A protein kinase (PK) was partially purified from NaCl extracts of the cell surface complex of Euglena using DEAE-cellulose chromatography. Tubulins extracted either from flagella or from the cell surface complexes of Euglena were readily phosphorylated when incubated with [γ-32P]-ATP and the PK. Protein kinase activity was augmented with 5 mM Mn2+ or Mg2+, and was inhibited or had greatly reduced activity with 5 mM Ca2+, Co2+, Cu2+ or Zn2+. Incorporation was much lower when [γ-32P]-GTP was the phosphate donor. Serine and threonine were the major radiolabeled phosphoamino acids in tubulins; label was also found in phosphotyrosine. Alpha-tubulin solubilized from flagella was a relatively poor substrate for the PK, but a Euglena α-tubulin cDNA overexpressed as a Trx-fusion protein incorporated [γ-32P]-ATP into serine and threonine when incubated with cell surface extracts. Alpha- and β-tubulins from cell surface complexes were equally good substrates for the PK. No incorporation was observed in intact microtubules either from the cell surface complex or from isolated flagella. In-gel assays identified a polypeptide of about 30 kDa that phosphorylated tubulins in extracts of both flagella and the cell surface complexes, and dephosphorylated casein was a competitive substrate for the partially purified kinase. In vivo incubation with [32P]-orthophosphate produced numerous radiolabeled bands in acrylamide gels of NaCl extracts of the cell surface complex, but none of these bands could be positively related to tubulins extracted from surface complex microtubules.46295104Bloodgood, R.A., Directed movements of ciliary and flagellar membrane components (1992) Biol. Cell, 76, pp. 291-301Bloodgood, R.A., Salamonsky, N.L., Phosphorylation of Chlamydomonas flagellar proteins (1995) Methods in Cell Biology, 47, pp. 121-127. , Dentier, W. & Witman, G. (ed.), Academic Press, New YorkCarlier, M.F., Dynamic Actin (1993) Curr. Biol., 3, pp. 321-323Cramer, M.L., Myers, J., Growth and photosynthetic characteristics of Euglena gracilis (1952) Arch. Mikrobiol., 17, pp. 384-402Diaz-Nido, J., Serrano, L., Lopez-Otin, C., Vandekerckhove, J., Avila, J., Phosphorylation of a neuronal β-tubulin isotype (1990) J Biol. Chem., 265, pp. 13949-13954Drewes, G., Ebneth, A., Preuss, U., Mandelkow, E.M., Mandelkow, E., MARK, a novel family of protein kinases that phosphorylate microtubule-associated proteins and trigger microtubule disruption (1997) Cell, 89, pp. 297-308Dubreuil, R.R., Bouck, G.B., The membrane skeleton of a unicellular organism consists of bridged, articulating strips (1985) J. Cell Biol., 101, pp. 1884-1896Dubreuil, R.R., Rosiere, T.K., Rosner, M.C., Bouck, G.B., Properties and topography of the major integral plasma membrane protein of a unicellular organism (1988) J. Cell Biol., 107, pp. 191-200Eddé, B., Rossier, J., Le Caer, J.P., Desbruyrès, E., Gros, F., Denoulet, P., Posttranslational glutamylation of α-tubulin (1990) Science, 247, pp. 83-85Edelman, A.M., Blumenthal, D.K., Krebs, E.G., Protein serine/ threonine kinases (1987) Ann. Rev. Biochem., 56, pp. 567-613Fazio, M.J., Marrs, J.A., Bouck, G.B., Protein kinases in protists (1994) Protoplasma, 181, pp. 283-290Fazio, M.J., Da Silva, A.C., Rosiere, R.K., Bouck, G.B., Membrane skeletal proteins are targets for tyrosine and threonine kinases in Euglena (1995) J. Euk. Microbiol., 42, pp. 570-580Fosket, D.E., Morejohn, L.C., Structural and functional organization of tubulin (1992) Ann. Rev. Plant Mol. Biol., 43, pp. 201-240Gard, D.L., Kirchner, M.W., A polymer-dependent increase in phosphorylation of β-tubulin accompanies differentiation of a mouse neuroblastoma cell line (1985) J. Cell Biol., 100, pp. 764-774Goode, B.L., Denis, P.E., Panda, D., Radeke, M.J., Miller, H.P., Wilson, L., Feinstein, S.C., Functional interactions between the proline-rich and repeat regions of tau enhanced microtubule binding and assembly (1997) Mol. Biol. Cell, 8, pp. 353-365Grandics, P., Miller, A., Schmidt, T.J., Mittman, D., Litwack, G., Purification of the unactivated glucocorticoid receptor and its subsequent in vitro activation (1984) J. Biol. Chem., 259, pp. 3173-3180Granger, B.L., Lazarides, E., Membrane skeleton protein 4.1 of avian erythrocytes is composed of multiple variants that exhibit tissue-specific expression (1984) Cell, 37, pp. 595-607Greer, K., Rosenbaum, J.L., Post-translational modifications of tubulin (1989) Cell Movements, 2, pp. 47-66. , Warner, J. D. & McIntosh, J. R. (ed.), Alan R. Liss, Inc., New YorkGurland, G., Gundersen, G.G., Protein