(Received for publication, December 11, 1995; and in revised form, January 17, 1996)
From the
Diacylglycerol (DAG) is a second messenger that activates
protein kinase C and also occupies a central role in phospholipid
biosynthesis. Conversion of DAG to phosphatidic acid by DAG kinase
regulates the amount of DAG and the route it takes. We used degenerate
primers to amplify polymerase chain reaction products from cDNA derived
from human endothelial cells. A product with a novel sequence was
identified and used to clone a 2.6-kilobase cDNA from an endothelial
cell library. When transfected with a truncated version of this cDNA,
COS-7 cells had a marked increase in DAG kinase activity, which
demonstrated clear selectivity for arachidonoyl-containing species of
diacylglycerol. The open reading frame of this clone has 567 residues
with a predicted protein of 64 kDa. This enzyme, which we designated
DGK, has two distinctive zinc finger-like structures in its
N-terminal region, but does not contain the E-F hand motifs found in
several other mammalian DGKs. The catalytic domain of DGK
, which
is related to other DGKs, contains two ATP-binding motifs. Northern
blotting demonstrated that DGK
is expressed predominantly in
testis. This unique diacylglycerol kinase may terminate signals
transmitted through arachidonoyl-DAG or may contribute to the synthesis
of phospholipids with defined fatty acid composition.
Diacylglycerol occupies a central position in the biosynthesis
of phospholipids and triglycerides. It also is an important
intracellular messenger because it can bind to and activate protein
kinase C, which, in turn, phosphorylates target proteins(1) .
This pathway has been implicated in many cellular response including
growth, differentiation, and other events such as secretion. The
mechanisms by which the signaling pathway and the synthesis of complex
lipids are differentially regulated is not clear, but the concentration
of DAG ()within the cell is almost certainly one important
component. In response to a variety of signals, the DAG level rises by
the activation of one or more phospholipases C and, in some cases, a
phospholipase D followed by phosphatidic acid phosphohydrolase. Either
pathway causes a rise in the amount of diacylglycerol by degrading
phospholipids. The level of DAG also is influenced by the rate at which
it is converted into other products. One pathway for decreasing DAG is
its conversion to phosphatidic acid, a reaction catalyzed by DAG
kinases (EC 2.7.1.107).
The stimulated rise in DAG levels is an integral component of the response of cells to a variety of stimuli that lead to growth or differentiation, and the effects of phorbol esters, which are tumor promoters, are through activation of protein kinase C. Thus, the level of DAG may be an important determinant of growth. In support of this, we found that rapidly growing endothelial cells have severalfold higher levels of DAG than quiescent cells, and others observed that transformation of cells by several oncogenes results in an increased content of DAG even in the absence of an additional stimulus(2, 3, 4) . The conversion of DAG to phosphatidic acid may dampen such signals, but the precise effects on cellular behavior are hard to predict because phosphatidic acid also may influence the growth response(5, 6, 7) . Another role of the DAG kinase reaction may be to resynthesize phosphatidylinositol, which, unlike most phospholipids, has a characteristic fatty acid composition: 1-stearoyl-2-arachidonoyl(8) . The mechanism for achieving this composition has never been elucidated although one possibility is that an enzyme(s) in the synthetic, or a salvage, pathway are specific for precursors with the appropriate molecular composition. Following the stimulated turnover of phosphatidylinositol, there is a later rise in phosphatidic acid, which has been thought to be the result of a DGK-catalyzed reaction. If this enzyme were specific for arachidonate-containing species of DAG, then multiple cycles might progressively enrich phosphatidylinositol with arachidonate.
The
first isoform of DAG kinase characterized at a molecular level,
DGK, has a molecular mass of about 80 kDa (9, 10) and is found predominantly in lymphocytes and
oligodendrocytes(11) . A second form, DGK
, was cloned from
brain where it is mainly expressed(12) . Kai et al.(13) isolated a cDNA for DGK
from a human liver
library, but subsequently found it to be expressed mostly in
retina(13) . A homologous rat cDNA is highly expressed in
cerebellar cells(14) . We recently identified DGK
from
human endothelial cells and showed that it has broad distribution, with
highest levels in brain and muscle. (
)None of these isoforms
exhibits a strong preference for substrates with specific fatty acids.
However, MacDonald et al.(15, 16) described
an activity that had marked preference for DAG species that contain an
arachidonoyl residue. Walsh et al.(17) reported the
purification of DAG kinase from bovine testis and found it to have a
molecular mass of 58 kDa. This activity was highest in testis, followed
by brain and spleen. The purified enzyme showed a marked preference (up
to 20-fold) for 1,2-DAG with arachidonate at the sn-2
position.
In the experiments reported here, we discovered a novel
isoform of DAG kinase by molecular cloning of a cDNA from an
endothelial cell library. When expressed in mammalian cells, it gives
an enzyme that is specific for arachidonate-containing DAG. This
enzyme, which we have named DGK, may have an important role in
signaling by regulating the concentration of DAG.
