RESUMEN
Caspase-8 is believed to play an obligatory role in apoptosis initiation by death receptors, but the role of its structural relative, caspase-10, remains controversial. Although earlier evidence implicated caspase-10 in apoptosis signaling by CD95L and Apo2L/TRAIL, recent studies indicated that these death receptor ligands recruit caspase-8 but not caspase-10 to their death-inducing signaling complex (DISC) even in presence of abundant caspase-10. We characterized a series of caspase-10-specific antibodies and found that certain commercially available antibodies cross-react with HSP60, shedding new light on previous results. The majority of 55 lung and breast carcinoma cell lines expressed mRNA for both caspase-8 and -10; however, immunoblot analysis revealed that caspase-10 protein expression was more frequently absent than that of caspase-8, suggesting a possible selective pressure against caspase-10 production in cancer cells. In nontransfected cells expressing both caspases, CD95L and Apo2L/TRAIL recruited endogenous caspase-10 as well as caspase-8 to their DISC, where both enzymes were proteolytically processed with similar kinetics. Caspase-10 recruitment required the adaptor FADD/Mort1, and caspase-10 cleavage in vitro required DISC assembly, consistent with the processing of an apoptosis initiator. Cells expressing only one of the caspases underwent ligand-induced apoptosis, indicating that each caspase can initiate apoptosis independently of the other. Thus, apoptosis signaling by death receptors involves not only caspase-8 but also caspase-10, and both caspases may have equally important roles in apoptosis initiation.
Asunto(s)
Apoptosis , Caspasas/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Western Blotting , Caspasa 10 , Caspasa 8 , Caspasa 9 , Caspasas/genética , Caspasas/inmunología , Electroforesis en Gel de Poliacrilamida , Humanos , Isoenzimas/inmunología , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Células Tumorales CultivadasRESUMEN
Metabolic labeling, immunoblotting and two-dimensional isoelectric focusing/SDS-PAGE are powerful techniques for characterizing endogenously expressed cellular proteins and their interactions. We achieved improved resolution and sensitivity for the detection of metabolically labeled proteins separated on two-dimensional gels by electroblotting the proteins onto polyvinylidene difluoride or nitrocellulose membranes and detecting the 35S signal on a bio-image analyzer. We obtained independent detection of specific proteins from the same blot by subsequent rehydration of the membrane and immunoblot analysis. The combination of these enhanced detection techniques with immunoprecipitation and two-dimensional electrophoresis on precast minigels provides a simple, sensitive method for detecting interactions between endogenous proteins in the cell.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Immunoblotting , Marcaje Isotópico , Proteínas/análisis , Proteínas/metabolismo , Animales , Anticuerpos/farmacología , Apoptosis , Autorradiografía , Proteínas Portadoras/análisis , Proteínas Portadoras/metabolismo , Línea Celular , Cisteína/metabolismo , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Proteína de Dominio de Muerte Asociada a Fas , Focalización Isoeléctrica , Metionina/metabolismo , Ratones , Fosforilación , Ratas , Transducción de Señal , Radioisótopos de Azufre , Receptor fas/análisis , Receptor fas/inmunología , Receptor fas/fisiologíaAsunto(s)
Apoptosis , Transducción de Señal , Receptor fas/fisiología , Línea Celular , Electroforesis en Gel Bidimensional/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Focalización Isoeléctrica/métodos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales Cultivadas , Receptor fas/química , Receptor fas/aislamiento & purificaciónRESUMEN
