The sphingolipid metabolites ceramide and sphingosine-1-phosphate are second messengers with opposing roles in mammalian cell growth arrest and survival; their relative cellular level has been proposed to be a rheostat that determines the fate of cells. This report demonstrates that this rheostat is an evolutionarily conserved stress-regulatory mechanism that inf luences growth and survival of yeast. Although the role of sphingosine-1-phosphate in yeast was not previously examined, accumulation of ceramide has been shown to induce G 1 arrest and cell death. We now have identified a gene in Saccharomyces cerevisiae, LBP1, that regulates the levels of phosphorylated sphingoid bases and ceramide. LBP1 was cloned from a yeast mutant that accumulated phosphorylated long-chain sphingoid bases and diverted sphingoid base intermediates from sphingolipid pathways to glycerophospholipid biosynthesis. LBP1 and its homolog, LBP2, encode very hydrophobic proteins that contain a novel-conserved sequence motif for lipid phosphatases, and both have long-chain sphingoid base phosphate phosphatase activity. In vitro characterization of Lbp1p shows that this phosphatase is Mg 2؉ -independent with high specificity for phosphorylated long-chain bases, phytosphingosine and sphingosine. The deletion of LBP1 results in the accumulation of phosphorylated long-chain sphingoid bases and reduced ceramide levels. Moreover, deletion of LBP1 and LBP2 results in dramatically enhanced survival upon severe heat shock. Thus, these phosphatases play a previously unappreciated role in regulating ceramide and phosphorylated sphingoid base levels in yeast, and they modulate stress responses through sphingolipid metabolites in a manner that is reminiscent of their effects on mammalian cells.Branching pathways of sphingolipid metabolism may mediate growth arrest, stress, or proliferative responses depending on the cell type and the nature of the stimulus. Ceramide is emerging as an important regulatory component of stress responses and programmed cell death, known as apoptosis (1-5). In contrast, another sphingolipid metabolite, sphingosine-1-phosphate (SPP), has been implicated as a second messenger in cellular proliferation (6) and antagonizes ceramide-mediated apoptosis (7). Thus, it has been suggested that the relative intracellular levels of ceramide and SPP are a critical factor for cell survival. Although the ceramide͞SPP rheostat may be an inherent characteristic of mammalian cells, external stimuli can reset this ratio (7-9). A variety of stress stimuli, including Fas ligand, TNF-␣, IL-1, growth factor withdrawal, anticancer drugs, oxidative stress, heat shock, and ionizing radiation, increase ceramide levels (1, 2, 10, 11), whereas platelet-derived growth factor and other growth factors stimulate rapid, transient elevations in SPP levels (6). The mechanisms that regulate the levels of these sphingolipid second messengers are under intense investigation with most of the attention focused on degradative pathways: sphingomyelinase, which...

Recent evidence suggests that branching pathways of sphingolipid metabolism may mediate either apoptotic or mitogenic responses depending on the cell type and the nature of the stimulus. While ceramide has been shown to be an important regulatory component of apoptosis induced by tumor necrosis factor alpha and Fas ligand, sphingosine-1-phosphate (SPP), a further metabolite of ceramide, has been implicated as a second messenger in cellular proliferation and survival induced by platelet-derived growth factor, nerve growth factor, and serum. SPP protects cells from apoptosis resulting from elevations of ceramide. Inflammatory cytokines stimulate sphingomyelinase, but not ceramidase, leading to accumulation of ceramide, whereas growth signals also leading to accumulation of ceramide, whereas growth signals also stimulate ceramidase and sphingosine kinase leading to increased SPP levels. We propose that the dynamic balance between levels of sphingolipid metabolites, ceramide, and SPP, and consequent regulation of different family members of mitogen-activated protein kinases (JNK versus ERK), is an important factor that determines whether a cell survives or dies.

