DEVELOPMENTAL GENETICS 12:147-153 (1991)
Ras-Related Genes in Dictyostelium discoideum STEPHEN M. ROBBINS, MEENAL KHOSLA, RICHARD THIERY, GERALD WEEKS, AND GEORGE B. SPIEGELMAN Departments of Microbiology (S.M.R., M.K., R.T., G.W., G.B.S.) and Medical Genetics (G.W., G.B.S.), University of British Columbia, Vancouver, British Columbia, Canada 19891. However, the detailed mechanism by which the activated ras proteins produce the malignant phenotype remains unknown. The ras genes are part of a large multigene family and numerous members have been found in a wide variety of organisms. The major sequence conservation within the superfamily is the preservation of the four domains associated with guanine nucleotide binding, but there are additional sequence similarities and the individual members of the ras gene superfamily vary in the amount of additional identity [Chardin, 19881. The cellular slime mold Dictyostelium discoideum has properties that make it attractive for examining ras gene function in both cellular proliferation and differentiation and studies on this organism may provide Key words: Eukaryotes, vegetative cells, rus proinsights into the function of ras genes in higher organteins isms. In Dictyostelium, growth and differentiation are distinct phenomena; in the presence of a bacterial food source amoeba proliferate, but upon starvation cell diINTRODUCTION vision ceases and a well-defined differentiation process Ras genes and members of the ras-related gene su- is initiated. During differentiation the amoeba are atperfamily have now been identified in eukaryotic or- tracted to one another by pulses of cyclic AMP (CAMP) ganisms as diverse a s yeast and man and their encoded forming a multicellular aggregate which eventually products are highly conserved [for review, see Barba- elongates to a migrating pseudoplasmodium. The ancid, 19871. This widespread phylogenetic conservation terior region of the pseudoplasmodium comprises suggests t h a t the proteins fulfill functions that are es- prestalk cells and the posterior region comprises presential to all eukaryotic cells. spore cells. Ultimately the cell mass evolves into the The mammalian ras genes encode proteins that bind mature fruiting body comprising two cell types, spore guanine nucleotides [Papageorge et al., 19821, exhibit and stalk cells. Differentiation is dependent upon siga n intrinsic GTPase activity [McGrath et al., 1984; nal transduction pathways t h a t require specific G proManne et al., 19851, and are attached to the inner sur- teins and may therefore be analogous to signal transface of the plasma membrane [Sefton et al., 1982; Shih duction pathways in higher organisms [for review, see et al., 1982; Willumsen et al., 19841. These biochemical Janssens and Van Haastert, 1987; Kessin, 1988; Firtel properties closely resemble those of the regulatory G et al., 19891. proteins [Gilman, 19871 and thus by analogy it is generally assumed that the ras proteins function a s reguRAS GENES IN DICTYOSTELIUM latory elements in a signal transduction pathway. DeTwo closely related ras genes have been identified spite the extensive biochemical knowledge, there are thus far in Dictyostelium, Ddras and DdrasG [Reymond only a few clues as to the physiological role of the ras
ABSTRACT Dicfyostelium discoideum, like other eukaryotes, has been shown to express several ras-related genes. Two gene products, Ddras and DdrasG, are highly conserved relative to the human ras proteins. Ddrus is expressed at the pseudoplasmodial stage of development, whereas DdrasG is expressed in vegetative cells and during early development. In addition, Dicfyostelium possesses three rus-related genes, SAS1, SAS2 and Ddrupl, whose gene products are only partially conserved relative to those of the rus genes. The expression of these three genes is also developmentally regulated.
proteins in normal growth and differentiation. The mammalian ras genes are expressed in both immature cells and in certain terminally differentiated cell types suggesting a role in both cellular proliferation and differentiation [Barbacid, 19871. Point mutations in either codons 12, 13, 59, or 61 result in a n activated ras gene product that produces a transformed cellular phenotype in a variety of cell lines [for review, see Bos,
0 1991 WILEY-LISS, INC.
Received for publication July 31, 1990. Address reprint requests to Dr. Gerald Weeks, Department of Microbiology, University of British Columbia, Vancouver, B.C., Canada V6T IW5.
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et al., 1984; Robbins et al., 19891. The deduced amino acid sequences for Ddras and DdrasG are 82% conserved and have 65%and 69% identity with the human ras proteins, respectively. Both of the predicted Ddras and DdrasG protein sequences have features characteristic of other ras proteins (Fig. 1).The amino half of the protein is over 90% conserved but the carboxyl region is variable with the exception of the tetrapeptide sequence at the extreme terminus. Both the Ddras and DdrasG proteins possess the four highly conserved domains (Figure 1)that in mammalian ras proteins are associated with guanine nucleotide binding or GTPase activity [Barbacid, 19871. In addition the Dictyostelium proteins contain the effector domain (Fig. 1) which in mammalian proteins is believed to interact with the GTPase-activating molecule (GAP) [McCormick, 19891 suggesting the possible existence of a GAP-like molecule in Dictyostelium.
