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. 1995 Oct 15;311(Pt 2):505–510. doi: 10.1042/bj3110505

Mutation of an EF-hand Ca(2+)-binding motif in phospholipase C of Dictyostelium discoideum: inhibition of activity but no effect on Ca(2+)-dependence.

A L Drayer 1, M E Meima 1, M W Derks 1, R Tuik 1, P J van Haastert 1
PMCID: PMC1136027  PMID: 7487887

Abstract

Phosphoinositide-specific phospholipase C (PLC) is dependent on Ca2+ ions for substrate hydrolysis. The role of an EF-hand Ca(2+)-binding motif in Ca(2+)-dependent PLC activity was investigated by site-directed mutagenesis of the Dictyostelium discoideum PLC enzyme. Amino acid residues with oxygen-containing side chains at co-ordinates x, y, z, -x and -z of the putative Ca(2+)-binding-loop sequence were replaced by isoleucine (x), valine (y) or alanine (z, -x and -z). The mutated proteins were expressed in a Dictyostelium cell line with a disrupted plc gene displaying no endogenous PLC activity, and PLC activity was measured in cell lysates at different Ca2+ concentrations. Replacement of aspartate at position x, which is considered to play an essential role in Ca2+ binding, had little effect on Ca2+ affinity and maximal enzyme activity. A mutant with substitutions at both aspartate residues in position x and y also showed no decrease in Ca2+ affinity, whereas the maximal PLC activity was reduced by 60%. Introduction of additional mutations in the EF-hand revealed that the Ca2+ concentration giving half-maximal activity was unaltered, but PLC activity levels at saturating Ca2+ concentrations were markedly decreased. The results demonstrate that, although the EF-hand domain is required for enzyme activity, it is not the site that regulates the Ca(2+)-dependence of the PLC reaction.

