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PubMed2000

Mutational analysis of op18/stathmin-tubulin-interacting surfaces. Binding cooperativity controls tubulin GTP hydrolysis in the ternary complex.

Oncoprotein 18 (Op18) is a microtubule regulator that forms a ternary complex with two tubulin heterodimers. Dispersed regions of Op18 are involved in two-site cooperative binding and subsequent modulation of tubulin GTPase activity. Here we have analyzed specific determinants of Op18 that govern both stoichiometry and positive cooperativity in tubulin binding and consequent stimulatory and inhibitory effects on tubulin GTPase activity. The data revealed that the central and C-terminal regions of Op18 contain overlapping binding-motifs contacting both tubulin heterodimers, suggesting that these regions of Op18 are wedged into the previously noted 1-nm gap between the two longitudinally arranged tubulin heterodimers. Both the N- and C-terminal flanks adjacent to the central region are involved in stabilizing the ternary complex, but only the C-terminal flank does so by imposing positive binding cooperativity. Within the C-terminal flank, deletion of a 7-amino acid region attenuated positive binding cooperativity and resulted in a switch from stimulation to inhibition of tubulin GTP hydrolysis. This switch can be explained by attenuated binding cooperativity, because Op18 under these conditions may block longitudinal contact surfaces of single tubulins with consequent interference of tubulin-tubulin interaction-dependent GTP hydrolysis. Together, our results suggest that Op18 links two tubulin heterodimers via longitudinal contact surfaces to form a ternary GTPase productive complex.

Animals↗
PubMed1999

Op18/stathmin mediates multiple region-specific tubulin and microtubule-regulating activities.

Oncoprotein18/stathmin (Op18) is a regulator of microtubule (MT) dynamics that binds tubulin heterodimers and destabilizes MTs by promoting catastrophes (i.e., transitions from growing to shrinking MTs). Here, we have performed a deletion analysis to mechanistically dissect Op18 with respect to (a) modulation of tubulin GTP hydrolysis and exchange, (b) tubulin binding in vitro, and (c) tubulin association and MT-regulating activities in intact cells. The data reveal distinct types of region-specific Op18 modulation of tubulin GTP metabolism, namely inhibition of nucleotide exchange and stimulation or inhibition of GTP hydrolysis. These regulatory activities are mediated via two-site cooperative binding to tubulin by multiple nonessential physically separated regions of Op18. In vitro analysis revealed that NH(2)- and COOH-terminal truncations of Op18 have opposite effects on the rates of tubulin GTP hydrolysis. Transfection of human leukemia cells with these two types of mutants result in similar decrease of MT content, which in both cases appeared independent of a simple tubulin sequestering mechanism. However, the NH(2)- and COOH-terminal-truncated Op18 mutants regulate MTs by distinct mechanisms as evidenced by morphological analysis of microinjected newt lung cells. Hence, mutant analysis shows that Op18 has the potential to regulate tubulin/MTs by more than one specific mechanism.

Allosteric Site↗
PubMed2005

A common pharmacophore for a diverse set of colchicine site inhibitors using a structure-based approach.

Modulating the structure and function of tubulin and microtubules is an important route to anticancer therapeutics, and therefore, small molecules that bind to tubulin and cause mitotic arrest are of immense interest. A large number of synthetic and natural compounds with diverse structures have been shown to bind at the colchicine site, one of the major binding sites on tubulin, and inhibit tubulin assembly. Using the recently determined X-ray structure of the tubulin:colchicinoid complex as the template, we employed docking studies to determine the binding modes of a set of structurally diverse colchicine site inhibitors. These binding models were subsequently used to construct a comprehensive, structure-based pharmacophore that in combination with molecular dynamics simulations confirms and extends our understanding of binding interactions at the colchicine site.

2-Methoxyestradiol↗
PubMed1999

OP18/stathmin binds near the C-terminus of tubulin and facilitates GTP binding.

