INTRODUCTION

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The sphingolipid metabolite, sphingosine-1-phosphate (SPP),¹ is emerging as a prototype of a new class of lipid second messengers, which has both intracellular and extracellular actions (1-4). Ample evidence indicates that SPP can serve as an intracellular second messenger; SPP modulates intracellular pathways important for diverse biological processes including cell growth, survival, motility, and cytoskeletal changes (reviewed in Ref. 5). Moreover, because SPP antagonizes apoptosis mediated by ceramide, a stress-induced sphingolipid metabolite (3, 6), we have proposed that the relative intracellular levels of these two sphingolipid metabolites is an important factor that determines whether cells will survive or die (3). In support of this idea, it has recently been shown that unfertilized mouse oocytes exposed to the anticancer drug doxorubicin undergo ceramide-mediated apoptosis that is inhibited by SPP (7). In addition, it seems that this ceramide/SPP rheostat is an evolutionarily conserved stress regulatory mechanism influencing growth and survival of yeast (8-10). Recently, it has been shown that SPP, a serum borne lipid, is the ligand for the G protein-coupled endothelial-derived receptor-1 (EDG-1), which regulates morphogenetic differentiation of endothelial cells (4). Taken together, these data suggest that SPP has dual actions (11).

Various stimuli, including platelet-derived growth factor and serum (1, 12), nerve growth factor (6, 13), activation of protein kinase C (14, 15), and cross-linking of FcR1 and FcR1 (16), increase cellular levels of SPP by activation of sphingosine kinase, the enzyme that catalyzes the phosphorylation of sphingosine. Competitive inhibitors of sphingosine kinase block formation of SPP and selectively inhibit cellular proliferation induced by platelet-derived growth factor and serum (1, 17), the cytoprotective effects of protein kinase C (3), and nerve growth factor (6), as well as FcRI- and FcR1-mediated calcium signaling (16), further supporting a role for endogenous SPP in cell growth, survival, and calcium mobilization.

Collectively, these results give new insights into the biological function of SPP and emphasize the importance of sphingosine kinase, the enzyme that regulates its formation. Recently, we have purified rat kidney sphingosine kinase 6 × 10⁵-fold to apparent homogeneity (18). Purified sphingosine kinase has an apparent molecular mass of approximately 49 kDa with K_m values of 5 and 93 µM for sphingosine and ATP, respectively (18). Based on peptide sequences, we report here the cloning and characterization of the first mammalian sphingosine kinases.

	EXPERIMENTAL PROCEDURES

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Materials-- SPP, sphingosine, and N,N-dimethylsphingosine were from Biomol Research Laboratory Inc. (Plymouth Meeting, PA). All other lipids were purchased from Avanti Polar Lipids (Birmingham, AL). [-³²P]ATP (3000 Ci/mmol) was purchased from Amersham Pharmacia Biotech. Poly L-lysine and collagen were from Boehringer Mannheim. Alkaline phosphatase from bovine intestinal mucosa, Type VII-NT, was from Sigma. Serum and medium were obtained from Biofluids, Inc. (Rockville, MD). Restriction enzymes were from New England Biolabs (Beverly, MA). Poly(A)⁺ RNA blots of multiple mouse adult tissues were purchased from CLONTECH (Palo Alto, CA). LipofectAMINE PLUS^TM and LipofectAMINE were from Life Technologies, Inc.

Protein Sequencing of Sphingosine Kinase-- Purified sphingosine kinase was electrophoresed on SDS-polyacrylamide gel electrophoresis and the Coomassie-stained 49-kDa band excised. After S-carboxyamidomethylation, this band was subjected to in-gel tryptic digestion as described (19). The resulting peptide mixture was separated by microbore high performance liquid chromatography on a Zorbax C18 1.0- × 150-mm reverse-phase column in a Hewlett-Packard 1090 HPLC with a 1040 diode array detector. Fractions were selected for sequencing based on differential UV absorbance at 205, 277, and 292 nm, and the peptide sequences were determined by automated Edman degradation (20). Complementary peptide sequence information was also obtained on 10% of the digest mixture by collisionally induced dissociation using microcapillary HPLC electrospray ionization/tandem mass spectrometry on a Finnigan TSQ7000 triple quadrupole mass spectrometer (19). Sequences of smaller peaks were determined on an Applied Biosystems Procise cLC 494 sequencer or by microcapillary HPLC-mass spectrometer as above or on a Finnigan LaserMat 2000 time-of-flight mass spectrometer matrix-assisted laser desorption ionization (21).

Cell Culture-- Human embryonic kidney cells (HEK293, ATCC CRL-1573), Swiss 3T3 cells (ATCC CCL-92), and NIH 3T3 (ATCC CRL-1658) cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum or 10% calf serum.

