scholarly journals N-Terminal Fragment of c-FLIP(L) Processed by Caspase 8 Specifically Interacts with TRAF2 and Induces Activation of the NF-κB Signaling Pathway

2004 ◽  
Vol 24 (7) ◽  
pp. 2627-2636 ◽  
Author(s):  
Takao Kataoka ◽  
Jürg Tschopp
Keyword(s):  
Immune System ◽  
Signaling Pathway ◽  
Death Receptor ◽  
Lymphocyte Activation ◽  
Dominant Negative ◽  
Caspase 8 ◽  
Caspase Inhibitor ◽  
Splice Form ◽  
Signaling Complex ◽  
Terminal Fragment

ABSTRACT Caspase 8 is required not only for death receptor-mediated apoptosis but also for lymphocyte activation in the immune system. FLIP(L), the long-splice form of c-FLIP, is one of the specific substrates for caspase 8, and increased expression of FLIP(L) promotes activation of the NF-κB signaling pathway. The synthetic caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) markedly blocked NF-κB activation induced by overexpression of FLIP(L). FLIP(L) is specifically processed by caspase 8 into N-terminal FLIP(p43) and C-terminal FLIP(p12). Only FLIP(p43) was able to induce NF-κB activation as efficiently as FLIP(L), and FLIP(p43)-induced NF-κB activation became insensitive to zVAD-fmk. In caspase 8-deficient cells, FLIP(p43) provoked NF-κB activation only when procaspase 8 or caspase 8(p43) was complemented. FLIP(p43)-induced NF-κB activation was profoundly blocked by the dominant-negative TRAF2. Moreover, endogenous TRAF2 interacted specifically with FLIP(p43), and the formation of the FLIP(p43)-caspase 8-TRAF2 tertiary complex was a prerequisite to induction of NF-κB activation. zVAD-fmk prevented the recruitment of TRAF2 into the death-inducing signaling complex. Thus, our present results demonstrate that FLIP(p43) processed by caspase 8 specifically interacts with TRAF2 and subsequently induces activation of the NF-κB signaling pathway.

scholarly journals Alpha/Beta Interferons Potentiate Virus-Induced Apoptosis through Activation of the FADD/Caspase-8 Death Signaling Pathway

2000 ◽  
Vol 74 (3) ◽  
pp. 1513-1523 ◽  
Author(s):  
Siddharth Balachandran ◽  
P. Christopher Roberts ◽  
Todd Kipperman ◽  
Kapil N. Bhalla ◽  
Richard W. Compans ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Infection ◽  
Sindbis Virus ◽  
Influenza Virus Infection ◽  
Dominant Negative ◽  
Caspase 8 ◽  
Signaling Complex ◽  
Dependent Protein ◽  
Induced Apoptosis ◽  
Dominant Negative Variant

ABSTRACT Interferon (IFN) mediates its antiviral effects by inducing a number of responsive genes, including the double-stranded RNA (dsRNA)-dependent protein kinase, PKR. Here we report that inducible overexpression of functional PKR in murine fibroblasts sensitized cells to apoptosis induced by influenza virus, while in contrast, cells expressing a dominant-negative variant of PKR were completely resistant. We determined that the mechanism of influenza virus-induced apoptosis involved death signaling through FADD/caspase-8 activation, while other viruses such as vesicular stomatitis virus (VSV) and Sindbis virus (SNV) did not significantly provoke PKR-mediated apoptosis but did induce cytolysis of fibroblasts via activation of caspase-9. Significantly, treatment with IFN-α/β greatly sensitized the fibroblasts to FADD-dependent apoptosis in response to dsRNA treatment or influenza virus infection but completely protected the cells against VSV and SNV replication in the absence of any cellular destruction. The mechanism by which IFN increases the cells' susceptibility to lysis by dsRNA or certain virus infection is by priming cells to FADD-dependent apoptosis, possibly by regulating the activity of the death-induced signaling complex (DISC). Conversely, IFN is also able to prevent the replication of viruses such as VSV that avoid triggering FADD-mediated DISC activity, by noncytopathic mechanisms, thus preventing destruction of the cell.

scholarly journals Model-based dissection of CD95 signaling dynamics reveals both a pro- and antiapoptotic role of c-FLIPL

2010 ◽  
Vol 190 (3) ◽  
pp. 377-389 ◽  
Author(s):  
Nicolai Fricker ◽  
Joel Beaudouin ◽  
Petra Richter ◽  
Roland Eils ◽  
Peter H. Krammer ◽  
...  
Keyword(s):  
Death Receptor ◽  
Caspase 8 ◽  
Converting Enzyme ◽  
Receptor Stimulation ◽  
Western Blots ◽  
Antiapoptotic Protein ◽  
Signaling Complex ◽  
Signaling Dynamics ◽  
Induced Apoptosis