EXPRESSION OF Ddras AND DdrusG DURING DICTYOSTELIUM DEVELOPMENT The Ddras and DdrasG genes are expressed at different times during Dictyostelium development (Fig. 2). The Ddras gene is expressed at the pseudoplasmodial stage as two mRNA species of 1.2 and 0.9 kb which are enriched in the prestalk cell population [Reymond et al., 1984; Robbins et al., 19891. Induction of only the 1.2 kb Ddras mRNA occurs precociously under in vitro conditions in response to the addition of CAMP.Precocious expression in response to cyclic AMP is characteristic for a number of other prestalk-enriched genes [Reymond et al., 19841. In contrast the DdrasG gene is expressed as a single mRNA species of 1.2 kb during growth and early development [Robbins et al., 19891. The amount of DdrasG mRNA increases approximately 2 fold during the first 2-3 hours of development and then declines reaching negligible levels by the aggregation stage [Khosla et al., 19901. The decline in DdrusG mRNA level requires a developmentally regulated gene product since it does not occur in the presence of the protein synthesis inhibitor, cycloheximide. During differentiation in shake suspension of DdrasG mRNA levels do not decline, unless cells are treated with pulses of CAMP (Fig. 3). These results suggest that the decline in mRNA levels observed during aggregation is a response of the cyclic AMP relay system to pulses of cyclic AMP. Consistent with this conclusion is the finding that the decrease in mRNA level did not occur when cyclic AMP pulses were applied in the presence of caffeine, a n inhibitor of the cyclic AMP signal relay response (Fig. 3). DICTYOSTELIUM rus GENE PRODUCTS The first indication of the existence of ras gene products in Dictyostelium came with the finding that a major protein of 23,000 M, (p23) was specifically precipitated from cell-free extracts using the ras-specific
monoclonal antibody, Y13-259 [Pawson and Weeks, 19841. The relative rates of p23 synthesis are maximal during vegetative growth and early development with a sharp decline thereafter. However, there is a small burst of synthesis during the pseudoplasmodial stage of development (Fig. 4). There is no specific degradation of p23 during differentiation suggesting that the decrease in synthesis of ras protein is solely responsible for the reduction of p23 levels that occurs [Pawson et al., 1985; Weeks and Pawson, 19871. Since the Y13-259 monoclonal antibody recognizes a sequence in the human ras proteins (amino acids 6273) [Sigal et al., 19861 that is conserved in both the Ddras and DdrasG proteins (Fig. 11, it would presumably immunoprecipitate both proteins. The combined expression of the Ddras and DdrasG genes [Robbins et al., 19891 can account for the changes in the rate of ras protein synthesis that are observed during the differentiation process [Pawson et al., 19851, although this does not preclude the possibility that there may be additional ras genes that remain to be discovered. In addition to p23 a minor protein of 24,000 M, (p24) is also immunoprecipitated from cell-free extracts of both vegetative and pseudoplasmodial cells. There is no evidence for a precursor-product relationship between p23 and p24 suggesting the possibility that they are distinct gene products, but the precise relationship between the two proteins remains to be determined [Weeks and Pawson, 19871. The Y13-259 precipitated protein, like its mammalian counterparts, is acylated and membrane bound [Weeks et al., 19871. When both vegetative and pseudoplasmodial mRNA preparations are in vitro translated, two Y13-259 immunoprecipitable proteins of similar but not identical molecular weights to p24 and p23 are detected [Pawson et al., 19851. The two products produced by in vitro translation of pseudoplasmodial mRNA are derived from the Ddras gene, since mRNA that was hybrid selected by the Ddras gene was in vitro translated into the two protein products [Robbins, unpublished observations]. However a detailed analysis of the in vitro synthesized protein products will be necessary to establish their exact relationship to each other and to p24 and p23.
CELL-TYPE SPECIFIC Ddrus EXPRESSION It was shown initially that Ddras is enriched in the prestalk cell population [Reymond et al., 19841 and this result has subsequently been confirmed [Jermyn et al., 19871. However the enrichment is only 3 fold whereas the DIF-induced genes pD63 and pD56 are enriched 12 fold [Jermyn et ul., 19871. In contrast, ras-related protein synthesis only occurs in the prespore cell population [Weeks and Pawson, 19871. In these latter experiments the prestalk and prespore cells were separated by a modification [Kwong, et al., 19881 of the Ratner and Borth [ 19811 Percoll gradient technique. We have
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Fig. 1. Comparison of the derived amino acid sequences of the human ras protein, Harasl [Capon et al., 19831, the two Dictyostelium ras proteins, Ddras [ Reymond et al., 19841 and DdrasG [Robbins et al., 19891, and the Dictyostelium ras-related proteins SASl and SASZ [Saxe and Kimmel, 19881 and Ddrapl [Robbins et al., 19901. Amino acids are represented by the single letter code and the letters with the
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black background indicate identity. Gaps have been inserted for optimal alignments and the arrow indicates a sequence from SASl (MIDTPNEQP)that has been omltted for the same reason. The open boxes above the sequences indicate regions involved in guanine nucleotide binding, the solid box Indicates the effector domain, and the hatched box demonstrates the Y13-259 antibody binding domain.
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Ddras Fig. 2. The expression of the DdrusG and Ddrus genes during Dictyostelium development. Poly(A)+ RNA (5 kg) from each of the indicated times (hours) after the onset of differentiation was separated on a 1.25% formaldehyde-agarose gel, transferred to nitrocellulose and probed with the DdrusG and Ddras cDNAs as indicated [Robbins et ul., 19891.
