RESUMEN
Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by insulin and a variety of growth factors, but its exact role in signal transduction remains unclear. We have used a novel, highly specific inhibitor of PT 3-kinase to dissect the role of this enzyme in insulin action. Treatment of intact 3T3-L1 adipocytes with LY294002 produced a dose-dependent inhibition of insulin-stimulated PI 3-kinase (50% inhibitory concentration, 6 microM) with > 95% reduction in the levels of phosphatidylinositol-3,4,5-trisphosphate without changes in the levels of phosphatidylinositol-4-monophosphate or its derivatives. In parallel, there was a complete inhibition of insulin-stimulated phosphorylation and activation of pp70 S6 kinase. Inhibition of PI 3-kinase also effectively blocked insulin- and serum-stimulated DNA synthesis and insulin-stimulated glucose uptake by inhibiting translocation of GLUT 4 glucose transporters to the plasma membrane. By contrast, LY294002 had no effect on insulin stimulation of mitogen-activated protein kinase or pp90 S6 kinase. Thus, activation of PI 3-kinase plays a critical role in mammalian cells and is required for activation of pp70 S6 kinase and DNA synthesis and certain forms of intracellular vesicular trafficking but not mitogen-activated protein kinase or pp90 S6 kinase activation. These data suggest that PI 3-kinase is not only an important component but also a point of divergence in the insulin signaling network.
Asunto(s)
Adipocitos/metabolismo , ADN/biosíntesis , Insulina/farmacología , Proteínas de Transporte de Monosacáridos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células 3T3 , Adipocitos/efectos de los fármacos , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Cromonas/farmacología , Replicación del ADN/efectos de los fármacos , Desoxiglucosa/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Cinética , Ratones , Modelos Biológicos , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas , Fosforilación , Proteínas Quinasas S6 Ribosómicas , Transducción de Señal/efectos de los fármacosRESUMEN
Activation of phosphatidylinositol 3-kinase (PI3K) and activation of the 70/85-kDa S6 protein kinases (alpha II and alpha I isoforms, referred to collectively as pp70S6k) have been independently linked to the regulation of cell proliferation. We demonstrate that these kinases lie on the same signalling pathway and that PI3K mediates the activation of pp70 by the cytokine interleukin-2 (IL-2). We also show that the activation of pp70S6k can be blocked at different points along the signalling pathway by using specific inhibitors of T-cell proliferation. Inhibition of PI3K activity with structurally unrelated but highly specific PI3K inhibitors (wortmannin or LY294002) results in inhibition of IL-2-dependent but not phorbol ester (conventional protein kinase C [cPKC])-dependent pp70S6k activation. The T-cell immunosuppressant rapamycin potently antagonizes IL-2-(PI3K)- and phorbol ester (cPKC)-mediated activation of pp70S6k. Thus, wortmannin and rapamycin antagonize IL-2-mediated activation of pp70S6k at distinct points along the PI3K-regulated signalling pathway, or rapamycin antagonizes another pathway required for pp70S6k activity. Agents that raise the concentration of intracellular cyclic AMP (cAMP) and activate cAMP-dependent protein kinase (PKA) also inhibit IL-2-dependent activation of pp70S6k. In this case, inhibition appears to occur at least two points in this signalling path. Like rapamycin, PKA appears to act downstream of cPKC-mediated pp70S6k activation, and like wortmannin, PKA antagonizes IL-2-dependent activation of PI3K. The results with rapamycin and wortmannin are of added interest since the yeast and mammalian rapamycin targets resemble PI3K in the catalytic domain.
