From the Department of Biochemistry, St. Jude
Children's Research Hospital, Memphis, Tennessee 38105 and the
§ Department of Biochemistry, University of Tennessee,
Memphis, Tennessee 38163
Received for publication, September 26, 2000, and in revised form, October 19, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ethanolamine kinase (EKI) is the first committed
step in phosphatidylethanolamine (PtdEtn) biosynthesis via the
CDP-ethanolamine pathway. We identify a human cDNA encoding an
ethanolamine-specific kinase EKI1 and the structure of the
EKI1 gene located on chromosome 12. EKI1
overexpression in COS-7 cells results in a 170-fold increase in
ethanolamine kinase-specific activity and accelerates the rate of
[3H]ethanolamine incorporation into PtdEtn as a function
of the ethanolamine concentration in the culture medium. Acceleration of the CDP-ethanolamine pathway does not result in elevated cellular PtdEtn levels, but rather the excess PtdEtn is degraded to
glycerophosphoethanolamine. EKI1 has negligible choline kinase activity
in vitro and does not influence phosphatidylcholine
biosynthesis. Acceleration of the CDP-ethanolamine pathway also does
not change the rate of PtdEtn formation via the decarboxylation of
phosphatidylserine. The data demonstrate the existence of separate
ethanolamine and choline kinases in mammals and show that ethanolamine
kinase can be a rate-controlling step in PtdEtn biosynthesis.
Ethanolamine kinase or
EKI1 (ATP:ethanolamine
O-phosphotransferase, EC 2.7.1.82) catalyzes the
first step of PtdEtn biosynthesis via the CDP-Etn pathway. ECT, a
cytidylyltransferase, and EPT, an amino alcohol
phosphotransferase, catalyze the subsequent two steps, and together
these three enzymes constitute the de novo pathway for
PtdEtn formation. The decarboxylation of PtdSer is an alternate route
for PtdEtn production and is functionally important in cultured cell
lines (1, 2), although the CDP-Etn pathway is considered a major route
for PtdEtn synthesis in most mammalian tissues (3-6). PtdEtn is an
abundant phospholipid in eucaryotic cell membranes, and the ECT
reaction is thought to be a major regulator of its synthesis (7). EKI
has been proposed as a regulatory step based on theoretical
considerations (8). However, experimental investigations result in
conflicting conclusions. Experiments performed with rat hepatocytes (4,
9) demonstrate that the supply of ethanolamine limits the rate of
PtdEtn production at concentrations below 30 µM. Only at
higher concentrations of ethanolamine does accumulation of
phosphoethanolamine occur, indicating ECT as the rate-limiting enzyme
of the pathway. On the contrary, McMaster and Choy (10) report that the
EKI step is rate-limiting with increased ethanolamine concentrations
using a hamster heart perfusion model. Both studies agree that the rate
of PtdEtn synthesis is dependent on the extracellular ethanolamine
concentration (9, 10) and that the stimulation of PtdEtn biosynthesis
occurs at physiological levels of 20-50 µM (9).
There is a long standing discussion as to the number and specificity of
the enzymes that catalyze the phosphorylation of ethanolamine and
choline. In yeast, there are two enzymes that are able to phosphorylate
choline and ethanolamine, and these enzymes are annotated based
on their preferred substrate specificities. The CKI enzyme has a
specific activity 3.6-fold higher with choline compared with
ethanolamine. The EKI enzyme has a specificity constant for
ethanolamine 2-fold higher than that for choline (11). CKIs have been
purified from several mammalian tissues. Preparations from rat kidney
(12), liver, and brain (13) exhibit significant ethanolamine kinase
activity with computed ratios of CKI to EKI activity of 1.4, 3.7, and
1.3, respectively. A mammalian CKI cDNA clone
also encodes an enzyme with dual specificity (14), and additional
highly homologous CKI clones have been identified (15, 16).
These results suggest that in mammals both activities reside on the
same protein.
However, the existence of mammalian ethanolamine-specific kinases
cannot be excluded by the data. Ethanolamine-specific kinase activities
have been identified (17-20) and partially purified. EKI activities 1 and 2 isolated from rat liver are separable distinct proteins
(21). EKI1 is estimated to be 36 kDa and does not exhibit CKI activity
(21). EKI2 is estimated to be 160 kDa and prefers ethanolamine but also
has significant CKI activity (21). An EKI of 42 kDa has been purified
from human liver with a 5-fold preference for ethanolamine compared
with choline (19), and also an EKI activity of 86 kDa has been
identified from rat liver and HeLa cells using an antibody raised
against the Drosophila EKI (20). The discovery of the
Drosophila EKI (22) encoded by the eas gene
establishes the existence of an ethanolamine-specific kinase in higher
eucaryotes. Expression of the eas gene product as a
glutathione S-transferase-fusion protein in
Escherichia coli reveals a kinase that is highly specific
for ethanolamine with negligible CKI activity (20). A defect in the
Drosophila eas results in a bang-sensitive
paralytic phenotype, although the molecular link between the absence of
the ethanolamine kinase activity and the physiological abnormality has
not been established (22).
