Epothilone and paclitaxel: unexpected differences in promoting the assembly and stabilization of yeast microtubules

Paclitaxel (Taxol) and the epothilones are antimitotic agents that promote the assembly of mammalian tubulin and stabilization of microtubules. The epothilones competitively inhibit the binding of paclitaxel to mammalian brain tubulin, suggesting that the two types of compounds share a common binding site in tubulin, despite the lack of structural similarities. It is known that paclitaxel does not stabilize microtubules formed in vitro from Saccharomyces cerevisiae tubulin; thus, it would be expected that the epothilones would not affect yeast microtubules. However, we found that epothilone A and B do stimulate the formation of microtubules from purified yeast tubulin. In addition, epothilone B severely dampens the dynamics of yeast microtubules in vitro in a manner similar to the effect of paclitaxel on mammalian microtubules. We used current models describing paclitaxel and epothilone binding to mammalian beta-tubulin to explain why paclitaxel apparently fails to bind to yeast tubulin. We propose that three amino acid substitutions in the N-terminal region and at position 227 in yeast beta-tubulin weaken the interaction of the 3'-benzamido group of paclitaxel with the protein. These results also indicate that mutagenesis of yeast tubulin could help define the sites of interaction with paclitaxel and the epothilones.     

Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.

We cloned the beta-tubulin gene of Neurospora crassa from a benomyl-resistant strain and determined its nucleotide sequence. The gene encodes a 447-residue protein which shows strong homology to other beta-tubulins. The coding region is interrupted by six introns, five of which are within the region coding for the first 54 amino acids of the protein. Intron position comparisons between the N. crassa gene and other fungal beta-tubulin genes reveal considerable positional conservation. The mutation responsible for benomyl resistance was determined; it caused a phenylalanine-to-tyrosine change at position 167. Codon usage in the beta-tubulin gene is biased, as has been observed for other abundantly expressed N. crassa genes such as am and the H3 and H4 histone genes. This bias results in pyrimidines in the third positions of 96% of the codons in codon families in which there is a choice between purines and pyrimidines in this position. Bias is also evident by the absence of 19 of the 61 sense codons. We demonstrated that benomyl resistance is due to the cloned beta-tubulin gene of strain Bml511(r)a and that this gene can be used as a dominant selectable marker in N. crassa transformation.     

Mutations at leucine 215 of beta-tubulin affect paclitaxel sensitivity by two distinct mechanisms

Paclitaxel resistance mutations in Chinese hamster ovary cells frequently alter a cluster of leucine residues in the H6-H7 loop region of beta-tubulin. To gain further insight into the role of this region in microtubule assembly and drug resistance, site-directed mutagenesis was used to systematically change amino acid L215. The mutated genes were cloned into a tetracycline-regulated expression vector and transfected into wild-type cells. Most of the mutations destabilized microtubule assembly, causing a decreased fraction of tubulin to appear in the microtubule cytoskeleton. In each case, the decreased level of assembly was associated with paclitaxel resistance and increased colcemid sensitivity. In two cases, however, the alteration did not significantly perturb the level of assembled tubulin or confer resistance to paclitaxel. One of these, L215V, produced little or no detectable phenotype, while the other, L215I, conferred increased sensitivity to paclitaxel. The increased drug sensitivity did not extend to epothilone A, a drug that binds to the same site and has a mechanism of action similar to that of paclitaxel, or colcemid, a drug with an opposing mechanism of action and a distinct binding site. Moreover, L215I conferred enhanced paclitaxel sensitivity at very low levels of expression, and sensitivity was not further enhanced in cells with higher levels of expression, implying that paclitaxel acts substoichiometrically. These properties, along with the proximity of L215 to the drug binding site, suggests that the L215I substitution may enhance the binding or effectiveness of paclitaxel. Our studies confirm the importance of the H6-H7 loop of beta-tubulin in microtubule assembly and resistance to antimitotic drugs. They also identify the first mammalian mutation shown to specifically increase sensitivity to paclitaxel.     