subsequently found that this modified procedure yields a prespore cell fraction contaminated with a subpopulation of prestalk cells [Kwong et al., 19901. This contaminating subpopulation of prestalk cells expresses the pDd63 and Ddras genes but does not express the pDd56 gene, suggesting that the Ddras gene is expressed in a subset of prestalk cells. This is not the first evidence for heterogeneity of the prestalk cell population. It has been shown recently that different areas of the prestalk region express difFig. 3. The effect of cAMP and caffeine on DdrusG expression. ferent genes [Gomer et al., 1986; Jermyn et al., 19891 cells were resuspended a t 5 x lo6 cells/ml in 20 mM potasand that the prestalk population can be functionally Washed sium phosphate buffer, pH 6.0, and (A) shaken at 250 rpm, (B) shaken subdivided into CAMP-independent and CAMP-depen- at 250 rpm and pulsed with cAMP to a concentration of 25 nM every dent cells on the basis of the requirements for stalk cell 5 minutes, or (C) shaken at 250 rpm in the presence of 2 mM caffeine formation in in vitro monolayers [Kwong et al., 19881. and pulsed with cAMP to a concentration of 25 nM every 5 minutes. The expression of the Ddras gene in a subset of prestalk RNA was extracted at the times (hours) indicated above each lane and hybridized with the DdrusG cDNA. cells provides further evidence for the heterogeneity within the prestalk zone of the migrating pseudoplasmodium. induced guanylate cyclase desensitization resulting in decreased intracellular cGMP accumulation [Van Ras GENE FUNCTION IN DICTYOSTELIUM Haastert et al., 19871. There is evidence that guanylate There is some evidence to suggest that in Dictyoste- cyclase regulation in Dictyostelium is coupled to the lium the Ddras gene is involved in cAMP transmem- inositol phosphate pathway [for review, see Newell et brane signal transduction. In Dictyostelium, cAMP sig- al., 1987; Janssens and Van Haastert, 19871 and the nal transduction involves two separate pathways regulation of this pathway is altered in the activated leading to adenylate cyclase and guanylate cyclase ac- Ddras transformants. In wild-type cells inositol tivation which are believed to control the signal relay triphosphate levels increase transiently in response to response and chemotactic response, respectively [for re- pulses of CAMP, whereas in the Ddras transformants views, see Newell et al., 1987; Janssens and Van there is a 3 fold higher basal level of inositol triphosHaastert, 19871. Dictyostelium transformants that phate and only a slight response to cAMP [Europeoverexpress a n activated Ddras gene (amino acid 12 Finner et al., 19881.These results suggest the possibilchanged from glycine to threonine) display a n aberrant ity that the Ddras gene product regulates the transient morphogenesis leading to multiple tipped aggregates activation of a n as yet undetected phospholipase C. In that do not form final fruiting bodies and this pheno- mammalian cells, the activation of phospholipase C ultype may be due to impaired regulation of the chemo- timately leads to the activation of protein kinase C tactic response [Reymond et al., 19861. The activated [Nishizuka, 19861 and it is possible therefore that proDdras transformants display reduced chemotactic sen- tein kinase C is involved in guanylate cyclase regulasitivity to cAMP and folic acid and a n enhanced CAMP- tion in Dictyostelium. Although there is as yet no direct
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signal transduction at the multicellular stage of development [Robbins et al., 19891. DdrasG gene expression is repressed by cAMP whereas the Ddras gene is induced by CAMP, a differential regulation that would ensure that the two gene products are not expressed at the same time during development. It has been found that the DdrasG gene is expressed throughout development in the axenic strain Ax2, providing a situation in which both the ras genes are expressed a t the pseudoplasmodial stage. The abnormal expression is unique to Ax2 since the expression in the parental strain NC4 resembled that observed in V12-M2 [Robbins and Khosla, unpublished observations]. Since the axenic strain develops relatively synchronously, culminating in a normal final fruiting body, i t does not appear that the continued expression of the DdrasG gene has a deleterious effect on development.
DEVELOPMENT
Fig. 4. Synthesis of D.discozdeum rus-related proteins during differentiation. Differentiating D.discoideum cells (10') were labeled for the indicated 2 hour periods with 250 FCi of [3"S1methionine. Cells were lysed and immunoprecipitated with Y 13-259 anti-p21 antibody, and the immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis. The label incorporated into rus-related proteins during these 2 hour pulses were determined by optically scanning the resulting fluorograms and integrating the peaks. The total incorporation into protein was measured by trichloroacetic acid precipitation of the original cell lysate. The extent of' rus-related protein synthesis for each 2 hour period is expressed as a fraction of the total protein synthesis in arbitrary units. Tip formation occurred at 10 hours of development in the experiment shown here [Pawson et al., 19851.
Ras GENE SUPERFAMILY IN DICTYOSTELIUM
In addition to DdrasG and Ddras, three ras-related genes, SAS1, SAS2, and Ddrapl, have thus far been characterized in Dictyostelium [Saxe and Kimmel, 1988; Robbins et al., 19901. The encoded products share the four guanine nucleotide binding domains, but do not possess lhe Y 13-259 niorioclonal antibody-binding site (Fig. 1). The encoded products of the SASl and SASB genes have similar sequences to those of the yeast YPT and SEC4 genes, which are involved in GTP-stimulated protein transport [Saxe and Kimmel, 19881, and the Dictyostelium gene products may perevidence for this involvement, indirect evidence sug- form similar functions. Comparisons of the SASl and gests that the activated Ddras gene product constitu- SASB gene product sequences with those of the other tively down regulates the cAMP receptors, a process Dictyostelium ras sequences are shown in Figure 1. which is normally under the control of protein kinase C We have recently isolated a gene, Ddrapl [Robbins et and the Ddras gene product [Luderus et al., 19881. al., 19901, that has approximately 80% sequence idenIt should be noted that in the above studies the ef- tity with the human rap genes [Pizon et al., 1988a; fects of the activated Ddras gene product on signal Pizon et al., 1988b; Kawata et al., 1988; Kitayama et transduction were performed on aggregation stage al., 19891. Within one of the four domains associated cells. Since Ddras is not expressed