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![SDS-PAGE of tubulin and MAPs incubated with [32P]NAD and cholera toxin. The reaction mixtures contained [32P]NAD 16.3 Ci/mmol and the protein to be labeled with or without cholera toxin as indicated. 10 l,g of labeled protein fraction were loaded in each lane, and the resulting autoradiographic pattern is shown. Lane 1, cholera toxin alone](profile/Yehudit-Zaltsman/publication/16434180/figure/fig1/AS:340574461808643@1458210687930/SDS-PAGE-of-tubulin-and-MAPs-incubated-with-32PNAD-and-cholera-toxin-The-reaction.png)
SDS-PAGE of tubulin and MAPs incubated with [32P]NAD and cholera toxin. The reaction mixtures contained [32P]NAD 16.3 Ci/mmol and the protein to be labeled with or without cholera toxin as indicated. 10 l,g of labeled protein fraction were loaded in each lane, and the resulting autoradiographic pattern is shown. Lane 1, cholera toxin alone
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![Fig. 1. SDS-PAGE of tubulin and MAPs incubated with [32P]NAD and...](profile/Yehudit-Zaltsman/publication/16434180/figure/fig1/AS:340574461808643@1458210687930/SDS-PAGE-of-tubulin-and-MAPs-incubated-with-32PNAD-and-cholera-toxin-The-reaction_Q320.jpg)
Incubation of purified rat brain tubulin with cholera toxin and radiolabeled [32P] or [8-3H]NAD results in the labeling of both alpha and beta subunits as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of these protein bands with snake venom phosphodiesterase resulted in quantitative release of labeled 5...
Citations
... § Human α-tubulin can be phosphorylated at S165 and pig brain β-tubulin at T409 and S420 (Yoshida et al., 2003;Abeyweera et al., 2009). References: (Amir-Zaltsman et al., 1982;Wandosell et al., 1987;Correia et al., 1993;Najbauer et al., 1996;Prasad and Dey, 2000;Hino et al., 2003;Yoshida et al., 2003;Plessmann et al., 2004;Starita et al., 2004;Rosas-Acosta et al., 2005;Iwabata et al., 2005;Dremina et al., 2005;Verhey and Gaertig, 2007;Wong et al., 2007;Ludueña and Banerjee, 2008a;Cicchillitti et al., 2008;Miller et al., 2008;Abeyweera et al., 2009;Del Duca et al., 2009;Song et al., 2009Song et al., ,2010Wloga and Gaertig, 2010;Xiao et al., 2010). ...
Tubulin, the protein subunit of microtubules (MTs), is an α/β heterodimer. In this chapter, a hypothesis on the evolution of the tubulin molecule is proposed, based in part on recent reports on the structures and functions of different forms of tubulin and its relatives. The concentration is on three main areas. 1) Evolution of the vertebrate β-tubulin isotypes. In addition to providing a clear idea about the relationships among these isotypes, recent data suggest that tubulin may have functions that do not involve being in a MT, namely, that it can function as an isolated α/β dimer or as a non-MT polymer. 2) Examination of the entire tubulin superfamily, which includes not only tubulins α, β, γ, δ, ε, η, and others but also a variety of prokaryotic proteins. The hypothesis is presented that the common ancestor of all these proteins formed a filamentous curving polymer that used the energy of GTP hydrolysis to apply force to nucleic acids and/or membranes and that this common ancestor may have been coeval with the first cells. A variety of chaperones, motors and MT-associated proteins may have coevolved with tubulin and their histories illuminate that of tubulin. The branched, highly negatively charged C-terminal domain present on α- and β-tubulin appears to be a relatively recent addition to tubulin. 3) The hypothesis is presented that the C-terminal domain may have been of prebiotic origin and that it gradually developed into a protein serving particular metabolic functions whose gene eventually became fused with those of α- and β-tubulin. Finally, some experiments are proposed that could illuminate the probability of these hypotheses.
