Post-translational incorporation of L-Dopa into the C-terminus of alpha-tubulin in living cells

DENTESANO Y; DITAMO Y; HANSEN C; ARCE CA; BISIG CG

Córdoba

Congreso; 52th Annual Meeting Argentine Society for Biochemistry and Molecular Biology (SAIB); 2016

Sociedad Argentina de Investigaciones Bioquímicas

C-terminal tyrosine of the α-tubulin chain is subjected to its removal and post-translational re-addition (detyrosination/tyrosination cycle). L-3,4-dihydroxy phenylalanine (L-Dopa) can be incorporated in place of tyrosine. We found that this L-Dopa incorporation occurs also in living cells. To detect and quantify Dopa-tubulin, we developed a procedure that consists in the determination of the ?tyrosination state? of tubulin using specific antibodies, before and after cells were incubated in the presence of Dopa. The presence of Dopa-tubulin (which is not recognized by Tyr- or Glu- antibodies) is assumed to arise from the difference between total tubulin and the sum of Tyr- and Glu-tubulin (subsequently confirmed by HPLC). Post-translational Dopa incorporation into tubulin was demonstrated in Neuro 2A cells and others cell lines. Dopa-tubulin had similar capability to form microtubules as Tyr- and Glu-tubulin. Viability, cell morphology and proliferation rate were not modified when 20% of total tubulin in the cells was Dopa-tubulin.