Skip to main content
NCBI home page
Molecular Biology of the Cell logoLink to Molecular Biology of the Cell
. 1996 Feb;7(2):261–272. doi: 10.1091/mbc.7.2.261

Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin.

U Gottwald 1, R Brokamp 1, I Karakesisoglou 1, M Schleicher 1, A A Noegel 1
PMCID: PMC275878  PMID: 8688557

Abstract

In search for novel actin binding proteins in Dictyostelium discoideum we have isolated a cDNA clone coding for a protein of approximately 50 kDa that is highly homologous to the class of adenylyl cyclase-associated proteins (CAP). In Saccharomyces cerevisiae the amino-terminal part of CAP is involved in the regulation of the adenylyl cyclase whereas the loss of the carboxyl-terminal domain results in morphological and nutritional defects. To study the interaction of Dictyostelium CAP with actin, the complete protein and its amino-terminal and carboxyl-terminal domains were expressed in Escherichia coli and used in actin binding assays. CAP sequestered actin in a Ca2+ independent way. This activity was localized to the carboxyl-terminal domain. CAP and its carboxyl-terminal domain led to a fluorescence enhancement of pyrene-labeled G-actin up to 50% indicating a direct interaction, whereas the amino-terminal domain did not enhance. In polymerization as well as in viscometric assays the ability of the carboxyl-terminal domain to sequester actin and to prevent F-actin formation was approximately two times higher than that of intact CAP. The sequestering activity of full length CAP could be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the activity of the carboxyl-terminal domain alone was not influenced, suggesting that the amino-terminal half of the protein is required for the PIP2 modulation of the CAP function. In profilin-minus cells the CAP concentration is increased by approximately 73%, indicating that CAP may compensate some profilin functions in vivo. In migrating D. discoideum cells CAP was enriched at anterior and posterior plasma membrane regions. Only a weak staining of the cytoplasm was observed. In chemotactically stimulated cells the protein was very prominent in leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-regulated manner.

