(Received for publication, October 10, 1995; and in revised form, February 5, 1996)
From the
Proliferation of T lymphocytes is triggered by the interaction
of interleukin 2 (IL-2) with its high affinity specific receptor that
is expressed on the cell surface following T lymphocyte activation.
Significant advances have recently been made in identifying the
multiple signals that follow IL-2 receptor occupancy, although the
exact mechanism responsible for IL-2-induced proliferation remains an
enigma. It has been shown previously that unique species of
phosphatidic acid are rapidly produced in vivo following IL-2
binding. It was then suggested that, in contrast to other eukaryotic
growth factor systems, phosphatidic acid was at least in part generated
through IL-2-induced diacylglycerol (DG) kinase activation. In the
present study we demonstrate IL-2-dependent activation of the
isoform of DG kinase. Confocal microscopy studies reveal that the
enzyme is located in the cytosol and nuclei of resting T cells.
Interleukin 2 stimulation induces translocation of the enzyme to the
perinuclear region. Furthermore, our results indicate that inhibition
of the
isoform of DG kinase has a profound effect on IL-2-induced
T cell growth. Studies on cell cycle distribution demonstrate that the
inhibition of IL-2-induced phosphatidic acid production induces arrest
in late G1 phase of IL-2 dependent cells. Altogether, these results
link previous observations of interleukin 2 and phosphatidic acid
production to activation of an specific isoform of DG kinase and
suggest that activation of this enzyme is part of a novel signaling
cascade that utilizes phosphatidic acid as an effector molecule.
Activation of T lymphocytes by cell-bound antigens induces the
expression of high affinity interleukin 2 (IL-2) ()receptors
on the cell surface. The binding of IL-2 to its high affinity receptor
triggers a complex signaling program that ultimately results in cell
proliferation. Although the transmembrane pathways activated upon IL-2
binding have been the subject of intensive studies, the exact mechanism
responsible for the IL-2-induced progression through the cell cycle
remains largely undefined. The functional high affinity IL-2 receptor
is a heterotrimeric complex composed of at least three distinct
polypeptide chains designated IL-2R
(p55), IL-2R
(p75), and
IL-2R
(p64) (1, 2, 3) . Although
IL-2R
is capable of low affinity binding to IL-2, high affinity
binding and biological response to IL-2 require association of the
three proteins. All the studies performed to date indicate that the
cytoplasmic domains of the
and
subunits are involved in
transducing the IL-2 proliferative signal(4, 5) . The
cytoplasmic domains of both proteins are devoid of any known intrinsic
catalytic activity; thus, the early responses to IL-2 stimulation must
be therefore transmitted by receptor-associated cytoplasmic enzymes. In
this regard, activation of Src family and Janus family tyrosine kinases
occupy a central role in the initiation of the IL-2-induced
proliferative signal(6, 7) . As has been described for
other receptors, tyrosine kinase activation would in turn induce
association and activation of other signaling molecules such as
phosphatidylinositol 3-kinase, p74 Raf kinase, or
p21
(8, 9, 10) .
In
our search for new molecules implicated in the transduction of the IL-2
proliferative signal, we have recently demonstrated that IL-2 induces
the rapid activation of a DG kinase (DGK) in T lymphocytes following
IL-2 binding to the high affinity receptor(11) . DGK
(ATP:1,2-diacylglycerol 3-phosphotransferase; EC 2.7.1.107) rapidly
converts DG to phosphatidic acid (PA). This activity has been largely
known as a regulator of the intracellular levels of DG, a second
messenger that, in turn, regulates cellular events through activation
of protein kinase C. In T cells, however, previous analysis indicates
that proliferation upon IL-2 binding does not require activation of the
classical and new protein kinase C family(12, 13) . In
fact, previous studies failed to reproducibly detect
phosphatidylinositol 4,5-bisphosphate hydrolysis or increases in
Ca flux as components of the IL-2 proliferative
response. Although IL-2 does not generate DG as a result of
phosphatidylinositol hydrolysis by phospholipase C
, accumulation
of DG and PA through hydrolysis of GPI molecules following IL-2 binding
has been reported(14, 15) . Activation of DGK could
therefore serve as a source of PA as an alternative mechanism to
GPI-phospholipase D activation. Several findings indicate that, rather
than being an intermediate in phosphatidylinositol turnover, the PA
generated upon IL-2 binding may itself modulate signaling pathways
responsible for the IL-2-induced lymphocyte proliferation. For
instance, addition of exogenous PA to CTLL2 cells is able to mimic the
IL-2 effect in proliferation as well as c-Myc
induction(11, 16) . The consecutive activation of a
GPI-PLC and a DGK would therefore constitute a novel pathway for the
generation of the bioactive lipid PA.
