Hostname: page-component-78c5997874-mlc7c Total loading time: 0 Render date: 2024-11-10T14:58:20.618Z Has data issue: false hasContentIssue false

Tight binding between a pool of the heterodimeric α/β tubulin and a protein kinase CK2 in Trypanosoma cruzi epimastigotes

Published online by Cambridge University Press:  07 December 2005

A. R. DE LIMA
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Caracas 1081-A, Venezuela Laboratorio de Protozoología, Centro BioMolP, Facultad de Ciencias de la Salud, Universidad de Carabobo, Valencia, Venezuela
R. MEDINA
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Caracas 1081-A, Venezuela
G. L. UZCANGA
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Caracas 1081-A, Venezuela
K. NORIS SUÁREZ
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Caracas 1081-A, Venezuela
V. T. CONTRERAS
Affiliation:
Laboratorio de Protozoología, Centro BioMolP, Facultad de Ciencias de la Salud, Universidad de Carabobo, Valencia, Venezuela
M. C. NAVARRO
Affiliation:
Laboratorio de Protozoología, Centro BioMolP, Facultad de Ciencias de la Salud, Universidad de Carabobo, Valencia, Venezuela
R. ARTEAGA
Affiliation:
Laboratorio de Protozoología, Centro BioMolP, Facultad de Ciencias de la Salud, Universidad de Carabobo, Valencia, Venezuela
J. BUBIS
Affiliation:
Departamento de Biología Celular, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Caracas 1081-A, Venezuela

Abstract

Tubulin is the predominant phosphoprotein in Trypanosoma cruzi epimastigotes and is phosphorylated by a protein kinase CK2. Interestingly, the presence or absence of divalent cations affected the solubilization of a pool of the parasite tubulin and the CK2 responsible for its phosphorylation. This fraction of tubulin and its kinase co-eluted using phosphocellulose, DEAE-Sepharose and Sephacryl S-300 chromatographies. Anti-α tubulin antibodies co-immunoprecipitated both tubulin and the CK2 responsible for its phosphorylation, and anti-CK2 α-subunit antibodies immunoprecipitated radioactively labelled α and β tubulin from phosphorylated epimastigote homogenates. Additionally, native polyacrylamide gel electrophoresis of the purified and radioactively labelled fraction containing tubulin and its kinase demonstrated the phosphorylation of a unique band that reacted with both anti-CK2 α-subunit and anti-tubulin antibodies. Together, these results establish a strong interaction between a pool of the heterodimeric α/β tubulin and a CK2 in this parasite. Hydrodynamic measurements indicated that the T. cruzi tubulin-CK2 complex is globular with an estimated size of 145·4–147·5 kDa.