... A rst overview revealed that the proteins tubulin, vimentin, nucleolin and heat shock cognate 71 protein were found to be interacting with ARTD9/BAL1 tandem (9Tm), ARTD7 and ARTD8 macro domains. ese proteins were previously described to be ADP-ribosylated and to be interaction partners of ARTD1 (11,37,127,128). is provided the rst evidence that the established experimental setup was working in the anticipated way. ...
Das B-aggressive Lymphomprotein (BAL1/ARTD9) wurde als prognostischen Marker für den Verlauf von diffus grosszelligem B-Zell Lymphom identifiziert, dem meistdiagnostizierten Lymphom bei Erwachsenen. Als macroARTD enthält ARTD9 zwei macro Domänen und eine ADP-ribosyltransferasen-Domäne, und gehört somit zu den diphterietoxin-ähnlichen ADP- ribosyltransferasen (ARTDs). Ziel dieser Arbeit war es, Interaktionspartner von macroARTD Proteinen zu identifizieren und funktionell zu charakterisieren. Unter Verwendung eines unvoreingenommenen Immunpräzipitationsverfahrens und mit Hilfe von Glutathion S- Transferase (GST) Pulldowns wurden Interaktionspartner der gesamtprotein- als auch der macro-Domänen-spezifischen BAL-Komplexe unter physiologischen Bedingungen untersucht. Anschliessend wurden interagierende Proteine mit Massenspektrometrie identifiziert und mittels Westernblot validiert. Das Protein p62, das an der selektiven Autophagie zentral beteiligt ist, wurde als spezifischer Interaktionspartner der ARTD9 macro Domänen gefunden. p62 ist in mehreren menschlichen Tumoren hochreguliert. Daher wurde der Effekt induzierter Autophagie auf ARTD9 untersucht. Wir stellten fest, dass die Menge an ARTD9 abnimmt, wenn Autophagie durch Hungern in A549 Zellen ausgelöst wird. Dieser Effekt konnte mit p62 siRNA aufgehoben werden, jedoch nicht mit beclin-1 siRNA. Diese Resultate zeigen, dass ARTD9 durch p62-abhängige aber beclin-1 unabhängige selektive Autophagie abgebaut wird. B-aggressive lymphoma 1 protein (BAL1/ARTD9) was described as prognostic marker for the progression of diffuse large B-cell lymphoma, the most common lymphoid cancer in humans. As a macroARTD, BAL1/ARTD9 contains 2 macro and an ADP-ribosyltransferase domain, the signature of diphteria toxin-like ADP-ribosyltransferases (ARTDs). The aim of this study was to identify and functionally characterize interaction partners of the macroARTD proteins. Using an unbiased glutathione S-transferase (GST) pull-down and co- immunoprecipitation approach, full length and macro domain specific interaction partners of BAL complexes were investigated under physiological conditions. Subsequently, protein interaction partners were identified by mass spectrometry and validated by western blot. Among other proteins, p62, a signaling hub involved in selective autophagy, was found as specific interaction partner of the ARTD9 macro domains. p62 is up-regulated in several human tumors. Therefore the effect of autophagy induction on ARTD9 was investigated. We discovered that ARTD9 levels are decreased upon induction of autophagy by starvation in A549 cells. is effect was abolished by p62 but not beclin-1 siRNA. These results revealed that ARTD9 is degraded by p62 dependent but beclin-1 independent selective autophagy.
... On the other hand tubulin is, like the G-proteins, a 1 Author for correspondence. substrate for pertussis or cholera toxin catalysed ADP-ribosylation (Amir-Zaltsman et al., 1982;Wang et al., 1990) and seems to control cellular Ca2" fluxes (Lim et al., 1985). Based on these and other (Taylor et al., 1973;Omann et al., 1987) observations that suggest the implication of the cytoskeleton in the signal transduction mechanism of plasma membrane receptors, we found it interesting to determine the role of the cytoskeleton on the hepatic al-adrenoceptor signalling pathway. ...
