THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 44, pp. 37118 –37129, November 4, 2005 Printed in the U.S.A.

SphK1 and SphK2, Sphingosine Kinase Isoenzymes with Opposing Functions in Sphingolipid Metabolism*
Received for publication, February 25, 2005, and in revised form, August 4, 2005 Published, JBC Papers in Press, August 23, 2005, DOI 10.1074/jbc.M502207200

Michael Maceyka‡, Heidi Sankala‡, Nitai C. Hait‡, Herve Le Stunff‡1, Hong Liu‡, Rachelle Toman‡, Claiborne Collier‡, ´ Min Zhang§, Leslie S. Satin§, Alfred H. Merrill, Jr.¶, Sheldon Milstien , and Sarah Spiegel‡2 From the Departments of ‡Biochemistry and §Pharmacology and Toxicology, Virginia Commonwealth University School of Medicine, Richmond, Virginia 23298, the ¶Georgia Institute of Technology, School of Biology, Atlanta, Georgia 30332, and the Laboratory of Cellular and Molecular Regulation, National Institutes of Mental Health, Bethesda, Maryland 20892
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 required for SphK2-induced apoptosis, as cell death and cytochrome c release was abrogated by inhibition of the mitochondrial Ca2 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 [3H]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. may also have intracellular functions important for calcium homeostasis (8), cell growth (9, 10), and suppression of apoptosis (11–14). Like other lipid mediators, S1P levels are tightly regulated by the balance between synthesis, catalyzed by sphingosine kinase (SphK), irreversible cleavage by S1P lyase, and reversible dephosphorylation to sphingosine by specific S1P phosphatases. Two distinct SphK isoforms, SphK1 and SphK2, have been cloned and characterized (15, 16). Diverse external stimuli, particularly growth and survival factors, stimulate SphK1 and intracellularly generated S1P has been implicated in their mitogenic and anti-apoptotic effects (10, 13, 17–24). Expression of SphK1 enhanced proliferation and growth in soft agar, promoted the G1-S transition, protected cells from apoptosis (10, 14, 17), and induced tumor formation in mice (17, 18). However, although SphK1 and intracellularly generated S1P can signal “inside-out” to regulate cytoskeletal rearrangements and cell movement, remarkably, cell growth stimulation and suppression of apoptosis induced by SphK1 are not mediated by S1P receptors (14). In contrast to SphK1, much less is known about SphK2. Although highly similar in amino acid sequence and possessing five evolutionarily conserved domains found in all SphKs (25), SphK2 diverges in its amino terminus and central region. These two isoenzymes have different kinetic properties and also differ in developmental and tissue expression (16), implying that they may have distinct physiological functions. Indeed, rather than promoting growth and survival, SphK2 suppressed growth and enhanced apoptosis that was preceded by cytochrome c release and activation of caspase-3 (26, 27). Moreover, SphK2-induced apoptosis was independent of activation of S1P receptors (26). Sequence analysis revealed that SphK2 contains a 9-amino acid motif similar to that present in Bcl2 homology domain 3 (BH3)-only proteins, a proapoptotic subgroup of the Bcl-2 family. As with other BH3-only proteins, co-immunoprecipitation demonstrated that SphK2 interacted with Bcl-xL (26). However, mutation of the conserved leucine residue present in all BH3 domains (28) only partially reduced apoptosis induced by SphK2 (26), suggesting that the SphK2 protein possesses additional determinants important for this function. In this study, we found that the catalytic activity of SphK2 and its localization to the ER during stress contribute to its apoptosis-inducing properties. Our results provide clues to how SphK1 and SphK2, two very closely related enzymes that use the same substrate and produce the same product, have opposite effects on cell survival and reveal their opposing functions in regulation of sphingolipid metabolism.

Downloaded from www.jbc.org at The University of Birmingham, on January 31, 2011

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),

* This work was supported by NCI National Institutes of Health Grants R01CA61774 (to
S. S.), R01DK46409 (to L. S. S.) and GM069338 (to A. H. M.), and a NRSA-Kirschstein postdoctoral fellowship (to M. M.). Confocal microscopy was supported in part by National Institutes of Health Grant P30 CA16059 to the Massey Cancer Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Present address: Laboratoire de Physiopathologie de la Nutrition, CNRS UMR 7059, 75251 Paris Cedex 05, France. 2 To whom correspondence should be addressed: 1101 E. Marshall St., Richmond, VA 23298-0614. Tel.: 804-828-9330; Fax: 804-828-8994; E-mail: sspiegel@vcu.edu. 3 The abbreviations used are: S1P, sphingosine 1-phosphate; BAPTA-AM, 1,2-bis(2aminophenoxy)ethane-N,N,N ,N -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.

EXPERIMENTAL PROCEDURES
Materials—[ -32P]ATP, [3H]palmitic acid, and L-3-[3H]serine were purchased from Amersham Biosciences. S1P and sphingosine were obtained from Biomol (Plymouth Meeting, PA). The internal standards for quantitation of the sphingolipids by ESI-MS/MS were obtained from

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VOLUME 280 • NUMBER 44 • NOVEMBER 4, 2005

