RESUMO
Tyrosine protein kinases (TPKs) play a major role in the transformation of cells. They are currently used as molecular targets for new generations of anticancer compounds. Numerous TPKs have been described from various tissues using either classical molecular biochemical techniques or cloning strategies. As a natural extension of these discoveries, a large number of "specific" inhibitors have been described in the literature. The major problem with these inhibitors is that there is no simple way to compare their specificity and/or selectivity from one report to another. We have set up a simple, straightforward technique to compare the inhibitory potency of 14 classical inhibitors towards six well-described and at least partially purified protein kinases. This technique is based on a new assay, easy to carry out and non-restrictive in terms of the type of protein substrate used. It permits direct comparisons between the results obtained from various sources. Data obtained showed that, when assessed in this integrated system, specificity and selectivity of many "classical" inhibitors are often weak, thus demonstrating that a universal technique such as ours is essential for the molecular screening of new protein kinase inhibitors. Compounds showing specificity for this panel of protein kinases will be more easily targeted to some defined types of oncogene and of transformed cells.
Assuntos
Inibidores Enzimáticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Inibidores de Proteínas Quinases , Alcaloides/farmacologia , Animais , Proteínas de Transporte/farmacologia , Humanos , Hidroquinonas/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Camundongos , Peptídeos/metabolismo , Proteínas Quinases/isolamento & purificação , Quercetina/farmacologia , Ratos , Estaurosporina , Suramina/farmacologiaRESUMO
A tyrosine protein kinase (TPK) partially purified and two co-purified proteins (pp39 and pp44) from HL-60 cells were used as a basis for a SDS-PAGE electrophoresis screening test of inhibitors of the activity catalyzed by this enzyme. Such inhibitors may constitute a new class of anticancer agents. TPK is a protein of about 32 to 35 kDa as measured by gel-sizing exclusion. This enzyme is apparently the main tyrosine protein kinase from the cytosol of HL-60 cells. The test was assessed using known inhibitors of various types of protein kinases. Erbstatin and Cibcron Blue--a nucleotide analog--were shown to be potent inhibitors of TPK activity.
Assuntos
Antineoplásicos/farmacologia , Leucemia Promielocítica Aguda/enzimologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Humanos , Hidroquinonas/farmacologia , Proteínas Tirosina Quinases/isolamento & purificação , Triazinas/farmacologia , Células Tumorais CultivadasRESUMO
Tyrosine as well as serine/threonine protein kinase inhibitors have potentially two sites of interaction with their targets: the protein-substrate binding site and the ATP binding site. The latter could be modelized by measuring the capacity of protein kinase inhibitors to inhibit ATPase activities. In order to do so, we assess a novel, highly sensitive HPLC method based on hydrophilic separation of [gamma-32P]ATP and [32P]Pi. The novel assay is presented. Furthermore, the potency of 13 protein kinase inhibitors was tested on two types of ATPase, namely: apyrase and partially purified liver mitochondria F1-ATPase. The method described for the assay of ATPase can be used with almost any type of enzyme catalyzing this activity. Only cibacron blue and suramin show interesting capacities in inhibiting these ATPase activities pointing out that several widely used protein kinase inhibitors are at least somewhat specific in that they do not inhibit these two ATPases.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Inibidores de Proteínas Quinases , Trifosfato de Adenosina/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Animais , Apirase/antagonistas & inibidores , Membrana Celular/enzimologia , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Cinética , Leucemia P388/enzimologia , Mitocôndrias Hepáticas/enzimologia , Fosfatos/isolamento & purificação , Fosfatos/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , ATPases Translocadoras de Prótons/antagonistas & inibidores , Ratos , Suramina/administração & dosagem , Suramina/farmacologia , Triazinas/administração & dosagem , Triazinas/farmacologiaRESUMO
Farnesylation is a key maturation step involved in the ras-dependent transformation of cells. This acylation step is catalyzed by protein: farnesyltransferase, a soluble enzyme. The present work describes the use of a new HPLC method of measurement of this enzymatic activity using the K-ras-derived CVIM tetrapeptide as substrate. The method is used to check the activity catalyzed by cytosols issued from various types of cancer cells. J82, a human bladder cancer cell line was retained for measurement of the inhibitory potency of a few peptide sequences and will be used as starting biological material for the purification of the enzyme. This HPLC method presented herein has the main advantages over other published methods of being automatisable and versatile, because it can be used with a wide spectrum of peptide substrates. Results presented herein are only first studies and need some more structural observations. The obtention of the cancer cell line-derived, partially purified farnesyltransferase will hopefully lead us to the discovery of specific inhibitors with potential non-cytotoxic anti-cancer activities.
