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PubMed1995

The N-terminal domain of c-Myc associates with alpha-tubulin and microtubules in vivo and in vitro.

The polymerization of alpha- and beta-tubulin into microtubules results in a complex network of microfibrils that have important structural and functional roles in all eukaryotic cells. In addition, microtubules can interact with a diverse family of polypeptides which are believed to directly promote the assembly of microtubules and to modulate their functional activity. We have demonstrated that the c-Myc oncoprotein interacts in vivo and in vitro with alpha-tubulin and with polymerized microtubules and have defined the binding site to the N-terminal region within the transactivation domain of c-Myc. In addition, we have shown that c-Myc colocalizes with microtubules and remains tightly bound to the microtubule network after detergent extraction of intact cells. These findings suggest a potential role for Myc-tubulin interaction in vivo.

Base Sequence↗
PubMed1996

Cofactor A is a molecular chaperone required for beta-tubulin folding: functional and structural characterization.

Actin and tubulin polypeptide chains acquire their native conformation in the presence of the chaperonin containing TCP-1 (CCT) and, in the case of alpha- and beta-tubulin additional protein cofactors. We recently identified one of these cofactors, termed cofactor A, that is required for the proper folding of the beta-tubulin chain [Gao et al. (1994) J. Cell. Biol. 125, 989-996]. We show here that cofactor A, a monomeric protein that has no measurable affinity for nucleotides, is a highly conserved protein among vertebrates. Its NH2-terminal region is essential for the structural integrity of the protein and consequently for its activity. We demonstrate that cofactor A does not interact with CCT nor does it affect the intrinsic ATPase activity of CCT, alone or in the presence of different target proteins. Thus, unlike GroES, cofactor A does not modulate or coordinate ATP hydrolysis. It does not act as a nucleotide exchange factor or a catalyst in tubulin folding. Rather, we demonstrate that cofactor A participates in the tubulin folding process by interacting with a folding intermediate of beta-tubulin that is released from CCT. Our data imply that cofactor A is a chaperone involved in tubulin folding.

Amino Acid Sequence↗
PubMed2003

Human immunoglobulins and Fc fragments promote microtubule assembly via tau proteins and induce conformational changes of neuronal microtubules in vitro.

The influence of human immunoglobulins (Ig) in neuronal cytoskeleton stability was studied in vitro. Here we show that human Ig and Fc fragments stimulate animal and human microtubule assembly by binding to microtubules via tau isoforms. In presence of Ig, microtubules show increased aggregation, twisting and rigidity. Non-immune Ig and Fc fragments promote microtubule assembly in temperature-dependent manner and stabilize microtubules at a molecular ratio of 1 Ig per 4 tubulin dimers. These in vitro data provide an experimental support for an immuno-mediated modulation of the cytoskeleton. In conjunction with previous neuropathological data, they suggest that Ig could participate in early stages of neurodegeneration by affecting the microtubule stability in vivo.

Aged↗
PubMed1977

Mitosis and microtuble assembly.

Microtubules reconstituted in vitro are identical in helical structure with tubules making up the mitotic spindle and both are formed by a helical condensation-polymerization mechanism. The protomer of microtubules is a heterodimer (alphabeta), of mol.wt. 110000 and sedimentation coefficient, SO25,w, 6S. This dimer has one binding site for colchicine or podophyllotoxin, two sites for Vinca alkaloids, two sites for guanine nucleotides and Ca2+-binding sites. There is also a beta-chain phosphoserine. Modulation of these properties is discussed as a possible way of regulating the competence of tubulin to polymerize. Reconstituted microtubules depolymerize to a mixture of 30-36S oligomeric tubulin and 6S dimer molecules. The 30-36S tubulin appears as a ring or disc when made visible in the electron microscope by negative staining. Three pathways of microtubule assembly have been proposed involving this ring as an intermediate: (1) the uncoiling of these rings into protofilaments; (2) the stacking of rings into macrotubules; (3) the rings as a scaffold for the assembly of a short segment of microtubule helix. Finally, the regulation of mitosis is discussed in terms of recent studies of tubulin and its polymerization, and studies in vitro and in vivo of the process of mitotic-spindle formation and disassembly.

Alkaloids↗
PubMed2000

Can isotype switch modulate antigen-binding affinity and influence clonal selection?

