Molecular and Cellular Biochemistry

, Volume 144, Issue 2, pp 109–116 | Cite as

A 205 kDa protein from non-neuronal cells in culture contains tubulin binding epitopes

  • Clarisa Vial
  • Rosario Armas-Portela
  • Jesús Avila
  • Mauricio González
  • Ricardo B. Maccioni
Article

Abstract

Microtubule-associated proteins (MAPs) interact with tubulinin vitro andin vivo. Despite that there is a large amount of information on the roles of these proteins in neurons, the data on non-neuronal MAPs or MAPs-related proteins is scarce. There is an increasing number of microtubule-interacting proteins that have been identified in different cultured cell lines, and some of them share common functional epitopes with the most well-known MAPs, MAP-2 and tau. In a search for tubulin-interacting proteins in non-neuronal cells we identified a 205 kDa protein in the monkey kidney Vero cells in culture, on the basis of immunological studies and affinity chromatography. This protein interacts with the C-terminal moiety of β-tubulin and cosediments with taxol assembled microtubules, but it was not recovered after successive cycles of assembly and disassembly. The presence of neuronal MAPs such as MAP-1, MAP-2 and tau was not detected in these cells. Interestingly, the studies showed that the 205 kDa protein contained a tubulin binding motif which was recognized by site-directed antibodies that also tag tubulin binding epitopes on MAP-2 and tau. This characteristic led us to designate this protein as MBD-205, a component that shares binding domains with these MAPs, rather than as a marker of the MAPs family. On the other hand, immunofluorescence experiments using site-specific antibodies, i.e. MAP-reacting monoclonal anti-idiotypic reagent MTB6.22 and a polyclonal antibody to the second tau repeat, revealed a MBD-205 co-localization with membrane structures and microtubule-organizing centers in Vero cells. Microinjection studies along with studies on the cell distribution suggest that MBD-205 appears to play a structural role at the level of the microtubule interactions in these cells.