Asunto(s)
AMP Cíclico/fisiología , Interleucina-2/farmacología , Linfocitos/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Androstadienos/farmacología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Activación Enzimática , Técnicas In Vitro , Ratones , Fosfatidilinositol 3-Quinasas , Fosforilación , Polienos/farmacología , Proteína Quinasa C/fisiología , Receptores de Interleucina-2/fisiología , Proteínas Quinasas S6 Ribosómicas , Transducción de Señal , Sirolimus , Tacrolimus/farmacología , Acetato de Tetradecanoilforbol/farmacología , WortmaninaRESUMEN
Treatment of human neutrophils with the peptide f-Met-Leu-Phe (FMLP) results in neutrophil activation concomitant with stimulation of phosphatidylinositol (PtdIns) 3-kinase activity as measured by production of PtdIns-3,4,5-P3 in [32P]orthophosphate labeled cells. Antiphosphotyrosine immunoprecipitates were assayed for PtdIns 3-kinase activity; essentially no activity was present in lysates from either stimulated or unstimulated cells. The 85 kDa regulatory subunit of PtdIns 3-kinase, which normally serves as a substrate for tyrosine kinases, was not detected by SDS-PAGE or Western blot analysis in antiphosphotyrosine immunoprecipitates. In addition, no radioactive band corresponding to PtdIns 3-kinase was observed by SDS-PAGE following antiPtdIns 3-kinase immunoprecipitations. However, immunoprecipitates using polyclonal antibodies against PtdIns 3-kinase showed high PtdIns 3-kinase activity in neutrophil lysates and the 85 kDa subunit of PtdIns 3-kinase was detected in Western blots; no differences in activity were observed in FMLP-stimulated and unstimulated cells. These results suggest that, in contrast to polypeptide growth factor signal transduction systems, the activation of PtdIns 3-kinase by FMLP does not require tyrosine phosphorylation.
Asunto(s)
Neutrófilos/enzimología , Fosfotransferasas/metabolismo , Transducción de Señal , Western Blotting , Cromatografía Líquida de Alta Presión , Activación Enzimática , N-Formilmetionina Leucil-Fenilalanina/farmacología , Fosfatidilinositol 3-Quinasas , Fosforilación , Tirosina/metabolismoRESUMEN
New C-11 esters of the fermentation product wortmannin have been synthesized, with some of them further derivatized at C-17. The new esters show greater inhibition of isolated phosphatidylinositol 3-kinase and increased cell cytotoxicity in a rapidly proliferating leukemia cell line, when compared to wortmannin. Reduction of the C-17 ketone caused a slight increase in activity, while acylation of this new alcohol caused severe loss of activity. With their increased activity, the new C-11 esters may be good candidates to explore the in vivo antitumor effects of phosphatidylinositol 3-kinase inhibitors.
Asunto(s)
Androstadienos/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Ésteres , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Fosfatidilinositol 3-Quinasas , Células Tumorales Cultivadas , WortmaninaRESUMEN
Wortmannin, a fungal metabolite, was identified as a potent inhibitor (IC50 = 4.2 nM) of phosphatidylinositol 3-kinase (PI 3-kinase). Due to the importance of PI 3-kinase in several intracellular signaling pathways, structure-activities studies on wortmannin analogs were performed in an effort to understand the structural requirements necessary for PI 3-kinase inhibition. Since wortmannin is an irreversible inhibitor of PI 3-kinase, it was postulated that covalent attachment at the electrophilic C-21 site was a possible mode of action for PI 3-kinase inhibition. We have prepared various wortmannin analogs which address the possibility of this mechanism. Of particular interest are compounds which affect the C-21 position of wortaminnin either sterically or electronically. Our results support the conclusion that nucleophilic addition by the kinase onto the C-21 position of wortmannin is required for inhibition of PI 3-kinase by wortmannin analogs. Additionally, we have prepared several D-ring analogs of wortmannin, and their activities are reported herein. We conclude that the wortmannin D ring is an important recognition site since modifications have such a dramatic effect on inhibitor potency. Finally, the identification of 17beta-hydroxywortmannin represents the first reported subnanomolar inhibitor of PI 3-kinase. These studies, along with in vivo antitumor experiments, suggest that the mechanism of PI 3-kinase inhibition correlates to the associated toxicity observed with wortmannin-based inhibitors of PI 3-kinase.
