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

DITAMO Y; ZORGNIOTTI A; ARCE CA; BISIG CG

Carlos Paz

Simposio; 1st Alexander von Humboldt Kolleg; 2017

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 ratbrain extracts that L-3,4- dihydroxyphenylalanine (L-Dopa) is incorporated into the samesite as Tyr. We now demonstrate that L-Dopa incorporation into tubulin also occurs inliving cells. We detected such incorporation by determining the ?tyrosination state? oftubulin before and after incubation of cells in the presence of L-Dopa. The presence ofa tubulin isospecies following L-Dopa incubation that was not recognized by antibodiesspecific 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-Dopaincorporation 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 intotubulin did not notably alter cell viability, morphology, or proliferation rate. CAD cells (aneuron-like cell line derived from mouse brain) are easily cultured under differentiatingand non-differentiating conditions, and can be treated with L-Dopa. Treatment of CADcells with L-Dopa resulted in reduction of microtubule dynamics in neurites, most likelydue to L-Dopa incorporation into α-tubulin, which may be associated with movementdisorders sometimes observed in patients treated with L-Dopa for prolonged periods.