RESUMEN
Rho and Rac, two members of the Ras superfamily of guanosine triphosphate (GTP)-binding proteins, regulate a variety of signal transduction pathways in eukaryotic cells. Upon stimulation of phagocytic cells, Rac enhances the activity of the enzyme nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) oxidase, resulting in the production of superoxide radicals. Activation of the NADPH oxidase requires the assembly of a multimolecular complex at the plasma membrane consisting of two integral membrane proteins, gp91phox and p21phox, and two cytosolic proteins, p67phox and p47phox. Rac1 interacted directly with p67phox in a GTP-dependent manner. Modified forms of Rac with mutations in the effector site did not stimulate oxidase activity or bind to p67phox. Thus, p67phox appears to be the Rac effector protein in the NADPH oxidase complex.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Fagocitos/enzimología , Fosfoproteínas/metabolismo , Sitios de Unión , Activación Enzimática , Guanosina Trifosfato/metabolismo , Humanos , NADPH Deshidrogenasa/metabolismo , NADPH Oxidasas , Proteínas Recombinantes de Fusión/metabolismo , Superóxidos/metabolismo , Proteínas de Unión al GTP racRESUMEN
The NADPH oxidase is an electron transport chain found in lymphocytes and in the wall of the endocytic vacuole of 'professional' phagocytic cells. It is so called because NADPH is used as an electron donor to reduce oxygen to superoxide and hydrogen peroxide. The redox components are provided by a very unusual flavocytochrome b from the membrane, which is dependent upon cytosolic factors (including two specialized proteins, p47phox and p67phox) for activation. The small GTP-binding protein, p21rac, is also implicated in this system, possibly as the switch that triggers electron transport. This system provides a key to our understanding of the way in which these GTP-binding proteins function.
Asunto(s)
NADH NADPH Oxidorreductasas/metabolismo , Fagocitos/enzimología , Secuencia de Aminoácidos , Animales , Activación Enzimática , Flavina-Adenina Dinucleótido/metabolismo , Humanos , Linfocitos/enzimología , Datos de Secuencia Molecular , NADP/metabolismo , NADPH Oxidasas , Estallido RespiratorioRESUMEN
BACKGROUND: Rac and Cdc42 are members of the Rho family of small GTPases. They modulate cell growth and polarity, and contribute to oncogenic transformation by Ras. The molecular mechanisms underlying these functions remain elusive, however. RESULTS: We have identified a novel effector of Rac and Cdc42, hPar-6, which is the human homolog of a cell-polarity determinant in Caenorhabditis elegans. hPar-6 contains a PDZ domain and a Cdc42/Rac interactive binding (CRIB) motif, and interacts with Rac1 and Cdc42 in a GTP-dependent manner. hPar-6 also binds directly to an atypical protein kinase C isoform, PKCzeta, and forms a stable ternary complex with Rac1 or Cdc42 and PKCzeta. This association results in stimulation of PKCzeta kinase activity. Moreover, hPar-6 potentiates cell transformation by Rac1/Cdc42 and its interaction with Rac1/Cdc42 is essential for this effect. Cell transformation by hPar-6 involves a PKCzeta-dependent pathway distinct from the pathway mediated by Raf. CONCLUSIONS: These findings indicate that Rac/Cdc42 can regulate cell growth through Par-6 and PKCzeta, and suggest that deregulation of cell-polarity signaling can lead to cell transformation.