Asunto(s)
Androstadienos/química , Androstadienos/farmacología , Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Androstadienos/uso terapéutico , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Humanos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Ratones , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Trasplante de Neoplasias , Fosfatidilinositol 3-Quinasas , Relación Estructura-Actividad , Termodinámica , Células Tumorales Cultivadas , WortmaninaRESUMEN
The insulin receptor substrate-1 (IRS-1) is expressed in 3T3-L1 adipocytes and is involved in at least some insulin responses, notably mitogenesis. Chronic exposure to insulin down regulates IRS-1 in these cells by stimulating its degradation (Rice, K.M., Turnbow, M.A. and Garner, C.W. (1993) Biochem. Biophys. Res. Commun. 190, 961-967). This insulin response was completely inhibited by wortmannin and LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), two inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase). Neither wortmannin nor LY294002 had any effect on the calcium-dependent degradation of IRS-1 in vitro nor did they inhibit the phosphorylation of IRS-1 in vitro. In addition, neomycin, a cationic aminoglycoside antibiotic that binds to phosphoinositides, inhibited the insulin-induced down-regulation of IRS-1 in 3T3-L1 adipocytes and, also, the C8-PIP3-stimulated degradation of IRS-1 in vitro. These results suggest that PI 3-kinase and its 3-phosphoinositide products mediate the insulin-induced down-regulation of IRS-1 in 3T3-L1 adipocytes.
Asunto(s)
Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Androstadienos/farmacología , Cromonas/farmacología , Insulina/farmacología , Morfolinas/farmacología , Fosfoproteínas/metabolismo , Células 3T3 , Animales , Proteínas de Unión al Calcio/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas Sustrato del Receptor de Insulina , Ratones , Neomicina/farmacología , Fosfatidilinositol 3-Quinasas , Fosfatos de Fosfatidilinositol/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , WortmaninaRESUMEN
The complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli has been established in the following manner. After being reduced with dithiothreitol, the purified aldolase was alkylated with iodoacetamide and subsequently digested with trypsin. The resulting 19 peptide peaks observed by high performance liquid chromatography, which compared with 21 expected tryptic cleavage products, were all isolated, purified, and individually sequenced. Overlap peptides were obtained by a combination of sequencing the N-terminal region of the intact aldolase and by cleaving the intact enzyme with cyanogen bromide followed by subdigestion of the three major cyanogen bromide peptides with either Staphylococcus aureus V8 endoproteinase, endoproteinase Lys C, or trypsin after citraconylation of lysine residues. The primary structure of the molecule was determined to be as follows. (formula; see text) 2-Keto-4-hydroxyglutarate aldolase from E. coli consists of 213 amino acids with a subunit and a trimer molecular weight of 22,286 and 66,858, respectively. No microheterogeneity is observed among the three subunits. The peptide containing the active-site arginine residue (Vlahos, C. J., Ghalambor, M. A., and Dekker, E. E. (1985) J. Biol. Chem. 260, 5480-5485) was also isolated and sequenced; this arginine residue occupies position 49. The Schiff base-forming lysine residue (Vlahos, C. J., and Dekker, E. E. (1986) J. Biol. Chem. 261, 11049-11055) is located at position 133. Whereas the active-site lysine peptide of this aldolase shows 65% homology with the same peptide of 2-keto-3-deoxy-6-phosphogluconate aldolase from Pseudomonas putida, these two proteins in toto show 49% homology.
