From the Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112
Many intracellular signaling
pathways are initiated by a simple reaction, the hydrolysis of
phosphatidylinositol 4,5-bisphosphate (PIP2)1
(1), which results in a transient rise in the amounts of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The initial
signaling has two predictable components: IP3 binds to
intracellular receptors to initiate calcium release from intracellular
stores (2), and DAG functions as an allosteric activator of protein
kinase C (PKC) (3). Both the polar product, IP3, and the
lipid messenger, DAG, are converted to inactive products to return the
cell to the basal state; the pathways that regulate inositol phosphate levels were reviewed in the fourth minireview in this series (22).
In addition to activating PKC, DAG participates in other cellular
events. For example, it is a potent activator of the guanine nucleotide
exchange factors vav (4) and Ras-GRP (5), indicating a
potential role for DAG in regulating Ras and Rho family proteins. In
addition to these signaling roles, DAG occupies a central position in
the synthesis of major phospholipids (phosphatidylcholine and phosphatidylethanolamine (6)) and triacylglycerols. Thus, to maintain
cellular homeostasis, intracellular diacylglycerol levels must be
tightly regulated. This is illustrated by evidence that inappropriate
accumulation of diacylglycerol contributes to cellular transformation.
For example, cell lines that overexpress PLC This review focuses on a family of enzymes, the diacylglycerol kinases
(DGKs) that phosphorylate diacylglycerol to phosphatidic acid (PA)
(Fig. 1), which also has signaling
functions; it stimulates DNA synthesis (13, 14) and modulates the
activity of several enzymes including phosphatidylinositol 5-kinases
(PI-5-K) (reviewed by Rameh and Cantley (84), first article in this
series), PAK1 (15), PKC DGKs have been identified in bacteria (17), Drosophila
melanogaster (18-20), Caenorhabditis
elegans,2 and plants (21), but this review will
focus on the mammalian isoforms. However, special note should be made
of the DGK from Escherichia coli; it is the best studied
member of a family of prokaryotic DGKs that have little structural
relationship to the eukaryotic enzymes. Because there is no evidence
that there is a signaling function for DAG in bacteria, the DGKs
presumably serve exclusively for the synthesis of complex lipids. The
E. coli DGK has also established a technological niche as a
reagent to determine DAG levels (10). Mammalian DGK activities have been identified in multiple cell types and a wide range of tissues, indicating their functional significance (23). Nine isoforms have been
cloned (Fig. 2), and all of them have a
conserved catalytic domain that likely functions similarly to the
protein kinase catalytic domains by presenting ATP as the phosphate
donor. One interesting feature of DGKs
INTRODUCTION
TOP
INTRODUCTION
The Diacylglycerol Kinase Gene...
Regulating Kinase Activity
Function(s) of DGKs
Perspectives and Future...
REFERENCES
have a malignant
phenotype (7). Also, cells transformed with one of several oncogenes
have elevated DAG levels (8-11), and growth factors that are
proto-oncogenes stimulate this pathway. Most of the evidence for this
pathological effect centers on excessive and/or prolonged activation of
PKC, which is a common feature of the transformed state, both in tumors
and in cell cultures (12). PKC function was identified, in part, by
virtue of being the target for phorbol esters; these tumor promoters
function in the same way as DAG to activate PKC but persist because
they are not metabolized (or at least this happens very slowly). Thus, these observations have led to the hypothesis that prolonged elevation of DAG functions as a tumor promoter, the equivalent of an endogenous phorbol ester.
, and Ras-GAP (16). Although the bulk of the
signaling "pool" of PA (it, too, is an intermediate in phospholipid
synthesis) is thought to derive from the action of phospholipase D
(16), DGKs likely contribute to it as well. Thus, DGKs catalyze a
reaction that removes DAG and would terminate the PKC-mediated signal
but yield a product, PA, that has other functions both in signaling and
phospholipid synthesis. The net result on cellular events is,
therefore, difficult to predict, but the potential outcomes all support
the conclusion that DGKs occupy an interesting niche.
View larger version (24K):
[in a new window]
Fig. 1.
Signaling functions of lipids involved in the
diacylglycerol kinase reaction. DGKs, using ATP as the phosphate
donor, catalyze the phosphorylation of diacylglycerol to produce
phosphatidic acid. Several downstream targets and potential functions
of both DAG and PA are indicated below each lipid. The fatty acyl
chains of these lipids may determine their signaling function(s).
The Diacylglycerol Kinase Gene Family
TOP
INTRODUCTION
The Diacylglycerol Kinase Gene...