Asunto(s)
Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteína Quinasa C/metabolismo , Proteínas/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Proteínas de Caenorhabditis elegans , Transformación Celular Neoplásica , Clonación Molecular , Activación Enzimática , Humanos , Datos de Secuencia Molecular , Proteínas/química , Proteínas/genética , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Técnicas del Sistema de Dos HíbridosRESUMEN
Actin polymerization at the cell cortex is thought to provide the driving force for aspects of cell-shape change and locomotion. To coordinate cellular movements, the initiation of actin polymerization is tightly regulated, both spatially and temporally. The Wiskott-Aldrich syndrome protein (WASP), encoded by the gene that is mutated in the immunodeficiency disorder Wiskott-Aldrich syndrome [1], has been implicated in the control of actin polymerization in cells [2] [3] [4] [5]. The Arp2/3 complex, an actin-nucleating factor that consists of seven polypeptide subunits [6] [7] [8], was recently shown to physically interact with WASP [9]. We sought to determine whether WASP is a cellular activator of the Arp2/3 complex and found that WASP stimulates the actin nucleation activity of the Arp2/3 complex in vitro. Moreover, WASP-coated microspheres polymerized actin, formed actin tails and exhibited actin-based motility in cell extracts, similar to those behaviors displayed by the pathogenic bacterium Listeria monocytogenes. In extracts depleted of the Arp2/3 complex, WASP-coated microspheres and L. monocytogenes were non-motile and exhibited only residual actin polymerization. These results demonstrate that WASP is sufficient to direct actin-based motility in cell extracts and that this function is mediated by the Arp2/3 complex. WASP interacts with diverse signaling proteins and may therefore function to couple signal transduction pathways to Arp2/3-complex activation and actin polymerization.
Asunto(s)
Actinas/metabolismo , Proteínas del Citoesqueleto , Proteínas/metabolismo , Síndrome de Wiskott-Aldrich/metabolismo , Proteína 2 Relacionada con la Actina , Biopolímeros , Proteína del Síndrome de Wiskott-AldrichRESUMEN
The p21-activated protein kinases (PAKs) are activated through direct interaction with the GTPases Rac and Cdc42Hs, which are implicated in the control of the mitogen-activated protein kinase (MAP kinase) c-Jun N-terminal kinase (JNK) and the reorganization of the actin cytoskeleton [1-3]. The exact role of the PAK proteins in these signaling pathways is not entirely clear. To elucidate the biological function of Pak2 and to identify its molecular targets, we used a novel two-hybrid system, the Ras recruitment system (RRS), that aims to detect protein-protein interactions at the inner surface of the plasma membrane (described in the accompanying paper by Broder et al. [4]). The Pak2 regulatory domain (PakR) was fused at the carboxyl terminus of a RasL61 mutant protein and screened against a myristoylated rat pituitary cDNA library. Four clones were identified that interact specifically with PakR and three were subsequently shown to encode a previously unknown homologue of the GTPase Cdc42Hs. This approximately 36 kDa protein, designated Chp, exhibits an overall sequence identity to Cdc42Hs of approximately 52%. Chp contains two additional sequences at the amino and carboxyl termini that are not found in any known GTPase. The amino terminus contains a polyproline sequence, typically found in Src homology 3 (SH3)-binding domains, and the carboxyl terminus appears to be important for Pak2 binding. Results from the microinjection of Chp into cells implicated Chp in the induction of lamellipodia and showed that Chp activates the JNK MAP kinase cascade.
Asunto(s)
Actinas/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Citoesqueleto/fisiología , Activación Enzimática , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/fisiología , Expresión Génica , Proteínas Quinasas JNK Activadas por Mitógenos , Datos de Secuencia Molecular , Mutagénesis , Ratas , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transcripción Genética , Proteína de Unión al GTP cdc42RESUMEN