Asunto(s)
Arginina , Escherichia coli/enzimología , Metaloendopeptidasas , Oxo-Ácido-Liasas , Secuencia de Aminoácidos , Carboxipeptidasas , Carboxipeptidasas A , Cromatografía Líquida de Alta Presión , Bromuro de Cianógeno , Ditiotreitol , Endopeptidasas , Yodoacetamida , Lisina , Datos de Secuencia Molecular , Oxo-Ácido-Liasas/análisis , Fragmentos de Péptidos/aislamiento & purificación , Homología de Secuencia de Ácido Nucleico , Serina Endopeptidasas , TripsinaRESUMEN
Pure 2-keto-4-hydroxyglutarate aldolase of Escherichia coli, a "lysine-type" trimeric enzyme which has the unique properties of forming an "abortive" Schiff-base intermediate with glyoxylate (the aldehydic product/substrate) and of showing strong beta-decarboxylase activity toward oxalacetate, binds any one of its substrates (2-keto-4-hydroxyglutarate, pyruvate, or glyoxylate) in a competitive manner. To determine whether the substrates bind at the same or different (juxta-positioned) sites and what degree of homology might exist between the active-site lysine peptide of this enzyme and that of other lysine-type (Class I) aldolases or beta-decarboxylases, the azomethine formed separately by this aldolase with either [14C]pyruvate or [14C]glyoxylate was reduced with CNBH3-. After each enzyme adduct was digested with trypsin, the 14C-labeled peptide was isolated, purified, and subjected to amino acid analysis and sequence determination. In each case, the same 14-amino acid lysine-peptide was isolated and found to have the following primary sequence: Glu-Phe-*Lys-Phe-Phe-Pro-Ala-Glu-Ala-Asn-Gly-Gly-Val-Lys (where * = the active-site lysine). Hence, glyoxylate competes for, and inhibits aldolase activity by reacting with, the one active-site lysine residue/subunit. This active-site lysine peptide has a high degree (65%) of homology with that of 2-keto-3-deoxy-6-phosphogluconate aldolase of Pseudomonas putida but is not similar to that of any Class I fructose-1,6-bisphosphate aldolase or of acetoacetate beta-decarboxylase of Clostridium acetobutylicum. Furthermore, it was found that extensive reaction of glyoxylate with the N-terminal amino group of this enzyme may well be general complicating factor in sequence studies with proteins plus glyoxylate.
Asunto(s)
Escherichia coli/enzimología , Lisina , Oxo-Ácido-Liasas/metabolismo , Piruvatos/metabolismo , Secuencia de Aminoácidos , Aminoácidos/análisis , Sitios de Unión , Borohidruros/farmacología , Glioxilatos/metabolismo , Modelos Químicos , Oxo-Ácido-Liasas/análisis , Fragmentos de Péptidos/análisis , Ácido Pirúvico , Tripsina/metabolismoRESUMEN
Treatment of pure 2-keto-4-hydroxyglutarate aldolase from Escherichia coli, a "lysine-type," Schiff-base mechanism enzyme, with the substrate analog bromopyruvate results in a time- and concentration-dependent loss of enzymatic activity. Whereas the substrates pyruvate and 2-keto-4-hydroxyglutarate provide greater than 90% protection against inactivation by bromopyruvate, no protective effect is seen with glycolaldehyde, an analog of glyoxylate. Inactivation studies with [14C] bromopyruvate show the incorporation of 1.1 mol of 14C-labeled compound/enzyme subunit; isolation of a radioactive peptide and determination of its amino acid sequence indicate that the radioactivity is associated with glutamate 45. Incubation of the enzyme with excess [14C]bromopyruvate followed by denaturation with guanidine.HCl allow for the incorporation of carbon-14 at cysteines 159 and 180 as well. Whereas the presence of pyruvate protects Glu-45 from being esterified, it does not prevent the alkylation of these 2 cysteine residues. The results indicate that Glu-45 of E. coli 2-keto-4-hydroxyglutarate aldolase is essential for catalytic activity, most likely acting as the amphoteric proton donor/acceptor that is required as a participant in the overall mechanism of the reaction catalyzed.
Asunto(s)
Escherichia coli/enzimología , Glutamatos , Oxo-Ácido-Liasas/metabolismo , Piruvatos/farmacología , Marcadores de Afinidad , Secuencia de Aminoácidos , Sitios de Unión , Ácido Glutámico , Ácidos Cetoglutáricos/metabolismo , Cinética , Modelos Estructurales , Datos de Secuencia Molecular , Oxo-Ácido-Liasas/antagonistas & inhibidores , Mapeo Peptídico , Conformación Proteica , TripsinaRESUMEN
A method is described to purify recombinant HIV-1 protease from soluble extracts of Escherichia coli. The isolation involves QAE-Sepharose anion exchange chromatography, hexyl agarose hydrophobic interaction chromatography, MonoS cation exchange chromatography, and Superose 6 size exclusion chromatography. Approximately 100 micrograms of protease was obtained from 18 g E. coli paste. The protein was judged to be homogeneous due to the presence of a single band on a silver-stained SDS polyacrylamide gel.
