Ceramide produced by sphingomyelinases (SMases) 1 has been recognized as an important second messenger in membrane receptor signaling. Through binding to the p55 TNF receptor (TNF-R55), TNF rapidly activates two distinct forms of SMases, a membrane-associated neutral (N-) SMase and an acid (A-) SMase (1), which reside in caveolae (2) and the endosomal-lysosomal compartment. Each type of SMase hydrolyzes the phosphodiester bond of sphingomyelin to yield the neutral lipid second messenger ceramide and phosphorylcholine. Ceramide generated by N-SMase at the plasma membrane directs the activation of ceramide-activated protein kinase (CAPK) (3) that phosphorylates and activates Raf-1 kinase (4, 5). Activated Raf-1 in turn phosphorylates and stimulates a dual specificity mitogen-activated kinase (MEK-1), which eventually phosphorylates and activates extracellular signal-regulated kinases (ERKs). The downstream targets of endosomal-derived ceramide produced by A-SMase are not yet defined but may include protein kinase C (PKC), JNK, and caspases (6 -8). N-SMase and A-SMase are activated independently by distinct cytoplasmic domains of TNF-R55 (1). N-SMase activation is mediated by neutral sphingomyelinase domain (NSD), spanning an 11-amino acid motif N-terminally adjacent to the death domain of TNF-R55 (9, 10). The NSD binds a WD-repeat protein, FAN, that mediates activation of N-SMase.The domain of TNF-R55 activating the A-SMase pathway strikingly corresponds to the death domain signaling the cytotoxic effects of TNF (1, 11). The cytoplasmic protein TRADD was recently identified to associate with the death domain of TNF-R55 in a TNF-dependent process (12, 13). TRADD serves as an adapter protein that recruits other proteins to the cytoplasmic TNF receptor complex (14,15). Overexpression of TRADD potently activates distinct signaling cascades leading to activation of NF-B and induction of cell death (13). FADD, which directly binds to TRADD, has been shown to mediate activation of a pro-apoptotic protease, caspase-8/a (FLICE/ MACH), eventually leading to apoptosis (16,17).Here we show that TNF-induced activation of A-SMase is mediated through TRADD and FADD. Neither caspase-8/a nor a dominant negative mutant of caspase-8/a alter the TNFinduced activation of A-SMase. However, several tetrapeptide caspase inhibitors as well as cytokine response modifier A (crmA) attenuated TNF-induced activation of A-SMase activity, suggesting that FADD mediates enhancement of A-SMase activity through activation of a protease distinct from FLICE. MATERIALS AND METHODSCell Culture and Biological Reagents-The human embryonic kidney cell line HEK 293 was kindly provided by Dr. M. Schmidt, Essen, FRG. Hela and COS7 cells were obtained from ATCC. Cells were maintained in high glucose Dulbecco's modified Eagle's medium (ICN) supplemented with 10% fetal calf serum, 10 mM glutamine, and 50 g/ml each of streptomycin and penicillin in a humidified incubator at 5% CO 2 . Highly purified recombinant human TNF (3 ϫ 10 7 units/mg) was kindly provided by...

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The magnesium-dependent, plasmamembrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homol-

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Ceramide, generated by the enzymatic function of sphingomyelinases (SMases) has emerged as an important signaling pathway transducing diverse biological effects of various cytokine receptors. The 55-kDa receptor for tumor necrosis factor (TNF-R55) activates two types of SMases through distinct cytoplasmic domains. The death domain that is responsible for the initiation of the apoptotic pathway also signals for the activation of an acid SMase (A-SMase). The adapter protein TRADD binds to TNF-R55 in a ligand-dependent manner and serves as anchor for the subsequent recruitment of other proteins into the signaling complex that directly lead to cell death or nuclear factor-kappaB (NF-kappaB) induction. Notably, the two pro-apoptotic adapter proteins TRADD and FADD are also involved in the activation of A-SMase. In contrast, the NF-kappaB-inducing adapters TRAF2 and RIP do not signal for A-SMase. Thus, activation of A-SMase appears to belong to signals leading to TNF-induced cell death. A second signaling domain (NSD) is located upstream of the death domain and directly links the TNF-R55 to the activation of a neutral SMase (N-SMase). A novel adapter protein, FAN, has been identified that specifically binds to the NSD. FAN contains five WD repeats at its carboxy terminus, while it shows significant sequence homology with the mouse beige protein and its human homolog, the CHS protein, in the center portion of the protein. Overexpression of full-length FAN enhanced N-SMase activity in TNF-treated cells, whereas truncated mutants of FAN produced dominant negative effects. FAN, however, did not interfere with any of the TNF responses signaled for by the death domain. Taken together, our data suggest that distinct cytoplasmic domains of TNF-R55 initiate independent signaling pathways by binding different adapter proteins.