Cellular FADD-like interleukin-1β–converting enzyme inhibitory proteins (c-FLIPs; isoforms c-FLIP long [c-FLIPL], c-FLIP short [c-FLIPS], and c-FLIP Raji [c-FLIPR]) regulate caspase-8 activation and death receptor (DR)–induced apoptosis. In this study, using a combination of mathematical modeling, imaging, and quantitative Western blots, we present a new mathematical model describing caspase-8 activation in quantitative terms, which highlights the influence of c-FLIP proteins on this process directly at the CD95 death-inducing signaling complex. We quantitatively define how the stoichiometry of c-FLIP proteins determines sensitivity toward CD95-induced apoptosis. We show that c-FLIPL has a proapoptotic role only upon moderate expression in combination with strong receptor stimulation or in the presence of high amounts of one of the short c-FLIP isoforms, c-FLIPS or c-FLIPR. Our findings resolve the present controversial discussion on the function of c-FLIPL as a pro- or antiapoptotic protein in DR-mediated apoptosis and are important for understanding the regulation of CD95-induced apoptosis, where subtle differences in c-FLIP concentrations determine life or death of the cells.

scholarly journals Rac-PAK Signaling Stimulates Extracellular Signal-Regulated Kinase (ERK) Activation by Regulating Formation of MEK1-ERK Complexes

2002 ◽  
Vol 22 (17) ◽  
pp. 6023-6033 ◽  
Author(s):  
Scott T. Eblen ◽  
Jill K. Slack ◽  
Michael J. Weber ◽  
Andrew D. Catling
Keyword(s):  
Protein Kinase ◽  
Signaling Pathway ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Erk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Signaling Complex ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT Utilizing mutants of extracellular signal-regulated kinase 2 (ERK2) that are defective for intrinsic mitogen-activated protein kinase or ERK kinase (MEK) binding, we have identified a convergent signaling pathway that facilitates regulated MEK-ERK association and ERK activation. ERK2-Δ19-25 mutants defective in MEK binding could be phosphorylated in response to mitogens; however, signaling from the Raf-MEK pathway alone was insufficient to stimulate their phosphorylation in COS-1 cells. Phosphorylation of ERK2-Δ19-25 but not of wild-type ERK2 in response to Ras V12 was greatly inhibited by dominant-negative Rac. Activated forms of Rac and Cdc42 could enhance the association of wild-type ERK2 with MEK1 but not with MEK2 in serum-starved adherent cells. This effect was p21-activated kinase (PAK) dependent and required the putative PAK phosphorylation sites T292 and S298 of MEK1. In detached cells placed in suspension, ERK2 was complexed with MEK2 but not with MEK1. However, upon replating of cells onto a fibronectin matrix, there was a substantial induction of MEK1-ERK2 association and ERK activation, both of which could be inhibited by dominant-negative PAK1. These data show that Rac facilitates the assembly of a mitogen-activated protein kinase signaling complex required for ERK activation and that this facilitative signaling pathway is active during adhesion to the extracellular matrix. These findings reveal a novel mechanism by which adhesion and growth factor signals are integrated during ERK activation.

scholarly journals Erythroid Differentiation Sensitizes K562 Leukemia Cells to TRAIL-Induced Apoptosis by Downregulation of c-FLIP

2003 ◽  
Vol 23 (4) ◽  
pp. 1278-1291 ◽  
Author(s):  
Ville Hietakangas ◽  
Minna Poukkula ◽  
Kaisa M. Heiskanen ◽  
Jarkko T. Karvinen ◽  
Lea Sistonen ◽  
...  
Keyword(s):  
Death Receptor ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Caspase 8 ◽  
Malignant Cells ◽  
Signaling Complex ◽  
Specific Effects ◽  
Organ Systems ◽  
Increased Sensitivity ◽  
Induced Apoptosis