Sphingosine kinase catalyzes the formation of the bioactive sphingolipid metabolite sphingosine 1-phosphate, which plays important roles in numerous physiological processes, including growth, survival, and motility. We have purified rat kidney sphingosine kinase 6 ؋ 10 5 -fold to apparent homogeneity. The purification procedure involved ammonium sulfate precipitation followed by chromatography on an anion exchange column. Partially purified sphingosine kinase was found to be stabilized by the presence of high salt, and thus, a scheme was developed to purify sphingosine kinase using sequential dye-ligand chromatography steps (since the enzyme bound to these matrices even in the presence of salt) followed by EAH-Sepharose chromatography. This 385-fold purified sphingosine kinase bound tightly to calmodulin-Sepharose and could be eluted in high yield with EGTA in the presence of 1 M NaCl. After concentration, the calmodulin eluate was further purified by successive high pressure liquid chromatography separations on hydroxylapatite, Mono Q, and Superdex 75 gel filtration columns. Purified sphingosine kinase has an apparent molecular mass of ϳ49 kDa under denaturing conditions on SDS-polyacrylamide gel, which is similar to the molecular mass determined by gel filtration, suggesting that the active form is a monomer. Sphingosine kinase shows substrate specificity for D-erythro-sphingosine and does not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, DL-threo-dihydrosphingosine, or N,N-dimethylsphingosine. However, the latter two sphingolipids were potent competitive inhibitors. With sphingosine as substrate, the enzyme had a broad pH optimum of 6.6-7.5 and showed Michaelis-Menten kinetics, with K m values of 5 and 93 M for sphingosine and ATP, respectively. This study provides the basis for molecular characterization of a key enzyme in sphingolipid signaling.Sphingolipid metabolites, such as ceramide, sphingosine, and sphingosine 1-phosphate (SPP), 1 are members of a novel class of lipid second messengers (1-4). Ceramide is an important regulatory component of stress responses and programmed cell death, known as apoptosis (2,5,6). In contrast, we have implicated a further metabolite of ceramide, SPP, as a second messenger in cellular proliferation and survival induced by platelet-derived growth factor, nerve growth factor, and serum (7-9). Previously, we showed that SPP protects cells from apoptosis resulting from elevations of ceramide (7, 9) and proposed that the dynamic balance between levels of the sphingolipid metabolites (ceramide and SPP) and consequent regulation of opposing signaling pathways is an important factor that determines whether a cell survives or dies (7). Recently, we demonstrated that this ceramide/SPP rheostat is an evolutionarily conserved stress regulatory mechanism influencing growth and survival of yeast (10). A variety of stress stimuli, including Fas ligand, tumor necrosis factor-␣, interleukin-1, growth factor withdrawal, anticancer drugs, oxidative stress, ...

Sphingosine 1-phosphate (SPP) is a lipid second messenger that also acts as a first messenger through the G protein-coupled receptor Edg-1. Here we show that SPP also binds to the related receptors H218 and Edg-3 with high affinity and specificity. SPP and sphinganine 1-phosphate bind to these receptors, whereas neither sphingosylphosphorylcholine nor lysophosphatidic acid compete with SPP for binding to either receptor. Transfection of HEK293 cells with H218 or edg-3, but not edg-1, induces rounded cell morphology in the presence of serum, which contains high levels of SPP. SPP treatment of cells overexpressing H218 cultured in delipidated serum causes cell rounding. A similar but less dramatic effect was observed in cells overexpressing Edg-3 but not with Edg-1. Cell rounding was correlated with apoptotic cell death, probably as a result of loss of attachment. Nerve growth factor-induced neuritogenesis in PC12 cells was inhibited by overexpression of H218 and to a lesser extent Edg-3. SPP treatment rapidly enhanced neurite retraction in PC12 cells overexpressing Edg-1, Edg-3, or H218. Thus, H218, and possibly Edg-3, may be the cell surface receptors responsible for cell rounding and neurite retraction induced by SPP. Moreover, the identification of these two additional SPP receptors indicates that a family of highly specific receptors exists that mediate different responses to SPP.The sphingolipid metabolite sphingosine 1-phosphate (SPP) 1 is emerging as a member of a new class of lipid second messengers (1, 2). SPP is mitogenic in diverse cell types (3-6) and suppresses programmed cell death or apoptosis (7-10). Various stimuli, including platelet-derived growth factor and serum (6, 11), nerve growth factor (NGF) (8, 12), vitamin D 3 (13), activation of protein kinase C (14, 15) or protein kinase A (10), cross-linking of the Fc⑀R1 (16) or Fc␥R1 (17) receptor by antigens, and binding of carbachol to m2 and m3 muscarinic acetylcholine receptors (18), increase cellular levels of SPP by activation of sphingosine kinase. Moreover, competitive inhibitors of sphingosine kinase eliminate the formation of SPP and selectively block cellular proliferation induced by platelet-derived growth factor and serum (11,19), the cytoprotective effects of protein kinase C and cAMP activators (7, 10), NGF (8), and vitamin D 3 (13) as well as Fc⑀R1-, Fc␥R1-, and muscarinic acetylcholine receptor-mediated calcium signaling (16, 17), further supporting a role for endogenous SPP in cell growth, survival, and calcium mobilization. In addition, microinjected SPP mobilizes calcium from internal sources (18) and is mitogenic for Swiss 3T3 fibroblasts (20), indicating that SPP acts intracellularly to regulate calcium homeostasis and proliferation.Several other responses to SPP are mediated through cell surface receptors, including platelet activation (21), inhibition of melanoma cell motility (22), activation of G i protein-gated inward rectifying K ϩ channels in atrial myocytes (23), and Rho-dependent neurite retraction and cell rounding o...