Tubulins in the primate retina: evidence that xanthophylls may be endogenous ligands for the paclitaxel-binding site

The xanthophylls-lutein, zeaxanthin, and meso-zeaxanthin (L&Z)-are found in the central region of the primate retina, which is called the macula lutea (yellow spot). How they are anchored there and what their function is has been debated for over 50 years. Here, we present evidence that they may be bound to the paclitaxel (Taxol) binding site of the beta-tubulin subunit of microtubules and that a major function may be to modulate the dynamic instability of microtubules in the macula. Also, we compare nucleic acid and amino acid sequences of tubulins that are in human brain with those we have isolated from human-retina and monkey-macula cDNA libraries. In so doing, we suggest that in primates, class I beta-tubulin consists of at least two subtypes (beta(Ia) and beta(Ib)). Alignment analysis of the sequences of the genes for beta(Ia) and beta(Ib) indicates that the corresponding mRNAs may have other functions in addition to that of coding for proteins. Furthermore, we show that there are at least five different types of beta-tubulin in the macula lutea of rhesus monkey.     

Heightened sensitivity to paclitaxel in Class IVa beta-tubulin-transfected cells is lost as expression increases

Stably transfected Chinese hamster ovary cell lines expressing increasing levels of beta4a, a class IV neuronal-specific beta-tubulin, were compared for effects on microtubule organization, assembly, and sensitivity to antimitotic drugs. It was found that beta4a reduced microtubule assembly in proportion to its abundance and thereby caused supersensitivity to microtubule disruptive drugs such as colcemid, vinblastine, and nocodazole. However, the response to paclitaxel was more complex. Low expression of beta4a caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that beta4a may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of beta4a expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by paclitaxel binding, and drug supersensitivity is lost. beta4a-Tubulin differs from the more ubiquitous beta4b isotype at relatively few amino acid residues, yet beta4b expression has little effect on microtubule assembly or drug response. To determine which amino acids mediate the effects of beta4a expression, beta4a and beta4b were altered by site-directed mutagenesis and expressed in Chinese hamster ovary cells. The introduction of N332S or N335S mutations into beta4b-tubulin was sufficient to confer microtubule disruption and increased colcemid sensitivity. On the other hand, mutation of Ala(115) to serine in beta4a-tubulin almost completely reversed heightened sensitivity to paclitaxel, but introduction of an S115A mutation into beta4b had no effect, suggesting that a complex interaction of multiple amino acids are necessary to produce this phenotype.     

Paclitaxel resistance in cells with reduced beta-tubulin

We previously described the isolation of colcemid resistant Chinese hamster ovary cell lines containing alpha- and beta-tubulin mutations that increase microtubule assembly and stability. By analyzing colcemid sensitive revertants from one of the beta-tubulin mutants, we now find that loss or inactivation of the mutant allele represents the most common mechanism of reversion. Consistent with this loss, the revertants have 35% less tubulin at steady state, no evidence for the presence of a mutant polypeptide, and a normal extent of tubulin polymerization. In addition to the loss of colcemid resistance, the revertant cells exhibit increased resistance to paclitaxel relative to wild-type cells. This paclitaxel resistance can be suppressed by transfecting the revertant cells with a cDNA for wild-type beta-tubulin, indicating that the reduction in tubulin in the revertant cells is responsible for the resistance phenotype. We propose that reducing tubulin levels may represent a novel mechanism of paclitaxel resistance.     

Intra-allelic suppression of a mutation that stabilizes microtubules and confers resistance to colcemid

