Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1994 Apr 1;13(7):1601–1609. doi: 10.1002/j.1460-2075.1994.tb06423.x

Role of phospholipase C in Dictyostelium: formation of inositol 1,4,5-trisphosphate and normal development in cells lacking phospholipase C activity.

A L Drayer 1, J Van der Kaay 1, G W Mayr 1, P J Van Haastert 1
PMCID: PMC394990  PMID: 8156999

Abstract

The micro-organism Dictyostelium uses extracellular cAMP to induce chemotaxis and cell differentiation. Signals are transduced via surface receptors, which activate G proteins, to effector enzymes. The deduced protein sequence of Dictyostelium discoideum phosphatidylinositol-specific phospholipase C (PLC) shows strong homology with the mammalian PLC-delta isoforms. To study the role of PLC in Dictyostelium, a plc- mutant was constructed by disruption of the PLC gene. No basal or stimulated PLC activity could be measured during the whole developmental programme of the plc- cells. Loss of PLC activity did not result in a visible alteration of growth or development. Further analysis showed that developmental gene regulation, cAMP-mediated chemotaxis and activation of guanylyl and adenylyl cyclase were normal. Although the cells lack PLC activity, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was present at only slightly lower concentrations compared with control cells. Mass analysis of inositol phosphates demonstrated the presence of a broad spectrum of inositol phosphates in Dictyostelium, which was unaltered in the plc- mutant. Cell labelling experiments with [3H]inositol indicated that [3H]Ins(1,4,5)P3 was formed in a different manner in the mutant than in control cells.