Since the full-length cDNA of DGK could not
be expressed in COS-7 cells (discussed under ``Results and
Discussion''), a PCR product of the DGK
cDNA lacking the
3`-untranslated region and part of the 5`-untranslated region was
amplified and cloned into pcDNAI/Neo (Invitrogen). The forward primer,
5`-GCATAAGCTCGATATCGAGGTATCGTCCTTG-3` (GP-3) contained 10 random
nucleotides, an EcoRV site (underlined), and 15 nucleotides
complementary to nucleotide -21 to -7 of DGK
. The
reverse primer, 5`-TTGTCTCGAGGTCGACATCTATTCAGTCGCC-3` (GP-4) contained
10 random nucleotides, a SalI site (underlined), and 15
nucleotides complementary to nucleotide 1692-1706 of DGK
.
The PCR amplification was performed as follows: 94 °C for 30 s, 50
°C for 30 s, and 72 °C for 2 min and 30 s for 5 cycles,
followed by 94 °C for 30 s, 65 °C for 30 s, and 72 °C for 2
min and 30 s for 30 cycles using Pfu (Stratagene) DNA
polymerase. The PCR product was gel-purified, cut with EcoRV/SalI, and cloned into the EcoRV/XhoI site of pcDNAI/Neo (Invitrogen).
The
octyl glucoside/PS mixed-micelle assay of DGK activity was performed as
described(17) . In brief, the assay mixture contained 50 mM MOPS, pH 7.2, 100 mM NaCl, 20 mM MgCl, 1 mM EGTA, 1 mM dithiothreitol, 2 mM diacylglycerol, 3.5 mM phosphatidylserine, 75 mM octyl-
-glucopyranoside,
500 µM [
-
P] ATP, and 20 µg
of cellular protein in a volume of 200 µl. The reaction was
initiated by the addition of [
-
P] ATP
(20-30 µCi/µmol) and processed for 10 min at 24 °C.
To stop the reaction, we added 1 ml of MeOH, 1 ml of CHCl
,
0.7 ml of 1% perchloric acid, and 50 µg of phosphatidic acid as
carrier. The lower phase of each sample was washed twice with 2 ml of
1% perchloric acid and then dried. They were resuspended in 100 µl
of 9:1 CHCl
/MeOH and separated by thin layer chromatography
(20
20 cm Silica 60A plates; Whatman). The plates were
developed in (325:75:25) CHCl
/MeOH/HOAc. The region
containing phosphatidic acid was scraped, and the radioactivity was
estimated by liquid scintillation spectrometry. The amount of
phosphatidic acid formed per reaction was calculated by dividing the
radioactivity in phosphatidic acid by the specific radioactivity of the
ATP in the reaction.
Figure 1:
Primary structure of a cDNA encoding
human diacylglycerol kinase . A, a map of clones encoding
DGK
. The full-length cDNA, DGKE13, was created by combining two
isolated clones, DGKE1 and DGKE3, at a BamHI site. The length
of each clone is denoted by the length of the labeled lines. The top diagram is the composite clone with the open box depicting the coding region and the solid line the
noncoding region, respectively. B, nucleotide sequence of the
composite cDNA and the deduced amino acid sequence of human DGK
.
The cysteine residues that comprise zinc finger-like structures are
indicated (
), and the predicted structural motifs are underlined. Residues characteristic of ATP-binding sites found
in other proteins are marked with an asterisk (*). The
positions of the PCR primers (GP1-GP4) described in the
text are also indicated. The sequence shown was constructed from that
of the individual clones.
The cDNA of DGK has an open reading frame encoding 567 amino
acids including the initiator methionine (calculated M
= 63,884) (Fig. 1B). The translation
initiation codon corresponds well with the Kozak sequence (23) . However, in the clones shown, we did not detect in-frame
stop codons in the 5`-untranslated region, nor were there typical
polyadenylation signals in the 3`-untranslated region. Thus, the
full-length messenger RNA for this enzyme is likely to be larger than
the clone we isolated (see below). In a subsequent experiment, we
screened a library from human testis (Clontech) and isolated another
DGK
clone with a longer 5` region, and an in-frame stop codon was
found at position -129 from the initiating methionine (data not
shown). DGK
has 34%, 36%, 36%, and 32% identity with human
DGK
(10) , rat DGK
(12) , human
DGK
(13) , and human DGK
,
respectively.
However, DGK
clearly differs from the other cloned DGKs as it does
not contain the N-terminal conserved region and E-F hand sequences
found in other mammalian DGKs (Fig. 2). Moreover, the two zinc
finger-like cysteine-rich sequences (residues
His
-Cys
and
His
-Cys
, Fig. 1B) found in
DGK
have distinctive patterns; the sequence of the first is
Cys-X
-Cys-X
-Cys-X
-Cys-X
-Cys-X
-Cys,
while the second has the sequence:
Cys-X
-Cys-X
-Cys-X
-Cys-X
-Cys-X
-Cys.
These precise zinc finger motifs are not found in any other DGKs, or in
protein kinases C. In particular, the number of amino acids separating
the last two cysteines in both the first and the second zinc
finger-like motifs is 9 in DGK
instead of 5-8 in most other
mammalian DGKs or protein kinases C (the other novel DGK that we
identified,
DGK
, also is unusual in that it has 9 and
10 amino acids separating the two final cysteines in the two motifs).
These features make DGK
unique among the known DGKs. However, the
catalytic domain, which is in the C-terminal region of DGK
,
contains two putative ATP binding motifs (Fig. 1B) and
is moderately conserved: the percent identity of amino acids in this
region is 41%, 42%, 42%, and 38% with human DGK
(10) , rat
DGK
(12) , human DGK
(13) , and human
DGK
,
respectively.
Figure 2:
Sequence comparison of human DGK with
other isoforms of diacylglycerol kinase. The conserved regions of
diacylglycerol kinases (designated C1-C4) are shown as shaded boxes. The amino acid identities with DGK
in each
region and overall are indicated as
percentages.
Figure 3:
Diacylglycerol kinase has marked
specificity for arachidonate-containing diacylglycerols. COS-7 cells
were transfected with vector alone (pcDNAI) or with a vector containing
the coding sequence of DGK
(pcDNAI/DGK
). Total lysates of the
cells were assayed for diacylglycerol kinase activity as described
under ``Experimental Procedures.'' A, DGK activity
was assayed with the following substrates (indicated on the right): 1,2-didecanoyl-sn-glycerol (10:0/10:0),
1-palmitoyl-2-oleoyl-sn-glycerol (16:0/18:1),
1-oleoyl-2-palmitoyl-sn-glycerol (18:1/16:0),
1,2-dioleoyl-sn-glycerol (18:1/18:1),
1-stearoyl-2-oleoyl-sn-glycerol (18:0/18:1),
1-stearoyl-2-linoleoyl-sn-glycerol (18:0/18:2), and
1-stearoyl-2-arachidonoyl-sn-glycerol (18:0/20:4). The data
were collected from two independent transfections, which are shown as
individual experiments. B, comparison of the substrate
specificity of different diacylglycerol kinases. We carried out
transfections as above, but, as a control, we also transfected cells
with DGK
(pcDNAI/DGK
). Additionally, assays were performed
with recombinant DGK from E. coli (1 µl of enzyme from the
Amersham diacylglycerol assay kit). The substrates used are indicated
using the abbreviations as above. For ease of comparison, the values
obtained with each DGK using
1-stearoyl-2-arachidonoyl-sn-glycerol (18:0/20:4) as the
substrate are shown as 100%. The activities measured using the other
substrates are expressed as relative to the arachidonate-containing
diacylglycerol. The values shown are the averages of two separate
experiments.
Figure 4:
Tissue-specific expression of DGK:
Northern blot analysis of mRNA from human tissues. Filters with
poly(A
) RNA from multiple human tissues were purchased
from Clontech and were hybridized with a
P-labeled 0.9-kb ApaI/SalI fragment of pBS/DGK
. Lanes: 1, heart; 2, brain; 3, placenta; 4,
lung; 5, liver; 6, skeletal muscle; 7,
kidney; 8, pancreas; 9, spleen; 10, thymus; 11, prostate; 12, testis; 13, ovary; 14, small intestine; 15, colon (mucosal lining); 16, peripheral blood leukocytes.
The enzyme encoded by the cDNA that we isolated, DGK, is
distinctive compared to other known DGKs as it is very selective for
arachidonoyl-DAG. The basis for this property is not clear. The
distinctive zinc finger-like structures, which likely are the sites for
DAG binding, may contribute to the substrate specificity, but DGK
,
which does not have the arachidonoyl-DAG specificity, has similar
sequences. A DAG kinase with such specificity might play an important
role in phospholipid metabolism by producing a precursor of
phosphatidylinositol, phosphatidic acid, that is enriched in
arachidonic acid. Multiple cycles of phosphatidylinositol hydrolysis
and resynthesis could lead to progressive enrichment in arachidonic
acid. However, DGK
seems unlikely to serve this function generally
as it has a very restricted pattern of expression. However, it is
possible that other tissues have very low level expression, below what
we could detect by Northern blotting, since the arachidonate-specific
DGK activity has been reported previously in aortic
endothelium(25) . Alternatively, there could be another isoform
with similar substrate preference.
Walsh et al.(17) recently reported purification of an
arachidonoyl-specific DGK from bovine testis, and it is likely that
DGK is its homolog(17) . For example, the bovine enzyme
has an apparent M
of 58,000 which is comparable to
the calculated M
of DGK
, they share the
substrate specificity, and both are highest in testis. However, unlike
the bovine enzyme, which is inhibited by PS and insensitive to DGK
inhibitors, DGK
is insensitive to PS and moderately inhibited by
R59949. These differences may reflect the assay conditions used or may
indicate that there are two isoforms of DGK with specificity for
diacylglycerol substrates containing arachidonic acid.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U49379[GenBank].
Note Added in Proof-During the
publication of this manuscript, the identification of another
diacylglycerol kinase () was reported. The nomenclature of this
manuscript was chosen to include this new member of the diacylglycerol
kinase family.