Regulating Kinase Activity
Function(s) of DGKs
Perspectives and Future...
REFERENCES
and
(and one
Drosophila DGK (19)) is that their catalytic domains are
bipartite, indicating that the two modules may act cooperatively. All
of the DGK catalytic domains have at least one presumed ATP binding
site with the consensus GXGXXG that is also found
in protein kinases (24). A mutation of the third glycine to aspartate
abolishes activity of DGKs
,
,2 and a
Drosophila DGK (20). However, this ATP binding motif differs
from that of the protein kinases, where there is an essential lysine
14-23 amino acids downstream of the glycines (24). All of the known
DGKs have a lysine in a similar position, but site-directed mutagenesis
of this amino acid in DGKs
(25, 26),
, or
2 does
not alter activity, indicating that the ATP binding pockets of
DGKs have a different conformation than the protein kinases.
View larger version (39K):
[in a new window]
Fig. 2.
Structural organization of the mammalian
diacylglycerol kinase family. Mammalian DGKs are divided into five
subtypes by the indicated structural motifs. All diacylglycerol kinases
have in common a catalytic domain with a putative ATP binding site and
two or three C1 motifs. Other structural motifs of unknown significance
have been identified but are not included in this figure.
All DGKs have at least two cysteine-rich regions homologous to the C1A
and C1B motifs of PKCs. DGK has three. These regions are also found
in several other proteins and in most cases are thought to bind
diacylglycerol or phorbol esters. However, C1 domains in several
proteins clearly do not bind DAG and in some cases serve as sites of
protein-protein interaction (27). Originally, these domains in DGKs
were thought to present diacylglycerol for phosphorylation. However, no
one has conclusively demonstrated that this occurs. In fact, Sakane
et al. (26) observed that DGK
was still active without
its C1 domains and that DGKs
,
, and
all failed to bind
phorbol esters. We have observed, however, that DGK
lacking either
of its two C1 domains was inactive.2 Thus, it is unclear
precisely what role these regions perform in DGKs, but in at least some
isoforms they are essential for activity. Hurley et al. (28)
recently examined the sequence homology of 54 C1 domains, including
those of six DGKs (
,
,
,
,
, and
). They proposed
that except for the C1A domains of DGKs
and
, all other DGK C1
domains may not bind DAG. The different functions of the proteins must
be considered, however; when PKCs bind DAG, they essentially exclude it
from an attacking phosphoryl group, which is sensible as the DAG is
functioning as an allosteric activator. In contrast, DGKs must present
DAG for phosphoryl transfer, which suggests that they might bind it differently; thus, an altered C1 conformation might serve such a purpose.
In addition to their catalytic and C1 domains, most DGKs have
structural motifs that form the basis for dividing them into five
subtypes and that likely play regulatory roles (Fig. 2). Type I DGKs
(29-31) have calcium binding EF hand motifs at their N termini, making
these isoforms calcium-responsive to slightly different extents (32).
Diacylglycerol kinases with pleckstrin homology (PH) domains at their N
termini are defined as type II (33, 34). Takeuchi et al.
(35) found that the PH domain of DGK could bind
phosphatidylinositols (PI). DGK
also has at its C terminus a region
homologous to the EPH family of receptor tyrosine kinases. The function
of this domain is unclear. DGK
(36) has the simplest structure in
that it does not have any identifiable regulatory domains and is the
sole member of group III to date. Paradoxically, although it is the
simplest in structure, it is the only DGK known to have substrate
specificity; it strongly prefers diacylglycerol substrates with an
arachidonoyl group at the sn-2 position. This preference
initially suggested that DGK
might be the component in the
phosphatidylinositol cycle that accounted for the enrichment of PI
with arachidonate (37); however, this isoform has a limited tissue
distribution and therefore cannot serve this function broadly. The
defining characteristic of type IV DGKs (38, 39) is that they have
ankyrin repeats at their C termini. One enzyme of this family, DGK
,
undergoes tissue-specific alternative splicing that results in an
enzyme with an elongated N terminus; it is found predominantly in
muscle (40). Both DGK
and
have a region homologous to the
phosphorylation site domain of the MARCKS protein. Finally, DGK
defines group V (41); it has three cysteine-rich domains and a PH
domain, as well as a region that is structurally similar to those in
other proteins that have been implicated as mediating association with
Ras, although this point is controversial (42). Thus, the complexity
and diversity of the DGK family strongly suggest that the DGKs perform
multiple roles in cellular functions.
