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Expression of an altered cAMP binding protein by rapid-developing strains of Dictyostelium discoideum
DEVELOPMENTAL
BIOLOGY
120,294-298 (19%‘)
BRIEF NOTES Expression of an Altered CAMP Binding Protein by Rapid-Developing Strains of Dictyostelium discoideum ADRIAN
S. TSANG, CAROLYN
A. KAY, AND MASAO TASAKA’
Department of Biology, McGiU University, Montreal, Quebec, Canada HsA lB1 Received June 17, 1986; accepted in revised form September 19, 1986 Immunoblotting with a monoclonal antibody raised against a novel CAMP binding protein termed CABPl revealed that the molecular weights of the two CABPl subunits are altered in certain strains of Dictyostelium disc&&urn Cellfree translation followed by immunoprecipitation showed that the altered CABPl polypeptides are derived from primary translation products. In addition, the affinity of the altered CABPl for CAMP is much higher than the wild-type form. Morphologically, these strains are indistinguishable from other wild-type strains except that their developmental phase is considerably shorter. The rapid developers also exhibit a precocious appearance of CABPl. These results indicate a good correlation between an altered CABPl and rapid development. o 1987 Academic PRESS, IW. INTRODUCTION
tion of the CAMP binding proteins is to examine the properties of these proteins in Dictyostelium strains In the cellular slime mold Dictgostelium disco&urn, CAMP acts as a chemotactic agent in aggregation, con- which show an altered pattern of developmental gene expression. We show here that some rapid-developing trols gene expression, and stimulates cell differentiation strains of D. disco&urn express a different form of (see Loomis, 1982). Controlling the expression of develCABPl. The implication of this result on the role of opmental genes is perhaps the mechanism underlying CABPl in development is discussed. the larger role for CAMP in the acquisition of aggregation competence and in the induction of cell differMATERIALS AND METHODS entiation. Thus, the addition of exogenous CAMP to early Strains. Except for HTlOO, all the strains examined developing cells simultaneously results in the premature appearance of developmental gene products and in re- in this report have been described previously. Strains ducing the time required to reach aggregation compe- NC4 and V12 originated from Dr. K. B. Raper of the tence (Darmon et a& 1975;Gerisch et a& 1975). Moreover, University of Wisconsin, Madison. Strains AX2, HC91, the activity of most, if not all, developmental genes is and XP55 are derivatives of NC4 which have been used extensively for biochemical and genetic studies. These modulated by CAMP (Williams et al, 1980; Chung et al, strains develop normally but they carry mutations which 1981). Since an elevation of intracellular CAMP level affects affect growth: AX2 can be cultured axenically (Ashworth the activity of some developmental gene products and Watts, 1970), XP55 cannot grow on Bacillus subtilis (Sampson et al, 1978), an intracellular CAMP receptor (Newell et al, 1977), while HC91 cannot grow at 27°C is presumed to mediate the effect of CAMP on gene ac- (Coukell, 1975). FR17 is a rapid-developing mutant of NC4 which forms aberrant fruiting bodies (Sussman, tivity. Recently, we have characterized two intracellular CAMP binding proteins termed CABPl and CABPZ 1955). The V12 clone which we obtained from Dr. M. B. (Tsang and Tasaka, 1986). CABPl is a novel CAMP bind- Coukell of York University, Toronto, also has a short ing protein which consists of two subunits while CABP2 developmental phase. V12M2 is a derivative of VI2 obappears to be the regulatory subunit of the CAMP de- tained from Dr. G. Gerisch of the Max-Planck-Institut pendent protein kinase which has been studied exten- f. Biochemie, Martinsried. HTlOO is a fast-developing sively by other workers (de Gunzburg and Veron, 1981, clone which we isolated during routine culturing. Cultural conditions. Cells were grown on SM agar 1982; de Gunzburg et a& 1984; Leichtling et d, 1981, (Sussman, 1966) in two-membered cultures with Enter1982,1984; Majerfeld et al, 1984; Rutherford et at, 1982). obacter aerogenee at 22°C. Before the bacterial lawn beThe role of these two proteins on developmental gene gan to clear, amoebae were harvested, washed free of activity is not clear. One approach in defining the funcbacteria, and then allowed to develop at 22°C on 1.5% agar containing 40 mM KHzP04-NaZHP04, 20 mM KCl, i Present address: National Institute for Basic Biology, Okazaki, Japan. 2.5 mM MgClz, pH 6.4. 6612-1606/W $3.00 Copyright All rights
0 198’7 by Academic Press. Inc. of reproduction in any form reserved.
