Inhibition of Sphingosine Kinase in Vitro and in Platelets

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 267, No. 5, Iasue of February 15, pp Printed in U. S. A. Inhibition of Sphingosine Kinase in Vitro and in Platelets IMPLICATIONS FOR SIGNAL TRANSDUCTION PATHWAYS* Benjamin M. Buehrer and Robert M. Bell$ From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina (Received for publication, August 29,1991) Sphingosine kinase was partially purified and char- ochromocytoma cells (8). Sphingosine has been shown to acterizedfromratbrainmicrosomes. A new assay, directly inhibit protein kinase C activity in vitro and in whole utilizing octyl-8-d-glucopyranoside and sphingosine platelets (9). mixed micelles, was developed to quantitate formation Sphingosine phosphate has been implicated in calcium re- of the sphingosine- 1-phosphate product. The was assay lease in permeabilized smooth muscle cells (10) and in Swiss proportional with respect to time and protein, dis- 3T3 fibroblasts (11). Sphingosine-1-phosphate has recently played Michaelis-Menten kinetics, and was subject to been shown to be a weak mitogen in Swiss 3T3 cells, and play surface dilution in regard to the sphingosine substrate. a role in cellular proliferation (11). Sphingoid bases have been Investigations into substrate specificity showed that reported to be cytotoxic at high concentrations (12). the enzyme is specific for the erythro-enantiomers of Dihydrosphingosine, a precursor of sphingosine, is synthesphingosine and dihydrosphingosine. Neither of the threo-enantiomers were phosphorylated in this system, sized by a condensation reaction between palmitoyl-cod and but both were found to be potent competitive inhibitors serine, followed by a reduction of the ketone product (Fig. 1). of sphingosine kinase activity. Human platelet sphingosine kinase activity displayed substrate and inhibitor specificities similar to the rat brain enzyme. A mixture of DL-threo-dihydro- sphingosine competitively inhibited sphingosine kinase activity in a dose dependent manner in isolated platelets. DL-Threo-dihydrosphingosine caused a pro- phorylation event on the primary hydroxyl. The second step longation of the inhibition of thrombin-induced protein is a lyase reaction catalyzed by sphingosine-1-phosphate alkinase C-dependent 40 (47)-kDa protein phosphoryl- dolase, generating the products through pyridoxal a phosphate ation in platelets. D-, L-, or DL-Threo-dihydrosphingosine may be useful as a tool to investigate D-Erythrosphingosine metabolism and the function of sphingosine- 1-phosphate in signal transduction processes. Complex sphingolipids including the lysosphingolipids have recently been implicated in many cellular functions (1). Gangliosides have long been known as markers for differentiation and have been shown to be specifically expressed in certain human melanomas. Sphingomyelin levels have been shown to be elevated in hairy cell leukemia treated with phorbol ester (2), and transiently decreased in HL-60 cells in response to vitamin D3-induced differentiation (3). In this same HL-60 cell system, ceramide levels increase transiently concomitant with a transient decrease in sphingomyelin; ceramide has been implicated as a lipid mediator of differentiation (4). Recent findings have raised the possibility that lysosphingolipids may function as second messengers or in the pathogenesis of the sphingolipidoses (5). Sphingosine has been shown to inhibit protein kinase C mediated processes in a variety of systems, including: the oxidative burst of neutrophils (6), phorbol ester-induced differentiation of HL-60 promyelocytic leukemia cells (7), and nerve growth factor-induced neurite outgrowth in PC-12 phe- * This work was supported by National Institutes of Health Grant DK 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. To whom correspondence should be addressed Generation of sphingosine is thought to occur by acylation of dihydrosphingosine to form ceramide, followed by insertion of the double bond and