. Cells were photographed before HL5 stimulation and then 5 and 10 minutes afterwards. To determine the effect of azide on cell substratum adherence, an adherent monolayer of cells was exposed to 2 mM Na azide (NaN3) in HL5 medium for 3 minutes with swirling at 60 rpm, in a modification of a previously described procedure (Pasternak et al, 1989; Springer et al, 1994). The plates were gently rinsed 3 times to remove floating cells. The coverslip was photographed before and after azide treatment. Between 200 to 600 cells were counted prior to azide treatment. Analysis of cell-cell adhesion Cell adhesion was measured using an agglutometer, which monitors light scattering (high values indicate small aggregates or single cells while small numbers indicate large aggregates). Cells were grown to a density of 2xl06 cells/ml, washed in PBS and assayed for cell agglutination over a 60 minute period in the presence and absence of cell adhesion. Flow cytometric analysis Cells were grown in conditioned medium for 24 hours in shake suspension and analyzed directly in medium using a flow cytometer (Becton-Dickinson) running Lysis II software. Five thousand events were analyzed for forward and side light scatter for each sample. Mean values were determined for forward scatter events above a cutoff point of 200. Transformation of D. discoideum D. discoideum Ax2 cells were transformed by the calcium phosphate precipitation technique (Nellen et al., 1984) in Bis-Tris HL5 (Egelhoff et al., 1989). After incubating the calcium phosphate DNA precipitate with the cells for 4 hours, the cells were given a 2 minute osmotic shock with 15% glycerol as previously described (Early and Williams, 1987). Transformants were selected in 10/tg/ ml G418 in HL5 and colonies were visible after approximately 10 days. Individual clones were transferred initially to 24 well plates, then to 100 mm plates. Established transformants were maintained in shake suspension as described above. The RasG, RasG-G12T, and RasG-SUN transformants were generated in a similar manner (Khosla et al., 1995). Dark field, Nomarski and fluorescence microscopy Vegetative cell morphology, the nutrient-induced rounding of starved cells and the effects of azide treatment were observed by dark field microscopy of an adherent layer of living cells on a glass coverslip. A two-sample t test for the two tailed hypothesis was used to test the significance of differences observed in the means between selected samples. Relative cell areas were determined by digitizing photographs of the cells (pdi Model DNA 35 scanner), manually tracing the outline of the cells on the digitized image and computing the areas with the PD Quest 4.1 program. To observe F-actin distribution, cells adherent to a glass coverslip were fixed with 3.7% formaldehyde in BS buffer for 10 minutes and then washed 3 rimes with PBS (8 g NaCl, 0.2 g KC1,1.44 g NaHP04,0.24 g K H 2 P O 4 in 1L H 2 O pH 7.4). Cells were then permeabilized for 5 minutes in -20°C acetone, rehydrated in PBS, overlaid with FITC-phalloidin solution (0.2 yM) and then washed with PBS to stain F-actin. To stain cell nuclei, the formaldehyde fixed cells were overlaid with 0.0005% Hoechst dye # 33258 for 5 minutes and then washed with PBS (Harlow and Lane, 1988). Cells were viewed using a Zeiss Axiophot microscope equipped with epifluorescence. All photographs were taken using TMAX 400 film. Molecular Biology Plasmid DNA preparation Plasmid DNA was isolated from DH5cxF' E. coli cells using a CTAB miniprep procedure (Del Sal et al., 1989) or a PEG-precipitation large scale procedure (Maniatis et al, 1989). Competent DH5ctF' E. coli cells were prepared using a rubidium chloride technique and transformations of competent cells were done as described (Maniatis et al, 1989). Transformed DH5cxF E. coli cells were selected on LB ampiciUin plates (10 g bacto-tryptone, 5 g bacto yeast extract, 10 g NaCl, 15 g bacto-agar pH 7.0, with 60 Mg/ml ampicillin). Southern blot analysis Genomic DNA was prepared as described (Maniatis et al, 1989) from isolated D. discoideum nuclei (Coccucci and Sussman, 1970). Briefly, isolated nuclei from 3.7 x 10^  cells were lysed in 1% SDS, treated with proteinase K (200/ig/ml), and extracted three times with an equal volume of phenol-chloroform. The sample was then ethanol precipitated, resuspended and treated with DNase-free RNase (10/ig/ ml). The sample was extracted with an equal volume of phenol-chloroform, ethanol precipitated and then resuspended in TE (10 mM Tris HCl 1 mM EDTA pH 7.4). The genomic DNA concentration was determined spectrophotometrically (Maniatis et al, 1989). Six Mg of genomic DNA were digested overnight with 25 units of restriction enzyme and the DNA was separated on a 0.8% agarose TBE gel (IX TBE is 89 mM Tris-borate, 89 mM boric acid 2 mM EDTA). The DNA was stained with ethidium bromide and the gel was photographed. The DNA was then denatured in 1.5 M NaCl, 0.5 M NaOH for 30 minutes and then neutralized in 1.5 M NaCl, 0.5 M Tris pH 8.0 for 30 minutes. The gel was soaked in 2X SSC and then transferred and fixed to nitrocellulose. Nitrocellulose filters were hybridized overnight at 37°C with the rapl cDNA probe in hybridization buffer (5X SSC (IX SSC is 150 mM NaCl, 15 mM Na citrate, pH 7.0), IX Denharts (0.1% ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 50 mM NaPC»4, 0.5% SDS and 30% formamide) (IX SSC is 0.15 M NaCl, 0.015 M Na citrate) and then washed twice at 65°C in 0.1X SSC and 0.1% SDS. Filters were then exposed to X-ray film at -70°C. Preparation of cDNA probes The cDNA fragment was purified by gel electrophoresis in a 2% low melt agarose gel. An aliquot of the low melt agarose containing the cDNA fragment was used directly for radiolabeling by nick translation (Maniatis et al., 1989). Nick translation was performed as described (Feinberg and Vogelstein, 1983). The radiolabeled probe was separated from unincorporated nucleotides with a Sephadex G-25 spin column (Maniatis et al, 1989). Sequencing Single stranded DNA was produced by infection with K07 M13 helper phage, followed by PEG precipitation, isolation on glass filters and elution into TE (Maniatis et ah, 1989). Double stranded DNA was sequenced directly from CTAB miniprep DNA. Sequencing reactions were performed according to the manufacturer's (United States Biochemical) protocol except that the Sequenase reaction buffer was added after DNA denaturation. DNA sequence was determined by the dideoxy chain termination method (Sanger et al., 1977) with modified T7 DNA polymerase. Electrophoresis and immunoblotting SDS-PAGE and immunoblotting techniques were performed as described (Robbins, 1991). For western blot analysis of transformants expressing genes under the control of the discoidin promoter, cells were inoculated at a density of 5 xlO^ cells/ml and grown for 24 hours in shake suspension in either conditioned medium or HL5 containing 1 mM folate. Cells were lysed in 1% SDS and diluted in an equal volume of 2X loading dye (20% glycerol, 10% p-mercaptoethanol, 4.6% SDS, 125 mM Tris HCl pH 6.8). Protein concentration was determined by UV absorbance (Harlow and Lane, 1988). About 10-15 ug of protein were electrophoresed on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose filter. Prestained molecular weight markers (BioRad) were used to estimate protein sizes. 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 IX TBS (8 g NaCl, 0.2 g KC1,3 g Tris HCl in 1 L H2O pH 7.4) 5% skim milk and 1% Tween-20. The Rapl protein was detected with a specific anti-Rapl peptide antibody at a 1:2000 dilution in IX TBS containing 0.5% skim milk and 1% Tween-20 (Robbins, 1991). The RasG protein was detected with a specific anti-RasG-GST protein antibody at a 1:5000 dilution (Robbins, 1991). A goat anti-rabbit antibody conjugated to horseradish peroxidase was used as a secondary antibody to generate a signal by ECL which was recorded on X-ray film. Protein phosphotyrosine western blot analysis was performed similarly using the anti-phosphotyrosine monoclonal antibody IgG2bk at a dilution of 1:2000 in IX TBS, 5% BSA, 0.1% Tween-20, 0.5 mM Na3V04 and 0.2 mM Na2Mo04-A goat anti-mouse IgG antibody conjugated to horseradish peroxidase was used as a secondary antibody. fr-galactosidase assay Growing cells were washed twice and 1x10? cells were plated in a 100 mm tissue culture plate in 10 ml KK2 buffer (20 mM KHPO4, pH 6.0) to initiate development. At the appropriate stages of development, the cells were rinsed in fresh KK2 buffer and resuspended in 0.5 ml 0.1 M phosphate buffer pH 7.0. Cell-free extracts were prepared by subjecting the cells to three cycles of freezing in a dry ice-ethanol bath and thawing at 37°C. The broken cell suspension was centrifuged (RCF = 13600) to remove insoluble debris and the supernatant fluid was assayed for (3-galactosidase activity using o-nitrophenyl-p-D-galactoside (ONPG) as the substrate (Miller, 1972; Dingermann et al., 1989; Knox et al., 1991). |3-galactosidase activities from samples were determined from duplicate reactions and averaged. Protein concentration was determined using the Bradford assay with BSA as a reference standard (Bradford, 1976). Isolation of the rapl genomic region A sucrose gradient from 10% to 40% sucrose was prepared with a gradient-forming device. Genomic DNA (50 /ig) was digested overnight with 50 units of Xbal and 50 units of BglTL, ethanol precipitated, dissolved in 100fd of TE buffer and then centrifuged on this gradient at 34000 rpm (RCF = 150 000) in an SW-41 swinging bucket rotor for 20 hours at room temperature. 1 ml fractions were collected and size analyzed by gel electrophoresis. DNA from the appropriate fraction (2-8 kb) was isolated by diluting the sample 4 fold in TE followed by ethanol precipitation. This fraction was predicted to contain the 6.6 kb 5' fragment and 4.0 kb 3' fragment of the rapl gene. The size-fractionated DNA was cloned into BamHI Xbal digested gel purified Bluescript vector, and approximately 35000 CFU were screened for inserts containing rapl DNA. The colonies were transferred to nitrocellulose using standard techniques and the colony blots were probed with a radiolabelled cDNA consisting of the full length rapl cDNA prepared by nick translation as described for the Southern blot procedure (Maniatis et al, 1989). Briefly, nitrocellulose filters were hybridized overnight at 37°C with the probe in hybridization buffer and then washed twice at 50°C in 2X SSC and 0.1% SDS. Filters were exposed to X-ray film and 15 positive clones were identified. The positive clones were picked, diluted, plated and the resulting colonies were probed as described above. Two colonies were still positive after probing and the presence of the predicted insert was confirmed by restriction digestion and Southern blot analysis. As both cloned inserts consisted of the 3' fragment of the rapl gene, a modified strategy was therefore used to isolate the remaining 5' fragment of the rapl gene. The size-fractionated genomic DNA was further digested with Hindi, reducing the size of the presumptive insert to 1.5 kb and the fragments were cloned into BamHI Hindi digested Bluescript vector. An EcoRI Bglll rapl cDNA fragment specific for the 5' region of the gene was used as a probe. Approximately 60000 clones were screened as described above. Five positive clones were identified, picked, diluted, plated out and the resultant colonies were probed as described above. Only one colony remained positive after this probing, but it was shown to contain the predicted insert by restriction digestion and Southern blot analysis. A missing internal 78 bp Bglll Bglll fragment was isolated by PCR. The presence of the predicted 600 bp fragment was confirmed by running 5ul of the PCR reaction on a 0.8% agarose TBE gel. The remaining 45/il of the PCR reaction was ethanol precipitated with ammonium acetate and resuspended in TE. The PCR product was digested with Bglll, extracted with phenol chloroform, ethanol precipitated and then resuspended in TE. The PCR product was ligated into BamHI digested Bluescript vector overnight at room temperature. The ligation mixture was further digested with BamHI to linearize Bluescript vector which did not contain insert DNA, heated at 65°C for 10 minutes and then transformed into competent DH5aF' E coli cells. Sequence analysis of the rapl genomic region A 1.8 kb Acc\ fragment from the 3' rapl fragment was cloned into the Bluescript vector in both orientations for sequence analysis. The 5' rapl fragment in Bluescript was removed by SstI Hindll digestion, was gel purified and cloned into SstI Hindll digested pTZ18U. Nested deletions (Maniatis et ah, 1989) of the genomic fragments were generated with exonuclease III in both orientations using the 3' rapl fragment in Bluescript, and in complementary orientations of the 5' rapl fragment in Bluescript and pTZ18U, respectively. Briefly, DNA was digested with two restriction enzymes to linearize the DNA and create a single end resistant to exonuclease IH. DNA was treated with exonuclease HI and the reactions terminated at 30 second intervals. DNA fragments of the desired size were treated with the Klenow large fragment, ligated and transformed into DH5aF'. Single stranded DNA was sequenced by the dideoxy chain-termination method (Sanger et al., 1977) with the universal or reverse primers and the remaining sequence was obtained using specific oligonucleotides as primers. The internal Bg/II PCR fragment was sequenced directly in the Bluescript vector in both orientations using a double stranded DNA template with the universal and reverse primers. Vector constructions pRaplGal: The 5' rapl Hindll EcoRI 1 kb fragment was isolated from the Bluescript vector, gel purified and ligated into pDdGall7 (Harwood and Drury, 1990) that had been digested with Xbal, filled in with Klenow fragment and digested with EcoRI. The junctions were confirmed by sequencing. pVEHRap: The pVEII vector (Blusch et al., 1992) was modified to remove the discoidin ATG translation start site by digestion with Xbal followed by treatment with E. coli DNA polymerase I in the presence of excess dATP in order to create a single stranded deletion which extended to the first internal adenosine nucleotide. Subsequently the vector was treated with SI nuclease and then the Klenow large fragment of £. coli DNA polymerase I to generate blunt ends before recircularization with DNA ligase. The deletion of the ATG in the modified vector was confirmed by DNA sequencing. This modified vector was then linearized with Kpnl and treated with T4 DNA polymerase to generate a blunt end. An EcoRI fragment containing a full length rapl cDNA was treated with the Klenow large fragment of £. coli DNA polymerase I to fill in the recessed ends, and then ligated into the modified vector in the sense orientation downstream of the discoidin promoter, recreating the flanking EcoRI sites. The junctions were confirmed by EcoRI digestion and by DNA sequencing. pVEII also contains the actin 15-Tn903 resistance cassette (Blusch et al, 1992) as a G418 selectable marker. Mutagenesis of the rapl cDNA and construction of pVEII vectors: The site-directed mutations in the rapl gene were generated by PCR. Because the D. discoideum rapl gene product contains two additional amino-terminal amino acids and two additional residues located 17 amino acids from the carboxyl terminus compared to the human rapl and ras gene products, amino acids have been numbered according to the consensus alignment of Ras proteins to facilitate comparison (Protein sequence alignments were performed with the CLUSTAL module of the PCGENE program). Mutations were introduced by PCR using the 674 bp D. discoideum rapl cDNA EcoRI fragment in the Bluescript vector as a template (Fig. 4) with the primers shown in Table 1, and either the reverse or universal primer as appropriate. Reaction conditions are described in detail below. Gel purified products of this reaction were subjected to a second PCR in the presence of universal and reverse primer using a BglR digested rapl cDNA as a template (this template is disrupted by an internal gap in the rapl cDNA). This PCR selectively regenerated a full length mutated rapl gene. cDNAs mutated at positions 17 and 38 were digested with Hindlll and Nsil and the region containing the mutation was cloned into Hindlll Nsil digested rapl in Bluescript. The cDNAs mutated at positions 61 and 157 were digested with SstI and Nsil, and the region containing the mutation was cloned into SstI Nsil digested rapl in Bluescript. The site-directed mutagenesis was confirmed by sequencing the entire region which had been amplified by PCR in order to confirm the mutation and check for any additional introduced mutations. The mutated rapl genes were then cloned into the pVEII vector (Blusch et al., 1992) which had been previously modified to remove the discoidin ATG translation start site as described above. The modified vector was linearized with Kpnl and treated with T4 DNA polymerase to generate blunt ends. An EcoRI fragment containing the full length rapl cDNA was treated with the large fragment of £. coli DNA polymerase I to fill in the recessed ends and then ligated into the modified vector in the sense orientation downstream of the discoidin promoter, recreating the flanking EcoRI sites. The junctions and the site-directed mutations were confirmed by DNA sequence analysis. First Reaction ^ 1 Extension ^ 2 Amplification Figure 4. PCR mutagenesis of the rapl gene The first reaction introduces the mutation into the rapl cDNA. The second reaction consists of 2 steps. The first step utilizes the PCR product of the first reaction as a primer with a rapl cDNA containing an internal deletion as a template in an extension reaction which results in a full length cDNA containing the desired mutation. The second step utilizes two oligonucleotide primers which are present in molar excess to amplify the full length product. Arrowheads indicate the orientation of the relevant polymerase reaction. Other reactions (not shown) can occur but do not result in as efficient an amplification. Second Reaction Table 1. Ol igonucleot ides used for site-directed mutagenesis of the rapl gene Mu ta t i on Ol igonucleot ide 3 S17N G T A G G T A A A A A T G C A T T G A C T G T G C D38E C C A A C C A T C G A A G A A T C C T A C A G D 3 8 N C C A A C C A T C G A A A A T T C C T A C A G T61Q G A T A C A G C T G G T C A A G A A C A A 111 A C F156L A A G T T A T A C A A A A 111 G T T C a Mutated bases are under l ined Polymerase chain reactions Dried oligonucleotides were dissolved in 30% NH4OH and purified by two cycles of precipitation with n-butanol followed by resuspension in water (Sawadogo and Van Dyke, 1990). The PCR reactions were performed on an Ericomp Twin Block Thermocycler. The PCR universal and reverse primers were 5' CGTTGTAAAACGACGGCCAGT 3' and 5' CAGGAAACAGCTATGAC CATG 3', respectively. The internal 76 bp BglR BglR rapl genomic fragment was isolated from genomic DNA by PCR amplification using the oligonucleotides RapE 5' AAACCAGATGCCTCTTAGAG 3' and JD13 5' AGCTGCAGA(C/A)ATC(G/A/ T)(C/G)(A/C)TTT(G/A)TTAAC 3' (bases in brackets indicate degenerate positions). The reaction consisted of 50 ng of Ax2 DNA, 25 pmoles of each oligonucleotide, 200 uM of each of dATP, dCTP, dGTP and dTTP, 50 mM Tris-HC1 pH 8,0.05% Tween 20, 0.05% NP-40 and 0.5 mM MgCl2 and 1 unit of Taq polymerase in a final volume of 50 ul. The amplification protocol consisted of an initial 'hot start' at 94°C for 5 minutes followed by 30 cycles of melting at 95°C for 1 minute, annealing at 47°C for 2 minutes and extension at 72°C for 1.5 minutes, with an additional 5 minute 72°C extension on the final cycle. Site-directed mutagenesis of the rapl cDNA was performed by two consecutive rounds of PCR according to the scheme shown in Figure 4. The oligonucleotides shown in Table 1 were used to introduce the mutations in a reaction with the universal primer (except for mutation F156L where the reverse primer was used) utilizing the rapl cDNA in Bluescript as a template. The first reaction consisted of 200 ng of template, 20 pmoles of each oligonucleotide, 50 uM of each of dATP, dCTP, dGTP and dTTP, 20 mM Tri&: HCl pH 8.4, 50 mM KC1, 0.05% Tween 20, 0.05% NP-40 and 2.0 mM MgCl2, and 1 unit of Taq polymerase in a final volume of 50 ul. The