294
295
BRIEF NOTES
Antibodies to CAMP binding proteins. The isolation and characterization of monoclonal antibody (B9) against CABPl was described in another paper (Tsang and Tasaka, 1986). Although this antibody does not cross-react with CABPZ, it reacts with at least three other polypeptides with molecular weights of 70,000, 34,000, and 31,000. Also, while the 34,000 M, and 31,000 M, polypeptides are detected at low levels by immunoblotting they are particularly prominent when in vitro translation products are immunoprecipitated with B9 antibody (Tsang and Tasaka, 1986). Immunoblotting. Cells were lysed directly in SDS sample buffer (Laemmli, 1975) at about lo8 cells/ml. The lysed cell suspensions were heated at 90°C immediately and the solubilized polypeptides were resolved on 12% SDS polyacrylamide gels. Unless specified otherwise, 10 pg of proteins from each sample were examined. Following electrophoresis, the resolved polypeptides were transferred to nitrocellulose filters (Schleicher and Schiill, BA 85) and probed with the monoclonal B9 as described (Tsang and Tasaka, 1986). Cell-free translation and immunoprecipitatio These procedures were performed as described (Tsang and Tasaka, 1986). Filter assay for CAMP binding activity. Crude extracts were prepared from V12M2 and NC4 cells which had been developed for 16 hr. CABPl activities were separated from CABPB by DEAE-Sephacel (Pharmacia) column chromatography and further purified by Affi-Gel Blue (Bio-Rad Laboratories) column chromatography. The CAMP binding activity was determined by the filter binding assay of Gilman (1970) as described (Tsang and Tasaka, 1986). Protein determinution. Protein concentration was determined by the method of Schaffner and Weissman (1973) using bovine serum albumin as a standard.
a
:lA :lB
:lA :lB
FIG. 1. Comparison of CABPl D. discoideum strains. (a) Immunoblotting. Cells were developed for 16 hr on nonnutrient agar at 22”C, and lysed directly in SDS sample buffer (Laemmli, 1970) at 90°C for 5 min. Solubilized proteins (from approximately 3 X 10 cells per sample) were resolved on a 12% SDS slab gel, transferred onto a nitrocellulose filter, and then reacted with B9 monoclonal antibodies. The antigen-antibody complexes were localized with [?]anti-mouse immunoglobulins (New England Nuclear) and visualized by autoradiography. (b) Immunoprecipitation of in vitro translation products. One microgram of poly(A)+RNA, extracted from 12-hr developingNC4 and V12M2 cells, was used to direct translation in vitro Samples containing 2 X lo6 cpm of acid precipitable material were immunoprecipitated with 1 pl of ascite fluid prepared from hybridoma B9 and Protein ASepharose (Pharmacia). The proteins bound to the Protein A-Sepharose were analyzed by SDS gel electrophoresis and fluorography (Laskey and Mills, 1975). The positions of CABPlA and CABPlB are marked 1A and lB, respectively.
With the exception of FR17, all strains tested form normal fruiting bodies-the final structures of develRESULTS AND DISCUSSION opment. The rate of development of these strains are, An Altered CABPl in Rapid Developers however, different. Strain V12M2 was described by Beug Previously, we had raised monoclonal antibodies et al. (1973) as a rapid developer. We isolated HTlOO against the CAMP binding proteins purified from devel- during routine subcloning of NC4 cells. Although strain oping D. discoideum cells. Monoclonal antibody B9 reacts V12 has been reported to have similar developmental with CABPl but not with CABPZ (Tsang and Tasaka, profile as NC4, the V12 clone which we examined was a 1986). We used this antibody to probe for changes in rapid developer. Besides exhibiting the same form of CABPl for a number of mutant and wild-type strains. CABPl, the strains Vl2, V12M2, and HTlOO we tested Figure la shows an immunoblot of crude extracts from have a shorter developmental phase than other wild16-hr developing cells of eight D. discoideum strains us- type strains. They aggregate and form contact sites A ing ascite fluid from hybridoma B9 as a probe. For in 3-4 hr instead of the 8 hr usually required by other strains AX2, NC4, FR17, HC91, and XP55, the two wild-type strains, and they form fruiting bodies in 1% CABPl subunits exhibited molecular weights of 43,000 20 hr while it takes other wild-type strains 24 hr to reach and 38,000 while their counterparts in strains V12, the same stage. However, HTlOO differs from V12M2 in V12M2, and HTlOO had molecular weights of 41,000 and two characteristics. While V12M2 cells differentiate into stalk cells on monolayers when the exogenous CAMP 36,000.