Four different monoclonal Ig (MIg) (IgA1kappa, IgG1kappa, IgG2kappa and IgG4kappa) displaying anti-tubulin activity were detected in the serum from a lymphoma patient. The complete sequence of three of these MIg showed identical V(H) and V(L) domains and the presence of mutations compatible with an antigen-driven process. Surprisingly, despite complete homology in their variable domains, IgA1kappa, IgG1kappa, or their Fab fragments bound to a common motif recognized in beta tubulin, with significant differences in affinity (IgA1kappa 1.52x10(-8) M, and IgG1kappa 2.09x10(-7) M). To substantiate these results, the V(H) and V(L) domains from IgA1kappa were cloned and introduced into expression vectors containing the constant kappa exon and either the mu or the gamma1 constant exon, and complete recombinant IgMkappa and IgG1kappa were obtained. Like the IgA1kappa, the IgMkappa construction bound to the tubulin epitope with consistent affinity (7.7x10(-9) M), whereas the IgG1kappa construction displayed a significantly lower affinity (3.28x10(-7) M). These results provide definitive evidence that isotype can influence binding affinity to antigen and suggest that malignant transformation occurred at the germinal center once the mutational process was achieved and the switch process was still active.

Amino Acid Sequence↗
PubMed2005

The TUBB1 Q43P functional polymorphism reduces the risk of cardiovascular disease in men by modulating platelet function and structure.

The discoid form of platelets is maintained by a marginal band of tightly coiled microtubules. beta1-tubulin is the major isoform within platelet and megakaryocyte microtubules. In 24.2% of 33 unrelated inherited macrothrombocytopenia patients and in 10.6% of 272 subjects of a healthy population a P for Q substitution in beta1-tubulin was found in the highly conserved residue 43. Heterozygous carriers of the Q43P variant showed a reduced platelet protein beta1-tubulin expression. Transfection of green fluorescent protein (GFP)-tagged Q43P beta1-tubulin in megakaryocytic MEG01 cells resulted in a disturbed tubulin organization. Electron microscopy revealed enlarged spherocytic platelets with a disturbed marginal band and organelle-free zones. In addition, platelets with the Q43P beta1-tubulin variant had reduced adenosine triphosphate (ATP) secretion, thrombin receptor activating peptide (TRAP)-induced aggregation and collagen adhesion. The prevalence of the Q43P beta1-tubulin variant was also 2 times higher (odds ratio, [OR] = 2.1;95% confidence interval [CI], 1.22-3.59) among control subjects than among patients with cardiovascular disease (10.4% versus 5.2%, P < .001). By analyzing this protective factor in men and women separately, this association was only found in men. This study thus presents the functional consequences of the platelet Q43P beta1-tubulin substitution that is frequent in the healthy population and may protect men against arterial thrombosis.

Adenosine Triphosphate↗
PubMed1997

Role of guanine nucleotides in the vinblastine-induced self-association of tubulin: effects of guanosine alpha,beta-methylenetriphosphate and guanosine alpha,beta-methylenediphosphate.

It is now well established that guanine nucleotides are allosteric effectors of the vinca alkaloid-induced self-association of tubulin. GDP enhances self-association for vinblastine-, vincristine- and vinorelbine-induced spiral assembly relative to GTP by 0.90 +/- 0.17 kcal/mol [Lobert et al. (1996) Biochemistry 35, 6806-6814]. Since chemical modifications of the vinca alkaloid structure are known to modulate the overall affinity of drug binding, it is very likely that, by Wyman linkage, chemical modifications of guanine nucleotide allosteric effectors also modulate drug binding. Here we compare the effects of the GTP and GDP alpha,beta-methylene analogues GMPCPP and GMPCP on vinblastine-induced tubulin association in 10 and 100 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes), 1 mM MgSO4, and 2 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), pH 6. 9, at different temperatures. We found that GMPCPP perfectly mimics GTP in its effect on spiral assembly under all ionic strength and temperature conditions. However, GMPCP in 10 mM Pipes behaves not as a GDP analogue, but as a GTP analogue. In 100 mM Pipes, GMPCP has characteristics that are intermediate between GDP and GTP. These data suggest that the alpha,beta methylene group in GMPCP and GMPCPP is sufficient to produce a GTP-like effect on vinblastine-induced tubulin self-assembly. This is consistent with previous observations that GMPCP-tubulin will assemble into microtubules in a 2 M glycerol and 100 mM Pipes buffer [Vulevic & Correia (1997) Biophys. J. 72, 1357-1375]. Our results demonstrate that an alpha,beta methylene modification of the guanine nucleotide phosphate moiety can induce a salt-dependent conformational change in the tubulin heterodimer that favors the GTP-tubulin structure. This has important implications for understanding allosteric interactions that occur in the binding of guanine nucleotides to tubulin.

