From the Department of Biochemistry and the Lucille P. Markey Cancer Center, University of Kentucky Medical Center, Lexington, Kentucky 40536-0084
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sphingolipid long chain bases (LCBs) and phosphorylated derivatives, particularly sphingosine 1-phosphate, are putative signaling molecules. To help elucidate the physiological roles of LCB phosphates, we identified two Saccharomyces cerevisiae genes, LCB4 (YOR171c) and LCB5 (YLR260w), which encode LCB kinase activity. This conclusion is based upon the synthesis of LCB kinase activity in Escherichia coli expressing either LCB gene. LCB4 encodes most (97%) Saccharomyces LCB kinase activity, with the remainder requiring LCB5. Log phase lcb4-deleted yeast cells make no LCB phosphates, showing that the Lcb4 kinase synthesizes all detectable LCB phosphates under these growth conditions. The Lcb4 and Lcb5 proteins are paralogs with 53% amino acid identity but are not related to any known protein, thus revealing a new class of lipid kinase. Two-thirds of the Lcb4 and one-third of the Lcb5 kinase activity are in the membrane fraction of yeast cells, a puzzling finding in that neither protein contains a membrane-localization signal. Both enzymes can use phytosphingosine, dihydrosphingosine, or sphingosine as substrate. LCB4 and LCB5 should be useful for probing the functions of LCB phosphates in S. cerevisiae. Potential mammalian cDNA homologs of the LCB kinase genes may prove useful in helping to understand the function of sphingosine 1-phosphate in mammals.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The sphingolipid metabolite sphingosine and its phosphorylated derivative, sphingosine 1-phosphate (SPP),1 are thought to be signaling molecules for regulating a variety of mammalian cellular processes including cell growth, motility, and death (for review, see Refs.1-3). Recently, SPP and sphingosylphosphorylcholine have been found to bind G protein-coupled receptors (for review, see Ref. 3) that may play roles in platelet activation (4), regulating heart rate (5), the oxidative burst (6), neurite retraction (7), suppression of ceramide-induced apoptosis (7, 8), and morphogenetic differentiation of endothelial cells (9). The finding of both intracellular and extracellular SPP receptors is a strong indication that SPP modulates physiological processes.
Most studies of SPP published to date have used cultured mammalian cells. Whole animal as well as tissue culture studies would benefit from the availability of a cDNA encoding sphingosine kinase which could be used to verify the physiological significance of SPP signaling and to determine how the level of SPP is regulated. No such cDNA is available, nor has any such gene been identified. Here we present evidence for the identification of two Saccharomyces cerevisiae genes encoding kinases, referred to as LCB (long chain base) kinases, because they phosphorylate several LCBs including sphingosine. These two genes should expedite future studies of the functions of LCB phosphates in Saccharomyces and in multicellular eukaryotes.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strains, Plasmids, and Media--
Strains used in these studies
are listed in Table I. Strain MSS200 was
made from strain JK9-3da by integrating the TPS2-LacZ reporter plasmid pTPS2-1 into the ura3 locus as described
previously (10). Strain MSS206 is a derivative of MSS200 made by
deleting DPL1 with the 1 allele followed by insertion of
TRP1 into LEU2 by a marker swapping technique
(11). The dpl1-
1 allele has the region between the
NheI and BglI restriction sites, codons 10-536,
replaced by LEU2. Strain RCD158 is a derivative of JK9-3d MAT
carrying lcb4-
1, a deletion allele with codons
1-560 replaced by a kanamycin resistance cassette. Strain RCD164 is a
derivative of JK9-3d MATa carrying lcb5-
1, which has
codons 3-683 replaced with a kanamycin resistance cassette. The
kanamycin deletion alleles were generated by using the PCR, pUG6 (12)
as a template, and a primer with 45 bases homologous to the sequence
upstream or downstream of the deleted codons. Cells transformed with
the deletion alleles were selected for G418 resistance (13). The
expected deletion event was verified by PCR analysis of chromosomal
DNA. RCD165 carries both lcb4-
1 and lcb5-
2
and was obtained as a meiotic segregant of diploid strain RCD167.
