Recent studies suggest that trimerization of Fas is insufficient for apoptosis induction and indicate that super-aggregation of trimerized Fas might be prerequisite. For many cell surface receptors, cross-linking by multivalent ligands or antibodies induces their lateral segregation within the plasma membrane and co-localization into "caps" on one pole of the cell. In this study, we show that capping of Fas is essential for optimal function and that capping is ceramide-dependent. In Jurkat T lymphocytes and in primary cultures of hepatocytes, ceramide elevation was detected as early as 15-30 s and peaked at 1 min after CH-11 and Jo2 anti-Fas antibody treatment, respectively. Capping was detected 30 s after Fas ligation, peaked at 2 min, and was maintained at a lower level for as long as 30 min in both cell types. Ceramide generation appeared essential for capping. Acid sphingomyelinase ؊/؊ hepatocytes were defective in Jo2-induced ceramide generation, capping, and apoptosis, and nanomolar concentrations of C 16 -ceramide restored these events. To further explore the role of ceramide in capping of Fas, we employed FLAG-tagged soluble Fas ligand (sFasL), which binds trimerized Fas but is unable to induce capping or apoptosis in Jurkat cells. Cross-linking of sFasL with M2 anti-FLAG antibody induced both events. Pretreatment of cells with natural C 16 -ceramide bypassed the necessity for forced antibody cross-linking and enabled sFasL to cap and kill. The presence of intact sphingolipid-enriched membrane domains may be essential for Fas capping since their disruption with cholesterol-depleting agents abrogated capping and prevented apoptosis. These data suggest that capping is a ceramide-dependent event required for optimal Fas signaling in some cells.The current model of Fas (CD95 or APO-1) signaling suggests that engagement of Fas by its ligand or anti-Fas antibody leads to receptor trimerization and recruitment of the cytoplasmic adapter protein FADD 1 (MORT-1) and pro-caspase 8 (Flice/ MACH-1), (1, 2), thus forming a death-inducing signaling complex. Assembly of the death-inducing signaling complex results in release of active caspase 8, initiating the apoptotic process (1, 2). Peter and co-workers (2) provide evidence that after caspase 8 activation, Fas signals apoptosis via two different mechanisms. In Type I cells, caspase 8 directly activates a hierarchical cascade of effector caspases, involving among others caspases 3 and 7. Apoptosis in Type II cells occurs after minimal caspase 8 activation and proceeds through an amplification cascade involving mitochondrial dysfunction, the release of mitochondrial cytochrome c, and activation of Apaf-1 and caspase 9. Commitment to apoptosis ensues subsequently via effector caspase activation. Whereas this model is widely accepted, recent data are incompatible with portions of this paradigm. Siegel et al. (3) reported recently that Fas may already exist in a trimerized state before ligand interaction and that preassembly may be required for binding Fas ligand and t...

Recent biophysical data suggest that the properties of ceramide observed in model membranes may apply to biological systems. In particular, the ability of ceramide to form microdomains, which coalesce into larger platforms or macrodomains, appears to be important for some cellular signaling processes. Several laboratories have now demonstrated similar reorganization of plasma membrane sphingolipid rafts, via ceramide generation, into macrodomains. This event appeared necessary for signaling upon activation of a speci¢c set of cell surface receptors. In this article, we review the properties and functions of rafts, and the role of sphingomyelinase and ceramide in the biogenesis and re-modeling of these rafts. As clustering of some cell surface receptors in these domains may be critical for signal transduction, we propose a new model for transmembrane signal transmission.