296
DEVELOPMENTALBIOLOGY
levels are kept high (Town et aL, 1976), cells from HTlOO do not differentiate under the same conditions. Also, like its NC4 parent, HTlOO cells form large fruiting bodies whereas V12M2 cells form relatively small fruiting bodies. The differences in the molecular weights of CABPl subunits displayed by the various strains tested are not likely to be caused by nonspecific proteolytic degradation or posttranslational modification. Cell-free translation of mRNA prepared from strains NC4 and V12M2 followed by immunoprecipitation with B9 monoclonal antibody showed that the various CABPl polypeptides detected by the antibody are derived from primary translation products (Fig. lb). We had also examined the structural integrity of the regulatory subunit of the CAMP dependent protein kinase in wild-type and rapid-developing strains by immunoblotting and by photoaffinity labeling. The molecular weight of this protein was 41,000in all strains tested (data not shown). Strain FR17, which carries a mutation in rdeA, and rdeB- strains can independently give rise to the phenotype of rapid development. In addition, these mutants form aberrant fruiting bodies and large aggregation territories (Kessin, 1977). The accelerated rate of development of FR17 has been attributed to the overproduction of CAMP (Coukell and Chan, 1980) and this strain expressed a normal form of CABPl (Fig. la). Strains VI2, V12M2, and HTlOO, which express an altered CABPl, produce normal fruiting bodies and aggregation territories. Thus, the mutations which cause the accelerated rate of development in these strains probably do not reside in the rdeA and r&B loci. Moreover, the above data taken together suggest that an altered pattern of development is closely correlated with a change either in the production of or the response to CAMP. The simultaneous change in molecular weights for both CABPl subunits in the rapid developers cannot be easily explained. One of several mutations can give rise to this phenomenon. The two subunits could be products of the same gene and a mutation in the structural gene encoding CABPl could result in the observed changes. On the other hand, the two subunits could be encoded by different genes. If this is the case, an alteration in the component regulating the transcription of CABPl genes could give rise to the same result. In addition, an anomaly in the processing of CABPl transcripts could produce changes in both subunits of CABPl. Kinetic Behavior of an Altered CABPl An altered CABPl can expedite development by elevating its affinity for CAMP or by modifying its association with developmental genes or by both of these mechanisms. We have determined the kinetics of CAMP
VOLUME120, 1987
binding of CABPl from strains NC4 and V12M2. Curvilinear Scatchard (1949) plots were obtained for both forms of CABPl. The CABPl of NC4 had a K= of lo-55 nM (Fig. 2a) whereas CABPl of V12M2 exhibited an apparent K,, of 0.2-l nM (Fig. 2b). We had analyzed the kinetic behavior of CABPl in several partially purified fractions prepared from V12M2 and NC4. The number of binding sites for CAMP varied significantly in different preparations (Fig. 2). This is probably caused by the different amounts of CABPl in various preparations and is not strain dependent. More importantly, the binding affinity of CABPl from V12M2 cells was consistently 50to loo-fold higher than that of CABPl from NC4 cells. The high affinity of its CABPl may allow V12M2 cells to respond to CAMP level much lower than that of NC4 cells which, in part, may explain the accelerated development displayed by V12M2 cells. Developwxntal Regulation of CABPl In addition to their differences in the structure and property of CABPl, the profile of accumulation of CABPl a
b
CAMP Bound
(pmollml)
FIG. 2. Scatchard (1949) plots of partially purified CABPl extracted from NC4 (a) and V12M2 (b) cells. Cells were developed for 16 hr on nonnutrient agar and CABPl was partially purified by DEAE-Sephacel and Affi-Gel Blue column chromatographies (Tsang and Tasaka, 1986). Binding assays were carried out with [8H]cAMP concentrations ranging from 0.1 to 400 n&f according to the method of Gilman (19’70).Each point on the graphs is an average of 5 determinations.
297
BRIEF NOTES
during development for the various strains was also different. Figure 3 shows that the levels of CABPl in vegetative and early developing cells for the rapid developers V12M2, HTlOO, and FR17 were higher than that of NC4. Moreover, the developmental profile of CABPl for strain AX2 showed a CABPl profile similar to that NC4 (data not shown). Since all the rapid developers tested including FR17, which does not express an altered CABPl, exhibit an early accumulation of CABPl, the precocious appearance of CABPl in these strains would appear to be the result of rapid development. Although the level of CABPl in early developing cells of the rapid developers is always higher than that of other wild-type strains, its level in late development (1424 hr) does not show significant differences. Quite often, the level of CABPl during late development is lower for the rapid developers than the other wild-type strains (Figs. la and 3). Possible Role of CABPl in Development
1978; Town and Gross, 1978). Since CAMP controls the expression of these gene products, it is reasonable to expect that an alteration in the mediator of the CAMP effect on gene expression can result in the phenotype of rapid development. The correlation between an altered CABPl and rapid development, therefore, implies that this protein plays an important role in mediating developmental gene expression. However, the present data cannot rule out the possibility that the expression of an altered CABPl is the result of rapid development. This work was supported by the National Cancer Institute of Canada and the Natural Sciences and Engineering Research Council. REFERENCES ASHWORTH,J. M., and WATX, D. J. (1970). Metabolism of the cellular slime mould Dictyostelium discddeum grown in axenic culture. Biochem J 19,175-182. BEUG,H., KATZ, F. E., and GERISCH,G. (1973). Dynamics of antigenic membrane sites relating to cell aggregation in Dictyostelium diswideurn
Despite its profound effect on developmental gene expression in D. discoideum, the mechanism mediating the action of CAMP is unknown. Earlier results suggest that the rapid development of V12M2 cells is the result of the precocious expression of developmentally regulated gene products (Beug et aZ., 1973; Sampson et al,
6
J. Cell Biol. 56, 647-658.
CHUNG,S., LANDFEAR,S., BLUMBERG,D., COHEN,N., and LODISH, H. (1981). Synthesis and stability of developmentally regulated Dietyostelium discoideum mRNAs are affected by cell-cell contact and CAMP. Cell 24,785-797. COUKELL,M. B. (1975). Parasexual genetic analysis of aggregationdeficient mutants of Dictyostelium diswideum. Mol. Gen. Genet. 142, 119-135. COUKELL,M. B., and CHAN, F. K. (1980). The precocious appearance and activation of an adenylate cyclase in a rapid developing mutant of Dictyostelium discoideum. FEBS L&t. 110,39-42. DARMON,M., BRACHET,P., and DA SILVA, L. H. (1975). Chemotactic signals induce cell differentiation in Dictyostelium discoideum. Proc. Natl. Acad Sci. USA 72,3163-3166. DE GUNZBURG,J., and VERON,M. (1981). Intracellular adenosine 3’,5’phosphate binding proteins in Dictyostelium disc&&urn: Partial purification and characterization in aggregation competent cells. Bie chemistry
20,4547-4554.
