The interactions between calcium-dependent regulator protein of cyclic nucleotide phosphodiesterase and microtubule proteins. II. Association of calcium-dependent regulator protein with tubulin dimer.

The Ca2+-dependent regulator protein (CDR) of cyclic nucleotide phosphodiesterase (PDE) was reported to be a Ca2+-dependent regulator of microtubule (MT) assembly in the preceding paper. In this paper, the binding of Ca2+-CDR complex to tubulin dimer was investigated in order to elucidate the Ca2+-dependent inhibitory action of CDR on MT assembly. Purified microtubular proteins (PMPs) isolated from porcine brain did not affect the ability of CDR to activate Ca2+-activatable PDE, and did not include any inhibitory protein of Ca2+-activatable PDE. The binding of CDR to the tubulin dimer was observed on Sephadex G-200 gel filtration and ammonium sulfate fractionation in a Ca2+-dependent manner. CDR did not bind to microtubule associated proteins. We now assume that Ca2+-dependent inhibition of MT assembly by CDR is due to the binding of CDR to tubulin dimer in a Ca2+-dependent manner.     

Calcium-independent, pH-regulated effects of S-100 proteins on assembly-disassembly of brain microtubule protein in vitro

At alkaline pH, Ca2+ is no longer required for S-100 proteins to inhibit the assembly and to promote the disassembly of brain microtubules in vitro, though the presence of Ca2+ significantly favors the S-100 effects. These effects are inversely related to the microtubule protein concentration and directly related to the S-100 concentration and the pH. Ca2+-independent, pH-regulated inhibition of assembly of phosphocellulose-purified tubulin by S-100 is also described. The microtubule disassembling effect of S-100 is additive to that of alkali (used to raise the pH), and S-100 further disassembles microtubules after alkalinization. Thus the larger inhibitory effect of S-100 on microtubule assembly at alkaline versus acid pH depends on both a decrease in the assembly rate and an increase in the disassembly rate. Together with previous data on this topic, the present findings indicate that S-100 proteins act on microtubule protein in vitro primarily by binding to tubulin, this event being Ca2+-regulated at a given pH, and pH-regulated at a given free Ca2+ concentration.     

Chlorpromazine inhibits the calcium-mediated effects of S-100 protein(s) on assembled brain microtubule proteins, but not those on microtubule protein assembly

We have examined the S-100-chlorpromazine interplay at the level of brain microtubule proteins in vitro. The results indicate that in the presence of 0.12 M KCl and 10 microM free Ca2+ the inhibitory effect of S-100 on microtubule assembly is additive to that of chlorpromazine, but S-100 fails to potentiate the disassembling effect of 0.1 mM Ca2+ if added to assembled microtubule proteins after chlorpromazine and Ca2+, probably because of inhibition of S-100 by the phenothiazine. Chlorpromazine does not compete with S-100 for binding to purified tubulin.     

Characteristics of the effect of S-100 proteins on the assembly-disassembly of brain microtubule proteins at alkaline pH in vitro

The ability of S-100 proteins to inhibit the assembly of brain microtubule proteins (MTPs) in the presence of microM levels of Ca2+ increases as a function of pH. This seems to be due to an increasingly larger inhibitory effect of S-100 on the nucleation and, probably, on the elongation of microtubules (MTs) as the pH raises. In the presence of microM Ca2+ levels, the ability of S-100 to disassemble MTs also increases linearly with the pH, suggesting that the larger inhibitory effect of S-100 on MTP assembly at alkaline than at acidic pH may depend on both a decrease in the assembly rate and an increase in the disassembly rate. Also, S-100 inhibits the assembly of phosphocellulose-purified tubulin to a larger and larger extent as the pH raises. S-100 brings about its effect on MT assembly-disassembly probably by sequestering soluble tubulin, though additional mechanisms cannot be excluded. The present data are briefly discussed in relation to the role attributed to changes in intracellular pH in the regulation of the state of assembly of cytoplasmic MTs.     

Microtubule assembly in the presence of adenosine triphosphate.

