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. 2004 Feb 15;378(Pt 1):63–72. doi: 10.1042/BJ20031147

Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi.

Maximiliano A D'Angelo 1, Santiago Sanguineti 1, Jeffrey M Reece 1, Lutz Birnbaumer 1, Héctor N Torres 1, Mirtha M Flawiá 1
PMCID: PMC1223918  PMID: 14556647

Abstract

Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites.

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Selected References

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

  1. Atienza J. M., Colicelli J. Yeast model system for study of mammalian phosphodiesterases. Methods. 1998 Jan;14(1):35–42. doi: 10.1006/meth.1997.0563. [DOI] [PubMed] [Google Scholar]
  2. Beard Matthew B., Huston Elaine, Campbell Lachlan, Gall Irene, McPhee Ian, Yarwood Stephen, Scotland Grant, Houslay Miles D. In addition to the SH3 binding region, multiple regions within the N-terminal noncatalytic portion of the cAMP-specific phosphodiesterase, PDE4A5, contribute to its intracellular targeting. Cell Signal. 2002 May;14(5):453–465. doi: 10.1016/s0898-6568(01)00264-9. [DOI] [PubMed] [Google Scholar]
  3. Boudreau R. J., Drummond G. I. A modified assay of 3':5'-cyclic-AMP phosphodiesterase. Anal Biochem. 1975 Feb;63(2):388–399. doi: 10.1016/0003-2697(75)90361-9. [DOI] [PubMed] [Google Scholar]
  4. Colicelli J., Birchmeier C., Michaeli T., O'Neill K., Riggs M., Wigler M. Isolation and characterization of a mammalian gene encoding a high-affinity cAMP phosphodiesterase. Proc Natl Acad Sci U S A. 1989 May;86(10):3599–3603. doi: 10.1073/pnas.86.10.3599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Colledge M., Scott J. D. AKAPs: from structure to function. Trends Cell Biol. 1999 Jun;9(6):216–221. doi: 10.1016/s0962-8924(99)01558-5. [DOI] [PubMed] [Google Scholar]
  6. D'Angelo Maximiliano A., Montagna Andrea E., Sanguineti Santiago, Torres Héctor N., Flawiá Mirtha M. A novel calcium-stimulated adenylyl cyclase from Trypanosoma cruzi, which interacts with the structural flagellar protein paraflagellar rod. J Biol Chem. 2002 Jul 16;277(38):35025–35034. doi: 10.1074/jbc.M204696200. [DOI] [PubMed] [Google Scholar]
  7. Ersfeld K., Gull K. Targeting of cytoskeletal proteins to the flagellum of Trypanosoma brucei. J Cell Sci. 2001 Jan;114(Pt 1):141–148. doi: 10.1242/jcs.114.1.141. [DOI] [PubMed] [Google Scholar]
  8. Flawiá M. M., Téllez-Iñn M. T., Torres H. N. Signal transduction mechanisms in Trypanosoma cruzi. Parasitol Today. 1997 Jan;13(1):30–33. doi: 10.1016/s0169-4758(96)10070-3. [DOI] [PubMed] [Google Scholar]
  9. Florio S. K., Prusti R. K., Beavo J. A. Solubilization of membrane-bound rod phosphodiesterase by the rod phosphodiesterase recombinant delta subunit. J Biol Chem. 1996 Sep 27;271(39):24036–24047. doi: 10.1074/jbc.271.39.24036. [DOI] [PubMed] [Google Scholar]
  10. Fraidenraich D., Peña C., Isola E. L., Lammel E. M., Coso O., Añel A. D., Pongor S., Baralle F., Torres H. N., Flawia M. M. Stimulation of Trypanosoma cruzi adenylyl cyclase by an alpha D-globin fragment from Triatoma hindgut: effect on differentiation of epimastigote to trypomastigote forms. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10140–10144. doi: 10.1073/pnas.90.21.10140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gietz R. D., Schiestl R. H. Applications of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier. Yeast. 1991 Apr;7(3):253–263. doi: 10.1002/yea.320070307. [DOI] [PubMed] [Google Scholar]