amplification protocol consisted of an initial 'hot start' at 95°C for 5 minutes followed by 25 cycles of melting at 95°C for 1 minute, annealing at 50°C for 1 minute and extension at 72°C for 1 minutes with an additional 5 minute 72°C extension on the final cycle. The second reaction consisted of 1 ul of the product of the first PCR reaction, 20 pmoles of universal and reverse primers, 20 ng of Bglll digested template, 50 uM of each of dATP, dCTP, dGTP and dTTP, 20 mM Tris-HCl pH 8.4, 50 mM KC1, 0.05% Tween 20, 0.05% NP-40 and 2.0 mM MgCl2, and 1 unit of Taq polymerase in a final volume of 50 ul. The amplification protocol was the same except 30 cycles were performed. Mutations D38N and F156L were generated with Vent polymerase which has greater fidelity than Taq polymerase. The first reaction consisted of 10 ng of template, 20 pmoles of each oligonucleotide, 400 JJM of each of dATP, dCTP, dGTP and dTTP, 10 mM KC1, 20 mM Tris-HCl pH 8.8, 10 mM (NH4)2S04 2 mM MgSC>4, 0.1% Triton X-100 and 1 unit of Vent polymerase in a final volume of 50 ul. The amplification protocol consisted of an initial 'hot start' at 95°C for 5 minutes followed by 20 cycles of melting 95°C for 30 seconds, annealing for 50 seconds and extension at 72°C for 38 seconds. The D38N mutation reaction was annealed at 40°C while the F156L mutation reaction was annealed at 45°C. The second reaction consisted of 2 ul of the product of the first PCR reaction, 20 pmoles of universal and reverse primers, 20 ng of Bg/II-digested template, 400 IIM of each of dATP, dCTP, dGTP and dTTP, 10 mM KC1,20 mM Tris-HCl pH 8.8,10 mM (NH4)2S04,2 mM MgS04, 0.1% Triton X-100 and 1 unit of Vent polymerase in a final volume of 50 ul. The amplification protocol consisted of an initial 'hot start' at 95°C for 5 minutes followed by 20 cycles of melting at 95°C for 30 seconds, annealing at 45°C for 50 seconds and extension at 72°C for 38 seconds. RESULTS The Dictyostelium discoideum rapl gene: isolation of the genomic sequence and characterization of the promoter region Introduction The D. discoideum rapl gene is expressed both during vegetative growth and during development. Steady state rapl mRNA levels increase during aggregation and then following a period of decline during pseudoplasmodial formation, increase again during the formation of the fruiting body (Robbins et al, 1990). During vegetative growth and early development, a rapl single mRNA 1.1 kb in size is detected. After 6-8 hours of development, this mRNA species is replaced by two mRNA species of 1.0 and 1.3 kb (Robbins et al., 1990). The 1.3 kb mRNA is positively regulated in response to pulses of cAMP (Robbins, 1991). To begin to elucidate the complex regulation controlling rapl mRNA expression in D. discoideum, genomic DNA encoding the rapl gene was isolated and sequenced. In addition, the ability of the upstream untranslated region to promote expression of a reporter gene was tested. Isolation of rapl genomic DNA Genomic DNA encoding the rapl gene was isolated by constructing and screening a plasmid library. Genomic DNA from D. discoideum strain Ax2 was digested with Xbal and Bglll, size fractionated on a sucrose gradient, and the fraction enriched in fragments between 2 and 8 kb in size was cloned into Xbal, BamHI digested Bluescript vector. Southern blot analysis using the rapl cDNA as a probe indicated that 2 fragments, 6.6 and 4.0 kb in size, corresponding to the 5' and 3 regions of the rapl gene would be present in this fraction (Fig. 5). Two positive clones which corresponded to the 3' region of the rapl gene were identified by hybridization with the rapl cDNA probe. There were two possible reasons why the 5' region of rapl was not isolated in the first screen. First, Southern blot analysis indicated that the full length probe gave a stronger signal with the 3' region compared to the 5' region (Fig 5). Secondly, given the large size of the fragment to be isolated, it was possible that the fragment was not stable in E. coli. To circumvent these potential problems, the 2-8 kb fraction of genomic DNA was further digested with Hindi and cloned into Hindi, BamHI digested Bluescript vector. One clone containing the 5' genomic region of the rapl gene was identified by hybridization with the EcoRI-Bglll 5' fragment of the rapl-c51 cDNA. The remaining internal 76 bp Bglll fragment was isolated by PCR using two oligonucleotides that corresponded to sequences within the flanking regions as described in the Materials and Methods. The PCR fragment was digested with Bglll releasing a 76 bp fragment which was then cloned into BamHI digested Bluescript vector. The three fragments are designated A, B and C, respectively (Fig. 6). Nucleotide sequence of the rapl genomic DNA including the 5' region Nested deletions (Maniatis et al., 1989) of fragments A and B were generated with exonuclease III and sequenced as described in the Materials and Methods. Both strands of fragments A and B were sequenced. The short Bglll fragment C was sequenced directly in the Bluescript vector in both orientations. The nucleotide sequence of the rapl genomic clone is shown in Figure 7. The coding region of the gene was found to be divided into two 1 2 3 4 5 Figure 5. Southern blot analysis of D. discoideum rapl genomic D N A G e n o m i c D N A (6f/g) from A x 2 was digested w i t h B g l l l (lane 1); B g l l l and H i n d l l l (lane 2); B g l l l and X b a l (lane 3); B g l l l and H i n d i (lane 4); and EcoRI (lane 5). The digested D N A was fractionated on a 0.8% T B E agarose gel and then transferred and fixed onto a nitrocellulose membrane. The filter was p robed w i t h a rapl c D N A r a d i o l a b e l e d as described in the materials and methods and washed in 0.1X SSC, 0.1% SDS at 65"C. Molecular size standards (kbp) are indicated. Figure 6. Genomic organization of the D. discoideum rapl gene. Exons are shown as hatched boxes. The isolation of fragments (A), (B) and (C) is described in the text. Restriction sites HincII, EcoRI, Bglll and Xbal are designated H, E, B and X, respectively. The gap in fragment A indicates a region omitted to compact the figure. aacactacacaaacattgac acaggcacacacacaaaata aaaaaccacccacttaatt t a a t t t a t t t t t a t t a t t a t t 80 a t a t t t t t t t t t t t t a t t t t t t t a t a t t t a t t t t t t t t t t a t t t a t t t c a a c t t t t t t t t t t t t t t t t t t t t t a a a a a a a 160 ataataatat tagtgataaa aataatatacaattaaaact a t t t t c c c a a a t t t c t t t t t cccc t tcc tacaaa t taa t t 240 t ta taataaccaat tcaaaa aaaaaaaaaaaaaatttatt t t t a t t t t t a t t t t t g t t t t t t t t t t t t t c t t t t t t t t t t 320 t t t t c t t t t t t t t t c a t t t t t a t t t t a a t t t t t t t t t t a t t t a t t t a t t a t t a c t a t t t t t t t t t t t t t a t a t a c a t c c t 400 tc t taaaacca t t t a t tag t a a g t a t t t t a t t t g t t t t t t t t t t t t t t t t t t t t a t t t a t t t t a t t t t t t t t a t t t t t t t 480 t t t c t c t a t a t a g g a t t t g t a a t c t t t t t t t a a t c a c t t t tttatacaagggtaaaaaaa aaaaaaaaaacgatggggag 560 ccacct t t tgat tcaaaaaa ataaaaaaaaaaaaaaaaaa gaaaaattatt t t t taaaaa a a a a t t a a a t t t t t t t t t t t 640 t t t t t t a a t t t t t t t t t t t t ttttccaacKaaaaattttt t t ca tcacaaaaa t t t t t t t t t t t t a t t t t t t a t t t t t a t 720 acacacatatatatataaca c t t t g t t g t t t t t a t t t a t t ta t t taataaccatcaccca accaaat tgtaat t taatac 800 at tcaactaat taaataat t t t t t t t a a t t a c a t a t t a t a t t t t t t aaa t ta t ta taaaa ataagaatttaaaaaaaaaa 880 aaaaaaaaaaaaaaaaaatt a tc ta t t t tcaaaaaact t t caaaactcaataaaactaaa taaaaggaaatatatattat 960 aaacaagATC CCTCTTOGAG AATTCMAMX^STCGmTA QGTTCMGTQGTCTAGGTAA ATCTGCrm^TSTOCAAT 1040 TTCTTCAflGGTAXlTlTUl'l' GAAAfiGT^GATCCAACCAT CGMGAITCCTACAGAAAAC AflGTCGAAGTTGAIAGCAftT 1120 CAftTGCftTGTTflGAAfllTrr ASAOaCAGgtaagtttaaat t t tactgt tataaagtgaac a t a a a a t c a t t t t t t t t t t t 1200 aataatgaagaaaaaaaaaa aaaaaaaaatcaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaatatcaaaa-tttt 1280 t t t taaagtggt taa t taa t t taac ta taactcaata t t t a t t c c t t t a c t a a t t t a t t t a t t a t t t t t a t a t a t a t t t t 1360 t t t t t t t a a a a a a a t t a t t a gCTOGTflCaXSaft^AarTTflC TQCAATCSflGfiGflTCITrflCA TGAAAAAIQGTCAfiGGTTTT 1440 GJTTTTflCTSffilOTCAMCAT TTOW^TCX^TTTTAflCG ftSTT^VJCAGRICTCCGTGAA CAAATTCTCACIflGTTftflGGA 1520 TTGTGAfiGATCTTCCAA3X3G TTCTTGTTGGTAflCAAAIGC GAaCTCC^aCOiflCGTGT TftriaGCflaV3AftCMGC3TC 1600 iWaAflCTCQCTCGTAAATTT GGTGaTTGTTMTTmaSA AGCATCTQCCAAGAAIAAAG TEARIGTTGAfiCAAATTTTC 1680 TftTAMITAATCCCTCAAAT CAACCGTAAAAAXCAGTTC GTCCftXAfiGCAAflGCTAAA TCAAAATCOXKTTTATTGta 1760 aacaatccatcaactctcca acaccct tccatactcaccc acccatttcaaatgtaacaa ttgaaaaacagaaaaaaaaa 1840 aaaaaaaaaaacaggaaaaa aaaaaaacacttt t t taaaa aaaaaaaaaaaaataataat aataataataataaccagta 1920 atatagtaaatatatatcgt aaagataccaaaatatgtaa t a a a t a a a t t t t t t t g t t t t t t t t t t a 1987 Figure 7. Nucleotide sequence encoding the rapl gene The initiating ATG is doubly underlined, nucleotides coding for the Rapl protein are in upper case, while flanking and intron sequences are in lower case. exons separated by a 234 bp intron. The 5' and 3' non-coding sequences and the intron contained a high adenine/thymine content (87%). The nucleotide sequence of approximately 1 kbp of the region upstream of the coding region was also determined. Analysis of the rapl promoter To determine if the isolated 5' untranslated fragment of the rapl was sufficient to promote expression from a reporter gene, the entire 5' fragment including the first 7 codons of the coding sequence was ligated in frame upstream of the (3-galactosidase reporter gene in the vector pDdGall7 (Harwood and Drury, 1990). Stable G418 resistant transformants were generated using this expression plasmid (RaplGal) as described in the Materials and Methods. Vegetative Rapl Gal cells expressed (3-galactosidase activity whereas the parental Ax2 cells had a very low level of endogenous |3-galactosidase activity (Fig. 8). p- galactosidase activity was detected both in vegetative cells and after 4.5 and 9 hours of development and there was a slight increase in p-galactosidase activity at 9 hours. Cells transformed with pDdGall7 had similar levels of p-galactosidase activity to the Ax2 cells (Fig. 8). These results indicate that the 1 kbp upstream region was sufficient to promote expression of p galactosidase. 12 Figure 8. p-galactosidase expression under the control of the 1 kb 5' untranslated region of the rapl gene. Cell free extracts were prepared from the RaplGal transformant cells at the indicated times and from Ax2 cells and pDdGall 7 cells. The height of the bar represents the mean and the error bars represent the standard deviation of P-galactosidase specific activity (nmoles of substrate hydrolyzed/ min/mg of protein) from four determinations for the RaplGal and Ax2 cells. The pDdGall 7 cells were analyzed only twice and no error is shown. Altered morphology of vegetative amebae induced by increased expression of the Dictuostelium discoideum rapl gene Introduction Rapl proteins have been implicated in diverse roles in various cell types. Kitayama et ah, (1989) isolated a rapl cDNA, Krev-1, based on its ability to suppress the transformed phenotype of K-ras transformed NIH 3T3 cells. Flattened, more adherent cells with reduced tumorigenicity were isolated following fransfection with the rapl cDNA. Rapl also prevents Ras induced germinal vesicle breakdown when microinjected into Xenopus laevis oocytes (Campa et ah, 1991). However, in other cell types, Rapl has effects that are not antagonistic to Ras. Microinjection of RaplB into Swiss 3T3 cells induces DNA synthesis and affects cell morphology (Yoshida et al., 1992). In S. cerevisiae, the Rapl homologue RSR1/BUD1 is required for control of bud orientation (Bender and Pringle, 1989), whereas Ras regulates adenylate cyclase activity (Kataoka et al., 1985; Toda et al., 1985). In addition RaplB has been identified in platelets (Siess et al, 1990) and upon platelet activation, it becomes phosphorylated by protein kinase A and associated with the cytoskeleton (Kawata et al, 1989; Fischer et al, 1990). Rapl has also been implicated in the oxidative burst process in B lymphocytes (Maly et al, 1994). In an attempt to identify a role for Rapl in D. discoideum, the consequences of expressing high levels of Rapl protein in D. Discoideum were studied. Effect of Rapl overexpression on cell morphology To determine the effects of high levels of the Rapl protein in -vegetative D. discoideum, the rapl cDNA was expressed from a folate repressible discoidin promoter. The rapl cDNA was cloned downstream of the discoidin promoter in the pVEII vector (Blusch et al, 1992) as described in Materials and Methods (Fig. 9) and the expression vector was introduced into D. discoideum by calcium phosphate precipitate transformation. Transformants were grown either in the presence or absence of folate and increased expression of the Rapl protein was detected by western blot analysis using the anti-Rapl specific antibody. Three of three transformants analyzed showed inducible expression of the Rapl protein (Fig. 10) (The increase in Rapl expression observed in the folate containing samples compared to Ax2 was due to incomplete repression by folate at high cell densities). Preliminary observations indicated that under inducing conditions the Rapl overexpressing transformants had an altered cell morphology not observed in the parental Ax2 cells (See Fig. 25 for AX2 morphology) and also failed to round up after HL5 stimulus (data not shown). Analysis of additional transformants derived independently, showed that 6/7 transformants exhibited an impaired response to HL5 stimulation. A transformant designated Rapl was selected for detailed analysis. As a control strain, a transformant containing pVEII Gal, which expresses (3 galactosidase under the control of the discoidin promoter, was generated. To examine the effect of overexpression of the rapl gene on cell morphology, Rapl was plated on a glass coverslip and incubated for 15 hours in conditioned medium or in HL5 with folate. Conditioned medium contains a pre-starvation factor (PSF) (Clarke et al, 1987; Clarke et al, 1988), which enhances expression from the discoidin promoter (Rathi et al, 1991). Rapl grown in conditioned medium contained morphologically aberrant cells which were absent when Rapl was grown in HL5 in the presence of folate (Fig 11). The aberrant cells were flat and spread out with occasional dark regions located around their periphery. 63 Figure 9. The pVEII Rapl expression vector The full length rapl cDNA EcoRI fragment was ligated into the modified pVEII vector in the sense orientation as described in the Materials and Methods, recreating the EcoRI sites (RI). The discoidin promoter transcriptional start site is indicated by the arrow. The 5' EcoRI junction sequence and the ATG of the initiating methionine of rapl are underlined. 12 3 4 5 6 7 - 80 -32.5 "*-18.5 Figure 10. Expression of Rapl protein in cells transformed with the pVEII Rapl vector Total protein (10 //g) from Ax2 cells (lane 1) and three independent transformants (lanes 2 and 3; 4 and 5, 6 and 7 respectively ) grown in HL5 medium to a density from 1x10° to 3x10° cells/ml either in the presence of folate (lane 2, 4 and 6) or the absence of folate (lanes 1, 3, 5 and 7) were subjected to SDS-PAGE and transferred to nitrocellulose. The blot was reacted with an anti-Rapl peptide antibody and the signal was detected by ECL as described in the Materials and Methods. The molecular masses of the size markers are indicated in kDa. The level of Rapl protein in Ax2 cells is unaffected by folate or conditioned media (S. Louis and M. Khosla, unpublished observations) 65 cell size Figure 11. Morphology of vegetative Rapl cells. Dark field micrographs of Rapl cells on a glass coverslip after (A) growth in conditioned medium for 15 hours and (B) growth in the HL5 medium containing 1 mM folate (B). (C) Comparison of the relative cell areas (n = 54 cells for inducing conditions and n = 81 for non-inducing conditions). Solid bars, cells under inducing conditions; hatched bars, cells under non-inducing conditions. Cell size is shown in arbitrary units. Ax2 cells grown in conditioned media have a similar appearance to the Rapl cells grown in the presence of folate (see Fig. 25) The mean and distribution of the relative cell areas were determined from digitized photographs of uninduced cells and cells induced with conditioned medium. The results from a representative analysis is shown in Figure 11 C. Uninduced cells showed a moderate distribution of cell sizes, while induced cells showed a broader distribution, ranging from normal to highly spread. The difference between the mean areas of cells grown under inducing and non-inducing conditions was statistically significant (p<0.01). Analysis of forward light scatter of Rapl cells growing in suspension under inducing conditions showed changes in the distribution of forward light scatter with a small but significant decrease in the mean (P<0.01) compared to Ax2 cells (Fig 12). These results indicate that although the light scattering properties of the cells are altered, cell volumes are not significantly increased, suggesting that the increased size on plastic is due to a spreading of the cells. Time course analysis of Rapl protein levels and cell morphology after induction of the discoidin promoter To determine if there was a correlation between levels of Rapl protein and the number of cells with an abnormal appearance, both parameters were analyzed at different times during induction with conditioned medium (Figs. 13 and 14). Western blots probed with an anti-Rapl peptide antibody showed a small increase in the levels of Rapl protein from Rapl cells after 8 hours of induction compared to the folate treated control prior to induction. The levels of the Rapl protein increased further after 16 and 24 hours of treatment with conditioned medium. When the cells were treated with conditioned medium, the number of abnormal cells was significantly increased after 8 hours (p<0.01) and reached a maximum after 24 hours (Fig. 13). 67 A B C D Figure 12. Forward and side light scatter analysis of vegetative Rapl cells. Ax2 (A and B) and Rapl cells (C and D) were analyzed for forward (x-axis) and side scatter (y-axis) (A and C). Shown is a single parameter histogram plotting cell number (Y axis) versus forward light scatter of Ax2 (B) and Rapl cells (D) in H L 5 . 3 0 % n Folate 4Hrs 8 hrs 16 hrs 24 hrs Figure 13. Time course analysis of cell morphology after induction of the discoidin promoter Rapl cells were incubated in HL5 medium containing 1 mM folate (folate) or in conditioned medium for the indicated times and the number of cells with a flat spread morphology was determined for 6 separate fields. Between 200 and 600 cells were analyzed for each timepoint. The height of the bar represents the mean and the error bars represent the standard deviation. Figure 14. T i m e course analysis of R a p l protein upon induc t ion of the discoidin promoter Total protein (10 //g) from the cells incubated in H L 5 in the presence of folate (lane 1) and in conditioned med ium (lanes 2-5) for 4 h (lane 2), 8 h (lane 3), 16 h (lane 4), and 24 h (lane 5) were subjected to S D S - P A G E and transferred to nitrocellulose. The blot was reacted wi th an ant i -Rapl peptide ant ibody and the signal was detected by E C L as described in the Materials and Methods. The molecular masses of the size markers are indicated in kDa. Localization of F-actin To determine if the observed morphological changes of the rapl transformant cells were associated with alterations of the cytoskeleton, the cells were fixed and stained with FLTC-phalloidin to visualize the distribution of F-actin. Under inducing conditions, abnormal Rapl cells had pronounced peripheral actin staining extending around most of the circumference of the cell (Fig. 15 A to D). The actin staining appeared to coincide either with flattened regions or thin ridges at the periphery of the cell that could be seen in the Nomarski image. In contrast, actin was distributed in clumps at the edges of uninduced Rapl cells (Fig. 15 E and F). This is consistent with previous reports that showed that F-actin in vegetative D. discoideum is typically punctate in distribution and present in pseudopods during cell translocation (Rubino et al, 1984; Hall et al, 1988). Effects of Rapl expression on growth ^ The Rapl cells grew slowly jvvith a doubling time of 12.0 hours when grown in HL5 medium without folate compared to a doubling time of 8.2 hours in the presence of folate (Fig. 16). The parental Ax2 cells had a doubling time of 8.6 hours. In addition, it was observed that the Rapl cells clumped together when grown in shake suspension at densities between 1x10° and 5x10^ and this cell-cell adhesion could be disrupted by the addition of 10 m M E D T A (data not shown). A quantitative analysis of the Rapl cells by the Gerish laboratory confirmed the observation of increased cell-cell adhesion and demonstrated that the cell-cell adhesion was disrupted by 10 m M E D T A (Fig 17). 