deacylation (13, 14). Breakdown of sphingosine has been shown to generate ethanolamine phosphate and hexadecanol through a two-step process (15, 16). The first step, mediated by sphingosine kinase, is a phos- requiring mechanism (17, 18). Sphingosine kinase activity has been observed from many sources, including: bovine brain (19), platelets (20), Tetruhymenu pyriformis (21, 22), and rat liver (23, 24). The kinase activity has been postulated to catalyze the rate-limiting step in the catabolism of sphingosine (25). Little is known about the enzyme, its structure, its substrate specificity, or its mechanism of regulation. The objectives of the studies reported here were to partially purify the enzyme, determine its substrate specificity, and to identify potential inhibitors. Sphingosine kinase was partially purified from rat brain and its kinetic parameters character- ized using a novel mixed micelle assay method. The specificity of the enzyme was investigated using various sphingosine and dihydrosphingosine isomers; one of the isomers (DL-threodihydrosphingosine) turned out to be a very poor substrate and to inhibit activity against the natural D-erythro substrate. The ability of DL-threo-dihydrosphingosine to act as a metabolic inhibitor of sphingosine kinase activity was demonstrated in whole platelets. MATERIALS AND METHODS D-Erythro-sphingosine, DL-threo-dihydrosphingosine, DL-erythrodihydrosphingosine, adenosine 5 -triphosphate-agarose, caproic anhydride, fatty acid-free BSA, prostaglandin I?, and acetylsalicylic acid were purchased from Sigma. D-Threo-, L-erythro-, and L-threosphingosine were generously provided by Dr. Alfred Merrill and Dr. Dennis Liotta. Unstripped rat brains were purchased from Pel-Freez The abbreviations used are: BSA, bovine serum albumin; MOPS, 4-morpholinepropanesulfonic acid, EGTA, [ethylenebis(oxyethylenenitri1o)ltetraacetic acid; SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

2 Inhibition Kinase of Sphingosine 3155 Biologicals. Octyl-8-D-glucopyranoside was purchased from Behring Diagnostics. Coarse grade (3-25 Sephadex and DEAE-Sephacel were obtained through Pharmacia LKB Biotechnology Inc. PI1 cellulose phosphate was purchased from Whatman. Phospholipids were purchased from Avanti Polar Lipids. [y-3zpjadenosine triphosphate and [32P]orthophosphate were purchased from Du Pont-New England Nuclear. Silica Gel 60 chromatography plates were obtained through VWR Scientific. Centriprep 30 concentrators were purchased from Amicon. A System 200 Imaging Scanner and an Auto Changer 3000 were obtained from Bioscan. Sphingosine Kinase Assay-Sphingosine kinase activity was measured in vitro using a mixed octyl-8-d-glucopyranoside (0-octyl glucoside)/sphingosine micellar system. Sphingosine substrate was delivered from an ethanol solution and dried under a stream of nitrogen. Substrate was resuspended by bath sonication in 50 plof buffer containing 255 mm P-octyl glucoside, 100 mm MOPS (ph 7.2), 5% glycerol, and 5 mm mercaptoethanol. Reaction buffer (125 pl) containing 200 mm MOPS (ph 7.2), 20 mm MgC12, 10% glycerol, and 10 mm mercaptoethanol was added after sonication. A 50-pl sample containing sphingosine kinase activity was used in the assay. The reaction was started by addition of 25 pl of 10 mm ATP ([32P]ATP added to give a specific activity of 100,000 cpm/nmol). The final assay concentrations were: 100 mm MOPS, 10 mm Mg, 51 mm p- octyl glucoside, 5% glycerol, 5 mm mercaptoethanol (ph 7.2) in 250 pl of total volume. Assay tubes were incubated at room temperature for 30 min and the reaction stopped by addition of 1.0 ml of methanol/ chloroform (2:l) (v/v) containing 5% triethylamine. Sphingosine-lphosphate was converted to N-caproyl-sphingosine-phosphate by addition of 20 p1 of caproic anhydride, followed by incubation at 37 "C for min. Alkaline hydrolysis of glycerophospholipids and excess caproic anhydride was carried out by addition of1.0 ml of0.2 N methanolic NaOH. After min at 37 "C, a biphasic system for lipid extraction was