RESUMEN
The Ras-related (R-Ras) isoforms TC21, R-Ras and M-Ras are members of the Ras superfamily of small GTPases. R-Ras family proteins are frequently overexpressed in human cancers, and expression of activated mutants of these GTPases is sufficient to induce cell transformation. Unlike Ras, few activating mutations of R-Ras proteins have been reported in human cancer, and very little is known about the regulation of their activity. In this study, we report that TC21 and R-Ras are phosphorylated on a conserved serine, Ser186 and Ser201, respectively, in intact cells. This residue is located in the C-terminal hypervariable region of the proteins and is not conserved in M-Ras. We show that the MAP kinases ERK1/2 phosphorylate TC21 and R-Ras on this C-terminal serine residue both in vitro and in vivo. Phosphorylation of R-Ras proteins does not affect their subcellular localization or stability but rather stimulates their activation. Phosphorylation-defective mutants of R-Ras and TC21 are compromised in their ability to promote cancer cell adhesion and migration/invasion, respectively. Importantly, we show that phosphorylation of TC21 and R-Ras potentiates their tumorigenic activity in immunodeficient mice. Our results identify a novel regulatory mechanism of the small GTPases TC21 and R-Ras that controls their oncogenic potential.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Secuencia de Aminoácidos , Humanos , Espacio Intracelular , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Unión al GTP Monoméricas/química , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Transporte de ProteínasRESUMEN
Ras proteins associate with cellular membranes by virtue of a series of post-translational modifications of their C-terminal CAAX sequences. The discovery that two of the three enzymes that modify CAAX proteins are restricted to the endoplasmic reticulum led to the recognition that all nascent Ras proteins transit endomembranes en route to the PM (plasma membrane) and that at steady-state N-Ras and H-Ras are highly expressed on the Golgi apparatus. To test the hypothesis that Ras proteins on internal membranes can signal, we developed a fluorescent probe that reports when and where in living cells Ras becomes active. We found that growth factors stimulated rapid and transient activation of Ras on the PM followed by delayed and sustained activation on the Golgi. We mapped one pathway responsible for this activity as involving PLCgamma (phospholipase Cgamma)/DAG (diacylglycerol)+Ca2+/RasGRP1. Using mammalian cells and fission yeast, we have shown that differential localization of activated Ras preferentially activates distinct signalling pathways. In very recent work, we have found that (i) the subcellular localization of K-Ras can be acutely modulated by phosphorylation of its C-terminal hypervariable region by PKC, (ii) among the membranes upon which phosphorylated K-Ras accumulates is the outer mitochondrial membrane and (iii) phosphorylated, internalized K-Ras promotes apoptosis. Thus the signalling output of Ras depends on its subcellular localization.
Asunto(s)
Membrana Celular/fisiología , Estructuras Celulares/fisiología , Transducción de Señal/fisiología , Proteínas ras/fisiología , Animales , Células Cultivadas , Aparato de Golgi/fisiologíaRESUMEN
The mammalian Rho family GTPases TC10 and Cdc42 share many properties. Activated forms of both proteins stimulate transcription mediated by nuclear factor kappaB, serum response factor, and the cyclin D1 promoter; activate c-Jun NH2-terminal kinase; cooperate with activated Raf to transform NIH-3T3 cells; and, by a mechanism independent of all of these effects, induce filopodia formation. In contrast, previously reported differences between TC10 and Cdc42 are not striking. We now present studies of TC10 and Cdc42 in cell culture that reveal clear functional differences: (a) wild-type TC10 localizes predominantly to the plasma membrane and less extensively to a perinuclear membranous compartment, whereas wild-type Cdc42 localizes predominantly to this compartment and less extensively to the plasma membrane; (b) expression of Rho guanine nucleotide dissociation inhibitor alpha results in a redistribution of wild-type Cdc42 to the cytosol but has no effect on the plasma membrane localization of wild-type TC10; (c) TC10 fails to rescue a Saccharomyces cerevisiae cdc42 mutation, unlike mammalian Cdc42; (d) dominant negative Cdc42, but not dominant negative TC10, inhibits neurite outgrowth in PC12 cells stimulated by nerve growth factor; and (e) activation of nuclear factor kappaB-dependent transcription by Cdc42, but not by TC10, is inhibited by sodium salicylate. These findings point to distinct pathways in which TC10 and Cdc42 may act and distinct modes of regulation of these proteins.