As the calreticulin antibody stained poorly after methanol fixation. and a photon counter (IonOptix. Mammalian expression constructs for SphK1. and images were collected separately using the appropriate laser excitation and then merged. IN). Lysates were then centrifuged at 14. The remaining supernatants were centrifuged at 100. and 10 mM HEPES (pH 7. OR). Subcellular Fractionation—Cells were suspended in buffer containing 20 mM HEPES (pH 7.5% Triton X-100 in PBS for 3 min. CA). 2 mM sodium orthovanadate. Alternatively. To analyze sphingolipids. Labeling with L-3-[3H]Serine and [3H]Palmitic Acid—Cells were serum starved for 18 h and then incubated without or with sphingosine (5 M) in the presence of [3H]palmitic acid (10 Ci/ml) or L-3[3H]serine (30 Ci/ml). 250 mM sucrose. fragmented nuclear regions exactly as described (26).25 M sucrose. on January 31. and phases were separated by addition of 600 l of 2 M KCl and 600 l of chloroform with vigorous vortexing. 2 mM MgCl2. SphK1 and SphK1-G82D were cloned into pCMV/myc/ER (Invitrogen) to generate constructs with ER targeting sequences. Molecular Probes) in buffer containing 116. aliquots of the organic phases (corresponding to 5 106 cpm) were separated by TLC on silica gel plates developed in chloroform/acetic acid (9:1. 1 mM phenylmethylsulfonyl fluoride. 2005 • VOLUME 280 • NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 37119 . For determination of ceramide. cytochrome c. Apoptosis Assays—Apoptosis was measured by staining nuclei with the Hoechst dye bisbenzimide and apoptotic cells were distinguished by condensed. The QuikChange site-directed mutagenesis kit (Stratagene) was used to prepare the G213-L219A SphK2 double mutant with the G213 primers using SphK2-L219A as template. and Golgi. Equal amounts (20 g) of proteins were separated by SDS-PAGE. 1. and rabbit secondary antibodies were from Jackson ImmunoResearch (West Grove. Sphingosine Kinase Activity—SphK2 activity in cytosol and 100. 1 mM EDTA. a galvanometer-driven mirror to select between 340 and 380 nm. 5 mM KCl. cytosolic fractions were prepared by resuspending cells in lysis buffer containing 75 mM NaCl. Alternatively. ER. cells were washed with ice-cold PBS and scraped in 500 l of buffer containing 50 mM HEPES (pH 7.4). fixed for 20 min at room temperature with 3. cells were scraped in 2 600 l of methanol/concentrated HCl (200:2. There was no fluorescence crossover between the channels. then for 30 min with the corresponding secondary antibodies. PA). Extracts were sonicated. a photomultiplier. 4 mM sodium pyrophosphate. Labeled sphingolipids were visualized by autoradiography after spraying with En3Hance (DuPont). Cells were washed with PBS. Monoclonal anti-HA antibody (3F10) was from Roche (Indianapolis. G418 was from Invitrogen. The postnuclear supernatants were centrifuged for 10 min at 5. Antibodies to calreticulin and calnexin were from Stressgen Biotechnologies (San Diego. v/v) as the developing solvent.SphK Isoenzymes in Sphingolipid Metabolism Avanti Polar Lipids (Alabaster.4). AL). and wild type and bax / /bak / mouse embryonic fibroblasts (a kind gift of Dr. Yuan (30). methanol.7% formalin. Coverslips were mounted on glass slides.2 M KOH in methanol for 2 h at 37 °C to degrade glycerolipids prior to TLC analysis. Where indicated. 1:500 protease inhibitor mixture (Sigma).000 g for 15 min to obtain the intracellular membrane fraction containing mitochondria. Mass Spectrometry of Sphingolipids and Metabolites—Cells were serum-starved for 18 h and incubated without or with sphingosine (5 Downloaded from www. Immunoblotting Analysis—Unless otherwise indicated. Alternatively. Cells and Plasmids—NIH 3T3 fibroblasts.000 g for 15 min.8 mM CaCl2. MA).org at The University of Birmingham. 31. CA). and ceramides were quantified with a Bioscan 2000 radiochromatogram scanner. Subcellular fractionation was performed by sequential centrifugation. Caspase-12 antibody was kindly provided by Dr. 150 mM NaCl.4) for 20 min at 37 °C (41). [Ca2 ]i was also measured with a SLM/Aminco fluorescence spectrophotometer (model AB2). apoptosis was measured with the Cell Death Detection ELISAPLUS kit (Roche Applied Science) that determines cytoplasmic histone-associated DNA fragments. Data were acquired and analyzed with Ionwizard software and [Ca2 ]i was calculated from a cell-free calibration curve. For analysis of cytochrome c. and HA (rabbit) were from Santa Cruz Biotechnology (Santa Cruz. 100 mM NaF. 600 l of chloroform and 500 l of H2O were added and vortexed. CA). 2 mM MgCl2. 1% Triton X-100. In some experiments. ERK2. v/v). The ratio of fluorescence emission (510 nm) of Ca 2-bound Fura-2 (excitation at 340 nm) to unbound Fura-2 (excitation at 380 nm) was measured in at least 25 fields of 1–5 cells each using a xenon arc excitation source. 1 mM NaH2PO4. Coverslips were broken. and perfused at 1 ml/min with the same buffer. a condition in which SphK2 activity is optimal and SphK1 is inhibited. postnuclear supernatants were directly centrifuged at 17. cells were fixed in methanol at 20 °C for 5 min. and images were collected either on a Nikon TE300 fluorescence microscope with a 60 objective and a CoolSnap CCD run with MetaMorph software or on a Zeiss LSM 510 laser confocal microscope. Intracellular Calcium Measurements—Cells plated on glass coverslips were loaded with 1 M Fura-2 acetoxymethyl ester (Fura-2AM. Real-time PCR was performed with pre-mixed primerprobe sets obtained from Applied Biosystems (Foster City. calnexin was used instead as a marker for the ER in these experiments. 1 mM EDTA. Horseradish peroxidase-conjugated and highly cross-adsorbed fluorophore conjugated anti-rat. Labeled phospholipids were also resolved by TLC with chloroform. Sequences of all constructs were confirmed by direct DNA sequencing. Polyclonal rabbit antibodies to SphK2 were raised against a unique peptide sequence (QALHIQRLRPKPEARPR) as previously described (29). Immunofluorescence and Confocal Microscopy—Transfectants grown on glass coverslips were treated as indicated in the figure legends. and immunopositive bands visualized by enhanced chemiluminescence (Pierce Chemical Co). SphK2.000 g to generate the heavy membrane mitochondria fraction. Milton.8 mM NaCl. Reverse Transcriptase-PCR—Total RNA was isolated with TRIzol reagent (Invitrogen) and was reverse transcribed with Superscript II (Invitrogen). an aliquot of the lipid extract (5 106 cpm) was subjected to alkaline hydrolysis in 0. v/v) (33).000 g for 1 h to obtain cytosol (S) and pelleted plasma membrane fractions. Serum and medium were from BioFluids (Rockville. J. and SphK2-L219A were described previously (10. 26. mouse. 6 h later. CA). Harada) were cultured and transfected as described (26). and 0. then blocked for 1 h with 4% bovine serum albumin. and permeabilized with 0.1 mM glucose. essentially as described (32). blots were scanned and densitometric analysis was performed with AlphaEaseFC software (Alpha Innotec. and 700 g/ml digitonin. 32). 2011 NOVEMBER 4. HEK 293 cells. The supernatants were then centrifuged at 17. Specific activity is expressed as picomoles of S1P formed per min/mg of protein. Olympus). San Leandro. SphK1-G82D. MD). 11. 10 g/ml each of aprotinin and leupeptin.000 g membrane fractions was determined in the presence of sphingosine complexed with 4 mg/ml bovine serum albumin and [ -32P]ATP in buffer containing 1 M KCl as previously described (16). shards placed in a perfusion chamber of an inverted fluorescence microscope (IX-50. After washing. 20 mM CaCl2 (60:20:4. H. transblotted to nitrocellulose. 10 mM KCl. Antibodies to myc (9E10). cells were incubated for 30 – 45 min with primary antibodies in PBS containing 1% bovine serum albumin.000 g for 15 min to obtain the light membrane fraction containing ER and Golgi. 8 mM Na2HPO4. 1 mM EDTA. Antibody to mitochondrial pyruvate dehydrogenase E1 was from Molecular Probes (Eugene.jbc.

