Article |
Address correspondence to Dr. Sarah Spiegel, Department of Biochemistry and Molecular Biophysics, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298-0614. Tel.: (804) 828-9330. Fax: (804) 828-8999. E-mail: sspiegel{at}vcu.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: S1P phosphaxtase-1; sphingosine-1-phosphate; ceramide; sphingosine; apoptosis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Many external stimuli, particularly growth and survival factors, activate sphingosine kinase, which phosphorylates sphingosine and increases cellular S1P levels. Intracellularly generated S1P can mobilize calcium from internal stores (Mattie et al., 1994; Meyer zu Heringdorf et al., 1998), suppress ceramide-mediated apoptosis (Cuvillier et al., 1996; Morita et al., 2000), or can act in a paracrine or autocrine fashion to activate S1P1 signaling, which is crucial for directed cell movement (Hobson et al., 2001). Thus, due to the pivotal role of S1P in regulating cellular processes, it is not surprising that its levels are low and tightly regulated in a spatialtemporal manner through its synthesis catalyzed by sphingosine kinase and degradation by an ER pyridoxal phosphatedependent S1P lyase and still not well-characterized phosphohydrolase activities.
As a first approach to identify specific mammalian S1P phosphatases, two genes were cloned from Saccharomyces cerevisiae, LBP1/YSR2/LCB3 and LBP2/YSR3, that encode specific sphingoid base phosphate phosphatases (Mao et al., 1997; Qie et al., 1997; Mandala et al., 1998). These S1P phosphatases belong to the family of magnesium-independent, N-ethylmaleimideinsensitive type 2 lipid phosphate phosphohydrolases (LPPs) (Stukey and Carman, 1997; Brindley and Waggoner, 1998). However, except for their conserved residues within three domains present in all LPPs (Stukey and Carman, 1997), they have little overall homology to other known LPPs. Genetic manipulations demonstrated that these S1P phosphohydrolases regulate the levels of phosphorylated sphingoid bases and ceramide and influence growth and survival of yeast after nutrient deprivation and heat stress (Dickson et al., 1997; Jenkins et al., 1997; Mandala et al., 1998; Mao et al., 1999; Skrzypek et al., 1999).
On the basis of sequence homology with LBP1, the first mammalian homologue, murine S1P phosphatase 1 (mSPP-1), was recently cloned. This hydrophobic enzyme contains 430 amino acids, 810 membrane-spanning regions, and degrades S1P, but not lysophosphatidic acid (LPA), a structurally related lysophospholipid (Mandala et al., 2000). By contrast, the other LPPs have broad substrate specificities and degrade LPA, phosphatidic acid (PA), diacylglycerolpyrophosphate, S1P, and ceramide-1-phosphate (Brindley and Waggoner, 1998). Previous studies have demonstrated that LPP1 is localized on the plasma membrane, with an externally oriented catalytic site, and functions as an ectophosphatase to attenuate the actions of LPA as an agonist of its cell surface receptors (Jasinska et al., 1999). However, more recently, it has been shown that the ability of LPP1 and LPP2 to attenuate GPCR signaling correlates with reduced intracellular PA and not with ectolipid phosphate phosphatase activity (Alderton et al., 2001). Moreover, although LPA-induced mitogenesis is regulated by LPPs, in an analogous manner to the intracellular actions of S1P (Van Brocklyn et al., 1998), it is also LPA receptor independent (Hooks et al., 2001). Thus, we examined the subcellular localization of SPP-1 in order to define its biological function and to determine whether it might play a role in regulating extracellular or intracellular levels of S1P. The results of this study indicate that this novel lipid phosphohydrolase is located mainly in the ER and that it functions in an unprecedented manner to regulate biosynthesis of ceramide, which plays a critical role in apoptosis.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