created similar to the method of Bligh and Dyer (26) ml of methanol, 1.66ml of chloroform, 1.0 ml of a 1% perchloric acid solution, and 150 yl of 70% perchloric acid were added, and the tubes vortexed. After centrifuging to separate phases, the upper phase was removed by aspiration. The chloroform phase was washed twice with 2.0 ml of the 1% perchloric acid solution, and the tubes vortexed and centrifuged between washes. Portions of the lower phase were transferred to scintillation vials, evaporated to dryness, and counted in a LKB Liquid Scintillation Counter. Additional portions were evaporated in a Savant Speedvac Concentrator and resuspended in 40 p1 of chloroform/methanol(4:1). All 40 pl of each sample were spotted on Silica G-60 plates and chromatographed in chloroform/pyridine/formic acid (60307). N- Caproyl-sphingosine-1-phosphate migrated with a RF = This system separates short-chain ceramide/phosphate from endogenous long-chain ceramide/phosphate and other phospholipids. The percentage of total label in the product spot was determined using a Bioscan imaging scanner. Phospholipid Dependence of Sphingosine Kinase Assay-Phospho- lipids were delivered in chloroform and dried under a stream of nitrogen to a final assay concentration of 10 mol % with respect to micellar P-octyl glucoside (25 mm critical micellar concentration). Substrate was delivered in ethanol and also dried under nitrogen. Both lipids were resuspended in 50 pl of buffer containing 255 mm p- octyl glucoside by bath sonication and assays were performed as described above. Sphingosine Kinase Activity in Whole Human Platelets-Human platelets were prepared from freshly drawn blood essentially as described by Seiss et al. (27), and were resuspended to 2.5 X 108platelets/ ml in modified Tyrode's buffer without EGTA. [32P]Orthophosphate was added to 0.6 mci/ml and the suspension was incubated for 75 L-Serine + c 3-Ketodihydm.phinpo~lne~ Delythm Dihydrosphlngosine Pelmitoyl-CoA POlIy ACvlZOA krythro Dlhydmceram(de 1 1 kmhro &ramide Ethanolamine Fatty AsycoA4kF.m Add phyhem -Sphingoslnbl-phoaph.m 4- k ~ Sphlngoslno m Hexadecenal ADP FIG. 1. Pathway of sphingosine metabolism. ATP min at 37 "C. Platelets were spun at 800 X g for 10 min and resuspended in the same volume of modified Tyrode's buffer without EGTA. Exogenous sphingolipids were added as 1:l BSA complexes. 100-pl portions were removed at the indicated times and added to 1.0 ml of methanol/chloroform (2:l) containing 5% triethylamine. The mixture was processed as previously stated. Thrombin-induced Phosphorylation of the Human Platelet 47-kDa Protein-Human platelets were prepared as stated above except aggregation was inhibited by incubatingplatelet-rich plasma with 100 pm aspirin for 10 min at 37 "c prior to the final pelleting step. Platelets were resuspended to 2.5 X 10' platelets/ml in modified Tyrode's buffer (without EGTA) and incubated with 400 pci/ml of [32P]orthophosphate as mentioned above. Platelets were pelleted by centrifugation at 800 X g for 10 min and resuspended to the same concentration in modified Tyrode's buffer (without EGTA). Exoge- nous sphingolipids were added in ethanol, maintaining a final concentration of 0.3%, and aliquots (100 pl) incubated at room temperature for varying times. Following the preincubation with sphingolipid, thrombin was added to a concentration of 1 unit/ml. Thrombin stimulation proceeded for 30 s and was terminated by adding an equal volume of 2 X sample buffer and boiling for 5-10 min. Samples (100 pl) were loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel and electrophoresis was performed according to the method of Laemmli (28). Gels were dried and autoradiographed. Gel slices containing the 47-kDa protein were cut out and the associated 32P counted. Data was normalized to the protein concentration of each loaded sample and expressed as a percent of maximal phosphorylation induced by thrombin. Sphingosine-1 -phosphute Production in Whole Human Platelets- Human platelets were isolated and