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Enhanced cell aggregation in Dictyostelium discoideum by ATP activation of cylic AMP receptors
DEVELOPMENTAL
BIOLOGY
Enhanced
47, 233-235 (1975)
Cell Aggregation in Dictyostelium discoideum ATP Activation of Cylic AMP Receptors
by
Jo& M. MATO AND THEO M. KONIJN Cell Biology
and Morphogenesis
Unit, Zoological Laboratory, the Netherlands
University
of Leiden, Kaiserstraat
63, Leiden,
Accepted June 23, 1975 Cell aggregation in the cellular slime mold Dictyostelium discoideum is mediated by cyclic AMP. In the presence of ATP the onset of cell aggregation is enhanced and cyclic AMP receptors are activated. The number of phosphorylation sites is species dependent, and two main phosphorylated proteins with MW of, respectively, 20,000 and 15,000 are localized. RESULTS
INTRODUCTION
Cyclic AMP induces the multicellular phase in the larger species of DictyosteZium (7). Cyclic AMP action on the single amoeba is extracellular (111, specific (5) and probably mediated by a cell-membrane-bound receptor (9, 10). The binding of cyclic AMP is limited to those species using this cyclic nucleotide as chemotactic agent (10). This article reports the earlier cell aggregation and cyclic AMP receptor activation in the presence of ATP and its possible mechanism via cell-membrane phosphorylation by an endogenous cyclic AMP-independent protein kinase. MATERIALS
AND
METHODS
Amoebae were grown and harvested as described previously (6). Cell aggregation was studied using small populations of amoebae on hydrophobic agar (6). Cyclic AMP binding was measured at 22°C on Millipore filters (10). Cells were incubated with 50 @l [Y-~~P]ATP (100 mCi/mmol, Amersham) and the phosphorylated proteins were separated from free [Y-~~P]ATP by repetitive washing with NaOH and icecold trichloroacetic acid (TCA) precipitation or by solubilization in 1% sodium dodecyl sulfate (SDS) containing 40 m&I dithiothreitol and 1 mM EDTA and subsequent electrophoresis (3) after incubation at 80°C for 30 min. Appropriate controls were treated the same way. I$> 1975 by Academic Press. of reproduction in any form
Inc. reserved
DISCUSSION
When small drops of an amoeba1 suspension of Dictyostelium discoideum were placed on hydrophobic agar plates containing 2 mM ATP, disodium salt, and 2 mM MgS04, aggregation started about 3-5 hr earlier than in control plates containing 2 mM MgSO, and 4 mM NaCl. Aggregation started in control plates about 10 hr after plating of the amoebae. Substitution of ATP by ADP or AMP did not affect the time of the onset of aggregation. Preaggregative amoebae became phosphorylated when incubated with [y-32P]ATP (Fig. 11, the precipitable radioactivity being stable in hot HCl but not in hot NaOH. Cyclic AMP in concentrations ranging from 10ey to 10e6M was without effect on the rate of amoeba1 phosphorylation. Cyclic AMP-independent protein kinase assayable with exogenous substrate has also been reported by Weinstein and Koritz (12). The lack of endogenous phosphorylation found by these authors might be explained by their measurement of phosphorylation in IO6 cells at lower [Y-~‘P]ATP specific activity (20 mCi/mmol) as well as to differences between strains. By SDS electrophoresis it was shown that two major species with molecular weights of, respectively, about 20,000 and 15,000 were preferentially phosphorylated (Fig. 2). Earlier cell aggregation needs to be ac-
233 Copyright All rights
AND
234
DEVELOPMENTAL
BIOLOGY
VOLUME 47, 1975
Molecular 16
weight
8
4
x104
2
1
I-
10
30
50 min
FIG. 1. Time course of phosphorylation in D. discoideum (0) and D. mucoroides (0). Phosphorylation was carried out in a final volume of 200 ~1 containing 50 fl [Y-~*P]ATP, 10 m&f NaF, 10 mM MgSO, and 10’ cells in a 1% saline solution, pH 7. After different incubation times at 20°C the reaction was stopped by adding 200 ~1 of ice-cold 10% TCA. After centrifugation the pellet was dissolved in 100 ~1 of 1 N NaOH and precipitated again by adding 1 ml of ice-cold 5% TCA. This step was repeated twice. The final pellet was washed twice with 5% TCA and dissolved in NaOH, and the Cerenkov radiation was measured.