Cells were serum starved for 24 h. **. including the interleukin-12 receptor 1 (37). The catalytically inactive mutant SphK2G213E also was cytosolic when cells were cultured in the presence of serum (Fig. D. is found in internal membranes. A and B). *. The double G213E/L219A mutation not only eliminated the enzymatic activity of SphK2 (Fig. M) for 6 h. 3A) and co-localization with calnexin was enhanced upon serum withdrawal (Fig. 1C). Golgi. Blots were stripped and re-probed with ERK2 antibody to demonstrate equal loading.org at The University of Birmingham.01. Specific enzyme markers were used to substantiate and define purity of the different compartments. electrospray ionization-tandem mass spectrometry as described previously (34). SphK2-L219A. we examined whether its enzymatic activity and formation of S1P was also important for its apoptotic effects. surprisingly. epitope-tagged SphK2 is distributed diffusely throughout the cytoplasm of NIH 3T3 fibroblasts cultured in the presence of serum (Fig. 2005 . site-directed mutagenesis of the equivalent residue in SphK2 (G213E) resulted in a complete loss of sphingosine phosphorylating activity (Fig. p 0. Similarly. A and B). 3D). as the levels of the mutant SphK2 proteins were essentially the same as the wild-type enzyme (Fig. Statistical Analysis—Experiments were repeated at least three times with consistent results. the enzymatic activities were mainly cytosolic (10.SphK Isoenzymes in Sphingolipid Metabolism Downloaded from www. suggesting that the catalytic activity of SphK2 was dispensable for translocation to the ER. C. lipids extracted. 1D). In stark contrast. 2A). but in all membrane fractions including internal membranes containing the ER and in the plasma membrane fraction (Fig. Confocal microscopy revealed that. calnexin (Fig. SphK2L219A also appeared to co-localize with the ER marker after serum starvation (Fig. 1. particularly 37120 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 44 • NOVEMBER 4. when overexpressed. 3B). 1. Similar results were obtained in at least three independent experiments. SphK2-G213E. p 0. 22–24). 2011 FIGURE 1. a highly conserved leucine present in all BH3 domains (28). Cells were then washed extensively with PBS and released by trypsinization. and probably endosomes (38.0.D. albeit much less than wild type SphK2 (Fig. 17. In NIH 3T3 cells transfected with V5-SphK2. SphK2 localization was examined in more detail. similar to SphK1. 16). Moreover. 1D). 4A). but not the mitochondria marker pyruvate dehydrogenase E1 . SphK2-induced apoptosis requires its BH3 domain and catalytic activity. and nuclei were stained with Hoechst. 27). Previously we have shown that mutation of leucine 219 in its putative BH3 domain. A and B). This is not so surprising as SphK2 also contains numerous putative transmembrane domains and has also been shown to interact with other membrane proteins. RESULTS Apoptosis Induced by SphK2 Requires Its Catalytic Activity—Many studies have shown that SphK1 promotes cell growth and inhibits apoptosis (10. 20. condensed nuclei indicative of apoptosis compared with the total number of green fluorescent protein-expressing cells (26). internal standards were added. A–C. Because the yeast homologue of SphK2. All members of the SphK superfamily have the ATP binding sequence (SGDGX17–21K(R)) present within the conserved C2 domain (35) and a single point mutation of the second conserved glycine residue to aspartate has been used to prepare a catalytically inactive SphK1 (35. An aliquot of cells was taken for protein and total lipid phosphate measurements. 19. SphK2-L219A. However. These effects did not result from differential expression. 1D). NIH 3T3 cells were co-transfected with green fluorescent protein and empty vector. fixed. albeit to a lesser extent than wild-type SphK2.jbc. 2B). including ER. To the rest. SphK2. protein expression was determined by immunoblotting with anti-HA antibody. For each experiment. Because SphK2-L219A with a mutated BH3 domain also had residual SphK activity (Fig. data from triplicate samples were calculated and expressed as the mean S. This suggests that SphK2 can be localized to the ER in BH3-dependent and independent manners. NIH 3T3 cells were transfected with empty vector. a fraction of SphK2 appeared to co-localize with the ER marker. Lysates were subcellularly fractionated by differential centrifugation and expression of SphK2 was determined by Western blotting. overexpression of SphK2 does the opposite. SphK2. or SphK2-G213E/L219A as indicated and SphK activity was measured in cytosol (A) and membrane (B) fractions. 39). serum deprivation for 24 h reduced expression of SphK2 in all subcellular fractions. or SphK2-G213E/L219A at a ratio of 1:5 as indicated. 1. reduced its ability to induce apoptosis (26) (Fig. suppressing cell growth and inducing apoptosis (26. and individual ceramide acyl chain species were quantified by liquid chromatography. SphK2 expression was detected not only in the cytosol. Lcb4. it totally abrogated its apoptotic activity (Fig. Apoptosis was determined by scoring the fraction of green fluorescent protein-expressing cells displaying fragmented. This catalytically inactive mutant slightly enhanced apoptosis in serum-starved NIH 3T3 fibroblasts.05. 36). on January 31. Statistics were performed using SigmaStat statistical software version 2. we noticed that in the absence of serum. Serum Starvation Increases SphK2 in the ER—Although SphK1 and SphK2 have opposite effects on apoptosis. SphK2-G213E. A minimum of 300 cells was scored in a double-blind manner. Moreover.