BACKGROUND: The negative factor (Nef) of human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV) is required for high levels of viremia and progression to AIDS. Additionally, Nef leads to cellular activation, increased viral infectivity and decreased expression of CD4 on the cell surface. Previously, we and others demonstrated that Nef associates with a cellular serine kinase (NAK) activity. Recently, it was demonstrated that NAK bears structural and functional similarity to p21-activated kinases (PAKs). RESULTS: In this study, we demonstrate that Nef not only binds to but also activates NAK via the small GTPases CDC42 and Rac1. First, the dominant-negative PAK (PAKR), via its GTPase-binding domain, and dominant-negative GTPases (CDC42Hs-N17 and Rac1-N17) block the ability of Nef to associate with and activate NAK. Second, constitutively active small GTPases (CDC42Hs-V12 and Rac1-V12) potentiate the effects of Nef. Third, interactions between Nef and NAK result in several cellular effector functions, such as activation of the serum-response pathway. And finally, PAKR, CDC42Hs-N17 and Rac1-N17 decrease levels of HIV-1 production to those of virus from which the nef gene is deleted. CONCLUSIONS: By activating NAK via small GTPases and their downstream effectors, Nef interacts with regulatory pathways required for cell growth, cytoskeletal rearrangement and endocytosis. Thus, NAK could participate in the budding of new virions, the modification of viral proteins and the increased endocytosis of surface molecules such as CD4. Moreover, blocking the activity of these GTPases could lead to new therapeutic interventions against AIDS.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Unión al GTP/genética , Productos del Gen nef/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Células 3T3 , Animales , Células COS , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proteínas de Unión al ADN/genética , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica , Productos del Gen nef/metabolismo , VIH-1/genética , VIH-1/metabolismo , VIH-1/fisiología , Humanos , Células Jurkat , Ratones , Proteínas Nucleares/genética , Ratas , Factor de Respuesta Sérica , Transducción de Señal , Replicación Viral , Proteína de Unión al GTP cdc42 , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Quinasas p21 Activadas , Proteínas de Unión al GTP racRESUMEN
The Rho family members Cdc42, Rac, and Rho play a central role in the organization of the actin cytoskeleton and regulate transcription. Whereas Rac and Rho have been implicated in transformation by oncogenic Ras, the role of Cdc42 in this process remains unknown. In this study, we found that Rat1 fibroblasts expressing constitutively active V12-Cdc42 were anchorage independent and proliferated in nude mice but failed to show enhanced growth in low serum. Similar to V12-Rac1-expressing Rat1 fibroblasts, V12-Cdc42 lines displayed a high frequency of multinucleated cells. Interestingly, coexpression of dominant negative N17-Rac1 blocked the V12-Cdc42-induced multinucleated phenotype but not growth in soft agar, indicating that Cdc42 controls anchorage independence in a Rac-independent fashion. We also showed that dominant negative N17-Cdc42 inhibited Ras focus formation and anchorage-independent growth and caused reversion of the transformed morphology, indicating that Cdc42 is necessary for Ras transformation. N17-Cdc42 caused only partial inhibition of Ras-induced low-serum growth, however. In contrast, whereas N17-Rac1 also effectively inhibited Ras-induced anchorage independence, it did not revert the morphology of Ras-transformed cells. N17-Rac1 strongly inhibited low-serum growth of Ras-transformed cells, however. Together, these data provide a novel function for Cdc42 in cell proliferation and indicate that Cdc42 and Rac play distinct roles in growth control and Ras transformation.
Asunto(s)
Adhesión Celular/genética , Proteínas de Ciclo Celular/metabolismo , Transformación Celular Neoplásica , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas ras/metabolismo , Células 3T3 , Animales , División Celular , Fibroblastos/metabolismo , Ratones , Microscopía Fluorescente , Ratas , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae , Proteínas de Unión al GTP racRESUMEN
The activation of Nef-associated kinase (NAK) by Nef from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. This induction occurs via the guanine nucleotide exchange factor Vav and the small GTPases Rac1 and Cdc42. In this study, we identified NAK as p21-activated kinase 1 (PAK1). PAK1 bound to Nef in vitro and in vivo. Moreover, the induction of cytoskeletal rearrangements such as the formation of trichopodia, the activation of Jun N-terminal kinase, and the increase of viral production were blocked by an inhibitory peptide that targets the kinase activity of PAK1 (PAK1 83-149). These results identify NAK as PAK1 and emphasize the central role its kinase activity plays in cytoskeletal rearrangements and cellular signaling by Nef.