Asunto(s)
Compartimento Celular/fisiología , Membrana Celular/enzimología , Células Cultivadas/enzimología , Transducción de Señal/fisiología , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Antiinflamatorios no Esteroideos/farmacología , Células COS , Membrana Celular/ultraestructura , Células Cultivadas/citología , Proteínas Fluorescentes Verdes , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Células HeLa , Humanos , Proteínas Luminiscentes/genética , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Factor de Crecimiento Nervioso/farmacología , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Células PC12/citología , Células PC12/efectos de los fármacos , Células PC12/metabolismo , Ratas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)alpha. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDI alpha in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDI alpha. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDI alpha binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDI alpha and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.
Asunto(s)
Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Transporte Biológico , Células CHO , Células COS , Línea Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Cricetinae , Perros , Proteínas Fluorescentes Verdes , Inhibidores de Disociación de Guanina Nucleótido/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Datos de Secuencia Molecular , Ácido Palmítico/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rho/genética , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
We show that Nras is transiently localized in the Golgi prior to the plasma membrane (PM). Moreover, green fluorescent protein (GFP)-tagged Nras illuminated motile, peri-Golgi vesicles, and prolonged BFA treatment blocked PM expression. GFP-Hras colocalized with GFP-Nras, but GFP-Kras4B revealed less Golgi and no vesicular fluorescence. Whereas a secondary membrane targeting signal was required for PM expression, the CAAX motif alone was necessary and sufficient to target proteins to the endomembrane where they were methylated, a modification required for efficient membrane association. Thus, prenylated CAAX proteins do not associate directly with the PM but instead associate with the endomembrane and are subsequently transported to the PM, a process that requires a secondary targeting motif.
Asunto(s)
Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Células COS , Línea Celular , Cricetinae , Perros , Proteínas Fluorescentes Verdes , Riñón , Cinética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Proteínas ras/química , Proteínas ras/genéticaRESUMEN
The anti-inflammatory effects of high-dose salicylates are well recognized, incompletely understood and unlikely due entirely to cyclooxygenase (COX) inhibition. We have previously reported a role for activation of the kinase Erk in CD11b/CD18 integrin-dependent adhesiveness of human neutrophils, a critical step in inflammation. We now report the effects of salicylates on neutrophil Erk and adhesion. Exposure of neutrophils to aspirin or sodium salicylate (poor COX inhibitor) inhibited Erk activity and adhesiveness of formylmethionyl-leucyl-phenylalanine- and arachidonic acid-stimulated neutrophils, consistent with anti-inflammation but not COX inhibition (IC50s = 1-8 mM). In contrast, indomethacin blocked neither Erk nor adhesion. Inhibition of Mek (proximal activator of Erk) also blocked stimulation of Erk and adhesion by formylmethionyl-leucyl-phenylalanineand arachidonic acid. Salicylate inhibition of Erk was independent of protein kinase A activation and generation of extracellular adenosine. These data are consistent with a role for Erk in stimulated neutrophil adhesion, and suggest that anti-inflammatory effects of salicylates may be mediated via inhibition of Erk signaling required for integrin-mediated responses.
