Posttranslational modifications of tubulin in cultured mouse brain neurons and astroglia

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 alpha and 10 beta isoforms), similar to that found in the brain at the same developmental stage. Astroglial tubulin exhibits a less complex pattern consisting of 4 alpha and 4 beta isoforms. After incubation of neuronal and glial cells with 3H-acetate in the presence of cycloheximide, a major posttranslational label was found associated with alpha-tubulin and a minor one with beta-tubulin. The acetate-labeled isotubulins of neurons were resolved by isoelectric focusing into as many as 6 alpha and 7 beta isoforms, while those of astroglia were resolved into only 2 alpha and 2 beta isoforms. The same alpha isoforms were also shown to react with a monoclonal antibody recognizing selectively the acetylated form(s) of alpha-tubulin. Whether acetate-labeling of alpha-tubulin in these cells corresponds to the acetylation of Lys40, as reported for Chlamydomonas reinhardtii, is discussed according to very recent data obtained by protein sequence analysis. Tubulin phosphorylation was analyzed by incubation of cell cultures with 32PO4. No phosphorylation of alpha-tubulin isoforms was detected. A single beta-tubulin isoform (beta'2), expressed only in neurons, was found to be phosphorylated. This isoform is similar to that previously identified in differentiated mouse neuroblastoma cells.     

Recognition of specific Physarum alpha-tubulin isotypes by a monoclonal antibody. Sequence heterogeneity around the acetylation site at lysine 40

The monoclonal antibody 6-11B-1 recognises specifically the acetylated form of alpha-tubulin. The acetylation event occurs on a unique lysine residue, lysine 40. Using 6-11B-1, acetylated alpha-tubulin was detected in myxamoebae but not plasmodia of Physarum polycephalum. Following chemical acetylation plasmodial alpha-tubulin was detected by 6-11B-1. The monoclonal antibody KMP-1 recognises certain Physarum alpha-tubulin isotypes but only in non-acetylated form. Whilst recognising all the non-acetylated fraction of myxamoebal alpha-tubulin only a proportion of plasmodial alpha-tubulin was recognised by KMP-1. Peptides were synthesised corresponding to the acetylation domains (containing lysine 40) of myxamoebal alpha-tubulin and the inferred acetylation domains of two plasmodial-specific alpha-tubulin isotypes. The only difference between the two peptides was at a single residue corresponding to amino acid 44 in the polypeptide. Tyrosine was present in myxamoebal alpha-tubulin and glycine was present in the plasmodial specific peptides; the peptides are referred to as the Tyr44 and Gly44 peptides respectively. Both peptides in acetylated form blocked 6-11B-1 reactivity towards acetylated myxamoebal alpha-tubulin. The Tyr44 but not the Gly44 peptide blocked KMP-1 reactivity towards non-acetylated myxamoebal alpha-tubulin. Tyrosine at position 44 is not found in any other known alpha-tubulin. Thus a unique antigenic determinant exists in certain Physarum alpha-tubulin isotypes, close to the acetylation site at lysine 40. This antigenic determinant forms part of the KMP-1 recognition epitope and explains the unique isotype selectivity of this monoclonal antibody.     

Acetylation of lysine 40 in alpha-tubulin is not essential in Tetrahymena thermophila

In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of alpha- and beta- tubulin heterodimer, precluding distinctions among microtubular systems based on tubulin primary sequence isotypes. Tetrahymena tubulins also are modified by several types of posttranslational reactions including acetylation of alpha-tubulin at lysine 40, a modification found in most eukaryotes. In Tetrahymena, axonemal alpha-tubulin and numerous other microtubules are acetylated. We completely replaced the single type of alpha-tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position. No acetylated tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40. Surprisingly, mutants lacking detectable acetylated tubulin are indistinguishable from wild-type cells. Thus, acetylation of alpha- tubulin at lysine 40 is non-essential in Tetrahymena. In addition, isoelectric focusing gel analysis of axonemal tubulin from cells unable to acetylate alpha-tubulin leads us to conclude that: (a) most or all ciliary alpha-tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated alpha-tubulin molecules in wild-type cells contain one or more additional charge-altering modifications.     