In the present study, we identify a human kinase that is highly
specific for ethanolamine (EKI1). Moreover, we demonstrate that EKI1
overexpression accelerates PtdEtn biosynthesis as a function of the
exogenous ethanolamine, whereas it has no effect on PtdCho formation.
Materials--
Sources of supplies were as follows:
L-[3-3H]serine (specific activity 30 Ci/mmol)
was from Amersham Pharmacia Biotech;
[methyl-3H]choline chloride (specific activity
80 Ci/mmol) and [1-3H]ethanolamine hydrochloride
(specific activity 55 Ci/mmol) were from American Radiolabeled
Chemicals (St. Louis, MO); LipofectAMINE reagent was from Life
Technologies, Inc.; restriction endonucleases and other molecular
biology reagents came from Promega; pcDNA3 plasmid came from
Invitrogen; cDNA clones AA598956 and R15326 were from American Type
Culture Collection; human liver poly(A)RNA was from
CLONTECH; ATP was from Roche Molecular
Biochemicals; thin layer chromatography plates came from
Analtech; choline, ethanolamine, phosphorylcholine,
phosphorylethanolamine, CDP-ethanolamine, and
glycerophosphorylethanolamine came from Sigma; and phospholipid standards were from Avanti Polar Lipids. All other supplies were reagent grade or better.
Cloning of the EKI cDNA and Construction of the Expression
Vector--
The expressed sequence-tagged data base was searched using
the published Drosophila ethanolamine kinase (easily
shocked) amino acid sequence (GenBankTM accession number
P54352). A clone from human Wilms tumor was identified
(GenBankTM accession number AA598956) and purchased from
American Type Culture Collection. The cDNA sequence was determined
on both strands using primers that flanked the multiple cloning sites
and synthesized internal primers. The cDNA contained a single open
reading frame we call EKI1. A 1.9-kb
HindIII-EcoRI fragment was excised and subcloned
into the mammalian expression vector pcDNA3.1 to generate plasmid
pPJ96, which expressed EKI1 from the constitutive cytomegalovirus promoter. The above cDNA lacked a stop codon upstream of the
postulated starting methionine. To obtain additional 5'-end sequence
information, we searched the high throughput genomic sequence data
base. We identified a contig that contained additional 5'-end
sequence (GenBankTM accession number AC027624). We
designed the primers and performed reverse transcriptase-polymerase
chain reaction using human liver poly(A)RNA. As a forward primer, we
used the oligo(5'-GTGACCGGAGCGAGAAACC), and as a reverse primer, we
used 5'-GAGAAAATTCCTGTTGTCGGAGC. The product of the polymerase
chain reaction was subcloned into the pCR2.1 vector, and the sequence
was confirmed. The 130-bp fragment of
HindIII-SacII was ligated with the ~6-kb
HindIII-SacII fragment of pPJ96 to construct the
pAL10. The pAL10 plasmid expresses a protein 452 amino acids long,
whereas pPJ96 expresses a protein 363 residues long starting from
methionine 89 of pAL10.
Transfections and Metabolic Labeling Experiments--
COS-7
cells were grown in 100-mm dishes to 80% confluency in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum and
1% glutamine. Transfections using the LipofectAMINE reagent were
performed according to the manufacturer's instructions. COS-7 cells
were transfected in 100-mm dishes with either plasmid pPJ96, pAL10, or
a control plasmid. Cells were harvested 24 h later, pooled, and
replated in 60-mm dishes to avoid variations due to transfection
efficiency. At 48 h posttransfection, the medium was changed, and
ethanolamine was added in the medium at the indicated concentrations.
Cells were labeled at 37 °C for 6 h with either: 1)
[3H]ethanolamine (specific activity 1.25 Ci/mmol), 2)
[3H]choline (specific activity 0.16 Ci/mmol), or 3)
[3H]serine (specific activity 1 Ci/mmol). The
[3H]ethanolamine-labeling experiment was performed in
normal COS-7 culture medium supplemented with the indicated
concentrations of ethanolamine (2-32 µM). The
[3H]choline-labeling experiment was performed in normal
COS-7 culture medium supplemented with 32 µM
ethanolamine. The [3H]serine-labeling experiment was
performed in Dulbecco's modified Eagle's medium containing with 30 µM choline, 5 µM serine, and 32 µM ethanolamine. Cells were washed with
phosphate-buffered saline, harvested, and stored as pellets at
The water-soluble metabolites from the
[3H]ethanolamine-labeling experiment were identified by
thin layer chromatography using two different solvent systems. For the
separation of ethanolamine and phosphoethanolamine, the aqueous phases
were spotted on Silica Gel G plates and were developed in 95%
ethanol,2% ammonium hydroxide, 5:1 (v/v). The RF
values for ethanolamine and phosphoethanolamine were 0.41 and 0.65, respectively. CDP-ethanolamine and glycerophosphoethanolamine were
separated on Silica Gel G plates developed in 95% ethanol,2% ammonium
hydroxide, 1:1 (v/v). The RF values for
CDP-ethanolamine and glycerophosphoethanolamine were 0.65 and 0.76, respectively.