Asunto(s)
Aspirina/análogos & derivados , Aspirina/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/sangre , Proteínas Quinasas Activadas por Mitógenos , Neutrófilos/fisiología , Salicilato de Sodio/farmacología , Acetaminofén/farmacología , Antígenos CD/fisiología , Ácido Araquidónico/farmacología , Antígenos CD18/fisiología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Agregación Celular/efectos de los fármacos , Agregación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/farmacología , Humanos , Técnicas In Vitro , Antígeno de Macrófago-1/fisiología , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
AA stimulates integrin-dependent neutrophil adhesion, a critical early step in acute inflammation. However, neither the signaling pathway(s) of AA-stimulated adhesion, nor whether AA acts directly or through the generation of active metabolites, has been elucidated. Previously, we have observed a tight association between neutrophil Erk activation and homotypic adhesion in response to chemoattractants acting through G protein-linked receptors. We now report a similar association between homotypic adhesion and Erk activation in response to AA. Erk activation was cyclooxygenase independent and required AA metabolism to 5(S)- hydroperoxyeicosatetraenoic acid (5-HpETE) via 5-lipoxygenase, but not the further lipoxygenase-dependent metabolism of 5-HpETE to leukotrienes. AA stimulation of Erk was accompanied by Raf-1 activation and was sensitive to inhibitors of Raf-1 and Mek. Whereas activation of Erk by AA was pertussis toxin sensitive, [3H]-AA binding to neutrophils was not saturable, suggesting that an AA metabolite activates a G protein. Consistent with this hypothesis, Erk activation by 5(S)-hydroxyeicosatetraenoic acid (5-HETE; lipoxygenase-independent metabolite of 5-HpETE) was also pertussis toxin sensitive. These data suggest that a 5-lipoxygenase metabolite of AA, e.g., 5-HETE, is released from AA-treated cells to engage a plasma membrane-associated, pertussis toxin-sensitive, G protein-linked receptor, leading to activation of Erk and adhesion via the Raf-1/Mek signal transduction pathway.
Asunto(s)
Araquidonato 5-Lipooxigenasa/fisiología , Ácido Araquidónico/farmacología , Integrinas/fisiología , Quinasa 1 de Quinasa de Quinasa MAP , Proteínas Quinasas Activadas por Mitógenos/fisiología , Proteínas del Tejido Nervioso/fisiología , Neutrófilos/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-raf/fisiología , Adhesión Celular , Agregación Celular/efectos de los fármacos , AMP Cíclico/fisiología , Activación Enzimática , Humanos , Ácidos Hidroxieicosatetraenoicos/farmacología , Proteína Básica de Mielina/metabolismo , Toxina del Pertussis , Fosforilación , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Prenylcysteine carboxyl methyltransferase (pcCMT) is the third of three enzymes that posttranslationally modify C-terminal CAAX motifs and thereby target CAAX proteins to the plasma membrane. Here we report the molecular characterization and subcellular localization of the first mammalian (human myeloid) pcCMT. The deduced amino acid sequence of mammalian pcCMT predicts a multiple membrane-spanning protein with homologies to the yeast pcCMT, STE14, and the mammalian band 3 anion transporter. The human gene complemented a ste14 mutant. pcCMT mRNAs were ubiquitously expressed in human tissues. An anti-pcCMT antiserum detected a 33-kDa protein in myeloid cell membranes. Ectopically expressed recombinant pcCMT had enzymatic activity identical to that observed in neutrophil membranes. Mammalian pcCMT was not expressed at the plasma membrane but rather restricted to the endoplasmic reticulum. Thus, the final enzyme in the sequence that modifies CAAX motifs is located in membranes topologically removed from the CAAX protein target membrane.
