Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1997 Jul 16;16(14):4317–4332. doi: 10.1093/emboj/16.14.4317

The Dictyostelium MAP kinase kinase DdMEK1 regulates chemotaxis and is essential for chemoattractant-mediated activation of guanylyl cyclase.

H Ma 1, M Gamper 1, C Parent 1, R A Firtel 1
PMCID: PMC1170058  PMID: 9250676

Abstract

We have identified a MAP kinase kinase (DdMEK1) that is required for proper aggregation in Dictyostelium. Null mutations produce extremely small aggregate sizes, resulting in the formation of slugs and terminal fruiting bodies that are significantly smaller than those of wild-type cells. Time-lapse video microscopy and in vitro assays indicate that the cells are able to produce cAMP waves that move through the aggregation domains. However, these cells are unable to undergo chemotaxis properly during aggregation in response to the chemoattractant cAMP or activate guanylyl cyclase, a known regulator of chemotaxis in Dictyostelium. The activation of guanylyl cyclase in response to osmotic stress is, however, normal. Expression of putative constitutively active forms of DdMEK1 in a ddmek1 null background is capable, at least partially, of complementing the small aggregate size defect and the ability to activate guanylyl cyclase. However, this does not result in constitutive activation of guanylyl cyclase, suggesting that DdMEK1 activity is necessary, but not sufficient, for cAMP activation of guanylyl cyclase. Analysis of a temperature-sensitive DdMEK1 mutant suggests that DdMEK1 activity is required throughout aggregation at the time of guanylyl cyclase activation, but is not essential for proper morphogenesis during the later multicellular stages. The activation of the MAP kinase ERK2, which is essential for chemoattractant activation of adenylyl cyclase, is not affected in ddmek1 null strains, indicating that DdMEK1 does not regulate ERK2 and suggesting that at least two independent MAP kinase cascades control aggregation in Dictyostelium.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Aubry L., Maeda M., Insall R., Devreotes P. N., Firtel R. A. The Dictyostelium mitogen-activated protein kinase ERK2 is regulated by Ras and cAMP-dependent protein kinase (PKA) and mediates PKA function. J Biol Chem. 1997 Feb 14;272(7):3883–3886. doi: 10.1074/jbc.272.7.3883. [DOI] [PubMed] [Google Scholar]
  2. Bardwell L., Cook J. G., Chang E. C., Cairns B. R., Thorner J. Signaling in the yeast pheromone response pathway: specific and high-affinity interaction of the mitogen-activated protein (MAP) kinases Kss1 and Fus3 with the upstream MAP kinase kinase Ste7. Mol Cell Biol. 1996 Jul;16(7):3637–3650. doi: 10.1128/mcb.16.7.3637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bardwell L., Thorner J. A conserved motif at the amino termini of MEKs might mediate high-affinity interaction with the cognate MAPKs. Trends Biochem Sci. 1996 Oct;21(10):373–374. [PubMed] [Google Scholar]
  4. Bokemeyer D., Sorokin A., Dunn M. J. Multiple intracellular MAP kinase signaling cascades. Kidney Int. 1996 May;49(5):1187–1198. doi: 10.1038/ki.1996.172. [DOI] [PubMed] [Google Scholar]
  5. Bominaar A. A., Kesbeke F., Van Haastert P. J. Phospholipase C in Dictyostelium discoideum. Cyclic AMP surface receptor and G-protein-regulated activity in vitro. Biochem J. 1994 Jan 1;297(Pt 1):181–187. doi: 10.1042/bj2970181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brott B. K., Alessandrini A., Largaespada D. A., Copeland N. G., Jenkins N. A., Crews C. M., Erikson R. L. MEK2 is a kinase related to MEK1 and is differentially expressed in murine tissues. Cell Growth Differ. 1993 Nov;4(11):921–929. [PubMed] [Google Scholar]
  7. Carr A. M., MacNeill S. A., Hayles J., Nurse P. Molecular cloning and sequence analysis of mutant alleles of the fission yeast cdc2 protein kinase gene: implications for cdc2+ protein structure and function. Mol Gen Genet. 1989 Jul;218(1):41–49. doi: 10.1007/BF00330563. [DOI] [PubMed] [Google Scholar]
