Incorporation of azatyrosine into the COOH-terrninus of α-tubulin.

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

Villa Carlos Paz, Cordoba, Argentina

Congreso; Sociedad Argentina de Investigaciones Bioquímicas y Biología Molecular (SAIB); 2002

The role of the tyrosination/detyrosination of a -tubulin at its COOH-terminus is not known. We found that the tyrosine analogue, azatyrosine (L-h-(5-p-hydtoxy-2-pyridyl)-alanine) can be incorporated, in vitro and in vivo, into a-tubulin in place of COOH-terminal tyrosine. In vitro, azatyrosine competed and prevented the incorporation of [14C]tyrosine. An antibody specific to tubulin-azatyrosine was developed in rabbits. By using immunoblots with this antibody, it was found that: a) the analogue is incorporated into the COOH-terminus of a-tubulin; b) the Km of azatyrosine is 2.3 x 10-4M: c) the incorporated azatyrosine can be released from tubulin by endogenous  "tubulin carboxypeptidase"; d) tubulin-azatyrosine can assemble into microtubules as efficiently as tyrosinated tubulin (both in vitro and in vivo). We used C6 cells (glioma-derived) to show that, when added to culture medium at 600 mM, azatyrosine could be reversibly incorporated into the COOH-terminus of a-tubulin in place of tyrosine. This was done by immunoblot and immunofluorescence. Microtubules containing tubulin-azatyrosine were indistinguishable from those containing tyrosinated tubulin. After 7 days at 600mM azatvrosine, cells stopped growth and differentiated. It is known that azatyrosine convert transformed cells to normal phenotype by a non-defined mechanism. Our results suggest the possibility that substitution of COOH-teminaI tyrosine by azatyrosine is involved in the reversion of the transformed phenotype.