Transient Expression of Sphingosine Kinase-- Transfections of various cell types were carried out using LipofectAMINE PLUS reagent, essentially as described by the manufacturer. For human embryonic kidney 293 cells, cells were seeded at 6 × 10⁵ per well in polylysine coated 6-cluster wells. After 24 h, cells were transfected with 1 µg of vector (pCMV-SPORT2) (Life Technologies, Inc.) alone or with vectors containing sphingosine kinase constructs (pCMV-SPORT2sphk1a or pCMV-SPORT2sphk1b), and 6 µl of LipofectAMINE PLUS reagent plus 4 µl of LipofectAMINE reagent per well. For Swiss 3T3 and NIH 3T3 fibroblasts, cells were seeded at 3 × 10⁵ per well in collagen-coated 6-well clusters. After 24 h, cells were transfected with a mixture of LipofectAMINE PLUS (8 µl) and LipofectAMINE (8 µl) and vector alone (2 µg), or sphingosine kinase constructs (2 µg). In some cases, as a measure of transfection efficiency, cells were co-transfected with 0.5 µg of pCEFLGFP (a kind gift of Dr. Silvio Gutkind), which encodes the green fluorescent protein, and were visualized with a fluorescence microscope.

Assay of Sphingosine Kinase Activity-- 1-3 days after transfection, cells were harvested and lysed by freeze-thawing in buffer A (20 mM Tris (pH 7.4), 20% glycerol, 1 mM -mercaptoethanol, 1 mM EDTA, 1 mM sodium orthovanadate, 40 mM -glycerophosphate, 15 mM NaF, 10 µg/ml leupeptin, aprotinin and soybean trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride, and 0.5 mM 4-deoxypyridoxine). In some experiments, cell lysates were fractionated into cytosol and membrane fractions by centrifugation at 100,000 × g for 60 min (4 °C). Sphingosine kinase activity was determined in the presence of 50 µM sphingosine, 0.25% Triton X-100, and [³²P]ATP (10 µCi, 1 mM) containing MgCl₂ (10 mM) in buffer A as described previously (18). The labeled SPP was separated by thin layer chromatography on silica gel G60 with 1-butanol/ethanol/acetic acid/water (80:20:10:20, v/v) and visualized by autoradiography. The radioactive spots corresponding to authentic SPP were identified as described (22) and quantified with a Molecular Dynamics Storm PhosphorImager (Sunnyvale, CA). Sphingosine kinase specific activity was expressed as picomoles of SPP formed/min/mg of protein.

Lipid Extractions-- Cells were washed with phosphate-buffered saline, scraped in 1 ml of 25 mM HCl/methanol, and lipids were extracted with 5 ml of chloroform/methanol/1 M NaCl (2:1:2, v/v) and 40 µl concentrated NH₄OH. Ceramide, sphingosine, and phospholipid levels were measured in the organic layer, whereas SPP levels were determined in the aqueous phase.

Measurement of Sphingosine-- Sphingosine was measured by the sphingosine kinase method as described previously (23). Briefly, aliquots of the organic phase containing 5-10 nmol of total phospholipids were dried under nitrogen and then resuspended in 0.25% Triton X-100. Reactions were started by the addition of [-³²P]ATP (10 µCi, 10 mM) containing 10 mM MgCl₂, 30,000 units of partially purified rat kidney sphingosine kinase (18), and then incubated for 30 min at 37 °C. Labeled SPP was quantified as described above. For each experiment, known amounts of sphingosine were used to generate a standard curve.

Measurements of SPP Levels-- Buffer B (200 mM Tris-HCl, pH 7.4, 1.2 M glycine buffer, pH 9.0, 75 mM MgCl₂) and 25-50 units of alkaline phosphatase (from bovine intestinal mucosa, Type VII-NT) were added to an aliquot of the aqueous phase containing extracted SPP. After 1 h at 37 °C, 40 µl concentration of HCl were added, and sphingosine was extracted and quantitated with the sphingosine kinase assay described above. For each experiment, known amounts of SPP were used to generate a standard curve.²

Mass Measurements of Ceramide-- Mass amounts of ceramide in cellular extracts were measured by the diacylglycerol kinase enzymatic method. Briefly, an aliquot of the organic phase from cellular lipid extracts or standard bovine brain type IV ceramides were resuspended in 40 µl of 7.5% (w/v) octyl--D-glucopyranoside, 5 mM cardiolipin in 1 mM DETPAC, 10 mM imidazole (pH 6.6) and solubilized by freeze-thawing and subsequent sonication. The enzymatic reaction was started by the addition of 20 µl of dithiothreitol (20 mM), 10 µl of Escherichia coli diacylglycerol kinase (0.88 units/ml), 20 µl [-³²P]ATP (10 µCi, 10 mM) and 100 µl of reaction buffer (100 mM imidazole (pH 6.6), 100 mM NaCl, 25 mM MgCl₂, and 2 mM EGTA). After incubation for 1 h at room temperature, lipids were extracted with 0.5 ml chloroform/methanol/concentrated HCl (100:100:1, v/v) and 85 µl of 1 M KCl. Labeled phosphatidic acid and ceramide-1-phosphate were resolved by thin layer chromatography with chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1, v/v) and quantified with a Molecular Dynamics Storm PhosphorImager.

