CD95 (also called APO-1; Fas; fas antigen; tumor necrosis factor receptor superfamily member 6, TNFRSF6) is a member of the DR family, a subfamily of the tumor necrosis factor receptor superfamily.1
All members of the DR family are characterized by a cytoplasmic region termed death domain (DD).2, 3
DD are 80–100 amino-acid long motifs involved in the transduction of the apoptotic signal. Crosslinking of CD95 with its natural ligand (L), CD95L (CD178)4
or with agonistic antibodies such as anti-APO-15
induces apoptosis in sensitive cells.
Stimulation of CD95 has been also reported to trigger non-apoptotic pathways.6, 7, 8, 9, 10, 11, 12
However, details of CD95-mediated non-apoptotic pathways remain largely unknown. Importantly, it has been shown that membrane-bound CD95L is essential for the cytotoxic activity, whereas soluble CD95L appears to promote autoimmunity and tumorigenesis via induction of non-apoptotic pathways, in particular NF-κ
Future studies should elucidate more details on the mechanism of non-apoptotic action of CD95L.
Binding of CD95L or agonistic antibodies to CD95 leads to formation of a receptor complex at the cellular membrane, which was named DISC.14
The DISC consists of oligomerized receptors, the DD-containing adaptor molecule FADD/MORT1 (Fas-associated DD), procaspase-8 (FLICE, MACHα
, Mch5), procaspase-10 and the c-FLIP ().15, 16, 17
The interactions between the molecules at the DISC are based on homotypic contacts. The 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 procaspases-8, -10 and c-FLIP. As a result of DISC formation procaspase-8 is activated at the DISC resulting in the formation of the active caspase-8, which leads to apoptosis.
Figure 1 The CD95 DISC and complex II. The DISC consists of CD95, (depicted in yellow), FADD, (depicted in light blue), procaspase-8/procaspase-10, (depicted in green) and c-FLIP (depicted in violet). Complex II comprises FADD, procaspase-8/10 and c-FLIP. DD are (more ...)
The initial events of DISC formation have not been clarified yet. Pre-oligomerization of CD95 via the pre-ligand assembly domain has been reported to have an important role in apoptosis initiation.18
Recently, there have been several new reports on the X-ray structure of the complex formed by isolated CD95 and FADD DDs.19, 20
Scott et al.19
have suggested that binding of CD95L leads to an opening of the CD95 DD, which exposes the FADD-binding site and simultaneously generates a bridge between two CD95 molecules. They show that a basic unit of this oligomeric CD95 network is composed of a tetramer, comprising four FADD DDs and four CD95 DDs. In contrast, Wang et al.20
have reported that a basic unit of the CD95 DISC comprises 5–7 CD95 DDs and 5 FADD DD. The reported X-ray structures contradict each other with respect to the CD95/FADD complex; nevertheless, they provide a basis for a model of DD interactions at the DISC leading to procaspase-8 recruitment and activation. Certainly, the future challenge should involve obtaining the X-ray structure of the CD95 DISC assembled from the full-length CD95, FADD and procaspases, which would provide further insights into the structure of the complex.
In the CD95 apoptotic pathway two types of cells and signaling pathways have been established.21
Type I cells are characterized by high levels of CD95 DISC formation and high amounts of active caspase-8. Activated caspase-8 directly leads to activation of downstream effectors caspases-3 and -7. In Type II cells, there are lower levels of CD95 DISC formation and, thus, lower levels of active caspase-8. In this case, signaling might require an amplification loop. This amplification loop involves the cleavage by caspase-8 of the Bcl-2-family protein Bid to generate truncated (t)Bid and subsequent bid-mediated release of cytochrome C
) from the mitochondria. The release of cyt C
from the mitochondria results in apoptosome formation followed by the activation of procaspase-9, which in turn cleaves downstream effector caspases-3, -6 and -7. Importantly, it has been shown that Bid is essential for CD95-induced apoptosis in hepatocytes but dispensable in thymocytes.22, 23
This indicates that CD95-mediated signaling in thymocytes can be considered as Type I, while hepatocytes can be classified as Type II cells. In addition, an important role of XIAP in the amplification of Type I signal has been reported recently.24
Jost et al.24
have shown that the levels of XIAP expression can define Type I versus
Type II signaling in a particular cell. They have demonstrated that BID/XIAP double-deficient mice are sensitive to CD95-induced apoptosis, whereas BID-deficient mice typically survive injection with CD95L or anti-CD95 antibodies.
Recently, we and others have shown that there is a second CD95 signaling complex formed upon CD95 stimulation, which was termed as complex II. Complex II comprises DED-proteins FADD, procaspase-8 and c-FLIP, and does not contain CD95 ().25, 26
Complex II is formed within minutes after receptor stimulation. Apparently, complex II contributes to the activation of caspase-8. However, the amount of procaspase-8, which is activated in complex II versus
in the DISC, and the mechanism of CD95 complex II formation remain unclear.25
Notably, the formation of a second complex was first discovered in TNF signaling for TNFR1 in Jürg Tschopp laboratory in 2003.27
Complex II in TNFR1 signaling does also not contain the receptor itself but comprises all the other components of the TNFR1 complex, for example TRADD, receptor-interacting protein 1 (RIP1), TRAF2 as well as the apoptosis-inducing components FADD, caspase-8 and caspase-10. In TNFR1 signaling, the generation of complex II has an essential role in caspase-8 activation and apoptosis induction. The formation of complex II was reported to be a characteristic feature of a number of TNFR family members including TRAIL-R1/R2 and CD95.25, 28
Therefore, the formation of complex II can be considered as a universal paradigm of TNFR signaling.
CD95 pro-and anti-apoptotic signaling is regulated at multiple levels: at the DISC, at complex II and at the mitochondria. Procaspase-8 and c-FLIP are two DED proteins, which are essential for the initiation of the apoptotic and non-apoptotic signals at the CD95 DISC. Below we describe in detail the DED-proteins procaspase-8 and c-FLIP, and the mechanism of their pro- and anti-apoptotic action.