Asunto(s)
Retículo Endoplásmico/enzimología , Proteína Metiltransferasas/química , Secuencia de Aminoácidos , Animales , Células CHO/citología , Clonación Molecular , Cricetinae , Bases de Datos Factuales , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes , Células HL-60 , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/inmunología , Ratones , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Proteínas ras/metabolismoRESUMEN
Neutrophil aggregation is mediated by the beta2 integrin CD11b/CD18, which has limited expression on the surface membrane of resting cells but is recruited from intracellular organelles after cell activation. We have previously found that CD11b/CD18 newly translocated to the plasma membrane does not contribute to adhesion but must be modified to be functional. Because neutrophil aggregation induced by phorbol myristate acetate (PMA) is accompanied by de novo phosphorylation of the CD18 cytoplasmic tail, we sought to determine whether CD11b/CD18 phosphorylation is separately regulated in the different cellular compartments. Accordingly, [32P]-labeled CD11b/CD18 was immunoprecipitated from purified neutrophil-specific granule or plasma membrane lysates. In plasma membrane fractions, as in whole cell lysates, CD18 became phosphorylated in cells exposed to PMA but not in untreated cells or cells treated with N-formyl-methionyl-leucyl-phenylalanine (fMLP). The alpha chain, CD11b, was phosphorylated under all conditions. In contrast, only marginal phosphorylation of specific granule-associated CD18 or CD11b was observed. Calyculin A, an inhibitor of serine/threonine phosphatases (pp1 > pp2a), induced strong phosphorylation of CD18 in the plasma membrane but not in the specific granules. Addition of intact specific granule membranes to the plasma membranes from PMA-treated neutrophils markedly decreased phosphorylation in both CD11b and CD18 subunits. These data suggest that the phosphorylation of CD11b/CD18, which accompanies neutrophil activation, is limited to plasma membrane-associated molecules. Phosphorylation, either constitutive or induced, is absent in the specific granule membranes. The difference may be due to a specific granule-associated phosphatase, probably distinct from ppl. Therefore adhesion-competent plasma membrane CD11b/CD18 and adhesion-incompetent specific granule CD11b/CD18 differ in their state of phosphorylation.
Asunto(s)
Antígenos CD18/metabolismo , Neutrófilos/metabolismo , Membrana Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Fosforilación , Fracciones Subcelulares/metabolismoRESUMEN
We employed neutrophils and enucleate neutrophil cytoplasts to study the activation of the mitogen-activated protein kinases (MAPKs) p44erk1 and p42erk2 in neutrophils by inflammatory agonists that engage G protein-linked receptors. Formyl-methionyl-leucyl-phenylalanine (FMLP) rapidly and transiently activated MAPK in neutrophils and cytoplasts, consistent with a role in signaling for neutrophil functions. FMLP stimulated p2lras activation in neutrophils and Raf-1 translocation from cytosol to plasma membrane in cytoplasts, with kinetics consistent with events upstream of MAPK activation. Insulin, a protein tyrosine kinase receptor (PTKR) agonist, stimulated neutrophil MAPK activation, demonstrating an intact system of PTKR signaling in these post-mitotic cells. FMLP- and insulin-stimulated MAPK activation in cytoplasts were inhibited by Bt2cAMP, consistent with signaling through Raf-1 and suggesting a mechanism for cAMP inhibition of neutrophil function. However, Bt2cAMP had no effect on FMLP-stimulated MAPK activation in neutrophils. The extent of MAPK activation by various chemoattractants correlated with their capacity to stimulate neutrophil and cytoplast homotypic aggregation. Consistent with its effects on MAPK, Bt2cAMP inhibited FMLP-stimulated aggregation in cytoplasts but not neutrophils. Insulin had no independent effect but primed neutrophils for aggregation in response to FMLP. Our studies support a p2lras-, Raf-1-dependent pathway for MAPK activation in neutrophils and suggest that neutrophil adhesion may be regulated, in part, by MAPK.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Proteínas Quinasas Activadas por Mitógenos , Neutrófilos/fisiología , Adhesión Celular , Agregación Celular , Activación Enzimática , Humanos , Insulina/farmacología , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-raf , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Superóxidos/metabolismoRESUMEN
Activation of the membrane-associated NADPH oxidase of neutrophils requires several cytosolic factors including p47phox, p67phox and p21rac2. We compared NADPH oxidase activity with the membrane translocation of p47phox, p67phox, and p21rac2. In a cell-free system, GTP gamma S stimulated translocation of p47phox and p67phox to the plasma membrane only in the presence of arachidonate, and this translocation correlated with NADPH oxidase activity of the reisolated plasma membranes (R = 0.94 and 0.97, respectively). In contrast, GTP gamma S-stimulated p21rac2 translocation with or without arachidonate, and the extent of translocation did not correlate with oxidase activity (R = 0.17). Neutrophil cytoplasts were used to relate membrane translocation of p47phox, p67phox and p21rac2 to membrane oxidase activity in response to the inflammatory agonists. Whereas N-formyl-methionyl-leucyl-phenylalanine stimulated equimolar, transient membrane translocation of p47phox and p67phox which kinetically paralleled NADPH oxidase activity, relatively little p21rac2 translocated (moles of p47phox/p21rac2 = 16.6). Moreover, although phorbol 12-myristate 13-acetate stimulated both the stable translocation of p47phox and p67phox and sustained NADPH oxidase activity, it did not stimulate p21rac2 translocation. From these data we conclude that membrane translocation of p21rac2 does not regulate NADPH oxidase activity stoichiometrically.
