guanosine-triphosphate and guanosine-5--(alpha-beta-methylene)triphosphate

Recently we reported that activation of Epac1, an exchange protein activated by cAMP, increases melanoma cell migration via Ca 2+ release from the endoplasmic reticulum (ER). G-protein βγ subunits (Gβγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gβγ in cell migration and Ca 2+ signaling in melanoma has not been well studied. Here we report that there is crosstalk of Ca 2+ signaling between Gβγ and Epac in melanoma, which plays a role in regulation of cell migration.. SK-Mel-2 cells, a human metastatic melanoma cell line, were mainly used in this study. Intracellular Ca 2+ was measured with Fluo-4AM fluorescent dyes. Cell migration was examined using the Boyden chambers.. The effect of Gβγ on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a Gβγ -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', α-β-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates Gβγ, also inhibited Epac-induced cell migration. In addition, co-overexpression of β1 and γ2, which is the major combination of Gβγ, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of β adrenergic receptor kinase (βARK-CT), an endogenous inhibitor for Gβγ, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for Gβγ. We next examined the effect of mSIRK on Epac-induced Ca 2+ response. When cells were pretreated with mSIRK, but not with L9A, 8-(4-Methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT), an Epac-specific agonist, failed to increase Ca 2+ signal. Co-overexpression of β1 and γ2 subunits inhibited 8-pMeOPT-induced Ca 2+ elevation. Inhibition of Gβγ with βARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDPβS), a GDP analogue that inactivates Gβγ, restored 8-pMeOPT-induced Ca 2+ elevation even in the presence of mSIRK. These data suggested that Gβγ inhibits Epac-induced Ca 2+ elevation. Subsequently, the mechanism by which Gβγ inhibits Epac-induced Ca 2+ elevation was explored. mSIRK activates Ca 2+ influx from the extracellular space. In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca 2+ elevation, and cell migration. These data suggest that, the mSIRK-induced Ca 2+ from the extracellular space inhibits the Epac-induced Ca 2+ release from the ER, resulting suppression of cell migration.. We found the cross talk of Ca 2+ signaling between Gβγ and Epac, which plays a major role in melanoma cell migration.

Topics: Amino Acid Sequence; beta-Adrenergic Receptor Kinases; Calcium Channel Blockers; Calcium Signaling; Calmodulin; Cell Line, Tumor; Cell Movement; GTP-Binding Protein beta Subunits; GTP-Binding Proteins; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Melanoma; Molecular Sequence Data; Neoplasm Proteins; Peptide Fragments; Peptides; Recombinant Fusion Proteins; Recombinant Proteins; Thionucleotides

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

Topics: Alkanesulfonic Acids; Animals; Brain; Calorimetry; Egtazic Acid; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Magnesium Sulfate; Piperazines; Swine; Tubulin; Vinblastine; Vincristine; Vinorelbine

Despite reduced affinity for the exchangeable nucleotide binding site of tubulin relative to GTP, 2',3'-dideoxyguanosine 5'-triphosphate (ddGTP) and guanosine 5'-[alpha, beta-methylene]triphosphate [pp(CH2)pG] are highly active in promoting tubulin assembly. Like the antimitotic drug paclitaxel, which interacts with the same part of the beta-tubulin molecule as exchangeable-site GTP, both analogs enhance nucleation reactions and promote formation hyperstable polymers. These observations led us to synthesize the doubly modified analog 2',3'-dideoxyguanosine 5'-[alpha, beta-methylene]triphosphate [pp(CH2)pddG]. We compared the effects of pp(CH2)pddG to those of ddGTP, pp(CH2)pG, and the three-cognate diphosphates in their interactions with tubulin. We found that pp(CH2)pddG was as active as ddGTP and pp(CH2)pG in supporting formation of polymer of increased stability, but that its affinity for the exchangeable site was lower than that of both singly modified analogs [relative affinities for the exchangeable site for pp(CH2)pddG:ddGTP:pp(CH2)-pG:GTP were 1:2.8:10:273]. There were significant differences in interactions of each of the three analogs with tubulin, and the behavior of pp(CH2)pddG was intermediate between that of ddGTP and that of pp(CH2)pG. Most importantly, under the reaction conditions studied, with heat-treated microtubule-associated proteins (MAPs) ddGTP-induced polymer consisted of short microtubules, while polymer formed with both pp(CH2)pddG and pp(CH2)pG consisted of short sheets. On the other hand, assembly without MAPs had a fivefold lower critical concentration for tubulin with ddGTP and pp(CH2)pddG (0.5 mg/ml) than with pp(CH2)pG (2.5 mg/ml). De novo assembly, which occurs readily with 2',3'-dideoxyguanosine 5'-diphosphate, was not observed with either alpha, beta-methylenediphosphate GDP analog.

