A 205 kDa protein from non-neuronal cells in culture contains tubulin binding epitopes
- 24 Downloads
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 wordsMAPs 205 kDa protein Vero cells immunofluorescence cell microinjection analysis
Unable to display preview. Download preview PDF.
- 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.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
- 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.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.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.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
- 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
- 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