Full text

PDF

Images in this article

1602Image
on p.1602
1603Image
on p.1603
1604Image
on p.1604
1605Image
on p.1605

Selected References

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

  1. Barklis E., Lodish H. F. Regulation of dictyostelium discoideum mRNAs specific for prespore or prestalk cells. Cell. 1983 Apr;32(4):1139–1148. doi: 10.1016/0092-8674(83)90297-0. [DOI] [PubMed] [Google Scholar]
  2. Bloomquist B. T., Shortridge R. D., Schneuwly S., Perdew M., Montell C., Steller H., Rubin G., Pak W. L. Isolation of a putative phospholipase C gene of Drosophila, norpA, and its role in phototransduction. Cell. 1988 Aug 26;54(5):723–733. doi: 10.1016/s0092-8674(88)80017-5. [DOI] [PubMed] [Google Scholar]
  3. Bominaar A. A., Kesbeke F., Snaar-Jagalska B. E., Peters D. J., Schaap P., Van Haastert P. J. Abberant chemotaxis and differentiation in Dictyostelium mutant fgdC with a defective regulation of receptor-stimulated phosphoinositidase C. J Cell Sci. 1991 Dec;100(Pt 4):825–831. doi: 10.1242/jcs.100.4.825. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Bominaar A. A., Van Haastert P. J. Phospholipase C in Dictyostelium discoideum. Identification of stimulatory and inhibitory surface receptors and G-proteins. Biochem J. 1994 Jan 1;297(Pt 1):189–193. doi: 10.1042/bj2970189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bominaar A. A., van der Kaay J., van Haastert P. J. Dynamics and function of the inositolcycle in Dictyostelium discoideum. Dev Genet. 1991;12(1-2):19–24. doi: 10.1002/dvg.1020120106. [DOI] [PubMed] [Google Scholar]
  7. Camps M., Carozzi A., Schnabel P., Scheer A., Parker P. J., Gierschik P. Isozyme-selective stimulation of phospholipase C-beta 2 by G protein beta gamma-subunits. Nature. 1992 Dec 17;360(6405):684–686. doi: 10.1038/360684a0. [DOI] [PubMed] [Google Scholar]
  8. Carozzi A., Camps M., Gierschik P., Parker P. J. Activation of phosphatidylinositol lipid-specific phospholipase C-beta 3 by G-protein beta gamma subunits. FEBS Lett. 1993 Jan 11;315(3):340–342. doi: 10.1016/0014-5793(93)81190-b. [DOI] [PubMed] [Google Scholar]
  9. De Lozanne A., Spudich J. A. Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination. Science. 1987 May 29;236(4805):1086–1091. doi: 10.1126/science.3576222. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Divecha N., Rhee S. G., Letcher A. J., Irvine R. F. Phosphoinositide signalling enzymes in rat liver nuclei: phosphoinositidase C isoform beta 1 is specifically, but not predominantly, located in the nucleus. Biochem J. 1993 Feb 1;289(Pt 3):617–620. doi: 10.1042/bj2890617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Drayer A. L., van Haastert P. J. Molecular cloning and expression of a phosphoinositide-specific phospholipase C of Dictyostelium discoideum. J Biol Chem. 1992 Sep 15;267(26):18387–18392. [PubMed] [Google Scholar]
  13. Europe-Finner G. N., Newell P. C. Cyclic AMP stimulates accumulation of inositol trisphosphate in Dictyostelium. J Cell Sci. 1987 Mar;87(Pt 2):221–229. doi: 10.1242/jcs.87.2.221. [DOI] [PubMed] [Google Scholar]
  14. Freund W. D., Mayr G. W., Tietz C., Schultz J. E. Metabolism of inositol phosphates in the protozoan Paramecium. Characterization of a novel inositol-hexakisphosphate-dephosphorylating enzyme. Eur J Biochem. 1992 Jul 1;207(1):359–367. doi: 10.1111/j.1432-1033.1992.tb17058.x. [DOI] [PubMed] [Google Scholar]
  15. Hadwiger J. A., Wilkie T. M., Strathmann M., Firtel R. A. Identification of Dictyostelium G alpha genes expressed during multicellular development. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8213–8217. doi: 10.1073/pnas.88.18.8213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Inoue H., Yoshioka T., Hotta Y. Membrane-associated phospholipase C of Drosophila retina. J Biochem. 1988 Jan;103(1):91–94. doi: 10.1093/oxfordjournals.jbchem.a122246. [DOI] [PubMed] [Google Scholar]
  17. Johnson R. L., Saxe C. L., 3rd, Gollop R., Kimmel A. R., Devreotes P. N. Identification and targeted gene disruption of cAR3, a cAMP receptor subtype expressed during multicellular stages of Dictyostelium development. Genes Dev. 1993 Feb;7(2):273–282. doi: 10.1101/gad.7.2.273. [DOI] [PubMed] [Google Scholar]
