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.     

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.     

α-tubulin tail modifications regulate microtubule stability through selective effector recruitment,not changes in intrinsic polymer dynamics

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Posttranslational tyrosination/detyrosination of tubulin

Tubulin can be posttranslationally modified at the carboxyl terminus of the alpha-subunit by the addition or release of a tyrosine residue. These reactions involve two enzymes, tubulin: tyrosine ligase and tubulin carboxypeptidase. The tyrosine incorporation reaction has been described mainly in nervous tissue but it has also been found in a great variety of tissues and different species. Molecular aspects of the reactions catalyzed by these enzymes are at present well known, especially the reaction carried out by the ligase. Several lines of evidence indicate that assembled tubulin is the preferred substrate of the carboxypeptidase, whereas nonassembled tubulin is preferred by the ligase. Apparently this posttranslational modification does not affect the capacity of tubulin to form microtubules but it generates microtubules with different degrees of tyrosination. Variation in the content of the carboxyterminal tyrosine of alpha-tubulin as well as changes in the activity of the ligase and the carboxypeptidase are manifested during development. Changes in the cellular microtubular network modify the turnover of the carboxyterminal tyrosine of alpha-tubulin. Different subsets of microtubules with different degrees of tyrosination have been detected in interphase cells and during the mitotic cycle. Data from biochemical, immunological, and genetic studies have been compiled in this review; these are presented, with pertinent comments, with the hope of facilitating the comprehension of this particular aspect of the microtubule field.     

Posttranslational Modifications of Axonemal Tubulin

Axonemal tubulin exhibits a high degree of heterogeneity mostly due to several posttranslational modifications (PTM). The aim of this work was to chemically characterize the different PTM occurring in the C-terminal tail of axonemal tubulin purified from sea urchin, Paracentrotus lividus, spermatozoa. After its purification, tubulin was enzymatically cleaved. The C-terminal peptides were chromatographically isolated, first by anion exchange and then by reverse-phase HPLC. Peptides were characterized by their sequence, determined by Edman degradation, and by their mass, determined by MALDI-TOF/MS. The two major conclusions are that the majority of the isolated C-terminal peptides were unmodified and that polyglycylation and polyglutamylation can occur simultaneously on one molecule of -tubulin.     

Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium

Summary— Ciliates are very good models for studying post-translationally generated tubulin heterogeneity because they exhibit highly differentiated microtubular networks in combination with reduced genetic diversity. We have approached the analysis of tubulin heterogeneity in Paramecium through extensive isolation and characterization of monoclonal antibodies using various antigens and several immunization protocols. Eight monoclonal antibodies and 10 hybridoma supernatants were characterized by: i) immunoblotting on ciliate and pig brain tubulins as well as on peptide maps of Paramecium axonemal tubulin; ii) immunoblotting on ciliate tubulin fusion peptides generated in E coli, a procedure which allows in principle to discriminate antibodies that are directed against tubulin sequence (reactive on fusion peptides) from those directed against a post-translational epitope (non-reactive); and iii) immunofluorescence on Paramecium, 3T3 and PtK2 cells. Twelve antibodies labeled all microtubules in Paramecium cells and were found to be directed against tubulin primary sequences (nine of them being located in the α N-terminal domain, one in the β C-terminal one, and two in α and β central stretches). The remaining ones decorated only a specific subset of microtubules within the cell and were presumably directed against post-translational modifications. Among these, three antibodies are directed against an N-terminal acetylated epitope of α-tubulin whereas the epitopes of three other ones (TAP 952°, AXO 58 and AXO 49°) apparently correspond to still unidentified post-translational modifications, located in the C-terminal domain of both α- and β-tubulins. The AXO 49° specificity is similar to that of a previously described polyclonal serum raised against Paramecium axonemal tubulin [2]. The results are discussed in terms of identification and accessibility of the epitopes and immunogenicity of ciliate tubulin with reference to mammalian and ciliate tubulin sequences.     

Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L. cells

Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L. was analysed using a panel of specific antibodies. Polyglutamylated, tyrosinated, nontyrosinated, acetylated and Δ2-tubulin variants were detected on α-tubulin subunits; polyglutamylation was also found on β-tubulin subunits. Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts. They were, however, located differently in the various microtubule structures. The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and Δ2-tubulin provided dot-like staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and Δ2-tubulins was strong in the pole regions of mitotic spindles. High-resolution isoelectric focusing revealed 22 tubulin charge variants in N. tabacum suspension cells. Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of α- and β-tubulin molecules, respectively, revealed that 11 isoforms belonged to the α-subunit and 11 isoforms to the β-subunit. Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several α-tubulin isoforms, antibodies against nontyrosinated and Δ2-tubulin reacted with only one. The combined data demonstrate that plant tubulin is extensively post-translationally modified and that these modifications participate in the generation of plant tubulin polymorphism. Received: 2 May 1996 / Accepted: 16 September 1996     

Post-translational tubulin modifications in spermatogeneous cells of the pteridophyteCeratopteris richardii