companied by earlier cyclic AMP binding and thus, when the cyclic AMP binding activity was measured in the presence of 1 n-N ATP and 1 mJ4 MgS04, this activity was maximal about 4 hr earlier than in controls (Fig. 3). Cyclic AMP secretion increases at the onset of aggregation (1) and therefore earlier aggregation should be accompanied by earlier increase in cyclic AMP secretion. However, this hypothesis needs to be proven. Whether any one of the two main phosphorylated species corresponds to the chemotactic receptor also needs further investigation. Autophosphorylation of cyclic AMP receptors by cyclic AMP-dependent protein kinase has been reported before (2, 4, 8), although a difference in cyclic AMP binding activity between phosphorylated and nonphosphorylated molecules was not demonstrated. In Fig. 1 it can be seen that the number of free phosphorylatable sites in D. discoideum is more than two times bigger than in D. mucoroides, which agrees with previously described differences in the number of cyclic AMP receptors per cell (10). However, whether in the
0.1
0.5
I.0
Relative
mobility
FIG. 2. Electropherogram of phosphorylated amoebae of D. discoideum. Amoebae (2 x 10’ cells) were incubated as described in Fig. 1. After 30-min incubation at 2O”C, the reaction was stopped with SDS as described in Materials and Methods. After electrophoresis, gels were cut into 1.5mm slices and the Cerenkov radiation measured in 8 ml of water. Hemoglobin, trypsin and bovine serum albumin were used as standards for the molecular weight determination. T.D., tracking dye.
2
4
6
8
10
hours FIG. 3. Binding of 8-“H-labeled cyclic AMP in D. discoideum. Cells (lo”) were deposited on Millipore filters and cyclic AMP binding determined, as described by Mato and Konijn (lo), at different developmental stages in the absence (a) and presence (a) of 1 mM ATP.
BRIEF
preaggregative stage most of the phosphorylation sites are free in both species and therefore the results in Fig. 1 comparable or some of the phosphorylation sites have been already phosphorylated in D. mucoroides, which species aggregates earlier than D. discoideum, needs to be established. J. M. Mato is the recipient of a grant from the Juan March Foundation. We thank Elsa Boon, F. A. Krens and P. J. M. van Haastert for technical assistance. REFERENCES 1. BONNER,
J. T., BARKLEY, D. S., HALL, E. KONIJN, T. M., MASON, J. W., O’KEEFE, III, and WOLFE, P. B. (1969). Deuelop. Biol. 72-87. J., ROSENFELD, R., and ROSEN, 2. ERLICHMAN, 0. (1974). J. Biol. Chem. 249, 5000-5003. 3. FAIRBANKS, G., STECK, T. L., and WALLACH,
M., G., 20, M. D.
NOTES
235
F. H. (1971) Biochemistry 10, 2606-2617. 4. GUTHROW, G. E., RASMUSSEN, H., BRUNSWICH, D. J., and COOPERMAN, B. S. (1973) Proc. Nut. Acad. Sci. USA 70, 3344-3346. 5. KONIJN, T. M. (1972). In “Advances in Cyclic Nucleotide Research” (P. Greengard, G. A. Robison, and R. Paoletti, eds.), Vol. 1, 17-31. Raven, New York. 6. KONIJN, T. M., and RAPER, K. B. (1961). Develop. Biol. 3, 725-756. 7. KONIJN, T. M., BARKLEY, D. S., CHANG, Y. Y., and BONNER, J. T. (1968). Amer. Natur. 102, 225-233. 8. MAENO, H., REYES, P. L., UEDA, T., RUDOLPH, S. A., and GREENGARD, P. (1974). Arch. Biothem. Biophys. 164, 551-559. 9. MALCHOW, D., and GERISCH, G. (1974). Proc. Nat. Acad. Sci. USA 71, 2423-2427. 10. MATO, J. M., and KONIJN, T. M. (1975). Biochim. Biophys. Acta 385, 173-179. 11. MOENS, P. B., and KONIJN. T. M. (1974) FEBS Lett. 45, 44-46. 12. WEINSTEIN, B. I., and KORITZ, S. B. (1973) Develop. Biol. 34, 159-162.