71 Of V . > - > 0 e IK Figure 15. Localization of F-actin in Rapl cells. Rapl cells on glass coverslips were incubated for 24 hours in conditioned medium (A to D) or in H L 5 medium containing 1 mM folate (E and F). Cells were fixed and stained with FITC-phalloidin and photographed using fluorescence microscopy (A, C and E) and Nomarski optics (B, D and F). Bar = 30/im. 1 0 8 io5-£ • 1 • 1 • 1 0 20 40 60 80 time (hours) Figure 16. Growth of the Rapl transformant Ax2 (O) or Rapl ( • , • ) cells were washed twice in BS buffer and inoculated into HL5 medium ( O, • ) or P1L5 medium containing 1 mM folate (• ) at a starting density of 5x10^ . The line of best fit for cells growing exponentially between 1x10^  and 5x10^  is shown. 0 TO 20, 30 40 50 60 [min] B 0.8 0.6 -0.4 -0 ' 10 20 30 40 50 60 [min] Figure 17. Cell-cell adhesion properties of the Rapl cells Rapl (V, • )and Ax2 cells ( ° , • ) were (A) grown in HL5 medium to 2xl()6 cells/ ml and (B) developed for 6 hours in shake suspension and then assayed for cell agglutination by measuring light scattering with an agglutometer in the presence ( • , • ) and absence ( V , ° ) of 10 mM EDTA. This data was provided by J. Faix of the Gerish laboratory. The response of cells to azide treatment It has been shown previously that wild type D. discoideum cells round up and detach when treated with azide, but that the effect of azide is abrogated in cells with a myosin II gene disruption (Patterson and Spudich, 1995). Since the morphological alterations induced by the expression of Rapl are associated with changes in the cytoskeleton (Rebstein et al., 1993), the response of Rapl expressing cells to azide was studied. It was observed that 57 % of wild type Rapl cells remained attached to the substratum after treatment with azide in contrast to the Ax2 cells which almost all detached from the substratum (Fig. 18). Determination of the number of nuclei in transformed cells The abnormal cell morphology of the activated Rapl cells was similar to that observed for the myosin II heavy chain and profilin mutants that exhibit defects in their ability to undergo cytokinesis (De Lozanne and Spudich, 1987; Knecht and Loomis, 1987; Haugwitz et al, 1994). The number of nuclei in the Rapl transformant cells was therefore determined. There was no increase observed in the number of cells with multiple nuclei. Both the Rapl transformant and the Ax2 cells had an average of 1.2 nuclei per cell. Cell motility analysis Since the vegetative Rapl transformant cells appeared to have an abnormal morphology with F-actin distributed around the cell periphery and were also impaired in their ability to regulate their morphology in response to a nutrient signal, the ability of the cells to translocate was evaluated. A field of cells was photographed and then rephotographed after a 30 minute interval (Fig 19). Both normal sized and highly spread Rapl cells were motile and the distances that they had translocated appeared similar to the distances translocated by the pVEII Gal transformant. However, a preliminary, more quantitative analysis by the Gerish laboratory using computer video analysis, suggested that there may be a slight reduction in the rate of cell translocation (personal communication). Analysis of morphology after HL5 stimulation When D. discoideum cells are transferred from starvation to growth conditions they rapidly round up and detach from the substratum (Schweiger et al., 1992; Howard et al, 1993). To determine whether Rapl cells were capable of this modulation of cell shape, Rapl cells and the parental Ax2 cells were plated on a glass coverslip and starved for 8 hours in BS buffer. The Rapl cells had a flat and spread appearance after starvation while the Ax2 cells had an elongated appearance characteristic of starved cells (Fig. 20 A and C respectively). The distribution of the relative areas of the starved cells was determined (Fig. 20 E), and the mean of the relative cell areas was significantly larger in Rapl cells compared to the Ax2 parental strain (p<0.01). Cells were then stimulated by replacing the BS buffer with nutrient HL5 medium. After 20 minutes a limited number of Rapl cells had rounded up, while all the Ax2 cells had rounded up (Fig. 20 B and D respectively). A time course analysis showed that all the Ax2 cells had rounded up by 10 minutes whereas the response for Rapl cells was strongly inhibited, with 29% of the cells rounded up after 20 minutes (Fig. 20 F). Figure 18. The effect of treating cells with azide Adhe ren t vegetative A x 2 (A and B), and Rapl (C and D) cells were incubated in conditioned m e d i u m for 24 hours (A, C,) and then treated with 2 m M s o d i u m azide in HL5 for 3 minutes and washed 3 times to remove detached cells (B, D). Figure 19. The motility of vegetative cells. Rapl cells (A) and pVEII Gal cells (B) were photographed at 30 minute intervals with dark field optics. The cell outlines were manually traced and the figures superimposed. The red outline indicates the original position of the cell and the blue outline indicates cell position after 30 minutes. Figure 20 Effect of HL5 stimulation on Rapl and Ax2 cells. Rapl (A, B) and the parental Ax2 cells (C, D) were plated at 3x10^ ' cells / cm2 on glass coverslips, incubated 24 hours in HL5 medium and then starved for 8 hours in BS buffer (A, C). HL5 nutrient medium was introduced for 20 minutes (B, D). (E) The relative cell areas of the cells prior to HL5 stimulation were determined. Solid bars, Rapl cells; hatched bars, Ax2 cells. Cell size is given in arbitrary units (n = 52 cells for Rapl and n = 94 for Ax2, respectively). (F) The time course of cell rounding in response to HL5 stimulation. 40 to 100 cells were counted at the indicated times and the percentage of round Rapl cells ( •) and Ax2 cells ( • ) was determined. Localization of F-actin in cells treated with HL5 after starvation To observe whether the HL5 stimulus altered the distribution of F-actin in cells with a flat spread appearance, the cells were fixed and stained with FLTC-phalloidin. After the reintroduction of HL5 medium, flat spread Rapl cells still exhibited pronounced peripheral actin staining (Fig. 21 A and B) which appeared to coincide with a highly flattened region around the edge of the cell that could be seen in the Nomarski image. By contrast the HL5 stimulated Ax2 cells were completely round with localized actin staining (Fig. 21C and D). Pattern of tyrosine phosphorylation of actin after HL5 stimulation The rounding of starved D . discoideum cells after the reintroduction HL5 medium correlates with a rapid tyrosine phosphorylation of a single major 45 kDa band previously shown to be actin (Schweiger et al, 1992). Since the Rapl cells did not respond to the HL5 stimulus, tyrosine phosphorylation was examined by western blot with an anti-phosphotyrosine antibody (Fig. 22). A dramatic increase in phosphorylation of a single major 45 kDa band (band A) was observed in both Rapl and Ax2 cells within 5 minutes after HL5 stimulation (Fig. 22 A and B, respectively). This phosphorylated band increased in intensity for 25 minutes after HL5 stimulation in both Rapl and Ax2 cells. This 45 kDa band corresponded to a prominent band in a Ponceau S stain of total protein on the same nitrocellulose blot and is likely to be actin. These data indicate that although tyrosine phosphorylation of actin may be necessary for cell rounding, it is not sufficient for it to occur. Some minor differences in the pattern of other tyrosine phosphorylated proteins were observed upon starvation of Rapl compared to the Ax2 cells (Fig 22 lane S panels A and B, respectively). Figure 21. Localization of F-actin in starved and H L 5 stimulated cells. Rapl cells (A and B) and the control A x 2 cells (C and D) were fixed 20 minutes after refeeding, stained w i t h F ITC-pha l lo id in and then photographed us ing fluorescence microscopy (A and C) and N o m a r s k i optics (B and D) . Bar = 30fim. Figure 22. Protein tyrosine phosphorylation after FIL5 stimulation. Total protein (10 Mg) from Rapl (A) and Ax2 cells (B) were subjected to SDS-PAGE and transferred to nitrocellulose. The protein was from vegetative cells (V), cells starved for 8 hours (S) and then exposed to HL5 medium for 5 min (5), 10 min (10), 15 min (15), 20 min (20) and 25 min (25). The blot was reacted with an anti-phosphotyrosine antibody and the signal was detected by ECL as described in the Materials and Methods. The molecular masses of the size markers are indicated in kDa. The letter A indicates the putative actin band. S 5 10 15 20 25 - 3 2 . 5 - 2 7 . 5 These differences have not been analyzed further. Analysis of the erasure response of Rapl cells Developing wild type cells are not committed to development and the reintroduction of nutrients leads to a return to normal growth. As the Rapl cells did not morphologically respond to the HL5 nutrient medium, it was possible that they would be unable to return to normal growth after initiating development. However, starvation for 8 hours did not affect the viability of the Rapl transformants. Both Rapl and control pVEII Gal transformant cells were fully viable and formed colonies at almost 100% efficiency in a colony formation assay. After nutrients have been reintroduced to cells, they retain the capacity to rapidly recapitulate development if starved again. However, after 2 hours in nutrients, this ability is rapidly erased by a mechanism that involves mRNA degradation (Soil and Waddell, 1975; Waddell and Soli, 1977; Kraft et al., 1989; Chandrasekhar et al, 1990). This 'erasure' response to nutrients was tested in the Rapl strain (Table 2). Rapl and pVEII Gal control transformant cells were allowed to develop in shake suspension for 8 hours and then HL5 medium was reintroduced for either 1 or 2 hours before plating the cells for development on filters. If cells were exposed to HL5 medium for 1 hour, fruiting bodies were observed after an additional 15 hours, while exposure to HL5 medium for 2 hours delayed the onset of the appearance of fruiting bodies by 3 additional hours. The erasure response was identical for both the Rapl cells and the pVEII Gal control transformant. Table 2. Erasure of the capacity for rapid developmental recapitulation3 HL5 exposure Rapl 1 2 pVEII Gal 1 2 Time 15 + - ± 17 + - + 19 + + + ± 21 + + + + a Transformant cells were starved for 8 hours and then returned to HL5 medium for 1 or 2 hours. The time of the appearance of fruiting bodies after plating cells on filters is indicated. + indicates that part of the population had formed fruiting bodies and + indicates the entire population had formed bruting bodies. Identification of conserved residues of the Dictyostelium discoideum Rapl protein required to alter cell morphology Introduction The tumor suppressor activity of human RaplA has been previously analyzed by site-directed mutagenesis and some of the amino acids necessary for the response were identified (Kitayama et al, 1990). Mutations previously shown to activate Ras enhanced tumor suppression by RaplA, and mutations in the effector domain which blocked Ras activation also blocked tumor suppression by RaplA. By contrast, in an analysis of the role of Rapl in oxidative burst in B lymphocytes, both activating and dominant negative mutations had an inhibitory effect, suggesting that in some cases, Rapl is required to cycle between GTP and GDP bound forms (Maly et al, 1994). Increased expression of the Rapl protein in vegetative D. discoideum cells from the discoidin gene promoter correlated with a flattened and spread cell morphology and an inhibition of morphological responses to external stimuli. To further analyze the role of Rapl in these processes, site-directed mutations of specific amino acids in the D. discoideum Rapl protein were generated. Amino acids conserved between the Rapl proteins or conserved between both the Rapl and Ras proteins were selected for mutagenesis and transformants overexpressing these mutated proteins were assessed for changes in their cell morphology and their responses to external stimuli. The effect of mutated rapl genes on cell morphology Site-directed mutations were constructed in the rapl gene which would encode proteins with following alterations G10V, G12V, S17N, D38E, D38N, T61Q and F156L (the first letter indicating the original amino acid, the number indicating the position of the amino acid in the protein sequence and the second letter indicating the mutated amino acid). The mutated rapl genes were cloned into the modified pVEII vector as described in the Materials and Methods (Fig. 23) and D. discoideum transformants were generated. None of the transformant clones expressing the G10V or S17N Rapl mutant proteins exhibited an abnormal morphology, while most of transformant clones expressing G12V, D38E, D38N, T61Q, and F156L Rapl mutant proteins contained cells that exhibited the flat and spread morphology (Table 3). To study transformants containing mutated Rapl proteins, a representative transformant which showed a significant proportion of abnormal cells was chosen. For the transformants expressing G10V and S17N mutant proteins (which did not exhibit the abnormal morphology), the transformant clone expressing the highest level of Rapl protein was identified by western blot analysis and selected for further analysis (Fig. 24). The appearance of the selected transformants is shown in Figure 25 and the proportion of cells with a flat spread morphology is shown in Table 4. The transformants Kapl-GUV, Rapl-D38N, Rapl-D38E, Rapl-T61Q and Rapl-F156L resembled the wild type Rapl transformant (compare Fig. 25, panels B, D, E, F, and G, respectively with H). In contrast, transformants Rapl-GlOV and Rapl-S17N resembled the parental Ax2 cells (compare Fig. 25 panels A and C respectively with I). The Rapl-G12V, Rapl-D38E, Rapl-T61Q and Rapl-F156L transformants (Fig. 26 A, lanes 4, 7, 8 and 9) expressed similar levels of Rapl protein to the wild type Rapl transformant (lane 1) while the transformant Rapl-D38N expressed somewhat more Rapl protein (lanes 6). As mentioned above, .86 S17N G12V D38N G10V D38E T61Q F156L Figure 23. Site-directed mutations of conserved amino acids of Rapl The positions of the introduced mutations are indicated (the first letter indicating the original amino acid, the number indicating the position of the amino acid in the protein sequence and the second letter indicating the mutated amino acid), the hatched boxes indicate regions proposed to be required for guanine nucleotide binding and hydrolysis, and the stippled box indicates the proposed effector domain. The arrow indicates the start of transcription from the discoidin promoter. Table 3. The effect of site-directed mutations in the rapl gene on cell morphology Mutation Clones showing cells with an abnormal cell morphology3 G10V 0/4 G12V 4/4 S17N 0/4 D38E 6/6 D38N 5/5 T61Q 6/7 F156L 9/10 none 4/6 3 Cells were transformed with mutated rapl genes under the control of the discoidin promoter. Independent clones were isolated and examined. The number of transformants with a flat spread morphology was determined by observing dark field micrographs of 3 independent fields. The transformed clones were categorized as having an abnormal cell morphology if >5% of the cells were flattened and spread. A 1 2 3 4 5 B 1 2 3 4 5 H u w H w - 32.5 - 27.5 - 18.5 - 32.5 - 27.5 I - 18.5 Figure 24. Expression of Rapl protein containing G10V and S17N substitutions A number of different transformants expressing Rapl proteins with substitutions G10V (A) and S17N (B) were incubated in conditioned medium for 24 hours. Cells were lysed in 1% SDS and 15//g of total cell protein was separated by SDS-PAGE, transferred to nitrocellulose and probed with an anti-Rapl peptide antibody. Lanes 1-4 represent independent transformants and lane 5 is the parental Ax2 strain. The molecular masses of the size markers are indicated in kDa. Figure 25. Cell morphology of transformants expressing mutated Rapl protein. Rapl-GWV (A), Rapl-GUV (B), Rapl-S17N (C), Rapl-D38E (D), Rapl-D387V (E), Rapl-T61Q(F), Rapl-F156L (G), and Rapl transformants (H) and the parental Ax2 cells (I) were photographed with dark field optics after incubation with conditioned media for 24 hours. Table 4. The proportion of vegetative cells with a flat spread morphology. Cell Type Flat Spread Cells3 (%) Rapl-GlOV 3 ± 2 (4) Rapl-GUV 18 ±4 (2) Rapl-S17N 2 + 1 (4) Rapl-D38E 17 + 5 (2) Rapl-D38N 14 + 5 (2) Rapl-T61Q 18 ±3 (3) Rapl-F156L 17 + 5 (2) Rapl 18 + 3 (6) Ax2 3 + 0 (6) a The number of flat spread cells in each transformant was determined by dark field microscopy of living cells in three separate fields of approximately 50 cells per field. The number of independent experiments is indicated in the brackets. The mean and standard error of the mean are shown. A B 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 - 8 0 - 8 0 -49 .5 -49.5 -27.5 -27.5 Figure 26. Rapl protein expression under inducing conditions. (A) Cells were incubated in shake suspension in conditioned medium for 24 hours and then (B) starved for 8 hours. Ax2 (lane 1), Rapl (lane 2), Rapl-GlUV (lane 3), Rapl-G12V (lane 4), Rapl-S17N (lane 5), Rapl-D38N (lane 6), Rapl-D38E (lane 7), Rapl-T61Q (lane 8) and Rnpl-F156L (lane 9) cells were lysed in 1% SDS and 15 //g of total cell protein was separated by SDS-P A G E , transferred to nitrocellulose and probed with an anti-Rapl peptide antibody. The molecular masses of the size markers are indicated in kDa Rapl-GlOV and Rapl-S17N were selected based on their expression of high levels of Rapl protein (lanes 3 and 5). F-actin distribution in transformed cells The effect of the mutated Rapl proteins on the distribution of F-actin was also evaluated since cells expressing Rapl showed an unusual pattern of F-actin staining. Rapl-GlOV and Rapl-S17N transformant cells were similar in appearance to Ax2 cells with punctate actin present at the cell periphery while the enlarged Rapl-GUV, Rapl-D38E, Rapl-D38N, Rapl-T61Q, Rapl-F156L and wild type Rapl transformant cells possessed regions of contiguous F-actin staining at the cell periphery (Fig. 27). Analysis of morphology after HL5 stimulation As shown in the previous section, Rapl overexpression impaired the ability of cells to change their morphology in response to the reintroduction of nutrients after the onset of starvation. The ability of the mutated Rapl proteins to similarly impair changes in cell morphology was therefore evaluated. Starvation for 8 hours did not appreciably change the levels of Rapl proteins from that observed in cells prior to starvation (compare Fig. 26 panel B with A). The Rapl-GlOV, Rapl-S17N and parental Ax2 cells rapidly rounded up and appeared retractile 5 minutes after the reintroduction of HL5 medium (Fig. 28 panels A, C and I, respectively). In contrast, many Rapl-GUV, Rapl-T61Q and wild type Rapl transformants cells remained flat and nonrefractile (Fig. 28 panels B, F and H, respectively). The Rapl-D38E, Rapl-D38N and Rapl-F156L transformants exhibited an intermediate phenotype with some refractile cells, some non-responding cells and some cells which appeared to contract but not become refractile (Fig. 28 panels D, E and G * E e ^| 1 1 1=! U % Figure 27. Cell morphology and F-actin distribution. Rapl-GlOV (A), Rapl-G12V (B), Rapl-SUN (C), Rapl-D38E (D), Rapl-D38N (E), Rapl-T61Q (F), Rapl-F156L (G), and Rapl transformants (H) and the parental Ax2 cells (panel I) were incubated in conditioned media for 24 h, fixed, stained with FITC phalloidin and photographed using fluorescence microscopy as described in the Materials and Methods. 