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. 1989 Jul;8(7):2039–2043. doi: 10.1002/j.1460-2075.1989.tb03612.x

Overproduction of the regulatory subunit of the cAMP-dependent protein kinase blocks the differentiation of Dictyostelium discoideum.

M N Simon 1, D Driscoll 1, R Mutzel 1, D Part 1, J Williams 1, M Véron 1
PMCID: PMC401086  PMID: 2551673

Abstract

During the aggregation of Dictyostelium discoideum extracellular cAMP is known to act as a chemotractant and as an inducer of cellular differentiation. However, its intracellular role as a second messenger remains obscure. We have constructed a fusion gene consisting of the cDNA encoding the regulatory subunit (R) of the cAMP-dependent protein kinase fused to the promoter and N-terminal-proximal sequences of a Dictyostelium actin gene. Stable transformants, containing multiple copies of this gene, overproduce the R subunit which accumulates prematurely relative to the endogenous protein. These transformants fail to aggregate. Detailed analysis has shown that they are blocked at interphase, the period prior to aggregation, and that they are severely defective in most responses to cAMP including the induction of gene expression. Our observations suggest that intracellular cAMP acts, presumably by activation of the catalytic subunit of the cAMP-dependent protein kinase, to facilitate early development.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alton T. H., Lodish H. F. Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoideum. Dev Biol. 1977 Oct 1;60(1):180–206. doi: 10.1016/0012-1606(77)90118-x. [DOI] [PubMed] [Google Scholar]
  2. Beug H., Katz F. E., Gerisch G. Dynamics of antigenic membrane sites relating to cell aggregation in Dictyostelium discoideum. J Cell Biol. 1973 Mar;56(3):647–658. doi: 10.1083/jcb.56.3.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boney C., Fink D., Schlichter D., Carr K., Wicks W. D. Direct evidence that the protein kinase catalytic subunit mediates the effects of cAMP on tyrosine aminotransferase synthesis. J Biol Chem. 1983 Apr 25;258(8):4911–4918. [PubMed] [Google Scholar]
  4. Brenner M. Cyclic AMP levels and turnover during development of the cellular slime mold Dictyostelium discoideum. Dev Biol. 1978 Jun;64(2):210–223. doi: 10.1016/0012-1606(78)90073-8. [DOI] [PubMed] [Google Scholar]
  5. Clegg C. H., Correll L. A., Cadd G. G., McKnight G. S. Inhibition of intracellular cAMP-dependent protein kinase using mutant genes of the regulatory type I subunit. J Biol Chem. 1987 Sep 25;262(27):13111–13119. [PubMed] [Google Scholar]
  6. Cohen S. M., Knecht D., Lodish H. F., Loomis W. F. DNA sequences required for expression of a Dictyostelium actin gene. EMBO J. 1986 Dec 1;5(12):3361–3366. doi: 10.1002/j.1460-2075.1986.tb04651.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Darmon M., Brachet P., Da Silva L. H. Chemotactic signals induce cell differentiation in Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1975 Aug;72(8):3163–3166. doi: 10.1073/pnas.72.8.3163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dinauer M. C., MacKay S. A., Devreotes P. N. Cyclic 3',5'-AMP relay in Dictyostelium discoideum III. The relationship of cAMP synthesis and secretion during the cAMP signaling response. J Cell Biol. 1980 Aug;86(2):537–544. doi: 10.1083/jcb.86.2.537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Early A. E., Williams J. G. Two vectors which facilitate gene manipulation and a simplified transformation procedure for Dictyostelium discoideum. Gene. 1987;59(1):99–106. doi: 10.1016/0378-1119(87)90270-8. [DOI] [PubMed] [Google Scholar]
  10. Flockhart D. A., Corbin J. D. Regulatory mechanisms in the control of protein kinases. CRC Crit Rev Biochem. 1982 Feb;12(2):133–186. doi: 10.3109/10409238209108705. [DOI] [PubMed] [Google Scholar]