Microtubule (MT) assembly was investigated in the presence of ATP and Ca ions using both crude extract (CE) and purified microtubular proteins (PMP) prepared from porcine brains. ATP inhibited MT assembly from CE prepared with the reassembly buffer containing 1 mM GTP. Half-maximal inhibition occurred at an ATP concentration of 0.4-0.5 mM. Calcium ions, on the contrary, cancelled the ATP-induced inhibition, 1-2 microM calcium ions supporting maximal MT assembly. The ATP-induced inhibition in PMP was not so prominent as in CE, but occurred significantly in the presence of RNA. In PMP dissolved in the reassembly buffer containing ATP and yeast tRNA, the content of the ring fraction decreased significantly as compared with PMP containing only RNA. Furthermore, microtubule-associated proteins were found to be capable of binding ATP. The significance of the ATP-induced inhibition of MT assembly and the release of the inhibition by Ca2+ was discussed.     

Detection of calcium-dependent regulatory protein binding components using 125I-labeled calcium-dependent regulatory protein.

The calcium-dependent regulatory protein (CDR) purified from bovine brain was iodinated with Na[125I]I using the lactoperoxidase-glucose oxidase system. The iodinated protein retained its ability to stimulate the Ca2+-sensitive CDR-depleted cyclic nucleotide phosphodiesterase from bovine heart. Stimulation of the phosphodiesterase by 125I-CDR was Ca2+-dependent and the labeled protein had a Ka for activation of cyclic nucleotide phosphodiesterase that was 4 times greater than unmodified CDR. 125I-CDR formed a Ca2+-dependent complex with the partially purified cyclic nucleotide phosphodiesterase which was detectable by autorradiography following electrophoresis of the complex on nondenaturing gels. This technique was used to detect CDR binding components in crude homogenates prepared from bovine heart and brain.     

Effect of S-100 protein on assembly of brain microtubule proteins in vitro

S-100 protein inhibits the assembly of brain microtubule proteins in vitro in the presence of 10 microM free Ca2+. The S-100 effect is generally greater on the rate than on the extent of assembly, and even greater as the microtubule protein concentration decreases and the time of preincubation between S-100 and microtubule proteins before GTP addition increases, at a given S-100/tubulin dimer molar ratio. The S-100 effect is greatly enhanced in the presence of physiological concentrations of K+ and is completely reversed by EGTA.     

Calcium-sensitivity of the microtubule reassembly system. Difference between crude brain extract and purified microtubular proteins

Microtubule reassembly in crude extracts of porcine brain was inhibited by 10(-5) M Ca2+, wherease 10(-3) M Ca2+ was required for inhibition of the reassembly from purified microtubular proteins. This accounts for the apparent discrepancies between the results reported by other investigators. Furthermore, the Ca-sensitivity of the purified microtubular proteins was nearly completely recovered on addition of a fraction obtained from crude brain extract.     

Calcium-sensitivity of brain microtubule proteins in the presence of S-100 proteins

In the presence of the usual 0.1 M Mes buffer, pH 6.7, mM free Ca2+ levels are required for half-maximal decrease in the rate and extent of brain microtubule protein (MTP) assembly in the absence of ox brain S-100, while microM free Ca2+ levels are sufficient in the presence of S-100. At the same pH 6.7, but in the presence of 0.12 M KCl, as low as 1.5 microM free Ca2+ is sufficient for S-100 to produce half-maximal reduction in the rate of assembly, while as high as 0.5 mM free Ca2+ is required in the absence of S-100. Similar results are obtained with rat brain S-100 (S-100b), indicating that single S-100 iso forms are equipotent in affecting the MTP assembly. At pH 7.5, MTPs are remarkably resistant to Ca2+ in the absence of S-100. In the presence of S-100, not only is the free Ca2+ concentration required for complete inhibition of assembly at least one order of magnitude smaller than that required in the absence of S-100, but significant S-100-dependent inhibition of assembly occurs in the absence of Ca2+. Under the two conditions where S-100 is particularly effective in inhibiting the assembly, i.e. at pH 6.7 in the presence of KCl and at pH 7.5, S-100 increases the disassembly rate even in the presence of microM Ca2+ levels. Our results suggest that the free Ca2+ concentration regulates the way S-100 disassembles microtubules (MTs): at microM Ca2+ levels, S-100 sequesters tubulin with concomitant increase in the disassembly rate; at mM Ca2+ levels, the S-100-Ca2+ complex probably interacts with MTs producing endwise disassembly.     