DE GUNZBURG,J., and VERON,M. (1982). A CAMP-dependent protein kinase is present in differentiating Dictyostelium disckdeum cells. EMBO J 1,1063-1068. DE GUNZBURG, J., PART,D., GUISO,N., and VERON,M. (1984).An unusual adenosine 3’,5’-phosphate dependent protein kinase from Didyostelium discoideum
0
6 Hours
16 of development
24
FIG. 3. Developmental profiles of CABPl. At times indicated developing cells were solubilized in SDS sample buffer. Ten micrograms of each sample were analyzed by immunoblotting and autoradiography. The radioactive bands on the filter, located by superimposing on the autoradiogram, were excised and the radioactivity was determined using a Beckman Gamma System. This method gave a linear relationship between the level of radioactivity and the amount of protein up to 30 cg for 16-hr developing cells; hence, 10 pg of protein were used. Each point on the graph represents the radioactivity associated with the two subunits of CAPBl. 0, NC4; A, V12M2; 0, HT100, and n , FR17.
Biochemistry
23,3805-3812.
GERISCH,G., FROMM,H., HUESGEN,A., and WICK, U. (1975). Control of cell contact sites by cyclic AMP pulses in differentiating Dictycstelium discoideum cells. Nature (London) 255,547-549. GILMAN,A. G. (1970). A protein binding assay for adenosine 3’:5’-cyclic phosphate. Proc. NatL Acad+ Sci. USA 67,305-312. KESSIN,R. H. (1977). Mutations causing rapid development of Dictyp stelium discoidewn. Cell 10, 703-708. LAEMMLI, U. K. (1970). Clevage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680685. LASKEY, R. A., and MILLS, A. D. (1975). Quantitative film detection of ‘H and “C in polyacrylamide gels by fluorography. Eur. J. B&hem. 56,335-341. LEICHTLING,B. H., TIHON, C., SPITZ,E., and RICKENBERG,H. V. (1981). A cytoplasmic cyclic AMP binding protein in Dictyostelium discoideum. Dev. Biol. 82.150-157.
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LEICHTLING,B. H., MAJERFELD,I. H., COFFMAN,D. S., and RICKENBERG, H. V. (1982). Identification of the regulatory subunit of a CAMP dependent protein kinase in Dictyostelium discoidearn B&hem Bie phys. Rex Cbmmun 105,949-955. LEICHTLING,B. H., MAJERFELD,I. H., SPIT, E., SCHWER, K. L., WOFFENDIN,C., KAKINUMA, S., and RICKENBERG,H. V. (1984). A cytosolic cyclic AMP-dependent protein kinase in LXct~ostclium disxideum II. Developmental regulation. J. Bid C&m. 259,662-668. LOOMIS,W. F. (1982). “The Development of Dictyostelium dkwideum. ” Academic Press, New York. MAJERFELD,J. H., LEICHTL.ING,B. H., MALIGENI, J. A., SPITZ,E., and RICKENBERG, H. V. (1984). A cytosolic cyclic AMP-dependent protein kinase in Dictyostelium discuideum I. Properties. J. Bid Chem 259, 654-661. NEWELL,P. C., HENDERSON,R. F., MOSSES,D., and RATNER,D. I. (1977). Sensitivity to BoxiUus subtilis, a novel system for selection of heterozygous diploids of Dictyostelium diswidewn. J. Gen Microbial. 100,207-212. RUTHERFORD, C. L., TAYLOR,R. D., FRANCE, L. T., and AUCK, R. L. (1982). A cyclic AMP dependent protein kinase in L?i&ost&um discoideum, Biochem. Biophys. Res. Cwmmun 108,1210-1220. SAMPSON, J., Tows, C., and GRO%S, J. (1978).Cyclic AMP and the control of aggregative phase gene expression in Dictyostelium discoideum Dev. Biol67,54-64.
VOLUME120, 1987 SCATCHARD,G. (1949). The attractions of proteins for small molecules and ions. Ann N. Y Acad 5’ci 51,660-672. SCHAFFNER,W., and WEISSMAN,C. (1973). A rapid, sensitive, and specific method for the determination of portein in dilute solution. Anal Biochem 56,502-514. SUSSMAN,M. (1955). “Fruity” and other mutants of the cellular slime mould, Dictyostelium di.scw&um: A study of developmental abberations. J. Gen Microbial. 13,295-309. SUSSMAN,M. (1966). Biochemical and genetic methods in the study of cellular slime mold development. In “Methods in Cell Physiology” (D. Prescott, Ed.), Vol. 2, pp. 397-410. Academic Press, New York. TOWN,C. D., GROSS,J. D., and KAY, R. R. (1976). Cell differentiation without morphogenesis in LXct~osteliumd&oi&um Nature (London) 262,717-719. TOWN,C., and GROSS,J. (1978). The role of cyclic nucleotides and cell agglomeration in postaggregative enzyme synthesis in LXctyostelium discoideum Dev. Biol 63,412-420. TSANG,A. S., and TASAKA, M. (1986). Identification of multiple cyclic AMP binding proteins in developing Dictyostelium discoideum cells. J. Biol. Chem. 261,10,753-10,759. WILLIAMS,J. G., TSANG,A. S., and MAHBUBANI,H. (1980). A change in the rate of transcription of a eukaryotic gene in response to cyclic AMP. Proc. Natl. AuuL Sci USA 77,7171-7175.
