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Post‐translational incorporation of 3,4‐dihydroxyphenylalanine into the C terminus of α‐tubulin in living cells
Author(s) -
Dentesano Yanela M.,
Ditamo Yanina,
Hansen Cristian,
Arce Carlos A.,
Bisig Carlos Gaston
Publication year - 2018
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.14386
Subject(s) - tubulin , microtubule , neurite , dihydroxyphenylalanine , tyrosine , biochemistry , cell culture , biology , cell division , cell , cricetulus , microbiology and biotechnology , incubation , chemistry , in vitro , dopamine , neuroscience , chinese hamster ovary cell , genetics , receptor
The C‐terminal tyrosine (Tyr) of the α‐tubulin chain is subjected to post‐translational removal and readdition 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 deTyr‐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 deTyr‐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 nondifferentiating conditions, and can be treated with l ‐Dopa. Treatment of CAD cells with l ‐Dopa and consequent increase in l ‐Dopa‐tubulin resulted in reduction of microtubule dynamics in neurite‐like processes.