94 9 i * D . f ** Figure 28. The response of starved cells to the reintroduction of HL5 media. Adherent cells on a glass coverslip were treated with conditioned media for 24 hours, starved for 8 hours in BS buffer, and then exposed to HL5 media for 5 minutes. The transformants analyzed were Rapl-GlOV (A), Rapl-GUV (B), Rapl-SUN (C), Rapl-D38E (D), Rapl-D38N (E), Rapl-T61Q (F) and Rapl-F156L (G), Rapl (H) and the parental Ax2 strain (I). respectively). To quantitate the response, the proportion of rounded refractile cells was determined 5 and 10 minutes after HL5 stimulation (Fig. 29). The Rapl-T61Q transformant was not significantly different from wild type Rapl transformant while the Rapl-G12V transformant exhibited a significant decrease in the proportion of responding cells (P<0.05). Almost all of the Rapl-GlOV, Rapl-SI7N and Ax2 cells were round and refractile after 5 minutes. The Rapl-D38E, Rapl-D38N and Rapl-F156L transformant cells exhibited an intermediate response, which was less than the Ax2 response but greater than the Rapl transformant response. The response of cells to azide treatment Wild type D. discoideum cells round up and detach when treated with azide, but the effect of azide is abrogated in cells overexpressing Rapl. Rapl-GUV and Rapl-T61Q transformant cells were highly resistant to azide treatment like the wild type Rapl transformant, while the Rapl-GlOV, Rapl-S17NRapl-D38E Rapl-D38N and Rapl-F156L transformant cells were considerably less sensitive to azide treatment (Table 5). Growth and development The Rapl-G12V, Rapl-S17N and the Rapl transformant grew more slowly than the Ax2 strain in the absence of folate (Table 6). The reduced growth rate did not correlate with increased cell-cell adhesion as the Rapl-GUV but not the Rapl-S17N transformant exhibited increased cell-cell adhesion (data not shown). The addition of folate increased the growth rate in all transformants but only the Rapl transformant grew at the same rate as Ax2 under these conditions. All transformants developed normally, both on filters and when plated on bacteria. > o > CM co LU CO ro Q Z co CO Q CO CO li"> ctf cr CM < Figure 29. The percentage of cells responding to the reintroduction of HL5 medium after 5 and 10 minutes. The number of round refractile cells on three plates was determined 5 min (black bars) and 10 min (hatched bars) after exposure to HL5 medium. The number of cells assessed at each time ranged from 125 to 445. The height of the bar represents the mean and the error bars represent the standard error of the mean from two independent experiments. Table 5. The percentage of cells remaining adherent after treatment with azide. Cell type % adherent cells3 Ax2 3± 1 (3) Rapl 57 + 10 (3) Rapl-GlOV 9± 4 (2) Rapl-GUV 66 + 14 (2) Rapl-S17N 1± 0 (2) Rapl-D38E 20 + 11 (2) Rapl-D38N 12+ 0 (2) Rapl-T61Q 85 + 15 (2) Rapl-F156L 24± 6 (2) 3 Mean + standard error of the mean are shown. The number of experiments is shown in brackets. Between 200-600 cells were present in the field of view prior to azide treatment. Table 6. Generation times of transformants expressing mutated Rapl proteins . Cell type Generation time (hr)a + folate -folate Ax2 ND 8.6 (0.98) Rapl 12.0 (0.92) 8.2 (0.98) Rapl-GUV 10.4(0.98) 9.6(0.98) Rapl-Sl 7N 14.3 (0.94) 11.0 (0.98) aGeneration times were determined as described in the Materials and Methods for populations between the densities of 1x10^  to 5x10^  cells/ ml since growth within this range was observed to be exponential. The R^ for the line of best fit is shown in brackets. Activation of the rasG gene alters cell morphology in D. discoideum Introduction Microinjection of an activated Ras protein into mammalian cells induces a characteristic transformed cell phenotype (Stacey and Kung, 1984; Feramisco et al, 1984; Bar-Sagi and Feramisco, 1986; Lloyd et al, 1989). The Ras protein induces morphological changes and increases membrane ruffling, reduces stress fiber formation and decreases the formation of focal adhesions (Feramisco et al., 1984; Stacey and Kung, 1984; Bar-Sagi and Feramisco, 1986). These effects of activated Ras on ruffling, stress fibers and focal adhesions are mediated by the ras-related Rac and Rho proteins, which remodel the actin cytoskeleton (Ridley et al, 1992; Ridley and Hall, 1992). D. discoideum expresses five ras genes during growth and development (Reymond et al, 1984; Robbins et al, 1989; Daniel et al, 1993b; Daniel et al., 1993a). One of these genes, rasG, is expressed in vegetative cells and encodes a protein which shares 69% overall amino acid identity with the human H-ras gene product (Robbins et al., 1989). Given the effects of activated Ras on mammalian cell morphology and the alteration of cell morphology by the D. discoideum Ras-related protein Rapl, it was of interest to determine the effects of "activated RasG on D. discoideum cell morphology. The effect of RasG protein on D. discoideum cell morphology Transformants generated by M. Khosla (Khosla et al, 1995) with increased levels of RasG, activated RasG-G12T and the putative dominant negative RasG-S17N proteins were analyzed for alterations in the regulation of cell morphology. Under inducing conditions, many RasG-G12T cells exhibited a flattened and spread out morphology with occasional dark regions around their periphery (Fig. 30 panel B). In contrast, RasG cells exhibited no differences in morphology compared to the parental Ax2 cells (panels A and D). It was observed that 30% of the RasG-G12T cells were abnormally flat and spread out, while 3-4% of the RasG, RasG-SUN and Ax2 cells were somewhat flat and spread out (Table 7). The RasG-S17N cells were more elongated than the Ax2 cells (Fig. 30 panel C). Under inducing conditions, higher RasG protein levels were present in the RasG, RasG-G12T and RasG-SUN transformants compared to the parental Ax2 cells (Fig. 31 panel A) with the RasG transformant expressing more protein than the other two transformants. Analysis of forward light scatter of RasG-G12T cells growing in suspension under inducing conditions showed changes in the distribution of forward light scatter with a significant decrease in the mean (P<0.01) compared to Ax2 cells (Fig. 32). These results indicate that although the light scattering properties of the cells are altered, cell volumes are not significantly increased, suggesting that the spread appearance of adherent cells may be due to a change in morphology and not an increase in cell volume. The localization of F-actin in D. discoideum cells expressing RasG To determine if the observed morphological changes correlated with alterations in the actin cytoskeleton, cells were stained to visualize the distribution of F-actin. RasG-G12T cells with an abnormal morphology exhibited pronounced contiguous peripheral F-actin staining while RasG and RasG-S17N cells exhibited a punctate peripheral F-actin that was characteristic of wild type Ax2 cells (Fig. 33) (see Fig. 27 for Ax2). F i g u r e 30. M o r p h o l o g y of vegeta t ive cells RasG ( A ) , RasG-GUT (B) , RasG-SUN ( C ) a n d A x 2 ( D ) c e l l s w e r e p h o t o g r a p h e d w i t h d a r k f i e ld op t i c s after i n c u b a t i o n w i t h c o n d i t i o n e d m e d i a for 24 hou r s . Figure 31. Induction of RasG protein expression RasG (lane 1) RasG-GUT (lane 2), RasG-SUN (lane 3) and Ax2 (lane 4) cells were incubated in shake suspension in conditioned medium for 24 hours (A) and then starved in suspension in Bonner's salts buffer for 8 hours (B). Cells were Iysed in 1% SDS and 15 ;/g of total cell protein was separated by SDS-PAGE, transferred to nitrocellulose and probed with an anti-RasG antibody. The molecular masses of the size markers are indicated in kDa. Figure 32. Forward and side light scatter analysis of RasG-GllT cells. Ax2 (A and B) and RasG-GllT cells (C and D) were grown in conditioned media for 24 hours and then analyzed for forward (x-axis) and side scatter (y-axis) (A and C). Shown is a single parameter histogram plotting cell number (Y axis) versus forward light scatter of Ax2 (B) and RasG-GlTT cells (D) in H L 5 . The Ax2 data is reproduced from Figure 12. Table 7. The proportion of cells with a flat spread morphology. Cell Type Flat Spread Cells3 (%) rasG 3 + 1 msG-Gm 30 + 2 rasG-S17N 4 ± 2 Ax2 3 + 1 3 The number of flat spread cells was determined in two independent experiments each consisting of three to five separate fields of approximately 50 cells per field. Cells were viewed with dark field microscopy. The mean and standard error of the mean are shown. 105 B ^ i j | | i fj 11 Figure 33. Localization of F-actin in Ras transformed cells Vegetative RiisG cells (A and B), RasG-Gl2T (C and D) and RasG-S17N (E and F) were incubated in conditioned media for 24 hours, fixed, stained with fluorescein isothiocyanate phalloidin and then photographed using either Nomarski optics (A, C, and E) or fluorescence microscopy (B, D and F). The response of cells to azide treatment Wild type D. discoideum cells round up and detach when treated with azide, but this response is abrogated in cells that overexpress Rapl. Given the fact that the altered appearance and changed F-actin distribution of the RasG-G22Tcells resembled that of Rapl cells, the response of the RasG, RasG-G12T, and RasG-S17N transformants to azide treatment was evaluated. As demonstrated previously, Ax2 cells were sensitive to azide treatment and detached from the substratum after azide treatment, while less than 5% of the RasG and RasG-S17N cells remained adherent after this regime. In contrast, 34 % of the RasG-GUT cells remained adherent (Fig. 34 and Table 8). Determination of the number of nuclei in transformed cells The abnormal cell morphology of the activated RasG-Gl2T cells was similar to that observed for the myosin II heavy chain and profilin mutants that exhibit defects in their ability to undergo cytokinesis (De Lozanne and Spudich, 1987; Knecht and Loomis, 1987; Haugwitz et al, 1994). The number of nuclei in the rasG, rasG-G12T and rasG-S17N transformant cells was therefore determined. Multinucleate cells were frequently observed in both the RasG and the RasG-G12T transformants whereas the Ax2 and RasG-S17N cells were mononucleate under inducing conditions (Fig. 35). Repression of the discoidin promoter by folate abolished the multinucleate phenotype of rasG-G12T and RasG transformants (Fig. 35 and Table 9). Both the RasG and the RasG-G12T cells had an average of 1.7 nuclei/cell which was significantly different from the values for Ax2 (P<05). Figure 34. The effect of treating cells with azide Adherent vegetative RasG (A and B), RasG-GUT (C and D), RasG-S17N (E and F) and Ax2 (G and H) cells were incubated in conditioned medium for 24 hours (A, C, E, and G) and then treated with 2 mM Na azide in HL5 medium for 3 minutes and washed 3 times to remove detached cells (B, D, F and H). Table 8. The proportion of cells remaining adherent after treatment with azide Cell type % adherent cells3 RasG 2 ± 0 (2) RasG-G12T 34 ± 7 (3) RasG-S17N 0 + 0 (2) Ax2 3 + 1 (3) Approximately 200-600 cells were present in the field of view prior to azide treatment in each trial. The number of independent experiments is indicated in the brackets. The mean and standard deviations are shown. E Figure 35. Nuclear staining of Ras transformed cells RasG (A, D), RasG-GUT (B), RasG-S17N (C) and Ax2 (E) cells were incubated in conditioned media for 24 hours (A, B, C, E) or in HL5 containing ImM folate (D). The cells were fixed and stained with Hoechst dye as described in the Materials and Methods. Table 9. The number of nuclei per cell Number of nuclei3 Strain Induced13 Repressedc RasG 1.7 + 0.2 (5) 1.1 (1) RasG-GUT 1.7 + 0.1 (5) 1.0 + 0.0 (2) RasG-SUN 1.1 + 0.0 (4) 1.1 (1) Rapl 1.2 + 0.1 (5) 1.0 + 0.0 (2) Ax2 1.2 + 0.0 (7) 1.1 (1) 3 Mean and + standard error of the mean of the number of experiments shown in brackets. An average of 200 cells were analyzed per experiment. bCells were grown in conditioned HL5 medium for 24 h. cCells were grown in HL5 medium with 1 mM folate for 24 h. Analysis of morphology after HL5 medium stimulation In view of the similar abnormal morphology of the RasG-G12T cells and the Rapl transformant, the capacity of the cells to respond to the HL5 stimulation was examined. The morphologies of the starved RasG, RasG-G12T, RasG-S17N and Ax2 cells are shown in Figure 36. The Ax2 cells and the RasG-S17N cells had the elongate morphology characteristic of starved amebae. In contrast, many of the RasG-G12T cells retained the extensively flattened and spread morphology previously observed when induced during vegetative growth. The RasG cells, which had a normal appearance when induced during vegetative growth, became more flattened and spread upon starvation, although not to the same extent as the activated RasG-G12T transformant. After starvation for 8 hours, high levels of RasG protein were still present in all three transformants (Fig. 31 panel B). Within 5 minutes of the reintroduction of the HL5 nutrient medium, the Ax2 and RasG-S17N cells became round and retractile and detached from the substratum (Fig. 36). In contrast, the majority of the RasG-G12T cells did not exhibit this characteristic response although there was some change in their appearance. RasG cells did respond to the HL5 stimulus, but not as rapidly or completely as for the Ax2 strain. The percentage of responding cells was determined 5 and 10 minutes after HL5 stimulation (Fig. 37). It was observed that 59 % of the RasG-Gl 2 T cells failed to respond within 5 minutes of the reintroduction of HL5 medium and there was only a small additional response after 10 minutes. In contrast, the majority of Ax2 cells and RasG-SUN cells responded within 5 minutes. RasG cells exhibited an intermediate response (Fig. 37). Figure 36. The response of starved cells to HL5 medium. Adherent RasG (A and B), RasG-GUT (C and D), RasG-SUN (E and F) and Ax2 (G and H) cells were incubated in conditioned medium for 24 hours, starved for 8 hours in Bonner's salts (A, C, E and G), and then exposed to HL5 medium for 5 minutes (B, D, F and H). 113 _co H> O o a. CO cr 100 i 80 -6 0 -40 -20 0 + " S17N Figure 37. The percentage of starved cells that respond to HL5 stimulation. The percentage of round refractile cells was determined 5 min (black bars) and 10 min (hatched bars) after the reintroduction of HL5 medium to starved cells of the indicated transformant strains and Ax2. The height of the bar represents the mean number of round refractile cells and the error bar represents the standard error of the mean for 3 independent plates of cells from a representative experiment. For each determination 200-400 cells were counted. Expression of Rapl protein in RasG-GUT cells and expression of RasG in Rapl cells Given the similar effects of overexpression of activated RasG and Rapl proteins on cell morphology and the inhibition of the response of cell morphology to external stimuli, western blot analysis was performed to determine if expression of activated RasG resulted in increased Rapl protein levels and likewise if expression of Rapl resulted in increased RasG protein levels. Neither protein caused an increase in expression of the other protein (Fig. 38). 1 2 3 32.5-27.5-18.5-B 1 2 3 32.5-27.5-18.5 Figure 38. Expression of Rapl protein in RasG-G12T cells and expression of RasG in Rapl cells Ax2 (lane 1) RasG-G12T (lane 2), and %>7(lane 3) were incubated in shake suspension in conditioned medium for 24 hours. Cells were lysed in 1% SDS and 15 /ig of total cell protein was separated by SOS-PAGE, transferred to nitrocellulose and probed with a anti-RasG (A) or anti-Rapl peptide antibody (B). The molecular masses of the size markers are indicated in kDa. GENERAL DISCUSSION Organization and expression of the rapl gene DNA sequence analysis of the genomic fragment encoding the rapl gene showed that the gene is divided into two exons separated by a 232 bp intron. The position of the intron is not conserved relative to those of the D. discoideum rasG and rasD genes (Robbins et al., 1992; Reymond et al., 1984). The 5' and 3' non-coding sequences and the intron contain a high adenine/thymine content, which is similar to that for other D. discoideum genes (Kimmel and Firtel, 1983). Different rapl mRNAs are expressed during vegetative growth and development (Robbins et al., 1990). During vegetative growth and early development, a single 1.1 kb mRNA is expressed. After 6-8 hours of development, this mRNA species is replaced by two mRNA species of 1.0 and 1.3 kb (Robbins et al, 1990). Since the rapl genomic DNA sequence,did not reveal any alternative exons, the existence of distinct mRNA species during growth and development must be due either to transcriptional initiation from alternative start sites, to distinct mRNA polyA processing sites, or to a combination of the two. Within the highly adenine/ thymine rich 5' untranslated region of the rapl gene, there are several clusters of cytosines and guanines that may be potential transcription regulatory sequences. Cytosine/ guanine rich sequences termed G boxes have been shown to be binding sites for the G box factor (GBF), which is the only D. discoideum transcription factor to be cloned and well characterized to date (Schnitzler et al, 1994). However, GBF mediates activation of cAMP dependent gene expression subsequent to aggregation (Schnitzler et al, 1994; Schnitzler et al, 1995) and since the rapl gene is expressed both during growth and development, expression of the rapl gene is likely to be regulated by additional transcription factors in addition to possible regulation by GBF. The roles of the cytosine/guanine rich sequences identified in the 5' region of the rapl gene could be determined by analyzing the effects of deletions on expression from the promoter and also by examining the ability of GBF to bind to the promoter. The 5' rapl fragment isolated in this study was tested for the ability to drive expression of p-galactosidase activity in D. discoideum. p-galactosidase activity was detected in vegetative cells and thus the isolated 5' untranslated fragment was sufficient for promoter activity during growth. p-galactosidase activity was also detected after 9 hours of development. As the stability of the Rapl/ p-galactosidase fusion protein in vegetative cells was not studied, it is possible that vegetative p-galactosidase protein may have persisted into development, p-galactosidase activity expressed under the control of the RasD promoter is detectable in vegetative cells but lost by 4 hours of development (Esch and Firtel, 1991), suggesting that at least some forms of expressed p-galactosidase enzyme are fairly rapidly degraded during early development, p-galactosidase expressed from the rasD promoter is also moderately unstable during late development (Esch and Firtel, 1991). In contrast, p-galactosidase activity expressed from the cotB, SP60 and PsA promoters is stable when expressed late in development (Detterbeck et al., 1994). The variation in the stability of the p-galactosidase fusion proteins means that the isolated 5' fragment of rapl has not been conclusively shown to promote expression during development. The effect of Rapl overexpression on D. discoideum cell morphology High levels of the D. discoideum Rapl protein were expressed in vegetative cells in an inducible manner from the discoidin promoter. High levels of the Rapl protein correlated with the appearance of amebae that were abnormally flattened and spread whereas the cells appeared normal when Rapl expression was repressed by folate. It was observed that 18 % of the cells in the population exhibited the abnormal morphology. The cytoskeleton of the flat spread cells was altered with increased F-actin localized to flat lamellipodial regions of the cell periphery. Usually F-actin staining in D. discoideum is punctate with the most intensely staining regions in the pseudopods of migrating or chemotaxing cells (Rubino et al., 1984; Hall et al., 1988). Analysis of forward light scatter of cells in suspension suggested that the average cell size was not increased. Rapl cells with an abnormal morphology were still capable of movement, although