![]() |
Regulating Kinase Activity |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Because DGKs influence cellular DAG and PA levels, control of their activity is essential; and this may be an important mechanism by which different functions are segregated, e.g. DAG that is used for complex lipid synthesis contrasted with DAG used as a signal. Activation of Type I DGK activity by calcium represents a potentially elegant way to increase DGK activity in situations in which DAG is elevated, because calcium usually increases in parallel because of the simultaneous signaling by IP3. This mechanism would attenuate the DAG signal, returning that arm of the signaling cascade to a quiescent state. Other than this effect, however, relatively little is known about how the activity is regulated. Several groups have observed compounds that can modulate DGK activity in cellular homogenates. These include arachidonic acid (43), vitamin E (44), sphingosine (45), 15-hydroxyeicosatetraenoic acid (46), ceramide (47), and several fatty acids (48, 49). In most cases it is not clear that these observations are physiologically relevant, but Reddy and co-workers (50) observed that dietary fatty acids induced marked alterations in DGK activity in colon tumors. Also, Walsh et al. (51) found that PIP2 was a potent inhibitor of arachidonoyl-specific DGK activity.
Several investigators have examined post-translational modifications
that may regulate DGK function. Schaap and co-workers (52) and Kanoh
et al. (53) have shown that DGK is phosphorylated by PKC
isoforms in vitro and in vivo. These findings
were consistent with previous observations that suggested that PKC
regulated the activity of DGK in cellular homogenates (54); however,
neither group could identify a functional consequence resulting from
the PKC-mediated phosphorylation.
A consistent observation from several groups is that DGKs shuttle
between the cytosol and membrane fractions of cells and that both DGK
activity and immunoreactivity become membrane-associated upon exposure
to chemotactic factors (55), adrenergic agonists (56), phorbol esters
(57), and diacylglycerol (58). These findings raise the distinct
possibility that DGKs are regulated by access to specific DAG pools at
the membrane, i.e. by moving the enzyme to its substrate. In
support of this possibility, van der Bend and co-workers (59) presented
experiments indicating that cellular DGK activity was topologically
restricted to DAG pools generated after receptor activation rather than
upon nonspecific generation of diacylglycerol by PLC treatment of
membranes. In agreement with this, Flores et al. (60)
recently observed that DGK translocates to the nuclear cytoskeleton
upon stimulation of cells via the IL-2 receptor. They went on to show
that the PA generated by a DGK (presumably
) following IL-2
treatment may be necessary for subsequent cellular proliferation. We
found that DGK
also translocates to the nucleus and that this
localization is negatively regulated by PKC-mediated phosphorylation of
the MARCKS phosphorylation site domain, which acts as the nuclear localization signal (61). These observations provide strong evidence
that cells regulate DGK activity (and thus DAG levels) by altering the
specific activity of DGKs and by translocating them to regions where
the DAG has accumulated. Because there is evidence of multiple DGK
isoforms within the same cell type, the different isozymes likely
perform specialized roles, perhaps by recognizing DAG pools generated
in response to different stimuli or that are found in specific membrane compartments.
![]() |
Function(s) of DGKs |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Lessons from Drosophila-- The diversity of the DGK family suggests that each isoform may perform a distinct function. However, this has been difficult to study because of a lack of isoform-specific inhibitors, and thus far, there has not been a description of a dominant negative DGK construct. If one turns to Drosophila, however, there has been demonstrated a clear role for activity of one isoform. Drosophila strongly relies on PLC activity to generate IP3 and DAG for its visual signal transduction (62). Several groups demonstrated that a Drosophila visual mutant, rdgA, was deficient in retinal DGK activity (63), and then Masai et al. (20) cloned the gene responsible for this defect. It was a DGK (dDGK2) with four C-terminal ankyrin repeats and is most similar to the mammalian type IV enzymes. They found a mutation in the ATP binding site that would presumably account for the loss of DGK activity. This group later showed that the rdgA phenotype could be rescued by introducing wild-type rdgA into the mutant strain (64). Together, these studies demonstrate that formation of phosphatidic acid by dDGK2 is necessary for proper visual signaling in Drosophila.