a t this time in wild- with guanine nucleotide binding, the human and the type cells [Reymond et al., 1984; Robbins et al., 19891, Dictyostelium rap genes encode a threonine a t position the activated ras protein might be interfering with 61, instead of the customary glutamine in other rassome other G protein rather than the normal Ddras related genes. The significance of this change is not gene product and thus ras proteins may not be involved known but it is interesting that the change of gluwith CAMP-mediated signal transduction during ag- tamine to threonine at position 61 in a ras gene is a n gregation. However, the Ddras protein may be involved activating mutation. In addition to the four domains in signal transduction a t the pseudoplasmodial stage of associated with guanine nucleotide binding, the effecdevelopment and require a specific cAMP receptor that tor domain that is believed to interact with the GAP is not expressed until this time. The existence of CAMP protein is highly conserved (Fig. 1).One of the interreceptor genes that are expressed a t the pseudoplasmo- esting features of the human raplA (also designated dial stage has recently been demonstrated [Saxe 111 et K-revl) gene is that it can suppress the transformed al., 19901. phenotype associated with a n activated ras gene in We have postulated that since the DdrasG and the NIH3T3 cells [Kitayama et al., 19891. The Ddrapl gene Ddras gene are expressed at distinctly different times is maximally expressed during the developmental during Dictyostelium development the two proteins stages where the levels of the DdrasG and Ddras mesmight perform different functions: DdrasG having a sages are declining [Robbins et al., 19901. The reciproregulatory role in cell proliferation and Ddras a role in cal nature of Ddrapl gene expression with respect to
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Kwong L, Sobolewski A, Atkinson L, Weeks G (1988): Stalk cell formation in monolayers from isolated prestalk and prespore cells of Dictyostelium discozdeum; evidence for two populations of prestalk cells. Development 104:121-127. Kwong L, Xie J, Daniel J , Robbins SM, Weeks G (1990): A DictyosteFUTURE PROSPECTS lium morphogen that is essential for stalk cell formation is generated by a subpopulation of prestalk cells. Development, in press. The discovery of a number of ras and ras-related Luderus MEE, Reymond CD, Van Haastert PJM, Van Driel R (1988): genes in many eukaryotes adds a n extra complexity in Expression of a mutated rus gene in Dictyostelium discoLdeum aldetermining the various functions of these genes. The ters the binding of cyclic AMP to its chemotactic receptor. J Cell Sci 90:701-706. availability of a DNA-mediated transformation system in Dictyostelium will allow the construction of defined Manne VE, Bekesi E, Kung H (1985):Ha-rus proteins exhibit GTPase activity: point mutations that activate Ha-rus genes result in demutants that can be used to directly address the biocreased GTPase activity. Proc Natl Acad Sci USA 82:376-380. logical functions of the ras and ras-related gene prod- McCormick F (1989): rus GTPase activating protein: signal transmitter and signal terminator. Cell 56:5-8. ucts. McGrath J P , Capon DJ, Goeddel DV, Levinson AD (1984): Comparative biochemical properties of normal and activated human ras p21 ACKNOWLEDGMENTS protein. Nature 310:644-649. The work cited from the Spiegelman and Weeks lab- Newell PC, Europe-Finner GN, Small NV (1987):Signal transduction during amoeba1 chemotaxis of Dictyostelium discoideum. Microbiol oratories was funded by grants from the Medical ReSci 4:5-12. search Council and the B.C. Health Care Research Nishizuka Y (1986):Studies and the perspectives of protein kinase C. Foundation. We wish to thank Don Henkelman of the Science 233:305-312. Cancer Control Agency of B.C. for assistance in illus- Papageorge AD, Lowy D, Scolnick EM (1982): Comparative biochemtrating the sequence identities shown in Figure 1. ical properties of p21 ras molecules coded for by viral and cellular ras genes. J Virol 44:509-519. Pawson T, Weeks G (1984): Expression of ras-encoded proteins in REFERENCES relation to cell growth and differentiation. In Bishop JM, Rowley Barbacid M (1987): ras genes. Annu Rev Biochem 56:779-827. JD, Greaves M (eds): Genes and Cancer. New York: Alan R. Liss, Bos J (1989): ras oncogenes in human cancer: a review. Cancer Res Inc. pp 461-470. 49:4682-4689. Pawson T, Amiel T, Hinze E, Auersperg N, Neave N, Sobolewski A, Capon DJ, Chen EY. Levinson AD, Seehurg PH, Cmeddel DV (1983): Weeks G (1985): Regulation of a ras-related protein during develComplete nucleotide sequence of the T24 human bladder carcinoma opment of Dictyostelium discoideum. Mol Cell Biol 5:33-39. oncogene and its normal homologue. Nature 302:33-37. Pizon V, Chardin P, Lerosey I, Olofsson B, Tavitian A (1988a): Human Chardin P (1988): The ras superfamily proteins. Biochimie 702365cDNAs rap1 and rap2 homologous to the Drosophda gene Dras3 868. encode proteins closely related to rus in the ‘effector’region. OncoEurope-Finner GN, Luderus MEE, Small NV, Van Driel R, Reymond gene 3:201-204. CD, Firtel RA, Newell PC (1988):Mutant ras gene induces elevated Pizon V, Lerosey I, Chardin P, Tavitian A (198813): Nucleotide selevels of inositol tris- and hexakisphosphates in Dictyostelium. J quence of a human cDNA encoding a ras-related protein (raplB). Cell Sci 89:13-20. Nucleic Acids Res 16:7719. Firtel RA, Van Haastert PJM, Kimmel AR, Devreotes PN (1989): G Reymond CD, Gomer RH, Mehdy M, Firtel RA (1984):Developmental protein linked signal transduction pathways in development: Dicregulation of a Dictyostelzumgene encoding a protein homologous to tyosteliun as a n experimental system. Cell 58:235-239. mammalian rus protein. Cell 39:141-148. Gilman A (1987): G proteins: transducers of receptor-generated sigReymond CD, Gomer RH, Nellen W, Theibert A, Devreotes P, Firtel nals. Annu Rev Biochem 56:615-650. RA (1986):Phenotypic changes induced by a mutated ras gene durGomer RH, Datta S, Firtel RA (1986): Cellular and subcellular dising the development of Dictyostelium transformants. Nature 323: tribution of a cyclic AMP-regulated prestalk protein and prespore 340-343. protein in Dictyostelium discoideum: A study of the ontogeny of Robbins SM, Williams JG, Jermyn KA, Spiegdman GB, Weeks G prestalk and prespore cells. J Cell Biol 103:1999-2015. (1989): Growing and developing Dictyostelium cells express differJanssens PMW, Van Haastert PJM (1987): Molecular basis of transent rus genes. Proc Natl Acad Sci USA 86:938-942. membrane signal transduction in Dictyostelium discoideum. MicroRobbins SM, Suttorp VV, Spiegelman GB, Weeks G (1990): A ras biol Rev 52:29-49. related