Inhibitory factor of microtubule assembly from sea urchin egg cortex. I. Preparation and characterization

A new inhibitory factor of the microtubule (MT) assembly system was isolated from unfertilized sea urchin egg cortex. This factor not only suppressed spontaneous brain MT assembly, but also induced depolymerization of the reconstituted MTs. The factor did not suppress initial MT growth initiated by ciliary outer fiber fragments but the assembled MTs were soon depolymerized with time. The inhibitory activity was heat-stable but sensitive to trypsin or urea. The mode of the inhibition was distinct from the inhibitory effects of RNA on the MT assembly. The inhibitory factor partially purified on DEAE-Sephadex A-50 completely inhibited tubulin polymerization in a factor: tubulin ratio of 0.013.     

Inhibition of microtubule assembly by poly(L-glutamic acid) and the site of its action

Poly(L-glutamic acid) (PGA) suppresses the polymerization of porcine brain microtubule proteins and induces the depolymerization in vitro in a concentration-dependent manner. The extent of inhibition increases with increasing molecular weight of the PGA tested. A 50% inhibition of the protein polymerization was observed at a PGA (molecular weight = 60,000) to microtubule protein ratio of 0.04 (w/w), and complete inhibition was obtained at a ratio of 0.07. Such an inhibition on the polymerization by PGA is greatly decreased when Mg2+ is present at a higher concentration. The addition of PGA raises the critical concentration of microtubule proteins necessary for assembly. During incubation with PGA, microtubule proteins retain the ability to assemble, i.e., substoichiometric amounts of taxol considerably relieve the inhibition of assembly by PGA. PGA interacts with microtubule-associated proteins (MAPs) preferentially, because the amount of MAPs binding to PGA-Sepharose 4B is much larger than that of tubulin. Tau proteins were observed only in adsorbed fractions, while MAP-2 was present in both unbound and adsorbed fractions.     

Inhibition of tubulin-dependent ATPase activity in microtubule proteins from porcine brain by S100 protein

Microtubule-associated proteins (MAPs) of brain microtubules exhibit an ATPase activity which is markedly enhanced by tubulin and Ca²⁺. Addition of S100 protein decreased the tubulindependent Ca²⁺-ATPase activity by about 85%, but did not affect the activity without tubulin. The inhibition by S100 protein was concentration-dependent and the apparent K_m value for ATP was not altered. A large amount of tubulin restored the inhibition, indicating that S100 protein acts through the binding to the tubulin molecule. Zn²⁺, which can bind both microtubule proteins and S100 protein, had little effect on the inhibitory action of S100 protein. The ATPase inhibition by S100 protein was partially restored by chlorpromazine or vinblastine. S100a is more effective than S100b on the inhibitory effect of tubulin-dependent ATPase activity. The results suggest that S100 protein may function as a regulatory factor of ATPase in brain microtubules.     

Calcium/calmodulin-dependent inhibition of microtubule assembly by brain synaptic junction

The effect of synaptic junction (SJ) on microtubule assembly was examined. After preincubation with ATP at 37°C, rat SJ decreased the initial velocity and the extent of the porcine brain microtubule assembly (initiated by the addition of GTP) in a Ca²⁺/calmodulin (CaM)-dependent manner. The degree of the inhibition reached 35% of the control assembly (0-min preincubation) after 20-min preincubation with ATP. There was no inhibition either with heat-treated SJ, at 0°C, or in the presence of EGTA or W-7 (CaM antagonist). The inhibition was due neither to protease(s) nor CaM contaminating the preparations. Free Ca²⁺ concentration level required for the inhibition of microtubule assembly was 10^–6 M. Phosphorylation of microtubule proteins was inhibited by SJ in a Ca²⁺/CaM-dependent manner, and the inhibition occurred in a physiological increase range of intracellular Ca²⁺ concentration (10^–6M) The heat-treated SJ caused no inhibition. The result suggested that the microtubule assembly in the postsynaptic region was regulated by a Ca²⁺/CaM-dependent protein kinase associated with SJ; i. e., major postsynaptic density protein.Abbreviations used CaM calmodulin - DTT dithiothreitol - MAPs microtubule-associated proteins - MES 2-(N-morphorino)ethanesulfonic acid - mPSDp major postsynaptic density protein - PSD postsynaptic density - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride     

Ca2+– and Calmodulin-Dependent Phosphorylation of Microtubule-Associated Protein 2 and t Factor, and Inhibition of Microtubule Assembly