Although there are differences in photoreceptor signaling between
Drosophila and mammals (65) these observations may provide insight into a function for some mammalian DGK isoforms. In fact, several groups have found evidence of light-dependent
activation of PIP2 hydrolysis and generation of PA in
vertebrate retina (66-68), and because PKC may participate in
vertebrate phototransduction (69) a role for DGK activity in mammalian
retina would not be surprising. To date, three mammalian isoforms have
been definitively localized to the retina. The first was DGK, a type
I enzyme. Kai and co-workers (31) found that this isoform is
predominantly localized in human retina whereas most other tissues had
inactive, truncated mutants. To date, they have not identified a
specific function for this protein in visual signaling. Rat DGK
has
also been found in both the outer and inner nuclear layers of the
retina (70), and we have observed that human and murine DGK
are
strongly expressed in the retina.2 Again, no specific
function in vision has been attributed to this isoform. Finally,
DGK
, a type IV enzyme, was cloned from a cDNA library prepared
from retina (39). It is the isoform most similar to dDGK2, and current
studies are under way to determine a role for this isozyme in retinal
signaling. In conclusion, no one has identified a role for DGK activity
in mammalian visual signaling, but several isoforms are strongly and
predominantly expressed in the retina, and their functions there have
not yet been elucidated.
Lessons from Tissue Distribution--
Mammalian DGK isoforms have
been observed in a variety of tissues by both Northern and Western blot
analysis. With the exception of DGKs and
, all isoforms
identified to date are found in the brain at a level equivalent to or
higher than any other tissue. In situ hybridization and
Northern blot analysis of specific brain regions have revealed high
levels of expression of several isoforms in the hippocampus (
,
,
,
,
), cerebellum (
,
,
,
), olfactory bulb (
,
,
,
), and the retina as noted above. It is interesting that
specific regions of the brain express several DGK isotypes, typically
representing many known subfamilies. This is strong evidence that the
enzymes within these subfamilies perform distinct functions. In fact,
we have observed four DGK isoforms in a glioblastoma cell line: DGKs
,
,
, and
.2 Notably, each isoform is from a
different DGK subfamily. Finally, Ding et al. (71) found
dramatic evidence of specific association of a DGK with neuronal
structures in the developing mouse. Together, these data strongly
suggest that DGKs are an integral part of the central nervous system
and peripheral neuronal function.
Two other tissues where DGKs appear to be enriched are white blood
cells and muscle. Both appear to express multiple DGK isoforms, usually
representing different families. Human DGK was purified and then
cloned using white cells, spleen, and thymus tissue (29, 72). DGKs
and
are expressed in HL60 cells,2 DGK
was identified
by Northern blotting in leukocytes (33), and mRNA for DGKs
and
have been observed in the spleen (34) and thymus (73),
respectively. Localization in these tissues is of interest because
several groups using DGK inhibitors have suggested that DGK activity
suppresses the respiratory burst (74) and chemotaxis (75) in
leukocytes. Both effects have been attributed to metabolism of DAG,
which would presumably suppress PKC activity. Finally, several DGK
isoforms have been identified in both striated muscle and cardiac
muscle. Using Northern blotting, Sakane et al. (33) found
DGK
mRNA predominantly expressed in skeletal muscle. DGKs
(30) and
2 are found in mRNA prepared from heart
tissue, and we have identified an alternatively spliced isoform of
DGK
in striated muscle (40). Again, no one has clearly identified a
role for DGK activity in muscle tissue, except that a DGK inhibitor can
suppress contractility in some smooth muscle cell types (76).
Lessons from Subcellular Distribution, the Nuclear
Story--
Informative experiments to determine functions of DGK
isotypes have been aimed at dissecting the subcellular distribution of
the isozymes. This approach has been very successful with DGK. We
observed that this isozyme is found in the nucleus (61). An emerging
body of evidence suggests that nuclear lipid signaling partly regulates
the cell cycle, and several groups have shown that there is a nuclear
phosphatidylinositol cycle that is regulated separately from its plasma
membrane/cytosolic counterpart (77). DAG has been found in nuclear
preparations, and the level fluctuates during the cell cycle (78),
suggesting that it has a regulatory role there. Because DAG is the
major physiologic activator of several PKCs, which translocate to the
nucleus following certain extracellular signals, one way to regulate
nuclear PKC activity would be to control the extent and duration of the
nuclear diacylglycerol accumulation. Nuclear DGKs are poised to
accomplish this. To clarify the nuclear role of DGK
, we identified
its nuclear localization signal. Interestingly, the MARCKS homology
domain was the predominant nuclear localization signal. We had
previously observed that this domain was likely phosphorylated by
protein kinase C isoforms, so we asked whether this modification
regulated the nuclear localization of DGK
. Indeed, phosphorylation
of the MARCKS domain by specific isoforms of PKC reduced nuclear
accumulation of DGK
. We also found that the consequence of
overexpressing DGK
was to increase cellular doubling time,
presumably because cells accumulate in the
G0/G1 phase of the cell cycle. Taken together,
our observations suggest that nuclear DAG is necessary for progression
of cells to S phase (possibly by activating nuclear PKCs) and that
nuclear DGK
regulates this lipid and, consequently, performs a role
in cell cycle control. Thus, PKC isoforms, in an elegant and surprising way, control their activation state within the nucleus by
phosphorylating the MARCKS domain of DGK
, which alters nuclear DAG
accumulation. We also found that the
other type IV DGK,
, was similarly regulated (71).