Alkanesulfonic Acids↗
PubMed1976

Mutants of Balb/c 3T3 fibroblasts defective in adhesiveness to substratum: evidence for alteration in cell surface proteins.

Two mutants of Balb/c 3T3 cells defective in adhesiveness to substratum have been isolated. After exposure to a mutagen a cell population was treated with prostaglandin E1 and methylisobutylxanthine to elevate the cells 3':5'-cyclic AMP levels and adhesion to the substratum; then, loosley adherent cells were harvested by gentle detachment. After four cycles of selection two clones were isolated. Both are characterized by a round morphology and a decreased adhesion to substratum. The defect in adhesion appears to be responsible for the change in cell shape, since the mutant cells become flattened and look similar to wild-type cells when treated with dibutyryl cyclic AMP even though their adhesiveness remains decreased. Further, the surface of both mutants is chemically altered. Lactoperoxidase-catalyzed iodination of the cell surface shows that two iodinated polypeptides (molecular weight, Mr, 90,000 and 135-140,000) present in wild-type cells are not detected on the surface of the mutants, and another iodinated band (110-120,000 Mr) is markedly decreased. In addition, one mutant is also missing the high-molecular-weight (230,000) cell surface protein that is sensitive to transformation. No changes have been found in the content of cyclic AMP or free or polymerized tubulin. The exponential growth rate and saturation density are not altered in these adhesion--eficient cells. This last result suggests that modulation of adhesion to substratum per se does not play an important role in the growth control of Balb/c 3T3 cells in vitro.

Cell Adhesion↗
PubMed1985

Colchicine-binding properties of hepatic tubulin. The role of "time-decay".

Determination of total and polymerized tubulin in the liver by the [3H]colchicine-binding assay is hampered by rapid loss of binding sites. One solution, which has been proposed to overcome this problem, is the use of a "time-decay" corrected assay that extrapolates to an initial binding capacity based on the results of testing multiple aliquots of liver supernatants over time. If such an approach is valid, it follows that the extrapolated initial value should be independent of the rate of time-decay. To test this relationship, we modulated the half-life (T1/2) for time-decay of a fixed amount of hepatic microtubule-derived tubulin using various concentrations of organic acids, sodium glutamate and glucose-1-phosphate. We found that the initial binding capacity based on simple first order decay did vary with the T1/2. Similar results were also obtained with bovine brain tubulin added to hepatic supernatants. These results indicate that the quantification of hepatic tubulin derived by extrapolated time-decay values is inaccurate and may overestimate the actual amount of tubulin present, especially when time-decay is rapid.

Animals↗
PubMed1998

Sulfide enhancement of PMN apoptosis.

Hydrogen sulfide is a toxic metabolite released by several bacterial agents under anaerobic conditions. In the present paper, we investigated the effects of sulfide on polymorphonuclear cell (PMN) apoptosis, a mechanism suggested for limiting the toxic potential of neutrophils in inflammatory sites. We showed that 1 mM sulfide (concentration not conditioning PMN viability) is able to enhance the apoptotic fate of human granulocytes by increasing: i) the number of cells containing pyknotic nuclei, ii) the internucleosomal cleavage, and, iii) the intensity of tubulin immunofluorescence staining. The sulfide effect is partially prevented by ionomycin and this finding is consistent with the hypothesis of the inhibiting role played by high levels of cytosolic calcium in PMN apoptosis modulating.

Apoptosis↗
PubMed1996

Influence of microtubule-associated proteins on the differential effects of paclitaxel and docetaxel.