. The cytoskeletal depolymerizing agent, colchicine, prevents the hepatic α 1 ‐adrenoceptor‐mediated stimulation of respiration, H ⁺ and Ca ²⁺ release to the effluent perfusate, intracellular alkalosis, and glycogenolysis. Unlike the other parameters, colchicine does not perturb the α 1 ‐agonist‐induced stimulation of gluconeogenesis or phosphorylase ‘a’ activation, and enhances the increase in portal pressure response. The lack of effect of colchicine on the hepatic α 2 ‐adrenoceptor‐mediated effects indicates that its actions are α 1 ‐specific.
. Colchicine enhances the acute α 1 ‐adrenoceptor‐mediated intracellular Ca ²⁺ mobilization and prevents the activation of protein kinase C. This differential effect on the two branches of the α 1 ‐adrenoceptor signalling pathway is a distinctive feature of the colchicine action.
. The lack of effect of colchicine in altering the α 1 ‐adrenoceptor ligand binding affinity suggests that it might interact with some receptor‐coupled regulatory element(s).
. The acuteness of the colchicine effect and the ability of its isomer β‐lumicolchicine to prevent all the α 1 ‐adrenoceptor‐mediated responses but the increase in vascular resistance, indicate that its action cannot be merely ascribed to its effects in depolymerizing tubulin.
. Colchicine perturbs the hepatic responses to vasoactive peptides. It enhances the vasopressin‐induced rise of cytosolic free Ca ²⁺ in isolated hepatocytes and prevents the sustained decrease of Ca ²⁺ in the effluent perfusate. It also inhibits the stimulation of glycogenolysis, without altering the stimulation of gluconeogenesis.
. It is concluded that there are at least two major α 1 ‐adrenoceptor signalling pathways. One is colchicine‐sensitive, independent of variations in free cytosolic Ca ²⁺ , and protein kinase C‐independent; the other one is colchicine‐insensitive, dependent on variations in free cytosolic Ca ²⁺ , and protein kinase C‐independent.
... To assay ADP-ribosyltransferase activity with histone as the ADPribose acceptor, samples were incubated as described (21) in a final volume of 0.1 ml, containing 100 mM potassium phosphate (pH 7.9, 10 mM thymidine, 0.1 mM GTP, 2.5 mM MgCI,, 1 mM EDTA, 1 mM D'IT, 20 pM NAD, 10 pCi [3ZP]NAD, and 10 p g arginine-rich histone (Sigma Chemical Co.; Cat. No. H4380). ...
This report demonstrates that incubation of cytotoxic T cells with NAD causes suppression of their ability to proliferate in response to stimulator cells or to lyse targets. Effects are evident after incubation for 3 h with concentrations of NAD as low as 1 microM and are sustained for many hours after removal of NAD from culture media. Suppression is a result of the failure of CTL to form specific conjugates with targets as well as a lower level of activation in response to TCR-mediated stimulation, although TCR-mediated transmembrane signaling is demonstrable. Metabolites of NAD such as nicotinamide, ADP-ribose, and cyclic-ADP-ribose have no detectable effect, indicating that NAD-glycohydrolase or ADP-ribose cyclase do not mediate suppression. Incubation of intact CTL with [32P]NAD leads to incorporation of 32P into a particulate, subcellular fraction, a reaction that is not inhibitable by ADP-ribose. Hydroxylamine, but not mercuric ion releases [32P]ADP-ribose, whereas phosphodiesterase releases [32P]AMP from the particulate subcellular fraction, suggesting that labeling is a result of enzymatic mono-ADP-ribosylation of arginines. In support of this, treatment of intact CTL with phosphatidylinositol-specific phospholipase C releases an arginine-specific ADP-ribosyltransferase and causes insensitivity to ecto-NAD suppression. These results suggest that a GPI-anchored ADP-ribosyltransferase uses ecto-NAD to ADP-ribosylate proteins that regulate CTL function.