BIOLOGY
120,294-298 (19%‘)
BRIEF NOTES Expression of an Altered CAMP Binding Protein by Rapid-Developing Strains of Dictyostelium discoideum ADRIAN
S. TSANG, CAROLYN
A. KAY, AND MASAO TASAKA’
Department of Biology, McGiU University, Montreal, Quebec, Canada HsA lB1 Received June 17, 1986; accepted in revised form September 19, 1986 Immunoblotting with a monoclonal antibody raised against a novel CAMP binding protein termed CABPl revealed that the molecular weights of the two CABPl subunits are altered in certain strains of Dictyostelium disc&&urn Cellfree translation followed by immunoprecipitation showed that the altered CABPl polypeptides are derived from primary translation products. In addition, the affinity of the altered CABPl for CAMP is much higher than the wild-type form. Morphologically, these strains are indistinguishable from other wild-type strains except that their developmental phase is considerably shorter. The rapid developers also exhibit a precocious appearance of CABPl. These results indicate a good correlation between an altered CABPl and rapid development. o 1987 Academic PRESS, IW. INTRODUCTION
tion of the CAMP binding proteins is to examine the properties of these proteins in Dictyostelium strains In the cellular slime mold Dictgostelium disco&urn, CAMP acts as a chemotactic agent in aggregation, con- which show an altered pattern of developmental gene expression. We show here that some rapid-developing trols gene expression, and stimulates cell differentiation strains of D. disco&urn express a different form of (see Loomis, 1982). Controlling the expression of develCABPl. The implication of this result on the role of opmental genes is perhaps the mechanism underlying CABPl in development is discussed. the larger role for CAMP in the acquisition of aggregation competence and in the induction of cell differMATERIALS AND METHODS entiation. Thus, the addition of exogenous CAMP to early Strains. Except for HTlOO, all the strains examined developing cells simultaneously results in the premature appearance of developmental gene products and in re- in this report have been described previously. Strains ducing the time required to reach aggregation compe- NC4 and V12 originated from Dr. K. B. Raper of the tence (Darmon et a& 1975;Gerisch et a& 1975). Moreover, University of Wisconsin, Madison. Strains AX2, HC91, the activity of most, if not all, developmental genes is and XP55 are derivatives of NC4 which have been used extensively for biochemical and genetic studies. These modulated by CAMP (Williams et al, 1980; Chung et al, strains develop normally but they carry mutations which 1981). Since an elevation of intracellular CAMP level affects affect growth: AX2 can be cultured axenically (Ashworth the activity of some developmental gene products and Watts, 1970), XP55 cannot grow on Bacillus subtilis (Sampson et al, 1978), an intracellular CAMP receptor (Newell et al, 1977), while HC91 cannot grow at 27°C is presumed to mediate the effect of CAMP on gene ac- (Coukell, 1975). FR17 is a rapid-developing mutant of NC4 which forms aberrant fruiting bodies (Sussman, tivity. Recently, we have characterized two intracellular CAMP binding proteins termed CABPl and CABPZ 1955). The V12 clone which we obtained from Dr. M. B. (Tsang and Tasaka, 1986). CABPl is a novel CAMP bind- Coukell of York University, Toronto, also has a short ing protein which consists of two subunits while CABP2 developmental phase. V12M2 is a derivative of VI2 obappears to be the regulatory subunit of the CAMP de- tained from Dr. G. Gerisch of the Max-Planck-Institut pendent protein kinase which has been studied exten- f. Biochemie, Martinsried. HTlOO is a fast-developing sively by other workers (de Gunzburg and Veron, 1981, clone which we isolated during routine culturing. Cultural conditions. Cells were grown on SM agar 1982; de Gunzburg et a& 1984; Leichtling et d, 1981, (Sussman, 1966) in two-membered cultures with Enter1982,1984; Majerfeld et al, 1984; Rutherford et at, 1982). obacter aerogenee at 22°C. Before the bacterial lawn beThe role of these two proteins on developmental gene gan to clear, amoebae were harvested, washed free of activity is not clear. One approach in defining the funcbacteria, and then allowed to develop at 22°C on 1.5% agar containing 40 mM KHzP04-NaZHP04, 20 mM KCl, i Present address: National Institute for Basic Biology, Okazaki, Japan. 2.5 mM MgClz, pH 6.4. 6612-1606/W $3.00 Copyright All rights
0 198’7 by Academic Press. Inc. of reproduction in any form reserved.