The analysis of the water-soluble metabolites from the
[3H]choline-labeling experiment was performed by spotting
the aqueous phase on a preadsorbent Silica Gel G plate, which was
developed in the solvent system in 95% ethanol,2% ammonium hydroxide,
1:1 (v/v). The RF values for choline,
phosphocholine, glycerophosphocholine, and CDP-choline are 0.06, 0.17, 0.35, and 0.59, respectively.
The analysis of the chloroform-soluble phospholipids from the labeling
experiments was performed by spotting the lipid phases on Silica Gel 60 plates and developing in the solvent system
CHCl3:CH3OH:NH4OH, 60:35:8 (v/v/v).
The RF values for PtdSer, sphingomyelin, PtdCho,
and PtdEtn were 0.22, 0.26, 0.45, and 0.76, respectively.
Choline and Ethanolamine Kinase Assays--
COS-7 cell pellets
transfected with either plasmid pPJ96, pAL10, or a control plasmid were
incubated in lysis buffer (20 mM Tris-HCl, pH 8.0, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 2 µg/ml aprotinin) for 1 h on
ice. The cells were disrupted by sonication, and the particulate
fraction was removed by low speed centrifugation. Ethanolamine or
choline kinase activities were determined essentially as described
previously (11, 24). The standard assays contained 100 mM
Tris-HCl, pH 8.0, 10 mM MgCl2, 10 mM ATP, 0.25 mM [3H]ethanolamine
(specific activity, 397 mCi/mmol), or 0.25 mM
[3H]choline (specific activity 398 mCi/mmol) in a final
volume of 50 µl. The reaction mixtures were incubated at 37 °C for
15 min. The reaction was stopped by the addition of 5 µl of 0.5 M Na3EDTA, and the tubes were vortexed and
placed on ice. Next, 35 µl of each sample was spotted on preadsorbent
Silica Gel G thin layer plates, which were developed with 2% ammonium
hydroxide,95% ethanol, 5:1 (v/v). Phosphoethanolamine and
phosphocholine were identified by comigration with standards that were
scraped from the plate and quantitated by liquid scintillation
counting. Protein was determined according to the Bradford method
(25).
Subcellular Fractionation--
COS-7 cells transfected with
either pAL10 or pPJ96 were incubated in lysis buffer (20 mM
Tris-HCl, pH 8.0, 2 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 2 µg/ml aprotinin) for 1 h on ice followed by sonication. Nuclei and
mitochondria were separated by centrifugation at 10,000 × g for 15 min. Microsomes were separated by centrifugation at
100,000 × g for 60 min. The pellets were resuspended
in buffer, and the ethanolamine kinase activity was determined as
described above.
Northern Blot Analysis--
Multiple human tissue Northern blots
were purchased from CLONTECH and were hybridized
and washed according to the manufacturer's instructions. The blots
were hybridized with 32P-labeled random primed probes
prepared from the 1.1-kb HindIII-NdeI fragment of
AA598956 in the case of EKI1, or in the case of EKI2 the blots were hybridized with a probe that was
prepared using the 1.5-kb HindIII-NotI fragment
of R15326 as template. Probes were made using [32P]dCTP
according to manufacturer's instructions.
Isolation of a Human EKI1 cDNA--
BLAST searches of the
public expressed sequence-tagged data base identified a cDNA clone
(GenBankTM accession number AA598956) with a sequence
similar to the Drosophila eas gene. The clone was obtained,
and the complete cDNA sequence was determined. In-frame stop codons
were not identified at the 5'-end of the cDNA; however, the clone
was 2040 bp long, and an open reading frame was identified between 252 and 1343 bp. Subsequent BLAST searches of the public high throughput
genomic sequence data base identified the genomic sequence upstream of the 5'-end of cDNA clone AA598956. Based on this information we
designed primers and performed reverse transcriptase-polymerase chain
reaction using human liver mRNA. The 133-bp product contained 103 additional nucleotides at the 5'-end. This additional sequence contained an in-frame methionine and an upstream stop codon. The starting methionine resides in a favorable Kozak translation initiation sequence (26, 27). We call the above product cDNA
EKI1. The EKI1 cDNA is predicted to
encode a protein of 452 amino acids with a molecular size of 49.7 kDa.
Searches of the data base revealed that expressed sequence-tagged
clones are highly similar to the sequence of EKI1. Using five clones (GenBankTM accession numbers AI798887,
AI972267, AW271947, AW960911, and U69210), we were able to reconstruct
in silico a 2488-bp cDNA with an open reading frame
specifying a protein 477 amino acids long, which we call
EKI2
A sequence highly similar to EKI2
BLAST searches of the high throughput genome sequences revealed that
the EKI1 gene is located on human chromosome 12. It spans ~60.5 kb, and it consists of 8 exons and 7 introns (Fig.