BIOLOGY
Enhanced
47, 233-235 (1975)
Cell Aggregation in Dictyostelium discoideum ATP Activation of Cylic AMP Receptors
by
Jo& M. MATO AND THEO M. KONIJN Cell Biology
and Morphogenesis
Unit, Zoological Laboratory, the Netherlands
University
of Leiden, Kaiserstraat
63, Leiden,
Accepted June 23, 1975 Cell aggregation in the cellular slime mold Dictyostelium discoideum is mediated by cyclic AMP. In the presence of ATP the onset of cell aggregation is enhanced and cyclic AMP receptors are activated. The number of phosphorylation sites is species dependent, and two main phosphorylated proteins with MW of, respectively, 20,000 and 15,000 are localized. RESULTS
INTRODUCTION
Cyclic AMP induces the multicellular phase in the larger species of DictyosteZium (7). Cyclic AMP action on the single amoeba is extracellular (111, specific (5) and probably mediated by a cell-membrane-bound receptor (9, 10). The binding of cyclic AMP is limited to those species using this cyclic nucleotide as chemotactic agent (10). This article reports the earlier cell aggregation and cyclic AMP receptor activation in the presence of ATP and its possible mechanism via cell-membrane phosphorylation by an endogenous cyclic AMP-independent protein kinase. MATERIALS
AND
METHODS
Amoebae were grown and harvested as described previously (6). Cell aggregation was studied using small populations of amoebae on hydrophobic agar (6). Cyclic AMP binding was measured at 22°C on Millipore filters (10). Cells were incubated with 50 @l [Y-~~P]ATP (100 mCi/mmol, Amersham) and the phosphorylated proteins were separated from free [Y-~~P]ATP by repetitive washing with NaOH and icecold trichloroacetic acid (TCA) precipitation or by solubilization in 1% sodium dodecyl sulfate (SDS) containing 40 m&I dithiothreitol and 1 mM EDTA and subsequent electrophoresis (3) after incubation at 80°C for 30 min. Appropriate controls were treated the same way. I$> 1975 by Academic Press. of reproduction in any form
Inc. reserved
DISCUSSION
When small drops of an amoeba1 suspension of Dictyostelium discoideum were placed on hydrophobic agar plates containing 2 mM ATP, disodium salt, and 2 mM MgS04, aggregation started about 3-5 hr earlier than in control plates containing 2 mM MgSO, and 4 mM NaCl. Aggregation started in control plates about 10 hr after plating of the amoebae. Substitution of ATP by ADP or AMP did not affect the time of the onset of aggregation. Preaggregative amoebae became phosphorylated when incubated with [y-32P]ATP (Fig. 11, the precipitable radioactivity being stable in hot HCl but not in hot NaOH. Cyclic AMP in concentrations ranging from 10ey to 10e6M was without effect on the rate of amoeba1 phosphorylation. Cyclic AMP-independent protein kinase assayable with exogenous substrate has also been reported by Weinstein and Koritz (12). The lack of endogenous phosphorylation found by these authors might be explained by their measurement of phosphorylation in IO6 cells at lower [Y-~‘P]ATP specific activity (20 mCi/mmol) as well as to differences between strains. By SDS electrophoresis it was shown that two major species with molecular weights of, respectively, about 20,000 and 15,000 were preferentially phosphorylated (Fig. 2). Earlier cell aggregation needs to be ac-
233 Copyright All rights
AND
234
DEVELOPMENTAL
BIOLOGY
VOLUME 47, 1975
Molecular 16
weight
8
4
x104
2
1
I-
10
30
50 min
FIG. 1. Time course of phosphorylation in D. discoideum (0) and D. mucoroides (0). Phosphorylation was carried out in a final volume of 200 ~1 containing 50 fl [Y-~*P]ATP, 10 m&f NaF, 10 mM MgSO, and 10’ cells in a 1% saline solution, pH 7. After different incubation times at 20°C the reaction was stopped by adding 200 ~1 of ice-cold 10% TCA. After centrifugation the pellet was dissolved in 100 ~1 of 1 N NaOH and precipitated again by adding 1 ml of ice-cold 5% TCA. This step was repeated twice. The final pellet was washed twice with 5% TCA and dissolved in NaOH, and the Cerenkov radiation was measured.