  18. Katz A., Wu D., Simon M. I. Subunits beta gamma of heterotrimeric G protein activate beta 2 isoform of phospholipase C. Nature. 1992 Dec 17;360(6405):686–689. doi: 10.1038/360686a0. [DOI] [PubMed] [Google Scholar]
  19. Kesbeke F., Snaar-Jagalska B. E., Van Haastert P. J. Signal transduction in Dictyostelium fgd A mutants with a defective interaction between surface cAMP receptors and a GTP-binding regulatory protein. J Cell Biol. 1988 Aug;107(2):521–528. doi: 10.1083/jcb.107.2.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kim H. K., Kim J. W., Zilberstein A., Margolis B., Kim J. G., Schlessinger J., Rhee S. G. PDGF stimulation of inositol phospholipid hydrolysis requires PLC-gamma 1 phosphorylation on tyrosine residues 783 and 1254. Cell. 1991 May 3;65(3):435–441. doi: 10.1016/0092-8674(91)90461-7. [DOI] [PubMed] [Google Scholar]
  21. Klein P. S., Sun T. J., Saxe C. L., 3rd, Kimmel A. R., Johnson R. L., Devreotes P. N. A chemoattractant receptor controls development in Dictyostelium discoideum. Science. 1988 Sep 16;241(4872):1467–1472. doi: 10.1126/science.3047871. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  24. Lee C. H., Park D., Wu D., Rhee S. G., Simon M. I. Members of the Gq alpha subunit gene family activate phospholipase C beta isozymes. J Biol Chem. 1992 Aug 15;267(23):16044–16047. [PubMed] [Google Scholar]
  25. Lilly P., Wu L., Welker D. L., Devreotes P. N. A G-protein beta-subunit is essential for Dictyostelium development. Genes Dev. 1993 Jun;7(6):986–995. doi: 10.1101/gad.7.6.986. [DOI] [PubMed] [Google Scholar]
  26. Mato J. M., Van Haastert P. J., Krens F. A., Rhijnsburger E. H., Dobbe F. C., Konijn T. M. Cyclic AMP and folic acid mediated cyclic GMP accumulation in Dictyostelium discoideum. FEBS Lett. 1977 Jul 15;79(2):331–336. doi: 10.1016/0014-5793(77)80814-4. [DOI] [PubMed] [Google Scholar]
  27. Mayr G. W. A novel metal-dye detection system permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens. Biochem J. 1988 Sep 1;254(2):585–591. doi: 10.1042/bj2540585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mohammadi M., Honegger A. M., Rotin D., Fischer R., Bellot F., Li W., Dionne C. A., Jaye M., Rubinstein M., Schlessinger J. A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1. Mol Cell Biol. 1991 Oct;11(10):5068–5078. doi: 10.1128/mcb.11.10.5068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Park D., Jhon D. Y., Lee C. W., Lee K. H., Rhee S. G. Activation of phospholipase C isozymes by G protein beta gamma subunits. J Biol Chem. 1993 Mar 5;268(7):4573–4576. [PubMed] [Google Scholar]
  31. 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]
  32. Pupillo M., Kumagai A., Pitt G. S., Firtel R. A., Devreotes P. N. Multiple alpha subunits of guanine nucleotide-binding proteins in Dictyostelium. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4892–4896. doi: 10.1073/pnas.86.13.4892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rhee S. G., Choi K. D. Regulation of inositol phospholipid-specific phospholipase C isozymes. J Biol Chem. 1992 Jun 25;267(18):12393–12396. [PubMed] [Google Scholar]
  34. Roos W., Gerisch G. Receptor-mediated adenylate cyclase activation in Dictyostelium discoideum. FEBS Lett. 1976 Oct 1;68(2):170–172. doi: 10.1016/0014-5793(76)80429-2. [DOI] [PubMed] [Google Scholar]
  35. Rotin D., Margolis B., Mohammadi M., Daly R. J., Daum G., Li N., Fischer E. H., Burgess W. H., Ullrich A., Schlessinger J. SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma. EMBO J. 1992 Feb;11(2):559–567. doi: 10.1002/j.1460-2075.1992.tb05087.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Saxe C. L., 3rd, Ginsburg G. T., Louis J. M., Johnson R., Devreotes P. N., Kimmel A. R. CAR2, a prestalk cAMP receptor required for normal tip formation and late development of Dictyostelium discoideum. Genes Dev. 1993 Feb;7(2):262–272. doi: 10.1101/gad.7.2.262. [DOI] [PubMed] [Google Scholar]
