E1B 19,000-Molecular-Weight Protein Interacts with and Inhibits CED-4-Dependent, FLICE-Mediated Apoptosis

Genetic studies of the nematode Caenorhabditis elegans (C. elegans) have identified several important components of the cell death pathway, most notably CED-3, CED-4, and CED-9. CED-4 directly interacts with the Bcl-2 homologue CED-9 (or the mammalian Bcl-2 family member Bcl-x_L) and the caspase CED-3 (or the mammalian caspases ICE and FLICE). This trimolecular complex of CED-4, CED-3, and CED-9 is functional in that CED-9 inhibits CED-4 from activating CED-3 and thereby inhibits apoptosis in heterologous systems. The E1B 19,000-molecular weight protein (E1B 19K) is a potent apoptosis inhibitor and the adenovirus homologue of Bcl-2-related apoptosis inhibitors. Since E1B 19K and Bcl-x_L have functional similarity, we determined if E1B 19K interacts with CED-4 and regulates CED-4-dependent caspase activation. Binding analysis indicated that E1B 19K interacts with CED-4 in a Saccharomyces cerevisiae two-hybrid assay, in vitro, and in mammalian cell lysates. The subcellular localization pattern of CED-4 was dramatically changed by E1B 19K, supporting the theory of a functional interaction between CED-4 and E1B 19K. Whereas expression of CED-4 alone could not induce cell death, coexpression of CED-4 and FLICE augmented cell death induction by FLICE, which was blocked by expression of E1B 19K. Even though E1B 19K did not prevent FLICE-induced apoptosis, it did inhibit CED-4-dependent, FLICE-mediated apoptosis, which suggested that CED-4 was required for E1B 19K to block FLICE activation. Thus, E1B 19K functions through interacting with CED-4, and presumably a mammalian homologue of CED-4, to inhibit caspase activation and apoptosis.     

Crystal structure of MC159 reveals molecular mechanism of DISC assembly and FLIP inhibition

The death-inducing signaling complex (DISC) comprising Fas, Fas-associated death domain (FADD), and caspase-8/10 is assembled via homotypic associations between death domains (DDs) of Fas and FADD and between death effector domains (DEDs) of FADD and caspase-8/10. Caspase-8/10 and FLICE/caspase-8 inhibitory proteins (FLIPs) that inhibit caspase activation at the DISC level contain tandem DEDs. Here, we report the crystal structure of a viral FLIP, MC159, at 1.2 Angstroms resolution. It reveals a noncanonical fold of DED1, a dumbbell-shaped structure with rigidly associated DEDs and a different mode of interaction in the DD superfamily. Whereas the conserved hydrophobic patch of DED1 interacts with DED2, the corresponding region of DED2 mediates caspase-8 recruitment and contributes to DISC assembly. In contrast, MC159 cooperatively assembles with Fas and FADD via an extensive surface that encompasses the conserved charge triad. This interaction apparently competes with FADD self-association and disrupts higher-order oligomerization required for caspase activation in the DISC.     

Regulation of the Fas death pathway by FLICE-inhibitory protein in primary human B cells

The Fas/Fas ligand (L) system plays an important role in the maintenance of peripheral B cell tolerance and the prevention of misguided T cell help. CD40-derived signals are required to induce Fas expression on virgin B cells and to promote their susceptibility to Fas-mediated apoptosis. In the current study, we have analyzed the early biochemical events occurring upon Fas ligation in CD40L-activated primary human tonsillar B cells with respect to Fas-associated death domain protein (FADD), caspase-8/FADD-like IL-1beta-converting enzyme (FLICE), and c-FLICE inhibitory protein (FLIP). We report here that Fas-induced apoptosis in B cells does not require integrity of the mitochondrial Apaf-1 pathway and that caspase-8 is activated by association with the death-inducing signaling complex (DISC), i.e., upstream of the mitochondria. We show that both FADD and the zymogen form of caspase-8 are constitutively expressed at high levels in virgin B cells, whereas c-FLIP expression is marginal. In contrast, c-FLIP, but neither FADD nor procaspase-8, is strongly up-regulated upon ligation of CD40 or the B cell receptor on virgin B cells. Finally, we have found that c-FLIP is also recruited and cleaved at the level of the DISC in CD40L-activated virgin B cells. We propose that c-FLIP expression delays the onset of apoptosis in Fas-sensitive B cells. The transient protection afforded by c-FLIP could offer an ultimate safeguard mechanism against inappropriate cell death or allow recruitment of phagocytes to ensure efficient removal of apoptotic cells.     