294
295
BRIEF NOTES
Antibodies to CAMP binding proteins. The isolation and characterization of monoclonal antibody (B9) against CABPl was described in another paper (Tsang and Tasaka, 1986). Although this antibody does not cross-react with CABPZ, it reacts with at least three other polypeptides with molecular weights of 70,000, 34,000, and 31,000. Also, while the 34,000 M, and 31,000 M, polypeptides are detected at low levels by immunoblotting they are particularly prominent when in vitro translation products are immunoprecipitated with B9 antibody (Tsang and Tasaka, 1986). Immunoblotting. Cells were lysed directly in SDS sample buffer (Laemmli, 1975) at about lo8 cells/ml. The lysed cell suspensions were heated at 90°C immediately and the solubilized polypeptides were resolved on 12% SDS polyacrylamide gels. Unless specified otherwise, 10 pg of proteins from each sample were examined. Following electrophoresis, the resolved polypeptides were transferred to nitrocellulose filters (Schleicher and Schiill, BA 85) and probed with the monoclonal B9 as described (Tsang and Tasaka, 1986). Cell-free translation and immunoprecipitatio These procedures were performed as described (Tsang and Tasaka, 1986). Filter assay for CAMP binding activity. Crude extracts were prepared from V12M2 and NC4 cells which had been developed for 16 hr. CABPl activities were separated from CABPB by DEAE-Sephacel (Pharmacia) column chromatography and further purified by Affi-Gel Blue (Bio-Rad Laboratories) column chromatography. The CAMP binding activity was determined by the filter binding assay of Gilman (1970) as described (Tsang and Tasaka, 1986). Protein determinution. Protein concentration was determined by the method of Schaffner and Weissman (1973) using bovine serum albumin as a standard.
a
:lA :lB
:lA :lB
FIG. 1. Comparison of CABPl D. discoideum strains. (a) Immunoblotting. Cells were developed for 16 hr on nonnutrient agar at 22”C, and lysed directly in SDS sample buffer (Laemmli, 1970) at 90°C for 5 min. Solubilized proteins (from approximately 3 X 10 cells per sample) were resolved on a 12% SDS slab gel, transferred onto a nitrocellulose filter, and then reacted with B9 monoclonal antibodies. The antigen-antibody complexes were localized with [?]anti-mouse immunoglobulins (New England Nuclear) and visualized by autoradiography. (b) Immunoprecipitation of in vitro translation products. One microgram of poly(A)+RNA, extracted from 12-hr developingNC4 and V12M2 cells, was used to direct translation in vitro Samples containing 2 X lo6 cpm of acid precipitable material were immunoprecipitated with 1 pl of ascite fluid prepared from hybridoma B9 and Protein ASepharose (Pharmacia). The proteins bound to the Protein A-Sepharose were analyzed by SDS gel electrophoresis and fluorography (Laskey and Mills, 1975). The positions of CABPlA and CABPlB are marked 1A and lB, respectively.
With the exception of FR17, all strains tested form normal fruiting bodies-the final structures of develRESULTS AND DISCUSSION opment. The rate of development of these strains are, An Altered CABPl in Rapid Developers however, different. Strain V12M2 was described by Beug Previously, we had raised monoclonal antibodies et al. (1973) as a rapid developer. We isolated HTlOO against the CAMP binding proteins purified from devel- during routine subcloning of NC4 cells. Although strain oping D. discoideum cells. Monoclonal antibody B9 reacts V12 has been reported to have similar developmental with CABPl but not with CABPZ (Tsang and Tasaka, profile as NC4, the V12 clone which we examined was a 1986). We used this antibody to probe for changes in rapid developer. Besides exhibiting the same form of CABPl for a number of mutant and wild-type strains. CABPl, the strains Vl2, V12M2, and HTlOO we tested Figure la shows an immunoblot of crude extracts from have a shorter developmental phase than other wild16-hr developing cells of eight D. discoideum strains us- type strains. They aggregate and form contact sites A ing ascite fluid from hybridoma B9 as a probe. For in 3-4 hr instead of the 8 hr usually required by other strains AX2, NC4, FR17, HC91, and XP55, the two wild-type strains, and they form fruiting bodies in 1% CABPl subunits exhibited molecular weights of 43,000 20 hr while it takes other wild-type strains 24 hr to reach and 38,000 while their counterparts in strains V12, the same stage. However, HTlOO differs from V12M2 in V12M2, and HTlOO had molecular weights of 41,000 and two characteristics. While V12M2 cells differentiate into stalk cells on monolayers when the exogenous CAMP 36,000.
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levels are kept high (Town et aL, 1976), cells from HTlOO do not differentiate under the same conditions. Also, like its NC4 parent, HTlOO cells form large fruiting bodies whereas V12M2 cells form relatively small fruiting bodies. The differences in the molecular weights of CABPl subunits displayed by the various strains tested are not likely to be caused by nonspecific proteolytic degradation or posttranslational modification. Cell-free translation of mRNA prepared from strains NC4 and V12M2 followed by immunoprecipitation with B9 monoclonal antibody showed that the various CABPl polypeptides detected by the antibody are derived from primary translation products (Fig. lb). We had also examined the structural integrity of the regulatory subunit of the CAMP dependent protein kinase in wild-type and rapid-developing strains by immunoblotting and by photoaffinity labeling. The molecular weight of this protein was 41,000in all strains tested (data not shown). Strain FR17, which carries a mutation in rdeA, and rdeB- strains can independently give rise to the phenotype of rapid development. In addition, these mutants form aberrant fruiting bodies and large aggregation territories (Kessin, 1977). The accelerated rate of development of FR17 has been attributed to the overproduction of CAMP (Coukell and Chan, 1980) and this strain expressed a normal form of CABPl (Fig. la). Strains VI2, V12M2, and HTlOO, which