Effect of overexpression of a neutral sphingomyelinase on CD95-induced ceramide production and apoptosis

We previously showed that ceramide (Cer) formed during the execution phase of apoptosis is derived from plasma membrane sphingomyelin (SM), most likel y by a neutral sphingomyelinase activity (Tepper et al, J. Cell Biol. 150, 2000, 155-164). In this study, we investigated the involvement of a cloned putative human neutral sphingomyelinase (nSMase1) in this process. Site-dir ected mutagenesis of predicted catalytic residues (Glu(49), Asn(180), and H is(272)) to Ala residues abolished the catalytic activity of nSMasel. Jurka t cells were retrovirally transduced with either wildtype or inactive (with all three point mutations) Myc-tagged nSMasel. Cells overexpressing wildty pe nSMasel showed dramatically elevated in vitro nSMase1 activity. However, nSMasel gene transduction (wildtype or mutant) did not alter steady-state levels of SM, Cer, or glucosylceramide. Moreover, the Cer response and apop tosis sensitivity to ligation of the CD95/Fas receptor in cells overexpress ing wildtype or mutant nSMasel were identical to vector-transduced cells. W e conclude that not nSMasel but a different, yet to be identified, nSMase a ccounts for the generation of Cer during the execution phase of death recep tor-induced apoptosis, (C) 2001 Academic Press.