Turnover of the carboxy-terminal tyrosine of alpha-tubulin and means of reaching elevated levels of detyrosination in living cells.
Monoclonal antibodies specific for either the tyrosinated (Tyr) or the detyrosinated (Glu) form of alpha-tubulin were elicited with synthetic peptides spanning the carboxy-terminal sequences of the two forms. While almost all microtubules (MTs) are usually of the Tyr-tubulin type (Tyr-rich MTs) some MTs containing noticeable amounts of Glu-tubulin (Glu-rich MTs) were found in many but not all cell lines studied. Glu-rich MTs seemed absent from proliferating CHO and N115 neuroblastoma cells. When differentiation of these cells was initiated by the addition of forskolin for CHO, or by serum deprivation for N115, elevated levels of microtubular Glu-tubulin were observed. In differentiated N115 cells Glu-tubulin was restricted to MT of elongated cell processes and was not found in growth cones and many MT of the cell body. Elevated levels of Glu-tubulin were also characteristic of other differentiated cell types, including neurones and myotubes but were not found in glial cells, astrocytes and fibroblasts in the same primary cultures. Additional experiments suggested that the restricted distribution of Glu-tubulin is the result of MT subsets with different stabilities. Results with mitotic drugs indicated that detyrosination occurs on MTs rather than on soluble tubulin and that stabilization of MTs usually favours the detyrosination process. Evidence for a functional alpha-tubulin cycle involving an inherent carboxypeptidase and a recharging ligase was apparent in 3T3 cells from the preponderance of Glu-rich MTs induced by taxol treatment or the micro-injection of certain antibodies either protecting the detyrosinated form (Glu-tubulin antibodies) or inhibiting retyrosination (ligase antibodies). As the same treatment of CHO cells resulted in comparable arrays of Glu-rich MTs only when forskolin was also present, different cell types may differ in the level of active carboxypeptidase. The results are discussed in terms of possible functions of the tyrosination/detyrosination cycle of alpha-tubulin. While most results can be explained on the basis of 'older' and, consequently, more detyrosinated MTs, others raise the possibility that cyclic-AMP-dependent events and certain environmental influences known to induce either a morphological transformation or a differentiation event may influence the carboxypeptidase inherent in the alpha-tubulin cycle.
Wehland
J
J
Max-Planck-Institute for Biophysical Chemistry, Goettingen, Federal Republic of Germany.
Weber
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002268:Carboxypeptidases / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D014404:Tubulin / Q000276:immunology / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D014404:Tubulin; D005973:Glutamine; D014443:Tyrosine; D000242:Cyclic AMP; D002268:Carboxypeptidases