In contrast to ISP-1, FB1 completely prevented the increase in intracellular ceramide levels resulting from treatment of mSPP-1expressing cells with S1P and did not affect the low levels of ceramide in dihydro-S1Ptreated cells (Fig. 9 A). In agreement, there was a concomitant large increase in sphingosine levels (Fig. 9 B). These results indicate that the recombinant mSPP-1 was indeed active in vivo, converting S1P to sphingosine that was then readily metabolized to ceramide via ceramide synthase. Remarkably, although no changes in the levels of ceramides could be detected when mSPP-1expressing cells were treated with dihydro-S1P, when added in the presence of FB1, there was a large accumulation of dihydrosphingosine (Fig. 9 B). These results confirm that dihydro-S1P is indeed cleaved by mSPP-1 intracellularly, yet there is no accompanying accumulation of ceramide, suggesting rapid metabolism of dihydroceramide to sphingomyelin or complex sphingolipids. Moreover, dihydro-S1P, which has no effect on the survival of vector- or mSPP-1transfected cells, induces apoptosis of the mSPP-1 transfectants, but not vector cells, only when added in the presence of FB1 (Fig. 9 B, inset). This is in agreement with previous studies showing that the accumulation of long chain sphingoid bases causes apoptosis (Sakakura et al., 1998; Perry et al., 2000; Cuvillier et al., 2001; Kagedal et al., 2001). It should be noted that FB1 caused a large increase in basal sphingosine levels in mSPP-1 compared with vector transfectants (Fig. 9 B). This result suggests that normal turnover of intracellular S1P is a dynamic process that can contribute to the regulation of ceramide levels.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SPP-1 regulates ceramide levels
Ceramide biosynthesis at the cytoplasmic face of the ER is initiated by condensation of L-serine with palmitoyl CoA, catalyzed by serine palmitoyltransferase (Futerman et al., 1990; Merrill et al., 1993). In two rapid reactions, the product, 3-ketosphinganine, is reduced to sphinganine (dihydrosphingosine) and subsequently acylated by ceramide synthase to form dihydroceramide (Fig. 11). Dihydroceramide is then converted to ceramide by a desaturase (Michel and van Echten-Deckert, 1997). Ceramide, or in some cases, dihydroceramide, is translocated from the ER to the Golgi apparatus by not well-defined mechanisms, and then converted to sphingomyelin by the enzyme phosphatidylcholine:ceramide cholinephosphotransferase (sphingomyelin synthase) on the lumenal side of the Golgi apparatus or to glucosylceramide on the cytosolic surface of the Golgi apparatus (for reviews see van Meer and Holthuis, 2000; Funato and Riezman, 2001). After translocation into the Golgi lumen, glucosylceramide is further converted to lactosylceramide and more complex glycosphingolipids.
|
ISP-1, an inhibitor of serine palmitoyltransferase, slightly reduced the elevation of ceramide levels, suggesting that the de novo pathway is not a major contributor. Moreover, both S1P and dihydro-S1P decreased [3H]serine incorporation into very long chain ceramides, whereas only S1P enhanced incorporation into long chain ceramides that comigrated with C16-ceramide. In agreement, S1P, and not dihydro-S1P, also increased the incorporation of [3H]palmitate into long chain ceramides. In addition, it appears that there is not a major defect in the transport of ceramide from its site of production at the ER to the Golgi apparatus, the site of synthesis of glycosphingolipids, because PDMP, an inhibitor of glucosylceramide synthase, enhanced ceramide levels induced by S1P. Collectively, our data suggest that the presence or absence of the trans double bond in the sphingoid bases dictates their function in the biosynthesis of ceramide.
Why does S1P, and not dihydro-S1P, regulate ceramide biosynthesis?
