The potent sphingolipid metabolite sphingosine 1-phosphate is produced by phosphorylation of sphingosine catalyzed by sphingosine kinase (SphK) types 1 and 2. In contrast to pro-survival SphK1, the putative BH3-only protein SphK2 inhibits cell growth and enhances apoptosis. Here we show that SphK2 catalytic activity also contributes to its ability to induce apoptosis. Overexpressed SphK2 also increased cytosolic free calcium induced by serum starvation. Transfer of calcium to mitochondria was

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... quired for SphK2-induced apoptosis, as cell death and cytochrome c release was abrogated by inhibition of the mitochondrial Ca 2؉ transporter. Serum starvation increased the proportion of SphK2 in the endoplasmic reticulum and targeting SphK1 to the endoplasmic reticulum converted it from anti-apoptotic to pro-apoptotic. Overexpression of SphK2 increased incorporation of [ 3 H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16ceramide, whereas SphK1 decreased it. Electrospray ionizationmass spectrometry/mass spectrometry also revealed an opposite effect on ceramide mass levels. Importantly, specific down-regulation of SphK2 reduced conversion of sphingosine to ceramide in the recycling pathway and conversely, down-regulation of SphK1 increased it. Our results demonstrate that SphK1 and SphK2 have opposing roles in the regulation of ceramide biosynthesis and suggest that the location of sphingosine 1-phosphate production dictates its functions. The sphingolipid metabolite, sphingosine 1-phosphate (S1P), 3 a ligand for a family of five specific G protein-coupled receptors, and regulates many important cellular processes including growth, survival, differentiation, cytoskeleton rearrangements, motility, angiogenesis, and immunity (reviewed in Refs. 1-3) . Although there is no doubt that S1P acts extracellularly, several studies suggest that this potent lipid, like its precursors sphingosine (4) and ceramide (N-acylsphingosine) (5-7), . 3 The abbreviations used are: S1P, sphingosine 1-phosphate; BAPTA-AM, 1,2-bis(2aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid tetrakis-acetoxymethyl ester; BH3, Bcl-2 homology domain 3; DKO, double knock-out; ESI-MS/MS, electrospray tandem mass spectrometry; SM, sphingomyelin; SphK, sphingosine kinase; ER, endoplasmic reticulum; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; PBS, phosphate-buffered saline. FIGURE 9. Effects of SphK1 and SphK2 on ceramide species. A, HEK 293 cells expressing vector, SphK1, or SphK2 were serum-starved for 18 h, then treated without or with 5 M sphingosine (Sph) for 6 h as indicated. Lipids were extracted and ceramide species were determined by ESI-MS/MS. B, data from A are presented in "pie" charts showing a proportion of each indicated ceramide species. Numbers indicate chain length followed by the number of double bonds in the fatty acid. Data are averages of triplicate determinations and are expressed as picomole of lipid/mg of protein. Similar results were found in two additional experiments.