Asunto(s)
Proteasa del VIH/aislamiento & purificación , Cromatografía en Agarosa , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Productos del Gen gag/metabolismo , Proteasa del VIH/química , Humanos , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Sefarosa/análogos & derivadosRESUMEN
Treatment of homogeneous preparations of Escherichia coli 2-keto-4-hydroxyglutarate aldolase with 1,2-cyclohexanedione, 2,3-butanedione, phenylglyoxal, or 2,4-pentanedione results in a time- and concentration-dependent loss of enzymatic activity; the kinetics of inactivation are pseudo-first order. Cyclohexanedione is the most effective modifier; a plot of log (1000/t 1/2) versus log [cyclohexanedione] gives a straight line with slope = 1.1, indicating that one molecule of modifier reacts with each active unit of enzyme. The kinetics of inactivation are first order with respect to cyclohexanedione, suggesting that the loss of activity is due to modification of 1 arginine residue/subunit. Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced structural alteration of the aldolase. The same Km but decreased Vmax values are obtained when partially inactivated enzyme is compared with native. Amino acid analyses of 95% inactivated aldolase show the loss of 1 arginine/subunit with no significant change in other amino acid residues. Considerable protection against inactivation is provided by the substrates 2-keto-4-hydroxyglutarate and pyruvate (75 and 50%, respectively) and to a lesser extent (40 and 35%, respectively) by analogs like 2-keto-4-hydroxybutyrate and 2-keto-3-deoxyarabonate. In contrast, formaldehyde or glycolaldehyde (analogs of glyoxylate) under similar conditions show no protective effect. These results indicate that an arginine residue is required for E. coli 2-keto-4-hydroxyglutarate aldolase activity; it most likely participates in the active site of the enzyme by interacting with the carboxylate anion of the pyruvate-forming moiety of 2-keto-4-hydroxyglutarate.
Asunto(s)
Arginina/análisis , Ciclohexanos/farmacología , Ciclohexanonas/farmacología , Escherichia coli/enzimología , Oxo-Ácido-Liasas/metabolismo , Aminoácidos/análisis , Sitios de Unión , CinéticaRESUMEN
Phosphatidylinositol (PtdIns) 3-kinase is an enzyme involved in cellular responses to growth factors. Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyrano-4-one), a naturally occuring bioflavinoid, was found to inhibit PtdIns 3-kinase with an IC50 of 1.3 micrograms/ml (3.8 microM); inhibition appears to be directed towards the ATP binding site of the kinase. Analogs of quercetin were also investigated as PtdIns 3-kinase inhibitors, with the most potent compounds exhibiting IC50's in the range of 1.7-8.4 micrograms/ml (5-19 microM). In contrast, genistein, a potent tyrosine kinase inhibitor of the isoflavone class, did not inhibit PtdIns 3-kinase significantly (IC50 greater than 30 micrograms/ml). These findings suggest that flavinoids may serve as potent inhibitors of PtdIns 3-kinase. Furthermore, the enzyme is much more sensitive to substituents at the 3-position of the flavinoid ring than are other protein and PtdIns kinases, suggesting that specific inhibitors of PtdIns 3-kinase can be developed to explore the biological role of the enzyme in cellular proliferation and growth factor response.
Asunto(s)
Flavonoides/farmacología , Fosfotransferasas/antagonistas & inhibidores , Quercetina/análogos & derivados , Quercetina/farmacología , Animales , Encéfalo/enzimología , Bovinos , Cinética , Estructura Molecular , Fosfatidilinositol 3-Quinasas , Fosfotransferasas/aislamiento & purificación , Relación Estructura-ActividadRESUMEN
In the present study, treatment of the PTEN negative U87MG human glioblastoma cell line with C2-ceramide resulted in a dose- and time-dependent decrease in the constitutive phosphorylation of Akt at threonine 308 and serine 473. The C2-ceramide induced dephosphorylation of Akt correlated with a 90-95% reduction in the Akt kinase activity. Exposure to C2-ceramide did not affect the basal or PDGF activated levels PtdIns-3,4-P(2) and PtdIns-3,4,5-P(3), indicating PI3-K activity was not inhibited. Additionally, treatment of cells with the PI3-K inhibitor wortmannin and C2-ceramide resulted in an enhanced rate of Akt dephosphorylation versus either agent alone. Finally, treatment of cells with the phosphatase inhibitors okadaic acid or calyculin A prevented the C2-ceramide induced dephosphorylation and inhibition of Akt activity. These data demonstrate the ability of C2-ceramide to inhibit the constitutive phosphorylation and activity of Akt in U87MG cells and implicate the activation of ceramide activated protein phosphatase, rather than decreased PI3-K activity, as the mechanism of inhibition.