  37. Saxe C. L., 3rd, Johnson R., Devreotes P. N., Kimmel A. R. Multiple genes for cell surface cAMP receptors in Dictyostelium discoideum. Dev Genet. 1991;12(1-2):6–13. doi: 10.1002/dvg.1020120104. [DOI] [PubMed] [Google Scholar]
  38. Smrcka A. V., Hepler J. R., Brown K. O., Sternweis P. C. Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq. Science. 1991 Feb 15;251(4995):804–807. doi: 10.1126/science.1846707. [DOI] [PubMed] [Google Scholar]
  39. Stahl M. L., Ferenz C. R., Kelleher K. L., Kriz R. W., Knopf J. L. Sequence similarity of phospholipase C with the non-catalytic region of src. Nature. 1988 Mar 17;332(6161):269–272. doi: 10.1038/332269a0. [DOI] [PubMed] [Google Scholar]
  40. Stephens L., Radenberg T., Thiel U., Vogel G., Khoo K. H., Dell A., Jackson T. R., Hawkins P. T., Mayr G. W. The detection, purification, structural characterization, and metabolism of diphosphoinositol pentakisphosphate(s) and bisdiphosphoinositol tetrakisphosphate(s). J Biol Chem. 1993 Feb 25;268(6):4009–4015. [PubMed] [Google Scholar]
  41. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  42. Suh P. G., Ryu S. H., Moon K. H., Suh H. W., Rhee S. G. Cloning and sequence of multiple forms of phospholipase C. Cell. 1988 Jul 15;54(2):161–169. doi: 10.1016/0092-8674(88)90548-x. [DOI] [PubMed] [Google Scholar]
  43. Suh P. G., Ryu S. H., Moon K. H., Suh H. W., Rhee S. G. Inositol phospholipid-specific phospholipase C: complete cDNA and protein sequences and sequence homology to tyrosine kinase-related oncogene products. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5419–5423. doi: 10.1073/pnas.85.15.5419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Taylor S. J., Chae H. Z., Rhee S. G., Exton J. H. Activation of the beta 1 isozyme of phospholipase C by alpha subunits of the Gq class of G proteins. Nature. 1991 Apr 11;350(6318):516–518. doi: 10.1038/350516a0. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Van Haastert P. J., De Vries M. J., Penning L. C., Roovers E., Van der Kaay J., Erneux C., Van Lookeren Campagne M. M. Chemoattractant and guanosine 5'-[gamma-thio]triphosphate induce the accumulation of inositol 1,4,5-trisphosphate in Dictyostelium cells that are labelled with [3H]inositol by electroporation. Biochem J. 1989 Mar 1;258(2):577–586. doi: 10.1042/bj2580577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Van Haastert P. J. Determination of inositol 1,4,5-trisphosphate levels in Dictyostelium by isotope dilution assay. Anal Biochem. 1989 Feb 15;177(1):115–119. doi: 10.1016/0003-2697(89)90024-9. [DOI] [PubMed] [Google Scholar]
  48. Van Haastert P. J., Janssens P. M., Erneux C. Sensory transduction in eukaryotes. A comparison between Dictyostelium and vertebrate cells. Eur J Biochem. 1991 Jan 30;195(2):289–303. doi: 10.1111/j.1432-1033.1991.tb15706.x. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Wu L. J., Devreotes P. N. Dictyostelium transiently expresses eight distinct G-protein alpha-subunits during its developmental program. Biochem Biophys Res Commun. 1991 Sep 30;179(3):1141–1147. doi: 10.1016/0006-291x(91)91690-e. [DOI] [PubMed] [Google Scholar]
  51. Wurster B., Schubiger K., Wick U., Gerisch G. Cyclic GMP in Dictyostelium discoideum, Oscillations and pulses in response to folic acid and cyclic AMP signals. FEBS Lett. 1977 Apr 15;76(2):141–144. doi: 10.1016/0014-5793(77)80139-7. [DOI] [PubMed] [Google Scholar]
  52. Yoko-o T., Matsui Y., Yagisawa H., Nojima H., Uno I., Toh-e A. The putative phosphoinositide-specific phospholipase C gene, PLC1, of the yeast Saccharomyces cerevisiae is important for cell growth. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1804–1808. doi: 10.1073/pnas.90.5.1804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yoshioka T., Inoue H., Hotta Y. Absence of phosphatidylinositol phosphodiesterase in the head of a Drosophila visual mutant, norpA (no receptor potential A). J Biochem. 1985 Apr;97(4):1251–1254. doi: 10.1093/oxfordjournals.jbchem.a135171. [DOI] [PubMed] [Google Scholar]

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

RESOURCES