(Received for publication, November 22, 1995; and in revised form, February 22, 1996)
From the
Diacylglycerol (DAG) occupies a central position in the
synthesis of complex lipids and also has important signaling roles. For
example, DAG is an allosteric regulator of protein kinase C, and the
cellular levels of DAG may influence a variety of processes including
growth and differentiation. We previously demonstrated that human
endothelial cells derived from umbilical vein express growth-dependent
changes in their basal levels of diacylglycerol and diacylglycerol
kinase activity (Whatley, R. E., Stroud, E. D., Bunting, M., Zimmerman,
G. A., McIntyre, T. M., and Prescott, S. M.(1993) J. Biol. Chem. 268, 16130-16138). To further explore the role of
diacylglycerol metabolism in endothelial responses, we used a
degenerate reverse transcription-polymerase chain reaction method to
identify diacylglycerol kinase isozymes expressed by human endothelial
cells. We report the isolation of a 3.5-kilobase cDNA encoding a novel
diacylglycerol kinase (hDGK) with a predicted molecular mass of
103.9 kDa. Human DGK
contains two zinc fingers, an ATP binding
site, and four ankyrin repeats near the carboxyl terminus. A unique
feature, as compared with other diacylglycerol kinases, is the presence
of a sequence homologous to the MARCKS phosphorylation site domain.
From Northern blot analysis of multiple tissues, we observed that
hDGK
mRNA is expressed at highest levels in brain. COS-7 cells
transfected with the hDGK
cDNA express 117-kDa and 114-kDa
proteins that react specifically with an antibody to a peptide derived
from a unique sequence in hDGK
. The transfected cells also express
increased diacylglycerol kinase activity, which is not altered in the
presence of R59949, an inhibitor of human platelet DGK activity. The
hDGK
displays stereoselectivity for 1,2-diacylglycerol species in
comparison to 1,3-diacylglycerol, but does not exhibit any specificity
for molecular species of long chain diacylglycerols.
1,2-Diacylglycerol (DAG) ()occupies a central
position in the biosynthesis of complex lipids and is a key
intracellular messenger by virtue of its ability to activate protein
kinase C (PKC). How DAG can serve two seemingly different, and
essential, roles in cellular processes is not clear, but one important
issue is likely to be the intracellular concentration at a given time.
The intracellular DAG levels are regulated by the rates of both
synthesis and degradation. Receptor-mediated activation of a
phospholipase C, or phospholipase D followed by a phosphatidate
phosphohydrolase, have been shown to increase intracellular DAG levels (1) . One potential fate of the DAG produced during these
responses is to be phosphorylated in a reaction that uses ATP as a
phosphate donor. This reaction is catalyzed by DAG kinase (DGK) (2) (EC 2.7.1.107) and yields phosphatidic acid. This enzymatic
conversion is thought to be a key mechanism by which PKC activation is
attenuated by virtue of lowering the intracellular concentration of
DAG. However, phosphatidic acid has been shown to be mitogenic (3) and to modulate the activity of intracellular proteins
including n-chimaerin (4) and NF-1(5) .
Further, it too is a central metabolite in complex lipid synthesis.
Thus, the conversion of DAG to phosphatidic acid may have complicated
net effects.
DGK activities have been identified from a wide range
of cellular sources. Further analysis of the activity from these
tissues and cell types by protein purification, nucleic acid
hybridization, and reverse transcription-PCR has identified several
isoforms of DAG kinase. An 80-kDa isozyme, DGK, was the first
isoform purified and cloned from porcine (6) and human
tissues(7) . This enzyme is primarily expressed in lymphocytes
and oligodendrocytes(8) . A cDNA for a second DGK isozyme,
DGK
, subsequently was isolated from a rat brain library and
encodes a protein with a predicted mass of 90 kDa. DGK
is
primarily expressed within adult brain cell populations including the
olfactory tubercle, nucleus accumbens, and the caudate
putamen(9) . Another form, DGK
, was cloned from a human
HepG2 library and found to be expressed predominantly in
retina(10) . A hDGK
cDNA containing a 25-amino acid
deletion within the catalytic domain was identified, suggesting that
this isoform may be regulated by alternative splicing. Recently, a rat
DGK cDNA displaying significant homology to hDGK
was isolated and
found to be highly expressed in cerebellar Purkinje cells (11) . All of these isoforms have a similar domain structure
including two E-F hands, two zinc fingers, and a catalytic domain
containing a predicted ATP binding site. Additional DGK activities have
been purified from a variety of sources. Some of these kinases display
unique characteristics that differ from DGK
, -
, and -
,
suggesting the presence of additional DGK isozymes. For example, a
unique 58-kDa DGK, which preferentially phosphorylates DAG species
containing arachidonate, recently was purified to homogeneity from
bovine testis(12) . Finally, several homologous cDNAs have been
identified in Drosophila(13, 14, 15) . Although
the enzymatic activities of the corresponding proteins have not been
well characterized, they represent another set of potentially important
kinases.