It is has been previously suggested that the protein Op18/stathmin may interact with tubulin via the alpha-tubulin subunit [Larsson, N., Marklund, U., Melander Gradin, H., Brattsand, G. & Gullberg, M. (1997) Mol. Cell. Biol. 17, 5530-5539]. In this study we have used limited proteolysis and cross-linking analysis to localize further the stathmin-binding site on alpha-tubulin. Our results indicate that such a binding site is in a region close to the C-terminus of the molecule comprising residues 307 to the subtilisin-cleavage site on the alpha-tubulin subunit. Based on a recent model of the structure of tubulin [Nogales, E., Wolf, S.G. & Dowing, D.H. (1998) Nature (London) 391, 199-203], we found that this region contained the same areas that may be involved in longitudinal contacts of alpha-tubulin subunits within the microtubule. We also observed that the binding of stathmin to tubulin can modulate the binding of GTP to tubulin, as a consequence of a conformational change in the beta-tubulin subunit that occurs upon interaction of stathmin with tubulin.

Adenosine Triphosphate↗
PubMed1985

Reconstruction of appropriate tubulin and actin gene regulation after transient transfection of cloned beta-tubulin and beta-actin genes.

Most animal cells rapidly depress the synthesis of new alpha- and beta-tubulin polypeptides in response to microtubule inhibitors that increase the pool of depolymerized subunits. This apparent autoregulatory control of tubulin synthesis is achieved through the modulation of tubulin mRNA levels. To begin to analyze the molecular mechanism responsible for such regulation, we have introduced exogenous beta-tubulin gene sequences into cultured mouse cells by DEAE-dextran-mediated DNA transfection. We find that the heterologous tubulin genes are expressed and that their RNA transcripts are accurately processed to mature mRNAs. Moreover, after drug-induced microtubule depolymerization, the expression of unintegrated tubulin gene sequences is regulated coordinately with the endogenous mouse alpha- and beta-tubulin RNA transcripts. Such regulation appears to be specific for transfected tubulin genes, since similar down-regulation is not observed in a contransfected beta-actin gene. Curiously, in response to microtubule depolymerization, the amount of RNA transcripts from a transfected beta-actin gene increases twofold, which qualitatively and quantitatively parallels that seen by the RNAs encoded by the endogenous actin genes. Thus, the transient DNA transfection approach may permit the unambiguous elucidation of regulatory sequences involved in establishing the proper level of expression of these two important cytoskeletal gene families.

Actins↗
PubMed1992

Neural cell adhesion molecules modulate tyrosine phosphorylation of tubulin in nerve growth cone membranes.

Triggering neural cell adhesion molecules of the immunoglobulin superfamily with specific ligands or antibodies inhibited the phosphorylation of tryosyl residues in a subpopulation of alpha- and beta-tubulin associated with membranes from a subcellular fraction of nerve growth cones from fetal rat brain. Preincubation of these membranes with purified extracellular fragments of L1, N-CAM, or myelin-associated glycoprotein, or with antibodies directed against the extracellular domains of L1 or N-CAM, inhibited pp60c-src-dependent phosphorylation of tubulin in an endogenous membrane kinase reaction. Other proteins that affect neurite outgrowth (fibronectin, laminin, antibodies against N-cadherin) had no effect. The results suggest that cell adhesion molecules transduce cell surface events to intracellular signals by modulating the activity of protein tyrosine kinases or phosphatases in axonal membranes to influence cytoskeletal dynamics at the growth cone.

Animals↗
PubMed1997

The association of tubulin carboxypeptidase activity with microtubules in brain extracts is modulated by phosphorylation/dephosphorylation processes.