RCD167 was made by crossing RCD165 carrying pRS315 (LEU2,
14) with RCD158, which had TPS2-LacZ reporter plasmid
integrated into the ura3 chromosomal locus to give
Ura+ cells.
|
Preparation of Yeast Extracts--
Yeast extracts were prepared
by vortexing (6 × 30 s in a 15-ml Corex tube) 50-75
A600 units of yeast cells in 1 ml of the extraction buffer (50 mM Hepes, pH 7.5; 5 mM
dithiothreitol; 1 mM phenylmethylsulfonyl fluoride; and 1 µg/ml each of leupeptin, pepstatin, and aprotinin) with 0.5 ml of
0.5-mm-diameter acid-washed glass beads. All steps were done at
4 °C. The lysate was centrifuged for 5 min at 1,000 × g in a Sorvall SS-34 rotor. The resulting supernatant fluid
was centrifuged at 100,000 × g for 15 min in a TLA
100.3 rotor (Beckman). The final supernatant fluid was frozen and
stored at 20 °C. The final pellet was washed twice with extraction buffer, resuspended in 400 µl of buffer containing 20% glycerol, and
frozen at
20 °C.
Assay of LCB Kinase Activity-- The LCB kinase assay was based on the method of Crowther and Lynch (19). Reactions contained 7 µM DL-erythro-[4,5-3H]DHS ((20), 30 cpm/pmol, 20,000 cpm total), 0.5 mM Triton X-100, 1 mM MgCl2, 1 mM ATP, 100 mM Tricine, pH 8.1, and 1-50 µg of yeast proteins in a final volume of 100 µl. The reaction mixture was prepared by drying DHS under a stream of nitrogen, adding Triton X-100, and vortexing, followed by the addition of the other components. Enzyme was added to initiate the reaction; after incubation at 30 °C for 30 min, the product was separated from the substrate by differential solvent extraction (19). The amount of product formed was determined by liquid scintillation counting in Ultima Gold LSC-mixture (Packard). The Bradford reagent (Bio-Rad Laboratories) was used to measure protein concentrations with bovine serum albumin as a standard.
Miscellaneous Procedures-- DL-erythro-Dihydrosphingosine, C2-ceramide (N-acetylsphingosine), L-threo-DHS, and D-erythro-sphingosine were purchased from Matreya. LCB phosphates were measured as 32P-labeled compounds by growing cells overnight in PYED medium containing 32Pi and lacking exogenous potassium phosphate.2
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Optimization of Assay Conditions for S. cerevisiae LCB Kinase Activity-- The assay conditions for LCB kinase activity have not been studied systematically in S. cerevisiae cells. We felt it was necessary to optimize the assay conditions as much as possible using cell extracts before isolation of mutants lacking enzyme activity. The components necessary for LCB kinase enzyme activity were determined using [3H]DHS as the LCB substrate. We verified that this substrate was converted to [3H]DHS-1-P by comparison of its mobility on a TLC plate with an authentic standard (data not shown).
As expected from previous studies of sphingosine kinase from mammals and other organisms (19, 21), the S. cerevisiae LCB kinase requires ATP and a detergent, Triton X-100, for activity (Fig. 1A). In the absence of detergent only about 10% of the [3H]DHS was solubilized in the reaction mixture. Complete Mg2+ dependence is evident from the partial loss of enzyme activity in the absence of exogenous Mg2+ and the complete inhibition of enzyme activity by EDTA in the absence of exogenous Mg2+. The reaction was not stimulated by 10 mM Ca2+ (data not shown). The reaction is linear with time for at least 60 min and with a protein concentration up to about 25 µg (data not shown).
|
|
Isolation of a Mutant Strain Lacking LCB Kinase Activity-- Our rationale for identifying a gene encoding LCB kinase activity was based upon the observation that cells deleted for the DPL1 gene are more sensitive to growth inhibition by treatment with sphingosine than are parental cells (22). DPL1 is necessary for the breakdown of SPP to phosphoethanolamine and hexadecenal and is thought to encode the SPP lyase activity present in S. cerevisiae cells (22). It seemed likely that sensitivity to sphingosine was caused by the accumulation of SPP and that inactivation of LCB kinase activity by mutation of the cognate gene would reverse the sphingosine-sensitive phenotype of the dpl1 deletion strain and allow cells to grow in the presence of an inhibitory concentration of sphingosine.
To be able to identify the mutated LCB kinase gene, MSS206 (relevant genotype is dpl1-
|
The LCB4 Gene Encodes Most of the LCB Kinase Activity--
To
verify that LCB4 was necessary for LCB kinase activity, we
deleted the gene in strain JK9-3d to give strain RCD158. RCD158 cells have only 2-3% of the LCB kinase activity found in parental JK9-3d
cells (Table II), demonstrating that Lcb4p constitutes 97-98% of the LCB kinase activity in wild type cells. The
distribution of the Lcb4 protein was examined by using RCD164
(
lcb5) cells. About one-third of the Lcb4 kinase activity
is in the soluble fraction after two washes of the 100,000 × g pellet, and the remainder is in the pellet fraction. This
distribution is reversed in strain RCD158 (
lcb4),
indicating that the Lcb5 protein has a different distribution of enzyme
activity (Table II). Because there is no detectable LCB kinase activity in the lcb4 lcb5 double
deletion strain RCD165, we conclude that these two genes encode all LCB kinase activity in JK9-3d cells, at least under our assay
conditions.
