Regulation of Phosphatidate Phosphatase Activity ... - Semantic Scholar

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 268, No. 19, Issue of July 5 , pp. 13830-13837, 1993 Printed in U.S.A.

0 1993 by The American Society for Biochemistry andMolecular Biology, Inc.

Regulation of Phosphatidate Phosphatase Activity from the Yeast Saccharomyces cerevisiaeby Sphingoid Bases* (Received for publication, February 12, 1993)

Wen-I Wu&Yi-Ping Lin& Elaine WangQ, Alfred H. Merrill, Jr.6, and George M. CarmanSlI From the $Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey08903 and the 6Deoartment of Biochemistry, Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia30322 I

.

The regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-snphosphatidate phosphohydrolase, EC 3.1.3.4) activity by sphingoid bases was examined using Triton X- loo/ lipid-mixed micelles. Sphingosine, phytosphingosine, and sphinganine inhibited purified preparations of the 104- and45-kDa forms of phosphatidate phosphatase in a dose-dependent manner. The structural requirements for the sphingoid base inhibition of phosphatidate phosphatase activity were a free amino group and a long chain hydrocarbon. A detailed kinetic analysis was performed to determine the mechanism of phosphatidate phosphatase inhibition by sphingoid bases. The phosphatidate phosphatase dependence on phosphatidate was cooperative(Hill numbers of -2) in the absence and presence of sphingoid bases. Sphingosine, phytosphingosine, andsphinganinewereparabolic competitive inhibitors of phosphatidate phosphatase activity. This indicated that more than one inhibitor molecule contributed to theexclusion of phosphatidate from the enzyme. The aKi values (inhibitor constants) for sphingosine, phytosphingosine, andsphinganine were 1.5,0.4, and0.2 mol %, respectively, and theK,,, value for phosphatidate was 2.2 mol %. The cellular concentrations of free phytosphingosine and sphinganine were0.16 and 0.53 mol %, respectively, relative to the total phospholipids in S. cerevisiae. The cellular concentrations of phytosphingosine and sphinganine were in the range of the aKi values for these sphingoid bases. These results raised the suggestion that phosphatidate phosphatase activity maybe regulated in vivo by sphingoid bases.

Phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate yielding diacylglycerol and Pi (1).In the yeast Saccharomyces cereukiae, the diacylglycerol derived from the phosphatidate phosphatase reaction is used for the synthesis of phosphatidylethanolamine and phosphatidylcholine via the CDP-ethanolamineand CDP-choline-based pathways, re-

* This work was supported by United States Public Health Service Grants GM-28140 (to G . M. C.) and GM-33369 (to A.H. M.) from the National Institutes of Health and National Science Foundation Grant MCB-9204588, New Jersey State funds, and the Charles and Johanna Busch Memorial Fund (to G. M. C.). This is New Jersey Agricultural Experiment Station Publication D-10531-1-93.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 18U.S.C. Section 1734 solelyto indicate this fact. ll To whom correspondence and reprint requests should be addressed.

spectively (2). Diacylglycerol is also used for the synthesis of triacylglycerol (2). Thus, phosphatidate phosphatase may be involved in regulating the proportional synthesis of these lipids (3-5). 104- and 45-kDa forms of phosphatidate phosphatase have been purified from S. cereuisiae membranes and have similar enzymological and kinetic properties (4, 6, 7). Mitochondrial membranes contain the 45-kDa enzyme, whereas the microsomal membranes contain both the 104and 45-kDa enzymes (4). In uiuo labeling experiments have shown that the 104-kDa phosphatidate phosphatase is not a precursor of the 45-kDa phosphatidate phosphatase (4). The levels of the 104- and 45-kDa forms of phosphatidate phosphatase increase as S. cereuisiae cells progress from the exponential phase to the stationary phase of growth (4). In contrast, the expression of the two forms of phosphatidate phosphatase is regulated differentially by inositol (4). The levels of the 45-kDa phosphatidate phosphatase are induced in cells supplemented with inositol whereas the levels of the 104-kDa enzyme are notaffected by inositol (4). The activities of the phosphatidate phosphatase isozymes are regulated differentially by phosphorylation (5). Only the 45-kDa enzyme is phosphorylated by CAMP-dependentprotein kinase in vitro and in vivo on a serine residue, which results in an activation of phosphatidate phosphatase activity (5). The regulation of phosphatidate phosphataseby the growth phase, inositol, and phosphorylation correlates with changes in lipid synthesis (3, 5, 8,9). Phosphatidate phosphatase is also an important enzyme for lipid synthesis in higher eucaryotic cells (10-12). Furthermore, the enzyme plays a role in mammalian cell signaling mechanisms as part of the phospholipase D-phosphatidate phosphatase pathway for the generation of diacylglycerol from phosphatidylcholine (13). The diacylglycerol derived by this pathway is responsible for the sustained activation of protein kinase C (13). The sphingoid base sphingosine has been suggested to be a regulator of this signaling pathway because it activates phospholipase D (14, 15) and inhibits phosphatidate phosphatase (16, 17) and protein kinase C (18-22). The structural requirements and mechanism of inhibition of protein kinase C by sphingosine have been determined in detail using purified protein kinase C (18, 21). However, definitive conclusions about the regulation of phospholipase D (14, 15) and phosphatidate phosphatase (16, 17) by sphingosine cannot be made because previous studies were performed with crude preparations of these enzymes. The availability of purified preparations of the 104- and 45kDa forms of phosphatidate phosphatase from S. cereuisiae allowed us to examine the regulation of phosphatidate phosphatase activity by sphingosine and other sphingoid bases in a well-defined system. We show here that sphingosine and the yeast counterpart, phytosphingosine, inhibited the 104-

