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 (ΔΨ m ). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-x L overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-x L 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 deathinducing 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 ΔΨ m . Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-x L , converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl-x L . 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).
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.
Viruses have evolved many distinct strategies to avoid the host's apoptotic response. Here we describe a new family of viral inhibitors (v-FLIPs) which interfere with apoptosis signalled through death receptors and which are present in several gamma-herpesviruses (including Kaposi's-sarcoma-associated human herpesvirus-8), as well as in the tumorigenic human molluscipoxvirus. v-FLIPs contain two death-effector domains which interact with the adaptor protein FADD, and this inhibits the recruitment and activation of the protease FLICE by the CD95 death receptor. Cells expressing v-FLIPs are protected against apoptosis induced by CD95 or by the related death receptors TRAMP and TRAIL-R. The herpesvirus saimiri FLIP is detected late during the lytic viral replication cycle, at a time when host cells are partially protected from CD95-ligand-mediated apoptosis. Protection of virus-infected cells against death-receptor-induced apoptosis may lead to higher virus production and contribute to the persistence and oncogenicity of several FLIP-encoding viruses.
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