Asunto(s)
GTP Fosfohidrolasas/sangre , Proteínas de Unión al GTP/metabolismo , Nucleótidos de Guanina/farmacología , NADH NADPH Oxidorreductasas/sangre , Neutrófilos/enzimología , Fraccionamiento Celular , Membrana Celular/metabolismo , Sistema Libre de Células , Citosol/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Cinética , N-Formilmetionina Leucil-Fenilalanina/farmacología , NADPH Oxidasas , Fosfoproteínas/sangre , Acetato de Tetradecanoilforbol/farmacología , Proteínas de Unión al GTP racRESUMEN
The gamma subunits of heterotrimeric guanine nucleotide-binding regulatory (G) proteins (G gamma) are post-translationally processed at their C termini by prenylation, proteolysis, and carboxyl methylation. Whereas prenylation of G gamma is required for membrane association of G proteins, the role of carboxyl methylation is unknown. Here we show that human neutrophils express G gamma 2 but not G gamma 3 or G gamma 7 and that carboxyl methylation of G gamma 2 is associated with signal transduction. In a reconstituted cell-free system, neutrophil G gamma 2 was labeled by the methyl donor S-[methyl-3H]adenosyl-L-methionine. Carboxyl methylation was confirmed by alkaline hydrolysis and quantitation of volatile [3H]methanol. Neutrophil G gamma 2 methylation was stimulated by activation of G protein with guanosine 5'-[beta, gamma-thio]triphosphate. We estimate that after 1 hr of G-protein activation at least 6% of the total pool of G gamma 2 was carboxyl-methylated. The inflammatory agonist fMet-Leu-Phe stimulated guanosine 5'-[beta,gamma-thio]triphosphate-dependent carboxyl methylation. Methylation of G gamma 2 was inhibited by the carboxyl methyltransferase inhibitor N-acetyl-S-trans,trans-farnesylcysteine at concentrations that affected signal transduction in neutrophils. These results demonstrate that activation of neutrophil Gi is associated with alpha-carboxyl methyl esterification of G gamma 2 and suggest that carboxyl methylation of G gamma may play a role in signal transduction.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Neutrófilos/metabolismo , S-Adenosilmetionina/metabolismo , Anticuerpos , Electroforesis en Gel de Poliacrilamida , Proteínas de Unión al GTP/aislamiento & purificación , Nucleótidos de Guanina/farmacología , Humanos , Immunoblotting , Técnicas In Vitro , Radioisótopos de Yodo , Sustancias Macromoleculares , Metilación , Peso Molecular , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , TritioAsunto(s)
Neutrófilos/enzimología , Proteína Metiltransferasas/sangre , Proteína Metiltransferasas/aislamiento & purificación , Prenilación de Proteína , Proteínas ras/sangre , Proteínas ras/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Autorradiografía/métodos , Encéfalo/enzimología , Fraccionamiento Celular/métodos , Membrana Celular/enzimología , Membrana Celular/ultraestructura , Centrifugación por Gradiente de Densidad/métodos , Citosol/enzimología , Detergentes/farmacología , Electroforesis en Gel de Poliacrilamida/métodos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Cinética , Metilación , Microsomas/enzimología , Microsomas Hepáticos/enzimología , Datos de Secuencia Molecular , Peso Molecular , Proteína Metiltransferasas/metabolismo , Ratas , S-Adenosilmetionina/metabolismo , Radioisótopos de AzufreAsunto(s)