13830

Regulation of Yeast Phosphatidate Phosphatase and 45-kDa forms of phosphatidate phosphatase. Sphinganine was also an inhibitor of the phosphatidate phosphatase activities. Detailed kinetic analyses showed that sphingosine and other sphingoid bases were parabolic competitive inhibitors. We also show that S. cereuisiae cells contained free phytosphingosine and sphinganine. The cellular concentrations of phytosphingosine and sphinganine were in the range of the inhibitorconstantsdetermined for these sphingoid bases using pure enzymes. These resultsraised the suggestion that phosphatidate phosphatase activity may be regulated in vivo by sphingoid bases.

13831

by two-dimensional paper chromatography (31).Total lipid phosphorus was determined by the method of Bartlett (32) with the modifications of Kates (33). RESULTS

Effect of Sphingoid Bases on Phosphatidate Phosphatase Actiuity-The availability of purified preparations of the 104and 45-kDa forms of phosphatidate phosphatase from S. cereuisiae allowed us to examine the effects of sphingosine and other sphingoid bases on phosphatidate phosphatase activity directly under well defined conditions. In Triton XlOO/lipid-mixed micelles, both forms of phosphatidate phosphatase (4,7) follow surface dilution kinetics with respect to EXPERIMENTALPROCEDURES phosphatidate(4, 7). Sphingoid bases also form uniform Materials mixedmicelles with Triton X-100 and phospholipids and All chemicals were reagent grade. ATP, neutral lipids, phospho- display surface dilution (18). Thus,the concentrations of lipids, D-erythro-sphingosine (4-sphingenine1, D-phytosphingosine phosphatidate and sphingoid bases in the TritonX-lOO/lipid(~-4-hydroxysphinganine), DL-erythro-sphinganine (DL-erythro-di- mixed micelles were expressed as surface concentrations (in hydrosphingosine), psychosine (galactosylsphingosine), sphingomye- mol %) as opposed to bulk concentrations (18).Phosphatidate lin, ceramide, and bovine serum albumin were purchased from Sigma. Dioleoyl-diacylglycerol and dioleoyl-phosphatidate were purchased phosphatase activity is independent of the bulk concentration X-100/lipid-mixed micelle from Avanti Polar Lipids. Escherichia coli diacylglycerol kinase was of phosphatidate at the Triton obtained from Lipidex Inc. Radiochemicals were purchased from Du concentrations used in this study(7). Pont-New England Nuclear and scintillation counting supplies were We examined the effect of sphingoid bases on the activities purchased from National Diagnostics. Triton X-100 was a gift from of the 104- and 45-kDa forms of phosphatidate phosphatase Rohm and Haas. Growth medium supplies were purchased from Difco at a saturating concentration of M$+ ions and a subsaturating Laboratories. concentration (3 mol %) of phosphatidate. By using a phosphatidate concentration nearthe K , value for these enzymes, Methods we could more readily observe inhibitory or stimulatory effects Purification of Enzymes-The 104- (4, 6) and 45-kDa (4)forms of phosphatidatephosphatase were purified to near homogeneity as on phosphatidate phosphataseactivity. Sphingosine inhibited described previously. The specific activities of the 104- and 45-kDa the 104- (Fig. 1A) and 45-kDa (Fig. 1B) phosphatidate phosphatase activities in a dose-dependent manner. This inhibienzymes were 15 and 2.3 pmol/min/mg, respectively. Enzyme Assays and Protein Determination-Phosphatidate phos- tion of yeast phosphatidate phosphataseactivity by sphingophatase activity was measured for 20 min by following the release of sine was similar to thatshown for phosphatidatephosphatase water-soluble ["PlPi from chloroform-soluble [32P]dioleoyl-phosphafrom mammalian cells using crude enzyme preparations (16, tidate (10,000 cpm/nmol) at 30 "C (3). The reaction mixture contained 50 mM Tris-maleate buffer (pH 7.0), 10 mM 2-mercaptoetha- 17). S. cereuisiae cells do not contain sphingosine as the long nol, 2 mM MgCIz, an appropriate dilution of phosphatidate phosphatase, andthe