Early events required for induction of apoptosis by CD95 are preassociation of CD95, the formation of the deathinducing signaling complex (DISC) and clustering of CD95 in distinct membrane domains. Here, we identify the molecular ordering of these events and show that the acid sphingomyelinase (ASM) functions upstream of the DISC to mediate CD95 clustering in ceramide-enriched membrane platforms, an event that is required for DISC formation. Experiments in ASM-deficient cells revealed that CD95 ligation, in the absence of ceramide generation, triggers o1% of full caspase 8 activation at the receptor. This event, however, is both necessary and sufficient to trigger translocation of ASM onto the outer leaflet of the plasma membrane, ASM activation and ceramide release, but insufficient for apoptosis induction. Ceramidemediated CD95 clustering then amplifies the primary CD95 signaling and drives the second step of CD95 signaling, that is, formation of the DISC yielding 100% caspase activity and apoptosis. These studies suggest that the most parsimonious interpretation of the molecular ordering of the earliest events in CD95 signaling, at least in some cells, is: CD95 ligation-1% of maximum caspase 8 activation-ASM translocation-ceramide generation-CD95clustering-DISC formation-100% of maximum caspase 8 activation-apoptosis.

Diacylglycerol pyrophosphate (DGPP) is involved in a putative novel lipid signaling pathway. DGPP phosphatase (DGPP phosphohydrolase) is a membrane-associated 34-kDa enzyme from Saccharomyces cerevisiae which catalyzes the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyzes the dephosphorylation of PA to yield diacylglycerol. Amino acid sequence information derived from DGPP phosphatase was used to identify and isolate the DPP1 (diacylglycerol pyrophosphate phosphatase) gene encoding the enzyme. Multicopy plasmids containing the DPP1 gene directed a 10-fold overexpression of DGPP phosphatase activity in S. cerevisiae. The heterologous expression of the S. cerevisiae DPP1 gene in Sf-9 insect cells resulted in a 500-fold overexpression of DGPP phosphatase activity over that expressed in wild-type S. cerevisiae. DGPP phosphatase possesses a Mg 2؉ -independent PA phosphatase activity, and its expression correlated with the overexpression of DGPP phosphatase activity in S. cerevisiae and in insect cells. DGPP phosphatase was predicted to be an integral membrane protein with six transmembrane-spanning domains. The enzyme contains a novel phosphatase sequence motif found in a superfamily of phosphatases. A dpp1⌬ mutant was constructed by deletion of the chromosomal copy of the DPP1 gene. The dpp1⌬ mutant was viable and did not exhibit any obvious growth defects. The mutant was devoid of DGPP phosphatase activity and accumulated (4-fold) DGPP. Analysis of the mutant showed that the DPP1 gene was not responsible for all of the Mg 2؉ -independent PA phosphatase activity in S. cerevisiae. Diacylglycerol pyrophosphate (DGPP)1 is a novel phospholipid that contains a pyrophosphate group attached to diacylglycerol (DG) (Fig. 1) (1). DGPP has been found in a variety of plants (2, 3) and in the yeast Saccharomyces cerevisiae (4). This phospholipid is synthesized from phosphatidate (PA) and ATP via the reaction catalyzed by the membrane-associated enzyme PA kinase (1) and is dephosphorylated to PA via the reaction catalyzed by the membrane-associated enzyme DGPP phosphatase ( Fig. 1) (4). The amounts of DGPP in wild-type S. cerevisiae and in plants are barely detectable (3, 4). For example, DGPP accounts for only 0.18 mol % of the major phospholipids in S. cerevisiae (4). The low abundance of DGPP is reminiscent of lipid signaling molecules such as the inositol-containing phospholipids (5-9). Recent studies indicate that the metabolism of DGPP is involved in a novel lipid signaling pathway. DGPP accumulates in plant tissues upon G protein activation through the stimulation of PA kinase activity (3), and metabolic labeling studies with Catharanthus roseus cells have shown that DGPP is metabolized rapidly to PA and then to DG (10). It has been suggested that DGPP may function as a signaling molecule (3, 4). Alternatively, the formation of DGPP may serve to attenuate the signaling functions of PA (11,12).DGPP phosphatase activity has been identified in S. cerevisiae, C. roseus, Escherichia coli, rat liver, pig l...

The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.