Fas (APO-1/CD95) and tumor necrosis factor receptor 1 (TNFR1) trigger apoptosis by recruiting the apoptosis initiator caspase-8 through the adaptor FADD. Fas binds FADD directly, whereas TNFR1 binds FADD indirectly, through TRADD. TRADD alternatively recruits the NF-kappaB-inducing adaptor RIP. The TNF homolog Apo2L/TRAIL triggers apoptosis through two distinct death receptors, DR4 and DR5; however, receptor over-expression studies have yielded conflicting results on the ligand's signaling mechanism. Apo2L/TRAIL induced homomeric and heteromeric complexes of DR4 and DR5 and stimulated recruitment of FADD and caspase-8 and caspase-8 activation in nontransfected cells. TRADD and RIP, which bound TNFR1, did not bind DR4 and DR5. Thus, Apo2L/TRAIL and FasL initiate apoptosis through similar mechanisms, and FADD may be a universal adaptor for death receptors.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis/inmunología , Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Glicoproteínas de Membrana/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Receptor fas/metabolismo , Proteínas Reguladoras de la Apoptosis , Caspasa 8 , Caspasa 9 , Espacio Extracelular/metabolismo , Proteína de Dominio de Muerte Asociada a Fas , Humanos , Ligandos , Linfoma de Células B/enzimología , Linfoma de Células B/inmunología , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Sustancias Macromoleculares , Glicoproteínas de Membrana/metabolismo , Modelos Inmunológicos , Proteínas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/inmunología , Factor 1 Asociado a Receptor de TNF , Ligando Inductor de Apoptosis Relacionado con TNF , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Fas ligand (FasL) is produced by activated T cells and natural killer cells and it induces apoptosis (programmed cell death) in target cells through the death receptor Fas/Apol/CD95. One important role of FasL and Fas is to mediate immune-cytotoxic killing of cells that are potentially harmful to the organism, such as virus-infected or tumour cells. Here we report the discovery of a soluble decoy receptor, termed decoy receptor 3 (DcR3), that binds to FasL and inhibits FasL-induced apoptosis. The DcR3 gene was amplified in about half of 35 primary lung and colon tumours studied, and DcR3 messenger RNA was expressed in malignant tissue. Thus, certain tumours may escape FasL-dependent immune-cytotoxic attack by expressing a decoy receptor that blocks FasL.
Asunto(s)
Neoplasias del Colon/genética , Neoplasias Pulmonares/genética , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Adulto , Secuencia de Aminoácidos , Apoptosis , Neoplasias del Colon/inmunología , Citotoxicidad Inmunológica , ADN Complementario , Etiquetas de Secuencia Expresada , Proteína Ligando Fas , Amplificación de Genes , Humanos , Células Jurkat , Células Asesinas Naturales/inmunología , Ligandos , Neoplasias Pulmonares/inmunología , Glicoproteínas de Membrana/antagonistas & inhibidores , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/aislamiento & purificación , Miembro 6b de Receptores del Factor de Necrosis Tumoral , Homología de Secuencia de Aminoácido , Distribución Tisular , Células Tumorales Cultivadas , Receptor fasRESUMEN
Phytohemagglutinin-activated peripheral CD95+ T cells (day 1 T cells) are resistant to CD95-mediated apoptosis. After prolonged interleukin-2 treatment, these T cells become CD95-mediated apoptosis-sensitive (day 6 T cells). To elucidate the molecular mechanism of apoptosis resistance, day 1 and day 6 T cells were tested for formation of the CD95 death-inducing signaling complex (DISC). DISC-associated active Fas-associated DD protein (FADD)-like interleukin-1 beta-converting enzyme-like protease (FLICE) also referred to as MACH/caspase 8 was only found in apoptosis-sensitive day 6 T cells. Further-analysis of mRNA and protein expression levels of apoptosis-signaling molecules FADD, receptor interacting protein, hematopoietic cell protein tyrosine phosphatase, Fas-associated phosphatase-1, FLICE, bel-2, bcl-xL, and, bax-alpha showed that only the expression level of bcl-xL correlated with T cell resistance to CD95-mediated apoptosis (day 1 T cells: bcl-xhiL; day 6 T cells: bcl-XloL). In T cells activated in vitro, up-regulation of bcl-xL, has previously been correlated with general apoptosis resistance. However, the experiments presented suggest that resistance to CD95-mediated apoptosis in T cells can also be regulated at the level of recruitment of FLICE to the DISC.