Alprostadil/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/sangre , Neutrófilos/fisiología , Receptores de Prostaglandina E/fisiología , Transducción de Señal , Adenosina Trifosfato/sangre , Bucladesina/farmacología , Activación Enzimática , Proteínas de Unión al GTP/metabolismo , Humanos , Técnicas In Vitro , Cinética , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Receptores de Prostaglandina E/efectos de los fármacos , Superóxidos/sangre , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Carboxylmethylation of ras-related proteins is stimulated immediately on exposure of myeloid cells to inflammatory agonists. When the methylation reaction is inhibited with prenylcysteine analogs, G-protein-mediated signal transduction responses are disrupted, but responses to phorbol ester, calcium ionophore, and phospholipase C (PLC) remain intact. Furthermore, prenylcysteine analogs block GTP gamma S-induced aggregation of permeabilized platelets. Together, these results suggest that protein prenylcysteine methylation can play a role in signal transduction. A number of studies with AdoMet antagonists have suggested a role for methylation in cell-cycle regulation and stimulus-response coupling. Because the compounds generally inhibit all cellular methylation events, however, their effects have been difficult to interpret. On the other hand, prenylcysteine analogs have proved to be specific inhibitors of protein prenylcysteine methylation, as opposed to other types of methylation reactions. This enables the segregation of the role of methylation at C-terminal prenylcysteine residues from methylation at other sites, such as the carboxyl terminus of the catalytic subunit of PP2A. It should be emphasized, however, that prenylcysteine tails of proteins may interact with other target sites in addition to the methyltransferase enzyme(s), and prenylcysteine analogs may compete for these sites as well. One cannot assume that the inhibition of a response by the drugs necessarily implicates the involvement of a prenylcysteine methylation reaction. Studies with the analogs must be interpreted in conjunction with other results to ascertain the locus of their effects.
Asunto(s)
Cisteína/análogos & derivados , Proteínas de Unión al GTP/metabolismo , Proteína Metiltransferasas/metabolismo , Transducción de Señal/efectos de los fármacos , Terpenos/farmacología , Secuencia de Aminoácidos , Animales , Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Agregación Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Cisteína/síntesis química , Cisteína/farmacología , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/efectos de los fármacos , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Metilación , Datos de Secuencia Molecular , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Proteína Metiltransferasas/antagonistas & inhibidores , Especificidad por Sustrato , Terpenos/síntesis químicaRESUMEN
Signal transduction in human neutrophils requires prenylcysteine-directed carboxyl methylation of ras-related low molecular weight GTP-binding proteins. We now report the subcellular localization and characterization of a neutrophil prenylcysteine alpha carboxyl methyltransferase. The highest carboxyl methyltransferase activity copurified with biotinylated neutrophil surface membranes, supporting a plasma membrane localization of the enzyme. Neutrophil nuclear fractions contained little or no methyltransferase activity. Methyltransferase activity was detergent-sensitive but could be reconstituted by removal of detergent in the presence of phosphatidyl choline and an anionic phospholipid. N-Acetyl-S-trans,trans-farnesyl-L-cysteine (AFC) and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine (AGGC) were effective substrates for neutrophil prenylcysteine-directed methyltransferase; Vmax values for AFC and AGGC (16.4 and 22.1 pmol of methylated/mg protein/min, respectively) are among the highest yet reported. Although both GTP gamma S and the chemoattractant fMet-Leu-Phe stimulated methylation of ras-related proteins, neither affected methylation of AFC. These data suggest that neutrophil plasma membranes contain a phospholipid-dependent, prenylcysteine-directed carboxyl methyltransferase of relatively high specific activity that modifies ras-related protein substrates in the GTP-bound, activated state.