Microtubule-associated proteins (MAPs) were phosphorylated by a Ca2+- and calmodulin-dependent protein kinase from rat brain cytosol. The maximal amount of phosphate incorporated into MAPs was 25 nmol of phosphate/mg protein. A Ka value of the enzyme for calmodulin was 57.0 nM, with MAPs as substrates. Among MAPs, MAP2 and tau factor were phosphorylated in a Ca2+- and calmodulin-dependent manner. The phosphorylation of MAPs led to an inhibition of microtubule assembly in accordance with its degree. This reaction was dependent on addition of the enzyme, Ca2+, and calmodulin, and had a greater effect on the initial rate of microtubule assembly rather than on the final extent. The critical tubulin concentration for microtubule assembly was unchanged by the MAPs phosphorylation. Therefore assembly and disassembly of brain microtubule are regulated by the Ca2+- and calmodulin-dependent protein kinase that requires only a nanomolar concentration of calmodulin for activation.     

Fission yeast Alp14 is a dose-dependent plus end-tracking microtubule polymerase

XMAP215/Dis1 proteins are conserved tubulin-binding TOG-domain proteins that regulate microtubule (MT) plus-end dynamics. Here we show that Alp14, a XMAP215 orthologue in fission yeast, Schizosaccharomyces pombe, has properties of a MT polymerase. In vivo, Alp14 localizes to growing MT plus ends in a manner independent of Mal3 (EB1). alp14-null mutants display short interphase MTs with twofold slower assembly rate and frequent pauses. Alp14 is a homodimer that binds a single tubulin dimer. In vitro, purified Alp14 molecules track growing MT plus ends and accelerate MT assembly threefold. TOG-domain mutants demonstrate that tubulin binding is critical for function and plus end localization. Overexpression of Alp14 or only its TOG domains causes complete MT loss in vivo, and high Alp14 concentration inhibits MT assembly in vitro. These inhibitory effects may arise from Alp14 sequestration of tubulin and effects on the MT. Our studies suggest that Alp14 regulates the polymerization state of tubulin by cycling between a tubulin dimer-bound cytoplasmic state and a MT polymerase state that promotes rapid MT assembly.     

Organization-dependent effects of toxic bivalent ions microtubule assembly and glycolysis.

The effects of bivalent ions on tubulin dynamics and the upper phase of glycolysis were investigated at different organization levels in vitro. Cu2+, Cd2+, Hg2+ and CrO4(2-) inhibit the tubulin polymerization at an IC50 of 14-24 microM with high cooperativity and also induce microtubule disassembly. The apparent binding constants of the ions to tubulin, estimated by fluorescence quenching, vary between 6 and 28 microM. BIAcore measurements for tubulin-tubulin interaction suggest that the presence of Cu2+ affects neither koff nor kon, but the amount of the bound tubulin. While the inhibitory effect of Cu2+ on tubulin polymerization is partially abolished by cross-linking of microtubules with substoichiometric amounts of phosphofructokinase or decoration of tubules with cytosolic proteins, in the presence of kinase but not with cytosolic proteins the tubules are resistant to CrO4(2-). No inhibitory effect of Cu2+ or CrO4(2-) on microtubule assembly was detected in the MAP-containing cytosolic fraction. Electron microscopy revealed that tubules assembled in the presence of Cu2+ or CrO4(2-) ions contain aggregates of thread-like oligomers that are less conspicuous in the presence of cytosolic proteins. Cu2+, Cd2+, and Hg2+ inhibit the glycolytic flux in the cytosolic fraction characterized at equilibrium by an IC50 of 10-14 microM with high cooperativity. Tubulin diminishes the inhibitory effect of the cations. These data indicate that the responses elicited by the bivalent ions are highly dependent on the supramolecular organization of the systems.     