indicated concentrations of Triton X-100 and chain base backbone of their sphingolipids (34). Instead, S. phosphatidate in a total volume of 0.1 ml. All assays were conducted cereuisiue has phytosphingosine (34), which differs from in triplicate with a typical standard deviation of f 5%. All assays sphingosine in that itlacks the 4,5-trans double bond present were linear with time and protein concentration. One unit of enzyin sphingosine and has a hydroxyl group at C-4 (Fig. 2). The matic activity was defined as the amount of enzyme that catalyzed the formation of 1 pmol of product/min. Specific activity was defined activities of the 104- (Fig. 1A)and 45-kDa (Fig. 1B) forms of as units/milligram of protein. Protein was determined by the method phosphatidate phosphatase were inhibited by phytosphingoof Bradford (23), using bovine serum albumin as thestandard. sine in a dose-dependent manner similar to that shown for Preparation of Triton X-lOO/Lipid-mixed Micelles-Lipids in chlo- sphingosine. roform were transferred to a test tube, and solvent was removed in We examined the effect of sphinganine on phosphatidate uacuo for 40 min. Triton X-100/lipid-mixed micelles were prepared phosphatase activity. Sphinganine differs from phytosphinby adding Triton X-100 to dried lipids. The total lipid concentration in Triton X-100/phospholipid-mixed micelles did not exceed 18 mol gosine in that it lacks the hydroxyl group at C-4 (Fig. 2). % to ensure that the structure of the mixed micelles were similar to Sphinganine inhibited 104- (Fig. lA)and 45-kDa (Fig. 1B) the structure of pure Triton X-100 (24,25). phosphatidatephosphatase activities in a dose-dependent Preparation of "P-labeled Phosphatidate-[32P]Dioleoyl-phospha- manner similar to that shown for sphingosine and phytotidate was synthesized enzymatically from dioleoyl-diacylglyceroland sphingosine. The unnatural threo-isomer of sphinganine also [y-32P]ATPusing E. coli diacylglycerol kinase (26) as described inhibited the phosphatidate phosphatase activities (data not previously (6). Growth Conditions-Wild-type strain ade5 MATa was used for the shown). Since the threo-sphinganine is not found in S. cerepurification of the 104- and 45-kDa forms of phosphatidate phospha- uisiae (35, 36) we did not further examine its effect on phostase and lipid analyses. Cells were maintained on 1%yeast extract, phatidate phosphatase activity. We also examined the effect 2% peptone, 2% glucose medium plates containing 2% Bacto-Agar. of sphingomyelin and ceramide on the 104- and 45-kDa phosCells were grown at 30 "C to the exponential phase of growth (1 x phatidate phosphatase activities (Fig. 1); neither had a signif10' cells/ml) in complete synthetic media containing 2% glucose (27, 28). Cell numbers were determined by microscopic examination with icant effect on the activities of the phosphatidate phosphatases. a hemacytometer or by absorbance a t 660 nm. Structural Requirements for Sphingoid Base Inhibition of Mass Analysis of Sphingoid Bases, Phosphatidate, and Total Lipid Phosphorus-Sphingoid bases (29) and totalphospholipids (30) were Phosphatidate PhosphataseActiuity-The structural features extracted from exponential phase cells (1 X 10' cells/ml) as previously common to thesphingoid bases are afree amino group, a long described. 0-Phthalaldehyde derivatives of sphingoid bases were pre- chain hydrocarbon, and free primary and secondary hydroxyl pared and analyzed by reverse-phase high performance liquid chromatography using a C18 column as described by Merrill et al. (29). groups (Fig. 2). Stearylamine, a long chain primary amine The identity of each sphingoid base was determined by comparing its (Fig. 2), inhibited the 104- (Fig. 3A) and 45-kDa (Fig. 3B) elution profile with that of authentic standards(29). C20 sphinganine forms of phosphatidatephosphatase in a dose-dependent was used as an internal standard (29). Phosphatidate was analyzed manner similar to theinhibitory effect displayed by sphingoid