gene from the lower eukaryote Dictyostelium that is highly Jermyn KA, Berks M, Kay RR, Williams J G (1987): Two distinct conserved relative to the human rap genes. Nucleic Acids Res 18: classes of prestalk enriched mRNA sequences in Dzctyostelium dzs5265-5269. coideum. Development 100:745-755. Saxe SA, Kimmel AR (1988):Genes encoding novel GTP-binding proJermyn KA, Duffy KTI, Williams J G (1989): A new anatomy of the teins in Dictyosteliurn. Dev Genet 9:259-265. prestalk zone in Dictyostelium. Nature 340:144-146. Saxe I11 CL, Johnson R, Devreotes PN, Kimmel AR (1990): Multiple Kawata M, Matsui Y, Kondo J, Hishida T, Teranishi Y, Takai Y genes for cell surface, CAMPreceptors in Dzctyostelium. Dev Genet (1988): A novel small molecular weight GTP-binding protein with 12:6-13. the same putative effector domain as the ras proteins in bovine brain membranes: purification, determination of primary structure, Sefton B, Trowbridge IS, Cooper J A (1982):The transforming protein of Rous sarcoma virus, Harvey sarcoma virus and Abelson virus and characterization. J Biol Chem 263:18965-18971. contains tightly bound lipid. Cell 31:465-474. Kessin RH (1988): Genetics of early Dictyostelium discoideum develShih TY, Weeks MO, Gruss P, Dahr R, Oroszlan S, Scolnick EM opment. Microbiol Rev 52:29-49. (1982):Identification of a precursor in the biosynthesis of the p21 Khosla M, Robbins SM, Spiegelman GB, Weeks G (1990): The regutransforming protein of Harvey murine sarcoma virus. 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that of the two ras genes suggests the possibility that the ras and rap gene products in Dictyostelium also have antagonistic roles.
RQS-RELATED GENES IN D. DISCOIDEUM Van Haastert PJM, Kesbeke F, Reymond CD, Firtel RA, Luderus E, Van Driel R (1987): Aberrant transmembrane signal transduction in Dictyostelium cells expressing a mutated ras gene. Proc Natl Acad Sci USA 84:4905-4909. Weeks G , Pawson T (1987): The synthesis and degradation of rusrelated gene products during growth and differentiation in Dzctyostelium discoideum.Differentiation 33:207-213.
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Weeks G, Lima AF, Pawson T (1987): A RAS-encoded protein in Dictyostelium discoideum is acylated and membrane associated. Mol Microbiol 1:347-354. Willumsen BM, Christensen A, Hubert NL, Papageorge AG, Lowy DR (1984): The p21 rus C-terminus is required for transformation and membrane association. Nature 310:583-586.
Ras-Related Genes in Dictyostelium discoideum STEPHEN M. ROBBINS, MEENAL KHOSLA, RICHARD THIERY, GERALD WEEKS, AND GEORGE B. SPIEGELMAN Departments of Microbiology (S.M.R., M.K., R.T., G.W., G.B.S.) and Medical Genetics (G.W., G.B.S.), University of British Columbia, Vancouver, British Columbia, Canada 19891. However, the detailed mechanism by which the activated ras proteins produce the malignant phenotype remains unknown. The ras genes are part of a large multigene family and numerous members have been found in a wide variety of organisms. The major sequence conservation within the superfamily is the preservation of the four domains associated with guanine nucleotide binding, but there are additional sequence similarities and the individual members of the ras gene superfamily vary in the amount of additional identity [Chardin, 19881. The cellular slime mold Dictyostelium discoideum has properties that make it attractive for examining ras gene function in both cellular proliferation and differentiation and studies on this organism may provide Key words: Eukaryotes, vegetative cells, rus proinsights into the function of ras genes in higher organteins isms. In Dictyostelium, growth and differentiation are distinct phenomena; in the presence of a bacterial food source amoeba proliferate, but upon starvation cell diINTRODUCTION vision ceases and a well-defined differentiation process Ras genes and members of the ras-related gene su- is initiated. During differentiation the amoeba are atperfamily have now been identified in eukaryotic or- tracted to one another by pulses of cyclic AMP (CAMP) ganisms as diverse a s yeast and man and their encoded forming a multicellular aggregate which eventually products are highly conserved [for review, see Barba- elongates to a migrating pseudoplasmodium. The ancid, 19871. This widespread phylogenetic conservation terior region of the pseudoplasmodium comprises suggests t h a t the proteins fulfill functions that are es- prestalk cells and the posterior region comprises presential to all eukaryotic cells. spore cells. Ultimately the cell mass evolves into the The mammalian ras genes encode proteins that bind mature fruiting body comprising two cell types, spore guanine nucleotides [Papageorge et al., 19821, exhibit and stalk cells. Differentiation is dependent upon siga n intrinsic GTPase activity [McGrath et al., 1984; nal transduction pathways t h a t require specific G proManne et al., 19851, and are attached to the inner sur- teins and may therefore be analogous to signal transface of the plasma membrane [Sefton et al., 1982; Shih duction pathways in higher organisms [for review, see et al., 1982; Willumsen et al., 19841. These biochemical Janssens and Van Haastert, 1987; Kessin, 1988; Firtel properties closely resemble those of the regulatory G et al., 19891. proteins [Gilman, 19871 and thus by analogy it is generally assumed that the ras proteins function a s reguRAS GENES IN DICTYOSTELIUM latory elements in a signal transduction pathway. DeTwo closely related ras genes have been identified spite the extensive biochemical knowledge, there are thus far in Dictyostelium, Ddras and DdrasG [Reymond only a few clues as to the physiological role of the ras
ABSTRACT Dicfyostelium discoideum, like other eukaryotes, has been shown to express several ras-related genes. Two gene products, Ddras and DdrasG, are highly conserved relative to the human ras proteins. Ddrus is expressed at the pseudoplasmodial stage of development, whereas DdrasG is expressed in vegetative cells and during early development. In addition, Dicfyostelium possesses three rus-related genes, SAS1, SAS2 and Ddrupl, whose gene products are only partially conserved relative to those of the rus genes. The expression of these three genes is also developmentally regulated.
proteins in normal growth and differentiation. The mammalian ras genes are expressed in both immature cells and in certain terminally differentiated cell types suggesting a role in both cellular proliferation and differentiation [Barbacid, 19871. Point mutations in either codons 12, 13, 59, or 61 result in a n activated ras gene product that produces a transformed cellular phenotype in a variety of cell lines [for review, see Bos,
0 1991 WILEY-LISS, INC.