Key words

MAPs 205 kDa protein Vero cells immunofluorescence cell microinjection analysis 

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References

  1. 1.
    Hiller G, Weber K: Radioimmunoassay for tubulins: a quantitative comparison of the tubulin content of different established tissue cultured cells and tissues. Cell 14: 795–804, 1978PubMedGoogle Scholar
  2. 2.
    Murphy D, Borisy G: Association of high molecular weight proteins with microtubules and their role in microtubule assemblyin vitro. Proc Natl Acad Sci, USA 72: 2696–2700, 1975Google Scholar
  3. 3.
    Sloboda R, Rudolph SA, Rosembaum JL, Greengard P: Cyclic AMP-dependent endogenous phosphorylation of a microtubule associated protein. Proc Natl Acad Sci, USA 72: 177–181, 1975Google Scholar
  4. 4.
    Weingarten M, Lockwood A, Hwo J, Kirschner M: A protein factor essential for microtubule assembly. Proc Natl Acad Sci 72: 1858–1862, 1975PubMedGoogle Scholar
  5. 5.
    Lee G, Cowan N, Kirschner M: The primary structure and heterogeneity of tau protein from mouse brain. Science 239: 285–288, 1988PubMedGoogle Scholar
  6. 6.
    Lewis SA, Wang D, Cowan N: Microtubule-associated protein MAP-2 shares a microtubule binding motif with tau protein. Science 242: 936–939, 1988PubMedGoogle Scholar
  7. 7.
    Kosik KS, Orechio L, Bakalis S, Neve R: Developmentally-regulated expression of specific tau sequences. Neuron 2, 1389–1397, 1989PubMedGoogle Scholar
  8. 8.
    Serrano L, Avila J, Maccioni RB: Controlled proteolysis of tubulin by subtilisin: Localization of the site for MAP-2 interaction. Biochemistry 23: 4675–4683, 1984PubMedGoogle Scholar
  9. 9.
    Serrano L, de la Torre J, Avila J: Involvement of the carboxyl-terminal of tubulin in microtubule assembly and regulation. Proc Natl Acad Sci, USA 81: 5989–5993, 1984Google Scholar
  10. 10.
    Littauer UZ, Giveon D, Thierauf M, Ginzburg I, Ponstingl H: Common and distinct tubuling binding sites for microtubule-associated proteins. Proc Nat Acad Sci, USA, 83: 7162–7166, 1986Google Scholar
  11. 11.
    Maccioni RB, Serrano L, Avila J, Cann J: Characterization and structural aspects of the enhanced assembly of tubulin after removal of its carboxyl-terminal domain. Eur J Biochem 156: 375–38, 1985Google Scholar
  12. 12.
    Maccioni RB, Rivas CI, Vera JC: Differential interaction of synthetic peptides from the carboxyl-terminal regulatory domain of tubulin with microtubule associated proteins (MAPs). EMBO J 7: 1975–1963, 1988Google Scholar
  13. 13.
    Vera JC, Rivas CI, Maccioni RB: Antibodies to synthetic peptides from the tubulin regulatory domain interact with tubulin and microtubules. Proc Natl Acad Sci USA, 85: 6763–6762, 1988PubMedGoogle Scholar
  14. 14.
    Cross D, Dominguez J, Maccioni RB, Avila J: MAP-1 and MAP-2 binding sites at the C-terminus of β-tubulin. Studies with synthetic tubulin peptides. Biochemistry 30: 4362–4366, 1991PubMedGoogle Scholar
  15. 15.
    Bulinski J, Borisy G: Self-assembly of microtubules in extracts of cultured HELA cells and the identification of HELA microtubule-associated proteins. Proc Natl Acad Sci USA, 76: 293–297, 1979PubMedGoogle Scholar
  16. 16.
    Olmsted JB, Lyon HD: A microtubule-associated protein specific to differentiated neuroblastoma cells. J Biol Chem 256: 3507–3511, 1981PubMedGoogle Scholar
  17. 17.
    Kotani S, Murofushi H, Maekawa S, Aizawa H, Sakai H: Isolation of rat liver microtubule-associated proteins. J Biol Chem 263: 5385–5389, 1988PubMedGoogle Scholar
  18. 18.
    Aizawa H, Emori Y, Murofushi H, Kagasaki H, Sakai H, Susuki K: Molecular cloning of a ubiquitously distributed microtubule-associated protein with Mr 190.000. J Biol Chem 265: 13849–13855, 1990PubMedGoogle Scholar
  19. 19.
    Aizawa H, Emori Y, Murofushi H, Sakai H, Susuki K: Functional analyses of the domain structure of microtubule associated protein-4 (MAP-U). J Biol Chem 266: 9841–9846, 1991PubMedGoogle Scholar
  20. 20.
    Chapin SJ, Bulinski JC: Non-neuronal microtubule-associated protein (MAP4) contains a domain homologous to the microtubule-binding domain of neuronal MAP2 and tau. J Cell Sci 98: 27–36, 1991PubMedGoogle Scholar
  21. 21.
    West R, Tembarge KM, Olmsted J: A model for microtubule-associated protein 4 structure. J Biol chem 266: 21886–21896, 1991PubMedGoogle Scholar