Tubulin carboxypeptidase, the enzyme which releases the COOH terminal tyrosine from the alpha-chain of tubulin, remains associated with microtubules through several cycles of assembly/disassembly (Arce CA, Barra HS: FEBS Lett 157: 75-78, 1983). Here, we present evidence indicating that in rat brain extract the carboxypeptidase/microtubules association is regulated by the relative activities of endogenous protein kinase(s) and phosphatase(s) which seem to determine the phosphorylation state of the enzyme (or another entity) and in some way the affinity of the enzyme for microtubules. The presence of 2.5 mM ATP during the in vitro microtubule formation resulted in a low recovery of carboxypeptidase activity in the microtubule fraction. This ATP-induced effect was not due to alteration of the enzyme activity or to inhibition of microtubule assembly but to a decrease of the association of the enzyme with microtubules. We found that the ATP-induced effect was not mediated by modifications on the microtubules but, presumably, on the enzyme molecule. The non-hydrolyzable ATP analogue, AMP-PCP, did not reproduce the effect of ATP. The inclusion of phosphatase inhibitors in the homogenization buffer also led to a decrease in the amount of tubulin carboxypeptidase associated with microtubules. Finally, we found that, in concordance with the mechanism hypothesized, the magnitude of the carboxypeptidase/microtubule association correlated well with the different incubation conditions created to favor maximal, minimal or intermediate protein phosphorylation states.

Adenosine Triphosphate↗
PubMed1991

The dynamic instability of microtubules is not modulated by alpha-tubulin tyrosinylation.

The tyrosinylation of chick brain alpha-tubulin and the effects of the tyrosinylation status on the assembly and dynamic instability of chick brain MAP2:tubulin microtubule protein have been examined. Each of the eight major alpha-isotypes can be tyrosinylated in vitro, irrespective of whether a C-terminal tyrosine is genetically encoded. The extent of tyrosinylation is however limited to congruent to 0.3 mol.mol-1. The tyrosinylation status (0 vs. 0.3 mol.mol-1) has no effect on either the assembly kinetics of chick brain microtubule protein or on the rate of length redistribution following assembly and shearing. It is therefore unlikely that the tyrosinylation status directly affects the intrinsic stability of assembled microtubules since the rate of length redistribution is both a sensitive assay and a function of the kinetic parameters governing dynamic instability.

Animals↗
PubMed1991

Differential modulation of yeast actin, tubulin, and YPT1 mRNA levels by cycloheximide.

We have examined the effects of cycloheximide (Chx) on transcription of genes encoding the yeast beta-actin (ACT), beta-tubulin (TUB) and yeast protein 1 (YPT1). As in mammalian cells, Chx caused an increase in levels of ACT, but not TUB, transcripts. The YPT1 gene was also activated. Induction of Chx was further studied by placing the promoter regions of the ACT and YPT1 genes in front of a globin (GLB)-encoding reporter gene (GLB) on yeast plasmids. Induction of GLB mRNA synthesis by Chx was not seen with either promoter; the YPT1 promoter was, however, strongly inducible by Chx if glucose was present. The YPT1 coding sequence conferred Chx inducibility on both the YPT1 and ACT promoters, suggesting that it may contain a transcription regulatory element.

Actins↗
PubMed1997

Organotypic spinal cord culture in serum-free fibrin gel: a new approach to study three-dimensional neurite outgrowth and of neurotoxicity testing: Effects of modulating the actin and tubulin dynamics and protein kinase activities.

Spinal cord explants from embryonic day seven (E7) chicken embryos were cultured without serum and in the presence of aprotinine, in a three-dimensional fibrin matrix. These conditions promote a robust, radial, unfasciculated outgrowth of neurites that are tipped by elaborate growth cones. Routinely after 5 days, the neurite outgrowth intensity (NOI) was determined by measuring the optical density of the immunostained neurites (image analysis program OPTIMAS version 5.2) within defined areas, extending radially for up to 3 mm from the explant border. A dose-dependent inhibition of NOI was determined for the cytoskeleton-affecting drugs nocodazole (half maximal inhibition ([I50]), 0.02 microM), taxol ([I50], 0.016 microM), cytochalasin D ([I50], 0.006 microM), and tetramethyl lead ([I50], 0.05 microM). Likewise, NOI was decreased in a dose-dependent fashion by ML-9 and RO-31-8220, inhibitors of myosin-light chain kinase and protein kinase C (PKC), respectively. The addition of 1,2-dioctanoyl-s,n-glycerol, a potent activator of PKC, led, at 5 microM, to an increase and at 30 and 60 microM to a decrease in NOI. The described system provides a rapid, reproducible, and quantitative assay for the effects of exogenous factors on the mode and intensity of neurite outgrowth.