Summary Acetylation and tyrosinization are post-translational modifications of tubulin generally associated, respectively, with highly stable or dynamic microtubule arrays in animals and protists. Little is known of these modifications in land plants, however. We examined the presence and distribution of post-translational tubulin modifications in developing spermatogenous cells of the pteridophyteCeratopteris richardii by immunofluorescence and immunogold, utilizing antibodies specific for acetylated and tyrosinated tubulin. Acetylated tubulin is found in mid to late stage spermatogenous cells in stable microtubule configurations: the spline, flagella, and basal bodies. Tyrosinated tubulin, a modification associated with dynamic microtubule arrays, is also present in these structures as well as all other microtubules in the cell. The lamellar strip of the multilayered structure, a body previously described as tubulin-containing, was not labelled by any of the tubulin antibodies or antiserum. Treatment of cultures with the microtubule stabilizer taxol results in the appearance of new arrays of microtubules, including bundles in the cytoplasm. Only those new taxol-induced microtubule arrays present in mid to late stage cells (i.e., those with other normally acetylated tubulin arrays) have acetylated domains. Younger spermatogenous cells had similar microtubule bundles but no acetylated tubulin. Tyrosinated tubulin was found in all these taxol-stabilized arrays. These data indicate that, although these pteridophyte cells have the ability to acetylate tubulin, that this ability is limited to stages after the final spermatogenous cell mitosis and is limited to the highly stable spline and flagella microtubules.Abbreviations LS lamellar strip of multilayered structure - MTOC microtubule organizing center     

纤毛微管蛋白翻译后修饰及其与纤毛病相关的研究进展

纤毛(cilia)是一种内含微管蛋白骨架、外被纤毛膜并从真核生物细胞表面凸起的毛发状结构;与生物体器官发育和稳态维持密切相关。纤毛微管蛋白经历了多种高度保守的翻译后修饰(posttranslational modifications;PTMs);包括乙酰化、谷氨酰化、甘氨酰化、去酪氨酸化等。研究表明;纤毛微管蛋白翻译后修饰能够精密调控纤毛组装/解聚的动态平衡、结构维持、运动功能以及信号转导等生理过程。纤毛微管蛋白翻译后修饰的异常调控会导致纤毛的功能障碍;从而引发包含纤毛病(ciliopathies)在内的人类疾病。本文综述了纤毛微管蛋白翻译后修饰调控纤毛形成与功能的研究进展;并重点探讨其与人类纤毛疾病的关联;以期为纤毛病的治疗干预提供新途径。     

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.     

Posttranslational chemistry of proteins of the GFP family

This review focuses on the current knowledge about posttranslational chemistry underlying the diverse optical properties of GFP-like proteins.     

Mammalian Sperm Tubulin: An Exceptionally Large Number of Variants Based on Several Posttranslational Modifications

Extraction of demembranated bull sperm flagella by SDS was used to maximize tubulin solubilization. The - and -tubulin separated by SDS-PAGE were treated with endoproteinases LysC and AspN, respectively. Carboxy-terminal fragments were isolated by Mono Q chromatography and reversed-phase HPLC. Automated sequencing and mass spectrometry revealed an astonishingly high number of tubulin variants. Many variants were due to polyglutamylation and in particular to polyglycylation. The number of side-chain glycyl residues ranged from 0 to 28 in and 0 to 15 in . Corresponding values for side-chain glutamyl residues were 0–6 in and 0–3 in . Additional variability was based on carboxy-terminal detyrosination and partial loss of the penultimate glutamate. A major glycylation site in - and -tubulin was mapped. Some variants seem to display both glycyl and glutamyl side chains.     

Posttranslational modifications of nerve cytoskeletal proteins in experimental diabetes

Axonal transport is known to be impaired in peripheral nerve of experimentally diabetic rats. As axonal transport is dependent on the integrity of the neuronal cytoskeleton, we have studied the way in which rat brain and nerve cytoskeletal proteins are altered in experimental diabetes. Rats were made diabetic by injection of streptozotocin (STZ). Up to six weeks later, sciatic nerves, spinal cords, and brains were removed and used to prepare neurofilaments, microtubules, and a crude preparation of cytoskeletal proteins. The extent of nonenzymatic glycation of brain microtubule proteins and peripheral nerve tubulin was assessed by incubation with³H-sodium borohydride followed by separation on two-dimensional polyacrylamide gels and affinity chromatography of the separated proteins. There was no difference in the nonenzymatic glycation of brain microtubule proteins from two-week diabetic and nondiabetic rats. Nor was the assembly of microtubule proteins into microtubules affected by the diabetic state. On the other hand, there was a significant increase in nonenzymatic glycation of sciatic nerve tubulin after 2 weeks of diabetes. We also identified an altered electrophoretic mobility of brain actin from a cytoskeletal protein preparation from brain of 2 week and 6 week diabetic rats. An additional novel polypeptide was demonstrated with a slightly more acidic isoelectric point than actin that could be immunostained with anti-actin antibodies. The same polypeptide could be produced by incubation of purified actin with glucose in vitro, thus identifying it as a product of nonenzymatic glycation. These results are discussed in relation to data from a clinical study of diabetic patients in which we identified increased glycation of platelet actin. STZ-diabetes also led to an increase in the phosphorylation of spinal cord neurofilament proteins in vivo during 6 weeks of diabetes. This hyperphosphorylation along with a reduced activity of a neurofilament-associated protein kinase led to a reduced incorporation of³²P into purified neurofilament proteins when they were incubated with³²P-ATP in vitro. Our combined data show a number of posttranslation modifications of neuronal cytoskeletal proteins that may contribute to the altered axonal transport and subsequent nerve dysfunction in experimental diabetes.     