Cmd 4 is a colcemid resistant beta-tubulin mutant of Chinese hamster ovary cells that exhibits hypersensitivity to paclitaxel and temperature sensitivity for growth. The mutant beta-tubulin allele in this cell line encodes a D45Y amino acid substitution that produces colcemid resistance by making microtubules more stable. By selecting revertants of the temperature sensitive and paclitaxel hypersensitive phenotypes, we have identified three cis-acting suppressors of D45Y. One suppressor, V60A, maps to the same region as the D45Y alteration, and a second suppressor, Q292H, maps to a distant location. Both appear to produce compensatory changes in microtubule assembly that counteract the effects of the original D45Y substitution. Consistent with this view, expression of the V60A mutation in transfected wild-type cells produced paclitaxel resistance and greatly decreased microtubule assembly. Additionally, it produced a paclitaxel-dependent phenotype in which cells grew normally in the presence, but not the absence, of the drug. The Q292H mutation caused even greater disassembly of microtubules such that cells were unable to proliferate when the transgene was expressed; but, unlike the V60A mutation, cell growth could not be rescued by paclitaxel. A third suppressor, A254V, maps to a region near the interface between alpha- and beta-tubulin that contains the colchicine binding site. Although it made transfected wild-type cells hypersensitive to colcemid, it did not affect paclitaxel or vinblastine sensitivity, nor did it reduce microtubule assembly. We suggest that this mutation acts by increasing tubulin's affinity for colcemid.     

Rationalization of paclitaxel insensitivity of yeast ß-tubulin and human ßIII-tubulin isotype using principal component analysis

ABSTRACT: BACKGROUND: The chemotherapeutic agent paclitaxel arrests cell division by binding to the hetero-dimeric protein tubulin. Subtle differences in tubulin sequences, across eukaryotes and among beta-tubulin isotypes, can have profound impact on paclitaxel-tubulin binding. To capture the experimentally observed paclitaxel-resistance of human betaIII tubulin isotype and yeast beta-tubulin, within a common theoretical framework, we have performed structural principal component analyses of beta-tubulin sequences across eukaryotes. RESULTS: The paclitaxel-resistance of human betaIII tubulin isotype and yeast beta-tubulin uniquely mapped on to the lowest two principal components, defining the paclitaxel-binding site residues of beta-tubulin. The molecular mechanisms behind paclitaxel-resistance, mediated through key residues, were identified from structural consequences of characteristic mutations that confer paclitaxel-resistance. Specifically, Ala277 in betaIII isotype was shown to be crucial for paclitaxel-resistance. CONCLUSIONS: The present analysis captures the origin of two apparently unrelated events, paclitaxel-insensitivity of yeast tubulin and human betaIII tubulin isotype, through two common collective sequence vectors.     

Multi-sited mutations of beta-tubulin are involved in benzimidazole resistance and thermotolerance of fungal biocontrol agent Beauveria bassiana

Fungicide resistance and thermotolerance of biocontrol agents in mitosporic fungi are of merits for enhancing fungal formulations against insect pests in the field. Among 20 wild strains of Beauveria bassiana (a well-known fungal biocontrol agent) tested in this study, 19 were sensitive or highly sensitive to carbendazim (methyl 2-benzimidazole carbamate), a typical benzimidazole fungicide, despite low resistance found in one strain. Sequential mutagenesis of a carbendazim-sensitive wild strain [minimal inhibitory concentration (MIC) = 1.32 microg ml(-1)] under artificial selection pressure generated 11 mutants sharing a common MIC of > 1000 microg ml(-1) without visible variation in colony growth and conidiation capacity. This represents at least 758-fold enhancement of the resistance among the mutants. However, accompanied with the enhanced resistance, all the mutants became less thermotolerable. Stressed at 48 degrees C, conidial LT(50)s of the mutants varied from 1.8 to 9.6 min and were lower than the parental LT(50) (36 min). Moreover, the contents of hydrophobin-like proteins in conidial walls declined significantly among the mutants compared with that of the wild parent. Mutations commonly relating to benzimidazole resistance in fungi were located at Q134, F167 and/or E198 around the taxol-binding site of beta-tubulin by sequencing the beta-tubulin of the mutants. Also, mutations of other 37 amino acid residues in the sequences (each having one to five residues mutated) were found for the first time and they were diverse in spatial structure. All mutations restricted to the half of beta-tubulin close to alpha-tubulin were likely involved in variation in each of the traits concerned but their interactions were complicated.     

