Microtubule capture by CENP-E silences BubR1-dependent mitotic checkpoint signaling.

The mitotic checkpoint is the major cell cycle control mechanism for maintaining chromosome content in multicellular organisms. Prevention of premature onset of anaphase requires activation at unattached kinetochores of the BubR1 kinase, which acts with other components to generate a diffusible "stop anaphase" inhibitor. Not only does direct binding of BubR1 to the centromere-associated kinesin family member CENP-E activate its essential kinase, binding of a motorless fragment of CENP-E is shown here to constitutively activate BubR1 bound at kinetochores, producing checkpoint signaling that is not silenced either by spindle microtubule capture or the tension developed at those kinetochores by other components. Using purified BubR1, microtubules, and CENP-E, microtubule capture by the CENP-E motor domain is shown to silence BubR1 kinase activity in a ternary complex of BubR1-CENP-E-microtubule. Together, this reveals that CENP-E is the signal transducing linker responsible for silencing BubR1-dependent mitotic checkpoint signaling through its capture at kinetochores of spindle microtubules.

Synthetic analogs of green tea polyphenols as proteasome inhibitors.

BACKGROUND: Animal, epidemiological and clinical studies have demonstrated the anti-tumor activity of pharmacological proteasome inhibitors and the cancer-preventive effects of green tea consumption. Previously, one of our laboratories reported that natural ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG] and (-)-gallocatechin-3-gallate [(-)-GCG], are potent and specific proteasome inhibitors. Another of our groups, for the first time, was able to enantioselectively synthesize (-)-EGCG as well as other analogs of this natural GTP. Our interest in designing and developing novel synthetic GTPs as proteasome inhibitors and potential cancer-preventive agents prompted our current study. MATERIALS AND METHODS: GTP analogs, (+)-EGCG, (+)-GCG, and a fully benzyl-protected (+)-EGCG [Bn-(+)-EGCG], were prepared by enantioselective synthesis. Inhibition of the proteasome or calpain (as a control) activities under cell-free conditions were measured by fluorogenic substrate assay. Inhibition of intact tumor cell proteasome activity was measured by accumulation of some proteasome target proteins (p27, I kappa B-alpha and Bax) using Western blot analysis. Inhibition of tumor cell proliferation and induction of apoptosis by synthetic GTPs were determined by G(1) arrest and caspase activation, respectively. Finally, inhibition of the transforming activity of human prostate cancer cells by synthetic GTPs was measured by a colony formation assay. RESULTS: (+)-EGCG and (+)-GCG potently and specifically inhibit the chymotrypsin-like activity of purified 20S proteasome and the 26S proteasome in tumor cell lysates, while Bn-(+)-EGCG does not. Treatment of leukemic Jurkat T or prostate cancer LNCaP cells with either (+)-EGCG or (+)-GCG accumulated p27 and IkappaB-alpha proteins, associated with an increased G(1) population. (+)-EGCG treatment also accumulated the pro-apoptotic Bax protein and induced apoptosis in LNCaP cells expressing high basal levels of Bax, but not prostate cancer DU-145 cells with low Bax expression. Finally, synthetic GTPs significantly inhibited colony formation by LNCaP cancer cells. CONCLUSIONS: Enantiomeric analogs of natural GTPs, (+)-EGCG and (+)-GCG, are able to potently and specifically inhibit the proteasome both, in vitro and in vivo, while protection of the hydroxyl groups on (+)-EGCG renders the compound completely inactive.

Isolation of high-affinity GTP aptamers from partially structured RNA libraries.

Aptamers, RNA sequences that bind to target ligands, are typically isolated by in vitro selection from RNA libraries containing completely random sequences. To see whether higher-affinity aptamers can be isolated from partially structured RNA libraries, we selected for aptamers that bind GTP, starting from a mixture of fully random and partially structured libraries. Because stem-loops are common motifs in previously characterized aptamers, we designed the partially structured library to contain a centrally located stable stem-loop. We used an off-rate selection protocol designed to maximize the enrichment of high-affinity aptamers. The selection produced a surprisingly large number of distinct sequence motifs and secondary structures, including seven different aptamers with K(d)s ranging from 500 to 25 nanomolar. The engineered stem-loop was present in the three highest affinity aptamers, and in 12 of 13 independent isolates with a single consensus sequence, suggesting that its inclusion increased the abundance of high-affinity aptamers in the starting pool.