Exposure of lumenal microtubule sites after mild fixation

High-resolution analysis of tubulin structure and docking the structure of tubulin dimer into a map of microtubules led to a prediction that sites for tubulin acetylation are in the interior of microtubules. This is somehow difficult to reconcile with their susceptibility to proteases and acetylation in assembled microtubules. To assess the availability of acetylated alpha-tubulin for antibodies, immunofluorescence on detergent-extracted cells, on cells fixed under various conditions and in microinjected cells was performed with monoclonal antibodies of known epitope locations. The presented data indicate that acetylated alpha:Lys40 is not exposed on unfixed microtubules but that this region of lumenal microtubule surface becomes easily exposed under mild fixation conditions.     

Acetylated and detyrosinated alpha-tubulins are co-localized in stable microtubules in rat meningeal fibroblasts

We have examined the distribution of acetylated alpha-tubulin using immunofluorescence microscopy in fibroblastic cells of rat brain meninges. Meningeal fibroblasts showed heterogeneous staining patterns with a monoclonal antibody against acetylated alpha-tubulin ranging from staining of primary cilia or microtubule-organising centers (MTOCs) alone to extensive microtubule networks. Staining with a broad spectrum anti-alpha-tubulin monoclonal indicated that all cells possessed cytoplasmic microtubule networks. From double-labeling experiments using an antibody against acetylated alpha-tubulin (6-11B-1) and antibodies against either tyrosinated or detyrosinated alpha-tubulin, it was found that acetylated alpha-tubulin and tyrosinated alpha-tubulin were often segregated to different microtubules. The microtubules containing acetylated but not tyrosinated alpha-tubulin were cold stable. Therefore, it appeared that in general meningeal cells possessed two subset of microtubules: One subset contained detyrosinated and acetylated alpha-tubulin and was cold stable, and the other contained tyrosinated alpha-tubulin and was cold labile. These results are consistent with the idea that acetylation and detyrosination of alpha-tubulin are involved in the specification of stable microtubules.     

Effects of sodium fluoride (NaF) on the cilia and microtubular system of Tetrahymena

The effect of the nucleophilic reagent NaF on the microtubular system of Tetrahymena was studied by using scanning electron microscopy (SEM), confocal microscopy, and flow cytometry. Treatments with 40 mM NaF significantly reduced the amount of alpha-tubulin while 80 mM treatment did not alter its quantity. One possible explanation for this alpha-tubulin overexpression is that the higher amount of alpha-tubulin enables this organism to carry out the appropriate function of the cytoskeleton under this undesirable influence of higher amounts of 80 nM NaF. However, the amount of acetylated tubulin increased in a dose-dependent manner. The cilia became fragile under the effect of 80 mM NaF. Confocal microscopy revealed that after 40 mM NaF treatment transversal microtubule bands (TMs) and longitudinal microtubule bands (LMs) as well as basal bodies (BBs) were extremely strong decorated with anti-acetylated tubulin antibody and TM-localization abnormalities were visible. In the 80 mM NaF-treated cells, the deep fiber of oral apparatus was very strongly labeled, while the TMs and LMs were less decorated with anti-acetylated tubulin antibody, and LM deformities were visible. It is supposed that post-translational tubulin modifications (e.g., acetylation) defend the microtubules against the NaF-induced injury. NaF is able to influence the activity of several enzymes and G-proteins, therefore is capable to alter the structure, metabolism, and the dynamics of microtubular system. The possible connection of signaling and cytoskeletal system in Tetrahymena is discussed.     

Tau – an inhibitor of deacetylase HDAC6 function

Analysis of brain microtubule protein from patients with Alzheimer's disease showed decreased alpha tubulin levels along with increased acetylation of the alpha tubulin subunit, mainly in those microtubules from neurons containing neurofibrillary tau pathology. To determine the relationship of tau protein and increased tubulin acetylation, we studied the effect of tau on the acetylation-deacetylation of tubulin. Our results indicate that tau binds to the tubulin-deacetylase, histone deacetylase 6 (HDAC6), decreasing its activity with a consequent increase in tubulin acetylation. As expected, increased acetylation was also found in tubulin from wild-type mice compared with tubulin from mice lacking tau because of the tau-mediated inhibition of the deacetylase. In addition, we found that an excess of tau protein, as a HDAC6 inhibitor, prevents induction of autophagy by inhibiting proteasome function.     