Interaction of calmodulin with histones. Alteration of histone dephosphorylation

The Ca2+-dependent regulator protein (CDR), also frequently termed "calmodulin" was determined to influence the dephosphorylation of mixed calf thymus histones or purified histones 1, 2A, or 2B by a partially purified bovine brain phosphoprotein phosphatase. CDR increase the rate of dephosphorylation of mixed histones more than 20-fold. With increasing concentrations of mixed histones as substrate, a proportionate increase of CDR concentration was required to maintain maximal expression of histone phosphatase activity. Mixed histones suppressed the activation by CDR of a bovine brain cyclic nucleotide phosphodiesterase activity, with activation being restored by increased quantities of CDR. Dephosphorylation of casein and phosphorylase alpha by the phosphatase preparation was not affected by CDR. These observations support the interpretation that the effects of CDR on histone dephosphorylation are substrate-directed. The rates of dephosphorylation of histones 1, 2A, and 2B by the phosphatase were 4- to 12-fold more rapid at low (sub-micromolar) concentrations of free Ca2+ than at high (200 microM) Ca2+ in incubations containing CDR, but they were unaffected by Ca2+ in incubations without CDR. The addition of stoichiometric quantities of calmodulin increased the apparent Km of the phosphatase for the various histones 2- to 6-fold, while maximal velocities were 4- to 12-fold higher at low than at high added Ca2+. The inhibitory effect of Ca2+ on histone dephosphorylation was immediately reversible by chelation of Ca2+ with EDTA. Ca2+-dependent inhibition of histone 1 or 2B phosphatase activities was also produced by rabbit skeletal muscle troponin C, but not by rabbit skeletal muscle parvalbumin, by poly(L-aspartate) or poly(L-glutamate). The phosphorylated fragment from the NH2-terminal region of either H2A (generated by treatment with N-bromosuccinimide) or H2B (generated by treatment with cyanogen bromide) was dephosphorylated by the phosphatase, with the rates of dephosphorylation being reduced 3- to 6-fold by Ca2+ in incubations containing CDR.     

Quantitative analysis of the interaction between S-100 proteins and brain tubulin

S-100 was shown to regulate the in vitro assembly of brain microtubule proteins (MTPs) in a Ca2+-mediated way by acting on both the nucleation and the elongation of microtubules (MTs). Here data will be shown suggesting that S-100 binds to tubulin. The binding is time-, temperature-, Ca2+-, and pH-dependent, and saturable with respect to S-100. At pH 6.75, the saturation curve is biphasic, displaying a high affinity component (dissociation constant, Kd1, approximately 0.1 microM) and a low affinity component (Kd2 approximately 3.8 microM). At pH 6.75, as the free Ca2+ concentration raises from 0 to 100 microM, the overall binding capacity increases from 0.065 to 0.66 mol S-100/mol tubulin dimer. This finding, together with the observation that the S-100 effect on MTP assembly is Ca2+-dependent at that pH, suggests that the S-100-induced inhibition of MTP assembly depends on S-100 binding to the low affinity sites on the tubulin molecule. The S-100 binding to tubulin is pH-dependent; as the pH raises from 6.75 to 8.3, both binding components are affected, the major changes consisting of an increase in the binding capacity and a decrease in the overall affinity. Moreover, as the pH raises, Ca2+ is no longer required for S-100 to bind to tubulin. S-100 also interacts with a component of whole MTPs (probably tubulin, on the basis of the above results). No S-100 binding to microtubule-associated proteins (MAPs) could be evidenced by the techniques employed in this study. On the contrary, some competition between S-100 and MAPs for binding sites or tubulin seems to occur.     

On the mechanism of calmodulin-induced inhibition of microtubule assembly in vitro

Binding of calmodulin to microtubule-associated proteins (MAPs) was analyzed by the equilibrium gel filtration method. The apparent dissociation constant (Kd) of calmodulin binding was found to be 2 microM for tau, and 5 microM for MAP2. These Kd values were similar to the Kd previously determined for calmodulin binding to tubulin. The inhibitory effect of increasing concentrations of calmodulin on the kinetics of microtubule assembly from tau and tubulin was not mimicked by decreasing the concentration of tau alone or tubulin alone. These results suggest that calmodulin inhibits microtubule assembly by its binding to both MAPs and tubulin.     

Microtubules and protein secretion in rat lacrimal glands. Inhibitory effect of the tubulin . colchicine complex isolated from lacrimal glands upon brain tubulin polymerization. Identification of the complex by gel electrophoresis.

The specific inhibitory effect of colchicine upon protein secretion by lacrimal glands could be related to the formation of a complex between colchicine and tubulin from the soluble fraction of the gland. By gel electrophoresis under nondissociating conditions, it is shown that this complex is similar to the colchicine . tubulin complex from brain. The complex isolated from lacrimal glands is highly inhibitory upon brain tubulin assembly since as low as 0.07 microM complex impedes the polymerization of 8 microM tubulin by 50%, compared to 3 microM for free colchicine. Therefore, a small percentage of complexed tubulin (0.9%) is enough for polymerization to be blocked. In lacrimal glands the complex might prevent the polymerization of tubulin, and colchicine shift the tubulin in equilibrium microtubules equilibrium to microtubules disassembly. The disorganization of the labile microtubular system could lead to a modification of the transport of the secretory granules and to a perturbation of secretion.