4A). C and D). After 48 h. culturing for 24 h in the absence of serum reduced their expression in the cytosol and a larger proportion appeared to be associated with membranes. many previous studies have shown that endogenous SphK1 is mainly cytosolic (10. 36. plasma membranes. Expression of all of the mutants was distributed between membrane fractions and the cytosol similar to wild type SphK2 (Fig. and serum starvation also reduced its level in the cytosol of these cells (Fig. we generated SphK1 and catalytically inactive SphK1-G82D mutants targeted to the ER. Right-hand panels show the superimposed merged pictures. possibly because of accumulation of misfolded protein in the ER. 5A). After serum starvation. 40). Intriguingly. The observation that expression of SphK2 in the ER increases upon serum deprivation (Figs. NIH 3T3 cells grown on coverslips were transfected with HA-tagged SphK2-G213E (A and B) and SphK2-L219A (C and D). After serum starvation. the yellow color indicates colocalization. these results suggest that the differences between pro-survival SphK1 and pro-apoptotic SphK2 are related in part to distinct subcellular localizations and spatially restricted production of S1P. In contrast to SphK2. which induced apoptosis to a similar extent as SphK2. 4C). when ER-SphK1 or SphK2 overexpressing HEK 293 cells were treated with sphingosine. Both ER-SphK1 (Fig. Although both ER-SphK1 and ER-SphK1-G82D were now membrane-associated proteins. as expected (Fig. Cells were visualized by confocal microscopy. 6D). 4C). 42). Much less is known of its intracellular actions. Representative cells of 100 cells examined are shown. FIGURE 3. 6A). To examine this possibility. targeting SphK1 to the ER converted it from anti-apoptotic into an apoptogenic protein (Fig. 6A). like the overexpressed protein. SphK2-induced Apoptosis Depends on Ca2 Release from Intracellular Stores—The role of S1P as a ligand of S1P receptors is well established (1. 38). decreased in the plasma membrane and cytosol and a much greater proportion was associated with internal membranes. noticeable in the cytosol and plasma membrane fractions. 41. 5C) co-localized with the ER marker calnexin. cells were cultured in the presence (A) or absence (B) of serum for 24 h. and stained with HA antibody (SphK2) or calnexin antibody (ER). These results indicate that SphK1 retains its catalytic activity when targeted to the ER. Collectively.jbc. fixed with methanol. Next. ER targeted SphK1 had similar SphK activity as wild type cytosolic SphK1 (Fig. Targeting SphK1 to the ER Promotes Apoptosis—Interestingly. or pyruvate dehydrogenase E1 (Mitochondria). cells were cultured for 24 h in the absence or presence of serum. although several studies have proposed that this bioactive sphingolipid may serve as a second messenger to mobilize calcium from internal sources in an inositol trisphosphate-independent manner (8. Translocation of SphK2 to the ER by serum starvation. total levels of SphK2 were reduced. whereas ER-SphK1-G82D had no enzymatic activity. 4. Localization of SphK2 mutants. 2011 NOVEMBER 4. Cells were visualized by confocal microscopy. After 48 h.org at The University of Birmingham. In contrast. on January 31. a larger proportion of the SphK2 was now present in light membrane fractions containing the ER and Golgi (Fig. which was almost as high as SphK2 (Fig. there was a 3– 4-fold increase in S1P levels. In HEK 293 cells. 5B) and ER-SphK1-G82D (Fig. This might explain the apparent greater localization of SphK2 in internal membranes of serum-starved cells detected by confocal microscopy (Fig. the yellow color indicates colocalization of SphK2 with the ER marker calnexin. fixed. 6C). and immunostained for SphK2 with antibodies to HA (left panels). 23. and cytosol (Fig. it was of interest to determine whether the different SphK2 mutants differ in their localizations and whether the BH3 motif plays a role in membrane targeting. However. endogenous SphK2. only wild-type ER-SphK1 had significant enzymatic activity. BH3-only pro- Downloaded from www. 6B). 2B). 4B). ectopically expressed SphK2 was also present in all membrane fractions and in the cytosol. Representative cells of 100 cells examined are shown. which remained predominantly cytosolic (Fig. NIH 3T3 cells grown on coverslips were transfected with HA-tagged SphK2. 2 and 3) and disappears from the cytosol suggests that its apoptotic effects might be related to production of S1P in the ER. whereas levels were not significantly increased by transfection with ER catalytically inactive SphK1 (Fig. 2005 • VOLUME 280 • NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 37121 . ER-targeted catalytically inactive SphK1-G82D only induced minimal apoptosis (Fig. Endogenous SphK2 is expressed in the plasma membrane and in internal membranes of HEK 293 cells and at much lower levels in the cytosol (Fig. serum starvation had no effect on localization of SphK1. Endogenous SphK2 was also present in internal membranes. In agreement. calnexin (ER). 6B). Right panels show the superimposed merged pictures.SphK Isoenzymes in Sphingolipid Metabolism FIGURE 2. As with wild type SphK2.

or anti-tubulin. anti-PDI. SphK2 activated pro-caspase-12 as shown by its processing (Fig. and apoptosis (43. Multiple death signals converge at mitochondria to release cytochrome c and initiate the caspase cascade (48).” Proteins (5 g from V5-SphK2 cells and 40 g from parental cells) were resolved by SDS-PAGE and immunoblotted with anti-V5 or anti-SphK2. respectively) were resolved by SDS-PAGE and immunoblotted with anti-V5 or anti-SphK2. this may be because of the high levels of expression and saturation of the ECL signal. fixed. 2005 . or SphK2-G213E/L219A.org at The University of Birmingham. Representative cells of more than 20 cells examined are shown.vintegrin. As previously reported (26). slightly enhanced apoptosis of vector transfectants. NIH 3T3 cells were transfected with empty vector. After 24 h. or parental cells were cultured in the presence or absence of serum. and cultured in the absence or presence of serum for 24 h. 7B). HA epitopetagged SphK2. Ru-360 drastically reduced SphK2-induced cell killing of serum-starved NIH 3T3 cells (Fig. an inhibitor of the mitochondrial calcium antiporter (47). as indicated. on January 31. a cell-permeant calcium chelator. Note that although SphK2 appears to be a doublet. Subcellular localization of SphK2. suggesting that mitochondrial calcium uptake may be important for SphK2-induced apoptosis. lysates from HEK 293 cells transfected with V5-SphK2 or from parental cells were subcellularly fractionated into mitochondria (HM). 7A). caspase-12 activation. mitochondria also take up and release calcium very efficiently and are strategically located close to calcium sources allowing mitochondria to shape calcium signals (44.v-integrin.5% Triton X-100. particularly that induced by the pro-apoptotic Bcl-2 protein Bax (46). Consistent with its effects on calcium. C. and cytosol (S). anti. and Golgi). Data are expressed as ratios of arbitrary optical density units. It was thus important to determine whether this increase in [Ca2 ]i was involved in propagation of the apoptotic cascade.jbc. which preceded the appearance of fragmented nuclei. Treatment with Ru-360 markedly reduced this cytochrome c release. 2011 FIGURE 4. FIGURE 5. and cytosol (S). Cells were immunostained with anti-myc antibody and visualized with fluorescein isothiocyanate-conjugated antimouse secondary antibody (left panels). 49). 7B). ER and Golgi (LM). To examine their involvement. Right-hand panels show the superimposed merged pictures. Equal amounts of proteins (5 g from SphK cells and 40 g from vector cells) from membrane fractions (M) and supernatants (S) were separated by SDS-PAGE and immunoblotted with anti-HA antibody. These DKO cells do not have defects in the handling of mitochondrial calcium but 37122 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 44 • NOVEMBER 4. Although the main intracellular Ca2 store is the ER. Because calcium uptake by mitochondria is important for cytochrome c release during apoptosis. Treatment of cells with BAPTA-AM. D. A. or ER-SphK1-G82D (C). Cells were then cultured for 24 h in serum-free medium (A) or in the presence of 10% serum (B and C). from V5-SphK2 and parental cells. blots were scanned and endogenous SphK2 levels in each fraction normalized with the appropriate loading control. Duplicate samples were immunoblotted with anti. NIH 3T3 cells transfected with V5-SphK2. Proteins (5 and 40 g. The ER marker calnexin was stained with anticalnexin antibody and visualized with Texas Red-conjugated anti-rabbit secondary antibody (center panels). SphK2 expression caused the release of cytochrome c from the mitochondria of cells cultured in the absence of serum (Fig. respectively. BAX and BAK are two pro-apoptotic proteins known to be involved in ER stress-induced calcium release. ER-SphK1 (B). Targeting cytosolic SphK1 to the ER. teins (43) have been implicated in ER calcium release and apoptosis. particularly because of perturbation of calcium homeostasis (30). Duplicate samples were immunoblotted with anti-protein-disulfide isomerase (PDI). 45). SphK2-L219A. which had no effect on calcium in empty vectortransfected cells (Fig. plasma membrane (PM). as described under “Experimental Procedures. Blots were stripped and reprobed with anti-calreticulin. SphK2-G213E. Previous studies have suggested the existence of a novel apoptotic pathway in which caspase-12 is specifically activated by ER stresses. 7D). cells were lysed and subcellularly fractionated into inner membrane (IM) (mitochondria. ER. 7C). while markedly reducing apoptosis induced by SphK2 in NIH 3T3 cells (Fig. we examined the effect of Ru-360. NIH 3T3 fibroblasts were transiently transfected with myc-tagged SphK1 (A). we utilized embryonic fibroblasts from bax / /bak / double knock-out (DKO) mice. B.SphK Isoenzymes in Sphingolipid Metabolism Downloaded from www. or anti-tubulin as specific organelle markers. In SphK2 transfectants intracellular calcium was increased within 6 –9 h after removal of serum. the yellow color indicates colocalization. and permeabilized with 0. plasma membrane (PM).

