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

DITAMO, Y; ZORGNIOTTI, A; ARCE, CA; BISIG, CG

Cordoba

Simposio; Alexander von Humboldt Kollege; 2017

Alexander von Humboldt Foundation

Abstract: The C-terminal tyrosine (Tyr) of the α-tubulin chain is subjected to post-translational removal and re-addition in a process termed the ?detyrosination/tyrosination cycle?. We showed in previous studies using soluble rat brain extracts that L-3,4-dihydroxyphenylalanine (L-Dopa) is incorporated into the same site as Tyr. We now demonstrate that L-Dopa incorporation into tubulin also occurs in living cells. We detected such incorporation by determining the ?tyrosination state? of tubulin before and after incubation of cells in the presence of L-Dopa. The presence of a tubulin isospecies following L-Dopa incubation that was not recognized by antibodies specific to Tyr- and Glu-tubulin was presumed to reflect formation of Dopa-tubulin. L-Dopa was identified by HPLC as the C-terminal compound bound to α-tubulin. L-Dopa incorporation into tubulin was observed in Neuro 2A cells and several other cell lines, and was not due to de novo protein biosynthesis. Dopa-tubulin had microtubule-forming capability similar to that of Tyr- and Glu-tubulin. L-Dopa incorporation into tubulin did not notably alter cell viability, morphology, or proliferation rate. CAD cells (a neuron-like cell line derived from mouse brain) are easily cultured under differentiating and non-differentiating conditions, and can be treated with L-Dopa. Treatment of CAD cells with L-Dopa resulted in reduction of microtubule dynamics in neurites, most likely due to L-Dopa incorporation into α-tubulin, which may be associated with movement disorders sometimes observed in patients treated with L-Dopa for prolonged periods.