Full text

PDF
261

Images in this article

265Image
on p.265
267Image
on p.267
268Image
on p.268
269Image
on p.269

Selected References

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

  1. André E., Brink M., Gerisch G., Isenberg G., Noegel A., Schleicher M., Segall J. E., Wallraff E. A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis. J Cell Biol. 1989 Mar;108(3):985–995. doi: 10.1083/jcb.108.3.985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Carlier M. F., Pantaloni D. Actin assembly in response to extracellular signals: role of capping proteins, thymosin beta 4 and profilin. Semin Cell Biol. 1994 Jun;5(3):183–191. doi: 10.1006/scel.1994.1023. [DOI] [PubMed] [Google Scholar]
  4. Cooper J. A., Walker S. B., Pollard T. D. Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization. J Muscle Res Cell Motil. 1983 Apr;4(2):253–262. doi: 10.1007/BF00712034. [DOI] [PubMed] [Google Scholar]
  5. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Eichinger L., Schleicher M. Characterization of actin- and lipid-binding domains in severin, a Ca(2+)-dependent F-actin fragmenting protein. Biochemistry. 1992 May 26;31(20):4779–4787. doi: 10.1021/bi00135a006. [DOI] [PubMed] [Google Scholar]
  8. Fedor-Chaiken M., Deschenes R. J., Broach J. R. SRV2, a gene required for RAS activation of adenylate cyclase in yeast. Cell. 1990 Apr 20;61(2):329–340. doi: 10.1016/0092-8674(90)90813-t. [DOI] [PubMed] [Google Scholar]
  9. Fenger U., Hofmann M., Galliot B., Schaller H. C. The role of the cAMP pathway in mediating the effect of head activator on nerve-cell determination and differentiation in hydra. Mech Dev. 1994 Aug;47(2):115–125. doi: 10.1016/0925-4773(94)90084-1. [DOI] [PubMed] [Google Scholar]
  10. Field J., Nikawa J., Broek D., MacDonald B., Rodgers L., Wilson I. A., Lerner R. A., Wigler M. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol. 1988 May;8(5):2159–2165. doi: 10.1128/mcb.8.5.2159-2165.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Field J., Vojtek A., Ballester R., Bolger G., Colicelli J., Ferguson K., Gerst J., Kataoka T., Michaeli T., Powers S. Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein. Cell. 1990 Apr 20;61(2):319–327. doi: 10.1016/0092-8674(90)90812-s. [DOI] [PubMed] [Google Scholar]
  12. Freeman N. L., Chen Z., Horenstein J., Weber A., Field J. An actin monomer binding activity localizes to the carboxyl-terminal half of the Saccharomyces cerevisiae cyclase-associated protein. J Biol Chem. 1995 Mar 10;270(10):5680–5685. doi: 10.1074/jbc.270.10.5680. [DOI] [PubMed] [Google Scholar]
  13. Gerisch G., Hülser D., Malchow D., Wick U. Cell communication by periodic cyclic-AMP pulses. Philos Trans R Soc Lond B Biol Sci. 1975 Nov 6;272(915):181–192. doi: 10.1098/rstb.1975.0080. [DOI] [PubMed] [Google Scholar]
  14. Gerisch G., Keller H. U. Chemotactic reorientation of granulocytes stimulated with micropipettes containing fMet-Leu-Phe. J Cell Sci. 1981 Dec;52:1–10. doi: 10.1242/jcs.52.1.1. [DOI] [PubMed] [Google Scholar]
  15. Gerst J. E., Ferguson K., Vojtek A., Wigler M., Field J. CAP is a bifunctional component of the Saccharomyces cerevisiae adenylyl cyclase complex. Mol Cell Biol. 1991 Mar;11(3):1248–1257. doi: 10.1128/mcb.11.3.1248-1257.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gerst J. E., Rodgers L., Riggs M., Wigler M. SNC1, a yeast homolog of the synaptic vesicle-associated membrane protein/synaptobrevin gene family: genetic interactions with the RAS and CAP genes. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4338–4342. doi: 10.1073/pnas.89.10.4338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gieselmann R., Mann K. ASP-56, a new actin sequestering protein from pig platelets with homology to CAP, an adenylate cyclase-associated protein from yeast. FEBS Lett. 1992 Feb 24;298(2-3):149–153. doi: 10.1016/0014-5793(92)80043-g. [DOI] [PubMed] [Google Scholar]