The generation of sphingosine, and not sphinganine, from the hydrolysis of sphingolipids is a consequence of the constitutive turnover of plasma membrane components through the endocytic pathway. Even if a very small fraction of membrane lipids are metabolized (Thilo, 1994), the absolute amount of sphingosine generated would be quite large relative to the small intracellular pools. Although sphingosine is solely a product of sphingolipid catabolism, it is also a substrate for ceramide synthase (Merrill et al., 1993). In view of the constitutive nature of the endocytic process/salvage pathway, cells can conserve energy by reutilizing sphingosine, minimizing the de novo synthesis of sphinganine. Indeed, it has been shown that slowly dividing cells rely on this recycling hydrolytic pathway for synthesis of the majority of glycosphingolipids and sphingomyelin (Gillard et al., 1998). Hence, sphingosine produced in late endosomes and lysosomes is phosphorylated on the cytosolic surface by sphingosine kinase (Fig. 11) and S1P thus formed, due its more hydrophilic nature, is readily transported to the ER and dephosphorylated by SPP-1 to sphingosine, where it is converted to ceramide, predominantly N-palmitoyl-sphingosine. It is tempting to speculate that S1P may play a critical role in the downregulation of the de novo pathway by affecting serine palmitoyltransferase or ceramide synthase (Fig. 11). Thus, when the salvage pathway is active, sphingosine, and concomitantly S1P, levels increase, leading to inhibition of the de novo pathway. Ectopic expression of SPP-1 and degradation of S1P results in the relief of this inhibition and, concomitantly, formation of sphingosine, the substrate of ceramide synthase. In support of this provocative idea, suppression of sphingoid base synthesis was observed after the addition of lipoproteins or free sphingoid bases, as a consequence of the downregulation of serine palmitoyltransferase by sphingoid base-1-phosphates (van Echten-Deckert et al., 1997). Moreover, we and others have convincingly demonstrated that mutations or deletions of LBP1, the yeast phosphatase, completely prevented the incorporation of exogenous dihydrosphingosine into sphingolipids (Mao et al., 1997; Qie et al., 1997; Mandala et al., 1998; Zanolari et al., 2000). This defect in sphingolipid synthesis supports the intriguing possibility that externally supplied sphingoid bases are phosphorylated after their entry into the cell and require dephosphorylation before they can be used for ceramide biosynthesis (Zanolari et al., 2000). Likewise, disruption of LCB4 and LCB5, the genes encoding the sphingosine kinases in yeast, markedly reduced the incorporation of exogenous dihydrosphingosine into sphingolipids (Zanolari et al., 2000).
SPP-1 and cell fate decisions
If S1P indeed plays a crucial role in regulating the key step in biosynthesis of ceramide, this might be the explanation for the opposing actions of S1P and ceramide on apoptosis and why their dynamic balance is tightly coupled and conversely regulated. Whereas ceramide levels increase in response to stress stimuli, suppression of apoptosis is associated with increases in S1P levels and decreases in ceramide (for reviews see Hannun, 1996; Kolesnick and Hannun, 1999; Spiegel and Milstien, 2000b). Likewise, the balance between the levels of ceramide and S1P, regulated by LBP1, is also critical for survival and resistance to environmental stress in yeast (Mao et al., 1997, 1999; Mandala et al., 1998; Skrzypek et al., 1999), suggesting that the sphingolipid rheostat is an evolutionarily conserved stress regulatory mechanism.
Insertion of the trans 4,5 double bond into ceramide by the desaturase is an important step because ceramide, and not dihydroceramide, induces apoptosis (Hannun, 1996; Kolesnick and Hannun, 1999; Scaffidi et al., 1999). Remarkably, although dihydro-S1P is also a substrate for recombinant SPP-1 in situ, forming dihydrosphingosine (60 pmol/nmol phospholipid), which is then converted by an FB1-sensitive ceramide synthase to dihydroceramide, our finding that it did not accumulate suggests that dihydroceramide may be more efficiently used for sphingomyelin and/or glycosphingolipid biosynthesis than ceramide. Alternatively, its translocation to the Golgi apparatus may be more rapid. In this regard, whereas glucosylceramide synthase in the cis-Golgi receives ceramide via vesicular transport, nonvesicular transport from the ER to the Golgi apparatus has been described in mammalian cells and yeast (Kok et al., 1998; Funato and Riezman, 2001). Hence, ceramide, or more likely, dihydroceramide, can be translocated from the ER to the trans-Golgi via membrane contact sites to sphingomyelin and glucosylceramide synthases (Funato and Riezman, 2001). Thus, we are suggesting that ceramide and dihydroceramide may have different biosynthetic trafficking and that vesicular and nonvesicular pathways of ceramide versus dihydroceramide transport may serve special functions (Fig. 11). Because membrane contact sites have often been observed between the ER and mitochondria (Marsh et al., 2001), it is also possible that these could be responsible for the delivery of apoptotic ceramide, but not dihydroceramide, to the mitochondria. Because dihydroceramides are much less potent than ceramides in the induction of apoptosis (Hannun, 1996; Kolesnick and Hannun, 1999; Scaffidi et al., 1999), our suggestion of divergence in the regulation of ceramide and dihydroceramide synthesis has important implications, not only for sphingolipid metabolism, but also for their distinct roles in apoptosis.