Multiple alpha-tubulin isoforms in cilia and cytoskeleton of Tetrahymena pyriformis generated by post-translational modifications. Studies during reciliation

alpha-Tubulin microheterogeneity was studied in Tetrahymena pyriformis. Using two-dimensional electrophoretic analysis, we found between five and seven alpha-tubulin and four beta-tubulin isoforms in cilia and four or five alpha-tubulins and two beta-tubulins in cytoskeleton. Immunoblotting assay with anti-(acetyl alpha-tubulin) monoclonal antibody 6-11B-1 and [3H]acetate labelling revealed that the alpha-tubulin isoforms are post-translationally modified by acetylation. Our results also show that tubulins in the soluble cytoplasmic fraction are not acetylated. Nevertheless, a slight reaction with the antibody 6-11 B-1 can be observed in the taxol and vinblastine-treated cytoplasmic pool. Pulse/chase experiments using [35S]methionine during cell reciliation have demonstrated that the basic alpha-tubulin isoforms are converted into acidic isoforms in the absence of protein synthesis, suggesting that the basic alpha-tubulin is the precursor of the acidic forms which are found in cilia and cytoskeleton. In-vivo-translation selection demonstrated the existence of a single precursor molecule which corresponded to the most basic alpha-tubulin. Taken together, our results provide evidence for the existence of post-translational modifications, namely acetylation. Nevertheless, other post-translational mechanisms involved in the biosynthesis of microtubules of cilia and cytoskeleton are required to explain the whole alpha-tubulin heterogeneity.     

Tubulins in Trichomonas vaginalis: molecular characterization of alpha-tubulin genes, posttranslational modifications, and homology modeling of the tubulin dimer

We have isolated and analysed an alpha-tubulin-encoding gene (atub1) in an early-diverging eukaryote, Trichomonas vaginalis. The complete atub1 open reading frame included 1.356 bp encoding a polypeptide of 452 amino-acyl residues. A second alpha-tubulin gene (atub2) was amplified by PCR using primers derived from consensus alpha-tubulin amino acid sequences. Both T. vaginalis alpha-tubulin sequences showed high identity to those described in other parabasalids (94.4%-97.3%), and exhibited a high degree of similarity to sequences from Metazoa (such as pig brain) and diplomonads (such as Giardia). Despite large evolutionary distances previously observed between trichomonads and mammals, the three-dimensional model of the T. vaginalis tubulin dimer was very similar to that of pig brain. Possible correlations between alpha-tubulin sequences and posttranslational modifications (PTMs) were examined. Our observations corroborated previous data obtained in T. vaginalis using specific anti-PTMs antibodies. As described in the related species Tritrichomonas mobilensis, microtubules are likely acetylated, non-tyrosinated, glutamylated, and non-glycylated in T. vaginalis. Evolutionary considerations concerning the time of appearance of these tubulin PTMs are also discussed since trichomonads are potentially one of the earliest diverging eukaryotic lineages.     

Posttranslational modifications of alpha-tubulin: acetylated and detyrosinated forms in axons of rat cerebellum

The distribution of acetylated alpha-tubulin in rat cerebellum was examined and compared with that of total alpha-tubulin and tyrosinated alpha-tubulin. From immunoperoxidase-stained vibratome sections of rat cerebellum it was found that acetylated alpha-tubulin, detectable with monoclonal 6-11B-1, was preferentially enriched in axons compared with dendrites. Parallel fiber axons, in particular, were labeled with 6-11B-1 yet unstained by an antibody recognizing tyrosinated alpha-tubulin, indicating that parallel fibers contain alpha-tubulin that is acetylated and detyrosinated. Axonal microtubules are known to be highly stable and the distribution of acetylated alpha-tubulin in other classes of stable microtubules suggests that acetylation and possibly detyrosination may play a role in the maintenance of stable populations of microtubules.     

Multiple histone deacetylases and the CREB-binding protein regulate pre-mRNA 3'-end processing

Trichostatin A (TSA), a specific inhibitor of histone deacetylases (HDACs), induces acetylation of various non-histone proteins such as p53 and alpha-tubulin. We purified several acetylated proteins by the affinity to an anti-acetylated lysine (AcLys) antibody from cells treated with TSA and identified them by mass spectrometry. Here we report on acetylation of CFIm25, a component of mammalian cleavage factor Im (CF Im), and poly(A) polymerase (PAP), a polyadenylating enzyme for the pre-mRNA 3'-end. The residues acetylated in these proteins were mapped onto the regions required for interaction with each other. Whereas CBP acetylated these proteins, HDAC1, HDAC3, HDAC10, SIRT1, and SIRT2 were involved in in vivo deacetylation. Acetylation of the CFIm25 occurred depending on the cleavage factor complex formation. Importantly, the interaction between PAP and CF Im complex was decreased by acetylation. We also demonstrated that acetylation of PAP inhibited the nuclear localization of PAP by inhibiting the binding to the importin alpha/beta complex. These results suggest that CBP and HDACs regulate the 3'-end processing machinery and modulate the localization of PAP through the acetylation and deacetylation cycle.     