Type
Research Article
Copyright
2005 Cambridge University Press

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Benaim, G., Losada, S., Gadelha, F. R. and Docampo, R. ( 1991). A calmodulin-activated (Ca2+-Mg2+) ATPase is involved in Ca2+ transport by plasma membrane vesicles from Trypanosoma cruzi. The Biochemical Journal 280, 715720.CrossRefGoogle Scholar
Bhatia, A., Sanyal, R., Paramchuk, W. and Gedamu, L. ( 1998). Isolation, characterization and disruption of the casein kinase II alpha subunit gene of Leishmania chagasi. Molecular and Biochemical Parasitology 92, 195206. DOI: 10.1016/S0166-6851(98)00002-4.CrossRefGoogle Scholar
Bloomfield, V., Dalton, W. O. and Van Holde, K. E. ( 1967). Frictional coefficients of multisubunit structures. I. Theory. Biopolymers 5, 135148.Google Scholar
Bradford, M. M. ( 1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248254.CrossRefGoogle Scholar
Burleigh, B. A. and Andrews, N. W. ( 1995). The mechanisms of Trypanosoma cruzi invasion of mammalian cells. Annual Review of Microbiology 49, 175200. DOI: 10.1146/annurev.mi.49.100195.001135.CrossRefGoogle Scholar
Calabokis, M., Kurz, L., Gonzatti, M. I. and Bubis, J. ( 2003). Protein Kinase CK1 from Trypanosoma cruzi. Journal of Protein Chemistry 22, 591599.CrossRefGoogle Scholar
Calabokis, M., Kurz, L., Wilkesman, J., Galán-Caridad, J. M., Möller, C., Gonzatti, M. I. and Bubis, J. ( 2002). Biochemical and enzymatic characterization of a partially purified casein kinase-1 like activity from Trypanosoma cruzi. Parasitology International 51, 2539. DOI: 10.1016/S1383-5769(01)00104-0.CrossRefGoogle Scholar
Camargo, E. P. ( 1964). Growth and differentiation in Trypanosoma cruzi. I. Origin of metacyclic trypanosomes in liquid media. Revista do Instituto de Medicina Tropical de São Paulo 6, 93100.Google Scholar
Casas, B., Calabokis, M., Kurz, L., Galán-Caridad, J. M., Bubis, J. and Gonzatti, M. I. ( 2002). Trypanosoma cruzi: in vitro phosphorylation of tubulin by a protein kinase CK2-like enzyme. Experimental Parasitology 101, 129137. DOI: 10.1016/S0014-4894(02)00110-8.CrossRefGoogle Scholar
Chapin, S. J. and Bulinski, J. C. ( 1991). Preparation and functional assay of pure populations of tyrosinated and detyrosinated tubulin. Methods in Enzymology 196, 254264.CrossRefGoogle Scholar
Chin, D. and Means, A. R. ( 2000). Calmodulin: a prototypical calcium sensor. Trends in Cell Biology 10, 322328. DOI: 10.1016/S0962-8924(00)01800-6.CrossRefGoogle Scholar
Crute, B. E. and Van Buskirk, R. G. ( 1992). A casein kinase-like kinase phosphorylates beta-tubulin and may be a microtubule-associated protein. Journal of Neurochemistry 59, 20172023.Google Scholar
De Moreno, M. R., Smith, J. F. and Smith, R. V. ( 1986). Mechanism studies of coomassie blue and silver staining of proteins. Journal of Pharmaceutical Sciences 75, 907911.CrossRefGoogle Scholar
Díaz-Nido, J., Armas-Portela, R. and Avila, J. ( 1992). Increase in cytoplasmic casein kinase II-type activity accompanies neurite outgrowth after DNA synthesis inhibition in NIA-103 neuroblastoma cells. Journal of Neurochemistry 58, 18201828.CrossRefGoogle Scholar
Diaz-Nido, J., Serrano, L., Mendez, E. and Avila, J. ( 1988). A casein-kinase II-related activity is involved in phosphorylation of microtubule-associated protein MAP-1B during neuroblastoma cell differentiation. Journal of Cell Biology 106, 20572065.CrossRefGoogle Scholar
Dolan, M. T., Reid, C. G. and Voorheis, H. P. ( 1986). Calcium ions initiate the selective depolymerization of the pellicular microtubules in bloodstream forms of Trypanosoma brucei. Journal of Cell Science 80, 123140.Google Scholar