Assuntos
Antineoplásicos/metabolismo , Dimetilaliltranstransferase/metabolismo , Animais , Carcinoma/patologia , Dimetilaliltranstransferase/farmacologia , Humanos , Leucemia/patologia , Leucemia Experimental/patologia , Células Tumorais Cultivadas/enzimologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
Myristoylation is a co-translational maturation process of proteins. It is extremely specific for the cosubstrate (myristoyl-CoA) and for the substrate protein that should bear a glycine at the N-terminus of the protein to be myristoylated. This acylation is catalyzed by the myristoyl-CoA:protein N-myristoyltransferase. Most of the molecular biochemistry and biology concerning this enzyme has been done on Saccharomyces cerevisiae. Because of the major importance of this pathway in several types of pathology, it is essential to study intensively the enzyme(s) isolated from mammalian tissue(s) to confirm that the enormous amount of work done on the yeast enzyme can be transposed to mammalian tissues. In earlier studies, we demonstrated the existence of a microsomal N-myristoyltransferase from the murine leukemia cell line L1210 [Boutin, J. A., Clarenc, J.-P., Ferry, G., Ernould, A. P., Remond, G., Vincent, M. & Atassi, G. (1991) Eur. J. Biochem. 201, 257-263], a feature which is not shared by yeast, and examined the N-myristoyltransferase activities associated with L1210 cytosol. In the present work, we purified to homogeneity one of the isoforms (A) of the transferase from L1210 cytosol. The purified enzyme showed on SDS/PAGE an apparent molecular mass of 67.5 kDa, distinct from the 53-kDa yeast cytosolic enzyme. The purified enzyme from L1210 cytosol could be labeled with [14C]myristoyl-CoA. Rabbit antibodies were raised against the A isoform and used to immunoprecipitate the enzyme and immunoinhibit the activity from the same source. A survey of the specificity of the partially and completely purified isoforms was performed using peptides derived from the NH2-terminus of 42 proteins which are potential substrates for myristoylation, including oncogene products and virus structural proteins. We synthesized a series of compounds capable of inhibiting the cytosol activities of the enzyme. For example, a myristoyltetrahydroquinolein derivative showed an IC50 of about 0.1 microM. Based on both biophysical and biochemical evidence, the N-myristoyltransferases extracted from mammalian cell cytosols seem to be different from the extensively studied yeast enzyme.
