Modification of the COOH-terrninal tyrosine of alpha-tubulin induced by low concentration of free tyrosine in the culture medium

PURRO, S.A., BISIG, C. G., CONTIN, M.A., BARRA, H.S. AND ARCE, C.A

Valdivia, Chile

Otro; Second Iberoamerican Forum on Cytoskeleton; 2002

Alpha-tubulin is biosynthesized with a tyrosine residue at its COOH-terminus that can be removed by tubulin carboxypeptidase and reincorporated by tubulin tyrosine ligase. Majority of microtubules in cells routinely grown in DMEM (which contains 400 uM tyrosine) are constituted mainly by tyrosinated tubulin (Tyr-tubulin) while detyrosinated tubulin (Glu-tubulin) is scarce. Data were obtained by using immunoblot and immunofluorescence techniques with antibodies specific to Glu- and Tyr-tubulin. When cells were cultured in Ham F 12K (60 uM tyrosine) during few days, immunoreactivity against Tyr-tubulin decreased practically to zero shilw total tubulin remained constant and Glu-tubulin did no increase. Treatment of tubulin with carboxypeptidase A produced a significan increase of Glu-tubulin suggesting that a compound was bound to glutamic acid through peptidic linkage at the COOH-terminus. This compound could be a modified tyrosine residue or an amino acid other than tyrosine that the antibody cannot recognize. When cells that had been grown in F12K were supplemented with tyrosine upo to 400 uM and growth continued for 24 hs, immunoreactivity to Tyr-tubulin was recovered. Taxol-stabilization of microtubules of cells cultured in F12K led to a significant increment of Glu-tubulin. These results indicate that even when Tyr-tubulin seems to be absent, the ligase and carboxypeptidase are still active. These results were found using several cell lines.