... Cells were maintained for 1 h at 37°C before being centrifuged at 500 x g. The permeabilization procedure described by Amir-Zaltsman et al. [23] was then followed. The cells were washed once in phosphate-buffered saline, pH 7.4 (NaCl/Pi), then suspended in a cold 5 m M potassium phosphate buffer (pH 7.4) containing 150 mM sucrose, 80 mM KCI, 35 mM Hepes (solution A). ...
Incubation of FRTL-5 rat thyroid cell membranes with [32P]NAD and pertussis toxin results in the specific ADP-ribosylation of a protein of about 40 kDa. This protein has the same molecular mass of the alpha i subunit of the adenylate cyclase regulatory protein Ni and is distinct from proteins ADP-ribosylated by cholera toxin in the same membranes. Prior treatment of FRTL-5 cells with pertussis toxin results in the ADP-ribosylation of Ni, as indicated by the loss of the toxin substrate in the ADP-ribosylation assay performed with membranes prepared from such cells. Preincubation of FRTL-5 cells with thyrotropin causes the same loss; cholera toxin has no such effect. Pertussis toxin, as do thyrotropin and cholera toxin, increases cAMP levels in FRTL-5 cells. Forskolin together with thyrotropin, cholera toxin or pertussis toxin causes a further increase in cAMP levels. Pertussis toxin and thyrotropin are not additive in their ability to increase adenylate cyclase activity, whereas both substances are additive with cholera toxin. A role of Ni in the thyrotropin regulation of the adenylate cyclase activity in thyroid cells is proposed.
Previous studies have demonstrated that dimeric tubulin, associated with synaptic membrane, is capable of activating the G-proteins Gs and G alpha i1 via transfer of GTP. To clarify the mechanism of intracellular interaction between tubulin and G alpha s as it refers to adenylyl cyclase activation, wild type and chimeric G alpha s/G alpha i2 proteins were transiently overexpressed in COS 1 cells. Effects of tubulin dimers with guanosine 5'-(beta, gamma-imido)triphosphate (Gpp(NH)p) bound (tubulin-Gpp(NH)p) or Gpp NH)p with/without isoproterenol on adenylyl cyclase were assessed in cells made permeable with saponin. In naive and wild type G alpha s-overexpressing COS 1 cells, the beta-adrenergic agonist isoproterenol potentiated significantly the stimulatory effects of Gpp(NH)p and, to an even greater extent, tubulin-Gpp(NH)p on adenylyl cyclase. In COS 1 cells expressing the chimera G alpha i(54)/s (G alpha i2 1-54, G alpha s 62-394 amino acids), tubulin-Gp-p (NH)p was more potent than Gpp(NH)p in the presence of isoproterenol, but the maximal activity was equal. In chimera G alpha s/i(38) (G alpha s 1-356, G alpha i2 357-392) tubulin-Gp-p(NH)p or Gpp(NH)p stimulated adenylyl cyclase activity 11-14 times above the control whether or not beta-adrenergic receptor was activated, suggesting that G alpha chimera and the beta-adrenergic receptor are uncoupled. The chimera G alpha i/s(Bam) (G alpha i2 1-212, G alpha s 213-292) was nearly identical to native COS 1 cells, but isoproterenol potentiated Gpp(NH)p but not the tubulin-Gpp(NH)p response. The construct G alpha i(Bam)/s/i(38) (G alpha i2 1-212, G alpha s 213-356, G alpha i2 357-392) was weakly responsive to Gpp(NH)p or tubulin-Gpp(NH)p and unresponsive to isoproterenol. In photoaffinity labeling studies with tubulin-[32P]azidoanilido-GTP (tubulin-[32P]AAGTP), isoproterenol increased the amount of tubulin associated with membranes and the transfer of [32P]AAGTP from tubulin to G alpha i(54)/s, G alpha s, and G alpha i/s(Bam), but not to G alpha i(Bam)/s/i(38) and very slightly to G alpha s/i(38). These results suggest that regions between the 54th and 212th amino acids of G alpha s are important for guanine nucleotide transfer from tubulin, while the 1st to 54th amino acids of G alpha s are required for the ability of tubulin to activate adenylyl cyclase. We speculate that the active G alpha s conformation provoked by nucleotide transfer from tubulin is stabilized by G alpha s-tubulin interaction leading to extended stimulation of adenylyl cyclase.