Single cell Ras-GTP analysis reveals altered Ras activity in a subpopulation of neurofibroma Schwann cells but not fibroblasts.

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by multiple neurofibromas, peripheral nerve tumors containing mainly Schwann cells and fibroblasts. The NF1 gene encodes neurofibromin, a tumor suppressor postulated to function in part as a Ras GTPase-activating protein. The roles of different cell types and of elevated Ras-GTP in neurofibroma formation are unclear. To determine which neurofibroma cell type has altered Ras-GTP regulation, we developed an immunocytochemical assay for active, GTP-bound Ras. In NIH 3T3 cells, the assay detected overexpressed, constitutively activated K-, N-, and Ha-Ras and insulin-induced endogenous Ras-GTP. In dissociated neurofibroma cells from NF1 patients, Ras-GTP was elevated in Schwann cells but not fibroblasts. Twelve to 62% of tumor Schwann cells showed elevated Ras-GTP, unexpectedly revealing neurofibroma Schwann cell heterogeneity. Increased basal Ras-GTP did not correlate with increased cell proliferation. Normal human Schwann cells, however, did not demonstrate elevated basal Ras activity. Furthermore, compared with cells from wild type littermates, Ras-GTP was elevated in all mouse Nf1(-/-) Schwann cells but never in Nf1(-/-) mouse fibroblasts. Our results indicate that Ras activity is detectably increased in only some neurofibroma Schwann cells and suggest that neurofibromin is not an essential regulator of Ras activity in fibroblasts.

A fluorometric assay for cyclic guanosine 3',5'-monophosphate incorporating a Sep-Pak cartridge and enzymatic cycling.

We developed a sensitive and nonradioactive fluorometric assay for cyclic guanosine 3',5'-monophosphate (cGMP). Guanine nucleotides except cGMP were enzymatically phosphorylated to GTP. cGMP, absorbed into a Sep-Pak amino propyl cartridge, was eluted separately from GTP. Purified cGMP was enzymatically converted to GTP, which was applied to the GTP-GDP cycle using succinic thiokinase and pyruvate kinase. When pyruvic acid produced by the GTP-GDP cycle was reduced by lactate dehydrogenase, a reduced form of nicotinamide adenine dinucleotide (NADH) was equivalently oxidized to NAD(+). NAD(+) was further converted into fluorescent compound, which was excited at 370 nm and emitted fluorescence at 460 nm, by a strong alkali. When 20 nmol NADH was used for this assay, the calibration curve over 50 to 500 fmol cGMP became sufficiently linear. The detection limit for cGMP was ca. 5 fmol (signal to noise ratio >3). Using this assay, we confirmed that the cGMP content in the left atrial strip of dog was changed from 11.4 +/- 3.8 to 19.3 +/- 2.6 fmol/mg wet wt of tissue (mean +/- SE, n = 6) by electrical driving at 1 Hz. Carbachol (1 microM) further increased the cGMP to 45.6 +/- 9.2 fmol/mg wet wt of tissue. From these results, it is suggested that this novel assay for cGMP is highly sensitive and can be applied to various biological samples.

A useful method for the synthesis of 8 azido-guanosine tri- and tetraphosphate: important substrates for many enzymatic reactions.

8-N3 GMP was synthesised from 8-BrGMP by the addition of LiN3. 8-N3GMP was then phosphorylated to N3-GDP and N3-GTP by controlled enzymatic reaction. 8-N3GDP can be converted to N3-ppGpp with crude Rel A, which phosphorylates the 3'-OH of GDP.

GTP consumption of elongation factor Tu during translation of heteropolymeric mRNAs.