express an altered CABPl, produce normal fruiting bodies and aggregation territories. Thus, the mutations which cause the accelerated rate of development in these strains probably do not reside in the rdeA and r&B loci. Moreover, the above data taken together suggest that an altered pattern of development is closely correlated with a change either in the production of or the response to CAMP. The simultaneous change in molecular weights for both CABPl subunits in the rapid developers cannot be easily explained. One of several mutations can give rise to this phenomenon. The two subunits could be products of the same gene and a mutation in the structural gene encoding CABPl could result in the observed changes. On the other hand, the two subunits could be encoded by different genes. If this is the case, an alteration in the component regulating the transcription of CABPl genes could give rise to the same result. In addition, an anomaly in the processing of CABPl transcripts could produce changes in both subunits of CABPl. Kinetic Behavior of an Altered CABPl An altered CABPl can expedite development by elevating its affinity for CAMP or by modifying its association with developmental genes or by both of these mechanisms. We have determined the kinetics of CAMP
VOLUME120, 1987
binding of CABPl from strains NC4 and V12M2. Curvilinear Scatchard (1949) plots were obtained for both forms of CABPl. The CABPl of NC4 had a K= of lo-55 nM (Fig. 2a) whereas CABPl of V12M2 exhibited an apparent K,, of 0.2-l nM (Fig. 2b). We had analyzed the kinetic behavior of CABPl in several partially purified fractions prepared from V12M2 and NC4. The number of binding sites for CAMP varied significantly in different preparations (Fig. 2). This is probably caused by the different amounts of CABPl in various preparations and is not strain dependent. More importantly, the binding affinity of CABPl from V12M2 cells was consistently 50to loo-fold higher than that of CABPl from NC4 cells. The high affinity of its CABPl may allow V12M2 cells to respond to CAMP level much lower than that of NC4 cells which, in part, may explain the accelerated development displayed by V12M2 cells. Developwxntal Regulation of CABPl In addition to their differences in the structure and property of CABPl, the profile of accumulation of CABPl a
b
CAMP Bound
(pmollml)
FIG. 2. Scatchard (1949) plots of partially purified CABPl extracted from NC4 (a) and V12M2 (b) cells. Cells were developed for 16 hr on nonnutrient agar and CABPl was partially purified by DEAE-Sephacel and Affi-Gel Blue column chromatographies (Tsang and Tasaka, 1986). Binding assays were carried out with [8H]cAMP concentrations ranging from 0.1 to 400 n&f according to the method of Gilman (19’70).Each point on the graphs is an average of 5 determinations.
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BRIEF NOTES
during development for the various strains was also different. Figure 3 shows that the levels of CABPl in vegetative and early developing cells for the rapid developers V12M2, HTlOO, and FR17 were higher than that of NC4. Moreover, the developmental profile of CABPl for strain AX2 showed a CABPl profile similar to that NC4 (data not shown). Since all the rapid developers tested including FR17, which does not express an altered CABPl, exhibit an early accumulation of CABPl, the precocious appearance of CABPl in these strains would appear to be the result of rapid development. Although the level of CABPl in early developing cells of the rapid developers is always higher than that of other wild-type strains, its level in late development (1424 hr) does not show significant differences. Quite often, the level of CABPl during late development is lower for the rapid developers than the other wild-type strains (Figs. la and 3). Possible Role of CABPl in Development
1978; Town and Gross, 1978). Since CAMP controls the expression of these gene products, it is reasonable to expect that an alteration in the mediator of the CAMP effect on gene expression can result in the phenotype of rapid development. The correlation between an altered CABPl and rapid development, therefore, implies that this protein plays an important role in mediating developmental gene expression. However, the present data cannot rule out the possibility that the expression of an altered CABPl is the result of rapid development. This work was supported by the National Cancer Institute of Canada and the Natural Sciences and Engineering Research Council. REFERENCES ASHWORTH,J. M., and WATX, D. J. (1970). Metabolism of the cellular slime mould Dictyostelium discddeum grown in axenic culture. Biochem J 19,175-182. BEUG,H., KATZ, F. E., and GERISCH,G. (1973). Dynamics of antigenic membrane sites relating to cell aggregation in Dictyostelium diswideurn
Despite its profound effect on developmental gene expression in D. discoideum, the mechanism mediating the action of CAMP is unknown. Earlier results suggest that the rapid development of V12M2 cells is the result of the precocious expression of developmentally regulated gene products (Beug et aZ., 1973; Sampson et al,
6
J. Cell Biol. 56, 647-658.
CHUNG,S., LANDFEAR,S., BLUMBERG,D., COHEN,N., and LODISH, H. (1981). Synthesis and stability of developmentally regulated Dietyostelium discoideum mRNAs are affected by cell-cell contact and CAMP. Cell 24,785-797. COUKELL,M. B. (1975). Parasexual genetic analysis of aggregationdeficient mutants of Dictyostelium diswideum. Mol. Gen. Genet. 142, 119-135. COUKELL,M. B., and CHAN, F. K. (1980). The precocious appearance and activation of an adenylate cyclase in a rapid developing mutant of Dictyostelium discoideum. FEBS L&t. 110,39-42. DARMON,M., BRACHET,P., and DA SILVA, L. H. (1975). Chemotactic signals induce cell differentiation in Dictyostelium discoideum. Proc. Natl. Acad Sci. USA 72,3163-3166. DE GUNZBURG,J., and VERON,M. (1981). Intracellular adenosine 3’,5’phosphate binding proteins in Dictyostelium disc&&urn: Partial purification and characterization in aggregation competent cells. Bie chemistry
20,4547-4554.