Tubulin glycylases are required for primary cilia,control of cell proliferation and tumor development in colon

TTLL3 and TTLL8 are tubulin glycine ligases catalyzing posttranslational glycylation of microtubules. We show here for the first time that these enzymes are required for robust formation of primary cilia. We further discover the existence of primary cilia in colon and demonstrate that TTLL3 is the only glycylase in this organ. As a consequence, colon epithelium shows a reduced number of primary cilia accompanied by an increased rate of cell division in TTLL3-knockout mice. Strikingly, higher proliferation is compensated by faster tissue turnover in normal colon. In a mouse model for tumorigenesis, lack of TTLL3 strongly promotes tumor development. We further demonstrate that decreased levels of TTLL3 expression are linked to the development of human colorectal carcinomas. Thus, we have uncovered a novel role for tubulin glycylation in primary cilia maintenance, which controls cell proliferation of colon epithelial cells and plays an essential role in colon cancer development.     

Calmodulin binds to a tubulin binding site of the microtubule-associated protein tau

Previous studies have demonstrated that the microtubule - associated proteins MAP-2 and tau interact selectively with common binding domains on tubulin defined by the low-homology segments a (430–441) and (422–434). It has been also indicated that the synthetic peptide VRSKIGSTENLKHQPGGG corresponding to the first tau repetitive sequence represents a tubulin binding domain on tau. The present studies show that the calcium-binding protein calmodulin interacts with a tubulin binding site on tau defined by the second repetitive sequence VTSKCGSLGNIHHKPGGG. It was shown that both tubulin and calmodulin bind to tau peptide-Sepharose affinity column. Binding of calmodulin occurs in the presence of 1 mM Ca 2+ and it can be eluted from the column with 4 mM EGTA. These findings provide new insights into the regulation of microtubule assembly, since Ca 2+/calmodulin inhibition of tubulin polymerization into microtubules could be mediated by the direct binding of calmodulin to tau, thus preventing the interaction of this latter protein with tubulin.     

The role of 26S proteasome-dependent proteolysis in the formation and restructuring of microtubule networks

In this review, we summarize the evidence pointing at the important role of 26S proteasome-dependent proteolysis in the regulation of microtubule synthesis and microtubule dynamics. Because most of the advances in this relatively unexplored research field originate from yeast and animal studies, we have considered those studies that describe the role of proteolysis in processes that are evolutionarily conserved and known to exist in plants. In addition, we place particular emphasis on the proteasome-dependent degradation of plant-specific microtubule-associated protein SPIRAL1 and its function in MT rearrangements associated with salt stress.     

Characterization and analysis of posttranslational modifications of the human large cytoplasmic ribosomal subunit proteins by mass spectrometry and Edman sequencing

The 60S ribosomal proteins were isolated from ribosomes of human placenta and separated by reversed phase HPLC. The fractions obtained were subjected to trypsin and Glu-C digestion and analyzed by mass fingerprinting (MALDI-TOF), MS/MS (ESI), and Edman sequencing. Forty-six large subunit proteins were found, 22 of which showed masses in accordance with the SwissProt database (June 2002) masses (proteins L6, L7, L9, L13, L15, L17, L18, L21, L22, L24, L26, L27, L30, L32, L34, L35, L36, L37, L37A, L38, L39, L41). Eleven (proteins L7, L10A, L11, L12, L13A, L23, L23A, L27A, L28, L29, and P0) resulted in mass changes that are consistent with N-terminal loss of methionine, acetylation, internal methylation, or hydroxylation. A loss of methionine without acetylation was found for protein L8 and L17. For nine proteins (L3, L4, L5, L7A, L10, L14, L19, L31, and L40), the molecular masses could not be determined. Proteins P1 and protein L3-like were not identified by the methods applied.     

Writing and Reading the Tubulin Code

Microtubules give rise to intracellular structures with diverse morphologies and dynamics that are crucial for cell division, motility, and differentiation. They are decorated with abundant and chemically diverse posttranslational modifications that modulate their stability and interactions with cellular regulators. These modifications are important for the biogenesis and maintenance of complex microtubule arrays such as those found in spindles, cilia, neuronal processes, and platelets. Here we discuss the nature and subcellular distribution of these posttranslational marks whose patterns have been proposed to constitute a tubulin code that is interpreted by cellular effectors. We review the enzymes responsible for writing the tubulin code, explore their functional consequences, and identify outstanding challenges in deciphering the tubulin code.