RegulationPhosphatidate of Yeast Phosphatase

13832 a0

Sphingosine

’

Phytosphingosine

0 5

4

10

15

Lipid. Mol %

0

5

10

Sphinganine

P 3

Psychosine

P 3

15

Lipid, Mol % FIG. 1. Effect of sphingoid bases and sphingolipids on 104and 46-kDa phosphatidate (PA) phosphatase activities. 104(panel A ) and 45-kDa (panel B ) activities were measured under standard assay conditions in the presence of the indicated surface concentrations of sphingosine (V),phytosphingosine (O), sphinganine (m), sphingomyelin (O),and ceramide (V).The surface concentration of phosphatidate was 3 mol % (bulk concentration of 0.1 mM).

Stearylamine FIG. 2. Structures of sphingoid bases and related compounds.

sentative of both the 104- and 45-kDa forms of phosphatidate phosphatase because the enzymological and kinetic properties of both enzymes are similar (4, 6, 7), and the inhibition of bases. Octylamine, oleic acid, and stearyl alcohol did not both enzymes by sphingoid bases was also similar. The phosinhibitphosphatidatephosphatase activity (Fig. 3). These phatidate dependence of 104-kDa phosphatidate phosphatase results raised the suggestion that a free amino group and a activity was examined in the absence and presence of several long chain hydrocarbon are required for the inhibition of fixed concentrations of sphingosine (Fig. 4.4). In the absence phosphatidate phosphatase activity. The requirement for the of sphingosine, the dependence of phosphatidate phosphatase free amino group was most likely due to its positive charge activity on phosphatidate was cooperative. The kinetic data (18,21). Thiswas consistent with the 104- and 45-kDa phos- were analyzed according to the Hill equation using the EZphatidate phosphatase activities being inhibited by propran- FIT computer program (37). A Hill number of 1.92was olol (4). Therequirement for a long chain hydrocarbon on the obtained for the phosphatidate dependence curve in the abmolecule is to partition the molecule into the Triton X-100/ sence of sphingosine. Phosphatidate phosphataseactivity was lipid mixed micelle or in vivo into themembrane lipid bilayer inhibited by sphingosine in adose-dependent manner at each concentration. Furthermore, the enzyme (18, 21). The primary hydroxyl group on the sphingoid base phosphatidate did not appear to be critical for inhibition since both steary- showed sigmoidal kinetics toward phosphatidate a t each lamine and psychosine inhibited phosphatidate phosphatase sphingosine concentration. The Hill number a t each sphinactivity (Fig. 3). Psychosine was a less potent inhibitor of the gosine concentration did not vary significantly from the value phosphatidatephosphatase activities when compared with of 2. The data were transformed to a double-reciprocal plot stearylamine and other sphingoid bases. This was presumably where the phosphatidate concentration was raised to theHill due to itsbulky galactosyl group (Fig. 2) which could sterically number of 2 (38). Sphingosine did not affect the apparent hinder the interaction of the enzyme with the amino group V,. value for the enzyme but did cause an increase in the apparent K,,, for phosphatidate (Fig. 4B). These results were on the sphingoid base molecule (21). Effect of Sphingoid Bases o n the Kinetics of Phosphutidate consistent with sphingosine being a competitive inhibitor of detailed kinetic analysis was per- phosphatidate phosphatase (38). A replot of the slopes from Phosphatase Activity-A formed on the 104-kDa phosphatidate phosphatase to exam- the double-reciprocal plot shown in Fig. 4B versus the sphinine the mechanism of sphingoid base inhibition on enzyme gosine concentration resulted in a parabolic curve (Fig. 4 0 . activity (Fig. 4). The 104-kDa enzyme was used as a repre- This was indicative of parabolic competitive inhibition where

Regu~ut~ of oYeast ~ P ~ o s p ~ ~t ~h uo ts e~ h u t a s e

13833

value for sphingosine was calculated to be 1.5 mol % (Table

I). The effects of phytosphingosine and sphinganine on the kinetic properties of phosphatidate phosphatase were examined by the same kinetic analysis used for sphingosine. Phytosphingosine (Fig. 5 A ) and sphinganine (Fig. 6 A ) inhibited phosphatidate phosphataseactivity in a dose-dependent manner at each phosphatidate concentration. Furthermore, phosphatidate phosphatase exhibited sigmoidal kinetics toward phosphatidate at each concentration of these sphingoid bases with little effect on the Hill number. Double-reciprocal plots were constructed from the data inFigs. 