the rate of cell movement may have been slightly reduced. These results suggest that although the cells were altered in appearance, processes underlying cell motility, such as pseudopod extension and attachment and detachment from the substratum were at most only slightly inhibited. Brief periods of exposure to azide cause D. discoideum cells to contract and detach from the substratum, and high levels of Rapl were found to reduce the response of cells to azide. The mechanism whereby Rapl acts to prevent the response to azide is not known. Azide disrupts respiration, causing a depletion of cellular ATP that is proposed to result in a 'rigor' contraction of the myosin filaments similar to that observed in skeletal muscle (Pasternak et al., 1989). Disruption of myosin II activity in D. discoideum cells has previously been shown to prevent this response (Pasternak et al, 1989; Springer et al, 1994; Patterson and Spudich, 1995) and the reduced azide response in cells that overexpress Rapl might be due to a specific alteration of the interaction of myosin II with the actin cytoskeleton. Alternatively, the effect of azide on D. discoideum cell morphology may simply reflect the requirements for a continuous supply of ATP in maintaining a normal cytoskeleton (Jungbluth et al., 1994). The effect of Rapl on the azide response may be due to an effect on some other component of the cytoskeleton. The possibility that Rapl acts in some way not related to the cytoskeleton to reduce the sensitivity of the cells to azide also cannot be excluded. HL5 addition to starved D. discoideum normally causes cells to rapidly round up and transiently detach from the substratum (Schweiger et ah, 1992; Howard et al, 1993). In contrast, addition of HL5 to Rapl transformant cells did not cause them to round up and detach. The inhibition of the response of Rapl cells to HL5 stimulation was not accompanied by loss of cell viability or an impaired erasure response, suggesting the cells were neither dying nor unable to revert to vegetative growth. Previously it was shown that D. discoideum cells round up in response to an inhibitor of tyrosine phosphatases, phenylarsine oxide (PAO) (Schweiger et al, 1992), suggesting that the cell rounding response is linked to phosphotyrosine levels. Furthermore, actin is rapidly tyrosine phosphorylated in response to HL5 stimulation (Schweiger et al, 1992) and overexpression of tyrosine phosphatase PTP1, but not PTP2, impairs both the cell rounding response and the amplitude and duration of actin phosphorylation (Howard et al, 1993; Howard et al, 1994). However, the Rapl transformant showed a normal pattern of actin tyrosine phosphorylation after HL5 stimulation despite its failure to rapidly round up. This result suggests that 1) the overexpression of Rapl does not block the tyrosine phosphorylation induced by HL5 stimulation; 2) the Rapl cells are still able to detect nutrients and 3) although tyrosine phosphorylation of actin may be necessary (Schweiger et ah, 1992; Howard et al, 1993) it is not sufficient to induce cell rounding in response to HL5 treatment. Given the appearance of the Rapl cells and their failure to respond to azide and HL5, it is possible that the phenotype of flat spread cells was caused by increased cell-substratum adhesion. In mammalian cells, a flatter, more spread cell morphology correlates with increased cell-substratum adhesion (Folkman and Moscona, 1978). However, since the Rapl cells exhibited normal motility, a property which requires the capacity to control adhesion and detachment from the substratum, it is unlikely that increased adhesion was responsible for the altered cell morphology. Rapl cells exhibited an increase in EDTA-sensitive cell-cell adhesion when grown in suspension. It is not clear whether this effect was related to the morphological and cytoskeletal changes discussed above. The increase in cell-cell adhesion did not appear to be due to an increase in expression the EDTA-sensitive adhesion molecule gp24 (data not shown) or to increased expression of the EDTA-resistant adhesion molecule gp80 (J. Faix, personal communication.) As detailed in the Introduction, Rapl proteins have been implicated in diverse roles in different cell types and some of these involve cell morphology or cytoskeletal changes. Transfection of a rapl A cDNA has been shown to suppress the transformed phenotype of Ki-ras transformed NTH 3T3 cells, producing flattened, more adherent cells with reduced tumorigenicity (Kitayama et al, 1989), whereas microinjection of RaplB into Swiss 3T3 cells induces membrane ruffling (Yoshida etal, 1992). The yeast Rapl homologue RSR1/BUD1 is required for the non-random positioning of the bud site (Chant and Herskowitz, 1991), a process which is linked to the spatial organization of polymerized actin. It is interesting to speculate that the effect of high expression of Rapl in D. discoideum, which resulted in a flattened and spread cell morphology, may be similar to the effects observed in the morphological reversion of the phenotype of Ras transformed mammalian cells. Likewise, it is possible that the extended flattened regions observed around the periphery of abnormal D. discoideum Rapl cells may be similar to the lamellipodia that are associated with the formation of ruffles in mammalian cells. The effects of mutant Rapl proteins on D. discoideum cell morphology Of all the Ras superfamily proteins, the effects of mutations on the proto-oncogenic Ras proteins have been most extensively studied, so the effects of mutations in amino acids conserved between Rapl and Ras will be discussed initially in the context of the model proposed for Ras (Lowy and Willumsen, 1993; McCormick, 1994; Marshall, 1993). The effects of conserved amino acid substitutions in Ras and Rapl are listed in Figure 10. Briefly, Ras is activated by a switching from a GDP bound state to a GTP bound state and deactivated by hydrolysis of GTP to GDP (Bourne et al, 1990; Lowy and Willumsen, 1993). Consequently, substitutions such as G12V, which result in a constitutively GTP bound protein, cause activation of Ras (Bourne et al, 1990; Lowy and Willumsen, 1993). In addition, substitutions such as F156L, which alter residues not directly involved in nucleotide binding, are also capable of activating Ras, albeit weakly (Quilliam et al, 1995). In contrast, an S17N substitution restricts Ras to a GDP bound state, producing a dominant negative form of Ras Table 10. The effects of conserved amino acid substitutions in Ras and Rapl proteins Substitution Biochemical Effect Effect on Ras Effect on Rapl G10V do not bind fails to impair activated hot done nucleotides1 Ras1 G12V binds GTP 2 activating 2 enhances tumor suppression activity 3 S17N binds GDP 4 dominant inhibitor 4 dominant inhibitor of oxidative burst5 D38E disrupts GAP activity 6 blocks transformation 7 not done D38N disrupts GAP activity 6 blocks transformation 7 reduces tumor suppression 3 T61Q increases rate of GTP not applicable reduce tumor hydrolysis by Rapl suppression3 Rapl is a substrate for Ras-GAP 8 F156L perturbs protein activating 9 disrupts D. structure 9 melanogaster eye 1 0 1 Clanton et al, 1987;2 Bourne et al, 1990;3 Kitayama et al., 1990;4 Feig and Cooper, 1988; 5 Maly et al, 1994.; 6 Cales et al, 1988;7 Stone and Blanchard, 1991; 8 Hart and Marshall, 1990;9 Quilliam et al, 1995;10 Hariharan et al, 91. (Feig and Cooper, 1988). The S17N mutated Ras protein have been proposed to act by sequestering guanine nucleotide exchange factors, thus blocking the exchange of GDP for GTP on wild type Ras (Feig and Cooper, 1988; Stacey et al, 1991; Farnsworth and Feig, 1991). A G10V substitution prevents the binding of both GTP and GDP (Clanton et al, 1987). Ras activity also requires an effector domain (amino acids 32-40) and substitutions such as D38E or D38N disrupt signal transduction by Ras (Cales et al, 1988; Farnsworth et al, 1991; Stone and Blanchard, 1991) by preventing the binding of effector proteins such as Raf (Vojtek et al., 1993; Warne et al., 1993; Zhang et al., 1993). The Ras superfamily proteins Rho, Rac and Cdc42Hs can be similarly activated or impaired by altering the amino acids equivalent to Ras amino acids G12 and S17, respectively, suggesting that these proteins are also regulated by GDP/ GTP exchange (Paterson et al, 1990; Ridley and Hall, 1992; Diekmann et al, 1991; Ridley et al, 1992; Qiu et al, 1995; Kozma et al, 1995; Nobes and Hall, 1995). Although the effector domains are not highly conserved in the Rho, Rac and Cdc42Hs proteins, mutation of their equivalent domains also blocks activity (Self et al, 1993; Diekmann et al, 1994; Freeman et al, 1994; Zheng et al, 1994). The expression of mutant Rapl proteins, Rapl-GlOV and Rapl-S17N, had no effect on D. discoideum cell morphology. In addition, such transformants responded to HL5 stimulation and to treatment with azide in the same way as the parental Ax2 cells. These results are consistent with the effects of mutations at position 17 in Ras superfamily proteins which block activity. However, the D. discoideum S17N Rapl protein did not have an effect on cell morphology, implying that either the S17N protein did not act in a dominant negative manner, or that interfering with the endogenous Rapl protein activity was not sufficient to alter cell morphology. Although high levels of S17N Rapl were obtained, it remains possible that further increases in expression would cause an abnormal phenotype. High levels of S17N Ras proteins were required in mammalian cells to cause an effect (Feig and Cooper, 1988; Stacey et al, 1991). The frequency and extent of the abnormal cell morphology was the same in cells expressing the Rapl-G12V protein as in cells overexpressing normal Rapl protein. Similarly, resistance to azide treatment did not differ significantly between the two transformants. However, the cells expressing the Rapl-G12V protein were 14% less responsive to HL5 stimulation than cells overexpressing normal Rapl protein (p<0.05). Together the data suggest that a G12V substitution at best only modestly enhanced the effects of Rapl in D. discoideum. The same substitution enhances tumor suppression by Rapl in mammalian cells, but again the effect is not large (Kitayama et al., 1990). This contrasts with the dramatic effect of the same substitution on transformation by Ras (Chang et al, 1982; Stacey and Kung, 1984). Possibly a high proportion of overexpressed Rapl in mammalian and D. discoideum cells is in the GTP bound state, and consequently the difference between the wild type and the activated protein is not great. The effects of mutations of amino acids 12 and 17 have been used to infer the requirements for GTP binding on protein activity in Ras superfamily proteins. The tumor suppressor activity of Rapl in Ras transformed mammalian cells was enhanced by a G12V substitution and decreased by a position 17 substitution (although a S17D rather than an S17N substitution was constructed) suggesting that the Rapl tumor suppressor activity required GTP binding (Kitayama et al, 1990). In contrast, both G12V and S17N mutant Rapl proteins, but not wild type Rapl, acted in an inhibitory manner in the oxidative burst process in B lymphocytes, suggesting that in this case it is necessary for Rapl to cycle between GTP bound and GDP bound states (Maly et al., 1994). This effect on the oxidative burst is reminiscent of the inhibition of Rab-mediated vesicular transport by a non-hydrolyzable GTP analog, which acts to inhibit the shuttling of Rab proteins between two cellular compartments (reviewed in Hall, 1990; Rothman and Orci, 1992). In an analysis of the S. cerevisiae Rapl homologue RSR1/BUD1, a G12V activating mutation was unable to rescue a rsrl/bndl deletion, again consistent with a requirement for cycling between a GTP and GDP form of the protein (Ruggieri et al, 1992). The contrasting effects of the Rapl-G12T and Rapl-S17N proteins on D. discoideum cell morphology suggest that Rapl in D. discoideum acts in a GTP dependent manner and that Rapl is not required to cycle between a GTP and GDP bound form. Overexpression of Rapl, Rapl-G12V and Rapl-S17N proteins resulted in reduced growth rates compared to the parental Ax2 cells. The reduction in the growth rate could be reversed by the addition of folate to repress the discoidin promoter. The similar inhibitory effects of all these proteins on growth contrasted with their markedly different effects on cell morphology. Since wild type, presumptive activating mutant and dominant negative mutant proteins all inhibited cell growth, there is no simple way to reconcile the data with models where Rapl acts in a GTP dependent manner or where Rapl is required to cycle between GTP and GDP bound forms. There was no attenuation of the Rapl-induced cell morphology in cells expressing Rapl-T61Q protein compared to cells overexpressing wild type Rapl protein in any of the assays I used. By contrast, the T61Q substitution reduces the tumor suppressor activity of mammalian RaplA (Kitayama et al, 1990). Residue 61 has been proposed to have at least two functions in mammalian RaplA. It is proposed to be involved in GTP hydrolysis and contributes to the lower intrinsic rate of GTP hydrolysis in mammalian Rapl relative to Ras (Freeh et al, 1990). In addition, residue 61 contributes to the interaction of RaplA with Ras-GAP. The attenuating effect of the T61Q substitution in mammalian RaplA has been hypothesized to be due an increased rate of GTP hydrolysis, possibly due to the enhanced activity of Ras-GAP on the mutated RaplA protein (Hart and Marshall, 1990). The lack of any disruptive effects of the T61Q substitution on D. discoideum Rapl activity suggests that increasing the intrinsic rate of GTP hydrolysis is not sufficient to reduce the effect of Rapl. In addition, the different effect of the T61Q substitution on D. discoideum Rapl compared to mammalian Rapl suggests that substitution of position 61 may not impair D. discoideum Rapl interaction with the presumptive GAPs that have yet to be characterized. In this regard, bovine Rapl-GAP (GAP3) activity is not inhibited by a T61Q RaplA substitution (Maruta et al, 1991). Expression of a Rapl protein containing the F156L substitution in D. discoideum resulted in cells with an abnormal cell morphology. However, the Rapl-F156L cells were more sensitive to treatment with azide compared to Rapl cells and also exhibited a reduced inhibition of the response to HL5 stimulation compared to wild type Rapl cells. These data suggest that the Rapl protein with a position F156L substitution has reduced activity compared to the wild type Rapl protein. This result contrasts with the weakly activating effect of this substitution in Ras and the dominant gain-of-function of this substitution in D. melanogaster Rapl (Hariharan et al, 1991; Quilliam et al, 1995). The basis of this difference is not known. Although position 38 mutations disrupt transformation by Ras and tumor suppression by Rapl (Sigal et al, 1986; Stone and Blanchard, 1991; Kitayama et al., 1990), Rapl-D38E and Rapl-D38N transformants cells exhibited the same abnormal cell morphology as the wild type Rapl transformant. However, the Rapl-D38E and Rapl-D38N cells were sensitive to azide treatment and they had a phenotype intermediate between that of the parental Ax2 strain and the Rapl transformant after HL5 stimulation. The Rapl-D38N cells had a smaller inhibitory effect on HL5 stimulation than the Rapl-D38E cells, despite the fact that the Rapl-D38N cells expressed somewhat more Rapl protein. This result is consistent with the fact that a D38N substitution is a more substantial change, substituting a basic side chain for an acidic side chain, than is D38E, which only introduces a single additional methyl group. Since these substitutions were in the core region of the effector domain that is conserved in all Ras and Rapl proteins (Bourne et al, 1991), it was anticipated that these mutated Rapl proteins would be inactive. The partial activity of the Rapl proteins with position 38 substitutions suggests that effector domain requirements for Rapl-induced changes in D. discoideum cell morphology differ from those required for tumor suppression by mammalian Rapl and transformation by Ras. Mutations D38E, D38N and F156L all attenuated the effect of Rapl expression on the response of cells to azide and the response of starved cells to HL5 stimulation, yet did not diminish the effect of Rapl on cell morphology. This raises the question of whether the effect on morphology, the response to azide and the response to HL5 stimulation represent the same or different phenomena. The response of Ax2 cells to HL5 and azide both involve a process of rapid cell contraction and detachment from the substratum in response to a stimulus, although it is not clear that the two responses involve identical cytoskeletal changes. Analysis of cells expressing mutant Rapl proteins showed a good correlation between resistance to azide treatment and the inhibition of the response to HL5 stimulation (Fig. 39), which suggests that these two assays 100% -8 0 % 6 0 % 4 0 % 2 0 % 0% Figure 39. Comparison of azide treatment with the HL5 stimulation assay The data for the proportions of cells resistant to azide treatment (open bars) is from Tables 5 and 8 and the data for HL5 stimulation for 10 minutes (black bars) is from Figures 29 and 37. Transformants have been arranged according to the extent of the inhibition of the response to HL5 stimulation. The height of the bar indicates the mean and the error bar indicates the standard error of the mean. may measure the same effect. The relationship between the flat spread morphology on the one hand and the azide and HL5 stimulation response on the other is more complex. An altered cell appearance and an inhibition of the responses to azide and HL5 stimulation were observed in cells expressing either activated RasG-G12T, Rapl, Rapl-G12V or Rap-T61Q proteins. However, the presence of flat, spread cells in transformants expressing similar levels of mutated Rapl-D38E, Rapl-D38N or Rapl-F156L protein did not predict the response to HL5 stimulation or the response to azide, suggesting that the flat spread cell phenotype is distinct from the two other responses. Analysis of additional mutations in the Rapl effector domain might clarify the relationship between the flat spread cell morphology and the responses to azide and HL5 stimulation. The role of R a s G in D. discoideum Under inducing conditions, many of the RasG-G12T cells exhibited an abnormal morphology: they appeared flattened and spread with increased F-actin located around the cell periphery, although there was no increase in cell volume as determined by an analysis of forward light scatter (this thesis) and Coulter counter analysis (G. Weeks, personal communication). By contrast, RasG cells had an appearance similar to that of Ax2 cells. Activated Ras has previously been shown to have a dramatic effect on mammalian cell morphology (Stacey and Kung, 1984; Feramisco et al, 1984; Bar-Sagi and Feramisco, 1986; Lloyd et al, 1989). Mammalian cells exhibit a characteristic transformed phenotype; becoming refractile, exhibiting increased membrane ruffling and a loss of stress fibers. Ras proteins have also been shown to regulate cell morphology in yeasts. In Schizosaccharomyces pombe, Rasl is required for normal cell shape, in a process involving cdc42sp a Rho-like protein (Chang et al, 1994) whereas in Saccharomyces cerevisiae RAS2 is involved in pseudohyphal growth, a process characterized by unipolar budding and an altered cell morphology (Gimeno et al, 1992). Expression of activated RasG-G12T increased the number of D. discoideum cells that remained adherent to the substratum after azide treatment and substantially inhibited the cell rounding response to HL5. By contrast, high expression of wild type RasG protein modestly inhibited the response to HL5 stimulation but had no effect on vegetative cell morphology or on the azide response. These results are also consistent with the G12V alteration causing an activation of RasG. The abnormal appearance of the RasG cells following starvation and the inhibited response to HL5 suggested that starved cells may be more sensitive than vegetative cells to the effects of overexpression of wild type RasG. However