El-Sayed, N. M., Myler, P. J., Bartholomeu, D. C., Nilsson, D., Aggarwal, G., Tran, A.-N., Ghedin, E., Worthey, E. A., Delcher, A. L., Blandin, G., Westenberger, S. J., Caler, E., Cerqueira, G. C., Branche, C., Haas, B., Anapuma, A., Arner, E., Aslund, L., Attipoe, P., Bontempi, E., Bringaud, F., Burton, P., Cadag, E., Campbell, D. A., Carrington, M., Crabtree, J., Darban, H., da Silveira, J. F., de Jong, P., Edwards, K., Englund, P. T., Fazelina, G., Feldblyum, T., Ferella, M., Frasch, A. C., Gull, K., Horn, D., Hou, L., Huang, Y., Kindlund, E., Klingbeil, M., Kluge, S., Koo, H., Lacerda, D., Levin, M. J., Lorenzi, H., Louie, T., Machado, C. R., McCulloch, R., McKenna, A., Mizuno, Y., Mottram, J. C., Nelson, S., Ochaya, S., Osoegawa, K., Pai, G., Parsons, M., Pentony, M., Petterss, U., Pop, M., Ramirez, J. L., Rinta, J., Robertson, L., Salzberg, S. L., Sanchez, D. O., Seyler, A., Sharma, R., Shetty, J., Simpson, A. J., Sisk, E., Tammi, M. T., Tarleton, R., Teixeira, S., Van Aken, S., Vogt, C., Ward, P. N., Wickstead, B., Wortman, J., White, O., Frasere, C. M., Stuart, K. D. and Andersson, B. ( 2005). The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease. Science 309, 409415. DOI: 10.1126/science.1112631.CrossRefGoogle Scholar
Faust, M. and Montenarch, M. ( 2000). Subcellular localization of protein kinase CK2. A key to its function? Cell Tissue Research 301, 329340. DOI: 10.1007/s004410000256.CrossRefGoogle Scholar
Faust, M., Schuster, N. and Montenarh, M. ( 1999). Specific binding of protein kinase CK2 catalytic subunits to tubulin. FEBS Letters 462, 5156. DOI: 10.1016/S0014-5793(99)01492-1.CrossRefGoogle Scholar
Galán-Caridad, J. M., Calabokis, M., Uzcanga, G., Aponte, F. and Bubis, J. ( 2004). Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi. Memorias do Instituto Oswaldo Cruz 99, 845854.CrossRefGoogle Scholar
Goldenring, J. R., Casanova, J. E. and Delorenzo, R. J. ( 1984). Tubulin-associated calmodulin-dependent kinase: evidence for an endogenous complex of tubulin with a calcium-calmodulin-dependent kinase. Journal of Neurochemistry 43, 16691679.CrossRefGoogle Scholar
Gull, K. ( 2001). The biology of kinetoplastid parasites: insights and challenges from genomics and post-genomics. International Journal for Parasitology 31, 443452. DOI: 10.1016/S0020-7519(01)00154-0.CrossRefGoogle Scholar
Hanna, D. E., Rethinaswamy, A. and Glower, C. V. C. ( 1995). Casein kinase II is required for cell cycle progression during G1 and G2/M in Saccharomyces cerevisiae. Journal of Biological Chemistry 270, 2590525914.CrossRefGoogle Scholar
Hathaway, G. M. and Traugh, J. A. ( 1979). Cyclic nucleotide-independent protein kinases from rabbit reticulocytes. Purification of casein kinases. Journal of Biological Chemistry 254, 762768.Google Scholar
Huang, L. J., Durick, K., Weiner, J. A., Chua, J. and Taylor, S. S. ( 1997). Identification of a novel protein kinase A anchoring protein that binds both type I and II regulatory subunits. Journal of Biological Chemistry 272, 80578064.CrossRefGoogle Scholar
Ikeda, M., Onda, T., Tomita, I. and Tomita, T. ( 1996). The differences in Ca2+-sensitivity of protein kinase C in platelets from Wistar Kyoto rat and stroke-prone spontaneously hypertensive rat. Thrombosis Research 82, 417427.CrossRefGoogle Scholar
Kohl, L. and Gull, K. ( 1998). Molecular architecture of the trypanosome cytoskeleton. Molecular and Biochemical Parasitology 93, 19.CrossRefGoogle Scholar
Laemmli, U. K. ( 1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, London 227, 680685.CrossRefGoogle Scholar
Lim, A. C., Tiu, S. Y., Li, Q. and Qi, R. Z. ( 2004). Direct regulation of microtubule dynamics by protein kinase CK2. Journal of Biological Chemistry 279, 44334439. DOI: 10.1074/jbc.M310563200.CrossRefGoogle Scholar
Litchfield, D. W. ( 2003). Protein Kinase CK2: structure, regulation and role in cellular decisions of life and death. The Biochemical Journal 369, 115. DOI: 10.1042/BJ20021469.CrossRefGoogle Scholar
Litchfield, D. W., Lozeman, F. J., Piening, C., Sommercorn, J., Takio, K., Walsh, K. A. and Krebs, E. G. ( 1990). Subunit structure of casein kinase II from bovine testis: demonstration that the α and α′ subunits are distinct polypeptides. Journal of Biological Chemistry 265, 76387644.Google Scholar
MacRae, T. H. ( 1997). Tubulin post-translational modifications. Enzymes and their mechanism of action. European Journal of Biochemistry 244, 265278.Google Scholar
Martin, R. and Ames, B. ( 1961). A method for determining the sedimentation behavior of enzymes: application to protein mixtures. Journal of Biological Chemistry 236, 13721379.Google Scholar
