In vivo incorporation of [14C]tyrosine into the C-terminal position of the α subunit of tubulin

In vitro incorporation of [¹⁴C]tyrosine into the C-terminal position of the α subunit of tubulin was not affected by 4 mm cycloheximide. This inhibitor of protein synthesis was used for in vivo experiments. The in vivo incorporation of [¹⁴C]tyrosine into soluble brain protein of cycloheximide-treated rats was 10% of that of untreated rats. Treatment with vinblastine sulfate of the soluble brain protein showed that the incorporation of [¹⁴C]tyrosine into tubulin was higher in cycloheximide-treated than in untreated rats with respect to the incorporation into the total soluble protein. In the case of cycloheximide-treated rats, about 60% of the radioactivity incorporated into protein was released by the action of carboxypeptidase A, whereas 10% was liberated from the protein of untreated rats. The radioactive compound released by the action of carboxypeptidase A was identified as [¹⁴C]tyrosine. The α and β subunits of tubulin from animals that received [¹⁴C]tyrosine were separated by polyacrylamide gel electrophoresis. The radiosactivity ratio of $\text{α}\text{β}$ subunits of tubulin from cycloheximide-treated rats was threefold higher than that of untreated rats. When a mixture of [¹⁴C]amino acids was injected, the radioactivity ratio of $\text{α}\text{β}$ subunits of tubulin was similar for cycloheximide-treated and untreated rats. The results reported are consistent with the assumption that the α subunit of tubulin can be tyrosinated in vivo.     

Zinc-induced self-assembly of goat brain tubulin: some novel aspects

Unlike normal microtubule assembly, the $\text{in vitro}$ assembly of DEAE-purified goat brain tubulin in presence of Zn(II) is not inhibited by suprastoichiometric concentrations of antimicrotubular drugs like colchicine and podophyllotoxin. However, assembly in the presence of Zn(II) is inhibited by vinblastine. Vinblastine sensitivity of the assembly process depends on the Mg(II) concentration in the assembly medium. Like normal microtubules, Zn(II)-induced polymers are sensitive to cold. The polymers assembled in presence of Zn(II) are readily disassembled on treatment with Zn(II)-chelators like EDTA or o-phenanthroline, indicating that the binding of Zn(II) to tubulin is essential for maintaining the polymeric structure.     

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.     

Resolution of cyclic AMP stimulated protein kinase from polymerization-purified brain microtubules.

Polymerization-deploymerization purified microtubules from mouse brain contain, in addition to tubulin, several minor proteins, including protein kinase activity. The protein kinase copurifies with microtubules in constant proportion to tubulin through two, three, or four cycles of polymerization; it can be resolved from tubulin by gel filtration chromatography and has an apparent molecular weight of 280,000. Its activity is stimulated 7-fold by cyclic AMP, and resembles the soluble brain protein kinase described by Miyamoto $\text{et al.}$ (1). The microtubule preparation serves as an endogenous substrate for this protein kinase; both 6S and 30S tubulin are substrates for phosphorylation to the extent of about 0.10 ± 0.05 moles/mole.     

The influence of microtubule associated proteins on the production of polymorphic products from tubulin and microtubules

Crude preparations of microtubule-associated proteins (MAPs), as well as purified MAP 2, influence the structure of products assembled from purified tubulin at low pH values. At pH 6.2, only 12% of the assembled products were microtubules (MTs) when assembly was conducted in 10% DMSO; 88% were large sheets of protofilaments. In the absence of DMSO, 28% of the structures were MTs. As the content of MAPs in the assembly reaction was increased, the proportion of MTs increased to 87% at a MAP/tubulin ($\text{w}\text{w}$) ratio of 0.67 in the presence of DMSO and to 98% at a MAP/tubulin ($\text{w}\text{w}$) ratio of 0.33 in the absence of DMSO. Purified MAP 2 was as effective as crude MAP preparations in promoting MT formation at pH 6.2. MTs formed from purified tubulin and MAP 2 were transformed into spirals of protofilaments upon the addition of Vinblastine (VLB). Spirals were also formed when VLB was added to a mixture of tubulin and MAP 2 at 4 ° C. It thus appears that MAP 2 is a causative factor in initiating spiral formation in the presence of VLB.     

Tubulin-like protein from Aspergillus nidulans

[³⁵S] labeled extracts of the fungus $\text{Aspergillus nidulans}$ were copolymerized with purified porcine brain tubulin. The [³⁵S] $\text{A. nidulans}$ protein which copurified with porcine microtubules was found to be similar to [³H] chick tubulin when the two were coelectrophoresed on several polyacrylamide gel electrophoresis systems. These results strongly suggest the presence in $\text{A. nidulans}$ of a tubulin-like protein.     