  12. Godsel L. M., Engman D. M. Flagellar protein localization mediated by a calcium-myristoyl/palmitoyl switch mechanism. EMBO J. 1999 Apr 15;18(8):2057–2065. doi: 10.1093/emboj/18.8.2057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gong K. W., Kunz S., Zoraghi R., Kunz Renggli C., Brun R., Seebeck T. cAMP-specific phosphodiesterase TbPDE1 is not essential in Trypanosoma brucei in culture or during midgut infection of tsetse flies. Mol Biochem Parasitol. 2001 Sep 3;116(2):229–232. doi: 10.1016/s0166-6851(01)00315-2. [DOI] [PubMed] [Google Scholar]
  14. Gómez M. L., Erijman L., Arauzo S., Torres H. N., Téllez-Iñn M. T. Protein kinase C in Trypanosoma cruzi epimastigote forms: partial purification and characterization. Mol Biochem Parasitol. 1989 Sep;36(2):101–108. doi: 10.1016/0166-6851(89)90182-5. [DOI] [PubMed] [Google Scholar]
  15. Henras A., Henry Y., Bousquet-Antonelli C., Noaillac-Depeyre J., Gélugne J. P., Caizergues-Ferrer M. Nhp2p and Nop10p are essential for the function of H/ACA snoRNPs. EMBO J. 1998 Dec 1;17(23):7078–7090. doi: 10.1093/emboj/17.23.7078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Houslay M. D., Milligan G. Tailoring cAMP-signalling responses through isoform multiplicity. Trends Biochem Sci. 1997 Jun;22(6):217–224. doi: 10.1016/s0968-0004(97)01050-5. [DOI] [PubMed] [Google Scholar]
  17. Jin S. L., Bushnik T., Lan L., Conti M. Subcellular localization of rolipram-sensitive, cAMP-specific phosphodiesterases. Differential targeting and activation of the splicing variants derived from the PDE4D gene. J Biol Chem. 1998 Jul 31;273(31):19672–19678. doi: 10.1074/jbc.273.31.19672. [DOI] [PubMed] [Google Scholar]
  18. Kanacher Tobias, Schultz Anita, Linder Jürgen U., Schultz Joachim E. A GAF-domain-regulated adenylyl cyclase from Anabaena is a self-activating cAMP switch. EMBO J. 2002 Jul 15;21(14):3672–3680. doi: 10.1093/emboj/cdf375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Li N., Florio S. K., Pettenati M. J., Rao P. N., Beavo J. A., Baehr W. Characterization of human and mouse rod cGMP phosphodiesterase delta subunit (PDE6D) and chromosomal localization of the human gene. Genomics. 1998 Apr 1;49(1):76–82. doi: 10.1006/geno.1998.5210. [DOI] [PubMed] [Google Scholar]
  21. Ma P., Wera S., Van Dijck P., Thevelein J. M. The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling. Mol Biol Cell. 1999 Jan;10(1):91–104. doi: 10.1091/mbc.10.1.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Martinez Sergio E., Wu Albert Y., Glavas Natalie A., Tang Xiao-Bo, Turley Stewart, Hol Wim G. J., Beavo Joseph A. The two GAF domains in phosphodiesterase 2A have distinct roles in dimerization and in cGMP binding. Proc Natl Acad Sci U S A. 2002 Sep 23;99(20):13260–13265. doi: 10.1073/pnas.192374899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mehats Celine, Andersen Carsten B., Filopanti Marcello, Jin S. L. Catherine, Conti Marco. Cyclic nucleotide phosphodiesterases and their role in endocrine cell signaling. Trends Endocrinol Metab. 2002 Jan-Feb;13(1):29–35. doi: 10.1016/s1043-2760(01)00523-9. [DOI] [PubMed] [Google Scholar]