|
LCB4 and Its Homolog LCB5 Encode LCB Kinase Activity-- Because the predicted Lcb4 and Lcb5 proteins do not show similarity to known proteins it was not clear whether they catalyzed phosphorylation of LCBs or regulated LCB kinase activity. To distinguish between these possibilities each gene was expressed in E. coli as a fusion protein with a His6-Xpress epitope-enterokinase cleavage sequence added to their amino terminus. The soluble protein fraction made from bacterial cells carrying either the LCB4 or the LCB5 fusion gene had LCB kinase activity as shown by the protein- and time-dependent formation of DHS-1P (Fig. 4). Control extracts made from cells carrying the vector had no detectable LCB kinase activity (Fig. 4). We conclude from these experiments that LCB4 and LCB5 encode LCB kinase activity.
|
RCD158 (lcb4) Cells Contain No Detectable LCB
Phosphates--
To determine which of the LCB genes is
responsible for synthesis of LCB phosphates we quantified the amount of
these compounds in mutant and parental cells. Cells were grown
overnight in the presence of 32Pi at 25 °C
to early log phase (A600 nm = 0.3) and then
shifted to 37 °C for 10 min. Lipids were extracted as described
previously (17), deacylated, and enriched for LCB phosphates by
chromatography on an AG4 column.2 The fractions containing
partially purified LCB phosphates were analyzed by thin layer
chromatography. Parental JK9-3da cells (Fig.
5, lane labeled WT)
make both DHS-1-P and PHS-1-P under these experimental conditions. The
lcb4 (RCD158) and
lcb4
lcb5 (RCD165)
cells contain what appears to be a very small amount of radiolabeled
compounds that migrate like the two LCB phosphates. However, when each
sample was chromatographed in a second dimension, the two radioactive
spots did not migrate like either PHS-1-P or DHS-1-P (data not shown).
Thus, neither strain makes any detectable LCB phosphates. The
lcb5 (RCD164) cells contain a normal level of PHS-1-P and
DHS-1-P compared with the wild type JK9-3da cells (Fig. 5). We conclude
from these data that the Lcb4 kinase is responsible for synthesis of
LCB phosphates under the conditions of these experiments; the Lcb5
kinase is unable to make any detectable LCB phosphates under these
experimental conditions.
|
Substrate Specificity of the Lcb4 and Lcb5 Kinases-- To begin to understand the function of each LCB kinase, we examined their substrate specificity because they might have different preferred substrates. In these experiments the concentration of substrate, [3H]DHS, was kept constant at about one-half its Km value, and the concentration of the nonradioactive competitor was added at a similar concentration (see Table III). If the competitor was a substrate of equal Km, then the reaction should be inhibited by 50%.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our data demonstrate that the LCB4 and LCB5 genes of S. cerevisiae encode LCB kinase activity, the first time that genes encoding this type of enzyme have been identified in any organism. This conclusion is based upon the complete lack of LCB kinase enzyme activity in an lcb4 lcb5 double deletion strain (Table II), upon the production of LCB kinase activity in E. coli cells expressing either LCB4 or LCB5 but not in cells carrying only the vector (Fig. 4), and finally, the demonstration that a lcb4 deletion mutant does not make any detectable DHS-1-P or PHS-1-P in vivo under conditions where the parental strain makes both compounds (Fig. 5).
Identification of the LCB4 gene was based upon the observation that a strain lacking the DPL1 gene, believed to encode LCB phosphate lyase activity, is more sensitive to and is growth-inhibited by lower concentrations of sphingosine than is the parental strain (22). In the presence of sphingosine the mutant cells accumulate SPP (22) which is believed to inhibit growth, although the mechanism is unclear (25). Our data support the idea that SPP is growth-inhibiting since mutation of lcb4 resulted in both the loss of more than 95% of cellular LCB kinase activity, and it allowed growth in the presence of an inhibitory concentration of sphingosine. Because SPP appears to inhibit growth it may be that under some physiological circumstances DHS-1-P, PHS-1-P, or both are used by yeast cells to modulate growth.
The predicted Lcb4 and Lcb5 proteins are paralogs that show no similarity to known proteins. They thus represent a new class of lipid kinase. They appear to be related to proteins with unknown functions including one from S. pombe (Fig. 3) and ones encoded by mouse and human expressed sequence tags (e.g. GenBank AA107451, D31133, R74736, and N55929). The two most conserved portions of the LCB kinases are in the middle and the COOH terminus of the proteins (Fig. 4). The functions of these regions and the less conserved NH2 terminus remain to be determined.