The stoichiometry of elongation factor Tu (EF-Tu) and GTP in the complex with aminoacyl-tRNA and the consumption of GTP during peptide bond formation on the ribosome were studied in the Escherichia coli system. The ribosomes were programmed either with two different heteropolymeric mRNAs coding for Met-Phe-Thr-Ile ... (mMFTI) or Met-Phe-Phe-Gly ... (mMFFG) or with poly(U). The composition of the complex of EF-Tu, GTP, and Phe-tRNA(Phe) was studied by gel chromatography. With equimolar amounts of factor and Phe-tRNA(Phe), a pentameric complex, (EF-Tu.GTP)2.Phe-tRNA(Phe), was observed, whereas the classical ternary complex, EF-Tu.GTP.Phe-tRNA(Phe), was found only when Phe-tRNA(Phe) was in excess. Upon binding of the purified pentameric complex to ribosomes carrying fMet-tRNA(fMet) in the peptidyl site and exposing a Phe codon in the aminoacyl site, only one out of two GTPs of the pentameric complex was hydrolyzed per Phe-tRNA bound and peptide bond formed, regardless of the mRNA used. In the presence of EF-G, the stoichiometry of one GTP hydrolyzed per peptide bond formed was found on mMFTI when one or two elongation cycles were completed. In contrast, on mMFFG, which contains two contiguous Phe codons, UUU-UUC, two GTP molecules of the pentameric complex were hydrolyzed per Phe incorporated into dipeptide, whereas the incorporation of the second Phe to form tripeptide consumed only one GTP. Thus, generally one GTP is hydrolyzed by EF-Tu per aminoacyl-tRNA bound and peptide bond formed, and more than one GTP is hydrolyzed only when a particular mRNA sequence, such as a homopolymeric stretch, is translated. The role of the additional GTP hydrolysis is not known; it may be related to frameshifting of peptidyl-tRNA during translocation.

Purification and crystallization of the ternary complex of elongation factor Tu:GTP and Phe-tRNA(Phe).

Elongation factor Tu (EF-Tu) is the most abundant protein in prokaryotic cells. Its general function in protein biosynthesis is well established. It is a member of the large family of G-proteins, all of which bind guanosine phosphates (GDP or GTP) as cofactors. In its active GTP bound state EF-Tu binds aminoacylated tRNA (aa-tRNA) forming the ternary complex EF-Tu:GTP:aa-tRNA. The ternary complex interacts with the ribosome where the anticodon on tRNA recognises a codon on mRNA, GTPase activity is induced and inactive EF-Tu:GDP is released. Here we report the successful crystallization of a ternary complex of Thermus aquaticus EF-Tu:GDPNP and yeast Phe-tRNA(Phe) after its purification by HPLC.

Incorporation of bisphosphonates into adenine nucleotides by amoebae of the cellular slime mould Dictyostelium discoideum.

Bisphosphonates are a class of synthetic pyrophosphate analogues. Some are known to be potent inhibitors of osteoclast-mediated bone resorption in vivo, but their mechanisms of action are unclear. The order of potency of bisphosphonates as inhibitors of bone resorption closely matches the order of potency as inhibitors of growth of amoebae of the slime mould Dictyostelium discoideum, indicating that bisphosphonates may have a mechanism of action that is similar in both osteoclasts and Dictyostelium. Methylenebisphosphonate and several halogenated derivatives, which have low potency as antiresorptive agents and as growth inhibitors of Dictyostelium, are metabolized intracellularly by Dictyostelium amoebae into methylene-containing adenine nucleotides. We have used a combination of n.m.r. and f.p.l.c. analysis to determine whether incorporation into nucleotides is a feature of other bisphosphonates, especially those that are potent antiresorptive agents. Only bisphosphonates with short side chains or of low potency are incorporated into adenine nucleotides, whereas those with long side chains or of high potency are not metabolized. Bisphosphonate metabolism in cell-free extracts of Dictyostelium was accompanied by inhibition of aminoacylation of tRNA by several aminoacyl-tRNA synthetases. These enzymes were barely affected by the bisphosphonates that were not metabolized. The results indicate that some bisphosphonates are not metabolically inert analogues of pyrophosphate and appear to be metabolized by aminoacyl-tRNA synthetases. The cellular effects of some bisphosphonates may be the result of their incorporation into adenine nucleotides or inhibition of aminoacyl-tRNA synthetases, although the potent bisphosphonates appear to act by a different mechanism.