Asunto(s)
Apoptosis/inmunología , Caspasas , Cisteína Endopeptidasas/fisiología , Activación de Linfocitos , Transducción de Señal/inmunología , Linfocitos T/inmunología , Receptor fas/fisiología , Secuencia de Aminoácidos , Caspasa 8 , Caspasa 9 , Muerte Celular/inmunología , Células Cultivadas , Cisteína Endopeptidasas/metabolismo , Humanos , Leucemia de Células T , Datos de Secuencia Molecular , Linfocitos T/metabolismo , Células Tumorales Cultivadas , Receptor fas/biosíntesisRESUMEN
Upon activation, the apoptosis-inducing cell membrane receptor CD95 (APO-1/Fas) recruits a set of intracellular signaling proteins (CAP1-4) into a death-inducing signaling complex (DISC). In the DISC, CAP1 and CAP2 represent FADD/MORT1. CAP4 was identified recently as an ICE-like protease, FLICE, with two death effector domains (DED). Here we show that FLICE binds to FADD through its N-terminal DED. This is an obligatory step in CD95 signaling detected in the DISC of all CD95-sensitive cells tested. Upon prolonged triggering of CD95 with agonistic antibodies all cytosolic FLICE gets proteolytically activated. Physiological FLICE cleavage requires association with the DISC and occurs by a two-step mechanism. Initial cleavage generates a p43 and a p12 fragment further processed to a p10 fragment. Subsequent cleavage of the receptor-bound p43 results in formation of the prodomain p26 and the release of the active site-containing fragment p18. Activation of FLICE is blocked by the peptide inhibitors zVAD-fmk, zDEVD-fmk and zIETD-fmk, but not by crmA or Ac-YVAD-CHO. Taken together, our data indicate that FLICE is the first in a cascade of ICE-like proteases activated by CD95 and that this activation requires a functional CD95 DISC.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis , Proteínas Portadoras/metabolismo , Caspasas , Cisteína Endopeptidasas/metabolismo , Proteínas Virales , Receptor fas/metabolismo , Animales , Caspasa 8 , Caspasa 9 , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática , Proteína de Dominio de Muerte Asociada a Fas , Humanos , Isoenzimas , Ratones , Modelos Biológicos , Pruebas de Precipitina , Unión Proteica , Serpinas/farmacología , Transducción de SeñalRESUMEN
Triggering of CD95 (APO-1/Fas) on different T- and B-cell lines resulted in the induction of a number of kinases (35 kDa, 38 kDa, 46 kDa and 54 kDa) that phosphorylate c-Jun and to a lesser extent Histone H1. Activation of these kinases was independent of protein biosynthesis and preceded apoptotic DNA degradation. The kinase activation pattern was specific for CD95 triggering since a variety of physical or chemical inducers of T- and B-cell apoptosis activated different kinases. The kinase activities at 46 and 54 kDa contained members of the stress-activated family of protein kinases (JNK/SAPK). Activation of the CD95-specific set of kinases was prevented by treating cells with the ICE-inhibiting peptide N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) or by overexpression of the cow pox virus serpin CrmA. However, despite inhibition of ICE-like proteases the death signal was readily initiated at the cell membrane since a CD95 death-inducing signaling complex (DISC) was formed. Thus, our results demonstrate that ICE-like proteases in the CD95 pathway function downstream of the DISC but upstream of SAP kinases.