Topics: Antineoplastic Agents; Binding, Competitive; Chromatography, High Pressure Liquid; Deoxyguanine Nucleotides; Dideoxynucleotides; Dose-Response Relationship, Drug; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Microtubule-Associated Proteins; Microtubules; Paclitaxel; Polymers; Temperature; Tubulin

The binding stability of the different nucleotide-dependent and -independent interactions between elongation factor 2 (EF-2) and 80S ribosomes, as well as 60S subunits, was studied and correlated to the kinetics of the EF-2- and ribosome-dependent hydrolysis of GTP. Empty reconstituted 80S ribosomes were found to contain two subpopulations of ribosomes, with approximately 80% capable of binding EF-2.GuoPP[CH2]P with high affinity (Kd less than 10(-9) M) and the rest only capable of binding the factor-nucleotide complex with low affinity (Kd = 3.7 x 10(-7) M). The activity of the EF-2- and 80S-ribosome dependent GTPase did not respond linearly to increasing factor concentrations. At low EF-2/ribosome ratios the number of GTP molecules hydrolyzed/factor molecule was considerably lower than at higher ratios. The low response coincided with the formation of the high-affinity complex. At increasing EF-2/ribosome ratios, the ribosomes capable of forming the high-affinity complex was saturated with EF-2, thus allowing formation of the low-affinity ribosome.EF-2 complex. Simultaneously, the GTPase activity/factor molecule increased, indicating that the low-affinity complex was responsible for activating the GTP hydrolysis. The large ribosomal subunits constituted a homogeneous population that interacted with EF-2 in a low-affinity (Kd = 1.3 x 10(-6) M) GTPase active complex, suggesting that the ribosomal domain responsible for activating the GTPase was located on the 60S subunit. Ricin treatment converted the 80S particles to the type of conformation only capable of interacting with EF-2 in a low-affinity complex. The structural alteration was accompanied by a dramatic increase in the EF-2-dependent GTPase activity. Surprisingly, ricin had no effect on the factor-catalyzed GTP hydrolysis in the presence of 60S subunits alone.

Topics: Animals; Binding Sites; GTP Phosphohydrolase-Linked Elongation Factors; Guanosine Triphosphate; Hydrolysis; Kinetics; Peptide Elongation Factor 2; Peptide Elongation Factors; Phosphoric Monoester Hydrolases; Rats; Ribosomal Proteins; Ribosomes

Injection of the alpha, beta-nonhydrolyzable GTP analog, guanosine 5'-[alpha, beta-methylene]triphosphate (pp[CH2]pG) into PtK2, A549, and Swiss 3T3 cells produced dramatic changes in the normal pattern of long radiating microtubules displayed by the cells before injection. Injection of pp[CH2]pG into cells growing in normal medium resulted in the formation of microtubule bundles resistant to depolymerization by Colcemid and calcium. Cells injected with pp[CH2]pG after incubation with Colcemid for 2 hr showed polymerization of tubulin into long wavy ribbons within 2 hr after injection. Removal of Colcemid 1 hr after the injection of pp[CH2]pG resulted in assembly of tubulin into short single randomly oriented microtubules. All cells injected with pp[CH2]pG showed impeded translocation and restriction or absence of intracellular movement. pp[CH2]pG also prevented the fragmentation of Golgi elements in A549 cells treated with Colcemid. Cells first treated with Colcemid and then injected with pp[CH2]pG failed to reassemble the Golgi elements after the removal of Colcemid. Cells in intimate membrane contact with cells injected with pp[CH2]pG showed similar changes in microtubule polymerization, cell movement, and organization of Golgi elements.

Topics: Animals; Cell Movement; Cells, Cultured; Golgi Apparatus; Guanosine Triphosphate; Microtubules; Protein Binding; Tubulin

Tubulin and the two microtubule-associated proteins MAP2 and tau were purified from pig brain. The ability of MAP2 and tau to induce tubulin polymerization in the presence of guanyl-5'-yl methylene diphosphonate was compared in parallel experiments. MAP2 at 0.2 mol/mol of tubulin induced the polymerization of tubulin exclusively into ribbons 0.45 +/- 0.05 micrometer long with an average of six protofilaments. At the same molar ratio, tau promoted the polymerizaton of tubulin exclusively into microtubules. These results strongly suggest that MAP2 and tau are functionally different proteins.

Topics: Animals; Brain; Guanosine Triphosphate; Macromolecular Substances; Microscopy, Electron; Microtubule-Associated Proteins; Microtubules; Proteins; Swine; tau Proteins; Tubulin

Substitution of pp(CH2)pT for GTP in the polymerization of microtubular protein results in a marked enhancement of both the rate and the extent of microtubule nucleation. Comparison of the kinetics of microtubule polymerization and pp(CH2)pG hydrolysis reveals that massive microtubule nucleation occurs in the absence of pp(CH2)pG hydrolysis. The shortest microtubule nuclei formed in the presence of pp(CH2)pG are curled ribbons on three protofilaments 0.15 to 0.2 micrometer long. No specific effect of pp(CH2)pG on microtubule propagation is observed. Nucleotide chase experiments suggest that the rings of microtubular protein present at 4 degrees C are not incorporated directly into the microtubule. Microtubules polymerized by pp(CH2)pG do not show the treadmill of tubulin protomers characteristic of the microtubules polymerized by GTP. Nucleotide analysis of microtubules polymerized by pp(CH2)pG and GTP reveals that 95% of the exchangeable nucleotide contained in the microtubules is p(CH2)pG and GDP, respectively. pp(CH2)pG blocks the treadmill of tubulin protomers from microtubules assembled by GTP by suppressing depolymerization at the depolymerization end of the microtubule. On the other hand, GTP promotes the treadmill of tubulin from microtubules assembled by pp(CH2)pG by reactivating the depolymerization end of the microtubule.

Topics: Animals; Brain; Guanosine Triphosphate; Kinetics; Macromolecular Substances; Microscopy, Electron; Microtubules; Rats; Tubulin