Ceramide generation and apoptosis
Ceramide is now emerging as an important component of mitochondrial-dependent apoptosis and in the regulation of stress responses (Hannun, 1996; Kolesnick and Hannun, 1999; Scaffidi et al., 1999). Although it was originally proposed that ceramide was generated from sphingomyelin by the activation of one or more sphingomyelinases, a flurry of recent studies have begun to implicate ceramide generated from de novo sphingolipid biosynthesis in apoptosis (Bose et al., 1995; Kolesnick and Hannun, 1999; Perry et al., 2000; Kroesen et al., 2001). There is convincing evidence that ceramide synthase as well as serine palmitoyltransferase are activated during apoptosis (Bose et al., 1995; Perry et al., 2000; Kroesen et al., 2001). In agreement, we found that whereas S1P induced ceramide biosynthesis, particularly C16-ceramide, and apoptosis in mSPP-1 transfectants, dihydro-S1P had no effect on ceramide levels nor did it induce apoptosis. Interestingly, induction of apoptosis through B cell receptor cross-linking occurs via de novogenerated C16-ceramide (Kroesen et al., 2001). In addition, in the presence of FB1, both S1P and dihydro-S1P increased levels of sphingoid bases and induced cell death. These results are in concordance with several recent reports suggesting that sphingoid bases are also apoptotic signaling molecules (Gudz et al., 1997; Cuvillier et al., 2000; Kagedal et al., 2001).
Recent studies suggest a crucial role for de novogenerated C16-ceramide in mitochondrial damage leading to downstream activation of caspases and apoptosis (Kroesen et al., 2001). However, to date, how de novogenerated ceramide is transported from the ER to mitochondria is not known, although as discussed above, close membrane contact sites (Marsh et al., 2001) could be involved. Alternatively, ceramide might be generated from sphingosine in mitochondria via a mitochondrial ceramide synthase (Shimeno et al., 1995). More recently, a novel mitochondrial ceramidase with reciprocal (ceramide synthase) activity that shows strong preference for sphingosine over dihydrosphingosine as substrate has been cloned (El Bawab et al., 2001). Thus, sphingosine, but not sphinganine, formed at the ER by mSPP-1 might be transported to the mitochondria and serve as substrate for either of these ceramide synthase activities (Fig. 11). This ceramide may induce apoptosis due to mitochondria damage, generation of reactive oxygen species, release of cytochrome C, and subsequent activation of caspase activity. Interestingly, C16-ceramide, but not dihydroceramide, can form stable channels in membranes that are large enough to allow cytochrome C permeation and could be involved in initiating mitochondrial-dependent apoptosis (Siskind and Colombini, 2000). In agreement, elevation of endogenous ceramide specifically in mitochondria, but not in other subcellular compartments, resulted in the induction of apoptosis (Birbes et al., 2001), further supporting a role of endogenous mitochondrial ceramide in regulating apoptosis.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture
NIH 3T3 fibroblasts (American Type Culture Collection [ATCC] accession No. CRL-1658) and human embryonic kidney cells (HEK 293; ATCC CRL-1573) were cultured in high glucose DME containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine supplemented with 10% calf serum or FBS, respectively (Olivera et al., 1999). Cells were transfected with Myc-pcDNA3 alone or with Myc-pcDNA3 containing mSPP-1 construct using Lipofectamine Plus (Life Technologies) as previously described (Mandala et al., 2000). Transfection efficiencies were typically 30% and 60% for NIH 3T3 and HEK 293 cells, respectively. Stable transfectants of HEK 293 cells were selected in medium containing 1 g/liter G418.