Assuntos
Aciltransferases/metabolismo , Proteínas do Capsídeo , Isoenzimas/metabolismo , Leucemia L1210/enzimologia , Processamento de Proteína Pós-Traducional , Aciltransferases/antagonistas & inibidores , Aciltransferases/isolamento & purificação , Sequência de Aminoácidos , Animais , Capsídeo/metabolismo , Citosol/enzimologia , Humanos , Isoenzimas/isolamento & purificação , Camundongos , Microssomos/enzimologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Poliovirus/metabolismo , Ratos , Saccharomyces cerevisiae/metabolismo , Especificidade da EspécieRESUMO
Using a partially purified HL-60 tyrosine protein kinase, we designed a new HPLC method for the measurement of tyrosylphosphorylation of angiotensin II. The present method uses reversed-phase chromatography and elution involving an acetonitrile gradient containing the counterion tetrabutylammonium phosphate. The peptide substrate, [gamma-32P]ATP, the cosubstrate, and 32P-labeled phosphorylated peptides were quantified online by measuring the Cerenkov effect. Injections, separation, and analysis were performed automatically. Furthermore, the method permits a direct visualization of peptide substrate phosphorylation and has a potentially universal application; i.e., it is usable with any kind of peptide in a given range of hydrophobicity. This assay was designed for specificity studies, which are of major importance at the molecular level, in order to understand active site topology and the biophysical requirements of tyrosine protein kinases. As examples, data on chromatography separations of angiotensin II analogs (five to ten amino acids in length) are presented, as well as for other peptide substrates such as RR-src, the pp60src autophosphorylation site-derived peptide, and minigastrin. We adapted our experimental conditions to accommodate crude extracts from HL-60 cells. Preliminary experiments clearly indicated that other biological sources can be used. Despite the existence of numerous methods published in the literature for the measurement of kinase activities, the method presented herein is the only one to the authors' knowledge that can be used in and has been assessed for specificity studies. Peptides do not require particular features such as charged residues (i.e., arginine) to be analyzed.
Assuntos
Proteínas Tirosina Quinases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Angiotensina II/análogos & derivados , Angiotensina II/biossíntese , Angiotensina II/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Estudos de Viabilidade , Humanos , Dados de Sequência Molecular , Peptídeos/metabolismo , Fosforilação , Tirosina/análogos & derivados , Tirosina/biossínteseRESUMO
A major peak of tyrosine protein kinase activity was partially purified from a Triton X100 extract of HL-60. This preparation submitted to high pressure gel filtration was eluted at a volume corresponding to a mass of 35/40 kD. This activity was insensitive to EGF and insulin. Autoradiographs of the preparations incubated with [gamma P32]-ATP and separated by electrophoresis do not give any evidence that autophosphorylation occurs for that particular tyrosine protein kinase. Furthermore, we failed to immunoprecipitate the enzyme with a specific antiphosphotyrosine antibody and anti v-src antibody. All the data presented herein suggest that this enzyme has not been previously purified.
Assuntos
Leucemia Promielocítica Aguda/enzimologia , Proteínas Tirosina Quinases/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Autorradiografia , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Ágar , Fator de Crescimento Epidérmico/farmacologia , Guanosina Trifosfato/metabolismo , Humanos , Técnicas de Imunoadsorção , Insulina/farmacologia , Peso Molecular , Octoxinol , Fosforilação , Polietilenoglicóis , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
The major tyrosine protein kinase, HPK40, isolated from HL-60, the preparation of which is described elsewhere (Ernould, A.P., Ferry, G., Barret, J.M., Genton, A. and Boutin, J.A., Eur. J. Biochem., 214, 503-514), was investigated as to its specificity on a number of peptides and proteins. It was found that HPK40 can phosphorylate histones (except histone H4), casein, acid-treated enolase, actin and tubulin but not calmodulin. Phosphorylation specificity of HPK40 was investigated using over a hundred peptidic structures. HPK40 is not related to the 'src' family and does not phosphorylate efficiently either the tetrapeptide NEYT derived from the pp60src autophosphorylation domain or the corresponding peptide RRsrc, RRLIED-NEYTARG. VALYDYESR from the SH3 domain of pp60c-src is recognized as a substrate with a high phosphorylation level. DEDYIQD, derived from the phosvitin/casein kinase II, was also highly phosphorylated. In order to determine the minimal recognition sequence of HPK40, the phosphorylation of about 60 dito tetrapeptides was investigated. Some of the tetrapeptides, such as *EEYE and NEYE, were well phosphorylated. Even some tripeptides, such as EYE, DYM, TYS and KYE, were recognized by HPK40, while none of the tested dipeptides was recognized as substrate. Sequences of peptides from DRVYHPF (angiotensin), LEEEEEAYGWMDF (minigastrin) and QEEYSAM (from H-ras1) were examined as substrates. The presence of one or several acidic residues on the N alpha-side of tyrosine residue was identified as the only apparently favorable determinant.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Leucemia Promielocítica Aguda/enzimologia , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Fosforilação , Fosfotransferases/metabolismo , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