Asunto(s)
Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Esfingosina/análogos & derivados , Esfingosina/farmacología , Proteínas Supresoras de Tumor , Androstadienos/farmacología , Western Blotting , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Humanos , Toxinas Marinas , Ácido Ocadaico/farmacología , Oxazoles/farmacología , Fosfohidrolasa PTEN , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositoles/metabolismo , Fosforilación/efectos de los fármacos , Pruebas de Precipitina , Proteínas Proto-Oncogénicas c-akt , Serina/química , Treonina/química , Factores de Tiempo , Células Tumorales Cultivadas , WortmaninaRESUMEN
The kringle-2 domain of tissue plasminogen activator, cloned and expressed in Escherichia coli (Wilhelm, O.G., Jaskunas, S.R., Vlahos, C.J., and Bang, N.U. (1990) J. Biol. Chem. 265, 14606-14611), was internally radiolabeled using [35S]methionine-cysteine. Following refolding and isolation, the labeled polypeptide was further purified by reverse-phase high performance liquid chromatography. The purified kringle-2 domain was digested with thermolysin, and the resulting peptides were purified by high performance liquid chromatography. Five major peptides containing 35S were obtained. Amino acid sequence analysis showed that these peptides represented various cleavage products containing one or more of the following disulfides: Cys180-Cys261, Cys201-Cys243, Cys232-Cys256 (sequence numbering based on Pennica et al. (Pennica, D., Holmes, W.E., Kohr, W.J., Hakins, R.N., Vehar, G. A., Ward, C.A., Bennett, W.F., Yelverton E., Seeburg, P.H., Heynecker, H.L., Goeddel, E.V., and Collen, D. (1983) Nature 301, 214-221)). These results confirm that the refolding methodology used produced kringle-2 with the predicted disulfide linkage and, thus, yielded material suitable for structural and functional studies.
Asunto(s)
Disulfuros/química , Escherichia coli/metabolismo , Fragmentos de Péptidos/química , Activador de Tejido Plasminógeno/química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Mapeo Peptídico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Termolisina , Activador de Tejido Plasminógeno/genéticaRESUMEN
The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis.
Asunto(s)
Escherichia coli/genética , Activador de Tejido Plasminógeno/metabolismo , Clonación Molecular , Fibrina/metabolismo , Humanos , Cinética , Lisina/metabolismo , Inactivadores Plasminogénicos/metabolismo , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Radioisótopos de Azufre , Activador de Tejido Plasminógeno/biosíntesis , Activador de Tejido Plasminógeno/genéticaRESUMEN
The cDNA encoding human preprorenin has been introduced into the adenovirus-transformed human kidney cell line 293. The recombinant 293 cells expressed and secreted prorenin; trypsin was used to activate the secreted prorenin to renin in vitro. The recombinant protein was purified to homogeneity by a single affinity chromatographic step. Using synthetic tetradecapeptide, the Km was 57.1 +/- 9.3 microM and the kcat was (7.48 +/- 1.57) x 10(3)/hr. Activation with trypsin resulted in a secondary cleavage between Arg53 and Leu54 generating a two chain form held together via a disulfide between Cys51 and Cys58. This secondary cleavage did not affect enzyme activity as determined by the ability of renin to degrade a synthetic tetradecapeptide substrate. Our paper demonstrates the potential for producing large quantities of renin from human kidney cells and also suggests that the use of trypsin, which has been widely used to convert prorenin to renin in vitro, causes a secondary cleavage in the renin peptide chain.