  11. Gerisch G. Cyclic AMP and other signals controlling cell development and differentiation in Dictyostelium. Annu Rev Biochem. 1987;56:853–879. doi: 10.1146/annurev.bi.56.070187.004225. [DOI] [PubMed] [Google Scholar]
  12. Gerisch G., Fromm H., Huesgen A., Wick U. Control of cell-contact sites by cyclic AMP pulses in differentiating Dictyostelium cells. Nature. 1975 Jun 12;255(5509):547–549. doi: 10.1038/255547a0. [DOI] [PubMed] [Google Scholar]
  13. Gerisch G., Hess B. Cyclic-AMP-controlled oscillations in suspended Dictyostelium cells: their relation to morphogenetic cell interactions. Proc Natl Acad Sci U S A. 1974 May;71(5):2118–2122. doi: 10.1073/pnas.71.5.2118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gomer R. H., Armstrong D., Leichtling B. H., Firtel R. A. cAMP induction of prespore and prestalk gene expression in Dictyostelium is mediated by the cell-surface cAMP receptor. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8624–8628. doi: 10.1073/pnas.83.22.8624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grove J. R., Price D. J., Goodman H. M., Avruch J. Recombinant fragment of protein kinase inhibitor blocks cyclic AMP-dependent gene transcription. Science. 1987 Oct 23;238(4826):530–533. doi: 10.1126/science.2821622. [DOI] [PubMed] [Google Scholar]
  16. Haribabu B., Dottin R. P. Pharmacological characterization of cyclic AMP receptors mediating gene regulation in Dictyostelium discoideum. Mol Cell Biol. 1986 Jul;6(7):2402–2408. doi: 10.1128/mcb.6.7.2402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Janssens P. M., Van Haastert P. J. Molecular basis of transmembrane signal transduction in Dictyostelium discoideum. Microbiol Rev. 1987 Dec;51(4):396–418. doi: 10.1128/mr.51.4.396-418.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kessin R. H. Genetics of early Dictyostelium discoideum development. Microbiol Rev. 1988 Mar;52(1):29–49. doi: 10.1128/mr.52.1.29-49.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Knecht D. A., Cohen S. M., Loomis W. F., Lodish H. F. Developmental regulation of Dictyostelium discoideum actin gene fusions carried on low-copy and high-copy transformation vectors. Mol Cell Biol. 1986 Nov;6(11):3973–3983. doi: 10.1128/mcb.6.11.3973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Krebs E. G., Beavo J. A. Phosphorylation-dephosphorylation of enzymes. Annu Rev Biochem. 1979;48:923–959. doi: 10.1146/annurev.bi.48.070179.004423. [DOI] [PubMed] [Google Scholar]
  21. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984 Dec;10(3-4):203–209. doi: 10.1016/0165-022x(84)90040-x. [DOI] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Leichtling B. H., Majerfeld I. H., Spitz E., Schaller K. L., Woffendin C., Kakinuma S., Rickenberg H. V. A cytosolic cyclic AMP-dependent protein kinase in Dictyostelium discoideum. II. Developmental regulation. J Biol Chem. 1984 Jan 10;259(1):662–668. [PubMed] [Google Scholar]
  24. Maeda Y., Gerisch G. Vesicle formation in Dictyostelium discoideum cells during oscillations of cAMP synthesis and release. Exp Cell Res. 1977 Nov;110(1):119–126. doi: 10.1016/0014-4827(77)90277-4. [DOI] [PubMed] [Google Scholar]
  25. Majerfeld I. H., Leichtling B. H., Meligeni J. A., Spitz E., Rickenberg H. V. A cytosolic cyclic AMP-dependent protein kinase in Dictyostelium discoideum. I. Properties. J Biol Chem. 1984 Jan 10;259(1):654–661. [PubMed] [Google Scholar]
  26. Mann S. K., Pinko C., Firtel R. A. cAMP regulation of early gene expression in signal transduction mutants of Dictyostelium. Dev Biol. 1988 Nov;130(1):294–303. doi: 10.1016/0012-1606(88)90435-6. [DOI] [PubMed] [Google Scholar]
