Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Nov 15;89(22):10701–10705. doi: 10.1073/pnas.89.22.10701

DdPK3, which plays essential roles during Dictyostelium development, encodes the catalytic subunit of cAMP-dependent protein kinase.

S K Mann 1, W M Yonemoto 1, S S Taylor 1, R A Firtel 1
PMCID: PMC50409  PMID: 1332055

Abstract

We have previously reported the analysis of DdPK3, a developmentally regulated putative serine/threonine kinase that shares approximately 50% amino acid sequence identity with metazoan cAMP-dependent protein kinase A (PKA) and protein kinase C, within their catalytic domains. Cells in which the DdPK3 gene has been disrupted do not aggregate but they are able to induce aggregation-stage genes in response to cAMP pulses and the prestalk-specific ras gene DdrasD in response to high continuous levels of cAMP but will not induce prespore gene expression. In this report, we present conclusive evidence that DdPK3 encodes the catalytic subunit of the Dictyostelium PKA. DdPK3 null cells lack kinase activity that phosphorylates a PKA-specific substrate and is specifically inhibitable by recombinant cAMP-dependent protein kinase inhibitor. DdPK3 expressed in Escherichia coli has PKA activity that is inhibitable by protein kinase inhibitor. When Ddpk3 null cells are complemented with DdPK3 expressed from an actin promoter on an extrachromosomal vector (low copy number), PKA activity is restored and the cells proceed to the slug stage but will not culminate, suggesting that properly regulated PKA activity is essential for culmination. Moreover, overexpressing DdPK3 in wild-type cells on integrating vectors (high copy number) from either an actin or prespore-specific promoter results in accelerated development and the ability to form mature spores in monolayer culture in the presence of high cAMP, a developmental potential lacking in wild-type cells.