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Selected References

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

  1. Bairoch A., Cox J. A. EF-hand motifs in inositol phospholipid-specific phospholipase C. FEBS Lett. 1990 Sep 3;269(2):454–456. doi: 10.1016/0014-5793(90)81214-9. [DOI] [PubMed] [Google Scholar]
  2. Bairoch A. The PROSITE dictionary of sites and patterns in proteins, its current status. Nucleic Acids Res. 1993 Jul 1;21(13):3097–3103. doi: 10.1093/nar/21.13.3097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bar-Sagi D., Rotin D., Batzer A., Mandiyan V., Schlessinger J. SH3 domains direct cellular localization of signaling molecules. Cell. 1993 Jul 16;74(1):83–91. doi: 10.1016/0092-8674(93)90296-3. [DOI] [PubMed] [Google Scholar]
  4. Bartfai T. Preparation of metal-chelate complexes and the design of steady-state kinetic experiments involving metal nucleotide complexes. Adv Cyclic Nucleotide Res. 1979;10:219–242. [PubMed] [Google Scholar]
  5. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  6. Berstein G., Blank J. L., Smrcka A. V., Higashijima T., Sternweis P. C., Exton J. H., Ross E. M. Reconstitution of agonist-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq/11, and phospholipase C-beta 1. J Biol Chem. 1992 Apr 25;267(12):8081–8088. [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Boy-Marcotte E., Vilaine F., Camonis J., Jacquet M. A DNA sequence from Dictyostelium discoideum complements ura3 and ura5 mutations of Saccharomyces cerevisiae. Mol Gen Genet. 1984;193(3):406–413. doi: 10.1007/BF00382076. [DOI] [PubMed] [Google Scholar]
  10. Camps M., Hou C., Sidiropoulos D., Stock J. B., Jakobs K. H., Gierschik P. Stimulation of phospholipase C by guanine-nucleotide-binding protein beta gamma subunits. Eur J Biochem. 1992 Jun 15;206(3):821–831. doi: 10.1111/j.1432-1033.1992.tb16990.x. [DOI] [PubMed] [Google Scholar]
  11. Cifuentes M. E., Honkanen L., Rebecchi M. J. Proteolytic fragments of phosphoinositide-specific phospholipase C-delta 1. Catalytic and membrane binding properties. J Biol Chem. 1993 Jun 5;268(16):11586–11593. [PubMed] [Google Scholar]
  12. Clark J. D., Lin L. L., Kriz R. W., Ramesha C. S., Sultzman L. A., Lin A. Y., Milona N., Knopf J. L. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP. Cell. 1991 Jun 14;65(6):1043–1051. doi: 10.1016/0092-8674(91)90556-e. [DOI] [PubMed] [Google Scholar]
  13. Drayer A. L., Van der Kaay J., Mayr G. W., Van Haastert P. J. Role of phospholipase C in Dictyostelium: formation of inositol 1,4,5-trisphosphate and normal development in cells lacking phospholipase C activity. EMBO J. 1994 Apr 1;13(7):1601–1609. doi: 10.1002/j.1460-2075.1994.tb06423.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Ellis M. V., Carne A., Katan M. Structural requirements of phosphatidylinositol-specific phospholipase C delta 1 for enzyme activity. Eur J Biochem. 1993 Apr 1;213(1):339–347. doi: 10.1111/j.1432-1033.1993.tb17767.x. [DOI] [PubMed] [Google Scholar]
  16. Emori Y., Homma Y., Sorimachi H., Kawasaki H., Nakanishi O., Suzuki K., Takenawa T. A second type of rat phosphoinositide-specific phospholipase C containing a src-related sequence not essential for phosphoinositide-hydrolyzing activity. J Biol Chem. 1989 Dec 25;264(36):21885–21890. [PubMed] [Google Scholar]
  17. Franke J., Kessin R. A defined minimal medium for axenic strains of Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1977 May;74(5):2157–2161. doi: 10.1073/pnas.74.5.2157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goldschmidt-Clermont P. J., Kim J. W., Machesky L. M., Rhee S. G., Pollard T. D. Regulation of phospholipase C-gamma 1 by profilin and tyrosine phosphorylation. Science. 1991 Mar 8;251(4998):1231–1233. doi: 10.1126/science.1848725. [DOI] [PubMed] [Google Scholar]
  19. Homma Y., Emori Y. A dual functional signal mediator showing RhoGAP and phospholipase C-delta stimulating activities. EMBO J. 1995 Jan 16;14(2):286–291. doi: 10.1002/j.1460-2075.1995.tb07002.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Howard P. K., Ahern K. G., Firtel R. A. Establishment of a transient expression system for Dictyostelium discoideum. Nucleic Acids Res. 1988 Mar 25;16(6):2613–2623. doi: 10.1093/nar/16.6.2613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Janmey P. A. Phosphoinositides and calcium as regulators of cellular actin assembly and disassembly. Annu Rev Physiol. 1994;56:169–191. doi: 10.1146/annurev.ph.56.030194.001125. [DOI] [PubMed] [Google Scholar]
  22. Katan M., Parker P. J. Purification of phosphoinositide-specific phospholipase C from a particulate fraction of bovine brain. Eur J Biochem. 1987 Oct 15;168(2):413–418. doi: 10.1111/j.1432-1033.1987.tb13435.x. [DOI] [PubMed] [Google Scholar]
  23. Kretsinger R. H., Nockolds C. E. Carp muscle calcium-binding protein. II. Structure determination and general description. J Biol Chem. 1973 May 10;248(9):3313–3326. [PubMed] [Google Scholar]
  24. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Maruyama K., Mikawa T., Ebashi S. Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis. J Biochem. 1984 Feb;95(2):511–519. doi: 10.1093/oxfordjournals.jbchem.a134633. [DOI] [PubMed] [Google Scholar]
  28. Moncrief N. D., Kretsinger R. H., Goodman M. Evolution of EF-hand calcium-modulated proteins. I. Relationships based on amino acid sequences. J Mol Evol. 1990 Jun;30(6):522–562. doi: 10.1007/BF02101108. [DOI] [PubMed] [Google Scholar]
  29. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature. 1988 Aug 25;334(6184):661–665. doi: 10.1038/334661a0. [DOI] [PubMed] [Google Scholar]
  30. Park D., Jhon D. Y., Lee C. W., Ryu S. H., Rhee S. G. Removal of the carboxyl-terminal region of phospholipase C-beta 1 by calpain abolishes activation by G alpha q. J Biol Chem. 1993 Feb 15;268(5):3710–3714. [PubMed] [Google Scholar]
  31. Pawson T., Schlessingert J. SH2 and SH3 domains. Curr Biol. 1993 Jul 1;3(7):434–442. doi: 10.1016/0960-9822(93)90350-w. [DOI] [PubMed] [Google Scholar]
  32. Payne W. E., Fitzgerald-Hayes M. A mutation in PLC1, a candidate phosphoinositide-specific phospholipase C gene from Saccharomyces cerevisiae, causes aberrant mitotic chromosome segregation. Mol Cell Biol. 1993 Jul;13(7):4351–4364. doi: 10.1128/mcb.13.7.4351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  34. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Streb H., Irvine R. F., Berridge M. J., Schulz I. Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate. Nature. 1983 Nov 3;306(5938):67–69. doi: 10.1038/306067a0. [DOI] [PubMed] [Google Scholar]
  36. Strynadka N. C., James M. N. Crystal structures of the helix-loop-helix calcium-binding proteins. Annu Rev Biochem. 1989;58:951–998. doi: 10.1146/annurev.bi.58.070189.004511. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Van Dijken P., Lammers A. A., Van Haastert P. J. In Dictyostelium discoideum inositol 1,3,4,5-tetrakisphosphate is dephosphorylated by a 3-phosphatase and a 1-phosphatase. Biochem J. 1995 May 15;308(Pt 1):127–130. [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Witke W., Hofmann A., Köppel B., Schleicher M., Noegel A. A. The Ca(2+)-binding domains in non-muscle type alpha-actinin: biochemical and genetic analysis. J Cell Biol. 1993 May;121(3):599–606. doi: 10.1083/jcb.121.3.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wu D., Jiang H., Katz A., Simon M. I. Identification of critical regions on phospholipase C-beta 1 required for activation by G-proteins. J Biol Chem. 1993 Feb 15;268(5):3704–3709. [PubMed] [Google Scholar]
  42. Wu D., Katz A., Simon M. I. Activation of phospholipase C beta 2 by the alpha and beta gamma subunits of trimeric GTP-binding protein. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5297–5301. doi: 10.1073/pnas.90.11.5297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]

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