Received for publication July 31, 1990. Address reprint requests to Dr. Gerald Weeks, Department of Microbiology, University of British Columbia, Vancouver, B.C., Canada V6T IW5.
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et al., 1984; Robbins et al., 19891. The deduced amino acid sequences for Ddras and DdrasG are 82% conserved and have 65%and 69% identity with the human ras proteins, respectively. Both of the predicted Ddras and DdrasG protein sequences have features characteristic of other ras proteins (Fig. 1).The amino half of the protein is over 90% conserved but the carboxyl region is variable with the exception of the tetrapeptide sequence at the extreme terminus. Both the Ddras and DdrasG proteins possess the four highly conserved domains (Figure 1)that in mammalian ras proteins are associated with guanine nucleotide binding or GTPase activity [Barbacid, 19871. In addition the Dictyostelium proteins contain the effector domain (Fig. 1) which in mammalian proteins is believed to interact with the GTPase-activating molecule (GAP) [McCormick, 19891 suggesting the possible existence of a GAP-like molecule in Dictyostelium.
EXPRESSION OF Ddras AND DdrusG DURING DICTYOSTELIUM DEVELOPMENT The Ddras and DdrasG genes are expressed at different times during Dictyostelium development (Fig. 2). The Ddras gene is expressed at the pseudoplasmodial stage as two mRNA species of 1.2 and 0.9 kb which are enriched in the prestalk cell population [Reymond et al., 1984; Robbins et al., 19891. Induction of only the 1.2 kb Ddras mRNA occurs precociously under in vitro conditions in response to the addition of CAMP.Precocious expression in response to cyclic AMP is characteristic for a number of other prestalk-enriched genes [Reymond et al., 19841. In contrast the DdrasG gene is expressed as a single mRNA species of 1.2 kb during growth and early development [Robbins et al., 19891. The amount of DdrasG mRNA increases approximately 2 fold during the first 2-3 hours of development and then declines reaching negligible levels by the aggregation stage [Khosla et al., 19901. The decline in DdrusG mRNA level requires a developmentally regulated gene product since it does not occur in the presence of the protein synthesis inhibitor, cycloheximide. During differentiation in shake suspension of DdrasG mRNA levels do not decline, unless cells are treated with pulses of CAMP (Fig. 3). These results suggest that the decline in mRNA levels observed during aggregation is a response of the cyclic AMP relay system to pulses of cyclic AMP. Consistent with this conclusion is the finding that the decrease in mRNA level did not occur when cyclic AMP pulses were applied in the presence of caffeine, a n inhibitor of the cyclic AMP signal relay response (Fig. 3). DICTYOSTELIUM rus GENE PRODUCTS The first indication of the existence of ras gene products in Dictyostelium came with the finding that a major protein of 23,000 M, (p23) was specifically precipitated from cell-free extracts using the ras-specific
monoclonal antibody, Y13-259 [Pawson and Weeks, 19841. The relative rates of p23 synthesis are maximal during vegetative growth and early development with a sharp decline thereafter. However, there is a small burst of synthesis during the pseudoplasmodial stage of development (Fig. 4). There is no specific degradation of p23 during differentiation suggesting that the decrease in synthesis of ras protein is solely responsible for the reduction of p23 levels that occurs [Pawson et al., 1985; Weeks and Pawson, 19871. Since the Y13-259 monoclonal antibody recognizes a sequence in the human ras proteins (amino acids 6273) [Sigal et al., 19861 that is conserved in both the Ddras and DdrasG proteins (Fig. 11, it would presumably immunoprecipitate both proteins. The combined expression of the Ddras and DdrasG genes [Robbins et al., 19891 can account for the changes in the rate of ras protein synthesis that are observed during the differentiation process [Pawson et al., 19851, although this does not preclude the possibility that there may be additional ras genes that remain to be discovered. In addition to p23 a minor protein of 24,000 M, (p24) is also immunoprecipitated from cell-free extracts of both vegetative and pseudoplasmodial cells. There is no evidence for a precursor-product relationship between p23 and p24 suggesting the possibility that they are distinct gene products, but the precise relationship between the two proteins remains to be determined [Weeks and Pawson, 19871. The Y13-259 precipitated protein, like its mammalian counterparts, is acylated and membrane bound [Weeks et al., 19871. When both vegetative and pseudoplasmodial mRNA preparations are in vitro translated, two Y13-259 immunoprecipitable proteins of similar but not identical molecular weights to p24 and p23 are detected [Pawson et al., 19851. The two products produced by in vitro translation of pseudoplasmodial mRNA are derived from the Ddras gene, since mRNA that was hybrid selected by the Ddras gene was in vitro translated into the two protein products [Robbins, unpublished observations]. However a detailed analysis of the in vitro synthesized protein products will be necessary to establish their exact relationship to each other and to p24 and p23.