Tubulin is phosphorylated at tyrosine by pp60c-src in nerve growth cone membranes

We show here that tubulin is the major in vivo substrate of the tyrosine-specific protein kinase pp60c-src in nerve growth cone membranes. Phosphotyrosine antibodies were used to demonstrate phosphotyrosyl residues in a subpopulation of alpha- and beta-tubulin that was highly enriched in a subcellular fraction of growth cone membranes from fetal rat brain. The presence of phosphotyrosine- modified isoforms of alpha- and beta-tubulin in vivo was confirmed by 32p labeling of rat cortical neurons in culture. Tubulin in growth cone membranes was phosphorylated at tyrosine in endogenous membrane phosphorylation reactions (0.068 mol phosphotyrosine/mol alpha-tubulin and 0.045 mol phosphotyrosine/mol beta-tubulin), and phosphorylation was specifically inhibited by antibodies directed against pp60c-src, which is localized in the growth cone membranes. pp60c-src was capable of directly phosphorylating tubulin as shown in immune complex kinase assays with purified brain tubulin. Phosphopeptide mapping revealed a limited number of sites of tyrosine phosphorylation in alpha- and beta- tubulin, with similar phosphopeptides observed in vivo and in vitro. These results reveal a novel posttranslational modification of tubulin that could regulate microtubule dynamics at the growth cone.     

Revertants of a Chinese hamster ovary cell mutant with an altered beta-tubulin: evidence that the altered tubulin confers both colcemid resistance and temperature sensitivity on the cell

We recently described the isolation of a mutant Chinese hamster ovary cell (Cmd 4) resistant to the cytotoxic effects of colcemid (Cabral et al., Cell 20:29-36, 1980). This mutant carries an altered beta-tubulin but still grows normally at 37 degrees C. In the present study we found that Cmd 4 is temperature sensitive for growth at 40.3 degrees C. A class of revertants selected for temperature resistance had simultaneously lost colcemid resistance and the altered beta-tubulin. In addition, we isolated a temperature-resistant revertant which carries a further alteration in the mutant beta-tubulin polypeptide. This second alteration appears to make the mutant beta-tubulin incompetent to assemble into microtubules, resulting in a strain which is again colcemid sensitive. These revertant cell lines provide strong evidence that a mutation in beta-tubulin can confer both colcemid resistance and temperature sensitivity on a mammalian cell line. Cellular microtubules studied by indirect immunofluorescence in both mutant and revertant cell lines had an apparently normal distribution at permissive and nonpermissive temperatures, yet mitosis appears to be abnormal in the mutant cell line. We conclude from these studies that incorporation of the altered beta-tubulin into microtubules does not affect their distribution but may affect their function during mitosis.     

Leishmania tarentolae: purification and characterization of tubulin and its suitability for antileishmanial drug screening

Previously, tubulin has been purified from Leishmania amazonensis and used to identify novel molecules with selective antimitotic activity. However, L. amazonensis is pathogenic and requires a relatively expensive medium for large-scale cultivation. Herein, the purification and characterization of tubulin from the non-pathogenic Leishmania tarentolae is reported, together with the sequence of alpha- and beta-tubulin from this organism. This protein was purified by sonication, diethylaminoethyl-Sepharose chromatography, and one assembly disassembly cycle in 1% overall recovery based on total cellular protein. Leishmania tarentolae tubulin was indistinguishable from the corresponding L. amazonensis protein in terms of binding affinity for dinitroaniline sulfanilamides and sensitivity to assembly inhibition by these compounds. The amino acid sequences derived from the L. tarentolae alpha- and beta-tubulin genes were 99.6 and 99.4% identical to the corresponding amino acid sequences from the Leishmania major Friedlin strain. These results indicate that tubulin from L. tarentolae is suitable for use in drug screening.     

Characterization of two divergent beta-tubulin genes from Colletotrichum graminicola

We have cloned and sequenced two beta-tubulin genes, TUB1 and TUB2, from the phytopathogenic fungus, Colletotrichum graminicola. The nucleotide sequences of the coding regions of the two genes are only 72.8% homologous. This divergence is reflected in the deduced amino acid (aa) sequences which differ at 94 aa residues. Comparison with the aa sequences of other fungal beta-tubulins indicates that the C. graminicola TUB2 gene encodes a conserved isotype, whereas the C. graminicola TUB1 product is highly divergent. Both genes contain six identically placed introns and the position of each intron is conserved in other fungal beta-tubulin genes. Also typical of other fungal beta-tubulin genes, there is a pronounced bias in codon usage in the C. graminicola TUB2 gene; there is a lesser codon bias in TUB1 from C. graminicola. Both C. graminicola beta-tubulin genes are transcribed and yield similar sized messages.     

