Acetylation of alpha-tubulin in cultured neurons and the induction of alpha-tubulin acetylation in PC12 cells by treatment with nerve growth factor.

alpha-Tubulin of cultured neurons undergoes a posttranslational modification that lowers its p/(Black and Kurdyla, 1983a). We show here that this modification is due to acetylation. Incubation of cultures with 3H-acetate results in the prominent labeling of a protein that comigrates with alpha-tubulin in 1-dimensional SDS gels, and in 2-dimensional isoelectric focusing X SDS gels, it comigrates with the more acidic alpha-tubulin forms. The labeled material can be immunoprecipitated by monoclonal antibodies to alpha-tubulin but not by antibodies to beta-tubulin. The reaction occurs to equal extent in the presence or absence of 0.1 mM cycloheximide or unlabeled amino acids. These observations indicate that the reaction is posttranslational and is not secondary to conversion of isotopic acetate into amino acids. The posttranslational nature of tubulin acetylation was further established by the demonstration that acetate is incorporated into alpha-tubulin in neurites, which lack the capacity for cytoplasmic protein synthesis. Finally, a monoclonal antibody that is specific for acetylated alpha-tubulin (Piperno and Fuller, 1985) recognizes the acidic variants of alpha-tubulin of cultured neurons that can be labeled by incubation with 3H-acetate. Collectively, these observations indicate that alpha-tubulin of cultured sympathetic neurons undergoes a posttranslational acetylation. Immunofluorescence analyses using a monoclonal antibody specific for acetylated alpha-tubulin revealed that it was present throughout the neuron and showed no obvious regional compartmentalization. We have also shown that alpha-tubulin is acetylated in the NGF-responsive PC12 line of pheochromocytoma cells using biochemical and immunological criteria. However, this modification was detected only in long-term (14 d) NGF-treated cells. The results are discussed in terms of the possible role of acetylation as a local mechanism for regulating microtubule stability.