labeled as stated for monitoring 47-kDa protein phosphorylation. Exogenous sphingolipids were added in ethanol to 1.0-ml suspensions of labeled platelets. Aliquots (100 pl) were transferred to separate tubes and preincubated for varying times prior to thrombin stimulation (1 unit/ml). The reactions were stopped by adding 1.0 ml of methanol/chloroform (2:l) containing 5% triethylamine and 25 pl of caproic anhydride. Base hydrolysis and extraction of the product were carried out as before. Purification of Rut Brain Sphingosine Kinase-Frozen unstripped rat brains were thawed and homogenized in buffer containing 20 mm MOPS (ph 7.2), 200 mm sucrose, 10 mm EDTA, 10 mm EGTA, 10 mm P-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride in dimethyl sulfoxide, and % leupeptin. The homogenate was centrifuged at 12,000 X g for 15 min to remove particulate matter. The supernatant was then centrifuged for 60 min at 10,000 X g. The resulting pellet was resuspended in half the original volume of homogenization buffer containing 1 M NaCl, placed on ice for min, and then centrifuged at 100,000 X g for 60 min. The supernatant was desalted by passage over an 80-ml Sephadex G-25 column using standard buffer (20 mm MOPS, ph 7.2, 10 mm p-mercaptoethanol, 1 mm EGTA, 1 mm EDTA, and 5% glycerol). This desalted fraction was batch bound to DEAE-Sephacel. The column was washed and then stepwise eluted with standard buffer containing 120 mm NaC1. The fractions containing maximal protein, determined by absorbance at 280 nm, were pooled. The pooled column fractions were diluted to 100 mm NaC1, and passed over a phosphocellulose column. The column was eluted stepwise (300 mm NaCI) and fractions containing sphingosine kinase activity were pooled. Association of enzyme activity with vesicles has been shown to be an effective way of isolating proteins (29). Phosphatidylcholine vesicles were added to the pooled phosphocellulose fractions, at a concentration of approximately 200 pg of lipid/ml of protein. This suspension was incubated on ice for min before it was centrifuged for 15 min at 500,000 X g. The supernatant was desalted with a Centriprep-30 concentrator and diluted with standard buffer to obtain a concentration of 100 mm NaCI. Phosphatidylcholine vesicles containing 13 mol % of DL-threo-dihydrosphingosine were added to a final concentration of approximately 200 pg/ml. This suspension was incubated on ice for min and then centrifuged for 15 min at 500,000 X g to pellet vesicles. Pelleted vesicles were resuspended in standard buffer containing 30 mm p-octyl glucoside, and passed over an ATP-agarose column equilibrated with the same buffer. The column was eluted with a mm NaCl gradient and fractions were assayed for activity as described above. Protein Determination-Protein concentrations were determined by a modification of the method by Lowry et al. (30). Determination of Protein Concentration for Samples Containing SDS-Proteins were precipitated by the method of Peterson (31) and

3 3156 Inhibition of Sphingosine Kinase quantitated by the method of Markwell et al. (30). Briefly, 75 pl of protein solution containing sample buffer was brought up to 1.0 ml, 10 ~l of 2% deoxycholate were added, the tubes vortexed, and incubated 10 min at room temperature. 100 p1 of 75% trichloroacetic acid was added and the tubes were spun in a microfuge for 10 min to pellet protein. The supernatant was aspirated and the pellet resuspended in 500 pl of water and 500 pl of reagent C (30). The samples were transferred to a larger tube and the original tubes were washed again with water and reagent C. Assays were continued as above. BSA standards were precipitated in the presence of sample buffer and displayed a proportional relationship. RESULTS Mixed Micelle Assay-Previous attempts at quantitatively measuring the activity of sphingosine kinase were hampered by the physical and chemical properties of the product, sphingosine-1-phosphate (24). The combination of hydrophobic and hydrophilic moieties, and the presence