Signaling by tumor necrosis factor (TNF) receptor 1 (TNF-R1), a prototypic member of the death receptor family, mediates pleiotropic biological outcomes ranging from inflammation and cell proliferation to cell death. Although many elements of specific signaling pathways have been identified, the main question of how these selective cell fate decisions are regulated is still unresolved. Here we identified TNF-induced K63 ubiquitination of TNF-R1 mediated by the ubiquitin ligase RNF8 as an early molecular checkpoint in the regulation of the decision between cell death and survival. Downmodulation of RNF8 prevented the ubiquitination of TNF-R1, blocked the internalization of the receptor, prevented the recruitment of the death-inducing signaling complex and the activation of caspase-8 and caspase-3/7, and reduced apoptotic cell death. Conversely, recruitment of the adaptor proteins TRADD, TRAF2, and RIP1 to TNF-R1, as well as activation of NF-B, was unimpeded and cell growth and proliferation were significantly enhanced in RNF8-deficient cells. Thus, K63 ubiquitination of TNF-R1 can be sensed as a new level of regulation of TNF-R1 signaling at the earliest stage after ligand binding. The cytokine tumor necrosis factor alpha (TNF-␣) is involved in a variety of cellular processes, such as inflammation, differentiation, control of cell proliferation, and initiation of apoptosis. TNF is known to bind to two receptors of the TNF receptor superfamily, TNF receptor 1 (TNF-R1) and TNF-R2. TNF-R1 is a member of the death receptor subgroup of this superfamily (1). The death receptors all have a death domain (DD) in the C-terminal tail that is necessary for activation of apoptosis. Selective recruitment of adaptor proteins to TNF-R1 decides whether nonapoptotic signaling pathways or cell death-inducing pathways will be initiated. Complex I is formed at the TNF-R1 DD by recruitment of TRADD, RIP1, TRAF2, and c-IAP1 (2). In the model of Micheau and Tschopp, induction of apoptosis is initiated by the ubiquitination of most complex I proteins, leading to their dissociation from TNF-R1. Binding of FADD to the DD of cytosolic TRADD facilitates the recruitment of caspase-8 and -10 via their DDs, forming complex II.Conflicting data exist regarding the complex formation that induces apoptosis after TNF stimulation. In contrast to the model described above (2), we previously reported that after recruitment of TRADD, RIP, and TRAF2 to TNF-R1 at the cell surface, the receptor is internalized and FADD and procaspase-8 are recruited, forming the death-inducing signaling complex (DISC) still associated with the TNF receptor at endosomal vesicles (TNF receptosomes) (3-5). Consecutively, caspase-8 is activated by autocleavage and induces caspase-3 activation either directly or indirectly via a mitochondrial amplification loop involving cytochrome c and APAF-1 release, forming the apoptosome with caspase-9.Recently, we found that within TNF receptosomes, caspase-8 activates caspase-7, which in turn cleaves A-SMase, initiating the production of...

Acid sphingomyelinase (A-SMase) plays an important role in the initiation of CD95 signaling by forming ceramide-enriched membrane domains that enable clustering and activation of the death receptors. In TNF-R1 and TRAIL-R1/R2 signaling, A-SMase also contributes to the lysosomal apoptosis pathway triggered by receptor internalization. Here, we investigated the molecular mechanism of CD95-mediated A-SMase activation, demonstrating that A-SMase is located in internalized CD95-receptosomes and is activated by the CD95/CD95L complex in a biphasic manner.Since several caspases have been described to be involved in the activation of A-SMase, we evaluated expression levels of caspase-8, caspase-7 and caspase-3 in CD95-receptosomes. The occurrence of cleaved caspase-8 correlated with the first peak of A-SMase activity and translocation of the A-SMase to the cell surface which could be blocked by the caspase-8 inhibitor IETD.Inhibition of CD95-internalization selectively reduced the second phase of A-SMase activity, suggesting a fusion between internalized CD95-receptosomes and an intracellular vesicular pool of A-SMase. Further analysis demonstrated that caspase-7 activity correlates with the second phase of the A-SMase activity, whereas active caspase-3 is present at early and late internalization time points. Blocking caspases-7/ -3 by DEVD reduced the second phase of A-SMase activation in CD95-receptosomes suggesting the potential role of caspase-7 or -3 for late A-SMase activation.In summary, we describe a biphasic A-SMase activation in CD95-receptosomes indicating (I.) a caspase-8 dependent translocation of A-SMase to plasma membrane and (II.) a caspase-7 and/or -3 dependent fusion of internalized CD95-receptosomes with intracellular A-SMase-containing vesicles.

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