Intracellular DAG levels have been shown to play a vital
role in cellular growth responses. Moreover, the tumor-promoting
effects of phorbol esters are thought to derive from their ability to
activate protein kinase C isozymes, and, therefore, an increased
intracellular concentration of DAG could be an endogenous tumor
promoter. Oncogenic transformation by ras(16) , sis(16) , src(17) , fms(17) , and erbB(18) all have been
demonstrated to elevate basal intracellular DAG. We showed that rapidly
growing human endothelial cells maintain a 2- to 3-fold higher level of
DAG than confluent cells and that PKC was activated in the dividing
cells(19) . Inversely, both DAG kinase and DAG lipase (20) activities increased as the endothelial cells reached
confluence suggesting that metabolism of DAG occurred through one or
both enzymes. Interestingly, the translocation of DAG kinase from the
cytosol to membranes observed in stimulated cells is diminished in ras- (21) , src-(22) , or erbB- (22) transformed fibroblasts. Further, the
elevated DAG levels in v-Ki-ras-transformed NIH/3T3 cells can
be lessened by overexpression of DGK(23) . To evaluate
more critically the role of DAG kinase in endothelial cell responses
and growth control, we conducted experiments to identify the isozymes
of DAG kinase expressed by human endothelial cells and, in the process,
discovered a novel isoform, which we have named hDGK
.
Figure 1:
Nucleic acid sequence and deduced amino
acid sequence of hDGK. Domain analysis and comparison to rdgA.
A, cDNA nucleic acid sequence and the deduced amino acid sequence
of hDGK
. The zinc fingers are underlined, and key
cysteine and histidine residues are marked with a
. Serine
residues within the MARCKS homology region are marked by an *. The
residues within the ATP-binding motif are double underlined.
The ankyrin motifs are displayed within boxes. B, a
comparison of the predicted protein sequences of hDGK
and rdgA. C, the alignment of hDGK
carboxyl-terminal
sequences with the consensus repeat found in ankyrin (47) .
Conservative substitutions were included in the alignment as
shown.
The domain structure of hDGK
differs significantly from the presently known DAG kinases,
particularly the mammalian isozymes
,
, and
. The most
intriguing difference is the presence of a sequence, KKKKRASFKRKSSKK (Fig. 1A), which is similar to the phosphorylation site
of the myristoylated, alanine-rich C-kinase substrate
(MARCKS)(29) . MARCKS is a major substrate for protein kinase
C, which is activated by DAG. In addition, among the known mammalian
isoforms, hDGK
uniquely lacks an E-F hand motif, a domain
implicated in calcium binding. Moreover, hDGK
contains four tandem
ankyrin repeats at the carboxyl terminus (Fig. 1C). In
contrast to hDGK
, ankyrin consistently contains an aspartic acid
or an asparagine at position 29 within this motif, whereas hDGK
has a methionine at this position in three of the four tandem repeats.
Finally, in hDGK
, the N-terminal sequence Leu
to
Glu
and internal sequence Pro
to Pro
scored favorably (9.5 and 16.1, respectively) in a PEST FIND
program(30) . PEST sequences are frequently observed in rapidly
degraded proteins, suggesting that hDGK
may be regulated by
protein degradation. Like the other DAG kinases, hDGK
contains two
zinc finger-like structures in a cysteine-rich region at the amino
terminus. The first zinc finger includes the sequence
HX
CX
CX
CX
CX
HX
CX
C
and the second
HX
CX
CX
CX
CX
HX
CX
C.
The hDGK
contains a single motif within the catalytic domain that
conforms to an ATP binding site (Gly
to
Lys
)(31) .
Figure 2:
Diacylglycerol kinase is expressed
in multiple tissues, but most strongly in brain. The 761-bp PCR product
corresponding to hDGK
(see ``Results'') was used as a
probe in Northern blots of 10 µg of total RNA extracted from
cultures of human endothelial cells (HUVEC) (A) or a human
multiple tissue Northern blot (Clontech) (B). In lane
C, we measured actin mRNA expression in the corresponding lanes of
the human multiple tissue Northern blot as a positive
control.
Figure 3:
Heterologous expression of hDGK
results in elevated levels of diacylglycerol kinase activity. COS-7
cells were transfected with either hDGK
(pcDNA1/AMP:DGK
; filled circles) or vector alone (pcDNA1/AMP; open
circles). After 48 h, the cells were harvested, and the indicated
amounts of cellular homogenate were assayed for DGK activity using
1,2-dioleoyl-sn-glycerol as the substrate. This experiment is
representative of results observed with cells from two different
transfections.