Actins↗
PubMed1994

Cosolvent modulation of the tubulin-colchicine GTPase-activating conformational change: strength of the enzymatic activity.

The locus of action of cosolvent additives in the activation of the tubulin-colchicine GTPase was investigated. The GDP off rates were slowed down by the cosolvents in a manner that parallels their specific viscosities, indicating that diffusion-controlled release of GDP may be rate-limiting under the conditions of these studies. Yet, the net effect of cosolvents was to increase the overall rate of GTP hydrolysis. Pre-steady-state kinetics of liganded tubulin in the presence of 1%, w/v, poly(ethylene glycol) 6000 (PEG-6000) exhibited a burst of inorganic phosphate release indicating that the cosolvents act at an early step in the process. A similar conclusion was drawn from measurements of the activation energy (Ea) of the reaction, which showed that 3.4 M glycerol decreased the value of Ea to 10.6 kcal mol-1 from 17.3 kcal mol-1 in its absence. The observed difference in apparent binding free energies of the colchicine analogues allo-colchicine (ALLO) and 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC, or des-ring B colchicine), when measured by fluorescence and enzyme activity titrations, identified the presence of a GTPase-activating protein conformational transition subsequent to the physicochemical binding of the ligands. The decrease of the apparent binding constant measured by enzyme activity in dilute buffer relative to that measured by fluorescence [for ALLO, Kb(fluor) = 1.46 x 10(6) M-1; Kb(enz act) = 1.1 x 10(5) M-1] yielded the value of the enzyme-activating conformational transition constant, K3 = 0.08.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

G beta gamma mediates the interplay between tubulin dimers and microtubules in the modulation of Gq signaling.

Agonist stimulation causes tubulin association with the plasma membrane and activation of PLC beta 1 through direct interaction with, and transactivation of, G alpha q. Here we demonstrate that G beta gamma interaction with tubulin down-regulates this signaling pathway. Purified G beta gamma, alone or with phosphatidylinositol 4,5-bisphosphate (PIP2), inhibited carbachol-evoked membrane recruitment of tubulin and G alpha q transactivation by tubulin. Polymerization of microtubules elicited by G beta gamma overrode tubulin translocation to the membrane in response to carbachol stimulation. G beta gamma sequestration of tubulin reduced the inhibition of PLC beta 1 observed at high tubulin concentration. G beta 1 gamma 2 interacted preferentially with tubulin-GDP, whereas G alpha q was transactivated by tubulin-GTP. Prenylation of the gamma 2 polypeptide was required for G beta gamma/tubulin interaction. Both confocal microscopy and coimmunoprecipitation studies revealed the spatiotemporal pattern of G beta gamma/tubulin interaction during carbachol stimulation of neuroblastoma SK-N-SH cells. In resting cells G beta gamma localized predominantly at the cell membrane, whereas tubulin was found in well defined microtubules in the cytosol. Within 2 min of agonist exposure, a subset of tubulin translocated to the plasma membrane and colocalized with G beta. Fifteen min post-carbachol addition, tubulin and G beta colocalized in vesicle-like structures in the cytosol. G beta/tubulin colocalization increased after pretreatment of cells with the microtubule-depolymerizing agent, colchicine, and was inhibited by taxol. Taxol also inhibited carbachol-induced PIP2 hydrolysis. It is suggested that G beta gamma/tubulin interaction mediates internalization of membrane-associated tubulin at the offset of PLC beta 1 signaling. Newly cytosolic G beta gamma/tubulin complexes might promote microtubule polymerization attenuating further tubulin association with the plasma membrane. Thus G protein-coupled receptors might evoke G alpha and G beta gamma to orchestrate regulation of phospholipase signaling by tubulin dimers and control of cell shape by microtubules.

Animals↗
PubMed2004

Modulation of the redox state of tubulin by the glutathione/glutaredoxin reductase system.

Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative diseases. We report that peroxynitrite-induced disulfides in porcine brain tubulin are repaired by the glutaredoxin reductase system composed of glutathione reductase, human or Escherichia coli glutaredoxin, reduced glutathione, and NADPH. Reduction of disulfide bonds between the alpha- and beta-tubulin subunits by the glutathione reductase system was assessed by Western blot. Tubulin cysteine oxidation and reduction was quantitated by monitoring the incorporation of 5-iodoacetamido-fluorescein, a thiol-specific labeling reagent. Tubulin disulfide bond reduction by the glutaredoxin reductase system restored tubulin polymerization activity that was lost following peroxynitrite addition. In support of redox modulations of tubulin by glutathione, thiol-disulfide exchange between tubulin and oxidized glutathione was detected and quantitated by HPLC. In addition, glutathionylation of tubulin was detected by dot blot using an anti-GSH antibody.

Animals↗
PubMed2005

Estrogen effects on tubulin expression and taxane mediated cytotoxicity in prostate cancer cells.

BACKGROUND: The present study was designed to determine if estrogens change microtubule polymerization and modulate cell cycle progression in vitro, related to modulation of tubulin expression and to determine if estrogens had antagonistic or synergistic effects with microtubule active agents. METHODS: cDNA array analysis of LNCaP cells treated with the estrogens, estradiol, estrone, diethylstilbestrol (DES), and 2-methoxyestradiol (2-ME) was carried out and the results confirmed by PCR and Western blotting. Microtubule arrays in cells treated with estrogens were assessed using indirect immunofluorescence. The effects of combining estrogens with taxane was assessed by MTT assay and flow cytometry for cell cycle kinetics. Human prostate cancer xenografts were treated with DES and docetaxel to assess the effects of combining estrogens and taxane in vivo. RESULTS: Treatment of LNCaP cells with DES and 2-ME suppressed transcripts and protein for beta-tubulin isotype IVa. This effect on tubulin synthesis was not blocked by estrogen or androgen receptor modulators. Other estrogens had no effect on beta-tubulin expression. 2-ME and DES decreased the density of microtubules. The administration of DES or 2-ME with paclitaxel enhanced cytotoxicity and G(2)-M arrest in vitro. DES enhanced tumor suppression in a human prostate cancer xenograft model when combined with the taxane docetaxel. CONCLUSION: The use of DES and 2-ME enhances the effects of taxanes and may be a novel and important means of increasing therapeutic efficacy of cytotoxic chemotherapy against prostate carcinoma.

Blotting, Western↗
PubMed1984

Developmental and biochemical analysis of chick brain tubulin heterogeneity.

Tubulin, isolated from brain tissue of chicks at different stages during late embryonic and early post-hatched development by ion-exchange chromatography and by in vitro microtubule reassembly, was analyzed by high-resolution isoelectric focusing and by two-dimensional polyacrylamide gel electrophoresis. Similar results were obtained with tubulins purified by the two methods. Sixteen isoelectric species of tubulin that differ in apparent net charge under denaturing conditions were detected by isoelectric focusing. By two-dimensional polyacrylamide gel electrophoresis, the chick brain tubulins were resolved into at least seven forms of alpha and 10 forms of beta tubulin. The number and relative proportions of the multiple brain tubulins were modulated during development. Since there are only four alpha tubulin and four beta tubulin genes in chickens, posttranslational modification of the tubulins must play a prominent role in the heterogeneity. Analysis of isotubulin distributions through cycles of microtubule assembly and disassembly indicated that the tubulins differ very little, if at all, in their capacity to assemble into microtubules. Therefore, the chemical differences that distinguish the multiple tubulins have very little structural impact on the protein surface areas involved in microtubule formation. Partial fractionation of the multiple tubulins during ion-exchange chromatography was observed, suggesting that it may be possible to isolate individual native tubulin variants for biochemical studies.

Aging↗
PubMed1989

Phosphorylation of tubulin by casein kinase II regulates its binding to a neuronal protein (NP 185) associated with brain coated vesicles.