CELL-TYPE SPECIFIC Ddrus EXPRESSION It was shown initially that Ddras is enriched in the prestalk cell population [Reymond et al., 19841 and this result has subsequently been confirmed [Jermyn et al., 19871. However the enrichment is only 3 fold whereas the DIF-induced genes pD63 and pD56 are enriched 12 fold [Jermyn et ul., 19871. In contrast, ras-related protein synthesis only occurs in the prespore cell population [Weeks and Pawson, 19871. In these latter experiments the prestalk and prespore cells were separated by a modification [Kwong, et al., 19881 of the Ratner and Borth [ 19811 Percoll gradient technique. We have
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Fig. 1. Comparison of the derived amino acid sequences of the human ras protein, Harasl [Capon et al., 19831, the two Dictyostelium ras proteins, Ddras [ Reymond et al., 19841 and DdrasG [Robbins et al., 19891, and the Dictyostelium ras-related proteins SASl and SASZ [Saxe and Kimmel, 19881 and Ddrapl [Robbins et al., 19901. Amino acids are represented by the single letter code and the letters with the
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black background indicate identity. Gaps have been inserted for optimal alignments and the arrow indicates a sequence from SASl (MIDTPNEQP)that has been omltted for the same reason. The open boxes above the sequences indicate regions involved in guanine nucleotide binding, the solid box Indicates the effector domain, and the hatched box demonstrates the Y13-259 antibody binding domain.
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subsequently found that this modified procedure yields a prespore cell fraction contaminated with a subpopulation of prestalk cells [Kwong et al., 19901. This contaminating subpopulation of prestalk cells expresses the pDd63 and Ddras genes but does not express the pDd56 gene, suggesting that the Ddras gene is expressed in a subset of prestalk cells. This is not the first evidence for heterogeneity of the prestalk cell population. It has been shown recently that different areas of the prestalk region express difFig. 3. The effect of cAMP and caffeine on DdrusG expression. ferent genes [Gomer et al., 1986; Jermyn et al., 19891 cells were resuspended a t 5 x lo6 cells/ml in 20 mM potasand that the prestalk population can be functionally Washed sium phosphate buffer, pH 6.0, and (A) shaken at 250 rpm, (B) shaken subdivided into CAMP-independent and CAMP-depen- at 250 rpm and pulsed with cAMP to a concentration of 25 nM every dent cells on the basis of the requirements for stalk cell 5 minutes, or (C) shaken at 250 rpm in the presence of 2 mM caffeine formation in in vitro monolayers [Kwong et al., 19881. and pulsed with cAMP to a concentration of 25 nM every 5 minutes. The expression of the Ddras gene in a subset of prestalk RNA was extracted at the times (hours) indicated above each lane and hybridized with the DdrusG cDNA. cells provides further evidence for the heterogeneity within the prestalk zone of the migrating pseudoplasmodium. induced guanylate cyclase desensitization resulting in decreased intracellular cGMP accumulation [Van Ras GENE FUNCTION IN DICTYOSTELIUM Haastert et al., 19871. There is evidence that guanylate There is some evidence to suggest that in Dictyoste- cyclase regulation in Dictyostelium is coupled to the lium the Ddras gene is involved in cAMP transmem- inositol phosphate pathway [for review, see Newell et brane signal transduction. In Dictyostelium, cAMP sig- al., 1987; Janssens and Van Haastert, 19871 and the nal transduction involves two separate pathways regulation of this pathway is altered in the activated leading to adenylate cyclase and guanylate cyclase ac- Ddras transformants. In wild-type cells inositol tivation which are believed to control the signal relay triphosphate levels increase transiently in response to response and chemotactic response, respectively [for re- pulses of CAMP, whereas in the Ddras transformants views, see Newell et al., 1987; Janssens and Van there is a 3 fold higher basal level of inositol triphosHaastert, 19871. Dictyostelium transformants that phate and only a slight response to cAMP [Europeoverexpress a n activated Ddras gene (amino acid 12 Finner et al., 19881.These results suggest the possibilchanged from glycine to threonine) display a n aberrant ity that the Ddras gene product regulates the transient morphogenesis leading to multiple tipped aggregates activation of a n as yet undetected phospholipase C. In that do not form final fruiting bodies and this pheno- mammalian cells, the activation of phospholipase C ultype may be due to impaired regulation of the chemo- timately leads to the activation of protein kinase C tactic response [Reymond et al., 19861. The activated [Nishizuka, 19861 and it is possible therefore that proDdras transformants display reduced chemotactic sen- tein kinase C is involved in guanylate cyclase regulasitivity to cAMP and folic acid and a n enhanced CAMP- tion in Dictyostelium. Although there is as yet no direct
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signal transduction at the multicellular stage of development [Robbins et al., 19891. DdrasG gene expression is repressed by cAMP whereas the Ddras gene is induced by CAMP, a differential regulation that would ensure that the two gene products are not expressed at the same time during development. It has been found that the DdrasG gene is expressed throughout development in the axenic strain Ax2, providing a situation in which both the ras genes are expressed a t the pseudoplasmodial stage. The abnormal expression is unique to Ax2 since the expression in the parental strain NC4 resembled that observed in V12-M2 [Robbins and Khosla, unpublished observations]. Since the axenic strain develops relatively synchronously, culminating in a normal final fruiting body, i t does not appear that the continued expression of the DdrasG gene has a deleterious effect on development.
DEVELOPMENT
Fig. 4. Synthesis of D.discozdeum rus-related proteins during differentiation. Differentiating D.discoideum cells (10') were labeled for the indicated 2 hour periods with 250 FCi of [3"S1methionine. Cells were lysed and immunoprecipitated with Y 13-259 anti-p21 antibody, and the immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis. The label incorporated into rus-related proteins during these 2 hour pulses were determined by optically scanning the resulting fluorograms and integrating the peaks. The total incorporation into protein was measured by trichloroacetic acid precipitation of the original cell lysate. The extent of' rus-related protein synthesis for each 2 hour period is expressed as a fraction of the total protein synthesis in arbitrary units. Tip formation occurred at 10 hours of development in the experiment shown here [Pawson et al., 19851.