starved RasG cells, unlike RasG-G12T cells, could develop normally (M. Khosla, personal communication). It is also worth noting that the effect of RasG overexpression on the azide and HL5 responses contrasted with the results of the Rapl mutant proteins, and was the only indication that the two responses might not be affected equally. Cells expressing RasG-S17N protein were more elongated than Ax2 cells whereas the RasG cells appeared normal. RasG-S17N may act as a dominant negative form of the protein causing an inhibition of the normal activity of the RasG protein as described previously for mammalian Ras S17N protein (Feig and Cooper, 1988). The contrasting effects of the G12T and S17N substitutions suggest that RasG affects vegetative cell morphology in a GTP dependent manner. No increased rate of rounding in response to HL5 stimulation could be detected in RasG-S17N cells (data not shown). This may be due to the difficulty of observing a response that is greater than the already large response of the parental Ax2 cells, or alternatively, as discussed for Rapl, the effect on HL5 stimulation and azide may be separate from the effect on cell morphology. Multinucleate cells were observed in both the RasG and the RasG-G12T transformants. However, expression of activated RasG did not increase the proportion of multinucleate cells or the average number of nuclei per cell compared to overexpression of wild type RasG. The similar effects of the two proteins contrasts with the enhanced effect that expression of the RasG-G12T protein had on cell morphology, suggesting that the effect on cytokinesis may be unrelated to changes in cell morphology. Furthermore, overexpression of Rapl altered cell morphology but had no affect on cytokinesis, again suggesting that the two phenomena are not related. Cells with a disruption of the myosin II heavy chain or the profilin genes are also multinucleate and have a flat spread cell morphology (Knecht and Loomis, 1987; De Lozanne and Spudich, 1987; Haugwitz et ai, 1994). However, the consequences of overexpressing RasG or an activated RasG-G12T protein differed from those reported for cells disrupted in myosin II or profilin in two respects. First, the proportion of multinucleate cells was not as great and number of nuclei per cell was considerably lower for the RasG and the RasG-G12T transformants. Secondly, disruption of the myosin II or profilin genes also results in cells which are unable to grow in suspension (Knecht and Loomis, 1987; De Lozanne and Spudich, 1987; de Hostos etal, 1993; Haugwitz et al, 1994). By contrast, RasG and RasG-G12T cells do grow in suspension, albeit at a reduced rate (M. Khosla, personal communication). H o w does overexpression of R a p l and RasG-G12T affect D. discoideum cells? In contrast to the apparently antagonistic roles that Rapl and Ras proteins play in mammalian cells, there are a number of striking similarities between the effects of Rapl and activated RasG overexpression on D. discoideum cells. Expression of either Rapl or activated RasG-G12T caused cells to become flattened and spread with a redistribution of F-actin around the cell periphery. In addition, expression of both Rapl and activated RasG reduce cell detachment after treatment with azide, and inhibit the rounding up of starved cells in response to a nutrient stimulus. However there are also significant differences between the actions of Rapl and RasG proteins. One difference is that overexpression of wild type and activated Rapl have similar effects on cell morphology and the responses to azide and HL5, whereas all three properties are more affected by expression of activated RasG than by overexpression of wild type RasG. Another difference is that RasG affects some processes which are unaffected by Rapl. Activated RasG expression blocks aggregation (M. Khosla personal communication) whereas wild type and activated Rapl have no effect on development. In addition, overexpression of either RasG or RasG-G12T but not Rapl protein resulted in a defect in cytokinesis. These results raise two questions for discussion. First, how does the overexpression of Rapl and RasG-G12T affect cell morphology, and the responses to azide and nutrient stimulation; and second, do Rapl and RasG act on the same or different regulatory pathway (s)? By analogy with the effects of Ras on morphology in mammalian cells, there are two non-mutually exclusive ways for Rapl and RasG to affect D discoideum cell morphology. Overexpression of Rapl and activated RasG-G 1 2 T may act to disrupt a signal transduction pathway controlled by Rapl and RasG that directly regulates the cytoskeleton. In mammalian cells, Ras affects actin-based processes via pathways involving Rho and Rac, as described in the Introduction. Several Rac and Rho genes have been isolated from D. discoideum (Bush et al, 1993a; Bush et al, 1993b), and it is possible that there is a network of Ras superfamily proteins regulating D. discoideum cell morphology. One cytoskeletal process that might be regulated by such a pathway is the contraction of the cytoskeleton by myosin II, since this process responds to azide. The assembly of myosin II onto the cytoskeleton is induced by stimuli such as cAMP and is regulated by phosphorylation (Berlot et al, 1985; Berlot et al, 1987; Egelhoff et al, 1993), and overexpression of Rapl and RasG-G12T could disrupt such a process. However, there are probably other signal transduction pathways that directly regulate the cytoskeleton that could be regulated by Rapl and RasG. The second way that overexpression of Rapl and RasG might act is to disrupt the transduction of a signal controlling gene expression which consequently results in an altered cell morphology. Activated Ras transduces a signal to the nucleus which regulates mammalian gene expression (reviewed in Gutman and Wasylyk, 1991; Lowy and Willumsen, 1993) and reduced expression of numerous actin associated genes has been observed in transformed mammalian cells, providing a precedent for such a proposal (Button et al, 1995; Janmey and Chaponnier, 1995). Recently, it has also been shown that other Ras superfamily proteins, Rho, Rac and Cdc42Hs, transduce signals that activate transcription (Minden et al, 1995; Coso et al, 1995); Hill, 1995 #497. There is currently is no direct evidence for activation of gene expression by a Rapl-specific pathway, but the Observation that microinjection of Rapl can stimulate mitogenesis in Swiss 3T3 cells (Yoshida et al, 1992) is consistent with a role for Rapl in transducing a signal to the nucleus. Since the effects of the Rapl and RasG proteins in this study were analyzed 24 hours after the induction of the discoidin promoter, sufficient time had elapsed to allow additional gene expression. Candidate D. discoideum genes that might be regulated by Rapl and RasG include those identified by gene disruption studies that cause similar alterations in cell morphology, such as profilin or coronin (Haugwitz et al., 1994; de Hostos et al., 1993). Actin and myosin II heavy chain levels did not appear to be altered in the Rapl and RasG-G12T cells (data not shown). Do Rapl and RasG act on the same of different regulatory pathway(s)? Given the very similar effects of overexpression of Rapl and activated RasG proteins, it is possible that the two proteins act on the same pathway. The additional effects of activated RasG-G12T on cytokinesis and aggregation could be due to another RasG-specific pathway, or to RasG acting upstream of Rapl and transducing two or more signals, only one of which is subsequently mediated by Rapl. The latter arrangement would be similar to the branched organization of Ras superfamily proteins involved in the regulation of mammalian cell morphology. Alternatively, Rapl and RasG could regulate two different pathways that converge on the cytoskeleton, perhaps at different regulatory sites, but with a similar phenotypic outcome. The relationship, if any, between Rapl and RasG might be addressed by coexpression experiments utilizing dominant negative forms of the proteins; for example, if one assumes that Rapl acts downstream of RasG then expression of Rapl-S17N should block some of the effects of activated RasG-G12T. The above discussion assumes that overexpression of Rapl and RasG-G12T disrupts a signaling pathway regulated by the endogenous Rapl and RasG proteins. Although expression of Rapl-S17N did not affect cell morphology, a role for endogenous RasG protein in directly regulating cell morphology is supported by the observation that the both RasG-G12T and the dominant negative RasG-S17N protein affected cell morphology. However, it is possible that Rapl and RasG-G12T disrupt a pathway that is normally regulated by another member of the Ras superfamily. It is conceivable that Rapl and activated RasG-G12T affect a pathway regulated by RasB or RasC, which are both expressed at significant levels during growth in D. discoideum (Robbins et al., 1989; Daniel et al., 1993a; Daniel et al., 1993b). Rapl and activated RasG-G12T are less likely to affect pathways regulated by other Ras superfamily proteins, such as Rac or Rho that share less amino acid identity with Ras. It has been proposed, based on studies in mammalian cells, that overexpression of Rapl may act in a manner distinct from that of an activated Ras protein. Rapl was proposed to revert the transformed phenotype of Ras transformed mammalian cells by competing for Ras effectors (Freeh et al, 1990; Zhang et ah, 1993) and it is possible that D . discoideum Rapl could similarly sequester effector or regulatory factors of other Ras superfamily proteins and switch on or off the pathways regulated by these molecules. Since Rapl does not affect cytokinesis and development, whereas RasG-G12T does affect these processes, it is unlikely that Rapl is activating RasG by sequestering negative regulatory factors. However, the effects of RasB and RasC on cell morphology are not known and it is possible that Rapl may act by stimulating or disrupting the effects of these or other as yet uncharacterized Ras proteins. In summary, it appears that D. discoideum morphology is regulated in part by RasG and possibly by Rapl too. The similar effects of RasG and Rapl on cell morphology may provide an experimentally tractable system to address the relationship between Ras and Rapl protein activities, that could prove to be relevant to other organisms. Finally, characterization of the effects of RasB and RasC on cell morphology is required to address some of the possible roles for Rapl and RasG discussed in this section. REFERENCES Altschuler, D. L., Peterson, S. N v Ostrowski, M . C. and Lapetina, E. G. (1995). Cyclic AMP-dependent activation of Raplb. J. Biol. Chem. 270:10373-10376. Anjard, G , Pinaud, S., Kay, R. R. and Reymond, C. D. (1992). Overexpression of DdPK2 protein kinase causes rapid development and affects the intracellular cAMP pathway of Dictyostelium discoideum. Development 115: 785-790. Avruch, J., Zhang, X. and Kyriakis, J. M. (1994). Raf meets Ras: completing the framework of a signal transduction pathway. Trends Biochem. Sci. 19:79-83. Bar-Sagi, D. and Feramisco, J. R. (1985). Microinjection of the ras oncogene product into PC12 cells induces morphological differentiation. Cell 42: 841-848. Bar-Sagi, D. and Feramisco, J. R. (1986). Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins. Science 233: 1061-1068. Barbacid, M . (1987). ras genes. Ann. Rev. Biochem. 56:779-827. Beitel, G. J., Clark, S. G. and Horvitz, H. R. (1990). Caenorliabditis elegans Ras gene Let-60 acts as a switch in the pathway of vulval induction. Nature 348: 503-509. Bement, W. M. , Hasson, T., Wirth, J. A., Cheney, R. E. and Mooseker, M . S. (1994). Identification and overlapping expression of multiple unconventional myosin genes in vertebrate cell types. Proc. Natl. Acad. Sci. USA 91:6549-6553. Bender, A. and Pringle, J. R. (1989). Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the 7-as-related gene RSR1. Proc. Natl. Acad. Sci. USA 86:9976-9980. Benito, M. , Pdrras, A., Nebreda, A. R. and Santos, E. (1991). Differentiation of 3T3-L1 fibroblasts to adipocytes induced by transfection of ras oncogenes. Science 253:565-568. Beranger, F., Goud, B., Tavitian, A. and Degunzburg, J. (1991). Association of the Ras-antagonistic Rapl/Krev-1 proteins with the Golgi complex. Proc Natl Acad Sci Usa 88:1606-1610. Berlot, C. H., Devreotes, P. N. and Spudich, J. A. (1987). Chemoattractant-elicited increases in Dictyostelium myosin phosphorylation are due to changes in myosin localization and increases in kinase activity. J. Biol. Chem. 262:3918-3926. Berlot, C. H., Spudich, J. A. and Devreotes, P. N. (1985). Chemoattractant-elicited increases in myosin phosphorylation in Dictyostelium. Cell 43: 307-314. Blumer, K. J. and Johnson, G. L. (1994). Diversity in function and regulation of MAP kinase pathways. Trends Biochem. Sci. 19:236-240. Blusch, J., Morandini, P. and Nellen, W. (1992). Transcriptional regulation by folate-inducible gene expression in Dictyostelium transformants during growth and early development. Nucleic Acids Res. 20:6235-6238. Boguski, M. S. and McCormick, F. (1993). Proteins regulating Ras and its relatives. Nature 366: 643-654. Bokoch, G. M. (1993). Biology of the Rap proteins, members of the ras superfamily of GTP binding proteins. Biochem. J. 289:17-24. Bokoch, G. M., Quilliam, L. A., Bohl, B. P., Jesaitis, A. J. and Quinn, M. T. (1991). Inhibition of RaplA binding to cytochrome-b558 of NADPH oxidase by phosphorylation of RaplA. Science 254:1794-1796. Bonner, J. T. (1947). Evidence for the formation of cell aggregates by chemotaxis in the development of the slime mold Dictyostelium discoideum. J. Exp. Zool. 106:1-26. Bos, J. L. (1989). ras oncogenes in human cancer: A review. Cancer Res. 49: 4682-4689. Bourne, H. R., Sanders, D. A. and McCormick, F. (1990). The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348:125-132. Bourne, H. R., Sanders, D. A. and McCormick, F. (1991). The GTPase superfamily: conserved structure and molecular mechanism. Nature 349: 117-127. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. Brunger, A. T., Milburn, M. V., Tong, L., De Vos, A. M., Jancarik, J., Yamaizumi, Z., Nishimura, S., Ohtsuka, E. and Kim, S. H. (1990). Crystal structure of an active form of RAS protein, a complex of a GTP analog and the HRAS p21 catalytic domain. Proc. Natl. Acad Sci. USA 87:4849-4853. Burdine, V. and Clarke, M. (1995). Genetic and physiologic modulation of the prestarvation response in Dictyostelium Discoideum. Mol. Biol. Cell 6: 311-325. Bush, J., Franek, K. and Cardelli, J. (1993a). Cloning and characterization of seven novel Dictyostelium discoideum rac-related genes belonging to the rho family of GTPases. Gene 136:61-68. Bush, J., Franek, K., Daniel, J., Spiegelman, G. B., Weeks, G. and Cardelli, J. (1993b). Cloning and characterization of five novel Dictyostelium discoideum rab-related genes. Gene 136:55-60. Buss, J. E., Quilliam, L. A., Kato, K., Casey, P. J., Solski, P. A., Wong, G., Clark, R., Mccormick, F., Bokoch, G. M. and Der, C. J. (1991). The COOH-terminal domain of the RaplA (Krev-1) protein Is isoprenylated and supports transformation by an H-Ras- RaplA chimeric protein. Mol Cell Biol 11:1523-1530. Button, E., Shapland, C. and Lawson, D. (1995). Actin, its associated proteins and metastasis. Cell Motil. Cytoskeleton 30:247-251. Cales, C , Hancock, J. F., Marshall, C. J. and Hall, A. (1988). The cytosplasmic protein GAP is implicated as the target for regulation by the ras gene product. Nature 332:548-551. Campa, M. J., Chang, K. J., Vedia, L. M. Y., Reep, B. R. and Lapetina, E. G. (1991). Inhibition of ras-induced germinal vesicle breakdown in Xenopus oocytes by rap1 IB. Biochem. Biophys. Res. Commun. 174:1-5. Cantley, L. C , Auger, K. R. , Carpenter, G, Duckworth, B., Graziani, A., Kapeller, R. and Soltoff, S. (1991). Oncogenes and signal transduction. Cell 64:281-302. Chandrasekhar, A., Rotman, M., Kraft, B. and Soli, D. R. (1990). Developmental mechanisms regulating the rapid decrease in a cohesion glycoprotein mRNA in Dictyostelium function primarily at the level of mRNA degradation. Dev. Biol. 141:262-269. Chang, E. G, Barr, M., Wang, Y., Jung, V., Xu, H. P. and Wigler, M. R (1994). Cooperative interaction of S. pombe proteins required for mating and morphogenesis. Cell 79:131-141. Chang, E. H., Furth, M. E., Scolnick, E. M. and Lowy, D. R. (1982). Tumorigenic transformation of mammalian cells induced by a normal human gene homologous to the oncogene of Harvey murine sarcoma virus. Nature 297:479-483. Chant, J. and Herskowitz, I. (1991). Genetic control of bud site selection in yeast by a set of gene products that constitute a morphogenetic pathway. Cell 65:1203-1212. Chant, J. and Stowers, L. (1995). GTPase cascades choreographing cellular behaviour: Movement, morphogenesis, and more. Cell 81:1-4. Clanton, D. J., Lu, Y., Blair, D. G. and Shih, T. Y. (1987). Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis. Mol. Cell. Biol. 7:3092-3097. Clarke, M., Kayman, S. and Riley, K. (1987). Density dependent induction of discoidin I synthesis in exponentially growing cells of Dictyostelium discoideum. Differentiation 34: 79-87. Clarke, M., Yang, J. and Kayman, S. (1988). Analysis of the prestarvation response in growing cells of Dictyostelium discoideum. Dev. Genetics 9: 315-326. Clarke, S. (1992). Protein isoprenylafion and methylation at carboxyl-terminal cysteine residues. Ann. Rev. Biochem. 61:355-386. Coccucci, S. M. and Sussman, M. (1970). RNA in cytoplasmic and nuclear fractions of cellular slime mold amebas. J. Cell Biol. 45: 399-407. Cook, S. J., Rubinfeld, B., Albert, I. and McCormick, F. (1993). RapV12 antagonizes Ras-dependent activation of ERK1 and ERK2 by LPA and EGF in Rat-1 fibroblasts. EMBOJ 12:3475-3485. Coso, O. A., Chiariello, M., Yu, J., Teramoto, H., Crespo, P., Xu, N., Miki, T. and Gutkind, J. S. (1995). The small GTP-binding proteins Racl and Cdc42 regulate the activity of the JNK/SAPK signalling pathway. Cell 81:1137-1146. Cote, G. P. and Bukiejko, U. (1987). Purification and characterization of a myosin heavy chain kinase from Dictyostelium discoideum. J. Biol. Chem. 262:1065-72. Cox, A. D., Brtva, T. R., Lowe, D. G. and Der, C. J. (1994). R-Ras induces malignant, but not morphologic, transformation of NIH 3T3 cells. Oncogene 9:3281-3288. Cox, D., Condeelis, J., Wessels, D., Soil, D., Kern, H. and Knecht, D. A. (1992). Targeted disruption of the ABP-120 gene leads to cells with altered mobility. J. Cell Biol. 116:943-955. D a n i e l , J . , B u s h , J . , C a r d e l l i , J . , S p i e g e l m a n , G . B. a n d W e e k s , G . (1993a). Iso lat ion of two nove l ras genes i n Dictyostelium discoideum: ev idence for a complex developmental ly regulated 7-as-gene subfamily. Oncogene 9: 501-508. D a n i e l , J . , S p i e g e l m a n , G . B. and W e e k s , G . (1993b). Character izat ion of a t h i r d ras gene, rasB, that is exp ressed t h r o u g h o u t the g r o w t h a n d deve lopment of Dictyostelium discoideum. Oncogene 8:1041-1047. D a n i e l , J . M . (1993). Three novel Dictyostelium ras genes. P h D Univers i ty of Br i t ish Co lumb ia . de Hostos, E. L., Rehfueb, C , Bradtke, B., Wadde l , D . R , Albrecht, R., M u r p h y , J . a n d G e r i s h , G . (1993). Dictyostelium mutants l ack ing the cy toske le ta l protein coronin are defective i n cytokinesis and cell moti l i ty. J . C e l l B i o l . 