Peter H. Krammer

FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex

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.

Two CD95 (APO‐1/Fas) signaling pathways

We have identified two cell types, each using almost exclusively one of two different CD95 (APO‐1/Fas) signaling pathways. In type I cells, caspase‐8 was activated within seconds and caspase‐3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for ∼60 min. However, both type I and type II cells showed similar kinetics of CD95‐mediated apoptosis and loss of mitochondrial transmembrane potential (ΔΨm). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl‐2 or Bcl‐xL overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl‐2 or Bcl‐xL blocked caspase‐8 and caspase‐3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase‐8 by the death‐inducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase‐8 and caspase‐3 occurred following the loss of ΔΨm. Overexpression of caspase‐3 in the caspase‐3‐negative cell line MCF7‐Fas, normally resistant to CD95‐mediated apoptosis by overexpression of Bcl‐xL, converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl‐xL. In summary, in the presence of caspase‐3 the amount of active caspase‐8 generated at the DISC determines whether a mitochondria‐independent apoptosis pathway is used (type I cells) or not (type II cells).

Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor.

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.

CD95's deadly mission in the immune system

Apoptosis in the immune system is a fundamental process regulating lymphocyte maturation, receptor repertoire selection and homeostasis. Thus, death by apoptosis is as essential for the function of lymphocytes as growth and differentiation. This article focuses on death receptor-associated apoptosis and the role of CD95 (Apo-1/Fas)-mediated signalling in T-cell and B-cell development and during the course of an immune response. Gaining an insight into these processes improves our understanding of the pathogenesis of diseases such as cancer, autoimmunity and AIDS, and opens new approaches to rational treatment strategies.

DEATH AND ANTI-DEATH: TUMOUR RESISTANCE TO APOPTOSIS

Every cell in a multicellular organism has the potential to die by apoptosis, but tumour cells often have faulty apoptotic pathways. These defects not only increase tumour mass, but also render the tumour resistant to therapy. So, what are the molecular mechanisms of tumour resistance to apoptosis and how can we use this knowledge to resensitize tumour cells to cancer therapy?

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.

The Role of c-FLIP in Modulation of CD95-induced Apoptosis

Upon stimulation, CD95 (APO-1/Fas) recruits the adapter molecule Fas-associated death domain protein (FADD)/MORT1 and caspase-8 (FADD-like interleukin-1β-converting enzyme (FLICE)/MACH/MCH5) into the death-inducing signaling complex (DISC). Recently, a molecule with sequence homology to caspase-8 was identified, termed cellular FLICE-inhibitory protein (c-FLIP). c-FLIP has been controversially reported to possess apoptosis-promoting and -inhibiting functions. Using c-FLIP-specific monoclonal antibodies, we now show that c-FLIP is expressed in two isoforms, both of which, like FADD and caspase-8, are recruited to the CD95 DISC in a stimulation-dependent fashion. In stably transfected BJAB cells, c-FLIP blocks caspase-8 activation at the DISC and thereby inhibits CD95-mediated apoptosis. During this process, both caspase-8 and c-FLIP undergo cleavage between the p18 and p10 subunits, generating two stable intermediates of 43 kDa that stay bound to the DISC. c-FLIP has been suggested to play a role in protecting activated peripheral T cells from CD95-mediated apoptosis (Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., Bodmer, J. L., Schroter, M., Burns, K., Mattmann, C., Rimoldi, D., French, L. E., and Tschopp, J. (1997) Nature 388, 190–195). In contrast to this hypothesis, neither caspase-8 nor c-FLIP were cleaved in these cells, ruling out c-FLIP as the main factor regulating DISC activity. Moreover, recruitment of FADD, caspase-8, and c-FLIP to the DISC was strongly reduced in the apoptosis-resistant but readily detectable in the apoptosis-sensitive T cells.

FADD/MORT1 and Caspase-8 Are Recruited to TRAIL Receptors 1 and 2 and Are Essential for Apoptosis Mediated by TRAIL Receptor 2

Apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO-2L) has been shown to exert important functions during various immunological processes. The involvement of the death adaptor proteins FADD/MORT1, TRADD, and RIP and the apoptosis-initiating caspases-8 and -10 in death signaling by the two death-inducing TRAIL receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) are controversial. Analysis of the native TRAIL death-inducing signaling complex (DISC) revealed ligand-dependent recruitment of FADD/MORT1 and caspase-8. Differential precipitation of ligand-stimulated TRAIL receptors demonstrated that FADD/MORT1 and caspase-8 were recruited to TRAIL-R1 and TRAIL-R2 independently of each other. FADD/MORT1- and caspase-8-deficient Jurkat cells expressing only TRAIL-R2 were resistant to TRAIL-induced apoptosis. Thus, FADD/MORT1 and caspase-8 are essential for apoptosis induction via TRAIL-R2.

Drug-induced apoptosis in hepatoma cells is mediated by the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wild-type p53.

Chemotherapeutic drugs are cytotoxic by induction of apoptosis in drug-sensitive cells. We investigated the mechanism of bleomycin-induced cytotoxicity in hepatoma cells. At concentrations present in the sera of patients during therapy, bleomycin induced transient accumulation of nuclear wild-type (wt) p53 and upregulated expression of cell surface CD95 (APO-1/Fas) receptor in hepatoma cells carrying wt p53 (HepG2). Bleomycin did not increase CD95 in hepatoma cells with mutated p53 (Huh7) or in hepatoma cells which were p53-/- (Hep3B). In addition, sensitivity towards CD95-mediated apoptosis was also increased in wt p53 positive HepG2 cells. Microinjection of wt p53 cDNA into HepG2 cells had the same effect. In contrast, bleomycin did not enhance susceptibility towards CD95-mediated apoptosis in Huh7 and in Hep3B cells. Furthermore, bleomycin treatment of HepG2 cells increased CD95 ligand (CD95L) mRNA expression. Most notably, bleomycin-induced apoptosis in HepG2 cells was almost completely inhibited by antibodies which interfere with CD95 receptor/ligand interaction. These data suggest that apoptosis induced by bleomycin is mediated, at least in part, by p53-dependent stimulation of the CD95 receptor/ligand system. The same applies to other anti-cancer drugs such as cisplatin and methotrexate. These data may have major consequences for drug treatment of cancer and the explanation of drug sensitivity and resistance.

Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis.

Activation of the cell surface receptor Fas/APO‐1 (CD95) induces apoptosis in lymphocytes and regulates immune responses. The cytoplasmic membrane protein Bcl‐2 inhibits lymphocyte killing by diverse cytotoxic agents, but we found it provided little protection against Fas/APO‐1‐transduced apoptosis in B lymphoid cell lines, thymocytes and activated T cells. In contrast, the cowpox virus protease inhibitor CrmA blocked Fas/APO‐1‐transduced apoptosis, but did not affect cell death induced by gamma‐radiation or serum deprivation. Signalling through Fas/APO‐1 did not down‐regulate Bcl‐2 or induce its antagonists Bax and Bcl‐xS. In Fas/APO‐1‐deficient lpr mice, Bcl‐2 transgenes markedly augmented the survival of antigen‐activated T cells and the abnormal accumulation of lymphocytes (although they did not interfere with deletion of auto‐reactive cells in the thymus). These data raise the possibility that Bcl‐2 and Fas/APO‐1 regulate distinct pathways to lymphocyte apoptosis.