phosphatase inhibitors induce the selective breakdown of stable microtubules in fibroblasts and epithelial cells (1993) Proc. Natl. Acad. Sci. (USA), 90, pp. 8827-8831Hasegawa, M., Morishima-Kawashima, M., Takio, K., Suzuki, M., Titani, K., Ihara, Y., Protein sequence and mass spectrometric analysis of tau in the Alzheimer's disease brain (1992) J. Biol. Chem., 267, pp. 17047-17054Hathaway, G.M., Traugh, J.A., Casein kinase II (1983) Methods in Enzymology, 99, pp. 317-331Hirano-Ohnishi, J., Watanabe, Y., Ca2+/calmodulin-dependent phosphorylation of ciliary β-tubulin in Tetrahymena (1989) J. Biochem., 105, pp. 858-860. , TokyoHoekstra, M.F., Dhillon, N., Carmel, G., DeMaggio, A.J., Lindberg, R.A., Hunter, T., Kuret, J., Budding and fission yeast casein kinase I isoforms have dual-specificity protein kinase actvity (1994) Mol. Biol. Cell, 5, pp. 877-886Jacobs, M., Smith, H., Taylor, E.W., Tubulin: Nucleotide binding and enzyme activity (1974) J. Mol. Biol., 89, pp. 455-468Kameshita, I., Fujisawa, H., A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate-acrylamide gel (1989) Anal. Biochem., 183, pp. 139-143Katagiri, K., Katagiri, T., Kajiyama, K., Yamamoto, T., Yoshida, T., Tyrosine-phosphorylation of tubulin during monocytic differentiation of HL-60 cells (1993) J. Immunol., 150, pp. 585-593Koontz, D.A., Choi, J.H., Evidence for phosphorylation of tubulin in carrot suspension cells (1993) Physiol. Plant., 87, pp. 576-583Levasseur, P.J., Meng, Q., Bouck, G.B., Tubulin genes in the algal protist Euglena gracilis (1994) J. Euk. Microbiol., 41, pp. 468-477Lewis, S.A., Tian, G., Vainberg, I.E., Cowan, N.J., Chaperonin-mediated folding of actin and tubulin (1996) J. Cell Biol., 132, pp. 1-4Laemmli, U.K., Cleavage of structural proteins during assembly of the head of bacteriophage T4 (1970) Nature, 277, pp. 680-685Little, M., An evaluation of tubulin as a molecular clock (1985) Biosystems, 18, pp. 241-247MacRae, T.H., Tubulin post-translational modifications. Enzymes and their mechanisms of action (1997) Eur. J. Biochem., 244, pp. 265-278Mandelkow, E.-M., Biernat, J., Drewes, G., Gustke, N., Trinczek, B., Mandelkow, E., Tau domains, phosphorylation, and interactions with microtubules (1995) Neurobiol. Aging, 16, pp. 355-362Markwell, M.A.K., A new solid-state reagent to iodinate proteins (1982) Anal. Biochem., 125, pp. 427-432Marrs, J.A., Bouck, G.B., The two major membrane skeletal proteins (articulins) of Euglena gracilis define a novel class of cytoskeletal proteins (1992) J. Cell Biol., 118, pp. 1465-1475Murofushi, H., Purification and characterization of a protein kinase from Tetrahymena cilia (1973) Biochim. Biophys. Acta, 327, pp. 354-364Redeker, V., Levilliers, N., Schmitter, J.M., Le Caer, J.P., Rossier, J., Adoutte, A., Bré, M.H., Polyglycylation of tubulin: A post-translational modification in axonemal microtubules (1994) Science, 266, pp. 1688-1691Rogalski, A.A., Bouck, G.B., Characterization and localization of a flagellar specific membrane glycoprotein in Euglena (1980) J. Cell Biol., 86, pp. 424-435Serrano, L., Diaz-Nido, J., Avila, J., Tubulin phosphorylation by casein kinase II is similar to that found in vivo (1987) J. Cell Biol., 105, pp. 1731-1739Silflow, C.W., Why do tubulin gene families lack diversity in flagellate/ciliate protists? (1991) Protoplasma, 164, pp. 9-11Silva, A.C., (1996) Caracterização Bioquímicas das Tubulinas do Complexo-membrana-citoesqueleto e Flagelo de Euglena Gracilis, com Ênfase Na Fosforilação, , Dissertation. Federal University of San Carlos, Brazil. 136 p. Available from authorTian, G., Lewis, S.A., Feierbach, B., Stearns, T., Rommelaere, H., Ampe, C., Cowan, N.J., Tubulin subunits exist in an activated conformational state generated and maintained by protein cofactors (1997) J. Cell Biol., 138, pp. 821-832Wandosell, F., Serrano, L., Avila, J., Phosphorylation of α-tubulin carboxy-terminal tyrosine prevents its incorporation into microtubules (1987) J. Biol. Chem., 262, pp. 8268-8273Wandosell, F., Serrano, L., Hernández, M.A., Avila, J., Phosphorylation of tubulin by a calmodulin-dependent protein kinase (1986) J. Biol. Chem., 261, pp. 10332-10339Wei, T., Tao, M., Human erythrocyte casein kinase II: Characterization and phosphorylation of membrane cytoskeletal proteins (1993) Arch. Biochem. Biophys., 307, pp. 206-216Zhou, R., Oskarsson, M., Paules, R.S., Schulz, N., Cleveland, D., Vande Woude, G.F., Ability of the c-mos product to associate with and phosphorylate tubulin (1991) Science, 251, pp. 671-675