ABSTRACT Regulation of the apoptotic threshold is of great importance in the homeostasis of both differentiating and fully developed organ systems. Triggering differentiation has been employed as a strategy to inhibit cell proliferation and accelerate apoptosis in malignant cells, in which the apoptotic threshold is often characteristically elevated. To better understand the mechanisms underlying differentiation-mediated regulation of apoptosis, we have studied death receptor responses during erythroid differentiation of K562 erythroleukemia cells, which normally are highly resistant to tumor necrosis factor (TNF) alpha-, FasL-, and TRAIL-induced apoptosis. However, upon hemin-mediated erythroid differentiation, K562 cells specifically lost their resistance to TNF-related apoptosis-inducing ligand (TRAIL), which efficiently killed the differentiating cells independently of mitochondrial apoptotic signaling. Concomitantly with the increased sensitivity, the expression of both c-FLIP splicing variants, c-FLIPL and c-FLIPS, was downregulated, resulting in an altered caspase 8 recruitment and cleavage in the death-inducing signaling complex (DISC). Stable overexpression of both c-FLIPL and c-FLIPS rescued the cells from TRAIL-mediated apoptosis with isoform-specific effects on DISC-recruited caspase 8. Our results show that c-FLIPL and c-FLIPS potently control TRAIL responses, both by distinct regulatory features, and further imply that the differentiation state of malignant cells determines their sensitivity to death receptor signals.

scholarly journals Death Receptor-Induced Signaling Pathways Are Differentially Regulated by Gamma Interferon Upstream of Caspase 8 Processing

2005 ◽  
Vol 25 (15) ◽  
pp. 6363-6379 ◽  
Author(s):  
Daniela Siegmund ◽  
Andreas Wicovsky ◽  
Ingo Schmitz ◽  
Klaus Schulze-Osthoff ◽  
Sebastian Kreuz ◽  
...  
Keyword(s):  
Cross Talk ◽  
Death Receptor ◽  
Gamma Interferon ◽  
Caspase 8 ◽  
Apoptosis Induction ◽  
Activated T Cells ◽  
Signaling Complex ◽  
Proinflammatory Responses ◽  
Ifn Γ ◽  
Induced Apoptosis

ABSTRACT FasL and gamma interferon (IFN-γ) are produced by activated T cells and NK cells and synergistically induce apoptosis. Although both cytokines can also elicit proinflammatory responses, a possible cross talk of these ligands with respect to nonapoptotic signaling has been poorly addressed. Here, we show that IFN-γ sensitizes KB cells for apoptosis induction by facilitating death-inducing signaling complex (DISC)-mediated caspase 8 processing. Moreover, after protection against death receptor-induced apoptosis by caspase inhibition or Bcl2 overexpression, IFN-γ also sensitized for Fas- and TRAIL death receptor-mediated NF-κB activation leading to synergistic upregulation of a variety of proinflammatory genes. In contrast, Fas-mediated activation of JNK, p38, and p42/44 occurred essentially independent from IFN-γ sensitization, indicating that the apoptosis- and NF-κB-related FasL-IFN-γ cross talk was not due to a simple global enhancement of Fas signaling. Overexpression of FLIPL and FLIPS inhibited Fas- as well as TRAIL-mediated NF-κB activation and apoptosis induction in IFN-γ-primed cells suggesting that both responses are coregulated at the level of the DISC.

scholarly journals Caspase Activation by Adenovirus E4orf4 Protein Is Cell Line Specific and Is Mediated by the Death Receptor Pathway

2001 ◽  
Vol 75 (2) ◽  
pp. 789-798 ◽  
Author(s):  
Adi Livne ◽  
Ronit Shtrichman ◽  
Tamar Kleinberger
Keyword(s):  
Death Receptor ◽  
Dominant Negative ◽  
Disulfonic Acid ◽  
Ros Generation ◽  
Caspase 8 ◽  
Dependent Manner ◽  
Caspase Activation ◽  
Apoptotic Pathway ◽  
Death Receptor Pathway ◽  
Induced Apoptosis

ABSTRACT Adenovirus E4orf4 protein has been shown to induce transformed cell-specific, protein phosphatase 2A-dependent, and p53-independent apoptosis. It has been further reported that the E4orf4 apoptotic pathway is caspase-independent in CHO cells. Here, we show that E4orf4 induces caspase activation in the human cell lines H1299 and 293T. Caspase activation is required for apoptosis in 293T cells, but not in H1299 cells. Dominant negative mutants of caspase-8 and the death receptor adapter protein FADD/MORT1 inhibit E4orf4-induced apoptosis in 293T cells, suggesting that E4orf4 activates the death receptor pathway. Cytochrome c is released into the cytosol in E4orf4-expressing cells, but caspase-9 is not required for induction of apoptosis. Furthermore, E4orf4 induces accumulation of reactive oxygen species (ROS) in a caspase-8- and FADD/MORT1-dependent manner, and inhibition of ROS generation by 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron) inhibits E4orf4-induced apoptosis. Thus, our results demonstrate that E4orf4 engages the death receptor pathway to generate at least part of the molecular events required for E4orf4-induced apoptosis.

scholarly journals Role of FLASH in caspase-8-mediated activation of NF-κB: dominant-negative function of FLASH mutant in NF-κB signaling pathway