Asunto(s)
Productos del Gen nef/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Células COS , Línea Celular , Citoesqueleto/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes , VIH-1/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Luminiscentes , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fragmentos de Péptidos/farmacología , Unión Proteica , Transducción de Señal , Transfección , Proteínas Virales/biosíntesis , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Quinasas p21 ActivadasRESUMEN
The ability of signaling via the JNK (c-Jun NH2-terminal kinase)/stress-activated protein kinase cascade to stimulate or inhibit DNA synthesis in primary cultures of adult rat hepatocytes was examined. Treatment of hepatocytes with media containing hyperosmotic glucose (75 mM final), tumor necrosis factor alpha (TNFalpha, 1 ng/ml final), and hepatocyte growth factor (HGF, 1 ng/ml final) caused activation of JNK1. Glucose, TNFalpha, or HGF treatments increased phosphorylation of c-Jun at serine 63 in the transactivation domain and stimulated hepatocyte DNA synthesis. Infection of hepatocytes with poly-L-lysine-coated adenoviruses coupled to constructs to express either dominant negatives Ras N17, Rac1 (N17), Cdc42 (N17), SEK1-, or JNK1- blunted the abilities of glucose, TNFalpha, or HGF to increase JNK1 activity, to increase phosphorylation of c-Jun at serine 63, and to stimulate DNA synthesis. Furthermore, infection of hepatocytes by a recombinant adenovirus expressing a dominant-negative c-Jun mutant (TAM67) also blunted the abilities of glucose, TNFalpha, and HGF to stimulate DNA synthesis. These data demonstrate that multiple agonists stimulate DNA synthesis in primary cultures of hepatocytes via a Ras/Rac1/Cdc42/SEK/JNK/c-Jun pathway. Glucose and HGF treatments reduced glycogen synthase kinase 3 (GSK3) activity and increased c-Jun DNA binding. Co-infection of hepatocytes with recombinant adenoviruses to express dominant- negative forms of PI3 kinase (p110alpha/p110gamma) increased basal GSK3 activity, blocked the abilities of glucose and HGF treatments to inhibit GSK3 activity, and reduced basal c-Jun DNA binding. However, expression of dominant-negative PI3 kinase (p110alpha/p110gamma) neither significantly blunted the abilities of glucose and HGF treatments to increase c-Jun DNA binding, nor inhibited the ability of these agonists to stimulate DNA synthesis. These data suggest that signaling by the JNK/stress-activated protein kinase cascade, rather than by the PI3 kinase cascade, plays the pivotal role in the ability of agonists to stimulate DNA synthesis in primary cultures of rat hepatocytes.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Ciclo Celular/metabolismo , ADN/biosíntesis , Proteínas de Unión al GTP/metabolismo , Hígado/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas ras/metabolismo , Animales , Células Cultivadas , ADN/genética , ADN/metabolismo , Glucosa/farmacología , Glucógeno Sintasa Quinasas , Guanosina Trifosfato/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , Hígado/citología , Hígado/efectos de los fármacos , Mutación , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-jun/genética , Ratas , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacología , Proteína de Unión al GTP cdc42Asunto(s)
Actinas/metabolismo , Proteínas del Citoesqueleto , Escherichia coli/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas/metabolismo , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Células HeLa , Humanos , Proteína del Síndrome de Wiskott-Aldrich , Proteína Neuronal del Síndrome de Wiskott-AldrichRESUMEN
OBJECTIVE: Aberrant expression of maspin protein related to DNA hypomethylation in the promoter region is frequently observed in gallbladder carcinomas, whereas the non-tumorous gallbladder epithelium is maspin negative. We investigated maspin expression in non-tumorous gallbladder epithelium in patients with cholelithiasis. METHODS: An immunohistochemical study of maspin expression was performed in 69 patients with cholelithiasis and 30 patients with gastric cancer without cholelithiasis. RESULTS: Immunoreactivity for maspin was observed in focal and patchy regions of the gallbladder epithelium. Positive immunoreactivity for maspin was significantly associated with the presence of intestinal metaplasia in patients with cholelithiasis (p<0.05). CONCLUSION: The high incidence of aberrant maspin expression in both intestinal metaplasia and carcinoma of the gallbladder supports the assumption that intestinal metaplasia of the gallbladder may predispose to gallbladder carcinoma.