of positive and negative charges, make sphingosine-1-phosphate virtually impossible to quantitatively extract into organic or aqueous solutions. Earlier assays used tritium-labeled sphingosine and extracted the phosphorylated product from unreacted substrate in an alkaline environment. We have developed a new assay which circumvents the unusual solubility properties of sphingosine-1-phosphate and the tedious preparation of labeled substrate. This assay system employs mixed micelles to present the lipid substrate to the enzyme. Sphingosine kinase activity was monitored by the transfer of 32P from y-labeled ATP to sphingosine in mixed micelles containing 0-octyl glucoside and sphingosine. To overcome the problem of sphingosine phosphate solubility, sphingosine- 1-phosphate was converted to a more easily extractable lipid, N-caproyl-sphingosine-1-phosphate. The acylation reaction proceeded to completion within 30 min at 37 "C. Following a mildmethanolic alkaline hydrolysis and acidification, the short-chain ceramide-phosphate product was extracted into chloroform, separated by thin layer chromatography, and quantitated directly from the TLC plate. The characteristics of this assay system are similar to those of other micellar assay systems (32, 33). This mixed micellar assay allowed for enhanced reproducibility, consistent delivery of the amphiphilic substrate, and the systematic variation of the concen- tration of surface active molecules. Sphingosine kinase activity was proportional with respect to time and protein (Fig. 2). Enzyme activity also showed Michaelis-Menten kinetics. Sphingosine exhibited surface dilution kinetics in the mixed micelles employed in this assay (Fig. 2). Therefore, sphingosine kinase activity is not dependent on the bulk concentration of sphingosine, but on the concentration of this substrate in micelles. Sphingosine kinase activity was not influenced by the presence of other phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, or phosphatidylserine (data not shown). Characteristics of Sphingosine Kinase Activity In Vitro- Other characteristics of sphingosine kinase activity were investigated. consistent with previous reports (19, 20), sphingosine kinase displayed a requirement for magnesium which could be partially overcome by manganese, a ph optimum of 7.2, and peripheral association with membranes. Sphingosine kinase localization correlates with that of sphingosine-l-phosphate lyase, the enzyme catalyzing the second step in sphin- gosine breakdown, which has been reported to be present only in membrane fractions (15, 18, 34). Sphingosine can exist in four different isomeric states (Fig. 3): D(+)-erythro (2S,3R), L(-)-erythro (2R,3S), D(+)-the0 (2RI3R), and L(-)-threo (2S,3S). Each of the stereoisomers and enantiomers was tested for its ability to serve as a substrate for sphingosine kinase under mixed micellar con time (min.) pg protein Datergent concenaatlon (mm) FIG. 2. Time dependence, protein dependence, and surface dilution characteristics of mixedmicelleassay. The mixed micelle assay was proportional with respect to time (130 pg of protein) (upper panel) and protein (30 min) (middle panel). Sphingosine displayed normal surface dilution characteristics in the micellar assay (lower panel). Substrate was held at a constant 2.6 mol % (triangles) and at a constant 50 mm bulk concentration (squares). ditions (Table I). Only the compounds containing the erythro conformation functioned as substrates. Because the threoisomers did not serve as substrates, they were tested for their ability to function as inhibitors. Both D-threo- and L-threosphingosine competitively inhibited activity with similar inhibitor constants (KJ (Fig. 4, Table I). Specificity of Platelet Sphingosine Kinase-Earlier work by

