Posttranslational modifications of tubulin in cultured mouse brain neurons and astroglia

Abstract Posttranslational modifications of tubulin were analyzed in mouse brain neurons and glia developing in culture. Purified tubulin was resolved by isoelectric focusing. After 3 weeks of culture neurons were shown to express a high degree of tubulin heterogeneity (8 α and 10 β isoforms), similar to that found in the brain at the same developmental stage. Astroglial tubulin exhibits a less complex pattern consisting of 4 α and 4 β isoforms. After incubation of neuronal and glial cells with 3 H-acetate in the presence of cycloheximide, a major posttranslational label was found associated with α-tubulin and a minor one with β-tubulin. The acetate-labeled isotubulins of neurons were resolved by isoelectric focusing into as many as 6 α and 7 β isoforms, while those of astroglia were resolved into only 2 α and 2 β isoforms. The same α isoforms were also shown to react with a monoclonal antibody recognizing selectively the acetylated forms(s) of α-tubulin [38]. Whether acetate-labeling of α-tubulin in these cells corresponds to the acetylation of Lys 40 , as reported for Chlamydomonas reinhardtii [30, 33, 34], is discussed according to very recent data obtained by protein sequence analysis. Tubulin phosphorylation was analyzed by incubation of cell cultures with 32 PO 4 . No phophorylation of α-tubulin isoforms was detected. A single β-tubulin isoform (β′2), expressed only in neurons, was found to be phosphorylated. This isoform is similar to that previously identified in differentiated mouse neuroblastoma cells [16, 18, 21].