S1P phosphohydrolase activity
HEK 293 cells (2 x 106) were seeded in 100-mm poly-D-lysinecoated plates and cultured to confluency. Cells were washed twice with ice-cold PBS and scraped on ice with 1 ml of buffer A (100 mM Hepes, pH 7.5, containing 10 mM EDTA, 1 mM DTT, and 10 µg/ml each leupeptin, aprotinin, and soybean trypsin inhibitor). Cells were freezethawed seven times and then centrifuged at 100,000 g for 1 h. Crude membranes were resuspended in buffer A and protein concentrations were determined by the Bradford assay (Bio-Rad Laboratories). 32P-labeled S1P was prepared with recombinant SPKH1a as previously described (Olivera et al., 2000). mSPP-1 activity was determined by adding 32P-labeled S1P (2 nmol, 100,000 cpm, 0.3% BSA complex) to membrane fractions (4 µg) in 200 µl buffer A, and S1P phosphohydrolase activity was determined as recently described (Le Stunff et al., 2002). Similar results were obtained by measurement of disappearance of labeled S1P or by formation of sphingosine (Le Stunff et al., 2002). Where indicated, 32P-labeled dihydro-S1P was used as substrate.
Immunofluorescence and confocal microscopy
Vector or MycSPP-1 transfectants grown on glass coverslips coated with collagen I were incubated overnight in DME supplemented with 2 µg/ml insulin, 2 µg/ml transferrin, and 20 µg/ml BSA. Cells were washed with PBS, fixed for 20 min at room temperature with 4% paraformaldehyde in 5% sucrose, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. After washing, cells were incubated for 45 min with primary antibodies for Myc and calnexin in PBS containing 0.1% BSA, and then for 45 min with the corresponding secondary antibodies conjugated with Texas red. In some experiments, Alexa Fluor®488 fluorescent phalloidin or Texas red WGA were also included in the secondary antibody incubation. To visualize mitochondria, cells were incubated with 500 nM MitoTracker green for 30 min at 37°C before fixation. Coverslips were mounted on glass slides using an Anti-Fade kit and examined by confocal microscopy. Images were collected by an Olympus Fluoview laser scanning microscope equipped with argon (488 nm) and krypton (568 nm and 647 nm) lasers and a 60X/1.4 NA PlanApo lens. Quantitative image analysis was performed using Metamorph image processing software. To avoid fluorescence crossover between the channels, FITC and Texas red images were collected separately using the appropriate laser excitation (488 nm and 568 nm, respectively) and then merged.
Subcellular fractionation
HEK 293 cells overexpressing Myc-tagged mSPP-1 were rinsed twice in ice-cold PBS, resuspended in buffer A containing 0.25 M sucrose, and homogenized in a Dounce homogenizer at 4°C. Subcellular fractionation was performed by sequential centrifugation: lysates were centrifuged at 1,000 g for 5 min at 4°C to remove unbroken cells and nuclei, postnuclear supernatants were further centrifuged for 15 min at 17,000 g at 4°C, and the pellet P1 (intracellular membrane fraction containing mitochondria, ER, and Golgi) was resuspended in the same buffer. The remaining supernatant was centrifuged at 100,000 g for 1 h at 4°C to obtain cytosol and pellet P2 containing the plasma membranes.
Western blot analysis
Equal amounts of protein were separated on 7.5 or 10% SDS-PAGE and electroblotted onto nitrocellulose membranes for 1 h at 100 V and 4°C. Blots were blocked in 5% nonfat dry milk in TBS containing 0.1% Tween-20 (TBST) for 2 h at room temperature and probed with anti-Myc mAb or with anticytochrome oxidase II, anti-PDI, or antiv-integrin antibodies as markers for mitochondria, ER, and plasma membrane, respectively. After washing three times with TBST, blots were incubated with secondary antibodies for 1 h at room temperature. Protein bands were visualized by ECL using Super Signal (Pierce Chemical Co.).
Measurement of mass levels of sphingosine and ceramide
HEK 293 cells (5 x 105) were seeded in six-well poly-L-lysinecoated plates. Cells were incubated with 5 µM S1P or dihydro-S1P for the indicated time periods. Mass levels of sphingosine and ceramide and total phospholipids in cellular lipid extracts were measured essentially as previously described (Edsall et al., 1997).