Measurement of Total Cellular Phospholipids-- Total phospholipids present in cellular lipid extracts were quantified as described previously (6). Briefly, to dried aliquots of cellular lipid extracts, 40 µl of a mixture of 10 N H₂SO₄, 60% perchloric acid (1:3, v/v) were added and samples were heated for 30 min at 210 °C. After cooling, 75 µl of water and 400 µl of 4.2% ammonium molybdate in 4 N HCl, 0.045% (w/v) malachite green (1:3 v/v) were added. Samples were incubated overnight at room temperature, and absorbances measured at 660 nm.

Northern Blotting Analysis-- Poly(A)⁺ RNA blots containing 2 µg of poly(A)⁺ RNA per lane from multiple mouse adult tissues were purchased from CLONTECH. Blots were hybridized with the 0.8-kilobase SalI fragment of pCMV-Sport2-mSPHK1, which was gel-purified and labeled with [³²P]dCTP by random priming. Hybridization in ExpressHyb^TM buffer (CLONTECH) at 65 °C overnight was carried out according to the manufacturer's protocol. Bands were quantified using the Molecular Dynamics PhosphorImager.

	RESULTS AND DISCUSSION

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Sequencing and Cloning of Sphingosine Kinase-- The 49-kDa sphingosine kinase polypeptide, purified from rat kidney, was excised from an SDS gel and subjected to trypsin digestion. The resulting peptides were separated by microcapillary reverse-phase HPLC and sequences of 8 peptides were determined by Edman degradation or matrix-assisted laser desorption ionization mass spectrometry (Table I). Homology searches (BLAST) against a comprehensive nonredundant data base revealed no matches to known proteins. However, when the data base of expressed sequence tags (dbEST) at NCBI was searched using the tBLASTn algorithm, an EST (GenBank^TM accession number AA011725) containing sequences homologous to 3 of the 8 peptides (peptides 5, 2, and 4) was retrieved. A further search with peptides 1, 3, and 7 yielded 4 additional ESTs (GenBank^TM accession numbers AA000819, AA107451, AA592274, and AA389543). The nucleotide sequences of mouse ESTs AA000819 and AA592274 were then used to search dbEST to obtain EST AA389187. Clones AA107451 and AA389187 were highly homologous at their 3'-ends, but were slightly divergent at their 5'-ends. Sequencing of the full-length cDNAs revealed apparent open reading frames coding for 381 and 388 amino acid polypeptides containing sequences highly homologous to 7 isolated peptides distributed throughout the protein, and these are thus designated SPHK1a and SPHK1b (Fig. 1). In addition, both contained a portion of peptide 8. SPHK1a and -1b have predicted pIs of 6.68 and 6.89 and molecular weights of 42,344 and 43,254, respectively, in agreement with the molecular weight of purified rat kidney sphingosine kinase (18). Because SPHK1b only differs by a few amino acids at the N terminus, it may arise by alternative splicing. However, both sequences lacked Kozak consensus sequences, suggesting that these cDNAs may not include the actual initiation sequences.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Predicted amino acid sequences of two murine sphingosine kinases. The numbers on the right refer to the amino acid sequence of SPHK1a. Sequences homologous to the 8 tryptic peptides (Table I) that were obtained from purified rat kidney sphingosine kinase are underlined. Calmodulin binding motifs are indicated by dashes. Potential protein kinase C and cAMP-dependent phosphorylation sites are indicated by (*) and (), respectively.

View larger version (70K): [in this window] [in a new window]   Fig. 2.   Domain structure of sphingosine kinase. Alignment of the conserved subdomains of sphingosine kinases from various species. The conserved regions of sphingosine kinase (designated C1-C5) are shown as boxes. Dashes represent gaps in sequences. Sequences were aligned by the CLUSTALW: Multiple Sequence Alignment program. The putative human sphingosine kinase sequence was obtained by assembling sequences from several human ESTs (accession numbers D31133, AA232791, W63556, AA081152, and AA026479).