Multiple effects of paclitaxel are modulated by a high c-myc amplification level

Paclitaxel affects microtubule stability by binding to beta-tubulin, thus leading to cell accumulation in the G(2)/M phase, polyploidization, and apoptosis. Because both cell proliferation and apoptosis could be somehow regulated by the protooncogene c-myc, in this work we have investigated whether the c-myc amplification level could modulate the multiple effects of paclitaxel. To this aim, paclitaxel was administered to SW613-12A1 and -B3 human colon carcinoma cell lines (which are characterized by a high and low c-myc endogenous amplification level, respectively), and to the B3mycC5 cell line, with an enforced exogenous expression of c-myc copies. In this experimental system, we previously demonstrated that a high endogenous/exogenous level of amplification of c-myc enhances serum deprivation- and DNA damage-induced apoptosis. Accordingly, the present results indicate that a high c-myc amplification level potentiates paclitaxel cytotoxicity, confers a multinucleated phenotype, and promotes apoptosis to a great extent, thus suggesting that c-myc expression level is relevant in modulating the cellular responses to paclitaxel. We have recently shown in HeLa cells that the phosphorylated form of c-Myc accumulates in the nucleus, as distinct nucleolar and extranucleolar spots; here, we demonstrated that, after the treatment with paclitaxel, phosphorylated c-Myc undergoes redistribution, becoming diffused in the nucleoplasm.     

Truncation of the carboxy-terminal domain of yeast beta-tubulin causes temperature-sensitive growth and hypersensitivity to antimitotic drugs [published erratum appears in J Cell Biol 1989 Feb;108(2):747]

beta-tubulin of budding yeast Saccharomyces cerevisiae is a polypeptide of 457 amino acids encoded by the unique gene TUB2. We investigated the function of the carboxy-terminal part of yeast beta-tubulin corresponding to the carboxy-terminal variable domain of mammalian and avian beta-tubulins. The GAA codon for Glu-431 of TUB2 was altered to TAA termination codon by using in vitro site-directed mutagenesis so that the 27-amino acid residues of the carboxyl terminus was truncated when expressed. The mutagenized TUB2 gene (tub2(T430)) was introduced into a haploid strain in which the original TUB2 gene had been disrupted. The tub2(T430) haploid strain grows normally less than 30 but not at 37 degrees C. The truncation of the carboxyl terminus caused hypersensitivity to antimitotic drugs and low spore viability at the permissive temperature for vegetative growth. Immunofluorescence labeling with antitubulin antibody and DNA staining with 4',6'-diamidino-2-phenylindole showed that in these cells at 37 degrees C, formation of spindle microtubules and nuclear division was inhibited and cytoplasmic microtubule distribution was aberrant. These results suggest that functions of the carboxy-terminal domain of yeast beta-tubulin are necessary for cells growing under suboptimal growth conditions although it is not essential for growth under the optimal growth conditions. Cells bearing tub2(411), a tub2 gene in which the GAA codon for Glu-412 was altered to TAA were no more viable at any temperature. In addition, a haploid strain carrying two functional beta-tubulin genes is not viable.     

Molecular analysis and characterization of the Cochliobolus heterostrophus beta-tubulin gene and its possible role in conferring resistance to benomyl

Cochliobolus heterostrophus Tub1 described here is the first beta-tubulin gene characterized from a naturally occurring benomyl-resistant ascomycete plant pathogen. The gene encodes a protein of 447 amino acids. The coding region of Tub1 is interrupted by three introns, of 116, 55, and 56 nt, situated after codons 4, 12, and 53, respectively. As a result of the preference for pyrimidines in the third position of the codons when a choice exists between purines and pyrimidines, codon usage in the Tub1 gene is biased. Tub1 shows high homology with beta-tubulin genes of other ascomycete species. However, Tub1 is exceptional in having Tyr(167), compared with Phe(167), possessed by beta-tubulin genes of other ascomycetes sequenced thus far. The Tyr(167) residue has been associated with benomyl resistance in other organisms. In contrast, all other benomyl-implicated residues of Tub1 correspond to sensitivity. Based on these results, we suggest that benomyl resistance in the fungus probably is attributed to Tyr(167).     