Post-translational modifications of tubulin in the nervous system

Many studies have shown that microtubules (MTs) interact with MT-associated proteins and motor proteins. These interactions are essential for the formation and maintenance of the polarized morphology of neurons and have been proposed to be regulated in part by highly diverse, unusual post-translational modifications (PTMs) of tubulin, including acetylation, tyrosination, detyrosination, Δ2 modification, polyglutamylation, polyglycylation, palmitoylation, and phosphorylation. However, the precise mechanisms of PTM generation and the properties of modified MTs have been poorly understood until recently. Recent PTM research has uncovered the enzymes mediating tubulin PTMs and provided new insights into the regulation of MT-based functions. The identification of tubulin deacetylase and discovery of its specific inhibitors have paved the way to understand the roles of acetylated MTs in kinesin-mediated axonal transport and neurodegenerative diseases such as Huntington's disease. Studies with tubulin tyrosine ligase (TTL)-null mice have shown that tyrosinated MTs are essential in normal brain development. The discovery of TTL-like genes encoding polyglutamylase has led to the finding that polyglutamylated MTs which accumulate during brain development are involved in synapse vesicle transport or neurite outgrowth through interactions with motor proteins or MT-associated proteins, respectively. Here we review current exciting topics that are expected to advance MT research in the nervous system.     

Synthesis of Nε-acetyl-L-homolysine by the Lossen rearrangement and its application for probing deacetylases and binding modules of acetyl-lysine

Lysine acetylation is a posttranslational protein modification mediating protein–protein interactions by recruitment of bromodomains. Investigations of bromodomains have focused so far on the sequence context of the modification site and acyl-modifications installed at lysine side chains. In contrast, there is only little information about the impact of the lysine residue that carries the modification on bromodomain binding. Here, we report a synthesis strategy for L-acetyl-homolysine from L-2-aminosuberic acid by the Lossen rearrangement. Peptide probes containing acetylated homolysine, lysine, and ornithine were generated and used for probing the binding preferences of four bromodomains from three different families. Tested bromodomains showed distinct binding patterns, and one of them bound acetylated homolysine with similar efficiency as the native substrate containing acetyl-lysine. Deacetylation assays with a bacterial sirtuin showed a strong preference for acetylated lysine, despite a broad specificity for N-acyl modifications.     

Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of alpha-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic alpha-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only alpha-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal alpha-tubulin isoform acetylated in vivo on the epsilon-amino group of lysine(s) (L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal alpha-tubulin, unassembled alpha-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic alpha-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated alpha-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of alpha-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated alpha-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo.     

Coordination of posttranslational modifications of bovine brain alpha-tubulin. Polyglycylation of delta2 tubulin

Microtubules participate in a large number of intracellular events including cell division, intracellular transport and secretion, axonal transport, and maintenance of cell morphology. They are composed of tubulin, a heterodimeric protein, consisting of two similar polypeptides alpha and beta. In mammalian cells, both alpha- and beta-tubulin occur as seven to eight different genetic variants, which also undergo numerous posttranslational modifications that include tyrosination-detyrosination and deglutamylation, phosphorylation, acetylation, polyglutamylation, and polyglycylation. Tyrosination-detyrosination is one of the major posttranslational modifications in which the C-terminal tyrosine residue in alpha-tubulin is added or removed reversibly. Although this modification does not alter the assembly activity of tubulin in vitro, these two forms of tubulin have been found to be distributed differently in vivo and are also correlated with microtubule stability (Gunderson, G. G., Kalnoski, M. H., and Bulinski, J. C. (1984) Cell 38, 779-789). Thus, the question arises as to whether these two forms of tubulin differ in any other modifications. In an effort to answer this question, the tyrosinated and the nontyrosinated forms of the alpha1/2 isoform have been purified from brain tubulin by immunoaffinity chromatography. matrix-assisted laser desorption/ionization-time of flight mass spectrometric analysis of the C-terminal peptide revealed that the tyrosinated form is polyglutamylated with one to four Glu residues, while the Delta2 tubulin is polyglycylated with one to three Gly residues. These results indicate that posttranslational modifications of tubulin are correlated with each other and that polyglutamylation and polyglycylation of tubulin may have important roles in regulating microtubule assembly, stability, and function in vivo.