Asunto(s)
Apoptosis/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Cisteína Endopeptidasas/metabolismo , Receptor fas/farmacología , Animales , Caspasa 1 , Línea Celular , ADN/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Linfoma de Células B/enzimología , Linfoma de Células T/enzimología , Ratones , Transducción de Señal , Estaurosporina/farmacología , Receptor fas/metabolismoRESUMEN
To identify CAP3 and CAP4, components of the CD95 (Fas/APO-1) death-inducing signaling complex, we utilized nano-electrospray tandem mass spectrometry, a recently developed technique to sequence femtomole quantities of polyacrylamide gel-separated proteins. Interestingly, CAP4 encodes a novel 55 kDa protein, designated FLICE, which has homology to both FADD and the ICE/CED-3 family of cysteine proteases. FLICE binds to the death effector domain of FADD and upon overexpression induces apoptosis that is blocked by the ICE family inhibitors, CrmA and z-VAD-fmk. CAP3 was identified as the FLICE prodomain which likely remains bound to the receptor after proteolytic activation. Taken together, this is unique biochemical evidence to link a death receptor physically to the proapoptotic proteases of the ICE/CED-3 family.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis/fisiología , Proteínas Portadoras/genética , Caspasas , Cisteína Endopeptidasas/genética , Receptor fas/fisiología , Secuencia de Aminoácidos , Neoplasias de la Mama , Proteínas de Caenorhabditis elegans , Carcinoma , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Caspasa 8 , Caspasa 9 , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Proteína de Dominio de Muerte Asociada a Fas , Granzimas , Proteínas del Helminto/química , Proteínas del Helminto/genética , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos , Unión Proteica , ARN Mensajero/análisis , Análisis de Secuencia , Homología de Secuencia de Aminoácido , Serina Endopeptidasas , Transducción de Señal/fisiología , Células Tumorales CultivadasRESUMEN
The CD95 (APO-1/Fas) receptor has attracted great interest in recent years because it transduces an apoptotic signal in a variety of different tissues. CD95 belongs to the NGF/TNF-receptor superfamily, members of which need to be trimerized by specific protein ligands in order to generate a signal. This review focuses on recent advances in the understanding of the proximal signal transduction mechanism of CD95. The cloning of numerous proteins that interact with CD95 and other members of this receptor family and the in vivo identification of several proteins that associate with CD95 in a ligand-dependent fashion opens the way to delineate the death pathway and to explain crosstalk among these receptors on a molecular basis.
RESUMEN
CD95 (Fas/APO-1) and tumor necrosis factor receptor-1 (TNFR-1) are related molecules that signal apoptosis. Recently, a number of novel binding proteins have been proposed to mediate the signaling of these death receptors. Here we report that an N-terminal truncation of one of these candidate signal transducers, FADD/MORT1, abrogates CD95-induced apoptosis, ceramide generation, and activation of the cell death protease Yama/CPP32. In addition, this dominant-negative derivative of FADD (FADD-DN) blocked TNF-induced apoptosis while not affecting NF- kappaB activation. FADD-DN bound both receptors, and in the case of CD95, it disrupted the assembly of a signaling complex. Taken together, our results functionally establish FADD as the apoptotic trigger of CD95 and TNFR-1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis , Proteínas Portadoras/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Receptor fas/fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos , Neoplasias de la Mama , Muerte Celular , Línea Celular , Supervivencia Celular , Ceramidas/metabolismo , Proteína de Dominio de Muerte Asociada a Fas , Femenino , Humanos , Inmunoglobulina M/farmacología , Cinética , Linfoma de Células B , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Conejos/inmunología , Proteínas Recombinantes/metabolismo , Transducción de Señal , Transfección , Células Tumorales CultivadasRESUMEN
APO-1 (Fas/CD95), a member of the tumor necrosis factor receptor superfamily, induces apoptosis upon receptor oligomerization. In a search to identify intracellular signaling molecules coupling to oligomerized APO-1, several cytotoxicity-dependent APO-1-associated proteins (CAP) were immunoprecipitated from the apoptosis-sensitive human leukemic T cell line HUT78 and the lymphoblastoid B cell line SKW6.4. CAP1-3 (27-29 kDa) and CAP4 (55 kDa), instantly detectable after the crosslinking of APO-1, were associated only with aggregated (the signaling form of APO-1) and not with monomeric APO-1. CAP1 and CAP2 were identified as serine phosphorylated MORT1/FADD. The association of CAP1-4 with APO-1 was not observed with C-terminally truncated non-signaling APO-1. In addition, CAP1 and CAP2 did not associate with an APO-1 cytoplasmic tail carrying the lprcg amino acid replacement. Moreover, no APO-1-CAP association was found in the APO-1+, anti-APO-1-resistant pre-B cell line Boe. Our data suggest that in vivo CAP1-4 are the APO-1 apoptosis-transducing molecules.