Three-dimensional reconstruction of tubulin in zinc-induced sheets.

The three-dimensional structure of porcine brain tubulin in planar sheets formed in the presence of zinc has been determined to a resolution of approximately 20 Å by electron microscopy and image reconstruction on negatively stained samples. The samples were prepared with a mica floatation technique, which yields tubulin sheets with 36 reciprocal space maxima on lattice lines at 21, 28, 42 and 84 Å^?1 in Fourier transforms of digitized images. In order to obtain three-dimensional data, sheets were tilted with the goniometer stage of the electron microscope to provide images at various angles between 0 ° and ± 60 °. Transforms of 33 tilted images plus the transform of untilted sheets based on an average of nine untilted images were combined to give the third dimension of reciprocal space ($\text{z}{}^1$). These data, were expressed in terms of the phases and amplitudes along the $\text{z}{}^1$ lattice line for each of the 36 maxima observed in untilted samples, as well as five additional lattice lines which have zero-amplitudes in the non-tilted central section of the three-dimensional transform. Home of these zero-amplitudes arise from systematic absences which are due to a 2-fold screw axis relating adjacent protofilaments of tubulin in the zinc-induced sheets. Thus in the three-dimensional reconstructions of the sheets a polarity of the protofilaments is apparent, with adjacent protofilaments aligned in opposite directions to give an antiparallel pattern, in contrast to normal microtubules composed of protofilaments in parallel alignment. Two classes of morphological units, each with a mass corresponding to a molecular weight of about 55,000, are found to alternate along the protofilaments. These distinct morphological units are identified as the α and β subunits of tubulin, confirming the representation of tubulin as an αβ heterodimer. Furthermore, the extensive internal contact between subunits within a dimer can readily be distinguished from the less extensive contact between dimer units. Such differences in contacts were not apparent in the earlier two-dimensional reconstructions. In addition, areas of excluded stain joining one class of subunits to the subunits of the other class in adjacent protofilaments have been resolved for tubulin polymerized in zinc-induced sheets. Of the two classes of subunits one is distinguished by a prominent cleft. Identification of which class of subunits is α and which is β is not yet possible.     

Autoregulation of tubulin expression is achieved through specific degradation of polysomal tubulin mRNAs

We have utilized protein synthesis inhibitors to investigate the autoregulatory mechanism that uses the concentration of unpolymerized tubulin subunits to specify tubulin mRNA content in animal cells. Puromycin and pactamycin, both of which remove RNAs from polysomes, completely unlink tubulin RNA content from the level of free subunits, whereas pretreatment of cells with cycloheximide, which traps mRNAs onto stalled polyribosomes, enhances the specific degradation of tubulin RNAs in response to increases in the subunit content. Moreover, in the absence of protein synthesis inhibitors, the tubulin RNAs that are lost from cells with elevated free tubulin subunit levels are those that are associated with polyribosomes. Further, beta-tubulin mRNAs encoding a truncated translation product of only 26 amino acids (and that cannot be polyribosomal) are not substrates for autoregulation. We conclude that autoregulation of tubulin synthesis is achieved by specifically altering the stability of tubulin RNAs that are bound to polyribosomes.     

Preferential action of a brain detyrosinolating carboxypeptidase on polymerized tubulin

A carboxypeptidase purified from brain catalyzes the release of COOH-terminal tyrosine without further digesting tubulin. It is distinct from previously described carboxypeptidases, and appears to have specificity for tubulin as it is not inhibited by peptides and proteins with COOH-terminal tyrosine, and because, unlike carboxypeptidase A (which by removing tyrosine from aldolase causes its inactivation), this enzyme does not decrease aldolase activity. The enzyme detyrosinolates both self-assembly-competent (cycle-purified) and -incompetent (phosphocellulose-purified) tubulin. However, under assembly conditions the rate was 2-3-fold higher for competent tubulin. Preincubation of assembly-competent tubulin with podophyllotoxin or colchicine resulted in a parallel concentration-dependent inhibition of tubulin polymerization and detyrosinolation. Similarly, when incompetent tubulin was induced to polymerize by preincubation with purified microtubule-associated protein 2 (an assembly-promoting protein) or taxol, the initial rate of its detyrosinolation increased 3-5-fold, and this increase was blocked if podophyllotoxin was also added along with microtubule-associated protein 2 or taxol during the preincubation. Oligomers induced by adding vinblastine to incompetent tubulin were also detyrosinolated more rapidly, and the stimulation was abolished by maytansine, which has been shown to disperse the vinblastine-induced oligomers. When polymerized and subunit fractions were separated after a steady state mixture had been partially digested with the carboxypeptidase, the former was found to have lost 2-3 times more COOH-terminal tyrosine. Although both polymer and monomer can be detyrosinolated by the enzyme, polymeric and oligomeric forms are the preferred substrates. Carboxypeptidase appeared to release tyrosine at the same rate from populations of short and long microtubules.     