5A and 6A where the phytosphingosine and sphinganineconcentrations, respectively, were raised to theHill number of 2. These plots showed that phytosphingosine (Fig. 5 B ) and sphinganine (Fig. 6 B ) were competitive inhibitors of phosphatidate phosphatase activity. Parabolic curves resulted from replots of the slopes from Figs. 5B and 6B versus the phytosphingosine (Fig. 5C) and sphinganine (Fig. 6 C ) concentrations, respectively. Thus, like sphingosine, phytosphingosine and sphinganine were parabolic competitive inhibitors of phosphatidate phosphatase value for each concentration activity. Accordingly, the Kinlope la, of phytosphingosine and sphinganine was calculated as described above. When the data were plotted as l/Kielowversus the phytosphingosine (Fig. 5L)) and sphinganine (Fig. 6 D ) concentrations, respectively, linearplots were obtained as 50 predicted by Equation 2. The aKi values for phytosphingosine and sphinganine were calculated to be 0.4 and 0.2 mol %, respectively (Table I). Cellular Concentrations of Sphingoid Bases-It is unclear 54 - 0 whether phytosphingosine or sphinganine existin S. cerevis5 10 15 $ 0 iae as free sphingoid bases. It was important for us to demonstrate that free phytosphingosine and sphinganine exist in Lipid, Mol% FIG.3. Effect lipid analogs of sphingoid bases on 104- and S. cerevisiae cells if these sphingoid bases were to have a role 46-kDa phosphatidate ( P A )phosphatase activities. 104- (panel in the regulation of phosphatidate phosphatase activity in A ) and 45-kDa (panel B ) activities were measured under standard vivo. The methods developed for the extractionand high assay conditions in the presence of the indicated surface concentra- performance liquid chromatography analysis of free sphingoid tions of stearylamine (II), psychosine (O), octylamine (V),stearyl alcohol (V),and oleic acid (e). The surface concentration of phos- bases from mammalian cells (29) were adapted for analysis of sphingoid bases from S. cerevisiae. A typical chromatogram phatidate was 3 mol % (bulk concentrationof 0.1 mM). of a sphingoid base analysis from S. cerevisiae cells is shown in Fig. 7. This analysis showed that S. cerevisiae cells did two inhibitor molecules contribute to the exclusion of the contain free phytosphingosine and sphinganine. As expected, substrate from the enzyme (38). The Ki value for sphingosine could not be determined from Fig. 4C because the curve was S. cerevisiae cells did not contain free sphingosine (Fig. 7 ) .In not linear. Fu~hermore,calculations of Ki values a t each addition to ph~osphingosineand sphinganine, the analysis showed the presence of other more minor sphingoid base sphingosine concentration determinedfrom the slopes of Fig. 4B would not be true Ki values (38). Since sphingosine was a species, which werenot identified. Care was taken so that the parabolic competitive inhibitor, the Ki values calculated from detection of free sphingoid bases in cells was not due to the Fig. 4B would actually be Kistopevalues (38). Kistopis not the hydrolysis of sphingolipids during the extraction procedure Ki for the dissociation of an enzyme-inhibitor complex (38). (29). The concentrations of phytosphingosine and sphinganInstead the Kis,opeis a more complex function of Ki which ine were calculated based on the C20 sphinganine internal varies with the inhibitor concentration (38). The KiSlw for standard, and theresults are shown in Table 11. The conceneach sphingosine concentration was calculated from the data tration of sphinganine was about %fold greater than that of phytosphingosine. Phytosphingosine and sphinganine acin Fig. 4B from Equation 1 (38) as follows: counted for 0.16 and 0.53 mol %, respectively, of the total slopell1 = I1 + ~ ~ I ~ / K ~ I ~ (Es. ~ ~1) ~ phospholipids K ~ / V ~(which ~ ~ included sphingolipids) in S. cerevisiae (Table 11). Sphingolipids account for about one-third of the where total phospholipids in S. cereuisiae (34). The concentrations of phytosphingosine and sphinganine were 18.5- and 5.7-fold Kidop = Ie/(IIl + 2aKA (Eq.21 lower, respectively, than that of phosphatidate (Table 11).As previously shown (391, phosphatidate accounted for 3 mol % Equation 2 takes into account two inhibitor sites and the term a in the uKi constant is an interaction factor for the of the totalphospholipids. inhibitor (38). Equation 2 predicts that a plot of l/Kislope DISCUSSION versus the inhibitor concentration should result in a straight line where the intercept of the inhibitor axis isequal to -2aKi Using purified preparations of phosphatidate phosphatase (38). When l/Kisiowwas plotted versus the sphingosine con- from the yeast S. cerevisiae, we examined in detail the inhicentration a straight line was obtained (Fig. 4 D ) . The aKi bition of phosphatidate phosphatase activity by sphingosine