4 Inhibition of Sphingosine Kinase 3157 w+)..rvulm (m3w HO H OH \: I CHdCH z),,ch=ch --C+--CH, W c \H HO H On \ 2 I CHr4CH r),z.chlch c-+-ch 2 Hfl k 9%, OH I CH r4ch,),zch=ch 4 - C C H z /1 HzN k mole% wmno m.35) Hq H OH CHACH a)tzch=ch -&)-CAS 2\ H*N H FIG. 3. Sphingosine isomers. The four stereoisomers of sphingosine and the orientations around carbons 2 and 3. TABLE I Kinetic parameters for sphingosine kinase from rat brain and human platelets wing sphingosine and dihydrosphingosine isomers K, values were determined from double reciprocal plots. The dissociation constants, Ki, for inhibition were determined from secondary plots of the slopes of double reciprocal plots uersw the concentration of inhibitor present. All values are expressed in terms of mole %. Platelet Rat brain SubstrateInhibitionSubstrateInhibition K, Ki K, Ki D-Erythro-sphingosine L-Erythro-sphingosine D-Threo-sphingosine L-Threo-sphingosine DL-Erythro-dihydrosphingo sine DL-Threo-dihydrosphingo sine C! ftrnole % FIG. 4. Competitive inhibition of rat brain sphingosine kinase by DL-threo-dihydrosphingosine in vitro. DL-Threo-dihydrosphingosine was added to tubes containing decreasing amounts of D-erythro-sphingosine. Inhibition of sphingosine kinase activity was monitored as described under Materials and Methods at five concentrations of dihydrosphingosine: no addition (closed circles), 0.19 mol % (open circles), 0.38 mol % (open sqwres) mol % (open triangles), and 1.52 mol % (open diamonds). lets-to determine whether DL-threo-dihydrosphingosine can be an effective modulator of sphingosine kinase activity in a model cell system, its effect on sphingosine-l-phosphate production in whole platelets was determined. Whole human Stoffel et al. (35) characterizing sphingosine kinase in human platelets showed that D-erythro-sphingosine, D-erythro-dihy- platelets are an ideal system in which to monitor sphingosinedrosphingosine, and L-threo-dihydrosphingosine functioned l-phosphate production, because no evidence exists for the as substrates. Whereas, L-erythro-dihydrosphingosine, L- presence of sphingosine-l-phosphate lyase in platelets. Isothreo-dihydrosphingosine, and D-threo-dihydrosphingosine lated human platelets were labeled with orthophosphate in inhibited activity. By using the mixed micellar assay, we were order to follow phosphorylation events. These platelets were able to characterize the substrate specificities of human plate- then exposed to both DL-threo-dihydrosphingosine/BSA and let sphingosine kinase. Results similar to those for rat brain D-erythro-sphingosine/BSA or to D-erythro-sphingosine/ sphingosine kinase were found in vitro (Table I). Both enan- BSA alone. D-Erythro-sphingosine, when added in a 1:l BSA tiomers of erythro-sphingosine were phosphorylated as well complex, was readily phosphorylated and was easily monias the mixture of erythro-dihydrosphingosines. The L-erythro- tored by conversion to a short-chain ceramide-phosphate. sphingosine had almost 100-fold less activity than the natu- This phosphorylation reaction, like that in vitro, was linear rally occurring isomer. Unlike the rat brain enzyme, platelet with time up to 20 min and followed saturation kinetics with sphingosine kinase does not differ in its ability to recognize respect to substrate. The addition of DL-threo-dihydrosphinthe erythro-sphingosines and -dihydrosphingosines, but dif- gosine complexed with BSA significantly inhibited the kinase fers greatly in ability to phosphorylate these species. DL- activity in a dose dependent manner (Fig. 5) and is consistent Erythro-dihydrosphingosine was approximately one-fifth as with competitive inhibition found in vitro. Therefore, sphinactive as D-erythro-sphingosine in the micellar assay. As with gosine kinase activity can be modulated by DL-threo-dihythe rat brain enzyme, threo-sphingosine was not phosphoryl- drosphingosine in the platelet model system. ated by the platelet enzyme. Upon further analysis, the threo- Inhibition of Thrombin-induced 40 (47)-kDa Protein Phosenantiomers of sphingosine and dihydrosphingosine were phorylation-sphingosine is known to inhibit thrombin-infound to be potent competitive inhibitors of platelet sphin- duced phosphorylation of the platelet 40 (47)-kDa protein by gosine kinase activity in vitro (Table I). inhibiting protein kinase C (9). Protein kinase C, in vitro, Inhibition of Sphingosine Kinase Activity in Whole Plate- shows no stereospecificity with regard to inhibition by sphin-