Full text

PDF
10701

Images in this article

Selected References

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

  1. Ahern K. G., Howard P. K., Firtel R. A. Identification of regions essential for extrachromosomal replication and maintenance of an endogenous plasmid in Dictyostelium. Nucleic Acids Res. 1988 Jul 25;16(14B):6825–6837. doi: 10.1093/nar/16.14.6825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bürki E., Anjard C., Scholder J. C., Reymond C. D. Isolation of two genes encoding putative protein kinases regulated during Dictyostelium discoideum development. Gene. 1991 Jun 15;102(1):57–65. doi: 10.1016/0378-1119(91)90538-m. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Denis C. L., Kemp B. E., Zoller M. J. Substrate specificities for yeast and mammalian cAMP-dependent protein kinases are similar but not identical. J Biol Chem. 1991 Sep 25;266(27):17932–17935. [PubMed] [Google Scholar]
  5. Devreotes P. Dictyostelium discoideum: a model system for cell-cell interactions in development. Science. 1989 Sep 8;245(4922):1054–1058. doi: 10.1126/science.2672337. [DOI] [PubMed] [Google Scholar]
  6. Durgerian S., Taylor S. S. The consequences of introducing an autophosphorylation site into the type I regulatory subunit of cAMP-dependent protein kinase. J Biol Chem. 1989 Jun 15;264(17):9807–9813. [PubMed] [Google Scholar]
  7. Dynes J. L., Firtel R. A. Molecular complementation of a genetic marker in Dictyostelium using a genomic DNA library. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7966–7970. doi: 10.1073/pnas.86.20.7966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Firtel R. A., Chapman A. L. A role for cAMP-dependent protein kinase A in early Dictyostelium development. Genes Dev. 1990 Jan;4(1):18–28. doi: 10.1101/gad.4.1.18. [DOI] [PubMed] [Google Scholar]
  9. Firtel R. A. Signal transduction pathways controlling multicellular development in Dictyostelium. Trends Genet. 1991 Nov-Dec;7(11-12):381–388. doi: 10.1016/0168-9525(91)90260-w. [DOI] [PubMed] [Google Scholar]
  10. Firtel R. A., van Haastert P. J., Kimmel A. R., Devreotes P. N. G protein linked signal transduction pathways in development: dictyostelium as an experimental system. Cell. 1989 Jul 28;58(2):235–239. doi: 10.1016/0092-8674(89)90837-4. [DOI] [PubMed] [Google Scholar]
  11. Gibbs C. S., Knighton D. R., Sowadski J. M., Taylor S. S., Zoller M. J. Systematic mutational analysis of cAMP-dependent protein kinase identifies unregulated catalytic subunits and defines regions important for the recognition of the regulatory subunit. J Biol Chem. 1992 Mar 5;267(7):4806–4814. [PubMed] [Google Scholar]
  12. Glass D. B., Feller M. J., Levin L. R., Walsh D. A. Structural basis for the low affinities of yeast cAMP-dependent and mammalian cGMP-dependent protein kinases for protein kinase inhibitor peptides. Biochemistry. 1992 Feb 18;31(6):1728–1734. doi: 10.1021/bi00121a021. [DOI] [PubMed] [Google Scholar]
  13. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  14. Gunzburg J., Veron M. A cAMP-dependent protein kinase is present in differentiating Dictyostelium discoideum cells. EMBO J. 1982;1(9):1063–1068. doi: 10.1002/j.1460-2075.1982.tb01297.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Haberstroh L., Firtel R. A. A spatial gradient of expression of a cAMP-regulated prespore cell-type-specific gene in Dictyostelium. Genes Dev. 1990 Apr;4(4):596–612. doi: 10.1101/gad.4.4.596. [DOI] [PubMed] [Google Scholar]
  16. Harwood A. J., Hopper N. A., Simon M. N., Bouzid S., Veron M., Williams J. G. Multiple roles for cAMP-dependent protein kinase during Dictyostelium development. Dev Biol. 1992 Jan;149(1):90–99. doi: 10.1016/0012-1606(92)90266-j. [DOI] [PubMed] [Google Scholar]
  17. Harwood A. J., Hopper N. A., Simon M. N., Driscoll D. M., Veron M., Williams J. G. Culmination in Dictyostelium is regulated by the cAMP-dependent protein kinase. Cell. 1992 May 15;69(4):615–624. doi: 10.1016/0092-8674(92)90225-2. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Kimmel A. R., Carlisle B. A gene expressed in undifferentiated vegetative Dictyostelium is repressed by developmental pulses of cAMP and reinduced during dedifferentiation. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2506–2510. doi: 10.1073/pnas.83.8.2506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kimmel A. R., Firtel R. A. cAMP signal transduction pathways regulating development of Dictyostelium discoideum. Curr Opin Genet Dev. 1991 Oct;1(3):383–390. doi: 10.1016/s0959-437x(05)80304-1. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Knighton D. R., Zheng J. H., Ten Eyck L. F., Ashford V. A., Xuong N. H., Taylor S. S., Sowadski J. M. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):407–414. doi: 10.1126/science.1862342. [DOI] [PubMed] [Google Scholar]
  23. Knighton D. R., Zheng J. H., Ten Eyck L. F., Xuong N. H., Taylor S. S., Sowadski J. M. Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):414–420. doi: 10.1126/science.1862343. [DOI] [PubMed] [Google Scholar]
  24. Kumagai A., Hadwiger J. A., Pupillo M., Firtel R. A. Molecular genetic analysis of two G alpha protein subunits in Dictyostelium. J Biol Chem. 1991 Jan 15;266(2):1220–1228. [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., Firtel R. A. A developmentally regulated, putative serine/threonine protein kinase is essential for development in Dictyostelium. Mech Dev. 1991 Sep;35(2):89–101. doi: 10.1016/0925-4773(91)90060-j. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Mehdy M. C., Firtel R. A. A secreted factor and cyclic AMP jointly regulate cell-type-specific gene expression in Dictyostelium discoideum. Mol Cell Biol. 1985 Apr;5(4):705–713. doi: 10.1128/mcb.5.4.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Richardson D. L., Hong C. B., Loomis W. F. A prespore gene, Dd31, expressed during culmination of Dictyostelium discoideum. Dev Biol. 1991 Apr;144(2):269–280. doi: 10.1016/0012-1606(91)90421-x. [DOI] [PubMed] [Google Scholar]
  31. Simon M. N., Driscoll D., Mutzel R., Part D., Williams J., Véron M. Overproduction of the regulatory subunit of the cAMP-dependent protein kinase blocks the differentiation of Dictyostelium discoideum. EMBO J. 1989 Jul;8(7):2039–2043. doi: 10.1002/j.1460-2075.1989.tb03612.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Simon M. N., Pelegrini O., Veron M., Kay R. R. Mutation of protein kinase A causes heterochronic development of Dictyostelium. Nature. 1992 Mar 12;356(6365):171–172. doi: 10.1038/356171a0. [DOI] [PubMed] [Google Scholar]
  33. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  34. Taylor S. S., Buechler J. A., Slice L. W., Knighton D. K., Durgerian S., Ringheim G. E., Neitzel J. J., Yonemoto W. M., Sowadski J. M., Dospmann W. cAMP-dependent protein kinase: a framework for a diverse family of enzymes. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 1):121–130. doi: 10.1101/sqb.1988.053.01.018. [DOI] [PubMed] [Google Scholar]
  35. Toda T., Cameron S., Sass P., Zoller M., Wigler M. Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase. Cell. 1987 Jul 17;50(2):277–287. doi: 10.1016/0092-8674(87)90223-6. [DOI] [PubMed] [Google Scholar]
  36. Williams J. G. Regulation of cellular differentiation during Dictyostelium morphogenesis. Curr Opin Genet Dev. 1991 Oct;1(3):358–362. doi: 10.1016/s0959-437x(05)80300-4. [DOI] [PubMed] [Google Scholar]
  37. Yonemoto W. M., McGlone M. L., Slice L. W., Taylor S. S. Prokaryotic expression of catalytic subunit of adenosine cyclic monophosphate-dependent protein kinase. Methods Enzymol. 1991;200:581–596. doi: 10.1016/0076-6879(91)00173-t. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES