Stabilization of post-translational modification of microtubules during cellular morphogenesis

This review discusses the possible role of alpha-tubulin detyrosination, a reversible post-translational modification that occurs at the protein's C-terminus, in cellular morphogenesis. Higher eukaryotic cells possess a cyclic post-translational mechanism by which dynamic microtubules are differentiated from their more stable counterparts; a tubulin-specific carboxypeptidase detyrosinates tubulin protomers within microtubules, while the reverse reaction, tyrosination, is performed on the soluble protomer by a second tubulin-specific enzyme, tubulin tyrosine ligase. In general, the turnover of microtubules in undifferentiated, proliferating cells is so rapid that the microtubules accumulate very little detyrosinated tubulin; that is, they are enriched in tyrosinated tubulin. However, an early event common to at least three well-studied morphogenetic events--myogenesis, neuritogenesis, and directed cell motility--is the elaboration of a polarized array of stable microtubules that become enriched in detyrosinated tubulin. The formation of this specialized array of microtubules in specific locations in cells undergoing morphogenesis suggests that it plays an important role in generating cellular asymmetries.     

微管蛋白酪氨酸连接酶类似物12通过干扰微管蛋白酪氨酸硝基化促进Hep-2细胞生长

硝基化酪氨酸与酪氨酸在结构上相似,它在病理情况下会出现,并在细胞内与微管蛋白结合,从而阻碍微管的正常功能. 硝基化酪氨酸在肿瘤中的作用,目前研究甚少.本文利用头颈鳞癌Hep-2细胞株,研究微管蛋白酪氨酸连接酶类似物12（tubulin tyrosine ligase like 12,TTLL12）和硝基化酪氨酸对头颈鳞癌Hep-2生长的影响,通过Western 印迹试验和MTT试验发现,随着硝基化酪氨酸的浓度升高,细胞内生成的硝基化酪氨酸微管蛋白含量也增高,同时细胞生长受抑制的程度显著增高; 对建立的TTLL12高表达细胞株加入硝基化酪氨酸培养,结果显示,TTLL12高表达细胞株内的硝基化酪氨酸微管蛋白含量明显低于对照组细胞;对照组细胞的生长明显受到抑制,而高表达细胞株的生长无明显改变,两者的细胞生长有显著性差异（P＜0.05）.本研究结果提示,TTLL12可通过阻碍硝基化酪氨酸与微管蛋白的结合,使头颈鳞癌Hep-2细胞逃避硝基化酪氨酸的打击. 对这一调控机制的进一步研究,必将有助于控制肿瘤细胞的生长,为治疗肿瘤寻找到新的治疗靶点.     

Rapid and reversible tubulin tyrosination in human neutrophils stimulated by the chemotactic peptide,fMet-Leu-Phe

Neutrophil activation by specific stimuli, such as the oligopeptide chemotactic factor fMet-Leu-(fMLF), is associated with an increased enzymatic addition of tyrosine to tubulin α -subunits, as measured by ¹⁴C tyrosine uptake. In studies using immunoblots we have found that this increased tyrosine uptake into tubulin in activated neutrophils reflects an increase in the proportion of cellular tubulin that is tyrosinated rather than simply an increase in the turnover of tyrosinated subunits. However, the increased accumulation of tyrosinated tubulin was also found to follow an initial depletion of tyrosinated tubulin and concomitant increase in detyrosinated tubulin between 0 and 60 sec following stimulation of neutrophils with fMLF. Immunogold electron microscopy studies of intact micro tubules recovered from activated neutrophils demonstrated that these rapid changes in the relative content of tubulin isoforms in the cells were not associated with the formation or disappearance of microtubule microdomains composed of only one form of tubulin. Previously, we have shown that under conditions of fMLF-stimulated exocytosis there is an increased binding of neutrophil granules to endogenous microtubules. Since neutrophil activation by fMLF is associated with increased tyrosination of α -tubulin subunits, we speculated that rapid changes in the levels of tyrosinated tubulin in the microtubules of activated neutrophils might have a role in the regulation of granule-microtubule interactions. When the binding of purified neutrophil granules to reconstituted rat brain microtubules containing approximately 50% tyrosinated tubulin was measured by electron microscopy and compared with granule binding to microtubules that contained no detectable tyrosinated tubulin, granule-microtubule associations were found to be significantly favored by detyrosinated vs. tyrosinated tubulin. These findings indicate that interactions between cytoplasmic granules and microtubules in activated neutrophils may be modulated by rapid changes in the relative content of detyrosinated and tyrosinated tubulin in the microtubule network of the cells. © 1993 Wiley-Liss, Inc.     

CAPABILITY OF TUBULIN AND MICROTUBULES TO INCORPORATE AND TO RELEASE TYROSINE AND PHENYLALANINE AND THE EFFECT OF THE INCORPORATION OF THESE AMINO ACIDS ON TUBULIN ASSEMBLY

Abstract— Incorporation of [¹⁴C]tyrosine into the C-terminal position of α-tubulin of rat brain cytosol was 10-fold higher for non-assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non-assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.
[¹⁴C]Tyrosine was released from assembled and non-assembled [¹⁴C]tyrosinated tubulin by the action of an endogenous carboxypeptidase. Release from non-assembled tubulin was shown by incubating a tubulinyl-[¹⁴C]tyrosine preparation in the presence of CaCl₂ at a concentration that abolished microtubule formation. Release from microtubules was inferred from the observation that the percentages of [¹⁴C]tyrosine released and the decrease of the specific radioactivity of the recovered microtubules were practically identical and did not change after a 10-fold dilution of the incubated microtubules.
[³H]Phenylalanine was released from a preparation of tubulinyl-[³H]phenylalanine also by an enzymatic activity.
The capacity of a tubulin preparation to incorporate tyrosine was increased 43% by pre-treatment with endogenous carboxypeptidase.
Tubulin tyrosinated in vitro was assembled to the same extent as native tubulin. After a mixture of tubulinyl-[¹⁴C]tyrosine and tubulinyl-[³H]phenylalanine was partially assembled, the ratio of ¹⁴C/³H found in the microtubules was the same as in the non-assembled tubulin fraction.     

Inhibitors of protein phosphatase 1 and 2A decrease the level of tubulin carboxypeptidase activity associated with microtubules.

The association of tubulin carboxypeptidase with microtubules may be involved in the determination of the tyrosination state of the microtubules, i.e. their proportion of tyrosinated vs. nontyrosinated tubulin. We investigated the role of protein phosphatases in the association of carboxypeptidase with microtubules in COS cells. Okadaic acid and other PP1/PP2A inhibitors, when added to culture medium before isolation of the cytoskeletal fraction, produced near depletion of the carboxypeptidase activity associated with microtubules. Isolation of the native assembled and nonassembled tubulin fractions from cells treated and not treated with okadaic acid, and subsequent in vitro assay of the carboxypeptidase activity, revealed that the enzyme was dissociated from microtubules by okadaic acid treatment and recovered in the soluble fraction. There was no effect by nor-okadaone (an inactive okadaic acid analogue) or inhibitors of PP2B and of tyrosine phosphatases which do not affect PP1/PP2A activity. When tested in an in vitro system, okadaic acid neither dissociated the enzyme from microtubules nor inactivated it. In living cells, prior stabilization of microtubules with taxol prevented the dissociation of carboxypeptidase by okadaic acid indicating that dynamic microtubules are needed for okadaic acid to exert its effect. On the other hand, stabilization of microtubules subsequent to okadaic acid treatment did not reverse the dissociating effect of okadaic acid. These results suggest that dephosphorylation (and presumably also phosphorylation) of the carboxypeptidase or an intermediate compound occurs while it is not associated with microtubules, and that the phosphate content determines whether or not the carboxypeptidase is able to associate with microtubules.     

Miniaturization and validation of a sensitive multiparametric cell-based assay for the concomitant detection of microtubule-destabilizing and microtubule-stabilizing agents

The authors describe a cell-based assay for anti-microtubule compounds suitable for automation. This assay allows the identification, in a single screening campaign, of both microtubule-destabilizing and microtubule-stabilizing agents. Its rationale is based on the substrate properties of the tubulin-modifying enzymes involved in the tubulin tyrosination cycle. This cycle involves the removal of the C-terminal tyrosine of the tubulin alpha-subunit by an ill-defined tubulin carboxypeptidase and its readdition by tubulin tyrosine ligase. Because of the substrate properties of these enzymes, dynamic microtubules, sensitive to depolymerizing drugs, are composed of tyrosinated tubulin, whereas non-dynamic, stabilized microtubules are composed of detyrosinated tubulin. Thus depolymerization or stabilization of the microtubule network can easily be detected with double-immunofluorescence staining using antibodies specific to tyrosinated and detyrosinated tubulin. The authors have scaled this assay to the 96-well plate format and adapted its process for an automated handling, including a readout using a microplate reader. They describe the different steps of this adaptation. This assay was validated using known compounds. This new cell-based assay represents an alternative to both global cytotoxicity assays and in vitro tubulin assembly assays commonly used for the detection of microtubule poisons.     

3-nitrotyrosine attenuates respiratory syncytial virus infection in human bronchial epithelial cell line

3-nitrotyrosine (NO2Tyr), an L-tyrosine derivative during nitrative stress, can substitute the COOH-terminal tyrosine of alpha-tubulin, posttranslationally altering microtubular functions. Because infection of the cells by respiratory syncytial virus (RSV) may require intact microtubules, we tested the hypothesis that NO2Tyr would inhibit RSV infection and intracellular signaling via nitrotyrosination of alpha-tubulin. A human bronchial epithelial cell line (BEAS-2B) was incubated with RSV with or without NO2Tyr. The release of chemokines and viral particles and activation of interferon regulatory factor-3 (IRF-3) were measured. Incubation with NO2Tyr increased nitrotyrosinated alpha-tubulin, and NO2Tyr colocalized with microtubules. RSV-infected cells released viral particles, RANTES, and IL-8 in a time- and dose-dependent manner, and intracellular RSV proteins coprecipitated with alpha-tubulin. NO2Tyr attenuated the RSV-induced release of RANTES, IL-8, and viral particles by 50-90% and decreased alpha-tubulin-associated RSV proteins. 3-chlorotyrosine, another L-tyrosine derivative, had no effects. NO2Tyr also inhibited the RSV-induced shift of the unphosphorylated form I of IRF-3 to the phosphorylated form II. Pre-exposure of the cells to NO(2) (0.15 ppm, 4 h), which produced diffuse protein tyrosine nitration, did not affect RSV-induced release of RANTES, IL-8, or viral particles. NO2Tyr did not affect the potential of viral spreading to the neighboring cells since the RSV titers were not decreased when the uninfected cells were cocultured with the preinfected cells in NO2Tyr-containing medium. These results indicate that NO2Tyr, by replacing the COOH-terminal tyrosine of alpha-tubulin, attenuated RSV infection, and the inhibition appeared to occur at the early stages of RSV infection.     

Tyrosination State of Tubulin and the Activity of Tubulin:Tyrosine Ligase and Tubulin Carboxypeptidase in the Developing Retina of the Chick

The tyrosination state of tubulin and the enzymes involved in the tubulin tyrosination/detyrosination cycle--tubulin:tyrosine ligase and tubulin carboxypeptidase--were determined in chick retina during development. The amount of tyrosinable (tyrosinated plus detyrosinated) tubulin increased approximately 110% from embryonic day 7 to 14. Then it decreased, and by day 19 it was similar to the value on day 7. This result did not change after hatching, at least up to day 20. The proportion of tyrosinated and detyrosinated tubulin significantly changed with the development of the animal. At embryonic day 7, these tubulin species were at a proportion of 70 and 30%, respectively, and after hatching, the values inverted, to 30 and 70%, respectively. This change did not correlate with the activity of the ligase relative to that of the carboxypeptidase, as measured in vitro. This observation suggested that a change in the turnover rate of microtubules, in the proportion of assembled and nonassembled tubulin pools, or in both had occurred. Coincident with the last possibility, the proportion of assembled tubulin was found to increase during the development of the animal. This finding suggests that the tyrosination state of tubulin may be determined, at least in part, by the assembly state.     

Differential Distribution of Posttranslationally Modified Microtubules in Osteoclasts

The differential distribution of microtubules in osteoclasts in culture was examined by using antibodies against acetylated, tyrosinated, or detyrosinated tubulins. Tyrosinated tubulin was found throughout the cytoplasmic microtubules in all cells examined. An expanding protrusion that contained tyrosinated tubulin but none of the detyrosinated or acetylated form was seen in the immature osteoclasts. Detyrosinated or acetylated tubulin was detectable in the peripheral cytoplasm of the mature osteoclasts displaying the loss of the expanding protrusion. Although most of the microtubules were derived from the centrosome, noncentrosomal microtubules were distributed in the expanding protrusion, which was predominantly positive for tyrosinated tubulin. By tracing single microtubules, the authors found that their growing ends were always rich in tyrosinated tubulin subunits. End binding protein 1 bound preferentially to the microtubule ends. Both acetylated and tyrosinated microtubules were shown to be closely associated with podosomes. Microtubules appeared to grow over or into the podosomes; in addition, the growing ends of single microtubules could be observed to target the podosomes. Moreover, a microtubule-associated histone deacetylase 6 was localized in the podosomes of the osteoclast. On the basis of these results, the authors conclude that posttranslational modifications of microtubules may correlate with characteristic changes in podosome dynamics in osteoclasts.     

Tyrosination of α‐tubulin controls the initiation of processive dynein–dynactin motility

Post‐translational modifications (PTMs) of α/β‐tubulin are believed to regulate interactions with microtubule‐binding proteins. A well‐characterized PTM involves in the removal and re‐ligation of the C‐terminal tyrosine on α‐tubulin, but the purpose of this tyrosination–detyrosination cycle remains elusive. Here, we examined the processive motility of mammalian dynein complexed with dynactin and BicD2 (DDB) on tyrosinated versus detyrosinated microtubules. Motility was decreased ~fourfold on detyrosinated microtubules, constituting the largest effect of a tubulin PTM on motor function observed to date. This preference is mediated by dynactin's microtubule‐binding p150 subunit rather than dynein itself. Interestingly, on a bipartite microtubule consisting of tyrosinated and detyrosinated segments, DDB molecules that initiated movement on tyrosinated tubulin continued moving into the segment composed of detyrosinated tubulin. This result indicates that the α‐tubulin tyrosine facilitates initial motor–tubulin encounters, but is not needed for subsequent motility. Our results reveal a strong effect of the C‐terminal α‐tubulin tyrosine on dynein–dynactin motility and suggest that the tubulin tyrosination cycle could modulate the initiation of dynein‐driven motility in cells.     

Assembly and turnover of detyrosinated tubulin in vivo

Detyrosinated (Glu) tubulin was prepared from porcine brain and microinjected into human fibroblasts and Chinese hamster ovary (CHO) cells. Glu tubulin assembled onto the ends of preexisting microtubules and directly from the centrosome within minutes of its microinjection. Incorporation into the cytoskeleton continued until almost all of the microtubules were copolymers of Glu and tyrosinated (Tyr) tubulin. However, further incubation resulted in the progressive and ultimately complete loss of Glu-staining microtubules. Glu tubulin injected into nocodazole-treated cells was converted to Tyr tubulin by a putative tubulin/tyrosine ligase activity. The observed decrease in staining with the Glu antibody over time was used to analyze microtubule turnover in microinjected cells. The mode of Glu disappearance was analyzed quantitatively by tabulating the number of Glu-Tyr copolymers and Tyr-only microtubules at fixed times after injection. The proportion of Glu-Tyr copolymers decreased progressively over time and no segmentally labeled microtubules were observed, indicating that microtubules turn over rapidly and individually. Our results are consistent with a closely regulated tyrosination-detyrosination cycle in living cells and suggest that microtubule turnover is mediated by dynamic instability.     

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 tubulin in teleost photoreceptor cytoskeletons

1. Posttranslational modifications of tubulin by acetylation and detyrosination have been correlated previously with microtubule stability in numerous cell types. 2. In this study, posttranslational modifications of tubulin and their regional distribution within teleost photoreceptor cones and rods are demonstrated immunohistochemically using antibodies specific for acetylated, detyrosinated, or tyrosinated tubulin. 3. Immunolocalization was carried out on isolated whole cones and mechanically detached rod and cone inner/outer segments. 4. Acetylated tubulin within rods and cones is found only in microtubules of the ciliary axoneme of the outer segment. Detyrosinated tubulin is also enriched in axonemes of both rod and cone outer segments. 5. Distributions of tyrosinated and detyrosinated cytoplasmic microtubules differ within cones and rods. In cones, detyrosinated and tyrosinated tubulins are both abundant throughout the cell body. In rods, the ellipsoid and myoid contain much more tyrosinated tubulin than detyrosinated tubulin. Comparisons between whole cones and cone fragments suggest that detyrosinated microtubules are more stable than tyrosinated microtubules in teleost photoreceptors. 6. Our findings provide further evidence that microtubules of teleost cones differ from rod microtubules in their stabilities and rapidity of turnover within the photoreceptor inner segment.     

Age-related changes in microtubules in the guinea pig organ of Corti

Biochemical and immunocytochemical analyses have been used to provide new insights into age-related changes in the sensory and supporting cells of the guinea pig organ of Corti. Quantitative densitometry of immunoblots showed that, while levels of alpha-tubulin remained relatively constant in guinea pigs from 3 weeks to 18 months old, there were progressive shifts in some tubulin isoforms. Levels of tyrosinated tubulin increased with age, nontyrosinatable tubulin (delta2-tubulin) showed a compensatory decrease, but detyrosinated tubulin did not change; acetylated, polyglutamylated, and glycylated tubulin levels also decreased. Immunolabeled tissue sections showed that cell type-specific distribution of tubulin seen in young guinea pigs (tyrosinated in the microtubules of the sensory cells, and post-translationally modified isoforms in the supporting cells) did not change as animals aged. However, there were age-related decreases in labeling for alpha-tubulin and all post-translationally modified isoforms. Biochemical and immunocytochemical results both support an age-related decrease in the number and/or length of microtubules as well as an increase in the pool of soluble tyrosinated and detyrosinated tubulin. They further suggest that microtubules containing nontyrosinatable tubulin from older animals are the sites for further modification of tubulin by acetylation, polyglutamylation, and glycylation. Changes in tubulin isoform levels and stability of microtubules in the organ of Corti may alter its micromechanical properties; the resulting changes in conduction of sound-induced vibration would provide one mechanism for age-related hearing loss.     

Tyrosinated, but not detyrosinated, α-tubulin is present in root tip cells

Summary The distribution of tyrosinated and detyrosinated tubulin in microtubule arrays of pine and onion cells was investigated by immunofluorescence techniques. Staining of isolated cells and methacrylate sections ofPinus radiata andAllium cepa root tips indicated that all microtubule structures contained tyrosinated tubulin but not the posttranslationally modified detyrosinated tubulin. The detyrosinated tubulin epitope was, however, created in vitro by treating both sections and fixed whole cells with carboxypeptidase A.     

Adenylate cyclase and cyclic AMP phosphodiesterase activity during the mitotic cycle of Physarum polycephalum.

Tyrosine, as well as small amounts of phenylalanine, were removed selectively and quantitatively from purified chick brain tubulin by enzymatic digestion with carboxypeptidase. The fraction of molecules containing hydrolyzable tyrosine changed with the stage of development and had the highest value (～0.5) around days 14–16 of the embryo. The increase in the fraction of tyrosinated molecules was found to be temporally correlated with an increased specific activity of the enzyme catalyzing the incorporation reaction. In addition, it was found that the availability of α-chain C-termini for $\text{in vitro}$ tyrosination also reached a maximum during the same period. Changes in the extent of modification of the C-terminus of tubulin may be relevant for the participation of the resultant microbubules in different developmental events.     

Enzymatic Detyrosination of Tubulin Tyrosinated in Rat Brain Slices and Extracts

Abstract: Tubulin was tyrosinated in slices and in extracts of brain of rats of 3, 25, and 120 days of age by successive incorporation of [¹⁴C]tyrosine and [³H]-tyrosine, respectively. The release of the incorporated amino acid was measured by using tubulinyl-tyrosine carboxypeptidase, carboxypeptidase A, and tubulin-tyrosine ligase. With the carboxypeptidases no differences in either the rates or the extents of the release of tyrosine between these two differently labeled tubulins were found. Differences were found when the detyrosination was catalyzed by the ligase and these were attributed to a higher inactivation of tubulin labeled in slices than of that labeled in extracts.     

Tyrosination of microtubules and non-assembled tubulin in brain slices

Brain slices were used to examine comparatively the incorporation of [14C]tyrosine into the C terminus of alpha-tubulin of the microtubule and non-assembled tubulin pools. We found that the incorporation of [14C]tyrosine from 5 min up to 60 min of incubation was higher in microtubules than in non-assembled tubulin. The possibility that this result was due to the activity of tubulin carboxypeptidase or tubulin:tyrosine ligase during the in vitro isolation of tubulin was discarded. We also found that tubulin:tyrosine ligase was mainly associated with microtubules when brain slices were homogenized under microtubule-preserving conditions. Conversely the enzyme behaved as a soluble entity when homogenization was performed under conditions that do not preserve microtubules. In addition, soluble tubulin:tyrosine ligase did not become sedimentable when in vitro conditions were changed to induce the formation of microtubules. The results presented in this work indicate the possibility that, in vivo, microtubules and not tubulin dimers are the major substrate for tubulin:tyrosine ligase. This is in contrast with previous findings from in vitro experiments, which showed a preference of the ligase for non-assembled tubulin.     

A tale of two α‐tubulin tails

Post‐translational modifications of tubulin, such as the removal of the C‐terminal tyrosine of α‐tubulin, have long been proposed to influence the ability of microtubule motors to walk along the microtubule surface. This hypothesis has now been tested for cytoplasmic dynein‐1 (dynein), revealing that active dynein–dynactin–adaptor complexes prefer to start moving on tyrosinated microtubules. This choice is governed by the p150 subunit of dynactin. Once moving, however, dynein is not choosy about whether the microtubule is tyrosinated or not.     

The contributions of microtubules and F-actin to the in vitro migratory mechanisms of Hydra nematocytes as determined by drug interference experiments.

In order to investigate the contributions of microtubules and of F-actin to the in vitro migration mechanisms of Hydra nematocytes we have studied the effects of agents directed against cytoskeletal structures. Disassembly of microtubules by treatment with the drug nocodazole in moving nematocytes resulted in the loss of all locomotory activity within 20 min after the onset of treatment and in the detachment from the substratum after about 30 min. Depolymerization of microtubules by exposure to low temperatures had the same effect but was reversible in this case. Locomoting cells treated with cytochalasin D, which disrupts the actin filaments, stopped movement 2 min after drug administration and detached from the substratum after 15 min. The pattern of F-actin, alpha-tubulin, and tyrosinated tubulin in drug- or cold-treated cells was determined by immunocytochemical techniques and confocal laser scanning microscopy. These patterns and the reactions of the cells to the various drug treatments suggest that both actin filaments and microtubules play a crucial role in nematocyte locomotion. Analysis of the cytoskeletal pattern in drug-treated cells shows that the microtubules which are involved in locomotion are mostly tyrosinated. Furthermore it is suggested that microtubules and actin filaments interact with each other during the locomotion of nematocytes.