Labeling with L-3-[3H]serine and [3H]palmitic acid
Vector- or mSPP-1transfected HEK 293 cells were incubated without or with 5 µM S1P or dihydro-S1P in the absence or presence of [3H]palmitic acid (10 µCi/ml) or L-3-[3H]serine (30 µCi/ml). 24 h later, cells were scraped twice in 600 µl of methanol/concentrated HCl (200:2). Extracts were sonicated and 600 µl of chloroform and 500 µl of H2O were added, mixed, and phases were separated by the addition of 600 µl 2 M KCL and 600 µl chloroform and vigorous vortexing. Aliquots of the organic phases (corresponding to 5 x 106 cpm) were spotted on silica gel plates and developed in chloroform/acetic acid (9:1). Labeled sphingolipids were visualized by autoradiography and [3H]ceramide was quantified by a radiochromatogram scanner (Bioscan).
Staining of apoptotic nuclei
Apoptosis was assessed by staining cells with 8 µg/ml bisbenzimide trihydrochloride (Hoechst 33258) in 30% glycerol/PBS for 10 min at room temperature. The percentage of apoptotic cells, distinguished by condensed, fragmented nuclear regions, was determined as previously described (Olivera et al., 1999). A minimum of 500 cells were scored in a double blinded manner.
Reproducibility of data
Experiments were repeated at least three times with consistent results and statistical differences were determined by ANOVA. Statistically different groups are indicated by an asterisk (P 0.05).
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
Submitted: 26 March 2002
Revised: 6 August 2002
Accepted: 6 August 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alderton, F., P. Darroch, B. Sambi, A. McKie, I.S. Ahmed, N. Pyne, and S. Pyne. 2001. G-protein-coupled receptor stimulation of the p42/p44 mitogen-activated protein kinase pathway is attenuated by lipid phosphate phosphatases 1, 1a, and 2 in human embryonic kidney 293 cells. J. Biol. Chem. 276:1345213460.
Birbes, H., S. El Bawab, Y.A. Hannun, and L.M. Obeid. 2001. Selective hydrolysis of a mitochondrial pool of sphingomyelin induces apoptosis. FASEB J. 15:26692679.
Brindley, D.N., and D.W. Waggoner. 1998. Mammalian lipid phosphate phosphohydrolases. J. Biol. Chem. 273:2428124284.
Cuvillier, O., L. Edsall, and S. Spiegel. 2000. Involvement of sphingosine in mitochondria-dependent fas-induced apoptosis of type II jurkat T cells. J. Biol. Chem. 275:1569115700.
Dickson, R.C., E.E. Nagiec, M. Skrzypek, P. Tillman, G.B. Wells, and R.L. Lester. 1997. Sphingolipids are potential heat stress signals in Saccharomyces. J. Biol. Chem. 272:3019630200.
Edsall, L.C., G.G. Pirianov, and S. Spiegel. 1997. Involvement of sphingosine 1-phosphate in nerve growth factor-mediated neuronal survival and differentiation. J. Neurosci. 17:69526960.
El Bawab, S., H. Birbes, P. Roddy, Z.M. Szulc, A. Bielawska, and Y.A. Hannun. 2001. Biochemical characterization of the reverse activity of rat brain ceramidase. A CoA-independent and fumonisin B1-insensitive ceramide synthase. J. Biol. Chem. 276:1675816766.
Funato, K., and H. Riezman. 2001. Vesicular and nonvesicular transport of ceramide from the ER to the Golgi apparatus in yeast. J. Cell Biol. 155:949959.
Futerman, A.H., B. Stieger, A.L. Hubbard, and R.E. Pagano. 1990. Sphingomyelin synthesis in rat liver occurs predominantly at the cis and medial cisternae of the Golgi apparatus. J. Biol. Chem. 265:86508657.
Gillard, B.K., R.G. Clement, and D.M. Marcus. 1998. Variations among cell lines in the synthesis of sphingolipids in de novo and recycling pathways. Glycobiology. 8:885890.
Gudz, T.I., K.Y. Tserng, and C.L. Hoppel. 1997. Direct inhibition of mitochondrial respiratory chain complex III by cell-permeable ceramide. J. Biol. Chem. 272:2415424158.