We
next examined the substrate specificity of the expressed hDGK (Table 1). A strong preference for 1,2-diacylglycerol over
1,3-diacylglycerol was observed, and hDGK
catalyzed the
phosphorylation of a short chain diacylglycerol (diC
) in
preference to all the long chain diacylglycerols examined. This result
likely reflects the fact that the short chain substrate is more soluble
than the other substrates we tested. Among the long chain
diacylglycerols examined, we observed a slight preference for
1-stearoyl-2-arachidonyl-sn-glycerol. Interestingly, the
expressed DGK activity did not distinguish between molecular species
that varied only in the relative position of the fatty acids:
1-palmitoyl-2-oleoyl-sn-glycerol versus 1-oleoyl-2-palmitoyl-sn-glycerol. The vector-transfected
cells did have detectable DGK activity, but it displayed a distinctly
different substrate specificity and did not contribute significantly to
the activity measured from hDGK
-transfected cells (Fig. 3).
For all substrates assayed, the DGK activity of vector-transfected
COS-7 cells was highest for
1-stearoyl-2-arachidonyl-sn-glycerol with a specific activity
of 9.05 nmol/mg/h.
Platelet DAG kinase is inhibited by the compound
R59949 which has been used to dissect the role(s) of DGK in cellular
responses(32) . We next asked whether R59949 could block the
activity of hDGK in vitro. We observed that the expressed
hDGK
was not significantly inhibited (94% of control) by 100
µM R59949. In contrast, the DGK activity of human
platelets was reduced to 54% of control by 100 µM R59949.
To confirm the predicted size of the hDGK protein, we made a
polyclonal antibody to a peptide based on the carboxyl-terminal
sequence: (C)LENRQHYQMIQREDQE. This antibody was used to probe a
Western blot containing protein from hDGK
- or vector-transfected
COS-7 cells (Fig. 4). Proteins with apparent masses of 117 kDa
and 114 kDa were recognized by the polyclonal antibody in
hDGK
-transfected cells, but were not present in control cells.
Furthermore, the recognition of these proteins was blocked by
preincubation of the antibody with the corresponding peptide antigen
confirming that the interaction of the antibody with these proteins was
specific. In subsequent studies, we have detected the expression of
endogenous DGK
in a glioblastoma-derived human cell line (A-172)
by Western blotting (data not shown). Similarly, A-172 cells expressed
two immunoreactive proteins which exhibited apparent molecular weights
indistinguishable from those detected from transfected COS-7 cells.
Figure 4:
COS-7 cells transfected with the hDGK
clone express novel proteins. Lanes 1 and 3 contain
25-µg samples of vector (pcDNA1/AMP) transfected COS-7 cells. Lanes 2 and 4 contain 25-µg samples of hDGK
(pcDNA1/AMP:DGK
) transfected COS-7 cells. Lanes 1 and 2 were probed with the carboxyl-terminal anti-peptide rabbit
antibody. Lanes 3 and 4 were probed with the
carboxyl-terminal anti-peptide rabbit antibody after preincubation with
the corresponding peptide as described under ``Experimental
Procedures.''
We report here the molecular cloning and characterization of
a new human diacylglycerol kinase. Similar to the previously identified
DGK isozymes, hDGK contains zinc finger-like sequences and an ATP
binding site consensus. In contrast to DGK
, -
, and -
,
the hDGK
isoform lacks an apparent E-F hand motif (33) and
its activity is not affected by calcium. This DGK is unique in that it
contains a sequence which is homologous to the MARCKS phosphorylation
site domain, which may have an important regulatory role(s). The
predicted protein sequence is 42% identical and 61% similar to another
DGK from Drosophila (dDGK2), which is encoded by the rdgA locus(13) . dDGK2 is the only other DGK that is known to
have ankyrin repeats, which are motifs implicated in a variety of
functions including protein-protein interactions. There are several
significant differences between hDGK
and dDGK2, however, including
the predicted length of the amino termini, the markedly different
patterns of tissue expression, and the presence of a sequence
homologous to the MARCKS phosphorylation site domain in hDGK
.
Northern blot analysis showed that hDGK
is expressed as a 3.7-kb
message in human endothelial cells, and in human tissues is expressed
at highest levels in brain. COS-7 cells transfected with hDGK
have
elevated levels of DGK activity that are not affected by
R59949(32) , a compound that inhibits DGK activity of human
platelets. Further analysis of the expressed hDGK
activity
demonstrated that it lacks substrate specificity among diacylglycerols
with long chain fatty acyl groups, but has a clear preference for
1,2-diacylglycerols compared with 1,3-diacylglycerols.
