Blockade of the COOH-terminus of alpha-tubulin by irreversible incorporation of L-dopa in cultured cells.

BISIG, CG, CARBAJAL, A, CHESTA, ME AND ARCE, CA

Mendoza

Congreso; Sociedad de Investigaciones en Bioquímica y Biología Molecular; 2012

We previously described that L-Dopa can be incorporated into the COOH-terminus of the α-chain of tubulin by using tubulin tyrosine ligase, one of the enzymes involved in the postranslational cyclic tyrosination/detyrosination of tubulin. Now, we found that after its incorporation into tubulin, Dopa cannot be released by the other enzyme involved in the tyrosination/detyrosination cycle, that is, tubulin carboxypeptidase. Dopa-tubulin, when mixed with Tyr-tubulin in a rat brain soluble extract, assembled into microtubules as well as Tyr-tubulin. The ability to disassemble by cold or dilution of microtubules formed from Tyr- or Dopa-tubulin was also similar. Due to the unavailability of an antibody specific to Dopa-tubulin, to monitor and to measure the amount of Dopa incorporated into tubulin we used a method based on Western blot analysis of the tyrosination state of samples before and after Dopa incorporation. Dopa incorporation into tubulin was also demonstrated to occur in cultured cells in the absence of “de novo” protein synthesis. Furthermore, once incorporated into tubulin of cultured cells, dopa could not be removed by subsequent incubation in medium lacking L-dopa even in the presence of added tyrosine reinforcing the idea that dopa binds irreversibly to the COOH-terminus of α-tubulin blocking the tyrosination/detyrosination cycle.