Asunto(s)
Renina/aislamiento & purificación , Secuencia de Aminoácidos , Línea Celular , Activación Enzimática , Precursores Enzimáticos/metabolismo , Vectores Genéticos , Humanos , Técnicas In Vitro , Riñón/enzimología , Datos de Secuencia Molecular , Peso Molecular , Proteínas Recombinantes/aislamiento & purificación , Renina/metabolismo , TransfecciónRESUMEN
In order to study the role of phosphatidylinositol-3-kinase (PI3K) in insulin action, we employed a specific inhibitor of PI3K, LY294002, and measured five biological functions of insulin in mouse 3T3 fibroblasts overexpressing human insulin receptors. LY294002 had no effect on tyrosine phosphorylation of both the insulin receptor beta-subunit and insulin receptor substrate 1 (IRS-1) and did not influence the association of the p85 subunit of PI3K with IRS-1. However, LY294002 partially inhibited insulin stimulated glucose uptake, amino acid uptake and protein synthesis, while it completely inhibited insulin stimulation of DNA synthesis and p70 S6 kinase activation. These data suggest that: 1) PI3K plays a crucial role in various functions of insulin; and 2) there exist multiple signaling pathways (both PI3K dependent and PI3K independent) for the insulin receptor.
Asunto(s)
Cromonas/farmacología , Morfolinas/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Receptor de Insulina/metabolismo , Transducción de Señal , Células 3T3 , Aminoácidos/metabolismo , Animales , ADN/biosíntesis , Activación Enzimática , Glucosa/metabolismo , Proteínas Sustrato del Receptor de Insulina , Ratones , Fosfatidilinositol 3-Quinasas , Fosfoproteínas/metabolismo , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas S6 Ribosómicas , Tirosina/metabolismoRESUMEN
Blocker-induced noise analysis of epithelial Na(+) channels (ENaCs) was used to investigate how inhibition of an LY-294002-sensitive phosphatidylinositol 3-kinase (PI 3-kinase) alters Na(+) transport in unstimulated and aldosterone-prestimulated A6 epithelia. From baseline Na(+) transport rates (I(Na)) of 4.0 +/- 0.1 (unstimulated) and 9.1 +/- 0.9 microA/cm(2) (aldosterone), 10 microM LY-294002 caused, following a relatively small initial increase of transport, a completely reversible inhibition of transport within 90 min to 33 +/- 6% and 38 +/- 2% of respective baseline values. Initial increases of transport could be attributed to increases of channel open probability (P(o)) within 5 min to 143 +/- 17% (unstimulated) and 142 +/- 10% of control (aldosterone) from baseline P(o) averaging near 0.5. Inhibition of transport was due to much slower decreases of functional channel densities (N(T)) to 28 +/- 4% (unstimulated) and 35 +/- 3% (aldosterone) of control at 90 min. LY-294002 (50 microM) caused larger but completely reversible increases of P(o) (215 +/- 38% of control at 5 min) and more rapid but only slightly larger decreases of N(T). Basolateral exposure to LY-294002 induced no detectable effect on transport, P(o) or N(T). We conclude that an LY-294002-sensitive PI 3-kinase plays an important role in regulation of transport by modulating N(T) and P(o) of ENaCs, but only when presented to apical surfaces of the cells.