  8. Chen M. Y., Insall R. H., Devreotes P. N. Signaling through chemoattractant receptors in Dictyostelium. Trends Genet. 1996 Feb;12(2):52–57. doi: 10.1016/0168-9525(96)81400-4. [DOI] [PubMed] [Google Scholar]
  9. Davis R. J. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993 Jul 15;268(20):14553–14556. [PubMed] [Google Scholar]
  10. Devreotes P. N. G protein-linked signaling pathways control the developmental program of Dictyostelium. Neuron. 1994 Feb;12(2):235–241. doi: 10.1016/0896-6273(94)90267-4. [DOI] [PubMed] [Google Scholar]
  11. Devreotes P. N., Zigmond S. H. Chemotaxis in eukaryotic cells: a focus on leukocytes and Dictyostelium. Annu Rev Cell Biol. 1988;4:649–686. doi: 10.1146/annurev.cb.04.110188.003245. [DOI] [PubMed] [Google Scholar]
  12. Devreotes P., Fontana D., Klein P., Sherring J., Theibert A. Transmembrane signaling in Dictyostelium. Methods Cell Biol. 1987;28:299–331. doi: 10.1016/s0091-679x(08)61653-2. [DOI] [PubMed] [Google Scholar]
  13. Errede B., Levin D. E. A conserved kinase cascade for MAP kinase activation in yeast. Curr Opin Cell Biol. 1993 Apr;5(2):254–260. doi: 10.1016/0955-0674(93)90112-4. [DOI] [PubMed] [Google Scholar]
  14. Gaskins C., Clark A. M., Aubry L., Segall J. E., Firtel R. A. The Dictyostelium MAP kinase ERK2 regulates multiple, independent developmental pathways. Genes Dev. 1996 Jan 1;10(1):118–128. doi: 10.1101/gad.10.1.118. [DOI] [PubMed] [Google Scholar]
  15. Hall A. L., Franke J., Faure M., Kessin R. H. The role of the cyclic nucleotide phosphodiesterase of Dictyostelium discoideum during growth, aggregation, and morphogenesis: overexpression and localization studies with the separate promoters of the pde. Dev Biol. 1993 May;157(1):73–84. doi: 10.1006/dbio.1993.1113. [DOI] [PubMed] [Google Scholar]
  16. Herskowitz I. MAP kinase pathways in yeast: for mating and more. Cell. 1995 Jan 27;80(2):187–197. doi: 10.1016/0092-8674(95)90402-6. [DOI] [PubMed] [Google Scholar]
  17. Howard P. K., Gamper M., Hunter T., Firtel R. A. Regulation by protein-tyrosine phosphatase PTP2 is distinct from that by PTP1 during Dictyostelium growth and development. Mol Cell Biol. 1994 Aug;14(8):5154–5164. doi: 10.1128/mcb.14.8.5154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hsu J. C., Perrimon N. A temperature-sensitive MEK mutation demonstrates the conservation of the signaling pathways activated by receptor tyrosine kinases. Genes Dev. 1994 Sep 15;8(18):2176–2187. doi: 10.1101/gad.8.18.2176. [DOI] [PubMed] [Google Scholar]
  19. Huang W., Kessler D. S., Erikson R. L. Biochemical and biological analysis of Mek1 phosphorylation site mutants. Mol Biol Cell. 1995 Mar;6(3):237–245. doi: 10.1091/mbc.6.3.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Insall R., Kuspa A., Lilly P. J., Shaulsky G., Levin L. R., Loomis W. F., Devreotes P. CRAC, a cytosolic protein containing a pleckstrin homology domain, is required for receptor and G protein-mediated activation of adenylyl cyclase in Dictyostelium. J Cell Biol. 1994 Sep;126(6):1537–1545. doi: 10.1083/jcb.126.6.1537. [DOI] [PMC free article] [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. Knetsch M. L., Epskamp S. J., Schenk P. W., Wang Y., Segall J. E., Snaar-Jagalska B. E. Dual role of cAMP and involvement of both G-proteins and ras in regulation of ERK2 in Dictyostelium discoideum. EMBO J. 1996 Jul 1;15(13):3361–3368. [PMC free article] [PubMed] [Google Scholar]
  23. Konijn T. M. Microbiological assay of cyclic 3',5'-AMP. Experientia. 1970 Apr 15;26(4):367–369. doi: 10.1007/BF01896891. [DOI] [PubMed] [Google Scholar]