Tissue Distribution of Sphingosine Kinase-- The tissue distribution of sphingosine kinase mRNA expression in adult mouse tissues was analyzed by Northern blotting (Fig. 3). In most tissues, including adult brain, heart, spleen, lung, kidney, and testis, a predominant 2.4-kilobase mRNA species was detected, indicating ubiquitous expression of sphingosine kinase. However, the level of expression was markedly variable among the different tissues. The mRNA levels were highest in adult lung and spleen and there were barely detectable levels in skeletal muscle and liver. The mSPHK1 mRNA detected by Northern blotting was slightly larger than the size of the cDNA (1.9 kilobases). Interestingly, expression of mSPHK1a mRNAs in various mouse tissues did not closely correlate with the relative sphingosine kinase activities in rat tissues, because we previously found that spleen and kidney have higher specific activities than liver, which has about twice the activity of brain (18).

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Tissue specific expression of sphingosine kinase. A sphingosine kinase probe was hybridized to poly(A)+ RNA from the indicated mouse tissues (2 µg/lane) as described under "Experimental Procedures." Lanes: 1, heart; 2, brain; 3, spleen; 4, lung; 5, skeletal muscle; 6, liver; 7, kidney; 8, testis. The numbers at the bottom indicate relative levels of expression compared with background as quantified with the Molecular Dynamics PhosphorImager.

Recombinant Sphingosine Kinase Activity-- To investigate whether SPHK1a and SPHK1b encode bona fide sphingosine kinases, HEK293 cells were transiently transfected with pCMVSPORT2 expression vectors containing either SPHK1a or SPHK1b cDNAs, and sphingosine kinase activity was measured. Modest levels of endogenous sphingosine kinase activity were present in control cells (either untransfected or transfected with an empty vector) (Fig. 4). Cells transfected with SPHK1a exhibited 300-fold increased sphingosine kinase activity 24 h after transfection that remained at this level for 4 days. In contrast, cells transfected with SPHK1b showed only 120-fold increased sphingosine kinase activity after 24 h and then decreased gradually to control levels after 4 days (Fig. 4). Transfection of either Swiss 3T3 or NIH 3T3 fibroblasts with SPHK1a or SPHK1b also resulted in marked increases in sphingosine kinase activity (Table II). As in HEK293 cells, transfection of 3T3 cells with SPHK1a led to much larger increases in sphingosine kinase activity than with SPHK1b. It should be noted that transfection efficiency was quite good and similar in all three cell lines (Table II).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Activity of sphingosine kinase expressed in HEK293 cells. HEK293 cells were transiently transfected with the pCMVSPORT2 expression vector alone () or containing either the SPHK1a cDNA (pCMVSPORT2sphk1a, ) or the SPHK1b cDNA (pCMVSPORT2sphk1b, ). At the indicated times, sphingosine kinase activity in cell lysates was measured as described under "Experimental Procedures." Data are means of duplicates and are representative of three independent experiments.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Distribution of sphingosine kinase in transfected HEK293 cells. HEK293 cells were transiently transfected with pCMVSPORT2 vector alone or containing either SPHK1a cDNA or SPHK1b cDNA. After 72 h, sphingosine kinase activity was measured in cytosol and particulate fractions as described under "Experimental Procedures." Data are means ± S.D. A, thin layer chromatography analysis of [32P]SPP formed by cytosolic (C) or membrane (M) fractions from vector- or sphingosine kinase-transfected cells. B, sphingosine kinase activity in cytosolic (open bars) and membrane (filled bars) fractions. The data are expressed as percent of the total activity.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Sphingosine kinase has marked specificity for D-(+)-erythro-sphingosine. A, HEK293 cells were transfected with pCMVSPORT2sphk1a (filled bars) or pCMVSPORT2sphk1b (open bars), and sphingosine kinase-dependent phosphorylation of various sphingosine analogs or other lipids (50 µM) was measured in cell lysates as described under "Experimental Procedures." Data are expressed as percentage of phosphorylation of D-erythro-sphingosine. Competitive inhibition of recombinant sphingosine kinase by N,N-dimethylsphingosine (B) and D,L-threo-dihydrosphingosine (C). Sphingosine kinase activity in HEK293 cell lysates after transfection with pCMVSPORT2sphk1a was measured with varying concentrations of D-erythro-sphingosine in the absence (), or presence of 20 µM () or 40 µM () N,N-dimethylsphingosine (B), or presence of 10 µM () or 20 µM () D,L-threo-dihydrosphingosine (C). Data are means ± S.D. Inserts are double reciprocal plots. Km (µM) and Vmax (nmol/min/mg) values: D-(+)-erythro-sphingosine, 2.15 and 46.7. Ki values are 2.12 for D,L-threo-dihydrosphingosine and 2.64 for N,N-dimethylsphingosine.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Changes in mass levels of sphingolipid metabolites in NIH 3T3 cells overexpressing sphingosine kinase. Mass levels of SPP (A), sphingosine (B), and ceramide (C) in NIH 3T3 cells transfected with empty vector (Control) or with pCMVSPORT2sphk1a were measured after 48 h as described under "Experimental Procedures."