Inhibition of the self-assembly of tubulin by diethylpyrocarbonate and photooxidation.

Chemical modification of tubulin by photooxidation and by reaction with diethylpyrocarbonate inhibits the $\text{in}\text{vitro}$ formation of microtubules. This inhibition apparently results from the modification of histidine residues, since the inhibition by diethylpyrocarbonate is reversed by hydroxylamine and the pH dependence of the rate of photooxidation shows the involvement of a group with a pKa value of about 6.5. The inhibition of self-assembly results from the modification of not more than three histidine residues. Sulfhydryl residues are not modified under the experimental conditions used. Colchicine and GTP binding by tubulin were not greatly affected under conditions which completely inhibited the polymerization.     

Interaction of oncodazole (R 17934), a new antitumoral drug, with rat brain tubulin.

Oncodazole (R 17934), methyl [5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl] carbamate (I), a new synthetic drug with anti-tumoral activity, inhibits the polymerization of rat brain tubulin $\text{in vitro}$. It has no depolymerizing effect on preformed microtubules $\text{in vitro}$. Binding studies by means of molecular sieving and equilibrium dialysis indicates that the drug binds to purified rat brain tubulin in a mole to mole ratio. Finally the drug competitively inhibits colchicine binding to purified rat brain tubulin. From these results the conclusion may be drawn that oncodazole is a true microtubule inhibitor.     

Tubulin tyrosylation in vivo and changes accompanying differentiation of cultured neuroblastoma-glioma hybrid cells.

Changes in a posttranslational modification of tubulin, which accompany differentiation, have been studied in neuroblastoma-glioma hybrid cultured cells. The modification consists of the reversible enzymatic addition of a tyrosine to the COOH terminus of the alpha chain. Cytoplasmic tubulin purified from undifferentiated cells resembled that from adult mammalian brain in that half was in a form which can not accept tyrosine; of the remainder, which is a substrate for tubulin-tyrosine ligase, a higher proportion had COOH-terminal tyrosine. In the tubulin from differentiated cells, in which there had been extensive assembly of axonal microtubules from a preformed pool of subunits, the nonsubstrate tubulin was almost entirely replaced by the species with COOH-terminal tyrosine. In living cells, in the absence of protein synthesis, there was fixation of labeled tyrosine into cytoplasmic alpha chains which was extensive enough to be consistent with turnover, during the course of an hour, of the pre-existing COOH-terminal tyrosine. The alpha chain in the particulate fraction of the cells was comparably labeled, along with some unidentified low molecular weight components.     

Interaction of Chlamydomonas dynein with tubulin

Studies were conducted to determine if dynein could bind to unpolymerized tubulin. Tubulin alone normally fractionated in the included volume of a molecular sieve Bio-Gel A-1.5m column. Incubated together, tubulin and dynein coeluted in the void volumn, suggesting that a complex had formed between the two. In addition, immunoelectron microscopy revealed preassembled microtubules were labeled with biotin antibody only when incubated in both dynein and biotinylated tubulin, evidence that dynein with bound biotinylated tubulin had decorated the microtubules. A fraction of the tubulin could be dissociated from dynein by addition of ATP and vanadate, as assayed by molecular sieve chromatography followed by densitometry of gels, suggesting that some tubulin bound to the B end of the dynein arm. Additional tubulin dissociated from the dynein under conditions of high salt. These studies, together with those indicating that tubulin blocked the A end of the dynein arm from binding to microtubules and promoted the interaction of two arms at their A ends, provide evidence that the A end of the arm also can bind tubulin. Thus, the tubulin subunits, themselves, on a microtubule rather than a particular surface lattice structure formed by adjacent protofilaments may provide the binding sites for both ends of the dynein arm.     

Release of C-terminal tyrosine from tubulin and microtubules at steady state.