Antidepressant drugs have been used clinically not only for depression but also for other psychiatric disorders. Despite extensive studies, the mechanisms of action of antidepressant drugs have not been clearly established. The classic monoamine hypothesis of depression suggests that depressive disorders are associated with subnormal monoamine release at certain synapses of the CNS. Antidepressant drugs are supposed to increase the availability of noradrenaline and serotonin, either by inhibiting amine reuptake or by blocking monoamine oxidase in presynaptic nerve terminals, and facilitate monoamine transmission (Schildkraut 1965). However, an acute effect of antidepressants on neurotransmission is inconsistent with the delayed onset of clinical efficacy of these drugs (Zemlan and Garver 1990). Furthermore, neuroleptic drugs such as amphetamine and cocaine that block reuptake or catabolism of monoamines do not have an antidepressant effect. Thus, an acute neurochemical effect of antidepressant drugs may not account for the mechanism of action of these drugs.
Very soon after the discovery that NAD was required for cholera toxin to activate adenylate cyclase in pigeon erythrocyte membranes, it was recognized that a second nucleotide was also essential. At first the requirement seemed highly unspecific, being satisfied by a variety of sugar phosphates as well as by the majority of nucleoside diphosphates and triphosphates. However, as it became possible to simplify the test system, it became clear that these compounds were only effective by virtue of their abilities to generate GTP from endogenous GDP and GMP. Under conditions in which such change was prevented, notably by the removal of the magnesium ions essential for transphosphorylation reactions, only GTP proved to be effective: not ATP and not GDP. The closest contender is ITP, a nucleotide closely related to GTP that is commonly found to substitute for GTP. The amount of GTP required was most relevantly measured in the presence of an excess of ATP in order to regenerate the GTP split by the nonspecific nucleoside triphosphatases present. An alternative method was to use the nonhydrolyzable analog of GTP, Gpp(NH)p, (the usual alternative, GTPγS, is in fact slowly hydrolyzed to GTP by lysed cells). Both methods gave Ka values of 1 to 3 μM [1].
ADP-ribosylation of proteins has been detected and characterized to occur as a posttranslational modification both on cytoplasmic and nuclear polypeptides. Usually, protein acceptors have been characterized by in vitro reactions, utilizing [32P]-NAD+ with high specific activity as substrate for enzyme-catayzed transfer of ADP-ribose. Several cytoplasmic acceptors have in this way been identified, including such diverse proteins as the protein synthesis elongation factor 2 [1, 2] and several cytoplasmic structural proteins [3,4]. However, few studies have tried to answer the question of which proteins act as in vivo acceptors, mainly because of a lack of a suitable radio-actively labeled precursor. Recently I showed that the by far most abundant intracellular acceptor for mono(ADP-ribose) in vivo is a polypeptide with a Mr of 83,000 and identified by several criteria as identical with the stress-inducible and glucose-regulated HSP-83 [5]. This was made possible by utilizing [3H]-adenosine as a precursor to intracellular [3H]-ATP and thus also [3H]-NAD+, and separating the total cell homogenate by two-dimensional isoelectric focusing/SDS Polyacrylamide gel electrophoresis (2D IEF/SDS-PAGE) followed by fluorography. It was further shown that both heat shock and glucose starvation can induce drastic changes in the incorporation of tritiated ADP-ribose into HSP-83, suggesting that this modification of the protein plays an important physiological function. However, so far no enzymatic activity or other specific function has been assigned to HSP-83, other than its enhanced transcription during stress situations induced in a variety of ways [6].
The α- and β-tubulins are the major polypeptide components of microtubules. In the immature nervous system, microtubules are involved in cell division, in the determination of cell shape, and in neuronal growth and migration. In the mature nervous system, microtubules are also involved in synaptogenesis and in axonal transport (Olmsted and Borisy, 1973; Dustin, 1978).