Asunto(s)
Biomarcadores de Tumor/análisis , Colelitiasis/química , Vesícula Biliar/química , Serpinas/análisis , Adulto , Estudios de Casos y Controles , Colelitiasis/patología , Progresión de la Enfermedad , Endotelio/química , Endotelio/patología , Femenino , Vesícula Biliar/patología , Genes Supresores de Tumor , Humanos , Inmunohistoquímica/métodos , Mucosa Intestinal/patología , Masculino , Metaplasia , Persona de Mediana Edad , Neoplasias Gástricas/químicaAsunto(s)
GTP Fosfohidrolasas/aislamiento & purificación , Proteínas de Unión al GTP/aislamiento & purificación , Inhibidores de Disociación de Guanina Nucleótido , Macrófagos Peritoneales/metabolismo , Neutrófilos/metabolismo , Fagocitos/metabolismo , Proteínas/aislamiento & purificación , Animales , Fraccionamiento Celular/métodos , Cromatografía en Gel/métodos , Cromatografía por Intercambio Iónico/métodos , Citosol/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , GTP Fosfohidrolasas/sangre , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/análisis , Proteínas de Unión al GTP/metabolismo , Proteínas Activadoras de GTPasa , Cobayas , Células HL-60 , Humanos , Indicadores y Reactivos , Sustancias Macromoleculares , Peso Molecular , Proteínas/análisis , Proteínas/metabolismo , Ultracentrifugación/métodos , Proteínas de Unión al GTP rac , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoAsunto(s)
Proteínas de Unión al GTP/metabolismo , NADH NADPH Oxidorreductasas/sangre , Neutrófilos/enzimología , Fraccionamiento Celular/métodos , Membrana Celular/enzimología , Membrana Celular/ultraestructura , Sistema Libre de Células , Grupo Citocromo b/sangre , Grupo Citocromo b/aislamiento & purificación , Citosol/enzimología , Citosol/ultraestructura , Proteínas de Unión al GTP/biosíntesis , Proteínas de Unión al GTP/aislamiento & purificación , Humanos , Sustancias Macromoleculares , Lípidos de la Membrana/sangre , Modelos Biológicos , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/aislamiento & purificación , NADPH Oxidasas , Neutrófilos/ultraestructura , Fosfolípidos/sangre , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Superóxidos/sangre , Proteínas de Unión al GTP racAsunto(s)
Proteínas de Ciclo Celular/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Biología Molecular/métodos , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Consenso , Guanosina Trifosfato/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Proteína del Síndrome de Wiskott-Aldrich , Proteína de Unión al GTP cdc42 , Quinasas p21 Activadas , Proteínas de Unión al GTP racRESUMEN
IREM-1 is an inhibitory cell surface receptor with an unknown function and is expressed on myeloid cell lineages, including cell lines derived from acute myeloid leukemia (AML) patients. We have generated a series of monoclonal antibodies (mAbs) against the extracellular domain of IREM-1 and further assessed its expression in normal and AML cells. IREM-1 was restricted to cells from myeloid origin and extensive expression analysis in primary cells obtained from AML patients showed IREM-1 expression in leukemic blasts of 72% (39/54) of samples. We therefore searched for specific IREM-1 mAbs with activity in functional complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Lead mAbs against IREM-1 showed specific cytotoxic activity against a variety of AML-derived cell lines and freshly isolated blasts from AML patients. Internalization of mAbs upon IREM-1 binding was also shown. In vivo anticancer activity of lead mAbs was observed in an established HL-60 xenograft model with a tumor growth delay of up to 40% and in a model using primary human AML cells, where treatment with anti-IREM-1 mAb resulted in a significant reduction of engrafted human cells. These results demonstrate IREM-1 as a potential novel target for immunotherapy of AML.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Receptores Inmunológicos/antagonistas & inhibidores , ADP-Ribosil Ciclasa 1/análisis , Animales , Anticuerpos Monoclonales/biosíntesis , Afinidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD34/análisis , Humanos , Ratones , Ratones Endogámicos BALB C , Receptores Inmunológicos/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder associated with lymphocytes and platelet abnormalities. The gene that encodes the Wiskott-Aldrich protein (WASP) was recently isolated, and shown to be defective in WAS patients. WASP contains multiple domains that interact with various signalling proteins, including the guanine triphosphatase (GTPase) Cdc42Hs and SH3 domain-containing proteins. Biochemical and genetic evidence strongly suggests that WASP is an important protein in the regulation of cell morphology. Recent progress in the identification of molecular partners for WASP suggests a molecular mechanism for the cellular abnormalities of WAS.