13834

Regulation of Yeast PhosphatidatePhosphatase a t

I

I

4

6

c '

FIG. 4. Effect of sphingosine on the kinetics of 104-kDa phosphatidate (PA)phosphatase activity with respect to the surface concentration of phosphatidate. Panel A, 104-kDa phosphatidate phosphatase activity was measured as a function of the surface concentration (mol %) of phosphatidate (bulk concentration of 0.1 mM) at set surface concentrations (mol %) of sphingosine: 0, 0; V, 2; V, 4.35; 0, 5; W, 7. Panel B, reciprocal plot of the data in panelA where the phosphatidate concentration was raised to the Hill number of 2. Panel C, replot of the slopes obtained frompanel B versm the sphingosine surface concentration. Panel D, replot of the l/KilOpobtained frompanel B versus the sphingosine surface concentration. The lines drawn in panels B and D were a result of a least-squares analysis of the data.

2

0

4

0'

6

0

PA, Mol %

I

I

2

I

Sphingosine,Mol %

2

B

3 -

-0.4

0.0

0.4

0.8

IPA, Mol %-2 TABLE I Kinetic constants for phosphatidatephosphatase

1

3

1.2

n "

-4

I 0

4

-

8

Sphingosine,Mol %

tase inhibition by the sphingoid bases was not straightforward since the enzyme did not follow typical saturation kinetics Phosphatidate (Figs. 4A, 5A, and 6A). Linear plots were obtained when the Substrate orphosphatase inhibitor kinetic data were plotted in double-reciprocal form where the phosphatidate concentration was raised to a Hill number of K, aKi 2 . These plots (Figs. 4B, 523, and 6 B ) resulted in a family of mol % lines consistent with the sphingoid bases being competitive 1.5 Sphingosine 0.4 inhibitors (38) of phosphatidate phosphatase activity. HowPhytosphingosine 0.2 Sphinganine ever, the sphingoid bases were not simple linear competitive 2.2 Phosphatidate inhibitors. Instead, replots of the kinetic data indicated a parabolic competitive inhibition mechanism (38) where two and other sphingoid bases including phytosphingosine and molecules of sphingoid base contributes to the exclusion of sphinganine. The structural requirements for the inhibition phosphatidate from the enzyme (Figs. 4C, 5C, and 6C). This, of enzyme activity by sphingoid bases were a free amino group coupled to the fact that phosphatidate phosphatase had two and along chain hydrocarbon. These structural requirements phosphatidate binding sites, indicated that themechanism of were similar to that shown for the sphingoid base inhibition enzyme inhibition by sphingoid bases was complex. We were, of protein kinase C purified from mammalian cells (18, 2 1 ) . however, able to determine inhibitor constants (aKi values) The detailed kinetic analysis performed here on the phospha- for the sphingoid bases by constructing replots of l/Kis,ow tidate phosphatase showed that thedependence of activity on versus the sphingoid base concentrations according to Segel phosphatidate was cooperative with a Hill number of 2. Our (38).These inhibitor constantswere useful for comparison of previous kinetic studies did not reveal this cooperative behav- the relative potency of the sphingoid base inhibitors andwith ior because the phosphatidateconcentrations used in the the cellular concentrations of sphingoid bases. The aKi values for sphingosine, phytosphingosine, and previous studies were not varied below 1.6 mol % (7). The cooperative behavior of phosphatidate phosphatase activity sphinganine were lower than theK,,, value for phosphatidate toward phosphatidate also occurred in the presence of the (Table I). Thus, when the concentration of these sphingoid sphingoid base inhibitors. Purified protein kinase C shows bases were high relative to the phosphatidate concentration, cooperative behavior toward its phospholipid cofactor phos- the bases had a dramaticeffect on phosphatidate phosphatase phatidylserine in the absence (40) and presence of sphingosine activity (Figs. 4A, 5A, and 6A). Sphingosine was the least potent of the sphingoid base inhibitors based on the aKi (18). Whereas sphingosine causes dramatic changes in the Hill number for the protein kinase C dependence on phos- values. The inhibition of phosphatidate phosphatase activity phatidylserine, the sphingoid bases did not significantly affect by sphingosine was not physiologically relevant in S. cerevisthe phosphatidate phosphatasedependence on phosphatidate. iae. Free sphingosine was not found in S. cerevisiae cells, and The analysis of the mechanism of phosphatidate phospha- it does not serve as the long chain base backbone of their

RegulationPhosphatidate of Yeast Phosphatase

FIG. 5. Effect of phytosphingosine on the kinetics of 104-kDa phosphatidate (PA)phosphatase activitywith respect to the surface concentration of phosphatidate. Panel A , 104-kDa phosphatidate phosphatase activity was measured as a function of the surface concentration (mol %) of phosphatidate (bulk concentration of 0.1 mM) at setsurface concentrations (mol %) of phytosphingosine: 0 , O ; V, 2; V, 4.35; 0, 6; B, 8. Panel B , reciprocal plot of the data in panel A where the phosphatidate concentration was raised to the Hill number of 2. Panel C, replot of the slopes obtained from panel B versus the phytosphingosine surface concentration. Panel D,replot of the l/Ki.. Iobtained from panel B versus the phytosphingosine surface concentration. The lines drawn in panels B and D were a result of a least-squaresanalysis of the data.

13835

0

D

3

3

2

B leO

1

0.5

0.0

0.4

I

0

t

-0.6 0.0 0.6

1.2

l P A , Mol %-2

I

I

6

PA, Mol %

9

1.5

0.8

l P A , Mol %-2

4

6

Phytosphingosine, Mol %

PA, Mol %

0 -0.4

2

3

Sphinganine. Mol %

1.8

Sphinganine, Mol %

1.2

0.0 -

3

0

3

6

9

Phytosphingosine, Mol %

FIG. 6. Effect of sphinganine on the kinetics of 104-kDa phosphatidate (PA)phosphatase activity with respect to the surface concentration of phosphatidate. Panel A , 104-kDa phosphatidate phosphatase activity was measured as a function of the surface concentration (mol %) of phosphatidate (bulk concentration of0.1mM) at set surface concentrations (mol %) of sphinganine: 0, 0; V, 2; V, 3; 0, 4.35; B, 6. Panel B, reciprocal plot of the data in panelA where the phosphatidate concentration was raised to the Hill number of 2. Panel C, replot of the slopes obtained from panel B versus the sphinganinesurface concentration. Panel D, replot of the l/Ki8Iw obtained frompanelB versus the sphinganine surface concentration. The lines drawn in panels B and D were a result of a least-squares analysis of the data.