Flores and colleagues (60) have studied the nuclear role of a different
diacylglycerol kinase, . They noted in T lymphocytes (where DGK
is the major isozyme) that this enzyme translocated to the perinuclear
space when the cells were treated with IL-2. Using nonselective
inhibitors of DGKs, they presented evidence that the phosphatidic acid
produced by this isozyme was necessary for progression to S phase of
the cell cycle. Although these data seem to contradict our findings
with DGK
, the Flores study did not specifically test whether DGK
altered nuclear lipids; rather, their assays for PA generation may have
also reflected changes occurring at the plasma membrane. Thus, the
consequence of the nuclear translocation of DGK
is still unclear. In
conclusion, although several issues need to be resolved, it appears
that some DGK isoforms play a prominent role in nuclear signaling (in
the case of DGK
, to regulate DAG).
DGKs and the Cytoskeleton--
In addition to attenuating cellular
DAG, DGKs may also function by increasing the amount of phosphatidic
acid. Although phospholipase D is thought to be the major source of PA,
these two enzymes generate PA with distinct fatty acid compositions
that may have differential effects on target enzymes (79). This
suggests that DGKs could generate a distinct phosphatidic acid pool
under specific conditions. In accordance with this, Tolias and
co-workers (80) identified DGK activity in the immunoprecipitates of
Rac, Rho, and Cdc42. They also noted PI-5-K activity and immunoreactive
Rho-GDI in the immunoprecipitates. The DGK activity in the
immunoprecipitates is novel and deserves further attention. If true,
this observation indicates a marriage of a group of molecules involved
in actin cytoskeletal rearrangement with an enzyme that can control
local availability of lipid mediators that can regulate their
activities, an elegant arrangement. For
example, PA stimulates PI-5-K activity 8-12-fold in vitro
(81), n-chimaerin (a Rac-GAP) is potentially regulated by
DAG levels because of its C1 domain (82), and Rho-GDI dissociates from
Rac in the presence of PIP2 or PA (83). These data strongly
indicate a potential role for DGKs in regulating actin cytoskeleton rearrangements.
![]() |
Perspectives and Future Directions |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The structural diversity and complex pattern of tissue expression
of DGKs is reminiscent of the PKC and PLC families; is it a coincidence
that these three families involved in lipid signaling exhibit this
diversity, or is there an underlying connection that has yet to be
identified? It is likely that mammalian DGKs will be shown to be
crucial in several cellular processes. In some cases their actions may
be the result of lowering the level of DAG and thereby serving as an
off signal for the allosteric activation of PKC, whereas at other times
the DGKs may serve as a source of phosphatidic acid that then functions
as a signal. It would not be surprising if DGKs, in some contexts,
served both of these functions simultaneously. That is, as an off
signal by metabolizing DAG and then as an on signal via the resulting
phosphatidic acid.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. G. A. Zimmerman, T. M. McIntyre, and M. Bunting for many helpful discussions, and T. Crotty and Dr. F. Sakane for critical reading of this manuscript.
![]() |
FOOTNOTES |
---|
* This minireview will be reprinted in the 1999 Minireview Compendium, which will be available in December, 1999. This is the fifth article of five in "A Thematic Series on Kinases and Phosphatases That Regulate Lipid Signaling."
Howard Hughes Medical Institute physician postdoctoral fellow.
§ To whom correspondence should be addressed: Huntsman Cancer Inst., University of Utah, 15 N. 2030 East, Rm. 4220, Salt Lake City, UT 84112. Tel.: 801-585-3773; Fax: 801-585-6345; E-mail: stephen.prescott{at}hci.utah.edu.
2 M. K. Topham, W. Tang, and S. M. Prescott, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PIP2, phosphatidylinositol 4,5-bisphosphate; DAG, diacylglycerol; DGK, diacylglycerol kinase; PA, phosphatidic acid; PKC, protein kinase C; PLC, phospholipase C; IP3, inositol 1,4,5-triphosphate; PI, phosphatidylinositol; IL-2, interleukin-2; PH, pleckstrin homology; PLD, phospholipase D; PI-5-K, phosphatidylinositol 5-kinase; MARCKS, myristoylated alanine-rich C kinase substrate; GDI, guanine nucleotide dissociation inhibitor.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|