Fas receptor clustering and involvement of the death receptor pathway in rituximab-mediated apoptosis with concomitant sensitization of lymphoma B cells to fas-induced apoptosis

Ab binding to CD20 has been shown to induce apoptosis in B cells. In this study, we demonstrate that rituximab sensitizes lymphoma B cells to Fas-induced apoptosis in a caspase-8-dependent manner. To elucidate the mechanism by which Rituximab affects Fas-mediated cell death, we investigated rituximab-induced signaling and apoptosis pathways. Rituximab-induced apoptosis involved the death receptor pathway and proceeded in a caspase-8-dependent manner. Ectopic overexpression of FLIP (the physiological inhibitor of the death receptor pathway) or application of zIETD-fmk (specific inhibitor of caspase-8, the initiator-caspase of the death receptor pathway) both specifically reduced rituximab-induced apoptosis in Ramos B cells. Blocking the death receptor ligands Fas ligand or TRAIL, using neutralizing Abs, did not inhibit apoptosis, implying that a direct death receptor/ligand interaction is not involved in CD20-mediated cell death. Instead, we hypothesized that rituximab-induced apoptosis involves membrane clustering of Fas molecules that leads to formation of the death-inducing signaling complex (DISC) and downstream activation of the death receptor pathway. Indeed, Fas coimmune precipitation experiments showed that, upon CD20-cross-linking, Fas-associated death domain protein (FADD) and caspase-8 were recruited into the DISC. Additionally, rituximab induced CD20 and Fas translocation to raft-like domains on the cell surface. Further analysis revealed that, upon stimulation with rituximab, Fas, caspase-8, and FADD were found in sucrose-gradient raft fractions together with CD20. In conclusion, in this study, we present evidence for the involvement of the death receptor pathway in rituximab-induced apoptosis of Ramos B cells with concomitant sensitization of these cells to Fas-mediated apoptosis via Fas multimerization and recruitment of caspase-8 and FADD to the DISC.     

FLICE is activated by association with the CD95 death-inducing signaling complex (DISC).

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.     

Structural and Biophysical Characterization of the Interactions between the Death Domain of Fas Receptor and Calmodulin

The extrinsic apoptotic pathway is initiated by cell surface death receptors such as Fas. Engagement of Fas by Fas ligand triggers a conformational change that allows Fas to interact with adaptor protein Fas-associated death domain (FADD) via the death domain, which recruits downstream signaling proteins to form the death-inducing signaling complex (DISC). Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells, suggesting a novel role of CaM in Fas-mediated signaling. CaM antagonists induce apoptosis through a Fas-related mechanism in cholangiocarcinoma and other cancer cell lines possibly by inhibiting Fas-CaM interactions. The structural determinants of Fas-CaM interaction and the underlying molecular mechanisms of inhibition, however, are unknown. Here we employed NMR and biophysical techniques to elucidate these mechanisms. Our data show that CaM binds to the death domain of Fas (FasDD) with an apparent dissociation constant (K_d) of ∼2 μm and 2:1 CaM:FasDD stoichiometry. The interactions between FasDD and CaM are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. We also show that both the N- and C-terminal lobes of CaM are important for binding. NMR and surface plasmon resonance data show that three CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, tamoxifen, and trifluoperazine) greatly inhibit Fas-CaM interactions by blocking the Fas-binding site on CaM. Our findings provide the first structural evidence for Fas-CaM interactions and mechanism of inhibition and provide new insight into the molecular basis for a novel role of CaM in regulating Fas-mediated apoptosis.     