13836

Regulation of Yeast PhosphatidatePhosphatase

consistent with this notion. However, morestudies areneeded to establish the sources of free long chain bases in S. cereuisiae. Sphinganine was a more potent inhibitor of phosphatidate phosphatase activity when compared with phytosphingosine. The aKi value for sphinganine (Table I) was 2.6-fold lower than the cellular concentration of sphinganine(Table 11) whereas the aKi value for phytosphingosine was2.5-fold higher than itscellular concentration. The uKi values for both phytosphingosine and sphinganine were in the range of the cellular concentrations of these sphingoid bases. Thus, phosphatidate phosphatase activity could be sensitive to changes in the phytosphingosine and/or sphinganine concentrations in vivo. We did not determine the membrane localization of phytosphingosine and sphinganine in S. cerevisiae cells. It is 0 5 10 15 20 25 known that the enzymes of sphingoid base synthesisare membrane-associated (42). Thus, the cellular localization of Retention Time, min membrane-associated phosphatidatephosphataseandits FIG. 7. C l 8 high performance liquid chromatography of sphingoid base inhibitors may be in proximity for the regufree sphingoid bases. Sphingoid bases were extracted from S. lation of activity in viuo. cereuisiue cells, and their0-phthalaldehyde derivatives were prepared In mammalian cells the inhibition of phosphatidate phosand analyzed by C18 high performance liquid chromatography as described in the text. The elution positions of phytosphingosine phatase activity by sphingosine is thought to play an indirect (PSo),sphinganine (Sa),and theC20 sphinganine internal standard role in the regulation of protein kinase C activity (16, 17). (C20) are indicated in the figure. The elution position of sphingosine The inhibition of phosphatidate phosphatase by sphingosine (So), which was not present in the sample, is also indicated. would prevent the generation of diacylglycerol via the phosphatidylcholine signaling pathway (13). Many of the enzymes TABLE I1 of the phosphatidylcholine signaling pathway, including proCellular concentrations of sDhinPoid bases and DhosDhatidate tein kinase C (48), exist in s. cerevisiae. However, it is not clear whether such a signaling mechanism involving sphingoid Lioid Cellular conc. bases exists in S. cerevisiae. The role of phosphatidate phospmol/lO' cells mol %" phatase inhibition by sphinganine and/or phytosphingosine 7.5 0.16 Phytosphingosine in S. cereuisiae may be manifested most directly in its effect 25 0.53 Sphinganine 140 3 on the proportional synthesis of phospholipids and triacylPhosphatidate 4630 Total DhosDholiDid glycerol.Accordingly, our futurestudies will address this "The concentrationin mol % was calculated by dividing the regulation. concentration of sphingoid base or phosphatidate by the concentration of total phospholipid. The reported values were the average of three determinations.

sphingolipids (34). In addition, sphingosine will not provide the sphingoid base requirement of long chain base (kbl mutant) auxotrophs (35) nor will it reverse the inhibitory effects of sphingofungin on the growth of wild-type cells (41). On the other hand, our kinetic studies on the inhibition of S. cerevisiae phosphatidate phosphatase by sphingosine should be useful in understandingthe mechanism of inhibition of phosphatidate phosphatase by sphingosine in higher eucaryotes. To our knowledge this is the first report that showed that S. cereuisiae cells contain free phytosphingosine and sphinganine. These sphingoid bases accounted for less than 1 mol % of the totalphospholipids in cells. Sphinganine is a precursor of phytosphingosine in S. cereuisiae (35, 42) and sphingosine and phytosphingosine in mammalian cells (43).In mammalian cells it appears that free long chain bases arise mostly from sphingolipid turnover (44). Sphinganine is mostly N-fatty acylated before being converted to sphingosine, and the levels of sphingosine in mammalian cells may be attributed to sphingolipid turnover as opposed to being used for sphingolipid synthesis (45, 46). The steps in the synthesis of phytosphingosine from sphinganine inS. cerevisiae is unclear (42). Similar to mammalian cells, free sphinganine in S. cereuisiae cells may represent some of the sphingoid base used for sphingolipid synthesis while free phytosphingosine may be the result of sphingolipid turnover. Angus and Lester (47) have suggested that there is little sphingolipid turnover in S. cereuisiae. The %fold lower cellular concentration of phytosphingosine relative to the concentration of sphinganine was

Acknowledgment-We thank Rosa J. Buxeda for helpful discussions concerning the kinetic analysis of phosphatidate phosphatase. REFERENCES 1. Smith, S. W., Weiss, S. B., and Kennedy, E. P. (1957)J. Biol. Chem. 228, 915-922 2. Carman, G . M., and Henry, S. A. (1989)Annu. Reu. Biochem. 68,,635-669 3. Morlock, K.R., Lin, Y.-P., and Carman, G. M. (1988)J. Bacterrol. 170, 'IRA1 """ -'IRA6 """