Asunto(s)
Proteínas/genética , Transducción de Señal , Síndrome de Wiskott-Aldrich/genética , Humanos , Activación de Linfocitos , Linfocitos T/inmunología , Síndrome de Wiskott-Aldrich/inmunología , Síndrome de Wiskott-Aldrich/patología , Proteína del Síndrome de Wiskott-AldrichRESUMEN
Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes in a cell-free system by anionic amphiphiles requires the participation of both membrane and cytosolic components. We reported that ammonium sulfate fractionation (Pick, E., Kroizman, T., and Abo, A. (1989) J. Immunol. 143, 4180-4187) and affinity chromatography on 2',5'-ADP-agarose (Shaag, D., and Pick, E. (1990) Biochim. Biophys. Acta 1037, 405-412) permit separation of cytosol in two fractions (sigma 1 and sigma 2) that support O2- production by solubilized membrane synergistically. We now describe the purification of sigma 1 to near homogeneity and demonstrate that it represents a cytosolic component distinct from p47-phox and p67-phox, that are both found in fraction sigma 2. Sigma 1 was absolutely required for the full expression of amphiphile-activated NADPH-oxidase activity. This requirement was evident whether sigma 1 was added to cell-free systems composed of: (a) solubilized membrane and a sigma 2-enriched cytosolic fraction, or (b) purified cytochrome b559, incorporated in liposomes, and purified sigma 2. Sigma 1 was purified by a sequence comprising ammonium sulfate fractionation, hydrophobic chromatography on phenyl-Superose, absorption with CM-Sepharose, anion exchange chromatography on DEAE-Sepharose, and gel filtration on Superose 12. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of sigma 1 of maximal purity, under both reducing and nonreducing conditions, demonstrated the presence of two proteins, of 24 and 22 kDa. On gel filtration, sigma 1 was eluted as a symmetrical peak of 46 kDa that by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed the presence of both 24- and 22-kDa bands. We suggest that, in its native form, sigma 1 might represent a complex of the 24- and 22-kDa proteins. The specific roles of each molecule in NADPH oxidase function remain to be determined.
Asunto(s)
Macrófagos/enzimología , NADH NADPH Oxidorreductasas/aislamiento & purificación , Complejo de Proteína del Fotosistema II , Superóxidos/metabolismo , Animales , Membrana Celular/enzimología , Sistema Libre de Células , Cromatografía por Intercambio Iónico , Grupo Citocromo b/metabolismo , Citosol/enzimología , Cobayas , Sustancias Macromoleculares , Peso Molecular , NADH NADPH Oxidorreductasas/metabolismo , NADPH OxidasasRESUMEN
The N-formyl peptide receptor is a G protein-coupled transmembrane receptor involved in stimulating a variety of differential responses in neutrophils including chemotaxis, degranulation, superoxide production, transcriptional activation, and actin reorganization. Although it is known that N-formyl-Met-Leu-Phe induces actin reorganization, the sequence of events from the receptor to the actin cytoskeleton is not well characterized. To study the signaling pathway from the N-formyl peptide receptor to the actin cytoskeleton, we developed a model system utilizing microinjection techniques with a nonhematopoietic cell line. An expression vector coding for the N-formyl peptide receptor was microinjected into porcine aortic endothelial cells and stimulated with N-formyl-Met-Leu-Phe to induce actin reorganization and membrane ruffling. The receptor-mediated signal was blocked by pertussis toxin and by a dominant negative Rac-N17, indicating the involvement of G(i)alpha subunit and the small guanosine triphosphatase Rac, respectively. Moreover, Gbetagamma subunits and membrane targeted forms of phosphatidylinositol (PI) 3-kinase alpha were sufficient to induce similar actin reorganization, and coexpression of various mutants of PI 3-kinase with the N-formyl peptide receptor identified a link to class Ia PI-3 kinase-mediated actin reorganization.