The protein
encoded by hDGK is 928 amino acids in length and has a predicted
molecular mass of 103.9 kDa. Following expression in COS-7 cells, we
observed two separate immunoreactive bands of 117 kDa and 114 kDa by
Western blot analysis, which conform reasonably well to the predicted
size. In other experiments, utilizing the anti-hDGK
peptide
antibody, we also detected the expression of two endogenous proteins
from a glioblastoma-derived human cell line (A-172) by Western blot
analysis that were indistinguishable in size from the immunoreactive
proteins expressed by the transfected COS-7 cells (data not shown). The
apparent increase in size on SDS-PAGE may be a result of aberrant
migration on the gel as the MARCKS protein consistently migrates at a
higher molecular weight than expected on SDS-PAGE(29) , and it
is possible that the homologous MARCKS phosphorylation site domain in
hDGK
decreases its mobility. Alternatively, the presence of two
bands on the Western blot may indicate post-translational processing or
reflect partial proteolysis of the larger band. We believe that the
latter is unlikely since the ratio of the two bands has remained
consistent in several experiments.
The N terminus of hDGK
contains two zinc finger-like sequences:
HX
CX
CX
CX
CX
HX
CX
C
and
HX
CX
CX
CX
CX
HX
CX
C.
Similar domains are represented in protein kinase C(34) , other
known DAG kinases (
,
, and
), Raf-1(35) ,
Vav(36) , n-chimaerin(37) ,
-chimaerin(38) , and Unc-13(39) . These regions
have been shown to confer binding properties for diacylglycerol,
phorbol esters, and phosphatidylserine as well as zinc (40, 41) . Thus, we conclude that these sequences in
hDGK
likely comprise the substrate binding regions.
Interestingly, hDGK also contains a sequence carboxyl-terminal
to the cysteine-rich region which is homologous to the phosphorylation
site domain of the MARCKS protein(29) . The phosphorylation
site domain of MARCKS has been demonstrated to bind calmodulin, promote
actin aggregation, and regulate intracellular localization in a
phosphorylation-dependent
manner(42, 43, 44) . hDGK
Ser
, Ser
, and Ser
represent
potential phosphorylation sites for PKC based on contextual identity to
the phosphorylated serines of the MARCKS protein. In related studies,
others have observed that a peptide with this sequence is a substrate
for PKC, but that it does not bind to calmodulin. (
)
The
catalytic domain of DGK displays strong homology to all previously
identified DGK isotypes. Homology between hDGK
and dDGK2 are
highest in this region displaying 72% similarity and 52% identity.
Internal to this domain lies the sequence
GXGXXGX
K, which conforms to an
ATP consensus binding motif(31) . Evidence that this site is
required for DGK
enzymatic activity derives from the analysis of a rdgA mutation in Drosophila which leads to retinal
degeneration. This mutant contains an amino acid substitution
(Gly
Asp) within the ATP-binding
motif(13) , and eye tissue from rdgA homozygotes
cannot synthesize phosphatidic acid, as measured by
P
incorporation(45, 46) .
Four sequential ankyrin
repeats were identified at the carboxyl terminus of hDGK. Ankyrin,
an integral component of the cytoskeleton, contains 22 N-terminal
tandem repeats conforming to a common consensus(47) . Proteins
containing ankyrin repeats are involved in a variety of cell regulatory
processes including: gene regulation (48, 49, 50) , cell cycle
control(51) , and cellular fate
determination(52, 53) . The ankyrin domain has been
shown to facilitate structural and regulatory protein-protein
interactions. The significance of these repeats has been demonstrated
by a Drosophila rdgA mutant, which has a nonsense mutation
carboxyl-terminal to the catalytic domain that results in a predicted
protein that lacks all four ankyrin repeats (13) . Although
which (if any) properties of DGK
are conferred by the ankyrin
repeats remain to be established, it is intriguing that some DGK
activities have been reported to be associated with the
cytoskeleton(11, 54, 55) .
The sequence of
hDGK reveals that it is most closely related to the Drosophila DGK2, derived from the rdgA gene (Fig. 1B). Mutants at this locus exhibit retinal
degeneration(46, 56) , and, although the
phototransduction cascade of mammalians and invertebrates appear to
differ markedly, light-dependent phosphatidylinositol metabolism has
been observed in mammalian retina (57) . This suggested that
the roles of the two enzymes might be similar. Consistent with this
observation, hDGK
has been demonstrated to be highly expressed in
human retina(10) . However, in contrast to the selective
expression of dDGK2 in Drosophila eye, hDGK
is expressed
in a variety of tissues, and there is no significant expression in
human retina. This suggests that dDGK2 and hDGK
have different
functions. The identification and molecular cloning of this new human
isoform of diacylglycerol kinase, hDGK
, will help dissect the role
of such kinases in signal transduction and lipid metabolism.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U51477[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.