Neuroprotectin D1 Induces Dephosphorylation of Bcl-xL in a PP2A-dependent Manner during Oxidative Stress and Promotes Retinal Pigment Epithelial Cell Survival

Retinal pigment epithelial (RPE) cell integrity is critical for the survival of photoreceptor cells. Bcl-x_L is a major anti-apoptotic Bcl-2 protein required for RPE cell survival, and phosphorylation of Bcl-x_L at residue Ser-62 renders this protein pro-apoptotic. In this study, we identify serine/threonine protein phosphatase 2A (PP2A) as a key regulator of Bcl-x_L phosphorylation at residue Ser-62 in ARPE-19 cells, a spontaneously arising RPE cell line in which Bcl-x_L is highly expressed. We found that either PP2A inhibitor okadaic acid or depletion of catalytic subunit α of PP2A (PP2A/Cα) by small interfering RNA enhanced Bcl-x_L phosphorylation when activated with hydrogen peroxide and tumor necrosis factor α-induced oxidative stress. Disruption of PP2A/Cα exacerbated oxidative stress-induced apoptosis. PP2A/Cα colocalized and interacted with S62Bcl-x_L in cells stressed with H₂O₂/tumor necrosis factor α. By contrast, the omega-3 fatty acid docosahexaenoic acid derivative, neuroprotectin D1 (NPD1), a potent activator of survival signaling, down-regulated oxidative stress-induced phosphorylation of Bcl-x_L by increasing protein phosphatase activity. NPD1 also attenuated the oxidative stress-induced apoptosis by knockdown of PP2A/Cα and increased the association of PP2A/Cα with S62Bcl-x_L as well as total Bcl-x_L. NPD1 also enhanced the heterodimerization of Bcl-x_L with its counterpart, pro-apoptotic protein Bax. Thus, NPD1 modulates the activation of this Bcl-2 family protein by dephosphorylating in a PP2A-dependent manner, suggesting a coordinated, NPD1-mediated regulation of cell survival in response to oxidative stress.     

Endogenous Bak inhibitors Mcl-1 and Bcl-xL: differential impact on TRAIL resistance in Bax-deficient carcinoma

Tumor necrosis factor (α)–related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that preferentially kills tumor cells with limited cytotoxicity to nonmalignant cells. However, signaling from death receptors requires amplification via the mitochondrial apoptosis pathway (type II) in the majority of tumor cells. Thus, TRAIL-induced cell death entirely depends on the proapoptotic Bcl-2 family member Bax, which is often lost as a result of epigenetic inactivation or mutations. Consequently, Bax deficiency confers resistance against TRAIL-induced apoptosis. Despite expression of Bak, Bax-deficient cells are resistant to TRAIL-induced apoptosis. In this study, we show that the Bax dependency of TRAIL-induced apoptosis is determined by Mcl-1 but not Bcl-x_L. Both are antiapoptotic Bcl-2 family proteins that keep Bak in check. Nevertheless, knockdown of Mcl-1 but not Bcl-x_L overcame resistance to TRAIL, CD95/FasL and tumor necrosis factor (α) death receptor ligation in Bax-deficient cells, and enabled TRAIL to activate Bak, indicating that Mcl-1 rather than Bcl-x_L is a major target for sensitization of Bax-deficient tumors for death receptor–induced apoptosis via the Bak pathway.     