SphK1. which is readily converted to ceramide in the salvage pathway. the rate-limiting step in the synthesis of sphingoid bases. including SM and monohexosylceramides. A. Because targeting SphK1 to the ER induced apoptosis similarly as overexpression of SphK2. p 0. 9A). HEK 293 cells were transfected as indicated. Targeting SphK1 to the ER converts it from anti-apoptotic to pro-apoptotic. ER-SphK1 also increased ceramide (Fig.jbc. 53). NIH 3T3 cells were co-transfected with ER-vector. Exogenous sphingosine increased the mass of all ceramide acyl chain species. treatment with sphingosine also increased the incorporation of [3H]palmitate primarily into the lower migrating monohexosylceramide band (containing long chain ceramides) in vector and SphK2 transfectants but not in SphK1 transfectants (Fig. whereas SphK2 increased all ceramide species (Fig. p 0. Lipids were extracted and S1P was determined by ESI-MS/MS. containing long chain ceramides (C16 and C18. To this end. 1. we observed that SphK1 decreased labeling of ceramide. Inset shows SphK1 expression by Western blotting of 100. Cells were then cultured in serum-free medium and apoptosis was scored as described in the legend to Fig. SphK1. whereas SphK2 increased it (Fig.SphK Isoenzymes in Sphingolipid Metabolism band).000 g. The increased ceramide levels caused by SphK2 were reflected in an increase of the mass of ceramide-based sphingolipids. whereas SphK2 increased ceramide mass levels (Fig. on January 31. 8D). Again. In agreement with the observation that SphK2 expression increased ceramide levels. C18-sphingosine was the predominant Downloaded from www. In agreement with previous studies (52. SphK2 activity was measured with sphingosine added as a bovine serum albumin complex in the presence of 1 M KCl. respectively. SphK2 expression also changed the relative ratio of the long chain to very long chain ceramides. C. NS. 10B). 10A). SphK2 expression markedly induced apoptosis of wild type mouse embryonic fibroblasts. sphingoid bases. as this is the best method to simultaneously determine the degree of saturation of the sphingoid base and the chain length of the fatty acids (34. NIH 3T3 fibroblasts were transiently transfected with ER-Vector. SphK1 expression decreased [3H]palmitate incorporation into ceramides relative to the vector transfectants by 40% (Fig. 54). 9B). B.01. 10A). D. 10A). 8F). or ER-SphK1-G82D. the BAX/BAK DKO cells transfected with SphK2 were almost completely resistant to apoptosis induced by serum withdrawal (Fig. 10A) and SM (Fig. lower band) and very long chain ceramides (C24 and C24:1. Expression of either SphK1 or SphK2 had no discernable effect on [3H]palmitate labeling of glycerophospholipids or sphingomyelin (SM) (Fig. ESI-MS/MS also enabled us to accurately determine levels of sphingoid bases and their phosphorylated counterparts (54). In agreement with our previous results (26). while SphK1 Decreases Intracellular Ceramide—Many studies have linked an increase in ceramide to cell growth arrest and induction of apoptosis (5–7).000 g supernatants (lanes 1 and 3) and pellets (lanes 2 and 4) from ER-SphK1. 2011 FIGURE 6. 7E).org at The University of Birmingham. Data are expressed as pmol/mg of protein. it also reduced the mass levels of monohexosylceramides (Fig. the substrates for SphKs. 24:0. including monohexosylceramides (Fig. B and C). 55). Equal loading was verified with anti-calnexin antibody. Therefore. whereas elevation of S1P has been shown to play a cytoprotective role (1). cannot deliver enough calcium to the mitochondria because their [Ca2 ]ER is too low and thus are resistant to a wide range of apoptotic stimuli (50) and many ER stresses. HEK 293 cells were transfected as indicated. not only did SphK1 reduce ceramide levels. and the monohexosylceramide levels were consistently higher in SphK2 and lower in SphK1 expressing cells relative to vector transfectants (Fig. B and C). ER-SphK1.and ER-SphK1-G82Dtransfected cells. Expression of SphK1 reduced levels of all of these ceramide species. but not SM (Fig. arachidonic acid. Data are expressed as -fold activity relative to vector transfectants (16 1 pmol/mg/min). These two bands co-migrated with authentic standards (data not shown). it was of interest to compare the effects of expression of SphK1 and SphK2 on ceramide levels. followed by 24:1. and as suggested from the labeling experiments (Fig. SphK2. upper NOVEMBER 4. 8B). as well as their various chain length species. 8. 10B). 8. Intriguingly. Because sphingoid bases are acylated with various acyl chains. Conversely. a substrate for serine palmitoyltransferase. and sphingosine kinase activity was measured in cell lysates (16). then treated with 5 M sphingosine for 6 h. radioactive serine was incorporated into two bands of ceramide (Fig. whereas SphK2 increased it (Fig. 9A). including H2O2. Thus. and these ceramides are not labeled with serine. with long chain ceramides incorporating significantly more radioactivity than very long chain ceramides (Fig. SphK1 expression inhibited total incorporation of serine into ceramides. most obviously enhancing the C16 and C18 species (Fig. 7E).05. 8A). particularly C16 and C18 (Fig. 9A). However. ‡. the most abundant ceramide species in HEK 293 cells was 16:0. 8E). cells were labeled with [3H]serine. However. and 18:0 (Fig. It is also evident that when the subspecies distributions are compared in pie diagrams. Cells also make ceramide and sphingolipids in a salvage pathway by re-utilizing sphingosine from degraded sphingolipids. treatment with sphingosine increased the proportion of long-chain ceramides. SphK2 Increases. When cells were treated with sphingosine. 8A). and ceramide (43). ESIMS/MS can also simultaneously determine the mass levels of sphingolipids derived from ceramide. Moreover. are readily converted to ceramides by ceramide synthase localized to the ER (52. A and B). cells were next labeled with [3H]palmitate. or ER-SphK1-G82D together with green fluorescent protein at a 5:1 ratio. *. 8A). serum-starved for 18 h. particularly in opposing ceramide-mediated apoptosis (11. 2005 • VOLUME 280 • NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 37123 . ERSphK1. treatment with sphingosine increased the monohexosylceramides (Fig. 9. expression of SphK1 reduced. 51). consistent with their opposite roles in apoptosis and cell growth regulation. lysed. 8B). which serves as a substrate for both serine palmitoyltransferase and ceramide synthase. not significant. these results suggest that SphK1 reduces and SphK2 increases ceramide. expression of SphK2 markedly increased incorporation of radioactivity into ceramide (Fig. the effects of ER-SphK1 on ceramide levels were examined. In agreement with a previous report (53). especially in the absence of serum (Fig. Collectively. and membrane fractions were prepared by centrifugation at 100. SphK1 and SphK2 Alter the Acyl Chain Distribution of Ceramides—We next examined the effects of SphK1 and SphK2 on sphingolipid species measured by high performance liquid chromatography ESI-MS/MS.