A new HPLC method has been developed to assay tyrosine protein kinase activity. Using hydrophilic interaction chromatography, it is possible to resolve the four components of the incubation medium: substrate peptide, [32P]phosphorylated peptide, unreacted [gamma-32P]ATP, and 32P-labelled inorganic phosphate. ATP interacts so strongly with the stationary phase material that it can be removed selectively from the incubation medium with solid-phase extraction cartridges packed with the same type of material. The three remaining components of interest can then be resolved by reversed-phase or hydrophilic interaction HPLC. This procedure permits the evaluation of almost every type of peptide as a substrate of tyrosine protein kinase.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Gastrinas/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Fosforilação , Espectrofotometria Ultravioleta , Especificidade por SubstratoRESUMO
The major tyrosine protein kinase from HL60 (a human non-differentiated promyelocytic cell line) has been purified almost to homogeneity as judged by silver-stained SDS/PAGE. The procedure involved four chromatographic steps: DEAE-Sepharose, casein-agarose, cibacron-blue--agarose and hexyl-agarose. The purification resulted in more than 1000-fold enrichment in angiotensin II phosphorylation activity. A gel-sizing experiment, labeling with [35S]ATP[gamma s] and autophosphorylation of the enzyme in the presence of [gamma-32P]ATP, all led to the identification of a single protein species with a molecular mass of about 40 kDa. Western blot experiments showed that this protein does not belong to the src family and is not related to the abl and fes oncogene products. Phosphorylation of angiotensin II and casein by this 40-kDa human promyelocytic kinase was stimulated by high ionic strength especially from class IA metal salts. The Km for ATP was 2 microM and the Vmax 3.1 nmol.min-1.mg-1 using angiotensin II as a substrate. The kinase requires the presence of either Mn2+ or Mg2+ for full activity and utilizes ATP or dATP but not GTP as phosphate donor. Based on numerous biochemical observations, it was possible to demonstrate that kinase is different from any other tyrosine protein kinases described in the literature. This 40-kDa protein was used as a molecular tool for testing some tyrosine protein kinase inhibitors described in the literature. It is one of the rare tyrosine protein kinases purified from human cancer cells to date.
Assuntos
Leucemia Promielocítica Aguda/enzimologia , Proteínas Tirosina Quinases/isolamento & purificação , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Angiotensina II/metabolismo , Ligação Competitiva , Western Blotting , Caseínas/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Magnésio/farmacologia , Manganês/farmacologia , Metais/farmacologia , Concentração Osmolar , Fosforilação , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Células Tumorais CultivadasRESUMO
The activity catalyzed by N-myristoyl transferase (NMT) is described for the first time in microsome-rich fractions from the murine leukemia cell line L1210, rat brain and mouse liver as biological sources. The enzyme from each source can accommodate various types of proteins (protein kinase A, virus structural gag protein or pp60src) as modelized by the use of their N-terminal derived peptides (GNAAAARR, GQTVTTPL and GSSKSKPKDP, respectively). As for some other types of membrane-bound enzymes, NMT activity can be enhanced by pretreatment with various types of detergents, amongst which Triton 770 and deoxycholate were the most potent. Further experiments on the L1210 microsome-rich fractions demonstrate that these two detergents were able to solubilize the microsomal enzyme, without modifying its substrate specificity. Finally, three compounds described in the literature to be inhibitors of NMT activity from other sources were tested for L1210 microsome-associated activity. None of them show any significant potency in inhibiting this activity. A new compound, myristoylphenylalanine, shows a slightly better inhibitory effect on the L1210 microsomal activity than the reference compounds with a median inhibitory concentration (IC50) of 0.2 mM.