Oncogene ◽  
2004 ◽  
Vol 24 (4) ◽  
pp. 688-696 ◽  
Author(s):  
Joon-Il Jun ◽  
Chul-Woong Chung ◽  
Ho-June Lee ◽  
Jong-Ok Pyo ◽  
Kee Nyung Lee ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Dominant Negative ◽  
Caspase 8 ◽  
Negative Function

scholarly journals Anticancer Drugs Induce Caspase-8/FLICE Activation and Apoptosis in the Absence of CD95 Receptor/Ligand Interaction

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 3053-3063 ◽  
Author(s):  
Sebastian Wesselborg ◽  
Ingo H. Engels ◽  
Evi Rossmann ◽  
Marek Los ◽  
Klaus Schulze-Osthoff
Keyword(s):  
Anticancer Drugs ◽  
De Novo ◽  
Death Receptor ◽  
Dominant Negative ◽  
Caspase 8 ◽  
Drug Induced ◽  
Ligand Interaction ◽  
Caspase Family ◽  
Death Receptor Signaling ◽  
Induced Apoptosis

Abstract Proteases of the caspase family are the critical executioners of apoptosis. Their activation has been mainly studied upon triggering of death receptors, such as CD95 (Fas/APO-1) and tumor necrosis factor-R1, which recruit caspase-8/FLICE as the most proximal effector to the receptor complex. Because apoptosis induced by anticancer drugs has been proposed to involve CD95/CD95 ligand interaction, we investigated the mechanism of caspase activation by daunorubicin, doxorubicin, etoposide, and mitomycin C. In Jurkat leukemic T cells, all drugs induced apoptosis and the cleavage of procaspase-8 to its active p18 subunit. However, cells resistant to CD95 were equally susceptible to anticancer drugs and activated caspase-8 with a similar kinetic and dose response as CD95-sensitive cells. The broad caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone prevented apoptosis and caspase-8 activation in response to CD95 and drug treatment, whereas a neutralizing CD95 decoy as well as a dominant-negative FADD construct selectively abrogated CD95, but not drug-induced effects. A potent activation of caspase-8 was also induced by cycloheximide, indicating that it was independent of protein synthesis. Our data, therefore, show that (1) anticancer drug-induced apoptosis does not require de novo synthesis of death ligands or CD95 interaction, and (2) that caspase-8 can be activated in the absence of a death receptor signaling.

scholarly journals Hypo-Expression of Flice-Inhibitory Protein and Activation of the Caspase-8 Apoptotic Pathways in the Death-Inducing Signaling Complex Due to Ischemia Induced by the Compression of the Asphyxiogenic Tool on the Skin in Hanging Cases

Diagnostics ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 938
Author(s):  
Aniello Maiese ◽  
Alessandra De Matteis ◽  
Giorgio Bolino ◽  
Emanuela Turillazzi ◽  
Paola Frati ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Active Form ◽  
Three Dimensional ◽  
Death Receptor ◽  
Cell Receptor ◽  
Control Group ◽  
Caspase 8 ◽  
Inhibitory Protein ◽  
Signaling Complex ◽  
Induced Apoptosis

The FLICE-inhibitory protein (c-FLIPL) (55 kDa) is expressed in numerous tissues and most abundantly in the kidney, skeletal muscles and heart. The c-FLIPL has a region of homology with caspase-8 at the carboxy-terminal end which allows the molecule to assume a tertiary structure similar to that of caspases-8 and -10. Consequently, c-FLIPL acts as a negative inhibitor of caspase-8, preventing the processing and subsequent release of the pro-apoptotic molecule active form. The c-FLIP plays as an inhibitor of apoptosis induced by a variety of agents, such as tumor necrosis factor (TNF), T cell receptor (TCR), TNF-related apoptosis inducing ligand (TRAIL), Fas and death receptor (DR). Increased expression of c-FLIP has been found in many human malignancies and shown to be involved in resistance to CD95/Fas and TRAIL receptor-induced apoptosis. We wanted to verify an investigative protocol using FLIP to make a differential diagnosis between skin sulcus with vitality or non-vital skin sulcus in hanged subjects and those undergoing simulated hanging (suspension of the victim after murder). The study group consisted of 21 cases who died from suicidal hanging. The control group consisted of traumatic or natural deaths, while a third group consisted of simulated hanging cases. The reactions to the Anti-FLIP Antibody (Abcam clone-8421) was scored for each section with a semi-quantitative method by means of microscopic observation carried out with confocal microscopy and three-dimensional reconstruction. The results obtained allow us to state that the skin reaction to the FLIP is extremely clear and precise, allowing a diagnosis of unequivocal vitality and a very objective differentiation with the post-mortal skin sulcus.

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