5 3158 Inhibition Kinase of Sphingosine I 120 I I time 2o 0 i Preincubation Time (min.) FIG. 6. Inhibition of thrombin-induced phosphorylation of the platelet 47-kDa protein by sphingosine. Washed platelets were obtained as described under Materials and Methods and preincubated with 10 pm DL-threo-dihydrosphingosine (open circles), 20 pm D-erythro-sphingosine (open squares), 10 pm DL-threo-dihydrosphingosine + 20 p~ D-erythro-sphingosine (closed triangles), or 30 pm sphingosine (open triangles). Samples were stimulated with 1 unit/ml of thrombin for 30 s and reactions were stopped with 2 X sample buffer. Aliquots of the protein samples were run on a 10% SDS-polyacrylamide gel. Slices of the dried gel containing the 47- kda protein were removed and the amount of associated radiation was counted and standardized to protein M D.L-threo dihydrosphingosine FIG. 5. Inhibition of sphingosine kinase activity in whole Dlatelets bs DL-threo-dihvdrosDhingosine. Washed Dlatelets were obtained as described ;nder- MaGrials and Methods and incubated with 10 p~ substrate/bsa alone (closed squares) or 10 pm substrate/bsa and 5 (open circles), 10 (open diamonds), or 20 p~ (open triangles) of DL-threo-dihydrosphingosine/BSA as described under Materials and Methods. The lower panel response represents inhibition at 15 min incubation time with increasing dihydrosphingosine. A ti l2o0o a r I I I gosine isomers (36). To test whether DL-threo-dihydrosphingosine could alter sphingosine metabolism and in turn thrombin-induced 40 (47)-kDa protein phosphorylation, whole human platelets were isolated and labeled with orthophosphate. The isolated platelets were preincubated with DL-threo-dihydrosphingosine, D-erythro-sphingosine, or both for up to 30 min prior to a 30-s stimulation with thrombin. The phos- phorylation of the 40 (47)-kDa protein was monitored by SDS-PAGE analysis. The results in Fig. 6 show that DLthreo-dihydrosphingosine (10 1M) alone did not affect thrombin-induced 40 (47)-kDa protein phosphorylation, whereas D- erythro-sphingosine inhibited this phosphorylation by approximately 20% at a concentration of 20 gm (and 60% at 30 pm). The inhibition by D-erythro-sphingosine diminished over the preincubation time, presumably by metabolism of D- erythro-sphingosine to sphingosine-l-phosphate. When both DL-threo-dihydrosphingosine and D-erythro-sphingosine were added to the platelet suspension, 40 (47)-kDa protein phos- phorylation was inhibited to a similar extent as that of 20 p~ D-erythro-sphingosine alone; however, there was no diminution of inhibition over the 30 min of preincubation. Fig. 7 shows the results of a similar experiment in which sphingosine phosphate generation was monitored under these same con- ditions. DL-Threo-dihydrosphingosine inhibited sphingosine- 1-phosphate production up to 25% over the time course of preincubation. These data taken together support the idea that DL-threo-dihydrosphingosine can inhibit sphingosine ki Preincubation Time (min.) FIG. 7. Production of sphingosine-1-phosphate in whole platelets in response to exogenous sphingosine and dihydrosphingosine. Washed platelets, treated with aspirin, were obtained as previously stated and preincubated with 10 pm DL-threo-dihydrosphingosine (closed circles), 20 pm D-erythro-sphingosine (open squares), or 10 pm DL-threo-dihydrosphingosine + 20 pm D-erythrOsphingosine (closed triangles). Samples were stimulated with 1 unit/ ml of thrombin for 30 s and reactions were stopped with 1.0 ml of methanol/chloroform (2:l) + 5% triethylamine (v/v). Sphingosine-lphosphate production was quantitated as described under Materials and Methods. nase activity, thus prolonging the time that inhibitory levels of D-erythro-sphingosine are present to influence protein kinase C activity. DISCUSSION In this paper, we describe a novel mixed micellar assay for monitoring sphingosine kinase activity in rat brain. This was done so that sphingosine could be reproducibly presented to the enzyme in an environment resembling the lipid bilayer of cellular membranes (37). The unfavorable solubility charac-