Asunto(s)
Actinas/metabolismo , Subunidades beta de la Proteína de Unión al GTP , Subunidades gamma de la Proteína de Unión al GTP , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP Heterotriméricas , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de Péptidos/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Animales , ADN Complementario/administración & dosificación , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Microinyecciones , Toxina del Pertussis , Plásmidos , Receptores de Formil Péptido , Receptores Inmunológicos/genética , Receptores de Péptidos/genética , Porcinos , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The superoxide-forming NADPH oxidase of resting macrophages can be activated in a cell-free system by certain anionic amphiphiles, most notably SDS. Activation requires the cooperation of membrane-associated and cytosolic components. We now report that at least two cytosolic factors are required for SDS-elicited activation of NADPH oxidase of guinea pig macrophages. Treatment of cytosol with ammonium sulfate at 37% saturation led to the partition of the two factors in the supernatant and precipitate fractions (termed components sigma 1 and sigma 2, respectively). Although each fraction by itself was inactive, recombining them resulted in complete recovery of the original ability of native cytosol to support SDS-elicited superoxide production by octyl-glucoside solubilized macrophage membranes. Both components are proteins, as shown by their susceptibility to trypsin and proteinase K, and were inactivated by heating at 60 degrees C. sigma 2, but not sigma 1, was inactivated by treatment with the covalent sulfhydryl reagent N-ethylmaleimide. On high-performance gel filtration, sigma 1 was found to have a molecular mass of 30 to 52 kDa, whereas sigma 2 eluted with molecules of 150 to 440 kDa. Component sigma 1 was partially purified from the ammonium sulfate supernatant fraction of cytosol by hydrophobic interaction chromatography followed by gel filtration. A material behaving like sigma 1 was also found to be present in the cytosol of guinea pig thymus cells, lymph node lymphocytes and brain and of the mouse myeloma cell line MOPC 315. However, sigma 2 appears to be strictly phagocyte specific. The molecular characteristics of sigma 1 components from nonphagocytic cells were similar to those of macrophage sigma 1, as shown by their presence in the supernatant, after treatment of cytosol with ammonium sulfate at 37% saturation, a molecular mass close to 30 to 52 kDa and a similar behavior on hydrophobic interaction chromatography. These findings raise the possibility that cytosolic component sigma 1 might be the bearer of a cellular function, more general than the one suggested by its role in the activation of NADPH oxidase of phagocytes.
Asunto(s)
Citosol/enzimología , Macrófagos/enzimología , NADH NADPH Oxidorreductasas/metabolismo , Superóxidos/metabolismo , Sulfato de Amonio , Animales , Encéfalo/enzimología , Línea Celular , Sistema Libre de Células , Activación Enzimática , Etilmaleimida , Precipitación Fraccionada , Cobayas , Ganglios Linfáticos/enzimología , Linfocitos/enzimología , Peso Molecular , NADPH Oxidasas , Péptido Hidrolasas , TemperaturaRESUMEN
Wiskott-Aldrich syndrome protein (WASP) plays a key role in cytoskeletal rearrangement and transcriptional activation in T-cells. Recent evidence links WASP and related proteins to actin polymerization by the Arp2/3 complex. To study whether the role of WASP in actin polymerization is coupled to T-cell receptor (TCR)-mediated transcriptional activation, we made a series of WASP deletion mutants and tested them for actin co-localization, actin polymerization, and transcriptional activation of NFAT. A WASP mutant with a deletion in the C-terminal region (WASPDeltaC) that is defective in actin polymerization potentiated NFAT transcription following TCR activation by anti-CD3 and anti-CD3/CD28 antibodies, but not by phorbol 12-myristate 13-acetate/ionomycin. Furthermore, cotransfection of a dominant-active mutant (WASP-WH2-C) for Arp2/3 polymerization did not inhibit NFAT activation. Finally, by analyzing a series of WASP double-domain deletion mutants, we determined that the WASP homology-1 domain is responsible for NFAT transcriptional activation. Our results suggest that WASP activates transcription following TCR stimulation in a manner that is independent of its role in Arp2/3-directed actin polymerization.