  24. Muradov Khakim G., Boyd Kimberly K., Martinez Sergio E., Beavo Joseph A., Artemyev Nikolai O. The GAFa domains of rod cGMP-phosphodiesterase 6 determine the selectivity of the enzyme dimerization. J Biol Chem. 2003 Jan 16;278(12):10594–10601. doi: 10.1074/jbc.M208456200. [DOI] [PubMed] [Google Scholar]
  25. Naula C., Seebeck T. Cyclic AMP signaling in trypanosomatids. Parasitol Today. 2000 Jan;16(1):35–38. doi: 10.1016/s0169-4758(99)01582-3. [DOI] [PubMed] [Google Scholar]
  26. Ouaissi A., Cornette J., Schöneck R., Plumas-Marty B., Taibi A., Loyens M., Capron A. Fibronectin cleavage fragments provide a growth factor-like activity for the differentiation of Trypanosoma cruzi trypomastigotes to amastigotes. Eur J Cell Biol. 1992 Oct;59(1):68–79. [PubMed] [Google Scholar]
  27. Paindavoine P., Rolin S., Van Assel S., Geuskens M., Jauniaux J. C., Dinsart C., Huet G., Pays E. A gene from the variant surface glycoprotein expression site encodes one of several transmembrane adenylate cyclases located on the flagellum of Trypanosoma brucei. Mol Cell Biol. 1992 Mar;12(3):1218–1225. doi: 10.1128/mcb.12.3.1218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pereira C. A., Alonso G. D., Paveto M. C., Iribarren A., Cabanas M. L., Torres H. N., Flawiá M. M. Trypanosoma cruzi arginine kinase characterization and cloning. A novel energetic pathway in protozoan parasites. J Biol Chem. 2000 Jan 14;275(2):1495–1501. doi: 10.1074/jbc.275.2.1495. [DOI] [PubMed] [Google Scholar]
  29. Pogacić V., Dragon F., Filipowicz W. Human H/ACA small nucleolar RNPs and telomerase share evolutionarily conserved proteins NHP2 and NOP10. Mol Cell Biol. 2000 Dec;20(23):9028–9040. doi: 10.1128/mcb.20.23.9028-9040.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rangel-Aldao R., Triana F., Fernández V., Comach G., Abate T., Montoreano R. Cyclic AMP as an inducer of the cell differentiation of Trypanosoma cruzi. Biochem Int. 1988 Aug;17(2):337–344. [PubMed] [Google Scholar]
  31. Rascón A., Viloria M. E., De-Chiara L., Dubra M. E. Characterization of cyclic AMP phosphodiesterases in Leishmania mexicana and purification of a soluble form. Mol Biochem Parasitol. 2000 Mar 5;106(2):283–292. doi: 10.1016/s0166-6851(99)00224-8. [DOI] [PubMed] [Google Scholar]
  32. Rascón Ana, Soderling Scott H., Schaefer Jonathan B., Beavo Joseph A. Cloning and characterization of a cAMP-specific phosphodiesterase (TbPDE2B) from Trypanosoma brucei. Proc Natl Acad Sci U S A. 2002 Apr 2;99(7):4714–4719. doi: 10.1073/pnas.002031599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rich T. C., Fagan K. A., Tse T. E., Schaack J., Cooper D. M., Karpen J. W. A uniform extracellular stimulus triggers distinct cAMP signals in different compartments of a simple cell. Proc Natl Acad Sci U S A. 2001 Oct 16;98(23):13049–13054. doi: 10.1073/pnas.221381398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schwartz J. H. The many dimensions of cAMP signaling. Proc Natl Acad Sci U S A. 2001 Nov 20;98(24):13482–13484. doi: 10.1073/pnas.251533998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Seebeck T., Gong K., Kunz S., Schaub R., Shalaby T., Zoraghi R. cAMP signalling in Trypanosoma brucei. Int J Parasitol. 2001 May 1;31(5-6):491–498. doi: 10.1016/s0020-7519(01)00164-3. [DOI] [PubMed] [Google Scholar]