methanol. 2011 FIGURE 7. SphK2-induced apoptosis proceeds through activation of caspase-12. or SphK2 were serum-starved for 18 h. NIH 3T3 cells expressing either vector or SphK2 were serum-starved overnight in the presence or absence of Ru-360 (10 M) and cytochrome c released to the cytosol was assessed by immunoblotting. B. in the presence of sphingo- 37124 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 44 • NOVEMBER 4.D. expression of SphK1 markedly increased dihydrosphingosine. cultured without or with 10% serum for 24 h. Data are mean S. F. Ru-360 reduces SphK2-induced cytochrome c release. as indicated. cells transfected with ER-vector or ER-SphK1 were labeled with [3H]palmitate (10 Ci/ml) in the absence or presence of 5 M sphingosine. However. phosphatidylserine. Ceramide was resolved by TLC (A–C).jbc. A. then incubated for 6 h with L-3-[3H]serine (A) (30 Ci/ ml) or [3H]palmitate (A–F) (10 Ci/ml) in the absence or presence of 5 M sphingosine. NIH 3T3 cells were co-transfected with vector or SphK2. which only occurs in central nervous system gangliosides (56). 2005 . on January 31. C. Similar results were obtained in three additional experiments. Cells were serum-starved for 6 h and [Ca2 ]i was determined in cells by epifluorescence microscopy after loading with Fura-2AM. PS. FIGURE 8. the percentage of apoptotic nuclei in cells expressing green fluorescent protein fluorescence was determined. and apoptosis determined by enzyme-linked immunosorbent assay for histone-associated DNA fragments. NIH 3T3 vector or SphK2 transfectants were deprived of serum for the indicated times. incorporation of serine and palmitate into ceramide. 24 h later. Cells were then washed and lipids extracted. SphK1. Equal loading was verified by blotting with anti-tubulin. whereas SphK1 reduces. HEK 293 cells expressing vector. Labeled lipids were visualized by autoradiography. and radioactivity quantified with a radiochromatogram scanner (C). v/v) before (D) or after (E) mild alkaline methanolysis to remove ester-linked glycerophospholipid lipids. Embryonic fibroblasts from wild type (WT) and bax / /bak / (DKO) mice were transfected with vector (open bars) or SphK2 (filled bars). monohexosylceramide.org at The University of Birmingham. NIH 3T3 cells stably expressing vector (open bars) or SphK2 (filled bars) were plated on coverslips. PI. HexCer. sphingoid base present. LPC. together with green fluorescent protein at 5:1 ratio. E. normalized to total lipid phosphate. lysophosphatidylcholine. there was a large increase in dihydro-S1P (TABLE ONE). visualized by autoradiography (A and B). SphK2 increases. with much lower levels of dihydrosphingosine (TABLE ONE). Similar results were obtained in four independent experiments. SphK2-induced apoptosis requires calcium uptake by mitochondria. Arrowheads indicate cleavage fragments. phosphatidylethanolamine. sphingomyelin. 20 mM CaCl2 (20:10:4. Glycerophospholipids and sphingolipids were resolved by TLC in a solvent system of chloroform. C20-sphingosine. Cells were then cultured in serum-free medium in the absence (None) or presence of BAPTA-AM (10 M) or Ru-360 (10 M). PC. and ceramide was extracted and resolved as above. as indicated. phosphatidylinositol.SphK Isoenzymes in Sphingolipid Metabolism Downloaded from www. Data are expressed as radioactivity incorporated into ceramide. Interestingly. PE. phosphatidylcholine. Cytosolic proteins were resolved by SDS-PAGE and analyzed by immunoblotting with antibody against caspase-12 and subsequently with anti-ERK2 antibody to show equal loading. and as a consequence.01). SphK2-induced apoptosis depends on Bax and Bak. was not detected. SphK2 expression increases [Ca2 ]i. SphK2 only modestly increased dihydro-S1P. Similar results were obtained in at least three independent experiments and statistical difference is indicated by the asterisk (p 0. D. SM. of measurements of at least 25 fields with 1–5 cells in each field.

Data are averages of triplicate determinations and are expressed as picomole of lipid/mg of protein. Numbers indicate chain length followed by the number of double bonds in the fatty acid. 2011 FIGURE 9. then treated without or with 5 M sphingosine (Sph) for 6 h as indicated. Similar results were found in two additional experiments.SphK Isoenzymes in Sphingolipid Metabolism Downloaded from www. B. data from A are presented in “pie” charts showing a proportion of each indicated ceramide species. Effects of SphK1 and SphK2 on ceramide species.jbc. on January 31. or SphK2 were serum-starved for 18 h. HEK 293 cells expressing vector.org at The University of Birmingham. A. 2005 • VOLUME 280 • NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 37125 . NOVEMBER 4. Lipids were extracted and ceramide species were determined by ESI-MS/MS. SphK1.