  24. Kuwayama H., Viel G. T., Ishida S., Van Haastert P. J. Aberrant cGMP-binding activity in non-chemotactic Dictyostelium discoideum mutants. Biochim Biophys Acta. 1995 Aug 31;1268(2):214–220. doi: 10.1016/0167-4889(95)00082-4. [DOI] [PubMed] [Google Scholar]
  25. Levin D. E., Errede B. The proliferation of MAP kinase signaling pathways in yeast. Curr Opin Cell Biol. 1995 Apr;7(2):197–202. doi: 10.1016/0955-0674(95)80028-x. [DOI] [PubMed] [Google Scholar]
  26. Lilly P. J., Devreotes P. N. Chemoattractant and GTP gamma S-mediated stimulation of adenylyl cyclase in Dictyostelium requires translocation of CRAC to membranes. J Cell Biol. 1995 Jun;129(6):1659–1665. doi: 10.1083/jcb.129.6.1659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lilly P. J., Devreotes P. N. Identification of CRAC, a cytosolic regulator required for guanine nucleotide stimulation of adenylyl cyclase in Dictyostelium. J Biol Chem. 1994 May 13;269(19):14123–14129. [PubMed] [Google Scholar]
  28. Maeda M., Aubry L., Insall R., Gaskins C., Devreotes P. N., Firtel R. A. Seven helix chemoattractant receptors transiently stimulate mitogen-activated protein kinase in Dictyostelium. Role of heterotrimeric G proteins. J Biol Chem. 1996 Feb 16;271(7):3351–3354. doi: 10.1074/jbc.271.7.3351. [DOI] [PubMed] [Google Scholar]
  29. Mann S. K., Brown J. M., Briscoe C., Parent C., Pitt G., Devreotes P. N., Firtel R. A. Role of cAMP-dependent protein kinase in controlling aggregation and postaggregative development in Dictyostelium. Dev Biol. 1997 Mar 15;183(2):208–221. doi: 10.1006/dbio.1996.8499. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Mann S. K., Firtel R. A. Cyclic AMP regulation of early gene expression in Dictyostelium discoideum: mediation via the cell surface cyclic AMP receptor. Mol Cell Biol. 1987 Jan;7(1):458–469. doi: 10.1128/mcb.7.1.458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mansour S. J., Matten W. T., Hermann A. S., Candia J. M., Rong S., Fukasawa K., Vande Woude G. F., Ahn N. G. Transformation of mammalian cells by constitutively active MAP kinase kinase. Science. 1994 Aug 12;265(5174):966–970. doi: 10.1126/science.8052857. [DOI] [PubMed] [Google Scholar]
  33. Mehdy M. C., Ratner D., Firtel R. A. Induction and modulation of cell-type-specific gene expression in Dictyostelium. Cell. 1983 Mar;32(3):763–771. doi: 10.1016/0092-8674(83)90062-4. [DOI] [PubMed] [Google Scholar]
  34. Nellen W., Datta S., Reymond C., Sivertsen A., Mann S., Crowley T., Firtel R. A. Molecular biology in Dictyostelium: tools and applications. Methods Cell Biol. 1987;28:67–100. doi: 10.1016/s0091-679x(08)61637-4. [DOI] [PubMed] [Google Scholar]
  35. Noegel A., Gerisch G., Stadler J., Westphal M. Complete sequence and transcript regulation of a cell adhesion protein from aggregating Dictyostelium cells. EMBO J. 1986 Jul;5(7):1473–1476. doi: 10.1002/j.1460-2075.1986.tb04384.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Okaichi K., Cubitt A. B., Pitt G. S., Firtel R. A. Amino acid substitutions in the Dictyostelium G alpha subunit G alpha 2 produce dominant negative phenotypes and inhibit the activation of adenylyl cyclase, guanylyl cyclase, and phospholipase C. Mol Biol Cell. 1992 Jul;3(7):735–747. doi: 10.1091/mbc.3.7.735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pagès G., Brunet A., L'Allemain G., Pouysségur J. Constitutive mutant and putative regulatory serine phosphorylation site of mammalian MAP kinase kinase (MEK1). EMBO J. 1994 Jul 1;13(13):3003–3010. doi: 10.1002/j.1460-2075.1994.tb06599.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pitt G. S., Milona N., Borleis J., Lin K. C., Reed R. R., Devreotes P. N. Structurally distinct and stage-specific adenylyl cyclase genes play different roles in Dictyostelium development. Cell. 1992 Apr 17;69(2):305–315. doi: 10.1016/0092-8674(92)90411-5. [DOI] [PubMed] [Google Scholar]