4. Morlock, K. R., McLaughlin, J. J., Lin, Y.-P., and Carman, G. M. (1991) J. Biol. Chem. 266,3586-3593 5. Quinlan, J. J., Nickels, J. T., Jr., Wu, W.-I., Lin, Y.-P., Broach, J. R., and Carman. G. M. (1992)J. Biol. Chem. 267. 18013-18020 6. Lin, Y.-P.,'and Carman; G. M. (1989)J. B{oj. Chem. 264,8641-8645 7. Lin, Y.-P., and Carman, G . M. (1990)J. Brol. Chem. 266,166-170 8. Hosaka, K., and Yamashita, S. (1984)Biochim. Biophys. Acta 796,110117 9. Taylor, F. R.,and Parks, L. W. (1979)Biochim. Biophys. Acta 676,204214 10. Brindley, D. N. (1984)Prog. Lipid Res. 23,115-133 11. Kennedy, E. P. (1986)in Lipids and Membranes: Past, Present and Future (Op den Kamp, J. A. F., Roelofsen, B., and Wirtz, K.W.A., eds) pp. 171-206,Elsevier Science Publishers B. V., Amsterdam 12. Moore, T. S. (1982)Annu. Reu. Plant Physiol. 33,235-259 13. Exton, J. H.(1990)J. Biol. Chem. 266, 1-4 14. Kiss, Z., and Anderson, W. B. (1990)J . Biol. Chem. 266,7345-7350 15. Lavie, Y., and Liscovitch, M. (1990)J. Biol. Chem. 266, 3868-3872 16. Mullmann, T. J., Siegel, M. I., Egan, R. W., and Billah, M. M. (1991)J . Biol. Chem. 266, 2013-2016 17. Lavie, Y., Piterman, O., and Liscovitch, M. (1990)FEBS Lett. 277, 7-10 18. Hannun, Y. A,, Loomis, C. R., Merrill, A. H., Jr., and Bell, R. M. (1986)J . Biol. Chem. 261,12604-12609 19. Hannun, Y. A,, and Bell, R. M. (1989)Science 243,500-507 20. Hannun, Y. A,, Greenberg, C. S., and Bell, R. M. (1987)J. Biol. Chem. 262,13620-13626 21. Merrill, A. H., Nimkar, S., Menaldino, D., Hannun, Y. A., Loomis, C., Bell, R. M., Tyagi, S. R., Lambeth, J. D., Stevens, V. L., Hunter, R., and Liotta, D. C. (1989)Biochemistry 28,3138-3145 22. Stevens, V. L., Nikmar, S., Jamison, W. C. L., Liotta, D. C., and Merrill, A. H. (1990)Biochim. Biophys. Acta 1061,37-45 23. Bradford, M. M. (1976)Anal. Brochem. 72,248-254 24. Robson, R. J., and Dennis, E. A. (1983)Acc. Chem. Res. 16,251-258 25. Lichtenberg, D., Robson, R. J., and Dennis, E. A. (1983)Biochim. Biophys. Acta 737,285-304 26. Walsh, J. P., and Bell, R.M. (1986)J. Biol. Chem. 261,6239-6247

RegulationPhosphatidate of Yeast Phosphatase 27. Henry, S. A., Atkinson, K. D., Kolat, A. J., and Culbertson, M. R. (1977) J. BacterioL 130,472-484 28. Culbertson, M. R., and Henry, S. A. (1975) Genetics 80,23-40 29. Merrill, A. H., Wang, E., Mullins, R. E., Jamison, W. C. L., Nimkar, S., and Liotta, D.C. (1988) Anal. Biochem. 171,373-381 30. Hanson, B. A., and Lester, R. L. (1980) J.Lipid Res. 21,309-315 31. Steiner, M. R., and Lester, R. L. (1972) Biochim. Biophys. Acta 260, 222243 32. Bartlett, G.R. (1959) J. Biol. Chem. 234,466-468 33. Kates, M. (ed) (1986) Techniques of Lipidology. Isolation, Analysis and Identification of Lipids, pp. 186-278, Elsevier, New York 34. Smith, S. W., and Lester, R. L. (1974) J.BWL Chem. 249,3395-3405 35. Wells, G . B., and Lester, R. L. (1983) J. Biol. Chem. 268,10200-10203 36. Pinto, W. J., Srinivasan, B., Shepherd, S., Schmidt, A., Dickson, R. C., and Lester, R. L. (1992) J. Bacteriol. 174,2565-2574 37. Perrella, F. (1988) Anal Biochem. 174,437-447 38. Segel, I. H. (ed) (1975) Enzyme Kinetics: Behavior and Analysis of Rapid

13837

Equilibrium and Steady-state Enzyme System, pp. 1-957, John Wiley and Sons New York 39. Kelley, M. d., Bailis, A. M., Henry, S. A., and Carman,G . M. (1988) J.Biol. Chem. 263,18078-18085 40. Hannun, Y.A., Loomis, C. R., and Bell, R. M. (1985) J. Biol. Chem. 260, 1 nnw-1*"nnAR 41. Zweerink, M. M., Edison, A. M., Wells, G. B., Pinto, W., and Lester, R. L. (1992) J. Biol. Chem. 267,25032-25038 42.Pi@,-W,J., Wells, G. W., and Lester, R. L. (1992) J. Bacteriol. 174,

"__"

. 1

Zb'lb-2581

Merrill, A. H., and Jones D. D. (1990) Biochim. Bio hys Acta 1044,l-12 Merrill A. H. (1991) J. dioener Bwmembr. 23 8 g 1 0 4 Merrili A. H. Jr. and Wang Ef(1986) J.Biol. h e m . 261,3764-3769 Wang E. Ndrred W. P B&on C. W. Rile R. T., and Merrill, A. H., Jr.,'(19h) J. B6l. Chem. 266,'14486-14498' 47. Angus, W. and Lester R. L. (1972) Arch. Bmchem. B W p Y 151,483-495 48. 0g1ta K h i amoto 4 Koide H. Iwal T. Oka M. An 0,K , Klshmoto, A. >kt& If. Fukim;, Y., and N'ishiz;ka:Y. (i9901 Proc. Natl. Acad. Set. U.'S. A. 87,k011-5015

43. 44. 45. 46.