Meggio, F., Boldyreff, B., Issinger, O-G. and Pinna, L. A. ( 1994). Casein kinase 2 down-regulation and activation by polybasic peptides are mediated by acidic residues in the 55–64 region of the beta subunit. A study with calmodulin as phosphorylatable substrate. Biochemistry 33, 43364342.Google Scholar
Nastainczyk, W., Schmidt-Spaniol, I., Boldyreff, B. and Issinger, O. G. ( 1995). Isolation and characterization of a monoclonal anti-protein kinase CK2 β-subunit antibody of the IgG class for the direct detection of CK2 β-subunit in tissue cultures of various mammalian species and human tumors. Hybridoma 14, 335339.CrossRefGoogle Scholar
Nelson, N. C. and Taylor, S. S. ( 1981). Differential labeling and identification of the cysteine-containing tryptic peptides of catalytic subunit from porcine heart cAMP-dependent protein kinase. Journal of Biological Chemistry 256, 37433750.Google Scholar
Ogueta, S., Intosch, G. M. and Téllez-Iñon, M. T. ( 1996). Regulation of Ca2+/calmodulin-dependent protein kinase from Trypanosoma cruzi. Molecular and Biochemical Parasitology 78, 171183.CrossRefGoogle Scholar
Ogueta, S. B., Macintosch, G. C. and Téllez-Iñón, M. T. ( 1998). Stage-specific substrate phosphorylation by a Ca2+/calmodulin-dependent protein kinase in Trypanosoma cruzi. Journal of Eukaryotic Microbiology 45, 392396.CrossRefGoogle Scholar
Park, J. H., Brekken, D. L., Randall, A. C. and Parsons, M. ( 2002). Molecular cloning of Trypanosoma brucei CK2 catalytic subunits: the α isoform is nucleolar and phosphorylates the nuclear protein Nopp44/46. Molecular and Biochemical Parasitology 119, 97106. DOI: 10.1016/S0166-6851(01)00407-8.CrossRefGoogle Scholar
Pinna, L. A. and Meggio, F. ( 1997). Protein kinase CK2 (“casein kinase-2”) and its implication in cell division and proliferation. Progress Cell Cycle Research 3, 7797.CrossRefGoogle Scholar
Robinson, D., Beattie, P., Sherwin, T. and Gull, K. ( 1991). Microtubules, tubulin and microtubule associated proteins of trypanosomes. Methods in Enzymology 196, 285299.CrossRefGoogle Scholar
Schagger, H., Cramer, W. A. and Von Jagow, G. ( 1994). Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Annals of Biochemistry 217, 22030. DOI:10.1006/abio.1994.1112.CrossRefGoogle Scholar
Serrano, L., Diaz-Nido, J., Wandosell, F. and Avila, J. ( 1987). Tubulin phosphorylation by casein kinase II is similar to that found in vivo. Journal of Cell Biology 105, 17311739.CrossRefGoogle Scholar
Serrano, L., Hernández, M. A., Díaz-Nido, J. and Avila, J. ( 1989). Association of casein kinase with microtubules. Experimental Cell Research 181, 263272.CrossRefGoogle Scholar
Siegel, L. M. and Monty, K. J. ( 1966). Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. Biochimica et Biophysica Acta 112, 346362.Google Scholar
Towbin, H., Staehelin, T. and Gordon, J. ( 1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Biotechnology 24, 145149.CrossRefGoogle Scholar
Uzcanga, G., Galán-Caridad, J. M., Noris Suárez, K. and Bubis, J. ( 2003). Divalent cations hinder the solubilization of a tubulin kinase activity from Trypanosoma cruzi epimastigotes. Biological Research 36, 367379.CrossRefGoogle Scholar
Uzcanga, G., Mendoza, M., Aso, P. and Bubis, J. ( 2002). Purification of a 64 kDa antigen from Trypanosoma evansi that exhibits cross-reactivity with Trypanosoma vivax. Parasitology 124, 287299.CrossRefGoogle Scholar
Uzcanga, G., Perrone, T., Noda, J. A., Perez-Pazos, J., Medina, R., Hoebeke, J. and Bubis, J. ( 2004). Variant surface glycoprotein from Trypanosoma evansi is partially responsible for the cross-reaction between Trypanosoma evansi and Trypanosoma vivax. Biochemistry 43, 595606. DOI: 10.1021/bi0301946.CrossRefGoogle Scholar
Vaughan, S., Attwood, T., Navarro, M., Scott, V., McKean, P. and Gull, K. ( 2000). New tubulins in protozoal parasites. Current Biology 10, R258R259. DOI: 10.1016/S0960-9822(00)00414-0.CrossRefGoogle Scholar
Zhou, R., Shen, R. L., Pinto Da Silva, P. and Vande Woude, G. F. ( 1991). In vitro and in vivo characterization of pp39mos association with tubulin. Cell Growth and Differentiation 2, 257265.Google Scholar