sphingosine-induced incorporation of [3H]palmitate into ceramide (Fig. HEK 293 cells expressing vector. Because ectopically expressed proteins may not always exactly mimic the localization and functions of their endogenous counterparts. 11C) were also markedly reduced. Lipids were extracted and monohexosylceramides (A) and sphingomyelin (B) species were determined by ESI-MS/MS. DISCUSSION It is now well established that S1P produced by SphK1 promotes cell growth and inhibits apoptosis.SphK Isoenzymes in Sphingolipid Metabolism Downloaded from www. and 60). we 37126 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 44 • NOVEMBER 4. have opposite effects on cell growth and apoptosis? A possible explanation for the opposing effects of SphK1 and SphK2 is that the two proteins are located in. was associated with inhibition of cell growth and was pro-apoptotic (26. However. 7. different compartments within the cell. down-regulation of SphK1 with small interfering RNA significantly reduced SphK1 protein levels. Conflicting data exists on the localization of ectopically expressed SphK2. 11A). 57–59). This raised the question: how can two enzymes that catalyze the same reaction and produce the same product. Thus. both SphK1 and SphK2 markedly increased S1P levels (4. 11A and data not shown). determined by immunoblotting with a specific SphK2 antibody (Fig. it was localized in the nuclei. Dihydro-Sph Vector SphK1 SphK2 Vector Sph SphK1 Sph SphK2 Sph 68 419 96 168 505 208 5 27 4 19 41 31 Dihydro-S1P 1 22 5 2 50 11 0. overexpressed SphK2 was found to associate with the interleukin-12 receptor. Igarashi and co-workers (27) reported that when SphK2 was transiently expressed in HeLa and COS7 cells. SphK2. and S1P were determined by ESI-MS/MS. In agreement with our recent study (32). it has been shown that SphK1 activity inversely correlates with ceramide levels (10. or translocated to. we utilized small interfering RNA targeted to a specific sequence of SphK1 that has been successfully used to down-regulate SphK1 protein and activity (19.jbc. Similar results were found in two independent experiments. it was surprising to discover that overexpression of the second isozyme. TABLE ONE Effect of SphK1 and SphK2 on levels of free sphingoid bases and phosphorylated forms HEK 293 cells expressing vector. 11E).5-fold. pSilencer targeted to SphK2 reduced SphK2 mRNA by 70% by quantitative PCR analysis. Data are averages of triplicate determinations and are expressed as picomole of lipid/mg of protein. 5. S1P. 11F). on January 31. SphK1. SphK1.1 3 1 0. As expected. and enzymatic activity (Fig. it was important to establish the involvement of endogenous SphK1 and SphK2 in regulation of ceramide levels. then treated without or with 5 M sphingosine (Sph) for 6 h as indicated. or SphK2 were serum-starved for 18 h.1 3 2 Sph 290 263 316 1844 1596 2253 17 25 11 106 86 242 S1P 17 21 15 93 397 250 2 2 4 7 12 121 sine. and that localized production of S1P has distinct functions. siSphK2 also slightly decreased both basal S1P levels and the increased S1P resulting from treatment with sphingosine (Fig. or SphK2 were serum-starved for 18 h. Opposing Functions of Endogenous SphK1 and SphK2 in Ceramide Formation—Consistent with the decreased ceramide levels induced by overexpression of SphK1. SphK1 down-regulation enhanced incorporation of [3H]palmitate into ceramide when cells were treated with sphingosine (Fig. further suggesting a role for SphK2 in reutilization of sphingosine and its conversion to ceramide. To this end. whereas in HEK 293 cells it was localized to the cytosol. Lipids were extracted and dihydrosphingosine.and 2. 2011 FIGURE 10. sphingosine. using a specific antibody. respectively). 32. In contrast. SphK2 protein. consistent with the inhibitory effect of overexpression of SphK1 (Fig.org at The University of Birmingham. In agreement. dihydro-S1P. Data are expressed as pmol/mg protein. 17–24). then treated without or with 5 M sphingosine (Sph) for 6 h as indicated. and enzymatic activity (Fig.1 at the plasma membrane in HEK 293 cells and T cell hybridomas (37). 11B). in part because of antagonism of ceramide-induced apoptosis (reviewed in Refs. Effects of SphK1 and SphK2 on monohexosylceramide and sphingomyelin species. 11D) and sphingosine-induced increases in the major ceramide species were sharply curtailed by downregulation of SphK2 (Fig. 27). without affecting expression or activity of SphK1 (data not shown). mRNA. Importantly. 2005 . 8C).

Blots were stripped and re-probed with anti-calnexin or anti-tubulin antibodies as loading controls. Similar results were obtained in three independent experiments. SphK1. The ER localization of SphK2 was partly dependent on its BH3 domain. we found that mutation of the conserved NOVEMBER 4. interfered with the ability of SphK2 to induce apoptosis (26). C. Moreover. we found that endogenous SphK2 in HEK 293 cells was mainly localized to the plasma membrane and internal membranes. nor deletion of S1P2 or S1P3. consistent with other studies showing that pro-apoptotic BH3-only proteins also localize to the ER via their BH3 domains. D. S1P made by SphK2 does not transactivate S1P receptors because neither treatment with pertussis toxin. and several growth factors induce its translocation to the plasma membrane (1). A. HEK 293 cells transfected with control small interfering RNA or small interfering RNA targeted to SphK1 were serum-starved for 18 h. duplicate cultures were serum starved for 18 h. and was present at much lower levels in the cytosol. E and F. on the other hand. the BH3-only protein BIK functions at the ER to stimulate cytochrome c release from mitochondria (62). on January 31. In agreement.SphK Isoenzymes in Sphingolipid Metabolism Downloaded from www. which inhibits the Gi-only coupled S1P1 receptor. Nevertheless. 2005 • VOLUME 280 • NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 37127 . possibly because of phosphorylation by protein kinase C or ERK2 (19. B. and expression of SphK2 protein was examined by immunoblot analysis with specific SphK2 antibodies. 31. [3H]Ceramide was resolved by TLC and radioactivity quantified with a radiochromatogram scanner. were unable to detect nuclear expression of endogenous SphK2 in either HEK 293 or MDA-MB-453 cells (32). in duplicate cultures.org at The University of Birmingham. For example. then incubated for 6 h with [3H]palmitate (10 Ci/ml) in the absence or presence of 5 M sphingosine. Lipids were extracted and S1P levels (E) and ceramide species (F) were determined by ESI-MS/MS. in this study. Effects of down-regulation of SphK1 and SphK2 on ceramide. B–F. [3H]Ceramide was resolved by TLC and radioactivity quantified with a radiochromatogram scanner. Unlike SphK1. S1P produced by translocation of SphK1 to the plasma membrane has been implicated in transactivation of cell surface S1P receptors (29.000 g pellets. duplicate cultures were lysed and equal amounts of proteins (20 g) were immunoblotted with anti-SphK1. 36). 61). we found that the proportion of SphK2 localized to the ER increased several hours after serum withdrawal. However. SphK2 activity was determined in the presence of 1 M KCl. HEK 293 cells expressing pSilencer vector or pSilencer-SphK2 were serum-starved for 18 h. then treated without or with 5 M sphingosine for 6 h. 23.jbc. Inset. Blots were stripped and reprobed with anti-tubulin to confirm equal loading. 2011 FIGURE 11. then incubated for 6 h with [3H]palmitate (10 Ci/ml) in the absence or presence of 5 M sphingosine. HEK 293 cells were transfected with either pSilencer control small interfering RNA (pSil-Vec) or pSilencer directed against SphK2 (pSil-K2). has been shown to be predominantly cytosolic. cell lysates were separated into supernatant and 100.