Microtubules are complex structures arising in part from the polymerization of tubulin dimers. Tubulin binds to a wide range of drugs which have been used as probes for tubulin conformation and assembly properties. There is some evidence that taxol and taxotere have differing effects on tubulin conformation. Previous work has shown that MAP2 and Tau, although they both induce microtubule assembly, have qualitatively different effects on tubulin's behavior. Since most microtubules in vivo are likely to be associated with MAPs, we decided to characterize the differential effects of MAP2, Tau, taxol, and taxotere on tubulin polymerization with the aim of understanding the mechanisms through which these agents stimulate microtubule assembly. Furthermore, the inhibitive effect of calcium has been used to elucidate the ability of the two drugs to force tubulin assembly. These observations suggest that docetaxel, in addition to its greater efficiency in tubulin assembly, may have the capacity to differently alter certain classes of microtubules. Tau and MAP2 accessory proteins may represent important cofactors modulating the effects of taxoids.

Animals↗
PubMed2000

Phosphorylation of human tau protein by microtubule-associated kinases: GSK3beta and cdk5 are key participants.

Microtubules (MTs), primarily composed of alpha and beta tubulin polymers, must often work in concert with microtubule-associated proteins (MAPs) in order to modulate their functional demands. In a mature brain neuron, one of the key MAPs that resides primarily in the axonal compartment is the tau protein. Tau, in the adult human brain, is a set of six protein isoforms, whose binding affinity to MTs can be modulated by phosphorylation. In addition to the role that phosphorylation of tau plays in the "normal" physiology of neurons, hyperphosphorylated tau is the primary component of the fibrillary pathology in Alzheimer's disease (AD). Although many protein kinases are known to phosphorylate tau in vitro, the in vivo players contributing to the hyperphosphorylation of tau remain elusive. The experiments in this study attempt to define which protein kinases and protein phosphatases reside in the associated network of microtubules, thereby being strategically positioned to influence the phosphorylation of tau. Microtubule fractions are utilized to determine which of the microtubule-associated kinases most readily impacts the phosphorylation of tau at "AD-like" sites. Results from this study indicate that PKA, CK1, GSK3beta, and cdk5 associate with microtubules. Among the MT-associated kinases, GSK3beta and cdk5 most readily contribute to the ATP-induced "AD-like" phosphorylation of tau.

Adenosine Triphosphate↗
PubMed2006

Protein lysine acetylation in normal and leukaemic haematopoiesis: HDACs as possible therapeutic targets in adult AML.

Several new therapeutic strategies are now considered for acute myelogenous leukaemia (AML), including modulation of protein lysine acetylation through inhibition of histone deacetylases (HDACs): a large group of enzymes that alters the acetylation and, thereby, the function of a wide range of nuclear and cytoplasmic proteins. Firstly, HDACs can deacetylate histones as well as transcription factors, and can modulate gene expression through both these mechanisms. Secondly, acetylation is an important post-translational modulation of several proteins involved in the regulation of cell proliferation, differentiation and apoptosis (e.g., p53, tubulin, heat-shock protein 90). The only HDAC inhibitors that have been investigated in clinical studies of AML are butyrate derivatives, valproic acid and depsipeptide. In the first studies, the drugs have usually been used as continuous therapy for several weeks or months, and in most studies the drugs were used alone or in combination with all-trans retinoic acid for treatment of patients with relapsed or primary resistant AML. Neurological toxicity and gastrointestinal side effects seem to be common for all three drugs. Complete haematological remission lasting for several months has been reported for a few patients (< 5% of included patients), whereas increased peripheral blood platelet counts seem more common and have been described both for patients with AML and myelodysplastic syndromes. Taken together, these studies suggest that HDAC inhibition can mediate antileukaemic effects in AML, but for most patients the clinical benefit seems limited and further studies of combination therapy are required.

Acetylation↗
PubMed1981

Inhibitory effect of interferon on cellular DNA synthesis: modulation by pure mitogenic factors.

Quiescent Swiss 3T3 cells can be stimulated to synthesize DNA by combinations of polypeptide hormones, retinoids and tubulin-disrupting agents. Low doses of mouse interferon are effective in inhibiting DNA synthesis induced by one, two or three factors. When more growth factors are added DNA synthesis is not further stimulated, although the dose response curve for interferon is shifted dramatically to the right. Even when added before the mitogenic stimulus, up to 2000 units of interferon have no effect on DNA synthesis stimulated by five factors. A change in the dose-response curve for interferon as an antiviral agent is also observed when the mitogenic stimulation is increased, but the effect is less dramatic than that observed on its function as an inhibitor or DNA synthesis.