  27. Mutzel R., Lacombe M. L., Simon M. N., de Gunzburg J., Veron M. Cloning and cDNA sequence of the regulatory subunit of cAMP-dependent protein kinase from Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1987 Jan;84(1):6–10. doi: 10.1073/pnas.84.1.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nellen W., Silan C., Firtel R. A. DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion. Mol Cell Biol. 1984 Dec;4(12):2890–2898. doi: 10.1128/mcb.4.12.2890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Oyama M., Blumberg D. D. Interaction of cAMP with the cell-surface receptor induces cell-type-specific mRNA accumulation in Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4819–4823. doi: 10.1073/pnas.83.13.4819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Part D., de Gunzburg J., Veron M. The regulatory subunit of cAMP-dependent protein kinase from Dictyostelium discoideum: cellular localization and developmental regulation analyzed by immunoblotting. Cell Differ. 1985 Nov;17(4):221–227. doi: 10.1016/0045-6039(85)90496-8. [DOI] [PubMed] [Google Scholar]
  31. Riabowol K. T., Fink J. S., Gilman M. Z., Walsh D. A., Goodman R. H., Feramisco J. R. The catalytic subunit of cAMP-dependent protein kinase induces expression of genes containing cAMP-responsive enhancer elements. Nature. 1988 Nov 3;336(6194):83–86. doi: 10.1038/336083a0. [DOI] [PubMed] [Google Scholar]
  32. Roesler W. J., Vandenbark G. R., Hanson R. W. Cyclic AMP and the induction of eukaryotic gene transcription. J Biol Chem. 1988 Jul 5;263(19):9063–9066. [PubMed] [Google Scholar]
  33. Schaap P., Van Lookeren Campagne M. M., Van Driel R., Spek W., Van Haastert P. J., Pinas J. Postaggregative differentiation induction by cyclic AMP in Dictyostelium: intracellular transduction pathway and requirement for additional stimuli. Dev Biol. 1986 Nov;118(1):52–63. doi: 10.1016/0012-1606(86)90072-2. [DOI] [PubMed] [Google Scholar]
  34. Schaap P., van Driel R. V. Induction of post-aggregative differentiation in Dictyostelium discoideum by cAMP. Evidence of involvement of the cell surface cAMP receptor. Exp Cell Res. 1985 Aug;159(2):388–398. doi: 10.1016/s0014-4827(85)80012-4. [DOI] [PubMed] [Google Scholar]
  35. Snaar-Jagalska B. E., Van Haastert P. J. Dictyostelium discoideum mutant synag 7 with altered G-protein-adenylate cyclase interaction. J Cell Sci. 1988 Oct;91(Pt 2):287–294. doi: 10.1242/jcs.91.2.287. [DOI] [PubMed] [Google Scholar]
  36. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Waterman M., Murdoch G. H., Evans R. M., Rosenfeld M. G. Cyclic AMP regulation of eukaryotic gene transcription by two discrete molecular mechanisms. Science. 1985 Jul 19;229(4710):267–269. doi: 10.1126/science.2990047. [DOI] [PubMed] [Google Scholar]
  38. Watts D. J., Ashworth J. M. Growth of myxameobae of the cellular slime mould Dictyostelium discoideum in axenic culture. Biochem J. 1970 Sep;119(2):171–174. doi: 10.1042/bj1190171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Williams J. G., North M. J., Mahbubani H. A developmentally regulated cysteine proteinase in Dictyostelium discoideum. EMBO J. 1985 Apr;4(4):999–1006. doi: 10.1002/j.1460-2075.1985.tb03730.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wurster B., Bumann J. Cell differentiation in the absence of intracellular cyclic AMP pulses in Dictyostelium discoideum. Dev Biol. 1981 Jul 15;85(1):262–265. doi: 10.1016/0012-1606(81)90257-8. [DOI] [PubMed] [Google Scholar]
  41. de Gunzburg J., Franke J., Kessin R. H., Véron M. Detection and developmental regulation of the mRNA for the regulatory subunit of the cAMP-dependent protein kinase of D. discoideum by cell-free translation. EMBO J. 1986 Feb;5(2):363–367. doi: 10.1002/j.1460-2075.1986.tb04220.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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