6 Inhibition of Sphingosine Kinase 3159 teristics of sphingosine-1-phosphate product were overcome by acylation to form an easily extractable short-chain ceramide-phosphate. The assay was proportional with time and protein, and also followed surface dilution kinetics with respect to sphingosine. Substrate and inhibition studies showed that rat brain sphingosine kinase is highly specific in its use of the four isomers of sphingosine. The natural isomer, D-erythro-sphingosine, was the most favored substrate tested. D-Threo-sphingosine was found to be the most potent inhibitory isomer of rat brain sphingosine kinase activity. Sphingosine kinase activity from platelets has been studied previously using different assay methods (20,35). Our studies on the platelet activity using the sphingosine isomers demonstrate a pattern of specificity and inhibition similar to that observed with the partially purified rat brain microsomal activity. Both erythro-enantiomers were found to be substrates and both threo-enantiomers were found to be competitive inhibitors of the platelet enzyme. Using the mixture of DL-threo-dihydrosphingosine, we were able to inhibit the production of sphingosine-1-phosphate in isolated whole platelets. This inhibition was dose dependent and followed competitive inhibition kinetics, and therefore, is unlikely to occur via an isomeric stimulation of a sphingosine-1-phosphate phosphatase. DL-Threo-dihydrosphingosine extended the time course of D-erythro-sphingosine inhibition of thrombininduced 40 (47)-kDa protein phosphorylation. This effect is presumably caused by an alteration in the rate of metabolism of sphingosine by sphingosine kinase. By inhibiting sphingosine kinase activity, DL-threo-dihydrosphingosine prolongs the time course of protein kinase C inhibition by D-erythrosphingosine. Therefore, the mixture of DL-threo-dihydrosphingosine may not only be a useful tool to investigate D- erythro-sphingosine metabolism, but may be useful to affect known signal transduction pathways, e.g. sphingosine-l-phosphate (11). By shutting down one of the known major pathways of sphingosine metabolism, it may also be possible to find less common sphingosine metabolites in systems such as whole platelets. The new finding that sphingosine-1-phosphate has a profound effect on cell growth in Swiss 3T3 fibroblasts (11) suggests that sphingosine kinase may have importance other than in the catabolism of sphingosine. Zhang et al. (11) found that sphingosine-1-phosphate was weakly mitogenic and also released intracellular calcium in fibroblasts. This evidence and that of Ghosh et al. (10) indicate that sphingosine not only has a possible negative regulatory role in relation to protein kinase C, but may also exert a positive effect by its metabolism to sphingosine-1-phosphate. Modulation of sphingosine kinase activity by inhibitors may allow the func- tion of D-erythro-sphingosine-1-phosphate to be elucidated. Such inhibitors will likely prove useful in defining the role of sphingosine metabolites in cellular regulation. Acknowledgments-We are appreciative of the invaluable technical assistance provided by Dr. Anders KalCn and many thoughtful discussions with Kevin Ramer during the preparation of this manuscript. REFERENCES 1. Hannun, Y. A., and Bell, R. M. (1989) Science 243, Lockney, M. W., Golomb, H. M., and Dawson, G. (1984) Biochim. Biophys. Acta 796, Okazaki, T., Bell, R. M., and Hannun, Y. A. (1989) J. Biol. Chem. 264, Okazaki, T., Bielawska, A., Bell, R. M., and Hannun, Y. A. (1990) J. Biol. Chem. 265, Hannun, Y. A., and Bell, R. M. (1987) Science 235, Wilson, E., Olcott, M.C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. 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