Novel C2-C3' N-peptide linked macrocyclic taxoids. Part 1: Synthesis and biological activities of docetaxel analogues with a peptide side chain at C3'

A series of novel docetaxel analogues possessing a peptide side chain at the C3'-N position was synthesized. These compounds were designed to mimic a region of the alpha-tubulin loop that is equivalent to the paclitaxel binding pocket in beta-tubulin. Eight new peptidic taxoids were obtained and evaluated as inhibitors of microtubule disassembly, as well as for their cytotoxicity.     

Identification of betaIII- and betaIV-tubulin isotypes in cold-adapted microtubules from Atlantic cod (Gadus morhua): antibody mapping and cDNA sequencing

Isolated microtubule proteins from the Atlantic cod (Gadus morhua) assemble at temperatures between 8 and 30 degrees C. The cold-adaptation is an intrinsic property of the tubulin molecules, but the reason for it is unknown. To increase our knowledge of tubulin diversity and its role in cold-adaptation we have further characterized cod tubulins using alpha- and beta-tubulin site-directed antibodies and antibodies towards posttranslationally modified tubulin. In addition, one cod brain beta-tubulin isotype has been sequenced. In mammals there are five beta-tubulins (betaI, betaII, betaIII, betaIVa and betaIVb) expressed in brain. A cod betaIII-tubulin was identified by its electrophoretic mobility after reduction and carboxymethylation. The betaIII-like tubulin accounted for more than 30% of total brain beta-tubulins, the highest yield yet observed in any animal. This tubulin corresponds most probably with an additional band, designated beta(x), which was found between alpha- and beta-tubulins on SDS-polyacrylamide gels. It was found to be phosphorylated and neurospecific, and constituted about 30% of total cod beta-tubulin isoforms. The sequenced cod tubulin was identified as a betaIV-tubulin, and a betaIV-isotype was stained by a C-terminal specific antibody. The amount of staining indicates that this isotype, as in mammals, only accounts for a minor part of the total brain beta-tubulin. Based on the estimated amounts of betaIII- and betaIV-tubulins in cod brain, our results indicate that cod has at least one additional beta-tubulin isotype and that beta-tubulin diversity evolved early during fish evolution. The sequenced cod betaIV-tubulin had four unique amino acid substitutions when compared to beta-tubulin sequences from other animals, while one substitution was in common with Antarctic rockcod beta-tubulin. Residues 221, Thr to Ser, and 283, Ala to Ser, correspond in the bovine tubulin dimer structure to loops that most probably interact with other tubulin molecules within the microtubule, and might contribute to cold-adaptation of microtubules.     

Cyclostreptin binds covalently to microtubule pores and lumenal taxoid binding sites

Cyclostreptin (1), a natural product from Streptomyces sp. 9885, irreversibly stabilizes cellular microtubules, causes cell cycle arrest, evades drug resistance mediated by P-glycoprotein in a tumor cell line and potently inhibits paclitaxel binding to microtubules, yet it only weakly induces tubulin assembly. In trying to understand this paradox, we observed irreversible binding of synthetic cyclostreptin to tubulin. This results from formation of covalent crosslinks to beta-tubulin in cellular microtubules and microtubules formed from purified tubulin in a 1:1 total stoichiometry distributed between Thr220 (at the outer surface of a pore in the microtubule wall) and Asn228 (at the lumenal paclitaxel site). Unpolymerized tubulin was only labeled at Thr220. Thus, the pore region of beta-tubulin is an undescribed binding site that (i) elucidates the mechanism by which taxoid-site compounds reach the kinetically unfavorable lumenal site and (ii) explains how taxoid-site drugs induce microtubule formation from dimeric and oligomeric tubulin.