DE GUNZBURG,J., and VERON,M. (1982). A CAMP-dependent protein kinase is present in differentiating Dictyostelium disckdeum cells. EMBO J 1,1063-1068. DE GUNZBURG, J., PART,D., GUISO,N., and VERON,M. (1984).An unusual adenosine 3’,5’-phosphate dependent protein kinase from Didyostelium discoideum
0
6 Hours
16 of development
24
FIG. 3. Developmental profiles of CABPl. At times indicated developing cells were solubilized in SDS sample buffer. Ten micrograms of each sample were analyzed by immunoblotting and autoradiography. The radioactive bands on the filter, located by superimposing on the autoradiogram, were excised and the radioactivity was determined using a Beckman Gamma System. This method gave a linear relationship between the level of radioactivity and the amount of protein up to 30 cg for 16-hr developing cells; hence, 10 pg of protein were used. Each point on the graph represents the radioactivity associated with the two subunits of CAPBl. 0, NC4; A, V12M2; 0, HT100, and n , FR17.
Biochemistry
23,3805-3812.
GERISCH,G., FROMM,H., HUESGEN,A., and WICK, U. (1975). Control of cell contact sites by cyclic AMP pulses in differentiating Dictycstelium discoideum cells. Nature (London) 255,547-549. GILMAN,A. G. (1970). A protein binding assay for adenosine 3’:5’-cyclic phosphate. Proc. NatL Acad+ Sci. USA 67,305-312. KESSIN,R. H. (1977). Mutations causing rapid development of Dictyp stelium discoidewn. Cell 10, 703-708. LAEMMLI, U. K. (1970). Clevage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680685. LASKEY, R. A., and MILLS, A. D. (1975). Quantitative film detection of ‘H and “C in polyacrylamide gels by fluorography. Eur. J. B&hem. 56,335-341. LEICHTLING,B. H., TIHON, C., SPITZ,E., and RICKENBERG,H. V. (1981). A cytoplasmic cyclic AMP binding protein in Dictyostelium discoideum. Dev. Biol. 82.150-157.
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DEVELOPMENTALBIOLOGY
LEICHTLING,B. H., MAJERFELD,I. H., COFFMAN,D. S., and RICKENBERG, H. V. (1982). Identification of the regulatory subunit of a CAMP dependent protein kinase in Dictyostelium discoidearn B&hem Bie phys. Rex Cbmmun 105,949-955. LEICHTLING,B. H., MAJERFELD,I. H., SPIT, E., SCHWER, K. L., WOFFENDIN,C., KAKINUMA, S., and RICKENBERG,H. V. (1984). A cytosolic cyclic AMP-dependent protein kinase in LXct~ostclium disxideum II. Developmental regulation. J. Bid C&m. 259,662-668. LOOMIS,W. F. (1982). “The Development of Dictyostelium dkwideum. ” Academic Press, New York. MAJERFELD,J. H., LEICHTL.ING,B. H., MALIGENI, J. A., SPITZ,E., and RICKENBERG, H. V. (1984). A cytosolic cyclic AMP-dependent protein kinase in Dictyostelium discuideum I. Properties. J. Bid Chem 259, 654-661. NEWELL,P. C., HENDERSON,R. F., MOSSES,D., and RATNER,D. I. (1977). Sensitivity to BoxiUus subtilis, a novel system for selection of heterozygous diploids of Dictyostelium diswidewn. J. Gen Microbial. 100,207-212. RUTHERFORD, C. L., TAYLOR,R. D., FRANCE, L. T., and AUCK, R. L. (1982). A cyclic AMP dependent protein kinase in L?i&ost&um discoideum, Biochem. Biophys. Res. Cwmmun 108,1210-1220. SAMPSON, J., Tows, C., and GRO%S, J. (1978).Cyclic AMP and the control of aggregative phase gene expression in Dictyostelium discoideum Dev. Biol67,54-64.
VOLUME120, 1987 SCATCHARD,G. (1949). The attractions of proteins for small molecules and ions. Ann N. Y Acad 5’ci 51,660-672. SCHAFFNER,W., and WEISSMAN,C. (1973). A rapid, sensitive, and specific method for the determination of portein in dilute solution. Anal Biochem 56,502-514. SUSSMAN,M. (1955). “Fruity” and other mutants of the cellular slime mould, Dictyostelium di.scw&um: A study of developmental abberations. J. Gen Microbial. 13,295-309. SUSSMAN,M. (1966). Biochemical and genetic methods in the study of cellular slime mold development. In “Methods in Cell Physiology” (D. Prescott, Ed.), Vol. 2, pp. 397-410. Academic Press, New York. TOWN,C. D., GROSS,J. D., and KAY, R. R. (1976). Cell differentiation without morphogenesis in LXct~osteliumd&oi&um Nature (London) 262,717-719. TOWN,C., and GROSS,J. (1978). The role of cyclic nucleotides and cell agglomeration in postaggregative enzyme synthesis in LXctyostelium discoideum Dev. Biol 63,412-420. TSANG,A. S., and TASAKA, M. (1986). Identification of multiple cyclic AMP binding proteins in developing Dictyostelium discoideum cells. J. Biol. Chem. 261,10,753-10,759. WILLIAMS,J. G., TSANG,A. S., and MAHBUBANI,H. (1980). A change in the rate of transcription of a eukaryotic gene in response to cyclic AMP. Proc. Natl. AuuL Sci USA 77,7171-7175.