Structural insight for the roles of fas death domain binding to fadd and oligomerization degree of the fas–fadd complex in the death‐inducing signaling complex formation: A computational study

Fas binding to Fas‐associated death domain (FADD) activates FADD–caspase‐8 binding to form death‐inducing signaling complex (DISC) that triggers apoptosis. The Fas–Fas association exists primarily as dimer in the Fas–FADD complex, and the Fas–FADD tetramer complexes have the tendency to form higher order oligomer. The importance of the oligomerized Fas–FADD complex in DISC formation has been confirmed. This study sought to provide structural insight for the roles of Fas death domain (Fas DD) binding to FADD and the oligomerization of Fas DD–FADD complex in activating FADD–procaspase‐8 binding. Results show Fas DD binding to FADD stabilized the FADD conformation, including the increased stability of the critical residues in FADD death effector domain (FADD DED) for FADD–procaspase‐8 binding. Fas DD binding to FADD resulted in the decreased degree of both correlated and anticorrelated motion of the residues in FADD and caused the reversed correlated motion between FADD DED and FADD death domain (FADD DD). The exposure of procaspase‐8 binding residues in FADD that allows FADD to interact with procaspase‐8 was observed with Fas DD binding to FADD. We also observed different degrees of conformational and motion changes of FADD in the Fas DD–FADD complex with different degrees of oligomerization. The increased conformational stability and the decreased degree of correlated motion of the residues in FADD in Fas DD–FADD tetramer complex were observed compared to those in Fas DD–FADD dimer complex. This study provides structural evidence for the roles of Fas DD binding to FADD and the oligomerization degree of Fas DD–FADD complex in DISC formation to signal apoptosis. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Identification of the Calmodulin-Binding Domains of Fas Death Receptor

The extrinsic apoptotic pathway is initiated by binding of a Fas ligand to the ectodomain of the surface death receptor Fas protein. Subsequently, the intracellular death domain of Fas (FasDD) and that of the Fas-associated protein (FADD) interact to form the core of the death-inducing signaling complex (DISC), a crucial step for activation of caspases that induce cell death. Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells and specifically interacts with FasDD to regulate the apoptotic/survival signaling pathway. Inhibition of CaM activity in DISC stimulates apoptosis significantly. We have recently shown that CaM forms a ternary complex with FasDD (2:1 CaM:FasDD). However, the molecular mechanism by which CaM binds to two distinct FasDD motifs is not fully understood. Here, we employed mass spectrometry, nuclear magnetic resonance (NMR), biophysical, and biochemical methods to identify the binding regions of FasDD and provide a molecular basis for the role of CaM in Fas–mediated apoptosis. Proteolytic digestion and mass spectrometry data revealed that peptides spanning residues 209–239 (Fas-Pep1) and 251–288 (Fas-Pep2) constitute the two CaM-binding regions of FasDD. To determine the molecular mechanism of interaction, we have characterized the binding of recombinant/synthetic Fas-Pep1 and Fas-Pep2 peptides with CaM. Our data show that both peptides engage the N- and C-terminal lobes of CaM simultaneously. Binding of Fas-Pep1 to CaM is entropically driven while that of Fas-Pep2 to CaM is enthalpically driven, indicating that a combination of electrostatic and hydrophobic forces contribute to the stabilization of the FasDD–CaM complex. Our data suggest that because Fas-Pep1 and Fas-Pep2 are involved in extensive intermolecular contacts with the death domain of FADD, binding of CaM to these regions may hinder its ability to bind to FADD, thus greatly inhibiting the initiation of apoptotic signaling pathway.     

A docking model of key components of the DISC complex: death domain superfamily interactions redefined

Apoptosis is mediated by a highly regulated signal transduction cascade that eventually leads to precisely directed cell death. The death-inducing signaling complex (DISC), composed of Fas, FADD, and caspase-8, is an apical signaling complex that mediates receptor-induced apoptosis. We have docked the experimentally determined structures of the Fas and FADD death domains into a model of a partial DISC signaling complex. The arrangement of Fas and FADD was determined using the interaction modes of the two heterodimer crystal structures determined to date, Pelle/Tube and Apaf-1/procaspase-9. The proposed model reveals that both interactions can be accommodated in a single multimeric complex. Importantly, the model is consistent with reported site-directed mutagenesis data indicating residues throughout the domain are critical for function. These results imply that members of the death domain superfamily have the potential for multivalent interactions, offering novel possibilities for regulation of apoptotic signaling.     