Hannun, Y. 1996. Functions of ceramide in coordinating cellular responses to stress. Science. 274:18551859.
Hla, T., M.J. Lee, N. Ancellin, J.H. Paik, and M.J. Kluk. 2001. Lysophospholipids-receptor revelations. Science. 294:18751878.
Hobson, J.P., H.M. Rosenfeldt, L.S. Barak, A. Olivera, S. Poulton, M.G. Caron, S. Milstien, and S. Spiegel. 2001. Role of the sphingosine-1-phosphate receptor EDG-1 in PDGF-induced cell motility. Science. 291:18001803.
Hooks, S.B., W.L. Santos, D.S. Im, C.E. Heise, T.L. Macdonald, and K.R. Lynch. 2001. Lysophosphatidic acid induced mitogenesis is regulated by lipid phosphate phosphatases and is Edg-receptor independent. J. Biol. Chem. 276:46114621.
Jenkins, G.M., A. Richards, T. Wahl, C. Mao, L. Obeid, and Y. Hannun. 1997. Involvement of yeast sphingolipids in the heat stress response of Saccharomyces cerevisiae. J. Biol. Chem. 272:3256632572.
Kok, J.W., T. Babia, K. Klappe, G. Egea, and D. Hoekstra. 1998. Ceramide transport from endoplasmic reticulum to Golgi apparatus is not vesicle-mediated. Biochem. J. 333:779786.[Medline]
Kroesen, B.J., B. Pettus, C. Luberto, M. Busman, H. Sietsma, L. de Leij, and Y.A. Hannun. 2001. Induction of apoptosis through B-cell receptor cross-linking occurs via de novo generated C16-ceramide and involves mitochondria. J. Biol. Chem. 276:1360613614.
Le Stunff, H., C. Peterson, R. Thornton, S. Milstien, S.M. Mandala, and S. Spiegel. 2002. Characterization of murine sphingosine-1-phosphate phosphohydrolase. J. Biol. Chem. 277:89208927.
Mandala, S.M., R. Thornton, Z. Tu, M.B. Kurtz, J. Nickels, J. Broach, R. Menzeleev, and S. Spiegel. 1998. Sphingoid base 1-phosphate phosphatase: a key regulator of sphingolipid metabolism and stress response. Proc. Natl. Acad. Sci. USA. 95:150155.
Mandala, S.M., R. Thornton, I. Galve-Roperh, S. Poulton, C. Peterson, A. Olivera, J. Bergstrom, M.B. Kurtz, and S. Spiegel. 2000. Molecular cloning and characterization of a lipid phosphohydrolase that degrades sphingosine-1-phosphate and induces cell death. Proc. Natl. Acad. Sci. USA. 97:78597864.
Mao, C., M. Wadleigh, G.M. Jenkins, Y.A. Hannun, and L.M. Obeid. 1997. Identification and characterization of Saccharomyces cerevisiae dihydrosphingosine-1-phosphate phosphatase. J. Biol. Chem. 272:2869028694.
Marsh, B.J., D.N. Mastronarde, K.F. Buttle, K.E. Howell, and J.R. McIntosh. 2001. Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography. Proc. Natl. Acad. Sci. USA. 98:23992406.
Mattie, M., G. Brooker, and S. Spiegel. 1994. Sphingosine-1-phosphate, a putative second messenger, mobilizes calcium from internal stores via an inositol trisphosphate-independent pathway. J. Biol. Chem. 269:31813188.
Meyer zu Heringdorf, D., H. Lass, R. Alemany, K.T. Laser, E. Neumann, C. Zhang, M. Schmidt, U. Rauen, K.H. Jakobs, and C.J. van Koppen. 1998. Sphingosine kinase-mediated Ca2+ signalling by G-protein-coupled receptors. EMBO J. 17:28302837.
Morita, Y., G.I. Perez, F. Paris, S.R. Miranda, D. Ehleiter, A. Haimovitz-Friedman, Z. Fuks, Z. Xie, J.C. Reed, E.H. Schuchman, et al. 2000. Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy. Nat. Med. 6:11091114.[CrossRef][Medline]
Olivera, A., T. Kohama, L.C. Edsall, V. Nava, O. Cuvillier, S. Poulton, and S. Spiegel. 1999. Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival. J. Cell Biol. 147:545558.