We recently described a new protein associated exclusively with neuronal clathrin-coated vesicles (CCVs), and characterized two monoclonal antibodies that react with it (S-8G8 and S-6G7). In this report, the association of neuronal protein of 185 kilodaltons (NP185) with CCV kinases and its interaction with tubulin are described. The affinity of NP185 for tubulin is significantly enhanced when tubulin is phosphorylated by CCV-associated casein kinase II. In contrast, phosphorylation of tubulin by a kinase activity associated with purified brain tubulin decreases its affinity for NP185. Together, these data suggest that the interaction of NP185 with tubulin is modulated by protein phosphorylation. Recent evidence has suggested that tubulin is phosphorylated by casein kinase II during neurite development. The enhanced affinity of NP185 for tubulin phosphorylated by casein kinase II could be important for proper intracellular sorting of this protein in the developing neuron.

Adaptor Proteins, Vesicular Transport↗
PubMed1990

Influence of anti-tubulin antibodies on muscarinic receptor modulation of G protein GTPase activity in rat striatum.

To understand the role of tubulin, an integral component of neural membranes, in signal transduction processes, the influence of anti-tubulin antibodies on the low Km GTPase activity associated with transducer G proteins was examined in rat striatum. Membranes were prepared from striatum by conventional procedures, and the low Km GTPase activity (EC 3.6.1.-) was determined using [gamma-32P]GTP at 37 degrees in an ATP-regenerating buffer containing 0.2 to 2.0 microM unlabeled GTP. GTPase activity was linear for up to 30 min and was directly proportional to protein concentration. Polyclonal anti-tubulin antibodies, anti-alpha-tubulin antibodies, and anti-beta-tubulin antibodies (10 micrograms) stimulated G protein GTPase activity. Anti-beta-tubulin antibody (10 micrograms) stimulated GTPase activity by about 60% at each time point, while 10 micrograms of either anti-alpha-tubulin or polyclonal anti-tubulin antibodies stimulated GTPase activity by only 20-30% at each time point. The Vmax/Km ratio, an index of the enzyme-substrate interaction, increased by only 26% with the anti-alpha-tubulin antibody and by 52% with anti-beta-tubulin antibody; polyclonal anti-tubulin antibodies did not affect this ratio. GTPase activity was stimulated by acetylcholine in an atropine-sensitive manner. At 100 microM, acetylcholine stimulated GTPase activity by about 50%. Polyclonal anti-tubulin, anti-alpha-tubulin, or anti-beta-tubulin antibodies (10 micrograms) potentiated acetylcholine stimulation of GTPase activity. Two possible mechanisms by which anti-tubulin antibodies could stimulate low Km GTPase activity and potentiate the stimulatory effects of acetylcholine are: (1) by inhibiting GTP binding to beta-tubulin, and (2) by eliminating a chronic inhibitory effect of tubulin on G protein or receptor-G protein interaction.

Acetylcholine↗
PubMed1994

Identification and characterization of a tubulin binding protein in rat brain plasma membrane.

Studies on the interaction of FITC-tubulin and 125I-tubulin with isolated plasma membrane of neural cells and with primary cultures of neuronal (N) and glial (G) cells of rat brain demonstrate the presence of specific, saturable, high affinity tubulin binding sites in these cells. The positive fluorescence of live unfixed primary cultures of N and G cells following incubation with FITC-tubulin indicate that the tubulin binding sites are located on the outer side of the plasma membrane. Such fluorescence was not observed with FITC-BSA, FITC-conalbumin or freshly dissociated cells from rat tissues or established cell lines. Binding of FITC-tubulin or 125I-tubulin is competed only by tubulin and not by other proteins. Scatchard analysis of the binding of 125I-tubulin to purified plasma membrane indicates very high affinity (Kd = 85 nM) with a Bmax of 7.4 pmol/mg protein. The putative tubulin receptor was partially purified by affinity chromatography on tubulin-sepharose column. Immunoprecipitation of the solubilized tubulin-receptor complex followed by SDS-PAGE analysis and autoradiography, revealed the presence of two components of molecular weights 70 and 45 kDa respectively, presumably representing the two nonidentical subunits of the putative receptor. In conjunction with several recent reports indicating the secretion of high molecular weight proteins from cultured neural cells and the ability of tubulin to modulate adenyl cyclase in synaptic membranes these findings suggest that the binding of exogenous tubulin to sites external to the plasma membrane may be involved in signal transduction.

Animals↗