  36. Shakur Y., Takeda K., Kenan Y., Yu Z. X., Rena G., Brandt D., Houslay M. D., Degerman E., Ferrans V. J., Manganiello V. C. Membrane localization of cyclic nucleotide phosphodiesterase 3 (PDE3). Two N-terminal domains are required for the efficient targeting to, and association of, PDE3 with endoplasmic reticulum. J Biol Chem. 2000 Dec 8;275(49):38749–38761. doi: 10.1074/jbc.M001734200. [DOI] [PubMed] [Google Scholar]
  37. Taylor M. C., Muhia D. K., Baker D. A., Mondragon A., Schaap P. B., Kelly J. M. Trypanosoma cruzi adenylyl cyclase is encoded by a complex multigene family. Mol Biochem Parasitol. 1999 Nov 30;104(2):205–217. doi: 10.1016/s0166-6851(99)00154-1. [DOI] [PubMed] [Google Scholar]
  38. Torphy T. J. Phosphodiesterase isozymes: molecular targets for novel antiasthma agents. Am J Respir Crit Care Med. 1998 Feb;157(2):351–370. doi: 10.1164/ajrccm.157.2.9708012. [DOI] [PubMed] [Google Scholar]
  39. Téllez-Iñn M. T., Ulloa R. M., Torruella M., Torres H. N. Calmodulin and Ca2+-dependent cyclic AMP phosphodiesterase activity in Trypanosoma cruzi. Mol Biochem Parasitol. 1985 Nov;17(2):143–153. doi: 10.1016/0166-6851(85)90013-1. [DOI] [PubMed] [Google Scholar]
  40. Walter R. D. 3':5'-cyclic-AMP phosphodiesterase from Trypanosoma gambiense. Hoppe Seylers Z Physiol Chem. 1974 Nov;355(11):1443–1450. doi: 10.1515/bchm2.1974.355.2.1443. [DOI] [PubMed] [Google Scholar]
  41. Walter R. D., Opperdoes F. R. Subcellular distribution of adenylate cyclase, cyclic-AMP phosphodiesterase, protein kinases and phosphoprotein phosphatase in Trypanosoma brucei. Mol Biochem Parasitol. 1982 Nov;6(5):287–295. doi: 10.1016/0166-6851(82)90061-5. [DOI] [PubMed] [Google Scholar]
  42. Wechsler Jeremy, Choi Young-Hun, Krall Judith, Ahmad Faiyaz, Manganiello Vincent C., Movsesian Matthew A. Isoforms of cyclic nucleotide phosphodiesterase PDE3A in cardiac myocytes. J Biol Chem. 2002 Aug 1;277(41):38072–38078. doi: 10.1074/jbc.M203647200. [DOI] [PubMed] [Google Scholar]
  43. Zaccolo Manuela, Magalhães Paulo, Pozzan Tullio. Compartmentalisation of cAMP and Ca(2+) signals. Curr Opin Cell Biol. 2002 Apr;14(2):160–166. doi: 10.1016/s0955-0674(02)00316-2. [DOI] [PubMed] [Google Scholar]
  44. Zoraghi R., Kunz S., Gong K., Seebeck T. Characterization of TbPDE2A, a novel cyclic nucleotide-specific phosphodiesterase from the protozoan parasite Trypanosoma brucei. J Biol Chem. 2000 Dec 27;276(15):11559–11566. doi: 10.1074/jbc.M005419200. [DOI] [PubMed] [Google Scholar]
  45. Zoraghi Roya, Seebeck Thomas. The cAMP-specific phosphodiesterase TbPDE2C is an essential enzyme in bloodstream form Trypanosoma brucei. Proc Natl Acad Sci U S A. 2002 Apr 2;99(7):4343–4348. doi: 10.1073/pnas.062716599. [DOI] [PMC free article] [PubMed] [Google Scholar]

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