2'-Deoxy-GTP in the microtubule cytoskeleton of neuronal cells cultured with nerve growth factor.

Tubulin, widely recognized as a GTP/GDP-binding protein, has been isolated in its polymerized state from rat PC12 cells and embryonic chick dorsal root ganglion neurons by Triton X-100 detergent extraction of the cytoskeletal fraction. Perchloric acid extraction and deproteinization of this fraction permitted subsequent analysis of nucleotide identity and content by high performance liquid chromatography. PC12 cells grown in the absence of nerve growth factor (NGF) contained ADP, ATP, GDP, and GTP at levels consistent with the actin and tubulin content of the cytoskeletal fraction. Microtubules from PC12 cells cultured in the presence of NGF contain an additional nucleotide that we have identified as dGTP. Analysis of whole cell nucleotide extracts from PC12 cells grown in the absence or presence of NGF revealed no evidence for the presence of dGTP at 4 and 14 days, respectively. We have determined that embryonic chick dorsal root ganglion neurons also contain this deoxyribonucleotide, and we found virtually no ADP or ATP in the extracted dorsal root ganglion cytoskeletal fraction. On the basis of metabolic labeling studies with [14C] guanine, we have inferred that the presence of dGTP in NGF-treated PC12 cells probably arises either from binding to the nonexchangeable nucleotide site of tubulin undergoing dynamic assembly/disassembly or from binding to the exchangeable site of tubulin subsequently incorporated into highly stabilized microtubules.

Purification and characterization of five different alpha subunits of guanine-nucleotide-binding proteins in bovine brain membranes. Their physiological properties concerning the activities of adenylate cyclase and atrial muscarinic K+ channels.

We have purified five different alpha subunits of guanine-nucleotide-binding proteins (G proteins) from bovine brain membranes as active forms bound to guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]). All the purified alpha subunits were interacted with beta gamma subunits and served as a substrate for pertussin-catalyzed ADP-ribosylation. Based on the findings of immunoblot analyses using specific antibodies raised against various alpha subunits of G proteins, three of them were identified as alpha i-1, alpha i-2 and alpha i-3, and the other two were classified into alpha o type. One of the alpha o-type proteins was the most abundant in the brain membranes (termed alpha o), and the other (alpha o2) appeared to differ from alpha o in its proteolytic digestion data. The physiological properties of these purified GTP[gamma S]-bound alpha subunits towards adenylate cyclase and atrial muscarinic K+ channels were studied. The nucleotide-bound forms of alpha i-1, alpha i-2, alpha i-3 and alpha o2 inhibited the adenylate cyclase activity of S49 cyc- membranes which had been reconstituted with GTP[gamma S]-treated Gs; this inhibition appeared to be mainly competitive with the activated Gs, alpha i-1 having the most potent inhibitory activity among them. GTP[gamma S]-bound alpha o, however, could not inhibit the Gs-stimulated activity at all. On the other hand, all the GTP[gamma S]-bound alpha subunits activated atrial muscarinic K+ channels, accompanied by a lag time, at picomolar concentrations. The beta gamma subunits resolved from G proteins also activated the K+ channels without a lag time at nanomolar concentration. The maximum activation by the beta gamma subunits appeared to be more potent than that by any of the alpha subunits. These results suggest that alpha and beta gamma subunits might activate the K+ channels by mechanisms different from each other.

Stereochemistry and lifetime of the GTP hydrolysis intermediate at the active site of elongation factor Tu from Bacillus stearothermophilus as inferred from the 17O-55Mn superhyperfine interaction.