Calcium/calmodulin-dependent protein kinase II regulation of c-FLIP expression and phosphorylation in modulation of Fas-mediated signaling in malignant glioma cells

Fas, upon cross-linking with Fas ligand (FasL) or Fas agonistic antibody, transduces apoptotic yet also proliferative signals, which have been implicated in tumor pathogenesis. In this study, we investigated the molecular mechanisms that control Fas-mediated signaling in glioma cells. Fas agonistic antibody, CH-11, induced apoptosis in sensitive glioma cells through caspase-8 recruitment to the Fas-mediated death-inducing signaling complex (DISC) where caspase-8 was cleaved to initiate apoptosis through a systematic cleavage of downstream substrates. In contrast, CH-11 stimulated cell growth in resistant glioma cells through recruitment of c-FLIP (cellular Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme (FLICE)-inhibitory protein) to the Fas-mediated DISC. Three isoforms of long form c-FLIP were detected in glioma cells, but only the phosphorylated isoform was recruited to and cleaved into a p43 intermediate form in the Fas-mediated DISC in resistant cells. Calcium/calmodulin-dependent protein kinase II (CaMK II) activity was up-regulated in resistant cells. Treatment of resistant cells with the CaMK II inhibitor KN-93 inhibited CaMK II activity, reduced c-FLIP expression, inhibited c-FLIP phosphorylation, and rescued CH-11 sensitivity. Transfection of CaMK II cDNA in sensitive cells rendered them resistant to CH-11. These results indicated that CaMK II regulates c-FLIP expression and phosphorylation, thus modulating Fas-mediated signaling in glioma cells.     

TCR-mediated up-regulation of c-FLIPshort correlates with resistance toward CD95-mediated apoptosis by blocking death-inducing signaling complex activity

To investigate apoptosis resistance upon restimulation in human peripheral blood T lymphocytes, we used the following in vitro model. This model represents the main features of T cell reactivity: freshly isolated PHA-activated T cells cultured in IL-2 for a prolonged period of time develop a CD95 (APO-1/Fas) apoptosis-sensitive phenotype. These T cells represent activation-induced cell death-sensitive T cells during the down phase of an immune response. A fraction of apoptosis-sensitive activated T cells becomes apoptosis resistant upon TCR/CD3 restimulation. CD95 apoptosis sensitivity requires formation of a functional receptor associated death-inducing signaling complex (DISC), i.e., a protein complex of CD95 receptors, the adaptor Fas-associated death domain protein (FADD)/MORT1 and caspase-8 (FADD-like IL-1ss-converting enzyme (FLICE), MACH, Mch5). We identified activation of procaspase-8 at the DISC as the main target for the protective activity of TCR/CD3 restimulation. We found that procaspase-8 cleavage is reduced in T cells after TCR/CD3 restimulation. In addition, we detected up-regulation of c-FLIP(S) (the short splice variant of the cellular FLICE inhibitory protein) and strongly enhanced recruitment of c-FLIP(S) into the DISC. These data suggest that the recruitment of c-FLIP(S) into the DISC results in reduced DISC and caspase-8 activity.     

