Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis

Abstract

Binding of Fas ligand or an agonistic anti-Fas antibody induces apoptosis in Fas-bearing cells. The interleukin-1Beta-converting enzyme (ICE) is a cysteine protease that is involved in apoptosis induced by various stimuli, including Fas-mediated apoptosis. Several ICE homologues have been identified, and these are subdivided into three groups (ICE-, CPP32-, and Ich-1-like proteases). We show here that specific inhibitors of ICE- or CPP32-like proteases can inhibit Fas-mediated apoptosis. Transient ICE-like activity was found in the cytosolic fraction of Fas-activated cells, whereas ICE-dependent, CPP32-like activity gradually accumulated in the cytosol. Cell lysates from mouse lymphoma supplemented with either recombinant ICE or CPP32 induced apoptosis of nuclei. The CPP32 inhibitor inhibited ICE- or CPP32-induced apoptosis in the cell-free system, whereas the ICE-inhibitor only inhibited ICE-induced apoptosis. Cell extracts from thymocytes from ICE-null mice induced apoptosis in the cell-free system when it was supplemented with CPP32. These results indicate that Fas sequentially activates ICE- and CPP32-like proteases, and that downstream CPP32, together with a component(s) in the cytoplasm, causes apoptosis of nuclei.

Evidence against a functional site for Bcl-2 downstream of caspase cascade in preventing apoptosis

“…Caspases have been suggested to constitute a protease cascade, based on observations that their active forms directly cleave the precursors of other family members in vitro to activate them (reviewed by Martin and Green, 1995). We and others have recently reported that a protease cascade involving caspase-1-like and caspase-3-like proteases operates to transduce apoptotic signals in vivo (Shimizu et al, 1996a;Enari et al, 1996).…”

Involvement of caspase-4(-like) protease in Fas-mediated apoptotic pathway

“…It has also been reported that thymocytes from caspase-1-de®cient mice are resistant to Fas-mediated apoptosis, indicating the direct involvement of caspase-1 itself in this process of thymocyte death (Kuida et al, 1995). Sequential activation of caspase-1(-like) and caspase-3(-like) proteases has been shown to occur in Fas-mediated apoptosis of mouse lymphoma cell line (Enari et al, 1996). Recently, a novel caspase family protease, caspase-8 (MACH/FLICE/Mch5),with two FADD-like domains has been described to be involved in transduction of death signals from Fas and TNF receptor directly to other caspase family proteases (Boldin et al, 1996;Muzio et al, 1996;FernandesAlnemri et al, 1996).…”

“…Ten members of caspase family proteases have recently been identi®ed , and among them caspase-1 (ICE)(-like), caspase-3 (CPP32/Yama/apopain)(-like) and caspase-2 (ICH-1) proteases are implicated in apoptosis (reviewed in Martin and Green, 1995). The caspase family proteases have been suggested to constitute a protease cascade (reviewed in Martin and Green, 1995;Enari et al, 1996;Shimizu et al, 1996).…”

JNK activation is not required for Fas-mediated apoptosis

“…The death domain recruits FADD upon Fas activation (Kischkel et al, 1995), this then recruits and activates caspase 8 (Muzio et al, 1996;Boldin et al, 1996). Other caspases, including 1 and 3, are subsequently activated (Enari et al, 1996) and caspase activation is necessary for Fas-mediated apoptosis (Tewari and Dixit, 1995).…”