Microtubule protein preparations purified by cycles of assembly-disassembly contain the enzyme tubulinyltyrosine carboxypeptidase (TTCPase). Using these preparations, containing tubulinyl[14C]tyrosine, we studied the release of [14C]tyrosine from assembled and non-assembled tubulin under steady-state conditions. It was found that both states of aggregation were detyrosinated at similar rates by the action of the endogenous TTCPase. However, practically no release of [14C]tyrosine from the non-assembled tubulin pool was found when microtubules were previously eliminated from the incubation mixture. These results indicated that non-assembled tubulin requires to interact with microtubules to be detyrosinated. This interaction seems to occur through the incorporation of dimers into microtubules, since when the capability of tubulin to incorporate into microtubules was diminished by binding of colchicine a concomitant decrease in the rate of release of tyrosine was observed. When detyrosination was accelerated by increasing the concentration of TTCPase relative to the microtubule protein concentration, microtubules were found to be detyrosinated faster than was non-assembled tubulin. Using exogenous TTCPase in an incubation system in which the formation of microtubules was not allowed, tubulinyl[14C]tyrosine and tubulinyl[14C]tyrosine-colchicine complex were shown to have similar capabilities to act as substrates for this enzyme. Free colchicine was shown not to affect the activity of TTCPase.     

Direct interaction between prion protein and tubulin

Recently published data show that the prion protein in its cellular form (PrP(C)) is a component of multimolecular complexes. In this report, zero-length cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) allowed us to identify tubulin as one of the molecules interacting with PrP(C) in complexes observed in porcine brain extracts. We found that porcine brain tubulin added to these extracts can be cross-linked with PrP(C). Moreover, we observed that the 34 kDa species identified previously as full-length diglycosylated prion protein co-purifies with tubulin. Cross-linking of PrP(C) species separated by Cu(2+)-loaded immobilized metal affinity chromatography confirmed that only the full-length protein but not the N-terminally truncated form (C1) binds to tubulin. By means of EDC cross-linking and cosedimentation experiments, we also demonstrated a direct interaction of recombinant human PrP (rPrP) with tubulin. The stoichiometry of cosedimentation implies that rPrP molecules are able to bind both the alpha- and beta-isoforms of tubulin composing microtubule. Furthermore, prion protein exhibits higher affinity for microtubules than for unpolymerized tubulin.     

Tyrosine phosphorylation of plant tubulin

Phosphorylation of αβ-tubulins dimers by protein tyrosine kinases plays an important role in the regulation of cellular growth and differentiation in animal cells. In plants, however, the role of tubulin tyrosine phosphorylation is unknown and data on this tubulin modification are limited. In this study, we used an immunochemical approach to demonstrate that tubulin isolated by both immunoprecipitation and DEAE-chromatography is phosphorylated on tyrosine residues in cultured cells of Nicotiana tabacum. This opens up the possibility that tyrosine phosphorylation of tubulin could be involved in modulating the properties of plant microtubules.     

Microtubule formation from maternal tubulins during sea urchin embryogenesis: measurement of soluble and insoluble tubulin pools

The mass of tubulin protein in developing embryos of the sea urchin Lytechinus pictus was measured using a radiodilution immunoassay based on densitometric analysis of immunoprecipitated tubulins resolved electrophoretically. The tubulins constitute an average of 360 +/- 35 pg per egg, or 0.66% of the total protein, and there is no significant change in their concentration during embryogenesis. The masses of soluble and polymerized tubulin were measured for extracts prepared under conditions that stabilize microtubules. In eggs, a maximum of 14% of the tubulin is insoluble, and this increases throughout embryogenesis to 67% at pluteus stage (72 hr). The concentration of tubulin in eggs is at least 500 micrograms/ml, well above the critical concentration for tubulin assembly in vitro, yet microtubules have not been observed in eggs. The mass of newly synthesized tubulin, estimated from the mass of tubulin mRNA per embryo, accounts for a small fraction of the total tubulin by the end of gastrulation but for over half of the tubulin by the 72-hr pluteus stage. These observations are consistent with a model in which the declining level of unpolymerized tubulin controls the stability of tubulin mRNa, providing an autogenous regulation of the ontogenetic pattern of tubulin synthesis during sea urchin embryogenesis (Gong and Brandhorst, Development 102: 31-43).     

Interaction of tyrosine hydroxylase with tubulin

Bovine adrenal medulla tyrosine hydroxylase associates with microtubules during tubulin assembly. Limited proteolytic digestion of tyrosine hydroxylase does not affect the enzymatic activity but prevents its association with tubulin. A possible interpretation is that an ionic interaction occurs between microtubules and a negatively charged region of the enzyme which is removed by the protease treatment. Tyrosine hydroxylase is able to induce purified tubulin assembly as do the microtubule associated proteins; however, the association induced by tyrosine hydroxylase corresponds to the formation of aggregates or organized structures different from microtubules. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and electron microscopy of proteins obtained from bovine adrenal medulla show the presence of tubulin in this tissue.     

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.