2). The exon sizes range between 77 and
828 bp. The intron sizes range between 0.3 and 18.1 kb. There is a
mouse homolog to EKI1 that also localizes to mouse
chromosome 12. In contrast, EKI2 as well as its mouse
homologue are located on chromosomes 1 in both species. The structure
of the EKI2 gene awaits the assembly of the raw genomic
sequence data.
Tissue Distribution of EKI1--
The relative abundance of
EKI1 and EKI2 mRNAs in a variety of human
tissues was addressed by Northern blot analysis (Fig. 3). The blots were probed with a
32P-labeled fragment from the 5'-end of the EKI1
cDNA. EKI1 is uniformly distributed in the tissues examined (Fig.
3A). A ~2.2-kb transcript was detected in several tissues.
This transcript corresponds to the size predicted from the cDNA
sequence (Fig. 2). An intense signal of ~1.7 kb was detected in the
testis, but it is not clear if it is due to degradation, alternative
splicing, or alternative polyadenylation of the mRNA. The band at
about 4.5 kb in several of the lanes corresponds to the
position of 28 S ribosomal RNA and is probably due to nonspecific
binding of the probe to rRNA or alternatively represents incompletely
spliced EKI1 mRNA.
EKI2 was selectively expressed in kidney, liver, ovary,
testis, and prostate, but the signal was below detectable levels in the
other tissues examined (Fig. 3B). Two sizes of
EKI2 transcripts were detected that probably correspond to
the two splice variants EKI2 Expression and Activity of EKI1--
COS-7 cells were transfected
with pAL10, pPJ96, or pcDNA3.1 vector control, and 48 h later
cell lysates were prepared. The lysates were assayed for ethanolamine
kinase and choline kinase activities. Ethanolamine kinase enzymatic
activity was significantly higher in lysates from both pAL10 and pPJ96
transfected cells (Fig. 4A).
Endogenous ethanolamine kinase activity was detected in control lysates
at protein concentrations above 35 µg (data not shown) with a basal
specific activity of 0.62 pmol/min/µg. Transfection of COS-7 cells
with pAL10 resulted in a 170-fold increase in specific activity to 106 pmol/min/µg. Cells transfected with pPJ96 exhibited a 147-fold
increase in ethanolamine kinase reaching a specific activity of 91.25 pmol/min/µg, which demonstrated that both cDNAs encoded a robust
EKI enzyme.
Endogenous choline kinase specific activity in control cell lysates was
determined to be 1.04 pmol/min/µg (Fig. 4B). The
overexpression of pAL10 did not increase choline kinase specific
activity (0.98 pmol/min/µg), and the overexpression of pPJ96 cDNA
resulted in a slight increase in choline kinase specific activity to
2.62 pmol/min/µg. The ratio of ethanolamine to choline kinase
specific activities in lysates overexpressing pAL10 was 108, and in
lysates overexpressing pPJ96 the ratio was 35. These data indicated
that EKI1 encoded a kinase that was highly selective for ethanolamine.
The amino terminus of EKI1 contains a region rich in hydrophobic
residues (amino acids 65-90), suggesting that these might constitute a
membrane interaction domain. Two independent predictive algorithms indicated that a transmembrane helix was likely found in amino acids 66-90. To test the possibility that the EKI1 is a
membrane-associated protein, we transfected COS-7 cells with either
pAL10 or pPJ96, which lacked the predicted transmembrane helix and
fractionated the cell extracts. In both cases, 96% of the total
ethanolamine kinase activity was recovered in the cytosolic fraction
with the remaining activity distributed almost equally between the
nuclear plus mitochondrial and microsomal fractions (data not shown).
The data indicated that the enzymes specified by the expression of the
EKI1 cDNAs were not associated with cell membranes.
Effect of EKI1 Expression on Phospholipid Metabolism--
To
investigate the contribution of the EKI step in the regulation of the
CDP-Etn pathway, we overexpressed pPJ96 in COS-7 cells, labeled the
cells with the appropriate precursor, and monitored the incorporation
of the label into the metabolites. All experiments were performed with
both the pAL10 and the pPJ96 expression vectors with identical results.
Two sets of COS-7 cells were transfected with either EKI1
expression vector or control vector. After 24 h, cells within a set were pooled and replated to avoid variation in transfection efficiency among replicated dishes. At 48 h after transfection, the medium was changed, and ethanolamine was added at the
concentrations indicated in Fig. 5. Cells
were radiolabeled with [3H]ethanolamine for 6 h at
37 °C and then harvested. The incorporation of
[3H]ethanolamine into PtdEtn increased in correspondence
with increasing concentrations of ethanolamine in the medium (more than
99% of the label incorporated into the lipid phase comigrated with a PtdEtn standard). Overexpression of ethanolamine kinase activity did
not affect the amount of the label incorporated into PtdEtn when the
concentration of ethanolamine in the medium was below 10 µM (Fig. 5). However, at higher concentrations of
ethanolamine (16 and 32 µM), EKI1 overexpression resulted
in increased radiolabeling of PtdEtn. At 16 µM we
observed a 1.5-fold increase (from 647,536 to 924,660 cpm/mg of cell
protein), whereas at 32 µM we detected an ~2.5-fold
increase in the incorporation of [3H]ethanolamine into
PtdEtn (from 539,519 to 1,393,556 cpm/mg of cell protein) (Fig. 5).
These data indicated that the phosphorylation of ethanolamine by EKI1
modulated the rate of PtdEtn biosynthesis at physiological
concentrations of ethanolamine.
The distribution of [3H]ethanolamine among the
water-soluble metabolites revealed that ECT was still the slow step in
the CDP-ethanolamine pathway (Fig. 6). At
32 µM ethanolamine, overexpression of EKI1 caused a
20-fold increase in the incorporation of the label into phosphoethanolamine, whereas it did not significantly affect the levels
of ethanolamine or CDP-ethanolamine. We also detected a 3-fold increase
in the levels of cellular glycerophosphoethanolamine. These data
indicated that the overexpression of EKI1 cDNA perturbed PtdEtn metabolism via the CDP-ethanolamine pathway, which caused an
accumulation of phosphoethanolamine, and that the additional PtdEtn produced was degraded to glycerophospho- ethanolamine.
To examine whether the overexpression of EKI1 cDNA
increased the cellular PtdEtn levels, we transfected COS-7 cells with
either EKI1 expression vector or a control vector labeled
with [32P]orthophosphate for 48 h to steady state
and quantitated the incorporation of the label into the lipid phase
(data not shown). Overexpression of the ethanolamine kinase activity
did not affect the incorporation of [32P]orthophosphate
into PtdEtn, indicating that the cellular levels of PtdEtn remained the
same although the rate of synthesis increased. This observation was
consistent with the enhanced glycerophosphoethanolamine levels we
observed with the overexpression of EKI (Fig. 6).
EKI1 exhibited some minimal choline kinase activity (Fig.
2B). We transfected COS-7 cells with either EKI1
expression vector or a control vector, and after 48 h the
transfected cells were labeled with [3H]choline to
examine whether EKI1 activity modulated PtdCho biosynthesis. EKI1
overexpression caused a 2-fold increase in the incorporation of the
label into phosphocholine but did not have an effect on the
incorporation of the label into PtdCho (Fig.
7). These data suggested that the EKI1
enzyme did not modify PtdCho biosynthesis.
An alternative route for PtdEtn formation is the decarboxylation of
PtdSer. We transfected cells with EKI1 and labeled with [3H]serine in the presence of 32 µM
ethanolamine in the culture medium to examine whether EKI acceleration
of the CDP-ethanolamine pathway affected the rate of decarboxylation of
PtdSer. The amount of radiolabeled serine incorporated into PtdEtn was
the same in control cells and in cells overexpressing EKI1 (20,805 ± 389 cpm/mg of cellular protein versus 23,773 ± 739 cpm/mg in cells transfected with pPJ96 and 20,789 ± 1217 cpm/mg
in cells transfected with pAL10, respectively), indicating that
increased rates of PtdEtn biosynthesis through the CDP-ethanolamine
pathway did not affect the rate of PtdEtn biosynthesis via the
decarboxylation of PtdSer. The amount of the label incorporated into
PtdSer was 34,202 ± 2390 cpm/mg protein in control cells,
33,600 ± 489 cpm/mg protein in cells transfected with pPJ96, and
28,966 ± 366 cpm/mg protein in cells overexpressing pAL10,
suggesting that the overexpression of EKI1 also did not cause an
accumulation of PtdSer.
The biochemical characterization of EKI1 reveals the
existence of a specific EKI enzyme in mammalian cells. The human
EKI1 cDNA encodes a protein with high specificity for
ethanolamine (Figs. 4, A and B). The EKI1
preference for ethanolamine holds true in vivo as well where
there is a dramatic stimulation of phosphoethanolamine formation (Fig.
6) and an increased PtdEtn radiolabeling (Fig. 5) in contrast to
the lack of any significant change in PtdCho biosynthesis (Fig. 7).
EKI1 may be identical to the mammalian ethanolamine-specific kinase
activities described previously (19, 21). Weinhold and Rethy (21)
partially purified an EKI activity with a molecular size of 36 kDa from
rat liver that exhibited no CKI activity. Draus et al. (19)
also reported the partial purification of a 42-kDa enzyme from human
liver that had a relatively high ratio (~5) of EKI to CKI activities.
The EKI1 clone reported in this paper encodes a protein with
a predicted molecular size of 49.7 kDa (Fig. 1) and may correspond to
the enzymes described above.
We also report the existence of a second gene that is highly similar to
EKI1, which we call EKI2. EKI1 and EKI2 EKI plays a role in controlling PtdEtn biosynthesis. The rate of PtdEtn
biosynthesis is a function of the availability of exogenous
ethanolamine (10), thus implicating a role for EKI. However, this
hypothesis has not been tested, and a prevalent view of the
CDP-ethanolamine pathway is that the ECT is the regulated step (30).
The stimulation of PtdEtn formation by EKI1 overexpression was not anticipated, and the data demonstrate that EKI as well as ECT
are important metabolic control points. The majority of the
ethanolamine radiolabel accumulates as phosphoethanolamine with a
20-fold increase over control, indicating that the ECT remains a key
rate-limiting enzyme. The stimulatory effect of EKI1
overexpression is evident when ethanolamine supplementation approaches
physiological concentrations (9), indicating that the level of
phosphoethanolamine is important in pushing the ethanolamine substrate
through to PtdEtn. The concentration of cellular phosphoethanolamine is
controlled by the availability of ethanolamine and EKI1 protein levels.
These data suggest that the regulation of EKI expression and/or activity may contribute to determining PtdEtn biosynthesis by
the CDP-ethanolamine pathway in different cell types, and this is
consistent with the reduced level of PtdEtn in the Drosophila eas mutant (22).
Our results indicate a mechanism for PtdEtn homeostasis that is similar
to the one that controls cellular PtdCho levels (31). Increased
glycerophosphoethanolamine was associated with stimulated PtdEtn
synthesis in response to EKI1 overexpression (Fig. 4). Earlier work from our laboratory indicated that excessive phospholipid synthesis is balanced by degradation to the glycerophosphobase to
maintain homeostatic levels of membrane lipid (31, 32). These results
are consistent with the lack of an effect on the radiolabeling of
PtdEtn with [3H]serine with EKI1 overexpression and
suggest that the contribution of the decarboxylation of PtdSer to
PtdEtn biosynthesis is not down-regulated by the activity of the
CDP-ethanolamine pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. Cell pellets were extracted using a two-phase system (23)
to separate the water-soluble and the chloroform-soluble metabolites,
and the amount of radiolabel incorporated into each phase was
determined by scintillation counting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Fig. 1). This open
reading frame does not have a stop codon upstream of the postulated
starting methionine; therefore, the possibility that EKI2
is longer cannot be excluded. However, the corresponding mouse cDNA
predicts a protein that starts from the methionine as shown in Fig. 1
and has an in-frame upstream stop codon verifying that this is the correct initiating methionine in this species (data not shown).
View larger version (106K):
[in a new window]
Fig. 1.
Alignment of the predicted amino acid
sequences of EKI1, EKI2 , Drosophila
EKI (eas gene product), and yeast EKI1. The
EKI1-predicted protein sequence (GenBankTM accession number AF207600)
is compared with the predicted amino acid sequences of EKI2
,
Drosophila EKI (GenBankTM accession number P 54352), and the
Saccharomyces cerevisiae EKI1 (GenBankTM accession number
NP_010431). Identical amino acid residues are boxed.
was identified in the
data base (GenBankTM accession number AK001623), which we
call EKI2
. EKI2
encodes a protein of 394 amino acids, and it is a splice variant of EKI2
. They are identical
in the first 338 amino acids, but they have a different carboxyl
terminus. Currently, there are no biochemical data establishing that
either EKI2
or EKI2
is an ethanolamine-specific kinase. Fig. 1
shows the similarity of the predicted EKI1 amino acid sequence to the
sequences of the EKI2
, Drosophila eas, and yeast EKI1.
View larger version (9K):
[in a new window]
Fig. 2.
Structure of the human EKI1
gene. Genomic sequence was derived from the data base entry
(GenBankTM accession number AC027624). The EKI1 gene spans
~60.5 kb. The length of each exon is as follows: exon 1, 407 bp; exon
2, 261 bp; exon 3, 145 bp; exon 4, 150 bp; exon 5, 87 bp; exon 6, 163 bp; exon 7, 77 bp; and exon 8, 828 bp. The sizes of the introns are
indicated on this figure. The gene is located on chromosome 12. Stars indicate stop codons.
View larger version (55K):
[in a new window]
Fig. 3.
Distribution of EKI1 and EKI2 in human
tissues. Human tissue Northern blots were hybridized and washed
according to the manufacturer's instructions. A, pattern of
EKI1 expression in human tissues. The blots were hybridized
with a 32P-labeled probe prepared from the 1.1-kb
HindIII-NdeI fragment of EKI1. B,
pattern of EKI2 expression in human tissues. The blots were
hybridized with a 32P-labeled probe prepared from the
1.1-kb HindIII-NotI fragment of the expressed
sequence-tagged clone R15326.
and EKI2
as
discussed above. We postulate that the larger transcript of ~3 kb
corresponds to EKI2
, whereas the smaller transcript of ~2.5 kb
corresponds to the
-isoform of EKI2.
View larger version (12K):
[in a new window]
Fig. 4.
EKI1 is an ethanolamine-specific kinase.
COS-7 cells were transfected with either pAL10 ( ), pPJ96 (
), or
the control plasmid pcDNA3 (
). After 48 h, the cells were
lysed, and the soluble extracts were evaluated for ethanolamine and
choline kinase activities. A, ethanolamine kinase assays
were performed as described under "Experimental Procedures."
B, choline kinase assays were performed as described under
"Experimental Procedures." The experiment was performed twice with
similar results. The data from one experiment are shown.
View larger version (17K):
[in a new window]
Fig. 5.
EKI1 overexpression accelerates PtdEtn
biosynthesis. COS-7 cells were transfected with either a control
vector ( ) or the EKI1 expression plasmid (pPJ96) (
) and labeled
with [3H]ethanolamine for 6 h in the presence of the
indicated concentrations of ethanolamine in the medium (specific
activity 1.25 Ci/mmol). Cells were harvested, lipids were extracted,
and the incorporation of [3H]ethanolamine into PtdEtn was
determined. More than 99% of the label is incorporated into PtdEtn.
Data represent the mean values from two experiments. The experiment was
also performed using the pAL10 expression construct with identical
results.
View larger version (24K):
[in a new window]
Fig. 6.
EKI1 overexpression increases intracellular
levels of phosphoethanolamine and glycerophosphoethanolamine
(GPE). COS-7 cells were transfected with either a control vector
or the EKI1 expression plasmid (pPJ96) and labeled with
[3H]ethanolamine for 6 h in the presence of 32 µM ethanolamine in the medium (specific activity 1.25 Ci/mmol). Intracellular 3H-labeled metabolites were
extracted and quantitated by thin layer chromatography as described
under "Experimental Procedures." Data are from the same experiment
described in Fig. 5. P-Etn, phosphoethanolamine. The
experiment was also performed using the pAL10 expression construct with
identical results.
View larger version (32K):
[in a new window]
Fig. 7.
Overexpression of EKI1 does not affect PtdCho
biosynthesis. COS-7 cells were transfected with either a control
vector or the EKI1 (pPJ96) expression plasmid and labeled with
[3H]choline for 6 h in the presence of 32 µM ethanolamine in the medium (specific activity 0.16 Ci/mmol). Intracellular 3H-labeled metabolites were
extracted and quantitated by thin layer chromatography as described
under "Experimental Procedures." The choline concentration was 27 µM in the medium. P-Cho, phosphocholine. The
experiment was repeated using the pAL10 expression construct with
identical results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
proteins are 47% identical and 59% similar. EKI1 is 32% identical
and 11% similar, whereas EKI2
is 29% identical and 38% similar to
the Drosophila Eas protein. Based on the above
comparison, it is difficult to definitely state which form is the
mammalian homologue of the eas gene. However, there is
emerging support for a family of EKI genes that is distinct
from the CKI gene family, which encodes dual specificity
enzymes. The existence of a mammalian ethanolamine-specific kinase has
been a point of discussion due to the characterization of CKI
activities that also exhibit significant EKI activity (12, 13, 24). The
physiological significance of this gene family remains to be
established, although the Drosophila bang-sensitive phenotype of the eas mutant points to a critical role for
PtdEtn or PtdEtn turnover via the CDP-ethanolamine pathway in neural function. Three cDNAs from rat tissues have been shown to encode CKI activities. Rat CKI1 protein (435 amino acids) and CKI2 protein (453 amino acids) are splice variants and are expressed in the liver
(15, 16). The rat liver CKI biochemically purified by Porter and Kent
(24) exhibits dual reactivity with choline and ethanolamine, has a
molecular mass of 47 kDa, and probably corresponds to one of the rat
CKI1 or CKI2 isoforms. A third cDNA, CKI3, isolated from the rat
kidney is the product of a second gene (14). Rat CKI3 has
been expressed in E. coli and demonstrated to encode an
enzyme that phosphorylates choline with a 6-fold higher specific activity than ethanolamine (14). Two CKI clones from mouse
embryo are homologous with the two rat genes (28). Therefore, there are
at least two genes encoding CKI in rat and mouse, although only one
human CKI has been characterized thus far (29). Also, the recently
reported yeast gene encoding the EKI protein has about twice the EKI
activity compared with CKI (11). Until now, only the Drosophila
eas gene has been demonstrated to encode a kinase with a high
specificity for ethanolamine (20).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Pam Jackson for excellent technical assistance and Charles Rock for critical comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM 45737 (S. J.), Cancer Center (CORE) Support Grant CA 21765, and the American Lebanese Associated Charities.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF207600.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, St. Jude Children's Research Hosp., 332 N. Lauderdale, Memphis, TN 38105-2794. Tel.: 901-495-3433; Fax: 901-525-8025; E-mail: suzanne.jackowski@stjude.org.
Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.M008794200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: EKI, ethanolamine kinase; Etn, ethanolamine; PtdEtn, phosphatidylethanolamine; PtdCho, phosphatidylcholine; PtdSer, phosphatidylserine; ECT, cytidylyltransferase; CKI, choline kinase; contig, group of overlapping clones; kb, kilobase pair(s) or kilobase; bp, base pair(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Voelker, D. R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 2669-2673 |
2. |
Miller, M. A.,
and Kent, C.
(1986)
J. Biol. Chem.
261,
9753-9761 |
3. | Zelinski, T. A., and Choy, P. C. (1982) Can. J. Biochem. 60, 817-823[Medline] [Order article via Infotrieve] |
4. | Sundler, R., and Åkesson, B. (1975) J. Biol. Chem. 250, 3359-3367 |
5. | Arthur, G., and Page, L. (1991) Biochem. J. 273, 121-125[Medline] [Order article via Infotrieve] |
6. |
Xu, Z. L.,
Byers, D. M.,
Palmer, F. B.,
Spence, M. W.,
and Cook, H. W.
(1991)
J. Biol. Chem.
266,
2143-2150 |
7. | Bladergroen, B. A., and van Golde, L. M. (1997) Biochim. Biophys. Acta 1348, 91-99[Medline] [Order article via Infotrieve] |
8. | Infante, J. P. (1977) Biochem. J. 167, 847-849[Medline] [Order article via Infotrieve] |
9. | Houweling, M., Tijburg, L. B., Vaartjes, W. J., and van Golde, L. M. (1992) Biochem. J. 283, 55-61[Medline] [Order article via Infotrieve] |
10. | McMaster, C. R., and Choy, P. C. (1992) Biochim. Biophys. Acta 1124, 13-16[Medline] [Order article via Infotrieve] |
11. |
Kim, K.,
Kim, K. H.,
Storey, M. K.,
Voelker, D. R.,
and Carman, G. M.
(1999)
J. Biol. Chem.
274,
14857-14866 |
12. | Ishidate, K., Furusawa, K., and Nakazawa, Y. (1985) Biochim. Biophys. Acta 836, 119-124[Medline] [Order article via Infotrieve] |
13. | Uchida, T., and Yamashita, S. (1990) Biochim. Biophys. Acta 1043, 281-288[Medline] [Order article via Infotrieve] |
14. | Aoyama, C., Nakashima, K., Matsui, M., and Ishidate, K. (1998) Biochim. Biophys. Acta 1390, 1-7[Medline] [Order article via Infotrieve] |
15. |
Uchida, T.,
and Yamashita, S.
(1992)
J. Biol. Chem.
267,
10156-10162 |
16. | Uchida, T. (1994) J. Biochem. (Tokyo) 116, 508-518[Abstract] |
17. | Upreti, R. K., Sanwal, G. G., and Krishnan, P. S. (1976) Arch. Biochem. Biophys. 174, 658-665[Medline] [Order article via Infotrieve] |
18. | Brophy, P. J., Choy, P. C., Toone, J. R., and Vance, D. E. (1977) Eur. J. Biochem. 78, 491-495[Abstract] |
19. | Draus, E., Niefind, J., Vietor, K., and Havsteen, B. (1990) Biochim. Biophys. Acta 1045, 195-204[Medline] [Order article via Infotrieve] |
20. | Uchida, T. (1997) Biochim. Biophys. Acta 1349, 13-24[Medline] [Order article via Infotrieve] |
21. | Weinhold, P. A., and Rethy, V. B. (1974) Biochemistry 13, 5135-5141[Medline] [Order article via Infotrieve] |
22. | Pavlidis, P., Ramaswami, M., and Tanouye, M. A. (1994) Cell 79, 23-33[Medline] [Order article via Infotrieve] |
23. | Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917 |
24. |
Porter, T. J.,
and Kent, C.
(1990)
J. Biol. Chem.
265,
414-422 |
25. | Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve] |
26. | Kozak, M. (1986) Cell 44, 283-292[Medline] [Order article via Infotrieve] |
27. | Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148[Abstract] |
28. | Aoyama, C., Nakashima, K., and Ishidate, K. (1998) Biochim. Biophys. Acta 1393, 179-185[Medline] [Order article via Infotrieve] |
29. | Hosaka, K., Tanaka, S., Nikawa, J., and Yamashita, S. (1992) FEBS Lett. 304, 229-232[CrossRef][Medline] [Order article via Infotrieve] |
30. | Tijburg, L. B., Geelen, M. J., and van Golde, L. M. (1989) Biochim. Biophys. Acta 1004, 1-19[Medline] [Order article via Infotrieve] |
31. |
Baburina, I.,
and Jackowski, S.
(1999)
J. Biol. Chem.
274,
9400-9408 |
32. | Barbour, S. E., Kapur, A., and Deal, C. L. (1999) Biochim. Biophys. Acta 1439, 77-88[Medline] [Order article via Infotrieve] |