S. cerevisiae cells contain a high level of LCB kinase activity in the 100,000 × g pellet fraction based upon a specific activity of 4,500 pmol/mg/min (Table II). This value is more than four times the value of 1,080 pmol/mg/min for LCB kinase activity in corn shoot microsomes (19) and 100 times greater than the value of 40 pmol/mg/min from bovine brain (26). About two-thirds of the Lcb4 and one-third of the Lcb5 enzyme activity are found in the pellet fraction of the cell (Table II). It remains to be determined if the proteins are retained within the lumen of an organelle or if they are bound to a membrane. Neither protein has a predicted transmembrane spanning domain nor an attachment site for a lipid anchor, so if the proteins are binding to a membrane, they must do so through protein-protein interaction or through a novel lipid anchor motif. In platelets, sphingosine kinase is primarily a soluble enzyme, whereas in rat brain and other tissues it is peripherally associated with membranes (21). About 80% of the activity in rat pheochromocytoma PC12 neuronal cells is soluble and the other 20% is in the particulate fraction (27), where it is presumed to be membrane-bound. It should now be possible to determine the mechanism and the circumstances under which Lcb4 and Lcb5 enzyme activity distribute between the membrane and soluble fractions. This information may help to elucidate why sphingosine kinase activity is in a similar fraction in mammalian cells. Other lipid kinases have been shown to bind to membranes in a regulated manner (28), and it will be important to determine if LCB kinases show similar behavior.
Most of the LCB kinase activity in JK9-3a cells is encoded by
LCB4 (lcb5, strain RCD164, Table II), and this
kinase synthesizes all of the DHS-1-P and PHS-1-P present in log phase
cells grown in rich medium because neither of these compounds was found
in cells making only the Lcb5 kinase (
lcb4, strain
RCD158, Fig. 5). Our data do not eliminate the possibility that LCB
phosphates made by the Lcb5 kinase are very short lived and present at
a very low concentration. The function of the Lcb5 kinase may be very
specialized and depend upon its cellular location. Alternatively, the
LCB5 gene may just be a remnant of the genome duplication event that occurred in S. cerevisiae (29). This possibility seems unlikely, however, because a nonessential gene would be expected
to become mutated and no longer encode an active enzyme. Mice and
humans also seem to have more than one species of LCB kinase based upon
our analysis of homologs predicted to be encoded by expressed sequence
tags. Retention of multiple species of LCB kinases over a long
evolutionary time span suggests that they perform specialized
functions.
All eukaryotes examined to date have LCB kinase activity, indicating that this enzyme provides survival value to cells. Besides its presumed role in generating signaling molecules, it is essential for catabolizing LCBs to yield phosphoethanolamine, which serves as a precursor for synthesis of phosphatidylethanolamine and phosphatidylcholine. The physiological importance of this metabolic conduit from sphingolipid to glycerophospholipid metabolism has not been determined. The LCB4 and LCB5 genes make it possible to examine the role of this metabolic pathway in S. cerevisiae cells. Cells lacking both genes grow normally in rich and synthetic medium (data not shown), indicating that LCB phosphates and flux through this metabolic pathway are not necessary for a normal rate of growth with glucose as the carbon source, at least not under short term laboratory conditions. Other phenotypes await analysis as does the role of LCB phosphates as second messengers during stress responses (30).
If the putative human and mouse expressed sequence tags do indeed encode homologs of the yeast LCB kinases, it should be possible to use them to manipulate the level of SPP in mammals and thereby establish the physiological functions of SPP.
![]() |
Addendum |
---|
After submission of this manuscript for publication, Olivera et al. (31) reported the purification and enzymatic characterization of sphingosine kinase from rat kidney.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM41302 (to R. L. L. and R. C. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The 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: Dept. of Biochemistry,
University of Kentucky Medical Center, 800 Rose St., Lexington, KY
40536-0084. Tel.: 606-323-6052; Fax: 606-257-8940; E-mail: bobd{at}pop.uky.edu.
1 The abbreviations used are: SPP, sphingosine 1-phosphate; LCB, long chain base; PCR, polymerase chain reaction; DHS, dihydrosphingosine; DHS-1-P, dihydrosphingosine 1-phosphate; PHS, phytosphingosine; PHS-1-P, phytosphingosine 1-phosphate.
2 M. Skrzypek, M. M. Nagiec, R. L. Lester, and R. C. Dickson, submitted for publication.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|