Caspase-2 was reported to be involved in a number of apoptotic pathways triggered by various stimuli. However, the molecular mechanism of procaspase-2 activation in the course of apoptosis remains poorly defined. In this report, we demonstrate that procaspase-2 is recruited to the CD95 (Fas/APO-1) deathinducing signaling complex (DISC) in human T-and B-cell lines. We show that procaspase-2 is activated at the DISC on CD95 stimulation. Despite its presence at the DISC, caspase-2 does not initiate apoptosis on CD95 stimulation in caspase-8-deficient cell lines. Taken together, our data reveal that caspase-2 is activated at the DISC but does not play an initiating role in the CD95-induced apoptosis. Introduction CD95 (APO-1/Fas) is a member of the death receptor family, a subfamily of the TNF-R superfamily. [1][2][3][4][5] Crosslinking of CD95 with its natural ligand CD95L (FasL/CD178) 6 or with agonistic antibodies such as anti-APO-1 7 induces apoptosis in sensitive cells. The death-inducing signaling complex (DISC) is formed within seconds after CD95 stimulation. 8 The DISC consists of oligomerized, probably trimerized, CD95 receptors; the adaptor molecule FADD; 2 isoforms of procaspase-8 (procaspase-8/a and procaspase-8/b); procaspase-10; and c-FLIP L/S/R . 5,9 The interactions between molecules at the DISC are based on homotypic contacts. The death domain (DD) of the receptor interacts with the DD of FADD, whereas the death effector domain (DED) of FADD interacts with the N-terminal tandem DEDs of procaspase-8, -10, and c-FLIP L/S/R . The binding of procaspase-8 to the DISC results in processing of the zymogen. As a result the active caspase-8 heterotetramer p10 2 -p18 2 is released into the cytosol to propagate the apoptotic signal. 10 Two CD95 signaling pathways were established. 11 Type I cells are characterized by high levels of CD95 DISC formation and increased amounts of active caspase-8. Activated caspase-8 directly leads to the activation of downstream effector caspase-3 and -7. Type II cells are characterized by lower levels of CD95 DISC formation and, thus, lower levels of active caspase-8. 11 In this case, signaling requires an additional amplification loop that involves the cleavage by caspase-8 of the Bcl-2-family protein Bid to generate truncated (t) Bid and subsequent tBid-mediated release of Cytochrome C from mitochondria. The release of Cytochrome C from mitochondria results in apoptosome formation, followed by activation of procaspase-9, which in turn cleaves downstream, effector caspases. 12 Caspase-2 is referred to as an initiator caspase. [13][14][15] It is characterized by the presence of a caspase activation recruitment domain (CARD) and is structurally related to the initiator caspase-9. 16,17 The mechanism of procaspase-2 activation in apoptosis in contrast to other initiator caspases remains poorly defined. It was reported that caspase-2 is implicated in Cytochrome C release and is essential for drug-induced apoptosis in several human cell lines. [18][19][20] On the basis of these dat...

Stimulation of CD95 (APO-1/Fas) by its natural ligand CD95L (APO-1L/FasL) leads to the formation of the death-inducing signaling complex. Here we report that upon CD95 stimulation in several T and B cell lines, a novel signaling complex is formed, which we term complex II. Complex II is composed of the death effector domain proteins as follows: procaspase-8a/b, three isoforms of c-FLIP (c-FLIP L , c-FLIP S , c-FLIP R ), and FADD. Notably, complex II does not contain CD95. Based on our findings we suggest that CD95 signaling includes two steps. The first step involves formation of the death-inducing signaling complex at the cell membrane. The second step involves formation of the cytosolic death effector domain protein-containing complex that may play an important role in amplification of caspase activation.Apoptotic cell death is common in multicellular organisms. Apoptosis can be triggered by a number of factors, including UV-or ␥-irradiation, chemotherapeutic drugs, and signaling from death receptor (1). CD95 (APO-1/Fas) is a member of the death receptor family, a subfamily of the tumor necrosis factor receptor superfamily (1-3). Cross-linking of CD95 with its natural ligand CD95L (CD178) (4) or with agonistic antibodies such as anti-APO-1 (5) induces apoptosis in sensitive cells.The death-inducing signaling complex (DISC) 3 is formed within seconds after CD95 stimulation. The DISC consists of oligomerized, probably trimerized CD95, the adaptor molecule FADD, two isoforms of procaspase-8 (procaspase-8/a and procaspase-8/b), procaspase-10, and c-FLIP L/S/R (6 -10). The interactions between the molecules at the DISC are based on homotypic contacts. The death domain of the receptor interacts with the death domain of FADD, whereas the death effector domain (DED) of FADD interacts with the N-terminal tandem DEDs of procaspases-8, -10, and c-FLIP L/S/R . The binding of procaspase-8 to the DISC results in processing of the zymogene, for which a two-step mechanism has been described (11). The first cleavage step generates the two subunits p43/p41 and p12. In a second cleavage step, the active enzyme subunits p18, p10, and the prodomains p26/p24 are produced. As a result the active caspase-8 heterotetramer p10 2 -p18 2 is released into the cytosol to propagate the apoptotic signal.Two CD95 signaling pathways were established (12). Type I cells are characterized by high levels of CD95 DISC formation and increased amounts of active caspase-8. Activated caspase-8 directly leads to the activation of downstream effector caspases-3 and -7. Type II cells are characterized by lower levels of CD95 DISC formation and thus lower levels of active caspase-8. In this case, signaling requires an additional amplification loop that involves the cleavage by caspase-8 of the Bcl-2 family protein Bid to generate truncated Bid and subsequent truncated Bid-mediated release of cytochrome c from mitochondria (10). The release of cytochrome c from mitochondria results in apoptosome formation followed by activation of procaspase-9, which in tur...