Electron paramagnetic resonance spectroscopy has been used to obtain information on the structure and stability of the products of GTP cleavage at the active site of elongation factor Tu (EF-Tu) from Bacillus stearothermophilus. Using stereospecifically labelled (Sp)-(Rp)-[beta-17O]GTP (prepared by modification of a previously published procedure which is now also suitable for guanine nucleotides), it was found that only one of the two possible diastereomers (Sp) led to detectable line-broadening of the EPR spectrum of Mn2+ at the active site of EF-Tu (linewidth 1.5 mT), whereas the Rp isomer caused the same linewidth as unlabelled nucleotide (1.3 mT). From our earlier work and from a demonstration that the lifetime of the state giving the broadened spectrum is too long to be assigned to the EF-Tu.GDP.Mn complex [the rate constant for decay as measured by displacement of GDP by the fluorescent 2'(3')-O-(N-methylanthraniloyl)-GDP is 6.2 x 10(-3) s-1 at 25 degrees C and pH 6.8], we conclude that the broadened signal arises from the EF-Tu.Mn.GDP.Pi complex, the predominant steady-state species. During the hydrolysis of GTP the Mn2+ remains bound to the beta-phosphate oxygen of GDP which arises from the beta pro-S oxygen of GTP, possibly until GDP dissociates and certainly until Pi dissociates. Addition of elongation factor Ts (EF-Ts) to this intermediate leads to rapid reduction of the linewidth to that expected for random distribution of interactions of one 17O and two 16O atoms of GDP with Mn2+, and is not distinguishable from that exhibited by (Rp)-[beta-17O]GTP in the corresponding complex in the presence of EF-Ts.

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VII. Chemical synthesis by phosphorylation of 6-thioguanosine 5'-monophosphate, 5'-diphosphate and 5'-triphosphate, and their purification and identification by reversed-phase/ion-pair high-performance liquid chromatography and by various enzymatic assays.

A fast and reliable two-step method has been established for the chemical synthesis of 6-thioguanosine 5'-monophosphate, 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate starting from the ribonucleoside. In the first step, 6-thioguanosine dissolved in triethyl phosphate, at high yield reacts with phosphorus oxide trichloride to 6-thioguanosine 5'-monophosphate which is purified by anion-exchange chromatography on DEAE-Sephadex using a step gradient of hydrochloric acid. In the second step, 6-thioguanosine 5'-monophosphate dissolved in water, reacts with phosphoric acid in the presence of pyridine/dicyclohexyl carbodiimide and is converted to 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate which are separated from each other and from the 6-thioguanosine 5'-monophosphate by anion-exchange chromatography on DEAE-Sephadex using a gradient of ammonium bicarbonate. Material from each step of the preparation procedure is separated by reversed-phase HPLC chromatography and analyzed for its free ribonucleoside content, 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and small amounts of unidentified phosphorylated compounds. The purity of the final preparations and the identity of each 6-thioguanosine 5'-phosphate are proven by highly specific enzymatic peak-shifting/HPLC analyses using alkaline phosphatase, 5'-nucleotidase, pyruvate kinase, nucleoside diphosphate kinase and combined hexokinase/glucose 6-phosphate dehydrogenase.

Functional and immunological evidence for stable association of solubilized vasoactive-intestinal-peptide receptor and stimulatory guanine-nucleotide-binding protein from rat liver.