Perry, D.K., J. Carton, A.K. Shah, F. Meredith, D.J. Uhlinger, and Y.A. Hannun. 2000. Serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis. J. Biol. Chem. 275:90789084.
Qie, L., M.M. Nagiec, J.A. Baltisberger, R.L. Lester, and R.C. Dickson. 1997. Identification of a Saccharomyces gene, LCB3, necessary for incorporation of exogenous long chain bases into sphingolipids. J. Biol. Chem. 272:1611016117.
Rosenfeldt, H.M., J.P. Hobson, M. Maceyka, A. Olivera, V.E. Nava, S. Milstien, and S. Spiegel. 2001. EDG-1 links the PDGF receptor to Src and focal adhesion kinase activation leading to lamellipodia formation and cell migration. FASEB J. 15:26492659.
Scaffidi, C., I. Schmitz, J. Zha, S.J. Korsmeyer, P.H. Krammer, and M.E. Peter. 1999. Differential modulation of apoptosis sensitivity in CD95 type I and type II cells. J. Biol. Chem. 274:2253222538.
Shimeno, H., S. Soeda, M. Yasukouchi, N. Okamura, and A. Nagamatsu. 1995. Fatty acyl-Co A: sphingosine acyltransferase in bovine brain mitochondria: its solubilization and reconstitution onto the membrane lipid liposomes. Biol. Pharm. Bull. 18:13351339.[Medline]
Siskind, L.J., and M. Colombini. 2000. The lipids C2- and C16-ceramide form large stable channels. Implications for apoptosis. J. Biol. Chem. 275:3864038644.
Skrzypek, M.S., M.M. Nagiec, R.L. Lester, and R.C. Dickson. 1999. Analysis of phosphorylated sphingolipid long-chain bases reveals potential roles in heat stress and growth control in Saccharomyces. J. Bacteriol. 181:11341140.
Spiegel, S., and S. Milstien. 2000b. Sphingosine-1-phosphate: signaling inside and out. FEBS Lett. 476:5567.[CrossRef][Medline]
Spiegel, S., and S. Milstien. 2002. Sphingosine 1-phosphate, a key cell signaling molecule. J. Biol. Chem. 277:2585125854.
Stukey, J., and G.M. Carman. 1997. Identification of a novel phosphatase sequence motif. Protein Sci. 6:469472.
Van Brocklyn, J.R., M.J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O. Cuvillier, D.M. Thomas, P.J.P. Coopman, S. Thangada, T. Hla, and S. Spiegel. 1998. Dual actions of sphingosine-1-phosphate: extracellular through the Gi-coupled orphan receptor edg-1 and intracellular to regulate proliferation and survival. J. Cell Biol. 142:229240.
van Echten-Deckert, G., A. Zschoche, T. Bar, R.R. Schmidt, A. Raths, T. Heinemann, and K. Sandhoff. 1997. cis-4-Methylsphingosine decreases sphingolipid biosynthesis by specifically interfering with serine palmitoyltransferase activity in primary cultured neurons. J. Biol. Chem. 272:1582515833.
Waggoner, D.W., J. Xu, I. Singh, R. Jasinska, Q.X. Zhang, and D.N. Brindley. 1999. Structural organization of mammalian lipid phosphate phosphatases: implications for signal transduction. Biochim. Biophys. Acta. 1439:299316.[Medline]
Xia, P., J.R. Gamble, K.A. Rye, L. Wang, C.S. Hii, P. Cockerill, Y. Khew-Goodall, A.G. Bert, P.J. Barter, and M.A. Vadas. 1998. Tumor necrosis factor- induces adhesion molecule expression through the sphingosine kinase pathway. Proc. Natl. Acad. Sci. USA. 95:1419614201.
Zanolari, B., S. Friant, K. Funato, C. Sutterlin, B.J. Stevenson, and H. Riezman. 2000. Sphingoid base synthesis requirement for endocytosis in Saccharomyces cerevisiae. EMBO J. 19:28242833.