acting in concert with S1P phosphatase (SPPase) to convert S1P back to sphingosine and then to ceramide. This raises another provocative possibility that cytosolic S1P formed by SphK1 might negatively regulate (dihydro)ceramide synthase. whereas calcium is necessary for cell growth and survival. It should be noted that sphingosine is not produced by de novo biosynthesis. we found that overexpressed SphK1 increases dihydrosphingosine and as a consequence dihydro-S1P. 12). For example. our results suggest that cytosolic S1P formed by SphK1 has a different function than S1P produced at the ER and/or other membranes by SphK2. In contrast.org at The University of Birmingham. S1P can be irreversibly cleaved to ethanolamine phosphate and hexadecenal by S1P lyase or dephosphorylated back to sphingosine by specific S1P phosphatases. or buffering of cytoplasmic calcium changes. but it has recently been suggested that this might be a mechanism to differentiate between sphingoid bases arising from de novo synthesis and recycling. Proposed model for opposing functions of SphK1 and SphK2 in sphingolipid metabolism. can prevent mitochondrial damage and protect cells from apoptosis induced by ceramide (69). Downloaded from www.jbc. 65. including ER. it is also possible that S1P formed by SphK1 negatively regulates ceramide synthesis as it has been suggested that phosphorylated long-chain sphingoid bases may inhibit serine palmitoyltransferase activity and de novo ceramide biosynthesis (68). 12). down-regulating this phosphatase increases S1P. and abrogates apoptosis (65). in yeast. These results provide further support for the importance of the sphingosine phosphorylating activity of SphK2 for its ability to induce apoptosis.and pro-apoptotic Bcl-2/BAX family members regulates [Ca2 ]ER (46). 2005 . How does SphK1 decrease and SphK2 increase ceramide levels? One of the most intriguing results of this study is that these two isoenzymes that catalyze the same reaction have opposite effects on ceramide and sphingolipid levels. S1P phosphohydrolase-1. SphK2 may interact with other membrane proteins (37). Consistent with the notion that localization of SphK2 to the ER and internal membranes is important for its function. Cells can conserve resources by reutilizing sphingosine. cytosolic S1P formed by SphK1 inhibits de novo ceramide biosynthesis as a cellular sensing mechanism to minimize unneeded biosynthesis of ceramide. this is the first evidence in support of the notion that SphK2. Similarly. Both ER ceramide (69) and BH3-only proteins have been connected to the ER-calcium apoptotic gateway (43). 64). 66). Collectively. reduces sphingosine levels. see text. it has been suggested that externally supplied sphingoid bases may be phosphorylated after their entry into cells and require dephosphorylation before they can be used for ceramide synthesis (33. and that the balance between anti. similar to yeast membrane-associated Lcb4. ceramide itself causes the release of calcium from the ER and thus increases both cytosolic and mitochondrial calcium. In yeast. was stimulated by intracellular S1P accumulation and required both Lcb4 and Lcb5 (70). Moreover. Our results support the notion that SphK2. The reduction of [Ca2 ]ER. The locations of the sphingolipid metabolizing enzymes depicted in this scheme are not meant to indicate topology as the active site topologies have not all been unequivocally identified. and can then be re-utilized for ceramide and complex sphingolipid synthesis or phosphorylated by SphKs to form S1P (Fig. but not Lcb5. and is required for ceramide synthesis from exogenously added sphingoid bases (39). particularly in response to stress. leading to apoptosis. 12). is localized to the cytosolic side of internal membranes. the substrate for ceramide synthase. yet reduces ceramide levels. important for replenishment of secretory organelles with calcium. Interestingly. Alternatively. similar to yeast membrane-associated Lcb4 (39). leucine in the SphK2 BH3 domain did not abolish its localization to the ER. is localized to the ER (33. they suggest that the difference between the effects of SphK1 and SphK2 on apoptosis may be linked to the location at which S1P is produced. BAK and BAX directly affect [Ca2 ]ER with subsequent sensitization of mitochondria to calciummediated fluxes and cytochrome c release (46). Our results strengthen the notion that S1P may also contribute to regulation of calcium homeostasis in mammalian cells. to the ER converted it from anti-apoptotic to pro-apoptotic. to phosphorylation and irreversible degradation by S1P lyase. More significantly. might play a role in a sphingosine salvage pathway of mammalian cells. calcium influx induced by ER stress is mediated by calcium channels that are similar to voltage-gated calcium channels. Increases in cytosolic free calcium have been intimately linked to both survival and apoptosis. reminiscent of its function in yeast (70) and plants (71) that lack S1P receptors. and probably endosomes (38. in agreement with our hypothesis. Thus. suggesting that its putative transmembrane domains might also contribute to its association with internal membranes. but not catalytically inactive SphK1. It has been reported previously that a specific S1P phosphohydrolase. cytosolic S1P formed by SphK1 inhibits ceramide biosynthesis possibly as a cellular sensing mechanism to regulate levels of ceramide (Fig. In support of this idea. Conversely. The significance of the phosphorylation/dephosphorylation of added sphingoid bases is unclear.SphK Isoenzymes in Sphingolipid Metabolism were labeled with [3H]palmitate. whereas down-regulation of SphK2 decreased incorporation by 50% and also reduced the mass level of ceramides determined by mass spectrometry. membrane-associated and cytosolic Lcb4 play distinct roles to differentially generate biosynthetic and signaling pools of phosphorylated long chain sphingoid bases (39). addition of sphingosine caused a 3-fold increase in incorporation of radioactivity into long-chain ceramides. minimizing the de novo synthesis of ceramide. the major yeast SphK. inappropriately increased intracellular levels may result in cell death (44). possibly for regulation of sphingolipid biosynthesis or signaling (67). Moreover. Previously. Several studies have shown that [Ca2 ]ER is an important determinant of the response of cells to stresses. on January 31. overexpression of S1P phosphohydrolase-1 increases ceramide levels and induces apoptosis (33). Lcb4. This calcium influx. Golgi. we found that when SphK2 transfectants 37128 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 44 • NOVEMBER 4. Rather. 39. SphK1 can reduce ceramide levels by routing sphingosine. 2011 FIGURE 12. For more details. Indeed. Ceramide generated in the ER has been linked to increased Ca2 release. it is derived from cleavage of ceramide by ceramidases in the sphingolipid degradative pathway. might play a role in the sphingosine salvage pathway of mammalian cells. Alternatively. including increased ceramide (43). 39). 63. Indeed. acting in concert with S1P phosphatase to convert S1P back to sphingosine and then to ceramide (Fig. targeting cytosolic SphK1.

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