Protein kinase C regulates FADD recruitment and death-inducing signaling complex formation in Fas/CD95-induced apoptosis

Activation of protein kinase C (PKC) triggers cellular signals that inhibit Fas/CD95-induced cell death in Jurkat T-cells by poorly defined mechanisms. Previously, we have shown that one effect of PKC on Fas/CD95-dependent cell death occurs through inhibition of cell shrinkage and K(+) efflux (Gómez-Angelats, M., Bortner, C. D., and Cidlowski, J. A. (2000) J. Biol. Chem. 275, 19609-19619). Here we report that PKC alters Fas/CD95 signaling from the plasma membrane to the activation of caspases by exerting a profound action on survival/cell death decisions. Specific activation of PKC with 12-O-tetradecanoylphorbol-13-acetate or bryostatin-1 induced translocation of PKC from the cytosol to the membrane and effectively inhibited cell shrinkage and cell death triggered by anti-Fas antibody in Jurkat cells. In contrast, inhibition of classical PKC isotypes with G?6976 exacerbated the effect of Fas activation on both apoptotic volume decrease and cell death. PKC activation/inhibition did not affect anti-Fas antibody binding to the cell surface, intracellular levels of FADD (Fas-associated protein with death domain), or c-FLIP (cellular FLICE-like inhibitory protein) expression. However, processing/activation of both caspase-8 and caspase-3 and BID cleavage were markedly blocked upon PKC activation and, conversely, were augmented during PKC inhibition, suggesting a role for PKC upstream of caspase-8 processing and activation. Analysis of death-inducing signaling complex (DISC) formation was carried out to examine the influence of PKC on recruitment of both FADD and procaspase-8 to the Fas receptor. PKC activation blocked FADD recruitment and caspase-8 activation and thus DISC formation in both type I and II cells. In contrast, inhibition of classical PKCs promoted the opposite effect on the Fas pathway by rapidly increasing FADD recruitment, caspase-8 activation, and DISC formation. Together, these data show that PKC finely modulates Fas/CD95 signaling by altering the efficiency of DISC formation.     

Homotypic FADD interactions through a conserved RXDLL motif are required for death receptor-induced apoptosis

Death receptors in the TNF receptor superfamily signal for apoptosis via the ordered recruitment of FADD and caspase-8 to a death-inducing signaling complex (DISC). However, the nature of the protein-protein interactions in the signaling complex is not well defined. Here we show that FADD self-associates through a conserved RXDLL motif in the death effector domain (DED). Despite exhibiting similar binding to both Fas and caspase-8 and preserved overall secondary structure, FADD RDXLL motif mutants cannot reconstitute FasL- or TRAIL-induced apoptosis and fail to recruit caspase-8 into the DISC of reconstituted FADD-deficient cells. Abolishing self-association can transform FADD into a dominant-negative mutant that interferes with Fas-induced apoptosis and formation of microscopically visible receptor oligomers. These findings suggest that lateral interactions among adapter molecules are required for death receptor apoptosis signaling and implicate self-association into oligomeric assemblies as a key function of death receptor adapter proteins in initiating apoptosis.     

Avicin D,a Plant Triterpenoid,Induces Cell Apoptosis by Recruitment of Fas and Downstream Signaling Molecules into Lipid Rafts

Avicins, a family of triterpene electrophiles originally identified as potent inhibitors of tumor cell growth, have been shown to be pleiotropic compounds that also possess antioxidant, anti-mutagenic, and anti-inflammatory activities. We previously showed that Jurkat cells, which express a high level of Fas, are very sensitive to treatment with avicins. Thus, we hypothesized that avicins may induce cell apoptosis by activation of the Fas pathway. By using a series of cell lines deficient in cell death receptors, we demonstrated that upon avicin D treatment, Fas translocates to the cholesterol- and sphingolipid-enriched membrane microdomains known as lipid rafts. In the lipid rafts, Fas interacts with Fas-associated death domain (FADD) and Caspase-8 to form death-inducing signaling complex (DISC) and thus mediates cell apoptosis. Interfering with lipid raft organization by using a cholesterol-depleting compound, methyl-β-cyclodextrin, not only prevents the clustering of Fas and its DISC complex but also reduces the sensitivity of the cells to avicin D. Avicin D activates Fas pathways independent of the association between extracellular Fas ligands and Fas receptors. A deficiency in Fas and its downstream signaling molecules leads to the resistance of the cells to avicin D treatment. Taken together, our results demonstrate that avicin D triggers the redistribution of Fas in the membrane lipid rafts, where Fas activates receptor-mediated cell death.     

