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 likely 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-directed mutagenesis of predicted catalytic residues (Glu(49), Asn(180), and His(272)) to Ala residues abolished the catalytic activity of nSMasel. Jurkat cells were retrovirally transduced with either wildtype or inactive (with all three point mutations) Myc-tagged nSMasel. Cells overexpressing wildtype 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 apoptosis sensitivity to ligation of the CD95/Fas receptor in cells overexpressing wildtype or mutant nSMasel were identical to vector-transduced cells. We conclude that not nSMasel but a different, yet to be identified, nSMase accounts for the generation of Cer during the execution phase of death receptor-induced apoptosis, (C) 2001 Academic Press.