  18. Goldschmidt-Clermont P. J., Janmey P. A. Profilin, a weak CAP for actin and RAS. Cell. 1991 Aug 9;66(3):419–421. doi: 10.1016/0092-8674(81)90002-7. [DOI] [PubMed] [Google Scholar]
  19. Haugwitz M., Noegel A. A., Karakesisoglou J., Schleicher M. Dictyostelium amoebae that lack G-actin-sequestering profilins show defects in F-actin content, cytokinesis, and development. Cell. 1994 Oct 21;79(2):303–314. doi: 10.1016/0092-8674(94)90199-6. [DOI] [PubMed] [Google Scholar]
  20. Haugwitz M., Noegel A. A., Rieger D., Lottspeich F., Schleicher M. Dictyostelium discoideum contains two profilin isoforms that differ in structure and function. J Cell Sci. 1991 Nov;100(Pt 3):481–489. doi: 10.1242/jcs.100.3.481. [DOI] [PubMed] [Google Scholar]
  21. Haus U., Hartmann H., Trommler P., Noegel A. A., Schleicher M. F-actin capping by cap32/34 requires heterodimeric conformation and can be inhibited with PIP2. Biochem Biophys Res Commun. 1991 Dec 16;181(2):833–839. doi: 10.1016/0006-291x(91)91265-e. [DOI] [PubMed] [Google Scholar]
  22. Hofmann A., Eichinger L., André E., Rieger D., Schleicher M. Cap100, a novel phosphatidylinositol 4,5-bisphosphate-regulated protein that caps actin filaments but does not nucleate actin assembly. Cell Motil Cytoskeleton. 1992;23(2):133–144. doi: 10.1002/cm.970230206. [DOI] [PubMed] [Google Scholar]
  23. Jungbluth A., von Arnim V., Biegelmann E., Humbel B., Schweiger A., Gerisch G. Strong increase in the tyrosine phosphorylation of actin upon inhibition of oxidative phosphorylation: correlation with reversible rearrangements in the actin skeleton of Dictyostelium cells. J Cell Sci. 1994 Jan;107(Pt 1):117–125. doi: 10.1242/jcs.107.1.117. [DOI] [PubMed] [Google Scholar]
  24. Kawamukai M., Gerst J., Field J., Riggs M., Rodgers L., Wigler M., Young D. Genetic and biochemical analysis of the adenylyl cyclase-associated protein, cap, in Schizosaccharomyces pombe. Mol Biol Cell. 1992 Feb;3(2):167–180. doi: 10.1091/mbc.3.2.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kerkut G. A., Oliver G., Rick J. T., Walker R. J. Biochemical changes during learning in an insect ganglion. Nature. 1970 Aug 15;227(5259):722–723. doi: 10.1038/227722a0. [DOI] [PubMed] [Google Scholar]
  26. Kouyama T., Mihashi K. Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labelled F-actin. Local structural change of actin protomer both on polymerization and on binding of heavy meromyosin. Eur J Biochem. 1981;114(1):33–38. [PubMed] [Google Scholar]
  27. Kreitmeier M., Gerisch G., Heizer C., Müller-Taubenberger A. A talin homologue of Dictyostelium rapidly assembles at the leading edge of cells in response to chemoattractant. J Cell Biol. 1995 Apr;129(1):179–188. doi: 10.1083/jcb.129.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lacombe M. L., Podgorski G. J., Franke J., Kessin R. H. Molecular cloning and developmental expression of the cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum. J Biol Chem. 1986 Dec 25;261(36):16811–16817. [PubMed] [Google Scholar]
  29. Langanger G., De Mey J., Adam H. 1,4-Diazobizyklo-[2,2,2]-Oktan (DABCO) verzögert das Ausbleichen von Immunfluoreszenzpräparaten. Mikroskopie. 1983 Sep;40(7-8):237–241. [PubMed] [Google Scholar]
  30. Lee S., Li M., Pollard T. D. Evaluation of the binding of Acanthamoeba profilin to pyrene-labeled actin by fluorescence enhancement. Anal Biochem. 1988 Jan;168(1):148–155. doi: 10.1016/0003-2697(88)90022-x. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. MacLean-Fletcher S. D., Pollard T. D. Viscometric analysis of the gelation of Acanthamoeba extracts and purification of two gelation factors. J Cell Biol. 1980 May;85(2):414–428. doi: 10.1083/jcb.85.2.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Malchow D., Nägele B., Schwarz H., Gerisch G. Membrane-bound cyclic AMP phosphodiesterase in chemotactically responding cells of Dictyostelium discoideum. Eur J Biochem. 1972 Jun 23;28(1):136–142. doi: 10.1111/j.1432-1033.1972.tb01894.x. [DOI] [PubMed] [Google Scholar]