Expression of genes that regulate Fas signalling and Fas-mediated apoptosis in colon carcinoma cells

The expression of genes that regulate Fas-induced apoptosis has been examined in 10 human cultured colon carcinoma cell lines with defined and varied sensitivity to the cytolytic anti-Fas MoAb CH-11. Four lines demonstrated sensitivity to CH-11 (HT29, GC3/c1, TS-, Thy4), and six were resistant to the induction of apoptosis vis Fas. In nine lines expressing Fas, PCR-sequencing indicated that the death domain contained wt sequences. Downstream of Fas, expression of FADD/MORT1 and FLICE, essential components of the DISC, and negative regulators of Fas signalling including sFas, FAP-1 and Bcl-2, showed no correlation between levels of expression and sensitivity to Fas-mediated cytotoxicity. However, levels of the Fas antigen varied by >1000-fold, and correlated with CH-11 sensitivity. Following fourfold elevation in Fas expression in HT29 cells treated with interferon-gamma, a synergistic effect on Fas-mediated apoptosis was obtained when CH-11 and interferon-gamma were combined.     

In vitro activation of procaspase-8 by forming the cytoplasmic component of the death-inducing signaling complex (cDISC)

Procaspase-8 is activated by forming a death-inducing signaling complex (DISC) with the Fas-associated death domain (FADD) and the Fas receptor, but the mechanism of its activation is not well understood. Procaspase-8 devoid of the death effector domain at its N-terminus (delta nprocaspase-8) was reported to be activated by kosmotropic salts, but it has not been induced to form a DISC in vitro because it cannot interact with FADD. Here, we report the production of full-length procaspase-8 and show that it is activated by adding the Fas death domain (Fas-DD) and the FADD forming the cytoplasmic part of the DISC (cDISC). Furthermore, mutations known to affect DISC formation in vivo were shown to have the same effect on procaspase-8 activation in vitro. An antibody that induces Fas-DD association enhanced procaspase-8 activation, suggesting that the Fas ligand is not required for low-level activation of procaspase-8, but that Fas receptor clustering is needed for high-level activation of procaspase-8 leading to cell death. In vitro activation of procaspase-8 by forming a cDISC will be invaluable for investigating activation of ligand-mediated apoptosis and the numerous interactions affecting procaspase-8 activation.     

Regulation of Fas-associated death domain interactions by the death effector domain identified by a modified reverse two-hybrid screen

The adapter protein FADD consists of two protein interaction domains and is an essential component of the death inducing signaling complex (DISC) that is formed by activated death receptors of the tumor necrosis factor (TNF) receptor family. The FADD death domain binds to activated receptors such as Fas or other adapters such as TRADD, whereas the FADD death effector domain binds to procaspase 8. Each domain can interact with its target in the absence of the other domain, and this has led to the idea that the two domains function independently. FADD death domain interactions with Fas and TRADD are thought to occur on the same surface; however, the regulation of these interactions is poorly understood. We developed a modified reverse two-hybrid method that can identify mutations, which inhibit some protein-protein interactions without affecting other interactions. Using this method, we identified mutations in FADD that prevent binding to Fas but do not affect binding to TRADD. Surprisingly, these mutations were in the death effector domain rather than the death domain. To test whether the mutants function in mammalian cells, we expressed wild type or mutant FADD molecules in FADD-deficient cells. Wild type FADD rescued both Fas ligand- and TNF-dependent signaling, whereas the FADD death effector domain mutants rescued only TNF signaling. These data indicate that in contrast to current models, the death effector domain of FADD is involved in interaction with Fas.