We have reported the solubilization of complexes between vasoactive intestinal peptide (VIP) and its receptor from rat liver in a GTP-sensitive form of Mr 150,000 [Couvineau, A., Amiranoff, B. & Laburthe, M. (1986) J. Biol. Chem. 261, 14482-14489]. In the present study, we demonstrate a stable association of solubilized VIP receptor and stimulatory guanine nucleotide-binding protein (Gs protein), taking advantage of the ability of the glycoproteic VIP receptor (Mr 48,000), and the inability of the Gs protein, to adsorb to wheat germ agglutinin (WGA). 125I-VIP-receptor complexes solubilized in Triton X-100 were adsorbed on WGA-Sepharose, extensively washed and the radioactivity retained was eluted with 1 mM GTP showing that: (a) radioactivity corresponds to free 125I-VIP and (b) alpha s (Mr 42,000) and beta (Mr 35,000) subunits of Gs protein are detectable in the GTP eluate by immunoblotting using antisera against these subunits. Such an effect of GTP implied that a stable ternary complex consisting of VIP, receptor and Gs protein had been adsorbed to WGA-Sepharose. When Triton-solubilized 125I-VIP-receptor complexes were adsorbed on WGA-Sepharose, then retained material was specifically eluted with 0.3 M N-acetylglucosamine, analysis of the sugar eluate showed the following results. (a) GTP induces the dissociation of 125I-VIP-receptor complexes of Mr 150,000 contained in the eluate indicating that 125I-VIP-receptor-G protein complexes had been adsorbed to the WGA column. (b) The Mr-42,000 alpha s subunit can be specifically ADP-ribosylated by cholera toxin. (c) Immunoblotting using antisera against the alpha s and beta subunits of Gs protein, reveals Mr-42,000 and Mr-35,000 components corresponding to alpha s and beta subunits, respectively. (d) Affinity cross-linking using dithiobis(succinimidyl-propionate) of 125-I-VIP-receptor complexes eluted from the WGA column reveals a major band corresponding to Mr 150,000. Immunoblotting using antisera against the beta-subunit shows the presence of the beta subunit (Mr 35,000) in this Mr-150,000 component. In conclusion, these data provide functional and immunochemical evidence for the physical association of solubilized VIP-receptor complexes with alpha s and beta subunits of Gs protein.

A firefly luciferase assay for determination of cytidine 5'-triphosphate in biological samples.

A routine assay for CTP in cell and tissue extracts using crude firefly lantern preparations is described. ATP, GTP, and UTP are removed by incubation of the samples with a mixture of 3-phosphoglycerate kinase, hexokinase, glucose-6-phosphate dehydrogenase, and UDP-glucose pyrophosphorylase in the presence of ADP. The method is sensitive (greater than 30 nM CTP), inexpensive, and reproducible. No chromatographic purification of the biological samples or of the firefly extract is necessary. Corrections must be made for some loss of CTP during the enzymatic incubation and for the background luminescence of ADP. The applicability of the assay is tested with extracts from the cyanobacterium Anacystis nidulans.

Updated protocols and comments on the purification without use of activating ligands of the coupling proteins Ns and Ni of the hormone sensitive adenylyl cyclase.

Ns and Ni have been purified without using NaF and Mg as stabilizing agents (Codina, J., Hildebrandt, J.D., Sekura, R.D., Birnbaumer, M., Bryan, J., Manclark, C.R. and Birnbaumer, L. [1984] J. Biol. Chem. 259, in press). Since the submission of that report, several modifications have been introduced to the purification procedure and additional fractions have been processed from which N proteins are obtained. This article describes the updated protocols and presents methodological details not included in the previous publication. The final products are Ns, the stimulatory N, Ni the inhibitory N, both of subunit structure alpha beta gamma, and a Mr = 40,000 protein of beta gamma composition. They are obtained from human erythrocytes.

Synthesis and affinity purification of beta-32P-labeled [gamma-S]GTP.

A method for the synthesis and purification of guanosine 5'-[gamma-S]triphosphate labeled with 32P in the beta-position is described. The first step in the synthesis involves the quantitative transfer of 32Pi from [gamma-32P]dATP to 5'-GMP catalyzed by GMP kinase. Further incubation of the beta-32P]GDP product with [gamma-S]GTP and nucleoside diphosphate kinase results in the synthesis of [beta-32P][gamma-S]GTP with a yield of 10 to 18%. The 32P-labeled [gamma-S]nucleotide is purified by binding to mercury-agarose and eluting with buffer containing beta-mercaptoethanol. Specific incorporation of 32P into the beta-position was demonstrated by treating [beta-32P][gamma-S]GTP with 7% formic acid to remove the gamma-thiophosphate and digesting the remaining [beta-32P]GDP with nucleotide pyro-phosphatase. Although 5'-GMP was released after pyrophosphatase digestion, the only 32P radioactivity detected was as inorganic phosphate.