Asunto(s)
Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Túbulos Renales Distales/metabolismo , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Sodio/metabolismo , Aldosterona/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Membrana Celular/metabolismo , Conductividad Eléctrica , Impedancia Eléctrica , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Canales Epiteliales de Sodio , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/fisiología , Túbulos Renales Distales/citología , Túbulos Renales Distales/efectos de los fármacos , Túbulos Renales Distales/fisiología , Probabilidad , Canales de Sodio/metabolismo , Factores de Tiempo , Xenopus laevisRESUMEN
Two glycoforms of a secretable human thrombomodulin mutant [TMD1-105 and TMD1-75; Parkinson, Grinnell, Moore, Hoskins, Vlahos & Bang (1990) J. Biol. Chem. 265, 12602-12610] were expressed in human 293 cells and used to study the role of glycosylation in the functions of this endothelial-cell thrombin receptor. Carbohydrate content analysis and intrinsic labelling with [3H]glucosamine and [35S]sulphate showed that TMD1-105 contained a chondroitin sulphate whereas TMD1-75 did not. Other than chondroitin sulphate, the carbohydrate contents of the two glycoforms were identical, indicating similar glycosylation patterns at other O-linked and N-linked sites in the two glycoforms. The properties of TMD1-105 were converted into those of TMD1-75 by chondroitin ABC lyase digestion. Trypsin digestion of labelled TMD1-105 permitted isolation of two overlapping peptides that contained chondroitin sulphate, spanned the entire O-glycosylation domain and had O-glycosylation sites at Ser-492, Ser-498, Thr-500, Thr-504 and Thr-506. The chondroitin sulphate-attachment site was assigned to Ser-492 as this residue is conserved in mouse and bovine thrombomodulin and lies within a sequence Ser-Gly-Ser-492-Gly-Glu-Pro, which has strong similarity to chondroitin sulphate attachment sites in other proteoglycans. Five peptides with N-linked carbohydrate were also isolated and contained glycosylation sites in the lectin-like domain (Asn-47, Asn-115, Asn-116) and in the fourth (Asn-382) and fifth (Asn-409) epidermal growth factor domains. The role of N-linked and simple O-linked carbohydrates in the functions of human thrombomodulin remain unclear. The present studies demonstrate, however, that the presence of chondroitin sulphate in human thrombomodulin has profound effects on all of the anticoagulant properties of this important anticoagulant thrombin receptor.
Asunto(s)
Glicosaminoglicanos/fisiología , Receptores de Superficie Celular/fisiología , Secuencia de Aminoácidos , Sitios de Unión , Metabolismo de los Hidratos de Carbono , Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , Sulfatos de Condroitina/fisiología , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Glicosilación , Humanos , Liasas/metabolismo , Datos de Secuencia Molecular , Proteína C/fisiología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Receptores de Trombina , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/fisiología , Relación Estructura-Actividad , Radioisótopos de Azufre , TritioRESUMEN
Phosphatidylinositol (PtdIns) 3-kinase is an enzyme implicated in growth factor signal transduction by associating with receptor and nonreceptor tyrosine kinases, including the platelet-derived growth factor receptor. Inhibitors of PtdIns 3-kinase could potentially give a better understanding of the function and regulatory mechanisms of the enzyme. Quercetin, a naturally occurring bioflavinoid, was previously shown to inhibit PtdIns 3-kinase with an IC50 of 1.3 microgram/ml (3.8 microM); inhibition appeared to be directed at the ATP-binding site of the kinase. Analogs of quercetin were investigated as PtdIns 3-kinase inhibitors, with the most potent ones exhibiting IC50 values in the range of 1.7-8.4 micrograms/ml. In contrast, genistein, a potent tyrosine kinase inhibitor of the isoflavone class, did not inhibit PtdIns 3-kinase significantly (IC50 > 30 micrograms/ml). Since quercetin has also been shown to inhibit other PtdIns and protein kinases, other chromones were evaluated as inhibitors of PtdIns 3-kinase without affecting PtdIns 4-kinase or selected protein kinases. One such compound, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (also known as 2-(4-morpholinyl)-8-phenylchromone, LY294002), completely and specifically abolished PtdIns 3-kinase activity (IC50 = 0.43 microgram/ml; 1.40 microM) but did not inhibit PtdIns 4-kinase or tested protein and lipid kinases. Analogs of LY294002 demonstrated a very selective structure-activity relationship, with slight changes in structure causing marked decreases in inhibition. LY294002 was shown to completely abolish PtdIns 3-kinase activity in fMet-Leu-Phe-stimulated human neutrophils, as well as inhibit proliferation of smooth muscle cells in cultured rabbit aortic segments. Since PtdIns 3-kinase appears to be centrally involved with growth factor signal transduction, the development of specific inhibitors against the kinase may be beneficial in the treatment of proliferative diseases as well as in elucidating the biological role of the kinase in cellular proliferation and growth factor response.