companied by earlier cyclic AMP binding and thus, when the cyclic AMP binding activity was measured in the presence of 1 n-N ATP and 1 mJ4 MgS04, this activity was maximal about 4 hr earlier than in controls (Fig. 3). Cyclic AMP secretion increases at the onset of aggregation (1) and therefore earlier aggregation should be accompanied by earlier increase in cyclic AMP secretion. However, this hypothesis needs to be proven. Whether any one of the two main phosphorylated species corresponds to the chemotactic receptor also needs further investigation. Autophosphorylation of cyclic AMP receptors by cyclic AMP-dependent protein kinase has been reported before (2, 4, 8), although a difference in cyclic AMP binding activity between phosphorylated and nonphosphorylated molecules was not demonstrated. In Fig. 1 it can be seen that the number of free phosphorylatable sites in D. discoideum is more than two times bigger than in D. mucoroides, which agrees with previously described differences in the number of cyclic AMP receptors per cell (10). However, whether in the
0.1
0.5
I.0
Relative
mobility
FIG. 2. Electropherogram of phosphorylated amoebae of D. discoideum. Amoebae (2 x 10’ cells) were incubated as described in Fig. 1. After 30-min incubation at 2O”C, the reaction was stopped with SDS as described in Materials and Methods. After electrophoresis, gels were cut into 1.5mm slices and the Cerenkov radiation measured in 8 ml of water. Hemoglobin, trypsin and bovine serum albumin were used as standards for the molecular weight determination. T.D., tracking dye.
2
4
6
8
10
hours FIG. 3. Binding of 8-“H-labeled cyclic AMP in D. discoideum. Cells (lo”) were deposited on Millipore filters and cyclic AMP binding determined, as described by Mato and Konijn (lo), at different developmental stages in the absence (a) and presence (a) of 1 mM ATP.
BRIEF
preaggregative stage most of the phosphorylation sites are free in both species and therefore the results in Fig. 1 comparable or some of the phosphorylation sites have been already phosphorylated in D. mucoroides, which species aggregates earlier than D. discoideum, needs to be established. J. M. Mato is the recipient of a grant from the Juan March Foundation. We thank Elsa Boon, F. A. Krens and P. J. M. van Haastert for technical assistance. REFERENCES 1. BONNER,
J. T., BARKLEY, D. S., HALL, E. KONIJN, T. M., MASON, J. W., O’KEEFE, III, and WOLFE, P. B. (1969). Deuelop. Biol. 72-87. J., ROSENFELD, R., and ROSEN, 2. ERLICHMAN, 0. (1974). J. Biol. Chem. 249, 5000-5003. 3. FAIRBANKS, G., STECK, T. L., and WALLACH,
M., G., 20, M. D.
NOTES
235
F. H. (1971) Biochemistry 10, 2606-2617. 4. GUTHROW, G. E., RASMUSSEN, H., BRUNSWICH, D. J., and COOPERMAN, B. S. (1973) Proc. Nut. Acad. Sci. USA 70, 3344-3346. 5. KONIJN, T. M. (1972). In “Advances in Cyclic Nucleotide Research” (P. Greengard, G. A. Robison, and R. Paoletti, eds.), Vol. 1, 17-31. Raven, New York. 6. KONIJN, T. M., and RAPER, K. B. (1961). Develop. Biol. 3, 725-756. 7. KONIJN, T. M., BARKLEY, D. S., CHANG, Y. Y., and BONNER, J. T. (1968). Amer. Natur. 102, 225-233. 8. MAENO, H., REYES, P. L., UEDA, T., RUDOLPH, S. A., and GREENGARD, P. (1974). Arch. Biothem. Biophys. 164, 551-559. 9. MALCHOW, D., and GERISCH, G. (1974). Proc. Nat. Acad. Sci. USA 71, 2423-2427. 10. MATO, J. M., and KONIJN, T. M. (1975). Biochim. Biophys. Acta 385, 173-179. 11. MOENS, P. B., and KONIJN. T. M. (1974) FEBS Lett. 45, 44-46. 12. WEINSTEIN, B. I., and KORITZ, S. B. (1973) Develop. Biol. 34, 159-162.