  34. Matviw H., Yu G., Young D. Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins. Mol Cell Biol. 1992 Nov;12(11):5033–5040. doi: 10.1128/mcb.12.11.5033-5040.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Noegel A., Metz B. A., Williams K. L. Developmentally regulated transcription of Dictyostelium discoideum plasmid Ddp1. EMBO J. 1985 Dec 30;4(13B):3797–3803. doi: 10.1002/j.1460-2075.1985.tb04150.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Ren R., Mayer B. J., Cicchetti P., Baltimore D. Identification of a ten-amino acid proline-rich SH3 binding site. Science. 1993 Feb 19;259(5098):1157–1161. doi: 10.1126/science.8438166. [DOI] [PubMed] [Google Scholar]
  38. Ridley A. J. Membrane ruffling and signal transduction. Bioessays. 1994 May;16(5):321–327. doi: 10.1002/bies.950160506. [DOI] [PubMed] [Google Scholar]
  39. Safer D., Nachmias V. T. Beta thymosins as actin binding peptides. Bioessays. 1994 Jul;16(7):473–479. doi: 10.1002/bies.950160706. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Schleicher M., Gerisch G., Isenberg G. New actin-binding proteins from Dictyostelium discoideum. EMBO J. 1984 Sep;3(9):2095–2100. doi: 10.1002/j.1460-2075.1984.tb02096.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Simpson P. A., Spudich J. A., Parham P. Monoclonal antibodies prepared against Dictyostelium actin: characterization and interactions with actin. J Cell Biol. 1984 Jul;99(1 Pt 1):287–295. doi: 10.1083/jcb.99.1.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  45. Stossel T. P. From signal to pseudopod. How cells control cytoplasmic actin assembly. J Biol Chem. 1989 Nov 5;264(31):18261–18264. [PubMed] [Google Scholar]
  46. Stossel T. P. On the crawling of animal cells. Science. 1993 May 21;260(5111):1086–1094. doi: 10.1126/science.8493552. [DOI] [PubMed] [Google Scholar]
  47. Theriot J. A., Mitchison T. J. The three faces of profilin. Cell. 1993 Dec 3;75(5):835–838. doi: 10.1016/0092-8674(93)90527-w. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Vojtek A. B., Cooper J. A. Identification and characterization of a cDNA encoding mouse CAP: a homolog of the yeast adenylyl cyclase associated protein. J Cell Sci. 1993 Jul;105(Pt 3):777–785. doi: 10.1242/jcs.105.3.777. [DOI] [PubMed] [Google Scholar]
  50. Vojtek A., Haarer B., Field J., Gerst J., Pollard T. D., Brown S., Wigler M. Evidence for a functional link between profilin and CAP in the yeast S. cerevisiae. Cell. 1991 Aug 9;66(3):497–505. doi: 10.1016/0092-8674(81)90013-1. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. Wegner A. Head to tail polymerization of actin. J Mol Biol. 1976 Nov;108(1):139–150. doi: 10.1016/s0022-2836(76)80100-3. [DOI] [PubMed] [Google Scholar]
  53. Weiner O. H., Murphy J., Griffiths G., Schleicher M., Noegel A. A. The actin-binding protein comitin (p24) is a component of the Golgi apparatus. J Cell Biol. 1993 Oct;123(1):23–34. doi: 10.1083/jcb.123.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Williamson M. P. The structure and function of proline-rich regions in proteins. Biochem J. 1994 Jan 15;297(Pt 2):249–260. doi: 10.1042/bj2970249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  56. Yin H. L., Janmey P. A., Schleicher M. Severin is a gelsolin prototype. FEBS Lett. 1990 May 7;264(1):78–80. doi: 10.1016/0014-5793(90)80769-f. [DOI] [PubMed] [Google Scholar]
  57. Yu G., Swiston J., Young D. Comparison of human CAP and CAP2, homologs of the yeast adenylyl cyclase-associated proteins. J Cell Sci. 1994 Jun;107(Pt 6):1671–1678. doi: 10.1242/jcs.107.6.1671. [DOI] [PubMed] [Google Scholar]
  58. Zelicof A., Gatica J., Gerst J. E. Molecular cloning and characterization of a rat homolog of CAP, the adenylyl cyclase-associated protein from Saccharomyces cerevisiae. J Biol Chem. 1993 Jun 25;268(18):13448–13453. [PubMed] [Google Scholar]

Articles from Molecular Biology of the Cell are provided here courtesy of American Society for Cell Biology

RESOURCES