Asunto(s)
Membrana Celular/metabolismo , Neutrófilos/enzimología , Proteína Metiltransferasas/sangre , Compartimento Celular , Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Técnicas In Vitro , Cinética , N-Formilmetionina Leucil-Fenilalanina/farmacología , Fracciones Subcelulares/enzimología , Especificidad por SustratoRESUMEN
In human neutrophils, as in other cell types, Ras-related guanosine triphosphate-binding proteins are directed toward their regulatory targets in membranes by a series of posttranslational modifications that include methyl esterification of a carboxyl-terminal prenylcysteine residue. In intact cells and in a reconstituted in vitro system, the amount of carboxyl methylation of Ras-related proteins increased in response to the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP). Activation of Ras-related proteins by guanosine-5'-O-(3-thiotriphosphate) had a similar effect and induced translocation of p22rac2 from cytosol to plasma membrane. Inhibitors of prenylcysteine carboxyl methylation effectively blocked neutrophil responses to FMLP. These findings suggest a direct link between receptor-mediated signal transduction and the carboxyl methylation of Ras-related proteins.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Inhibidores de Disociación de Guanina Nucleótido , Neutrófilos/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/fisiología , Membrana Celular/metabolismo , Citosol/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanosina Trifosfato/farmacología , Humanos , Metionina/análogos & derivados , Metionina/metabolismo , Metilación , N-Formilmetionina Leucil-Fenilalanina/farmacología , Proteína Metiltransferasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , S-Adenosilmetionina/metabolismo , Tritio , Proteínas de Unión al GTP rap , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
Degranulation of neutrophils involves the differential regulation of the exocytosis of at least two populations of granules. Low molecular weight GTP-binding proteins (LMW-GBPs) have been implicated in the regulation of vesicular traffic in the secretory pathways of several types of cells. In the present study we identify distinct subsets of LMW-GBPs associated with the membranes of neutrophil-specific and azurophilic granules. Ninety-four percent of total [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was equally distributed between the plasma membrane and cytosol with the remaining 6% localized in the granules. In contrast, the cytosol contained only 10% of the total GTPase activity while the specific granules accounted for 13%. [alpha-32P]GTP binding to proteins transferred to nitrocellulose revealed LMW-GBPs in all fractions except the azurophilic granules. The specific granules contained three out of four bands which were found in the plasma membrane; these ranged from 20 to 23 kDa and all were resistant to alkaline extraction. Photoaffinity labeling with [alpha-32P]8-azido-GTP in the presence of micromolar Al3+ identified proteins of 25 and 26 kDa unique to azurophilic granules; these could not be labeled with [alpha-32P]8-azido-ATP and could be extracted by acidic but not alkaline pH. Botulinum C3-mediated [32P]ADP-ribosylation identified proteins of 16, 20, and 24 kDa both in plasma membranes and those of specific granules. An anti-ras monoclonal antibody, 142-24E5, recognized a 20-kDa protein localized to the plasma and specific granule membranes which could not be extracted by alkaline pH, was not a substrate for botulinum C3 ADP-ribosyltransferase, and was translocated from specific granules to plasma membrane after exposure of neutrophils to phorbol myristate acetate. We conclude that neutrophil-specific and azurophilic granules contain distinct subsets of LMW-GBPs which are uniquely situated to regulate the differential exocytosis of these two compartments.