  39. Richardson D. L., Loomis W. F., Kimmel A. R. Progression of an inductive signal activates sporulation in Dictyostelium discoideum. Development. 1994 Oct;120(10):2891–2900. doi: 10.1242/dev.120.10.2891. [DOI] [PubMed] [Google Scholar]
  40. Ross F. M., Newell P. C. Streamers: chemotactic mutants of Dictyostelium discoideum with altered cyclic GMP metabolism. J Gen Microbiol. 1981 Dec;127(2):339–350. doi: 10.1099/00221287-127-2-339. [DOI] [PubMed] [Google Scholar]
  41. Salomon Y. Adenylate cyclase assay. Adv Cyclic Nucleotide Res. 1979;10:35–55. [PubMed] [Google Scholar]
  42. Schaap P., Wang M. cAMP induces a transient elevation of cGMP levels during early culmination of Dictyostelium minutum. Cell Differ. 1985 Feb;16(1):29–33. doi: 10.1016/0045-6039(85)90604-9. [DOI] [PubMed] [Google Scholar]
  43. Schnitzler G. R., Fischer W. H., Firtel R. A. Cloning and characterization of the G-box binding factor, an essential component of the developmental switch between early and late development in Dictyostelium. Genes Dev. 1994 Feb 15;8(4):502–514. doi: 10.1101/gad.8.4.502. [DOI] [PubMed] [Google Scholar]
  44. Schulkes C., Schaap P. cAMP-dependent protein kinase activity is essential for preaggregative gene expression in Dictyostelium. FEBS Lett. 1995 Jul 17;368(2):381–384. doi: 10.1016/0014-5793(95)00676-z. [DOI] [PubMed] [Google Scholar]
  45. Segall J. E., Kuspa A., Shaulsky G., Ecke M., Maeda M., Gaskins C., Firtel R. A., Loomis W. F. A MAP kinase necessary for receptor-mediated activation of adenylyl cyclase in Dictyostelium. J Cell Biol. 1995 Feb;128(3):405–413. doi: 10.1083/jcb.128.3.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Snaar-Jagalska B. E., Kesbeke F., Van Haastert P. J. G-proteins in the signal-transduction pathways of Dictyostelium discoideum. Dev Genet. 1988;9(4-5):215–226. doi: 10.1002/dvg.1020090404. [DOI] [PubMed] [Google Scholar]
  48. Sutoh K. A transformation vector for dictyostelium discoideum with a new selectable marker bsr. Plasmid. 1993 Sep;30(2):150–154. doi: 10.1006/plas.1993.1042. [DOI] [PubMed] [Google Scholar]
  49. Valkema R., Van Haastert P. J. A model for cAMP-mediated cGMP response in Dictyostelium discoideum. Mol Biol Cell. 1994 May;5(5):575–585. doi: 10.1091/mbc.5.5.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Van Haastert P. J. Transduction of the chemotactic cAMP signal across the plasma membrane of Dictyostelium cells. Experientia. 1995 Dec 18;51(12):1144–1154. doi: 10.1007/BF01944732. [DOI] [PubMed] [Google Scholar]
  51. Williams J. G., Ceccarelli A., McRobbie S., Mahbubani H., Kay R. R., Early A., Berks M., Jermyn K. A. Direct induction of Dictyostelium prestalk gene expression by DIF provides evidence that DIF is a morphogen. Cell. 1987 Apr 24;49(2):185–192. doi: 10.1016/0092-8674(87)90559-9. [DOI] [PubMed] [Google Scholar]
  52. Wu L., Hansen D., Franke J., Kessin R. H., Podgorski G. J. Regulation of Dictyostelium early development genes in signal transduction mutants. Dev Biol. 1995 Sep;171(1):149–158. doi: 10.1006/dbio.1995.1267. [DOI] [PubMed] [Google Scholar]
  53. Wu L., Valkema R., Van Haastert P. J., Devreotes P. N. The G protein beta subunit is essential for multiple responses to chemoattractants in Dictyostelium. J Cell Biol. 1995 Jun;129(6):1667–1675. doi: 10.1083/jcb.129.6.1667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yashar B., Irie K., Printen J. A., Stevenson B. J., Sprague G. F., Jr, Matsumoto K., Errede B. Yeast MEK-dependent signal transduction: response thresholds and parameters affecting fidelity. Mol Cell Biol. 1995 Dec;15(12):6545–6553. doi: 10.1128/mcb.15.12.6545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Zheng C. F., Guan K. L. Activation of MEK family kinases requires phosphorylation of two conserved Ser/Thr residues. EMBO J. 1994 Mar 1;13(5):1123–1131. doi: 10.1002/j.1460-2075.1994.tb06361.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES