(Received for publication, November 20, 1995)
From the
A fourth member of the diacylglycerol kinase (DGK) gene family
termed DGK was cloned from the human testis cDNA library. The cDNA
sequence contains an open reading frame of 3,507 nucleotides encoding a
putative DGK protein of 130,006 Da. Interestingly, the new DGK isozyme
contains a pleckstrin homology domain found in a number of proteins
involved in signal transduction. Furthermore, the C-terminal tail of
this isozyme is very similar to those of the EPH family of receptor
tyrosine kinases. The primary structure of the
-isozyme also has
two cysteine-rich zinc finger-like structures (C3 region) and the
C-terminal C4 region, both of which have been commonly found in the
three isozymes previously cloned (DGKs
,
and
).
However, DGK
lacks the EF-hand motifs (C2) and contains a long
Glu- and Ser-rich insertion (317 residues), which divides the C4 region
into two portions. Taken together, these structural features of
DGK
indicate that this isozyme belongs to a DGK subfamily distinct
from that consisting of DGKs
,
, and
. Inc
reased DGK
activity without marked preference to arachidonoyl type of
diacylglycerol was detected in the particulate fraction of COS-7 cells
expressing the transfected DGK
cDNA. The enzyme activity was
independent of phosphatidylserine, which is a common activator for the
previously sequenced DGKs. Northern blot analysis showed that the
DGK
mRNA (
6.3 kilobases) is most abundant in human skeletal
muscle but undetectable in the brain, thymus, and retina. This
expression pattern is different from those of the previously cloned
DGKs. Our results show that the DGK gene family consists of at least
two subfamilies consisting of enzymes with distinct structural
characteristics and that each cell type probably expresses its own
characteristic repertoire of DGKs whose functions may be regulated
through different signal transduction pathways.
Diacylglycerol kinase (DGK, ()EC 2.7.1.107)
phosphorylates diacylglycerol to produce phosphatidic acid (1) . DGK is thought to play an important role in the signal
transduction linked to phospholipid turnover. The roles of
diacylglycerol and phosphatidic acid as lipid second messengers have
been attracting much attention. Diacylglycerol is known to be an
activator of protein kinase C(2, 3) , and phosphatidic
acid has been reported in a number of studies to modulate a ras GTPase-activating protein(4) , phosphatidylinositol
(PI)-4-phosphate kinase (5) and many other important enzymes.
Phosphatidic acid is also known to have mitogenic effects in a variety
of cells(6) . Thus, DGK is thought to be one of the key enzymes
involved in the cellular signal transduction(1, 7) .
For instance, DGK was found to be involved in interleukin-2 production
in T-lymphocytes (8) and retinal degeneration of Drosophila
melanogaster(9) .
Many DGK isozymes have been purified
from various animal sources, such as two isozymes from porcine brain
and thymus(10, 11) , three from human
platelets(12) , two from rat brain(13) , and one from
bovine testis(14) . These isozymes have been described to
differ from each other with respect to molecular masses, enzymological
properties, activators, and substrate specificity. It is thus likely
that these isozymes are operated under distinct regulatory mechanisms.
To date, three mammalian DGK genes, DGKs
(15, 16, 17) ,
(18) , and
(19, 20) have been isolated. The protein
products of these genes contain four conserved regions, i.e. the N-terminal region (C1), two sets of
Ca
-binding EF-hand motifs (C2), two cysteine-rich
zinc finger-like structures (C3), and the C-terminal C4 regions. These
isozymes have been shown to exhibit different tissue- and cell-specific
modes of expression. Namely, DGK
is most abundant in thymus (15) and oligodendrocytes of brain(17) , and DGK
is particularly enriched in rat neuron(18) . DGK
, on the
other hand, is highly expressed in the human retina (19) and
rat cerebellar Purkinje cells(20) . Despite different
expression patterns, the structural and enzymatic properties of the
three cloned DGKs are very similar to each other, and all of them are
characteristically expressed in the central nervous system. Thus, it
seems quite likely that DGK gene family may possibly include additional
hitherto unknown members with distinct structural features.
Identification of novel DGK isozymes and determination of the complete
DGK repertoire represent an important and necessary step for
elucidating the exact physiological role of DGK.
In the present
investigation, we cloned a novel DGK isozyme, DGK, that apparently
belongs to a subfamily distinct from that consisting of DGKs previously
cloned. Interestingly, the novel isozyme contains a pleckstrin homology
(PH) domain at the N terminus and a C-terminal tail similar to those of
EPH family of protein-tyrosine kinases. Moreover, its expression
pattern was markedly different from those of other DGKs.
General manipulation of DNA and RNA was carried out according to the standard procedures(21) .
To obtain DGK-related core sequences, reverse
transcriptase PCR amplification was carried out using GeneAmp(TM)
RNA PCR kit (Perkin-Elmer Corp.). In brief, first strand cDNA template
was synthesized from 0.1 µg of poly(A) RNA
prepared from human testis in 20 µl of reverse transcriptase buffer
containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5
mM MgCl
, 1 mM each dNTP, 1 unit/µl
RNase inhibitor, 2.5 units/µl Moloney murine leukemia virus reverse
transcriptase, and 2.5 µM random hexamers. The reaction
mixture was incubated for 10 min at 25 °C and then for 30 min at 42
°C. After heat treatment (97 °C for 5 min), the mixture was
added with 80 µl of Taq polymerase buffer containing 10
mM Tris-HCl (pH 8.3), 50 mM KCl, 1.25 mM MgCl
, 2.5 units of Taq polymerase, and 1 nmol
each of primers P1 and P2. Amplification reactions were carried out for
40 cycles on a GeneAmp PCR System 2400 (Perkin-Elmer Corp.) under the
following conditions: 94 °C for 1 min, 52 °C for 2 min, and 72
°C for 2 min, followed by a final elongation step at 72 °C for
7 min. Amplified PCR products were subsequently separated by
preparative agarose gel electrophoresis. Bands in the position
(
500 bp) predicted by reference to the cDNA sequences of DGKs so
far cloned were recovered, digested with XhoI, and subcloned
into the XhoI site of pBluescript II SK+ (Stratagene).
Figure 1:
Cloning strategy and restriction map of
human DGK cDNA. The four representative clones (DGKD core, DGKD3,
DGKD10, and DGKD11) isolated are shown by thick bars. These
sequences were combined to construct the composite cDNA (DGKD103, top) in which the open box and lines indicate the coding and noncoding sequences, respectively. Horizontal arrows denote primers used for reverse
transcriptase PCR and 5`-RACE amplification. The construct (DGKD104)
used for cDNA expression in COS-7 cells is shown at the bottom. In this case the nucleotide substitutions were done as
indicated to fulfill Kozak consensus translation initiation sequence
and also to create EcoRI sites for
subcloning.
The octyl glucoside mixed micellar assay of DGK activity was done as
described previously(31) . In brief, the assay mixture (50
µl) contained 50 mM MOPS (pH 7.4), 50 mM octyl
glucoside, 1 mM dithiothreitol, 100 mM NaCl, 20
mM NaF, 10 mM MgCl, 1 mM EGTA,
1.5 mM diacylglycerol, and 1 mM [
-
P]ATP (100,000 cpm/nmol). The
reaction was initiated by adding the particulate or soluble fraction
(10 µg of protein) from COS-7 cells and continued for 5 min at 30
°C. Lipids were extracted from the mixture, and phosphatidic acid
separated by thin layer chromatography (32) was scraped and
counted by a liquid scintillation spectrophotometer.
The soluble and
particulate fractions of COS-7 cells (10 µg of protein each) were
separated on an SDS, 7.5% polyacrylamide gel(34) . The proteins
were transferred to a nitrocellulose membrane (Schleicher and Schuell)
and blocked for 1 h in phosphate-buffered saline (pH 7.4) containing
0.1% (v/v) Tween 20 and 20% (w/v) skim milk. The membrane was incubated
with the anti-DGK antibody (0.7 µg/ml) in the same buffer for
1 h. The immunoreactive bands were visualized using
peroxidase-conjugated, anti-rabbit IgG antibody (Jackson ImmunoResearch
Laboratories) and enhanced chemiluminescence (ECL kit, Amersham Corp.).
This
cDNA clone (the DGKD core sequence, Fig. 1) was used to screen
800,000 clones of the testis library and
1,000,000 clones of
the HepG2 cell library. Seven positive clones (DGKD1-7) and three
positive clones (DGKD8-10) were isolated from the testis and the
HepG2 libraries, respectively (Fig. 1). Only the most 5`- and
3`-stretched clones, DGKD10 and -3, respectively, were indicated in Fig. 1. However, sequence analysis revealed that these clones
lacked the N-terminal part of the open reading frame including the
initiation methionine codon. Although we further screened the same
libraries with DGKD10 cDNA as a probe, we could not obtain clones
covering the 5`-region. We, therefore, carried out the RACE-anchored
PCR procedure using poly(A)
RNA isolated from HepG2
cells as a template. The 5`-anchored PCR employing primers R1 and R2 ( Fig. 1and Fig. 2) gave rise to three clones
(DGKD11-13) with the same length, all of which were found to
cover the complete 5`-region.
Figure 2:
Nucleotide sequence and deduced amino acid
sequence of DGK. Nucleotides and amino acids are numbered at the right, respectively. An in-frame stop codon in the
5`-untranslated sequence and a putative polyadenylation signal in the
3`-untranslated sequence are dotted underlined, respectively.
The positions of the reverse transcriptase PCR primers and the 5`-RACE
primers are indicated by underlines below the nucleotide
sequences. The PH domain is thick underlined. The zinc
finger-like cysteine-rich sequences are gray underlined. In
this sequence the conserved cysteine and histidine residues are marked
by asterisks. The C4-a and C4-b subregions are indicated by double underlines. The C-terminal tail similar to those of the
EPH family of receptor tyrosine kinase is dashed
underlined.
The composite DGK cDNA sequence
obtained from clones DGKD3, DGKD10, and DGKD11 (DGKD103, 6214 bp, Fig. 1) has an open reading frame starting from nucleotide 81 to
nucleotide 3590 (Fig. 2). The initiation ATG of the open reading
frame was identified as the first ATG sequence following an in-frame
termination codon at nucleotide 66 to 68. The open reading frame is
flanked by 80- (5`-) and 2624-bp (3`-) noncoding nucleotide sequences.
A polyadenylation signal (AATAAA) is found 22 nucleotides upstream of
the poly(A) sequence at the 3`-end of the cDNA.
Figure 3:
Sequence comparison of DGK with other
DGKs, the members of the EPH receptor family and several proteins
containing the PH domain. A, the schematic structure of
DGK
is shown with reference to previously sequenced porcine
DGK
(15) , rat DGK
(18) , human
DGK
(19) , Drosophila DGK1 (23) , and Drosophila DGK2(9) . The mammalian DGK gene family is
proposed to consist of at least two enzyme subfamilies tentatively
designated Type I and Type II. B, amino acid alignment of the
cysteine-rich sequences of DGK
and other DGK isozymes. Asterisks indicate the conserved cysteine and histidine
residues. Since the motifs of the cysteine-rich sequences in Drosophila DGK2 (9) are considerably different, their
alignments are not shown. C, alignment of the C4-a of DGK
and C4 regions of other DGK isozymes and Drosophila homologs. D, alignment of the C4-b of DGK
and C4 regions of other
DGK isozymes and Drosophila homologs. E, alignment of
the PH domains of DGK
and several proteins containing the PH
domain(s)(38, 39, 40) . The consensus
sequence(38, 39, 40) , including
alternatives, is shown below; the most highly conserved residues are highlighted. F, alignment of the C-terminal tails of
DGK
and the members of the tyrosine kinase EPH receptor family (42, 43, 44, 45
, 46, 47, 48) . Highlighted letters in the consensus sequence indicate
invariant residues found in all members of the EPH family. B-F, the residue number of the first amino acid is given
in parentheses for each sequence. Identical amino acids are
indicated in reverse type. Similar amino acids are hatched. The groups of similar amino acids were defined as
follows: small R groups with near neutral polarity (G, A, P, S, and T);
acidic and uncharged polar R groups (D, E, N, and Q); basic polar R
groups (H, R, and K); nonpolar chain R groups (M, I, L, and V); and
aromatic nonpolar or uncharged R groups (F, W, and Y). The extent of
identity (I) and similarity (S) are shown at the end
of each column. Dashes indicate gaps inserted to maximize
alignment.
One of the striking structural features of DGK is that there is
a long insertion (317 residues) that divides the C4 region into two
portions, designated C4-a and C4-b subregions (Fig. 3A). A similar division of the C4 region was also
noted previously for Drosophila DGK1. However, both sequences
of the C4 subregions are very similar to the corresponding parts of the
C4 region of other DGKs, exhibiting 51-55% (C4-a) and
38-44% (C4-b) identities, respectively (Fig. 3, C and D). Since the C4 region is well conserved in all
mammalian DGK isozymes and Drosophila homologs, it seems to be
the catalytic domain. Indeed, we recently found that a DGK
mutant
containing only the C4 region showed phospholipid-dependent DGK
activity. (
)The insert separating the two C4 subregions
displayed no significant sequence similarity to that of Drosophila DGK1, and the effect of this insert on the catalytic activity
remains unknown. The insertion contains a Glu- and Ser-rich sequence
(residues 416-538). In this case, 15 (12.2%) and 22 (17.9%) out
of 123 residues are Glu and Ser residues, respectively. The additional
characteristic of the sequence of DGK
in comparison with other
DGKs is that DGK
has neither the N-terminal C1 region nor the
EF-hand motifs (C2) ( Fig. 2and Fig. 3A).
Interestingly, this novel DGK isozyme contains the PH
domain(38, 39, 40) (residues 10-100)
at the N terminus ( Fig. 2and Fig. 3E). The PH
domain, comprising about 100 amino acids, was originally detected as an
internal repeat in pleckstrin, a 47-kDa protein that is the major
substrate of protein kinase C in platelets(41) . At present,
the PH domains have been found in a number of proteins involved in
signal transduction (38, 39, 40) such as a ras-GTPase activating protein, AKT/RAC protein kinase,
and
types of PI-specific phospholipase C, a vav oncogene
product and protein kinase Cµ. All of the most highly conserved
residues (Gly
, Leu
, Leu
,
Leu
, Phe
, and Trp
in DGK
)
of the PH domains (38, 40) are completely conserved
also in DGK
(Fig. 3E). The extents of identity and
similarity between the PH domain of DGK
and the corresponding
regions of several proteins such as pleckstrin, AKT1, protein kinase
Cµ and phospholipase Cs, are 17-31% and 35-52%,
respectively (Fig. 3E). The C-terminal tail (residues
1105-1169) of DGK
is unexpectedly similar to those of the
EPH family of receptor tyrosine kinases(42, 43, 44, 45, 46, 47, 48) ( Fig. 2and 3F). The C terminus of the EPH family, which
follows the catalytic domain, is thought to be a regulatory domain. The
EPH family (the fourth family of receptor protein-tyrosine kinases) is
distinct from the epidermal growth factor receptor, insulin receptor,
and platelet-derived growth factor receptor
families(42, 43, 44, 45, 46, 47, 48) .
Most of the invariant residues (Trp
, Leu
,
Phe
, Asp
, Gly
, and
Ile
, except for Met
, in DGK
) found
in all EPH members cloned so far (48) are conserved also in
DGK
. The extents of identity and similarity between DGK
and
the receptor family in the C-terminal tail are 26-32% and
48-57%, respectively, as shown in Fig. 3F.
Figure 4:
Transient expression of DGK in COS-7
cells. A, immunoblot analysis of translation product of
DGK
cDNA in COS-7 cells. Particulate (P) and soluble (S) fractions (10 µg of protein each) from COS-7 cells
transfected with pSRE-DGK
(DGK
) or the vector alone (pSRE) were separated by SDS-polyacrylamide gel
electrophoresis and transferred to a nitrocellulose membrane. The
DGK
protein was detected with anti-DGK
antibodies as
described under ``Experimental Procedures.'' B, particulate (ppt) and soluble (sup) fractions
were separated from COS-7 cells transfected with pSRE-DGK
or pSRE
vector alone. DGK activity was measured as described under
``Experimental Procedures.'' The results are means ±
S.D. of three independent experiments.
When assayed with different molecular
species of diacylglycerol including 1-stearoyl-2-arachidonoylglycerol,
1-stearoyl-2-linoleoylglycerol, 1,2-dioleoylglycerol, and
1,2-didecanoylglycerol as substrate, DGK showed no marked
specificity with regard to the acyl compositions of diacylglycerol
(data not shown). PS is known to be a common activator of DGKs
,
,
, and other enzymes purified(1) . However, PS (18.0
mol%) did not activate DGK
but was rather inhibitory (Fig. 5). The PH domain of pleckstrin was found to bind to
PI-4,5-bisphosphate (49) . Although 7.0 mol% of
PI-4,5-bisphosphate was added to the assay mixture, no increased
activity was detected (Fig. 5). Ca
, which is
an activator of DGK
and
(18, 31) , also h
ad
no effect on the activity of DGK
(data not shown).
Figure 5:
Effects of PS and PI-4,5-bisphosphate on
the activity of DGK. DGK activity was measured in the presence of
18.0 mol% of PS or 7.0 mol% of PI-4,5-bisphosphate (PIP2). To
facilitate comparison, background activities (those of the control
cells transfected with the vector alone) were subtracted, and the
results were expressed as percent of the value obtained in the absence
of activators. The values are the average of duplicate determination,
which differed by less than 10% of the mean. Similar results were
obtained in two repeated experiments.
Figure 6:
Northern hybridization analysis of human
tissues and cells. Poly(A) RNA (2 µg each) was
analyzed as described under ``Experimental Procedures.''
Except for poly(A)
RNAs from retina, HL-60, Jurkat,
and HepG2 cells, commercially available multiple tissue Northern Blot I
and II (Clontech) were employed. The filters were hybridized with the XhoI-NcoI fragment of DGKD3 labeled with
P and exposed for 60 h with intensifying screens. Almost
equal amount of RNA had been loaded, as judged from hybridization of
the same filter with a human
-actin cDNA probe (data not
shown).
Our work adds a new member, DGK, to the growing list of
mammalian DGK genes. Although rat DGK-III cDNA has recently been
cloned(20) , this isozyme shared 88% identical amino acid
sequence with human DGK
(19) . We thus believe that the
DGK-III should be a rat counterpart of human DGK
. It is of
particular interest to note that DGK
contains the PH domain at the
N terminus and the C-terminal tail very similar to those of the
protein-tyrosine kinases of EPH receptor family ( Fig. 2and Fig. 3A). Such structural features enabled us to
classify DGK
into the new DGK subfamily (type II), which is
distinct with respect to domain structures from that (type I)
consisting of DGKs
,
, and
previously cl
oned. It should
be noted here that DGKs belonging to the type I subfamily possess
basically the same domain structures despite their markedly different
expression pattern. Moreover, DGK
is highly expressed in the
skeletal muscle with a very low expression in the brain (Fig. 6). This expression pattern of DGK
is quite different
from those of the type I DGK isozymes, which are typically expressed in
the central nervous system. It is interesting to see if the type II DGK
subfamily also consists of multiple isozymes with related structural
characteristics. We may hypothesize that the DGK gene family probably
consists of several different subfamilies, as has been known for
protein kinase C (3, 35) and PI-specific
phospholipase C(50) . The divergence of DGK isozymes may
reflect their physiological importance and the needs to respond to a
variety of signaling pathways operating under distinct regulatory
mechanisms.
The distribution of the PH domain has so far been
limited to cytoskeletal proteins and other proteins involved in
cellular signal transductions, many of which are associated with the
cell surface and intracellular membranes(40) . This domain is
thought to serve as the site of protein-protein interaction, which is
crucial to the function of these proteins(40) . The presence of
the PH domain in DGK suggests that this enzyme would share similar
regulatory mechanisms with the signaling and cytoskeletal proteins.
Lefkowitz and co-workers (51) have recently found that the PH
domains in a number of proteins such as
-adrenergic receptor
kinase and PI-specific phospholipase C
can bind the
complexes of heterotrimeric G proteins. On the other hand, the PH
domains of pleckstrin (49) and the AKT protein kinase family (52) have been found to bind PI-4,5-bisphosphate and
PI-3-phosphate, respectively. Although it is possible that the activity
of DGK
may be regulated by binding to regulatory proteins or
phospholipids, we could not detect at present the effects of at least
PI-4,5-bisphosphate on the DGK
activity.
The EPH family with a
dozen members is currently known to be the largest subfamily of
receptor protein-tyrosine kinases(53) . Oncogenic activities of
this receptor family have also been described previously (54) .
Recently, one of their ligands has been found to be B61, the protein
product of an early response gene induced by tumor necrosis
factor-(55) . It is also found that PI-3-kinase serves as
a downstream target of this receptor family(56) . Our work is
the first to report that the homology region to the C-terminal tail of
the EPH protein-tyrosine kinases exists in the C terminus of an
apparently unrelated enzyme protein, DGK. This finding led us to
speculate that this domain may be distributed in a wider range of
proteins than has been previously anticipated. We thus tentatively
propose that the conserved C-tail region is designated an EPH
C-terminal tail homology domain. Although the function of the EPH
tyrosine kinases has been suggested to be regulated by phosphorylation
of the conserved tyrosine residue of the C-terminal tail (tyrosine 1117
in the case of DGK
) (44) , the exact function(s) of this
domain remains to be explored. We are currently investigating the
possibility that the action of DGK
is regulated by tyrosine
phosphorylation.
DGK has a long insertion composed of 317
residues between the C4-a and C4-b subregions. This insertion occurs
only in DGK
among the sequenced mammalian DGKs, but a similar
insertion was described previously for Drosophila DGK1.
However, there was no significant sequence homology between the
insertions of DGK
and the Drosophila homolog. Moreover,
the insertion did not have significant similarities to other sequences
deposited in protein data base. We noted that the insertion of DGK
contains Glu- and Ser-rich sequences. It is interesting to note that
casein kinases I, II and mammary gland casein kinase phosphorylate Ser
residues in the sequences, Glu-X-X-Ser,
Ser-X-X-Glu, and Ser-X-Glu,
respectively(57) . DGK
contains several of these sequences (Fig. 2). It is therefore possible that the insertion of
DGK
serves as a phosphorylation site(s) of such protein kinases.
The sequences of C4 region were most highly conserved in all DGK
isozymes and Drosophila homologs. This region is therefore
quite likely to serve as the catalytic domain common to all DGKs. There
are several sequences perfectly conserved in all DGKs (Fig. 3, C and D), and these are probably important for the
catalytic action including DG and ATP bindings. Although our earlier
study noted in the C4 region of DGK
a putative ATP-binding site
with a motif of
Gly-X-Gly-X
-Gly-Lys(15) , this
motif is not conserved in other DGKs including DGK
. A comparison
of the amino acid sequence of the C4 region with other
nucleotide-binding proteins (58) did not reveal candidate
ATP-binding site(s) of DGK. The present work showed that the zinc
finger structures are conserved also in DGK
, the domain structure
of which is markedly deviated from those of the type I isozymes. The
zinc fingers together with the highly conserved C4 region thus appear
to be essential for the action of different DGKs. It was previously
shown that the zinc fingers of human DGK
is incapable of phorbol
ester binding(59) . Furthermore, we found that a DGK
mutant lacking the zinc fingers exhibited a phospholipid-dependent DGK
activity.
These results indicate that the DGK zinc fingers
are not the sites of phorbol ester or DG binding. The function of this
motif is thus entirely unknown for DGKs. Recently the zinc fingers of
protein kinase C
and Raf-1 kinase have been shown to be a
subcellular localization domain (60) and an interaction site
with p21
(61, 62) , respectively. It is
therefore likely that some novel and unexpected functions would be
ascribed to the DGK zinc fingers in future studies.
The DGK
cDNA gave only 2.5-fold increase of DGK activity in the transfected
COS-7 cells (Fig. 4B). This contrasts with the high
enzyme activities of other DGKs transfected under the same experimental
conditions(19) . This low level of enzyme activity hindered us
from defining detailed enzymological properties of DGK
. This may
be partly due to the low expression of the enzyme protein or to
proteolytic degradation of the expressed enzyme as noted in
immunoblotting (Fig. 4A). In view of the apparent lack
of phosphatidylserine dependence of the DGK
activity (Fig. 5), the enzyme assay conditions developed for the type I
enzymes may not be suitable for this isozyme. It is also possible that
DGK
requires unknown activator(s) or its phosphorylation on serine
or tyrosine residues to exert full catalytic activity. In this respect,
we could not deny the possibility that DGK
might prefer substrates
other than DG. However, we could not detect the enzyme activity when
the COS cell extracts were tested with PI, 1- or 2-monoacylglycerol,
ceramide, and sphingosine as substrates. The enzymological studies
using purified DGK
are needed to define the regulatory mechanisms
of this isozyme.
Despite interesting domain structures, the function
of DGKs in cellular phospholipid metabolism is largely unknown. The
present work further points to the potential importance of DGKs as
signaling molecules by disclosing the existence of a novel isozyme
possessing the PH and EPH C-terminal tail homology domains in addition
to the previously identified functional regions. The expression pattern
of DGK, when taken together with those of other DGKs, suggests
that each DGK species may play a specialized role in differentiated
cells and that each cell type probably expresses its own characteristic
repertoire of DGK isozymes. Elucidation at the molecular level of the
regulatory mechanisms of DGKs would reveal the physiological
significance of these proteins with unique structures.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D73409[GenBank].