120: 163-173. D e L o z a n n e , A . a n d S p u d i c h , J . A . (1987). D i s rup t i on of the Dictyostelium m y o s i n heavy chain gene by homologous recombination. Science 236:1086-1091. D e V o s , A . M . , T o n g , L., M i l b u r n , M . V . , Mat ias , P. M . , Jancar ik, J . , N o g u c h i , S . , N i s h i m u r a , S . , M i u r a , K. , O h t s u k a , E. a n d K i m , S. H . (1988). Three-d imens iona l structure of an oncogene protein: Catalyt ic d o m a i n of h u m a n c-H-ras p21. Science 239:888-893. D e W i t , R. J . W . (1982). T w o dist inct types of surface fo l ic a c i d - b i n d i n g proteins i n Dictyostelium discoideum. F E B S Lett. 150:445-448. D e W i t , R. J . W . , Bu lgakov , R., R i n k e D e Wi t , T. F. a n d K o n i j n , T . M . (1985). Relat ionships between the l igand specificity of cell surface folate b ind ing sites, fo late d e g r a d i n g e n z y m e s a n d ce l l u l a r responses i n Dictyostelium discoideum. B i o c h i m . B iophys . Ac ta 814: 214-226. DeFeo-Jones, D., Tatchel l , K., Rob inson, L. C , S iga l , I. S., Vass, W . C , L o w y , D. R. a n d S c o l n i c k , E . M . (1985). M a m m a l i a n and yeast ras gene p roduc ts : biological funct ion i n their heterologous systems. Science 306:179-184. D e l S a l , G . , M a n f i o l e t t i , G . a n d S c h n e i d e r , C . (1989). The C T A B - D N A prec ip i ta t ion method : A c o m m o n mini -scale preparat ion of template D N A f rom phagemids, phages or plasmids suitable for sequencing. B ioTechn iques 7:514-519. De r , C . J . (1989). The ras fami ly of oncogenes. Oncogenes. C . Benx and E. L i u , K l u w e r Academic Publishers: 73-119. Det terbeck, S . , M o r a n d i n i , P., Wetterauer, B., Bachmai r , A . , F ischer , K . a n d M a c W i l l i a m s , H . K . (1994). The 'prespore- l ike cel ls ' of Dictyostelium have ceased to express a prespore gene: analysis us ing short l i ved fi-galactosidases as reporters. Development 120:2847-2855. D i e k m a n n , D . , A b o , A . , J o h n s t o n , C , S e g a l , A . W . a n d H a l l , A . (1994). Interaction of Rac wi th p67phox and regulation of phagocytic N A D P H oxidase activity. Science 265:531-3. D i e k m a n n , D., B r i l l , S., Garrett, M . D., Totty, N . , H s u a n , J . , Monf r ies , C , H a l l , C . , L i m , L. a n d H a l l , A . (1991). Bcr encodes a GTPase-act ivat ing prote in for p21rac. Nature 351:400-402. D i n g e r m a n n , T. , R e i n d l , N . , Werne r , H . , H i l d e b r a n d t , M . , N e l l e n , W . , H a r w o o d , A . , W i l l i a m s , J . a n d N e r k e , K . (1989). O p t i m i z a t i o n and i n s i tu de tec t i on of Escherichia coli beta-galactosidase gene e x p r e s s i o n i n Dictyostelium discoideum. G e n e 85: 353-362. D o r i n g , V . , Schle icher , M . and Noege l , A . (1991). Dic tyoste l ium annexin VII (synexin). c D N A sequence and isolation of a gene d isrupt ion mutant. J . B i o l . Chem. 266:17509-17515. E a r l y , A . a n d W i l l i a m s , J . (1987). T w o vectors w h i c h faci l i tate gene m a n i p u l a t i o n a n d a s imp l i f i ed t ransformat ion procedure for Dictyostelium discoideum. G e n e 59: 99-106. Ege lho f f , T. , B r o w n , S. , M a n s t e i n , D. and S p u d i c h , J . (1989). H y g r o m y c i n resistance as a selectable marker i n Dictyostelium discoideum. M o l . C e l l . B i o l . 9:1965-1968. Ege lho f f , T . T. , L e e , R. J . a n d S p u d i c h , J . A . (1993). Dictyostelium m y o s i n heavy cha in phosphory la t ion sites regulate m y o s i n f i lament assembly a n d local izat ion i n v ivo. C e l l 75:363-371. Esch , R. K . a n d F i r te l , R. A . (1991). c A M P and cell sorting control the spatial exp ress ion of a deve lopmen ta l l y essent ia l ce l l - type-spec i f i c ras gene i n Dictyostelium. Genes Dev . 5: 9-21. Fa rnswo r th , C . L. a n d F e i g , L. A . (1991). Dominan t inh ib i tory mutat ions i n the M g 2 + - b i n d i n g site of R a s ^ prevent its activation by G T P . M o l . C e l l . B i o l . 11:4822^829. Farnswor th , C . L., M a r s h a l l , M . S., G i b b s , J . B., Stacey, D . W . a n d Fe ig , L. A . (1991). Preferential inh ib i t ion of the oncogenic fo rm of R a s H by mutat ions i n the G A P binding/'effector' domain. Ce l l 64:625-633. Fa r re l , F. X . , O h m s t e d e , C , R e e p , B. R. a n d L a p e t i n a , G . (1990). c D N A sequence of a new ras-related gene (rap2b) isolated f rom human platelets w i t h sequence homology to rap2. Nuc le ic Ac ids Res. 18:4281. Fe ig , L. A . a n d Cooper , G . M . (1988). Inhibit ion of N I H 3T3 cel l prol i ferat ion by a mutant ras protein w i th preferential affinity for G D P . M o l . C e l l . B i o l . 8: 3235-3243. F e i g , JL. A . a n d E m k e y , R. (1993). Ra l gene products and their regulat ion. The ras super fami ly of GTPases. C . Lacal and F. McCormick. Boca Raton, Fla., C R C Press: 247-258. Fe inberg , A . P. and Voge ls te in , B. (1983). A technique for radiolabel l ing D N A restr ict ion endonuclease fragments to h igh specific activity. A n a l . B i o c h e m . 132:6-13. Feramisco, J . R., Gross, M . , Kamata, T., Rosenberg, M . and Sweet, R. W . (1984). M i c r o i n j e c t i o n of the oncogenic f o r m of the h u m a n H-ras (T24) p ro te in results i n rap id proliferation of quiescent cells. C e l l 38:109-117. F i r t e l , R. A . (1995). Integration of s ignal ing in format ion i n cont ro l l ing cel l -fate decisions i n Dictyostelium. Genes Dev . 9:1427-1444. F i r t e l , R. A . a n d C h a p m a n , A . L. (1990). A role for cyc l ic A M P - d e p e n d e n t protein kinase A i n early Dictyostelium development. Genes D e v . 4:18-28. F ischer , T . H. , G a t l i n g , M . N . , Laca l , J . a n d W h i t e II, G . C . (1990). r a p l B , a c A M P - d e p e n d e n t p ro te in k inase substrate, associates w i t h the platelet cytoskeleton. J . B io l . Chem. 265:19405-19408. F o l k m a n , J . a n d M o s c o n a , A . (1978). Role of cel l shape i n g r o w t h contro l . Nature 273:345-349. Freeh, M . , J o h n , J . , P i z o n , V . , Cha rd in , P., Tav i t ian, A . , C la rk , R., M c C o r m i c k , F. a n d W i t t i n g h o f e r , A . (1990). Inh ib i t ion of G T P a s e ac t i va t ing p ro te i n st imulat ion of Ras-p21 GTPase by the Krev-1 gene product. Science 249:169-171. Freeman, J . L. R., K reck , M . L., Uh l inger , D . J . and Lambeth , J . D . (1994). Ras e f fec tor -homologue reg ion o n Rac regulates p ro te in associat ions i n the neutophi l respiratory burst oxidase complex. Biochemistry 33:13431-13435. F u t r e l l e , R. P . , T rau t , J . a n d M c K e e , W . G . (1982). C e l l behav iou r i n Dictyostelium: preaggregation reponses to local ized c A M P pulses. J . C e l l B i o l . 92:807-821. G i m e n o , C J . , L j ungdah l , P. O. , Styles, C A . and F i n k , G . R. (1992). Un ipo lar cel l d iv is ions i n the yeast S cerevisiae lead to f i lamentous g rowth - regulat ion by starvation and R A S . Ce l l 68:1077-1090. Gomer, R. H. and Firtel, R. A. (1987). Cell-autonomous determination of cell-type choice in Dictyostelium development by cell-cycle phase. Science 237: 758-762. Graham, S. M., Cox, A. D., Drivas, G., Rush, M G., Deustachio, P. and Der, C J. (1994). Aberrant function of the Ras-related protein TC21 / R-Ras2 triggers malignant transformation. Mol Cell Biol 14:4108-4115. , Griffith, L. M., Downs, S. M. and Spudich, J. A. (1987). Myosin light chain kinase and myosin light chain phosphatase from Dictyostelium: effects of reversible phosphorylation on myosin structure and function. J. Cell Biol. 104:1309-1323. Gross, J. D. (1994). Developmental decisions in Dictyostelium discoideum. Microbiol. Rev. 58:330-351. Guierrez, L., Magee, A. I., Marshall, C. J. and Hancock, J. F. (1989). Post-translational processing of p21 ras is two-step and involves carboxy-methylation and carboxy-terminal proteolysis. EMBOJ. 8:1093-1098. Gutman, A. and Wasylyk, B. (1991). Nuclear targets for transcription regulation by oncogenes. Trends Genetics 7:49-54. Hadwiger, J. A., Lee, S. and Firtel, R. A. (1994). The Ga subunit Ga4 couples to pterin receptors and identifies a signaling pathway that is essential for multicellular development in Dictyostelium. Proc. Natl. Acad. Sci. USA 91: 10566-10570. Hall, A. (1990). The cellular function of small GTP-binding proteins. Science 249:635-640. Hall, A., Marshall, C. J., Spurr, N. K. and Weiss, R. A. (1983). Identification of transforming gene in two human sarcoma cell lines as a new member of the ras gene family located on chromosome 1. Nature 303:396-400. Hall, A. L., Schlein, A. and Condeelis, J. (1988). Relationship of pseudopod extension to chemotactic hormone-induced actin polymerization in amoeboid cells. J. Cell. Biochem. 37:285-299. Hammer III, J. A. (1994). Regulation of Dictyostelium myosin II by phosphorylation: what is essential and what is important? J. Cell Biol. 127: 1779-1782. Hancock, J. F., Magee, A. I., Childs, J. E. and Marshall, C. J. (1989). All ras proteins are polyisoprenylated but only some are palmitoylated. Cell 57: 1167-1177. Hariharan, I. K v Carthew, R. W. and Rubin, G. M. (1991). The Drosophila Roughened mutation - activation of a rap homolog disrupts eye development and interferes with cell determination. Cell 67: 717-722. Harlow, E. and Lane, D. (1988). Antibodies: A laboratory manual. Cold Spring Harbor, N Y , Cold Spring Harbor Laboratory. Hart, P. A. and Marshall, C. J. (1990). Amino acid 61 is a determinant of sensitivity of rap proteins to the GTPase activating protein. Oncogene 5: 1099-1101. Harwood, A. J. and Drury, L. (1990). New vectors for expression of the E. coli lacZ gene in Dictyostelium. Nucleic Acids Res. 18:4292. Harwood, A. J., Hopper, N. A., Simon, M. N., Bouzid, S., Veron, M. and Williams, J. G. (1992a). Multiple roles for cAMP-dependent protein kinase during Dictyostelium development. Dev. Genetics 149: 90-99. Harwood, A. J., Hopper, N. A., Simon, M. N., Driscoll, D. M., Veron, M. and Williams, J. G. (1992b). Culmination in Dictyostelium is regulated by the cAMP-dependent protein kinase. Cell 69:615-624. Haugwitz, M., Noegel, A. A., Karakesisoglou, J. and Schleicher, M. (1994). Dictyostelium amoebae that lack G-actin-sequestering profilins show defects in F-actin content, cytokinesis, and development. Cell 79: 303-314. Heidemann, S. R. (1993). A new twist on integrins and the cytoskeleton. Nature 260:10804081. Hitt, A. L., Hartwig, J. H. and Luna, E. J. (1994). Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444. Hofer, F., Fields, S., Schneider, C. and Martin, G. S. (1994). Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator. Proc. Natl. Acad. Sci. USA 91:11089-11093. Hong, H. J., Hsu, L. and Gould, M. N. (1990). Molecular cloning of rat Krev-1 cDNA and analysis of the mRNA levels in normal and NMU-induced mammary carcinomas. Carcinogenesis 11:1245-1247. Hopper, N. A., Sanders, G. M., Fosnaugh, K. L., Williams, J. G. and Loomis, W. F. (1995). Protein kinase A is a positive regulator of spore coat gene transcription in Dictyostelium. Differentiation 58:183-188. Howard, P. K., Gamper, M., Hunter, T. and Firtel, R. A. (1994). Regulation by protein tyrosine phosphatase PTP2 is distinct from that by PTP1 during Dictyostelium growth and development. Mol. Cell. Biol. 14:5154-5164. Howard, P. K., Sefton, B. M. and Firtel, R. A. (1993). Tyrosine phosphorylation of actin in Dictyostelium associated with cell-shape changes. Science 259:241-244. Itoh, T., Kaibuchi, K., Sasaki, T. and Takai, Y. (1991). The smg GDS-induced activation of smg p21 Is initiated by cyclic AMP-dependent protein kinase-catalyzed phosphorylation of smg p21. Biochem. Biophys. Res. Commun. 177:1319-1324. Janmey, P . A. and Chaponnier, C. (1995). Medical aspects of the actin cytoskeleton. Curr. Opinion Cell Biol. 7:111-117. Jermyn, K. and Williams, J. (1995). Comparison of the Dictyostelium RasD and ecmA genes reveals two distinct mechanisms whereby an mRNA may become enriched in prestalk cells. Differentiation 58:261-267. Jermyn, K. A., Duffy, T. I. and Williams, J. G. (1989). A new anatomy of the prestalk zone in Dictyostelium. Nature 340:144-146. Jimenez, B., Pizon, V., Lerosey, I., Beranger, F., Tavitian, A. and Degunzburg, J. (1991). Effects of the ras-related rap2 protein on cellular proliferation. Int. J. Cancer 49:471-479. Jung, G., Fukui, Y., Martin, B. and Hammer III, J. A. (1993). Sequence, expression pattern, intracellular localization, and targeted disruption of the Dictyostelium myosin ID heavy chain isoform. J. Biol. Chem. 268: 14981-14990. Jung, G. and Hammer III, J. A. (1990). Generation and characterization of Dictyostelium cells deficient in a myosin I heavy chain isoform. J. Cell Biol. 110:1955-1964. Jungbluth, A,, von Arnim, V., Biegelmann, E., Humbel, B., Schweiger, A. and Gerisch, G. (1994). Strong increase in the tyrosine phosphorylation of actin upon inhibition of oxidative phosphorylation: correlation with reversible rearrangements in the actin skeleton of Dictyostelium cells. J. Cell Sci. 107: 117-125. Kahn, R. A., Der, G J. and G.M., B. (1992). The ras superfamily of GTP-binding proteins: guidelines on nomenclature. FASEB J. 6: 2512-2513. Kaibuchi, K., Mizuno, T., Fujioka, H., Yamamoto, T., Kishi, K., Fukumoto, Y., Hori, Y. and Takai, Y. (1991). Molecular cloning of the cDNA for stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like small GTP- binding proteins) and characterization of stimulatory GDP / GTP exchange protein. Mol. Cell. Biol. 11:2873-2880. Kataoka, T., Powers, S., Cameron, S., Fasano, O., Goldfarb, M., Broach, J. and Wigler, M. (1985). Functional homology of mammalian and yeast ras genes. Cell 40:19-26. Kawata, M., Kikuchi, A., Hoshijima, M., Yamamoto, K., Hashimoto, E., Yamamura, H. and Takai, Y. (1989). Phosphorylation of smg p21, a ras p21-like GTP-binding protein, by cyclic AMP-dependent protein kinase in a cell-free system and in response to prostaglandin E^ in intact human platelets. J. Biol. Chem. 264:15688-15695. Kawata, M., Matsui, Y., Kondo, J., Hishida, T., Teranishi, Y. and Takai, Y. (1988). A novel small molecular weight GTP-binding protein with the same putative effector domain as the ras proteins in bovine brain membranes. J. Biol. Chem. 263:18965-18971. Kay, R. R. (1994). Differentiation and patterning in Dictyostelium. Curr. Opin. Genetics Dev. 4:637-641. Khosla, M., Robbins, S. M., Spiegelman, G. B. and Weeks, G. (1990). Regulation of DdrasG Gene expression during Dictyostelium development. Mol. Cell. Biol. 10:918-922. Khosla, M., Spiegelman, G. B. and Weeks, G. (1995). Effects of rasG overexpression on the growth and differentiation of Dictyostelium discoideum. submitted Mol. Cell. Biol. . Kimmel, A. and Firtel, R. (1983). Sequence organization in Dictyostelium: unique structure at the 5' ends of protein coding genes. Nucleic Acids Res 11: 541-552. Kitayama, H., Matsuzaki, T., Ikawa, Y. and Noda, M. (1990). Genetic analysis of the Kirsten-ras-revertant 1 gene: Potentiation of its tumor suppressor activity by specific point mutations. Proc Natl. Acad. Sci. USA 87:4284-4288. Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y. and Noda, M. (1989). A 7'as-related gene with transformation suppressor activity. Cell 56: 77-84. Knecht, D . A. and Loomis, W. F. (1987). Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. Science 236:1081-1086. Knox, J. J., Rebstein, P. J., Manoukian, A. and Gronostajski, R. M. (1991). In vivo stimulation of a chimeric promoter by binding sites for Nuclear Factor I. Mol. Cell. Biol. 11:2946-2951. Koide, H., Satoh, T., Nakafuku, M. and Kaziro, Y. (1993). GTP-dependent association of Raf-1 with Ha-Ras: Identification of Raf as a target downstream of Ras in mammalian cells. Proc. Natl. Acad. Sci. USA 90:8683-8686. Kopachik, W., Oohata, A., Dhokia, B., Brookman, J. J. and Kay, R. R. (1983). Dictyostelium mutants lacking DIF, a putative morphogen. Cell 33:107-114. Kozma, R., Ahmed, S., Best, A. and Lim, L. (1995). The Ras-related protein Cdc42hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. Mol. Cell. Biol. 15:1942-1952. Kraft, B., Chandrasekhar, A., Rotman, M., Klein, C. and Soil, D. R. (1989). Dictyostelium erasure mutant H14 abnormally retains development-specific mRNAs during dedifferentiation. Dev. Biol. 136:363-371. Kumagai, A., Hadwinger, J. A., Pupillo, M. and Firtel, R. A. (1991). Molecular genetic analysis of two G a protein subunits in Dictyostelium. J. Biol. Chem. 266:1220-1228. Labadia, M. E., Bokoch, G. M. and Huang, C. (1993). The RaplA protein enhances protein kinase C activity in vitro. Biochem. Biophys. Res. Commun. 195:1993. Liu, G., Kuwayayama, H., Ishida, S. and Newell, P. C (1993). The role of cyclic GMP in regulating myosin during chemotaxis of Dictyostelium: evidence from a mutant lacking the normal cyclic GMP response to cyclic AMP. J. Cell Sci. 106:591-596. Liu, G. and Newell, P. C. (1991). Evidence that cyclic GMP may regulate the association of myosin II heavy chain with the cytoskeleton by inhibiting its phosphorylation. J. Cell Sci. 98:483-490. Liu, G. and Newell, P. C. (1994). Regulation of myosin regulatory tight chain phosphorylation via cyclic GMP during chemotaxis of Dictyostelium. J. Cell Sci. 107:1737-1743. Liu, T., Williams, J. G. and Clarke, M. (1992). Inducible expression of calmodulin antisense RNA in Dictyostelium cells inhibits the completion of cytokinesis. Mol. Biol. Cell 3:1403-1413. Lloyd, A. C, Paterson, H. F., Morris, J. D. R, Hall, A. and Marshall, C J. (1989). p21Hras-induced morphological transformation and increases in c-mxjc expression are independent of functional protein kinase C. EMBO J. 8:1099-1104. Lowy, D. R. and Willumsen, B. M. (1993). Function and regulation of Ras. Ann. Rev. Biochem. 62:851-891. Lu, X., Chou, T. B., Williams, N. G., Roberts, T. and Perrimon, N. (1993). Control of cell fate determination by p21ras/Rasl, an essential component of torso signalling in Drosophila. Genes Dev. 7: 621-632. Luna, E. J. and Condeelis, J. S. (1990). Actin-associated proteins in Dictyostelium discoideum. Dev. Genetics 11: 328-332. Maeda, Y., Ohmori, T., Abe, T., Abe, F. and Amagai, A. (1989). Transition of starving Dictyostelium cells to differentiation phase at a particular position of the cell cycle. Differentiation 41:169-175. Maly, F., Quilliam, L. A., Dorseuil, O., Der, C. J. and Bokoch, G. M. (1994). Activated or dominant inhibitory mutants of RaplA decrease the oxidative burst of Epstein-Barr virus-transformed human B lymphocytes. J. Biol. Chem. 269:18743-18746. Mangues, R., Seidman, I., Gordon, J. W. and Pellicer, A. (1992). Overexpression of the N-ras proto-oncogene, not somatic mutational activation, associated with malignant tumors in transgenic mice. Oncogene 7:2073-2076. Mangues, R., Seidman, I., Pellicer, A. and Gordon, J. W. (1990). Tumorigenesis and male sterility in transgenic mice expressing a MMTV/N-ras oncogene. Oncogene 5:1491-1497. Maniatis, T., Sambrook, J. and Fritsch, E. F. (1989). Molecular cloning: A laboratory manual. Cold Spring Harbor, Cold Spring Harbor Press. Mann, S. K. O. and Firtel, R. A. (1993). cAMP-dependent protein kinase differentially regulates prestalk and prespore differentiation during Dictyostelium development. Development 119:135-146. Mann, S. K. O., Richardson, D. L., Lee, S., Kimmel, A. R. and Firtel, R. A. (1994). Expression of cAMP-dependent protein kinase in prespore cells is sufficient to induce spore cell differentiation in Dictyostelium. Proc. Natl. Acad. Sci. USA 91:10561-10565. Marshall, M. S. (1993). The effector interactions of p21ras. Trends Biochem. Sci. 18:250-254. Marshall, M. S., Davis, L. J., Keys, R. D., Mosser, S. D., Hill, W. S., Scolnick, E. M. and Gibbs, J. B. (1991). Identification of amino acid residues required for Ras-p21 target activation. Mol. Cell. Biol. 11:3997-4004. Martin, G. A., Viskochil, D., Bollag, G., McCabe, P. C, Crosier, W. J., Haubruck, H., Conroy, L., Clark, R., O'Connell, P., Cawthon, R. M., et al. (1990). The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell 63:843-849. Martin, G. A., Yatani, A., Clark, R., Conroy, L., Polakis, P., Brown, A. M. and Mccormick, F. (1992). G A P domains responsible for Ras p21-dependent inhibition of muscarinic atrial K + channel currents. Science 255:192-194. Maruta, H., Baltes, W., Dieter, P., Marme, D. and Gersih, G. (1983). Myosin heavy chain kinase inactivated by Ca^ +/ calmodulin from aggregating cells of Dictyostelium discoideum. EMBO J. 2. Maruta, FL, Holden, J., Sizeland, A. and D'Abaco, G. (1991). The residues of Ras and Rap proteins that determine their G A P specificities. J. Biol. Chem. 266:11661-11668. Matsudaira (1991). Modular organization of actin crosslinking proteins. Trends Biochem. Sci. 16:87-92. McCormick, F. (1994). Activators and effectors of ras p21 proteins. Curr. Opin. Genetics Dev. 4:71-76. McGlade, J., Brunkhorst, B., Anderson, D., Mbamalu, G., Settleman, J., Dedhar, S., Rozakis-Adcock, M., Chen, L. B. and Pawson, T. (1993). The N -terminal region of G A P regulates cytoskeletal structure and cell adhesion. EMBO J. 12:3073-3081. McRobbie, S. J. and Newell, P. C. (1983). Changes in actin association with cytoskeleton following chemotactic stimulation of Dictyostelium discoideum. Biochem. Biophys. Res. Commun. 