Animals↗
PubMed1986

Goldfish retina and tectum influence each other's growth activity during regrowth of the retinotectal projection.

Variations in retinal and tectal growth activity, during regrowth of the goldfish retinotectal projection, were monitored by measuring the rates of incorporation of [14C]leucine into soluble protein and tubulin-enriched fractions at different times after crushing the optic nerves. Other experiments tested for growth-modulating interactions between tectum and retina. Here we studied how the absence of one of these structures (i.e. tectal ablation or eye removal) affected the profile of biosynthetic activity in the other. Experiments were also conducted on groups of fish in which the tectum was reinnervated by a half-retina (either half-nasal (1/2 N) or half-temporal (1/2 T) retina). This was done to ascertain if growth interactions between retina and tectum display any position-dependent differences that may be relevant to retinotopic ordering during regeneration. Our studies have revealed that: the retina and tectum of 1/2 T and 1/2 N groups differ in their growth responses during regeneration of the visual pathway: the tectum may exert a stimulatory and at other times an inhibitory influence on retinal protein synthesis; and retina and tectum display a bimodal profile of biosynthetic activity during regeneration that coincides with two stages of increased cell division (primarily glia) which other workers have found occurs in the tectum and tract during regeneration of the retinotectal projection. Indeed it seems there may be a link between this glial proliferation and the neurotrophic and guiding influences which tectum and retina exert upon one another during regeneration.

Animals↗
PubMed1994

Different stability of posttranslationally modified brain microtubules isolated from cold-temperate fish.

Microtubule proteins were isolated by a temperature-dependent assembly-disassembly method from brain tissue of for cold-temperature fish; one fresh water fish (Oncorhynchus mykiss), and three marine fish (Labrus berggylta, Zoarces viviparus and Gadus morhua). The alpha-tubulins from all four fish species were acetylated. The alpha-tubulins from the marine fish were composed of a mixture of tyrosinated and detyrosinated tubulin, while the fresh water fish tubulin only reacted with an antibody against detyrosinated tubulin. The isolated microtubules had a similar MAP composition. A 400 kD protein and a MAP2-like protein were found, but MAP1 was missing. All microtubules disassembled upon cooling to 0 degrees C. In spite of these common characteristics, the assembly of microtubules from Labrus berggylta was inhibited by colchicine and calcium, in contrast to the assembly of microtubules from Oncorhynchus mykiss and Zoarces viviparus. For the latter, colchicine was not completely inhibitory even at a concentration as high as 1 mM, and calcium induced the formation of both loosely and densely coiled ribbons. The effects of calcium and colchicine on microtubules from Oncorhynchus mykiss and Zoarces viviparus were modulated by either fish or cow MAPs, indicating that the effects are due to intrinsic properties of the fish tubulins and not the MAPs. In view of these findings, our results suggest that there is no correlation between colchicine sensitivity, inability of calcium to inhibit microtubule assembly, and acetylation and detyrosination.

Acetylation↗
PubMed1992

Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization.

Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of [14C]NAD+ and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the alpha and beta chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight mirotubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated [14C]ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD+ resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.

ADP Ribose Transferases↗
PubMed1996

Modulation of microtubule shape in vitro by high molecular weight microtubule associated proteins MAP1A, MAP1B, and MAP2.

The effect of microtubule associated proteins on microtubule shape has been investigated in reconstitution experiments using purified tubulin and purified MAP1A, MAP1B, and MAP2. Microtubules assembled in the presence of these MAPs were fixed with 0.1% glutaraldehyde and, after negative staining, were examined by electron microscopy. The results show that MAP1A microtubules were generally short and "straight' while those assembled with MAP1B were longer and "bendy'. MAP2 microtubules showed both types of morphologies even though straight microtubules were more abundant. These data suggest that MAPs may modulate not only microtubule dynamics but also microtubule shape which may be important in their spatial distribution and/or role in specific neuronal areas.

Animals↗