  22. 22.
    Schneider A, Hemphill A, Wyler T, Seebeck T: Large microtubule associated protein ofT. brucei has tandemly repeated, near identical sequences. Science 241: 459–462, 1988PubMedGoogle Scholar
  23. 23.
    Pierre P, Scheel J, Richard JE, Kreis TE: Clip-170 links endocytic vesicles to microtubules. Cell 70: 887–900, 1992PubMedGoogle Scholar
  24. 24.
    Prior P, Schmitt B, Grenningloh G, Pribilla I, Multhaup G, Beyreuther K, Maulet Y, Werner P, Langosh D, Kirsch J, Betz H: Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein. Neuron 8: 1161–1170, 1992PubMedGoogle Scholar
  25. 25.
    Farias G, Vial C, Maccioni RB: Specific macromolecular interactions between tau and the microtubule system. Mol Cell Biochem 112: 81–88, 1992PubMedGoogle Scholar
  26. 26.
    Cross D, Vial C, Maccioni RB: A tau-like protein interacts with stress fibers and microtubules in human and rodent cultured cell lines. J Cell Sci 105: 51–60, 1993PubMedGoogle Scholar
  27. 27.
    Klymkowsky MW: Intermediate filaments in 3T3 cells collapse after the microinjection of a monoclonal anti-intermediate filament antibody. Nature 291: 249–251, 1981PubMedGoogle Scholar
  28. 28.
    Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970PubMedGoogle Scholar
  29. 29.
    Towbin H, Stahelin T, Gordon JL: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354, 1979PubMedGoogle Scholar
  30. 30.
    Maccioni RB, Vera J, Dominguez J, Avila J: A discrete repeated sequence defines a tubulin binding domain on microtubule-associated protein tau. Arch Biochem Biophys 275: 568–579, 1989PubMedGoogle Scholar
  31. 31.
    Joly J, Flynn G, Purich D: The microtubule-binding fragment of microtubule-associated protein-2: localization of the protease accessible site and identification of an assembly-promoting peptide J Cell Biol 109: 2289–2294, 1989PubMedGoogle Scholar
  32. 32.
    Vallee RB, Bloom GS: Isolation of sea urchin egg microtubules with taxol and identification of mitotic spindle microtubule-associated proteins with monoclonal antibodies. Proc Natl Acad Sci USA 80: 6259–6263, 1983PubMedGoogle Scholar
  33. 33.
    Lee YC, Wolff J: Calmodulin binds to both microtubule-associated protein 2 and τ protein. J Biol Chem 259: 1226–1230, 1984PubMedGoogle Scholar
  34. 34.
    De Brabander MJ, Van der Veil R, Aerts F, Borges M, Janssen PA: The effects of methyl (5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl) carbamate, a new synthetic anti-tumoral drug interfering with microtubules on mammalian cells culturedin vitro. Cancer Res 36: 905–916, 1976PubMedGoogle Scholar
  35. 35.
    Masson D, Kreis TE: Identification and molecular characterization of E-MAP-115, a novel microtubule-associated protein predominantly expressed in epithelial cells. J Cell Biol 123: 357–371, 1993PubMedGoogle Scholar
  36. 36.
    González M, Cambiazo V, Maccioni RB: MIP-90 a novel microtubule-interacting interacting protein in cultured cells. Mol Biol Cell 4: 51a, 1993Google Scholar
  37. 37.
    Wang y, Loomis P, Zinkowsky R, Binder L: A novel tau transcript in cultured human neuroblastoma cells expressiong nuclear tau. J Cell Biol 121: 257–267, 1993PubMedGoogle Scholar
  38. 38.
    Vandré DD, Centonze VE, Peloquin J, Tombes RM, Borisy GG: Proteins of the mammalian mitotic spindle: phosphorylation/dephosphorylation of MAP-4 during mitosis. J Cell Sci 98: 577–588Google Scholar
  39. 39.
    Vandekerckhove J: Structural principles of actin binding proteins. Curr Opin Cell Biol 1: 15–22, 1989PubMedGoogle Scholar
  40. 40.
    Way M, Pope B, Weeds AG: Are the conserved sequences in segment 1 of gelsolin important for binding actin. J Cell Biol 116: 1135–114, 1992PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1995

Authors and Affiliations

  • Clarisa Vial
    • 1
  • Rosario Armas-Portela
    • 2
  • Jesús Avila
    • 2
  • Mauricio González
    • 1
  • Ricardo B. Maccioni
    • 1
    • 3
  1. 1.International Center for Cancer and Developmental Biology (ICC)Santiago 7Chile
  2. 2.Centro de Biología Molecular ‘Severo Ochoa’SantiagoChile
  3. 3.Department of Biology, Faculty of SciencesUniversity of ChileSantiagoChile

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