115:351-359. McRobbie, S. J. and Newell, P. C (1984). Chemoattractant-mediated changes in cytoskeletal actin of cellular slime moulds. J. Cell Sci. 68:139-151. Milburn, M. V., Tong, L., De Vos, A. M., Brunger, A., Yamaizumi, Z., Nishimura, S. and Kim, S. H. (1990). Molecular switch for signal transduction: Structural differences between active and inactive forms of protooncogenic ras proteins. Science 247:939-945. Miller, J. H. (1972). Experiments in molecular genetics. Cold Spring Harbor, Cold Spring Harbor Laboratory: 352-355. Minden, A., Lin, A., Claret, F., Abo, A. and Karin, M. (1995). Selective activation of the JNK signalling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell 81:1147-1157. M i u r a , Y . , K a i b u c h i , K. , I toh, T. , C o r b i n , J . D., Franc is , S . H . a n d T a k a i , Y . (1992). Phosphory la t i on of smg p 2 1 B / raplB p21 by cycl ic G M P - d e p e n d e n t protein kinase. F E B S Lett. 297:171-174. M i z u n o , T., K a i b u c h i , K., Yamamoto, T., Kawamura , M . , Sakoda , T., Fu j i oka , H . , M a t s u u r a , Y . a n d T a k a i , Y . (1991). A st imulatory G D P / G T P exchange prote in for smg-p21 is active on the post-translationally processed fo rm of c-K i - ras-p21 and rhoA-p21. Proc Nat l Acad Sci Usa 88:6442-6446. M o o d i e , S . A . , W i l l u m s e n , B. M . , W e b e r , M . J . a n d W o l f m a n , A . (1993). C o m p l e x e s of R a s - G T P w i t h Raf-1 and mi togen-act ivated p ro te in k inase kinase. Science 260:1658-1661. M o o d i e , S. A . a n d W o l f m a n , A . (1994). The 3Rs of life: Ras, Raf and g rowth regulation. Trends Genetics 10:44-48. M u l c a h y , L. S. , S m i t h , M . R. a n d Stacey, D . W . (1985). Requirement for ras proto-oncogene funct ion du r ing serum-st imulated g rowth of N I H 3T3 cells. Nature 313: 241-243. N a d i n - D a v i s , S., N a s i n , A . and Beach, D. (1986). Involvement of ras i n sexual d i f ferent iat ion but not i n g rowth control i n f iss ion yeast. E M B O J . 5: 2963-2971. N a s s a r , M . , H o r n , G . , H e r r m a n n , C , Scherer , A . , M c C o r m i c k , F. a n d Wi t t inghofer , A . (1995). The 2.2 angstrom crystal structure of the Ras-b ind ing d o m a i n of the serine threonine kinase c -Raf l i n complex w i t h R a p l A a n d a G T P analogue. Nature 375:554-560. N e l l e n , W . , S i l a n , C . and F i r te l , R. A . (1984). D N A - m e d i a t e d transformation i n Dictyostelium discoideum: regulated express ion of an act in gene fus ion . M o l . Ce l l . B io l . 4:2890-2898. N e w e l l , P. C . a n d L i u , G . (1992). Streamer F mutants and chemotax is of Dictyostelium. Bioessays 14:473-479. N o b e s , C . a n d H a l l , A . (1994). Regulat ion and funct ion of the Rho subfami ly of smal l GTPases. Curr . B i o l . 4:77-81. N o b e s , C . D . a n d H a l l , A . (1995). Rho, rac, and cdc42 GTPases regulate the assembly of mult imolecular focal complexes associated w i th actin stress fibers, lamel l ipodia and f i lopodia. Ce l l 81: 53-62. N o d a , M . (1993). Structures and funct ions of the Krev-1 t rans fo rmat ion suppressor gene and its relatives. B ioch im. B iophys. Ac ta 1155:97-109. Nur-E-Kamal, M. S. A., Sizeland, A., D'Abaco, G. and Maruta, H. (1992). Asparagine 26, glutamic acid 31, valine 45, and tyrosine 64 of Ras proteins are required for their oncogenicity. J. Biol. Chem. 267:1415-1418. Ohmstede, C , Farrel, F. X., Reep, B. R., Clemetson, K. J. and Lapetina, E. G. (1990). RAP2B: A RAS-related GTP-binding protein from platelets. Proc. Natl. Acad. Sci. USA 87:6527-6531. Pai, E. F., Krengel, U., Petsko, G. A., Goody, R. S., Kabsch, W. and Wittinghofer, A. (1990). Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBOJ. 9:2351-2359. Pan, P., Hall, E. M. and Bonner, J. T. (1972). Folic acid as a second chemotactic substance in the cellular slime moulds. Nature New Biology 237:181-182. Pan, P., Hall, E. M. and Bonner, J. T. (1975). Determination of the active portion of the folic acid molecule in cellular slime mold chemotaxis. J. Bacterid. 122:955-959. Pasternak, C , Spudich, J. A. and Elson, E. L. (1989). Capping of surface receptors and concomitant cortical tension are generated by conventional myosin. Nature 341:549-551. Paterson, H., Reeves, B., Brown, R., Hall, A., Furth, M., Bos, J., Jones, P. and Marshall, C. (1987). Activated N-ras controls the transformed phenotype of HT1080 human fibrosarcoma cells. Cell 51:803-812. Paterson, H. F., Self, A. J., Garrett, M. D., Just, I., Aktories, K. and Hall, A. (1990). Microinjection of recombinant pllrho induces rapid changes in cell morphology. J. Cell Biol. Ill: 1001-1007. Patterson, B., Ruppel, K. M. and Spudich, J. A. (1991). Molecular genetic approaches to the cytoskeleton in Dictyostelium. Curr. Opin. Genetics Dev. 1: 378-382. Patterson, B. and Spudich, J. A. (1995). A novel positive selection for identifying cold-sensitive myosin II mutants in Dictyostelium. Genetics 140: 505-515. Pawson, T. (1995). Protein modules and signalling networks. Nature 373: 573-580. Peterson, M. D., Novak, K. D., Reedy, M. C , Ruman, J. I. and Titus, M. A. (1995). Molecular genetic analysis of myoC, a Dictyostelium myosin I. J. Cell Sci. 108:1093-1103. Pizon, V., Chardin, P., Lerosey, I., Olofsson, B. and Tavitian, A. (1988a). Human cDNAs rapl and rapl homologous to the Drosophila gene Dras3 encode proteins closely related to ras in the 'effector' region. Oncogene 3:201-204. Pizon, V., Desjardins, M., Bucci, C , Parton, R. G. and Zerial, M. (1994). Association of Rapla and Raplb proteins with late endocytic/ phagocytic compartments and Rap2a with the Golgi complex. J. Cell. Sci 107:1661-1670. Pizon, V., Lerosey, I., Chardin, P. and Tavitian, A. (1988b). Nucleotide sequence of a human cDNA encoding a ras-related protein (raplB). Nucleic Acids Res. 16:7719. Polakis, P., Rubinfeld, B. and Mccormick, F. (1992). Phosphorylation of raplGAP in vivo and by cAMP-dependent kinase and the cell cycle p34cdc2 kinase in vitro. J. Biol. Chem. 267:10780-10785. Qiu, R., Chen, J., Kirn, D., McCormick, F. and Symons, M. (1995). An essential role for Rac in Ras transformation. Nature 374:457-459. Quilliam, L. A., Zhong, S., Rabun, K. M., Carpenter, J. W., South, T. L., Der, C J. and Campbell-Burk, S. (1995). Biological and structural characterization of a Ras transforming mutation at the phenylalanine-156 residue, which is conserved in all members of the Ras superfamily. Proc. Natl. Acad. Sci. USA 92:1272-1276. Raleigh, E. A., Lech, K. and Brent, R. (1989). .Current protocols in Molecular Biology. F. M. Ausubel. New York, Publishing Associates and Wiley Interscience. Unit 1.4. Raper, K. (1940). Pseudoplasmodium formation and organization in Dictyostelium discoideum. J. Elisha. Mitchell. Sci. Soc. 59:241-282. Rathi, A., Kayman, S. C. and Clarke, M. (1991). Induction of gene expression in Dictyostelium by prestarvation factor, a factor secreted by growing cells. Dev. Genetics 12:82-87. Ravid, S. and Spudich, J. A. (1989). Myosin heavy chain kinase from developed Dictyostelium cells. Purification and characterization. J. Biol. Chem. 264:15144-50. Ravid, S. and Spudich, J. A. (1992). Membrane-bound Dictyostelium mysoin heavy chain kinase: purification and characterization. Proc. Natl. Acad. Sci. USA 89:5877-5881. Rebstein, P. J., Spiegelman, G. B. and Weeks, G. (1993). Altered morphology of vegetative amoebae induced by increased expression of the Dictyostelium discoideum ras-related gene rapl. Dev. Genetics 14: 347-355. Rey, I., Harris-Taylor, P., van Erp, H. and Hall, A. (1994). R-ras interacts with rasGAP, neurofibromin and c-raf but does not regulate cell growth or differentiation. Oncogene 9:685-692. Reymond, C. D., Gomer, R. H., Mehdy, M. C. and Firtel, R. A. (1984). Developmental regulation of a Dictyostelium gene encoding a protein homologous to mammalian ras protein. Cell 39:141-148. Reymond, C. D., Gomer, R. H., Nellen, W., Theibert, A., Devreotes, P. and Firtel, R. A. (1986). Phenotypic changes induced by a mutated ras gene during the development of Dictyostelium transformants. Nature 323: 340-343. Ridley, A. J. and Hall, A. (1992). The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70:389-399. Ridley, A. J., Paterson, H F., Johnston, C L., Diekmann, D. and Hall, A, (1992). The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70:401-410. Robbins, S. M. (1991). Characterization of a ras and a ras-related gene and their developmental expression in the cellular slime mould Dictyostelium discoideum. PhD Thesis University of British Columbia. Robbins, S. M., Suttorp, V. V., Weeks, G. and Spiegelman, G. B. (1990). A ras-related gene from the lower eukaryote Dictyosteliwn that is highly conserved relative to the human rap genes. Nucleic Acids Res. 18:5265-5269. Robbins, S. M., Williams, J. G., Jermyn, K. A., Spiegelman, G. B. and Weeks, G. (1989). Growing and developing Dictyostelium cells express different ras genes. Proc Natl. Acad. Sci. USA 86:938-942. Robbins, S. M., Williams, J. G., Spiegelman, G. B. and Weeks, G. (1992). Cloning and characterization of the Dictyostelium discoideum rasG genomic sequences. Biochim. Biophys. Acta 1130:85-89. Rothman, J. E. and Orci, L . (1992). Molecular dissection of the secretory pathway. Nature 355:409-415. Rubinfeld, B., Munemitsu, S., Clark, R., Conroy, L. , Watt, K., Crosier, W. J., McCormick, F. and Polakis, P. (1991). Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rapl. Cell 65:1033-1042. Rubino, S., Fighetti, M., Unger, E. and Cappuccinelli, P. (1984). Location of actin, myosin, and microtubular structures during directed locomotion of Dictyostelium amebae. J. Cell Biol. 98:382. Ruggieri, R., Bender, A., Matsui, Y., Powers, S., Takai, Y., Pringle, J. R. and Matsumoto, K. (1992). RSR1, a 7as-like gene homologous to Krev-1 (smg21A/ rapl A): Role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae. Mol. Cell. Biol. 12: 758-766. Ruppel, K. M. and Spudich, J. A. (1995). Myosin motor function: structural and mutagenic approaches. Curr. Opinion Cell Biol. 7:89-93. Russell, M., Langecarter, C. and Johnson, G. L. (1995). Direct interaction between Ras and the kinase domain of mitogen-activated protein kinase kinase kinase (MEKK1). J. Biol. Chem. 270:11757-11760. Sanger, F., Nicklen, S. and Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl. Acad. Sci. USA 74:5463-5467. Satoh, T., Endo, M., Nakafuku, M., Akiyama, T., Yamamoto, T. and Kaziro, Y. (1990a). Accumulation of p21 r a s-GTP in response to stimulation with epidermal growth factor and oncogene products with tyrosine kinase activity. Proc Natl. Acad. Sci. USA 87:7926-7929. Satoh, T., Endo, M., Nakafuku, M., Nakamura, S. and Kaziro, Y. (1990b). Platelet-derived growth factor stimulates formation of active p21 r a s-GTP complex in Swiss mouse 3T3 cells. Proc. Natl. Acad. Sci. USA 87: 5993-5997. Satoh, T., Nakamura, S. and Kaziro, Y. (1987). Induction of neurite formation in PC12 cells by microinjection of proto-oncogenic Ha-ras protein preincubated with guanosine-5'-0 -(3-thiotriphosphate). Mol. Cell. Biol. 7: 4553-4556. Sawadogo, M. and Van Dyke, M. W. (1990). A rapid method for the purification of deprotected oligodeoxynucleotides. Nucleic Acids Res. 19:674. Schejter, E. D. and Shilo, B. (1985). Characterization of functional domains of p21 ras by use of chimeric genes. EMBO J. 4:407^ 412. Schlessinger, J. (1994). SH2/SH3 signaling proteins. Curr. Opin. Genetics Dev. 4:25-30. Schnitzler, G. R., Briscoe, G, Brown, J. M. and Firtel, R. A. (1995). Serpentine cAMP receptors may act through a G protein-independent pathway to induce postaggregative development in Dictyostelium.. Cell 81: 737-745. Schnitzler, G. R., Fischer, W. H. and Firtel, R. A. (1994). Cloning and characterization of the G-box binding factor, an essential component of the developmental switch between early and post-aggregative development in Dictyostelium. Genes Dev. 8:502-514. Schweiger, A., Mihalache, O., Ecke, M. and Gerisch, G. (1992). Stage-specific tyrosine phosphorylation of actin in Dictyostelium discoideum cells. J. Cell Sci. 102:601-609. Self, A. J., Paterson, H. F. and Hall, A. (1993). Different structural organization of Ras and Rho effector domains. Oncogene 8:655-661. Shaulsky, G. arid Loomis, W. F. (1993). Cell type regulation in response to expression of ricin A in Dictyostelium. Dev. Biol. 160: 85-98. Siess, W., Winegar, D. A. and Lapetina, E. G. (1990). Rapl-B is phosphorylated by protein kinase A in intact human platelets. Biochem. Biophys. Res. Commun. 170: 944-950. Sigal, I. S., Gibbs, J. B., D/Alonzo,J. S. and Scolnick, E. M. (1986). Identification of effector residues and a neutralizing epitope of Ha-7'fls-encoded p21. Proc. Natl. Acad. Sci. USA 83:4725-4729. Simon, M. A., Bowtell, D. D. L., Dodson, G. S., Laverty, T. R. and Rubin, G. M. (1991). Rasl and a putative guanine nucleotide exchange factor perform crucial steps in signalling by the sevenless protein tyrosine kinase. Cell 67: 701-716. Simon, M. N., Driscoll, D., Mutzel, R., Part, D., Williams, J. and Vreron, M. (1989). Overproduction of the regulatory subunit of the cAMP-dependent protein kinase blocks the differentiation of Dictyostelium discoideum. EMBO J. 8:203943. Simon, M. N., Pelegrini, O., Veron, M. and Kay, R. R. (1992). Mutation of protein kinase-A causes heterochronic development of Dictyostelium. Nature 356:171-172. Soil, D. R. and Waddell, D. R. (1975). Morphogenesis in the slime mold Dictyostelium discoideum I. The accumulation and erasure of 'morphogenetic information'. Dev. Biol. 47:292-302. Spaargaren, M., Martin, G. A., McCormick, F., Fernandez-Sarabia, M. J. and Bischoff, J. R. (1994). The Ras-related protein R-ras interacts directly with Raf-1 in a GTP-dependent manner. Biochem. J. 300:303-307. Spandidos, A. D. and Wilkie, N. M. (1984). Malignant transformation of early passage rodent cells by a single mutated human oncogene. Nature 310: 469-475. Springer, M. L., Patterson, B. and Spudich, J. A. (1994). Stage-specific requirement for myosin II during Dictyostelium development. Development 120:2651-2660. Spudich, J. A. (1994). How molecular motors work. Nature 372:515-518. Stacey, D. W., Feig, L. A. and Gibbs, J. B. (1991). Dominant inhibitory Ras mutants selectively inhibit the activity of either cellular or oncogenic Ras. Mol. Cell. Biol. 11:4053-4064. Stacey, D. W. and Kung, H. F. (1984). Transformation of NIH 3T3 cells by microinjection of Ha-ras p2l protein. Nature 310:508-511. Stone, J. C. and Blanchard, R. A. (1991). Genetic definition of ras effector elements. Mol. Cell. Biol. 11:6158-6165. Sudhbf, T. C. (1995). The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature 375:645-653. Tan, J. L., Ravid, S. and Spudich, J. A. (1992). Control of nonmuscle myosins by phosphorylation. Annu. Rev. Biochem. 61:721-759. Titus, M. A., Kuspa, A. and Loomis, W. F. (1994). Discovery of myosin genes by physical mapping in Dictyostelium. Proc Natl Acad Sci USA 91:9446-9450. Toda, T., Uno, I., Ishikawa, T., Powers, S., Kataoka, T., Broek, D., Cameron, S., Broach, J. B., Matsumoto, K. and Wigler, M. (1985). In yeast RAS proteins are controlling elements of adenylate cyclase. Cell 40:27-36. Trahey, M. and McCormick, F. (1987). A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science 238:542-545. Traynor, D., Kessin, R. H. and Williams, J. G. (1992). Chemotactic sorting to cAMP in the multicellular stages of Dictyostelium development. Proc. Natl. Acad. Sci. USA 89:8303-8307. Valencia, A., Chardin, P., Wittinghofer, A. and Sander, C. (1991). The ras protein family: Evolutionary tree and role of conserved amino acids. Biochemistry 30:46374668. Viciana, P. R., Warne, P. H , Dhand, R., Vanhaesebroek, B., Gout, I., Fry, M. J., Waterfield, M. D. and Downward, J. (1994). Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370:527-532. Vithalani, K., Larochelle, D. and DeLozanne, A. (1995). Disruption by REMI of a Rho/Rac-like protein in Dictyostelium Discoideum cells impairs cytokinesis. International Dictyostelium Conference, France. Vojtek, A. B., Hollenberg, S. M. and Cooper, J. A. (1993). Mammalian Ras interacts directly with the serine /threonine kinase Raf. Cell 74:205-214. W a d d e l l , D . R. a n d S o i l , D . R. (1977). A character izat ion of the erasure phenomenon i n Dictyostelium discoideum. D e v . B i o l . 60: 83-92. W a n g , N . , But ler , J . P. and Ingber, D . E. (1993). Mechanotransduct ion across the cell surface and through the cytoskeleton. Science 260:1124-1127. Warne, P. H . , V i c i ana , P. R. and D o w n w a r d , J . (1993). Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature 364: 352-355. Wat ts , D . a n d A s h w o r t h , J . (1970). G r o w t h of myxamoebae of the ce l lu lar s l ime m o u l d Dictyostelium discoideum i n axenic cul ture. B i o c h e m . J . 119: 171-174 W e e k s , G . a n d Gross , J . D . (1991). Potential morphogens invo lved i n pattern format ion du r ing Dictyostelium differenfion. B iochem. C e l l B i o l . 69: 608-617. W i l l i a m s , J . G . , Ceccarel l i , A . , M c R o b b i e , S., M a h b u b a n i , H . , K a y , R. R., Ear ly , A . , B e r k s , M . a n d J e r m y n , K . A . (1987). Direct i nduc t i on of Dictyostelium prestalk gene expression by DIF provides evidence that DIP is a morphogen. Ce l l 49:185-192. W i l l i a m s , J . G . , D u f f y , K. T. , Lane , D . P. , M c R o b b i e , S. J . , H a r w o o d , A . J . , T r a y n o r , D . , K a y , R. R. a n d J e r m y n , K . A . (1989). O r i g i ns of the prestalk-prespore pattern in Dictyostelium development. C e l l 59:1157-1163. W i l l i a m s e n , B. M . , Chr i s tensen , A . , Hubbe r t , N . L., Papageorge, A . G . a n d L o w y , D . R. (1984). The p21 ras C-terminus is required for transformation and membrane association. Nature 310:583-586. W i t k e , W. , N e l l e n , W . a n d Noege l , A . (1987). Homologous recombinat ion i n the D ic t yos te l i um a-ac t in in gene leads to an altered m R N A and the lack of the protein. E M B O J. 6:4143-4148. W u , L., V a l k e m a , R., V a n Haastert, P. J . M . and Devreotes, P. N . (1995). The G pro te in p subuni t is essential for mu l t ip le responses to chemoattractants i n Dictyostelium. J . C e l l B i o l . 129:1667-1675. X u , H . , W a n g , Y . , R i g g s , M . , Rodgers , L. a n d W i g l e r , M . (1990). B io log ica l activity of the mammal ian Rap genes in yeast. Ce l l Regu la t ion 1: 763-769. Yatani , A . , Okabe , K., Polak is , P., Halenbeck, R., McCormick , F. and B rown , A . M . (1990). ras p21 and G A P inhibit coupl ing of muscur in ic receptors to atr ial K + channels. Ce l l 61:769-776. Y a t a n i , A . , Q u i l l i a m , L. A . , B r o w n , A . M . and B o k o c h , G . M . (1991). R a p l A antagonizes the abil i ty of Ras and Ras-Gap to inhibit muscar inic K+ channels. J B io l C h e m 266:22222-22226. Yoshida, Y., Kawata, M., Miura, Y., Musha, T., Sasaki, T., Kikuchi, A. and Takai, Y. (1992). Microinjection of smg/rapl /Krev-1 p21 into Swiss 3T3 cells induces DNA synthesis and morphological changes. Mol. Cell. Biol. 12: 3407-3414. Yuen, I. S.Jain, R. , Bishop, J. D., Lindsey, D. F., Deery, W. J., Van Haastert, P. J. M. and Gomer, R . H . (1995). A density-sensing factor regulates signal transduction in Dictyostelium. J. Cell Biol. 129:1251-1262. Zhang, K., Noda, M., Vass, W. C , Papageorge, A. G. and Lowry, D. R . (1990). Identification of small clusters of divergent amino acids that mediate the opposing effects of ras and Krev-1. Science 249:162-165. Zhang, K., Papageorge, A. G., Martin, P., Vass, W. C , Olah, Z., Polakis, P. G., McCormick, F. and Lowy, D. R. (1991). Heterogeneous amino acids in Ras and RaplA specifying sensitivity to GAP proteins. Science 254:1630-1634. Zhang, X. F., Settleman, J., Kyriakis, J. M., Takeuchisuzuki, E., Elledge, S. J., Marshall, M. S., Bruder, J. T., Rapp, U. R . and Avruch, J. (1993). Normal and oncogenic P21(ras) proteins bind to the amino-terminal regulatory domain of C-Raf-1. Nature 364:308-313. Zheng, Y., Bagrodia, S. and Cerione, R . A. (1994). Activation of phosphoinositide 3-kinase activity by Cdc42Hs binding to p85. J. Biol. Chem. 269:18727-18730. 

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