Asunto(s)
Proteínas de Unión al GTP/análisis , Neutrófilos/química , Adenosina Difosfato Ribosa , Marcadores de Afinidad , Anticuerpos Monoclonales/inmunología , Western Blotting , Toxinas Botulínicas/metabolismo , Fraccionamiento Celular , Membrana Celular/química , Complemento C3/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/inmunología , Humanos , Peso Molecular , Neutrófilos/enzimologíaRESUMEN
Cocaine and its derivatives blunted responses of neutrophils (cell/cell aggregation, up-regulation of the receptor for C3bi (CR3, CD11b/CD18), generation of superoxide anion (O2-) and degranulation to various stimuli. The order of potency of these agents was the same as that for local anesthesia: tetracaine greater than bupivacaine greater than cocaine greater than lidocaine. Neutrophil aggregation elicited by the chemoattractant FMLP (10(-7) M) was inhibited by cocaine (10 mM) to 13.6 +/- 6% of control (p less than 0.002); the IC50 was approximately 4 mM. Cocaine and the other local anesthetics not only inhibited the upregulation of CR3 and O2- generation, but also blocked degranulation of cytochalasin B-treated cells. Cocaine (10 mM) reduced beta-glucuronidase and lysozyme secretion to 4.3 +/- 0.7 and 13 +/- 2.2% controls, respectively; its IC50 was 4 mM. Local anesthetics added after ligand/receptor engagement (FMLP) interrupted aggregation and halted generation of O2-. Moreover, local anesthetics rapidly inhibited aggregation, O2- generation, and degranulation elicited by PMA (1 microgram/ml) or the Ca ionophore A23187 (10 microM): the effects of cocaine could therefore not be attributed to unique actions at the FMLP receptor. Peak levels of intracellular Ca2+ ([Ca]i) at 5 to 10 s, and levels of [Ca]i 120 s after FMLP in Fura 2-loaded cells were significantly lower in cells treated with lidocaine, findings that could be explained by enhanced 45Ca2+ efflux from neutrophils. In cells loaded with bis(carboxyethyl)carboxyfluorescine (pH indicator) local anesthetics failed to affect the initial FMLP-induced (0 to 15 s) drop of pHi but inhibited the later (120 s) realkalinization of the cytosol (lidocaine, bupivacaine). Most remarkably, autoradiographs of SDS gels prepared from stimulated, 32P-labeled neutrophils treated with local anesthetics showed no difference from resting cells, either with respect to patterns of phosphorylation and dephosphorylation or their kinetics. Labeling of a 47-kDa protein, a component of the reduced nicotinamide-adenine dinucleotide phosphate-oxidase system, was unchanged. The effects of local anesthetics, which blunt neutrophil responses without affecting protein phosphorylation, suggest that protein phosphorylation is an insufficient signal for neutrophil activation. Inasmuch as cocaine and its derivatives affect cell functions at sites distal to activation of protein kinase C, these agents should prove useful in uncoupling protein phosphorylation from functional responses.
Asunto(s)
Anestésicos Locales/farmacología , Cocaína/farmacología , NADH NADPH Oxidorreductasas/metabolismo , Neutrófilos/efectos de los fármacos , Calcimicina/farmacología , Agregación Celular/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Glucuronidasa/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Muramidasa/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/metabolismo , Receptores de Complemento/metabolismo , Transducción de Señal/efectos de los fármacos , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Nonsteroidal antiinflammatory drugs (NSAIDs) inhibit neutrophil functions via mechanisms separate from their capacity to inhibit prostaglandin synthesis. We have studied discrete events in the process of signal transduction: NSAIDs but not a related analgesic drug (acetaminophen), inhibited aggregation in response to the chemoattractants f-Met-Leu-Phe (FMLP), leukotriene B4, and C5a. NSAIDs, but not acetaminophen, inhibited binding of radiolabeled FMLP to purified neutrophil membranes. Gpp(NH)p, a GTPase insensitive analog of GTP, also inhibited the binding of FMLP but, paradoxically, enhanced superoxide anion generation and lysozyme release. The inhibition of ligand binding by NSAIDs did not correlate with their capacity to inhibit FMLP-induced increments in diacylglycerol (DG): piroxicam, but not salicylate effectively inhibited appearance of label ([3H]arachidonate, [14C]glycerol) in DG. Finally, NSAIDs exerted differential effects on the viscosity of neutrophil plasma membranes and multilamellar vesicles (liposomes): membrane viscosity was increased by piroxicam and indomethacin, decreased by salicylate, and unaffected by acetaminophen. Thus, the different effects of NSAIDs on discrete pathways are not due to their shared capacity to reduce ligand binding but rather to a capacity to uncouple postreceptor signaling events that depend upon the state of membrane fluidity.