Ras GENE SUPERFAMILY IN DICTYOSTELIUM
In addition to DdrasG and Ddras, three ras-related genes, SAS1, SAS2, and Ddrapl, have thus far been characterized in Dictyostelium [Saxe and Kimmel, 1988; Robbins et al., 19901. The encoded products share the four guanine nucleotide binding domains, but do not possess lhe Y 13-259 niorioclonal antibody-binding site (Fig. 1). The encoded products of the SASl and SASB genes have similar sequences to those of the yeast YPT and SEC4 genes, which are involved in GTP-stimulated protein transport [Saxe and Kimmel, 19881, and the Dictyostelium gene products may perevidence for this involvement, indirect evidence sug- form similar functions. Comparisons of the SASl and gests that the activated Ddras gene product constitu- SASB gene product sequences with those of the other tively down regulates the cAMP receptors, a process Dictyostelium ras sequences are shown in Figure 1. which is normally under the control of protein kinase C We have recently isolated a gene, Ddrapl [Robbins et and the Ddras gene product [Luderus et al., 19881. al., 19901, that has approximately 80% sequence idenIt should be noted that in the above studies the ef- tity with the human rap genes [Pizon et al., 1988a; fects of the activated Ddras gene product on signal Pizon et al., 1988b; Kawata et al., 1988; Kitayama et transduction were performed on aggregation stage al., 19891. Within one of the four domains associated cells. Since Ddras is not expressed a t this time in wild- with guanine nucleotide binding, the human and the type cells [Reymond et al., 1984; Robbins et al., 19891, Dictyostelium rap genes encode a threonine a t position the activated ras protein might be interfering with 61, instead of the customary glutamine in other rassome other G protein rather than the normal Ddras related genes. The significance of this change is not gene product and thus ras proteins may not be involved known but it is interesting that the change of gluwith CAMP-mediated signal transduction during ag- tamine to threonine at position 61 in a ras gene is a n gregation. However, the Ddras protein may be involved activating mutation. In addition to the four domains in signal transduction a t the pseudoplasmodial stage of associated with guanine nucleotide binding, the effecdevelopment and require a specific cAMP receptor that tor domain that is believed to interact with the GAP is not expressed until this time. The existence of CAMP protein is highly conserved (Fig. 1).One of the interreceptor genes that are expressed a t the pseudoplasmo- esting features of the human raplA (also designated dial stage has recently been demonstrated [Saxe 111 et K-revl) gene is that it can suppress the transformed al., 19901. phenotype associated with a n activated ras gene in We have postulated that since the DdrasG and the NIH3T3 cells [Kitayama et al., 19891. The Ddrapl gene Ddras gene are expressed at distinctly different times is maximally expressed during the developmental during Dictyostelium development the two proteins stages where the levels of the DdrasG and Ddras mesmight perform different functions: DdrasG having a sages are declining [Robbins et al., 19901. The reciproregulatory role in cell proliferation and Ddras a role in cal nature of Ddrapl gene expression with respect to
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Kwong L, Sobolewski A, Atkinson L, Weeks G (1988): Stalk cell formation in monolayers from isolated prestalk and prespore cells of Dictyostelium discozdeum; evidence for two populations of prestalk cells. Development 104:121-127. Kwong L, Xie J, Daniel J , Robbins SM, Weeks G (1990): A DictyosteFUTURE PROSPECTS lium morphogen that is essential for stalk cell formation is generated by a subpopulation of prestalk cells. Development, in press. The discovery of a number of ras and ras-related Luderus MEE, Reymond CD, Van Haastert PJM, Van Driel R (1988): genes in many eukaryotes adds a n extra complexity in Expression of a mutated rus gene in Dictyostelium discoLdeum aldetermining the various functions of these genes. The ters the binding of cyclic AMP to its chemotactic receptor. J Cell Sci 90:701-706. availability of a DNA-mediated transformation system in Dictyostelium will allow the construction of defined Manne VE, Bekesi E, Kung H (1985):Ha-rus proteins exhibit GTPase activity: point mutations that activate Ha-rus genes result in demutants that can be used to directly address the biocreased GTPase activity. Proc Natl Acad Sci USA 82:376-380. logical functions of the ras and ras-related gene prod- McCormick F (1989): rus GTPase activating protein: signal transmitter and signal terminator. Cell 56:5-8. ucts. McGrath J P , Capon DJ, Goeddel DV, Levinson AD (1984): Comparative biochemical properties of normal and activated human ras p21 ACKNOWLEDGMENTS protein. Nature 310:644-649. The work cited from the Spiegelman and Weeks lab- Newell PC, Europe-Finner GN, Small NV (1987):Signal transduction during amoeba1 chemotaxis of Dictyostelium discoideum. Microbiol oratories was funded by grants from the Medical ReSci 4:5-12. search Council and the B.C. Health Care Research Nishizuka Y (1986):Studies and the perspectives of protein kinase C. Foundation. We wish to thank Don Henkelman of the Science 233:305-312. Cancer Control Agency of B.C. for assistance in illus- Papageorge AD, Lowy D, Scolnick EM (1982): Comparative biochemtrating the sequence identities shown in Figure 1. ical properties of p21 ras molecules coded for by viral and cellular ras genes. J Virol 44:509-519. Pawson T, Weeks G (1984): Expression of ras-encoded proteins in REFERENCES relation to cell growth and differentiation. In Bishop JM, Rowley Barbacid M (1987): ras genes. Annu Rev Biochem 56:779-827. JD, Greaves M (eds): Genes and Cancer. New York: Alan R. Liss, Bos J (1989): ras oncogenes in human cancer: a review. Cancer Res Inc. pp 461-470. 49:4682-4689. Pawson T, Amiel T, Hinze E, Auersperg N, Neave N, Sobolewski A, Capon DJ, Chen EY. 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