Although increases in DGK
activity following IL-2 binding were previously described, the identity
of the enzyme responsible for this activity was not known. Several
mammalian DGKs have been purified from various tissues so far, and some
of them have already been cloned. At the present time the best
characterized is the one having a mass of 80 kDa (DGK) originally
purified from porcine brain cytosol(17) . This isoform, which
is highly tissue-specific, is only present in brain and lymphoid
tissue. This enzyme is extremely abundant in the cytosol of lymphocytes
and comprises more than 0,2% of the total soluble protein in T
cell-enriched pig splenocytes(18) . The aa sequences described
for this DGK isozyme from porcine, human, and rat tissue are more than
80% identical to each other. The primary structure contains two sets of
EF-hand motifs and a cysteine-rich zinc finger-like
sequence(19) . Although lymphocytes contain very high levels of
DGK
isoform, the role of this enzyme on the regulation of the
immune response has never been addressed. In the present study we have
investigated the activation of the
DGK isoform upon IL-2 binding
on T lymphocytes. Immunoprecipitation studies using a specific antibody
against
DGK demonstrate IL-2-dependent activation of this enzyme.
By using inhibitors of this enzyme, we demonstrate that this isoform is
responsible for the majority of PA generated in response to IL-2
through DGK activation. Confocal microscopy studies reveal that the
enzyme, located in the cytosol and nuclei of resting cells,
translocates to the perinuclear space in response to IL-2 binding.
Using
DGK inhibitors we demonstrate the essential role of the PA
generated through
DGK activation on T cell growth. Finally, cell
cycle analysis demonstrates that inhibition of PA production prevents
the cells from entering S-phase. These results further demonstrate that
activation of the
DGK isoform and the subsequent accumulation of
PA play a significant role in IL-2-dependent control of cell cycle
progression in T lymphocytes.
Figure 1:
DGK inhibitor prevents
IL-2-induced DGK activation. The effect of the inhibitor R59022 on
total cellular DGK activity was determined. Resting cells were
stimulated with recombinant IL-2 at the times indicated, and DGK
activity in cell lysates was assayed by measuring the incorporation of
P into PA in the absence (upper panel) or
presence (lower panel) of the
DGK inhibitor R59022. The
formation of PA was further determined by ascending thin layer
chromatography and autoradiography. Autoradiograms were scanned, and a
quantification of four independent assays is represented. IL-2, open symbol; IL-2 + R59022, closed
symbol.
Figure 2:
IL-2-stimulated DGK activity is
immunoprecipitated by anti-DGK antibodies. Quiescent cells were
stimulated with IL-2 for the indicated times, and, after lysis in
detergent-containing buffer,
DGK was immunoprecipitated with an
specific anti-
DGK polyclonal antibody. A, immunoblot
analysis of immunoprecipitates. B, autoradiograms of the thin
layer chromatography separation of the [
P]PA
formed when the immunoprecipitates are subjected to the phosphorylation
assay performed in the absence or presence of the
DGK inhibitor
R59022. Autoradiograms of three independent phosphorylation experiments
were scanned and represented. IL-2, open circles; IL-2 +
R59022, closed circles.
Figure 3:
Effect of IL-2 binding on DGK
distribution. Serum-starved cells were treated without (Control) or with 100 units/ml IL-2 for 10 min in the absence
(IL-2) or presence of R59022 at 10 µM final (IL-2 +
R59022). Cells were fixed as described under ``Experimental
Procedures'' and stained with anti-
DGK polyclonal antibody.
The primary antibody was detected by using and anti-rabbit secondary
antibody coupled to FITC. Confocal images from the middle of the cells
were obtained. Three different fields of each condition are shown. Bar, 10 µm.
Figure 4:
Effect of DGK inhibitors on IL-2-induced
CTLL-2 proliferation. Proliferation of IL-2-dependent CTLL-2 cells in
the presence of increasing concentrations of DGK inhibitors was
determined by [
H]thymidine incorporation into
DNA. Cells were incubated for 20 h at 37 °C in flat-bottomed
96-well plates either in the absence of IL-2 (control) or with
50 units/ml IL-2 (IL-2). The
DGK inhibitors were added at
the concentrations indicated. [
H]Thymidine was
added for the final 4 h, and cells were harvested on glass-fiber
filters. Results of three experiments performed in quadruplicate are
presented. Histograms show [
H]thymidine
incorporation as percentage of maximal proliferation (x axis)
plotted versus cell treatment (y axis).
Figure 5: Cell cycle distribution analysis CTLL2 cells were cultured for 12 h in basal medium without IL-2. The IL-2-deprived cells were exposed to the indicated stimuli, and cell cycle distributions were determined by propidium iodide staining and flow cytofluorometry. Quiescent cells (A) were cultured under exponential growth conditions in the presence of saturating concentrations of IL-2 alone (B) or plus R59022 (C) or R59949 (D) or rapamycin (E). Histograms show relative DNA content (x axis) plotted versus cell number (y axis). Insets indicate the percentage of cells in each phase of the cell cycle.
Figure 6:
S-phase entry after IL-2 stimulation.
Growth factor deprived cells were restimulated with 50 units/ml IL-2
minus or plus DGK inhibitors at time 0. Quadruplicate samples were
subsequently pulsed at 2-h intervals with
[
H]thymidine. Data represent mean
[
H]thymidine incorporation into DNA, plotted at
the end of each 2-h pulse-labeling interval. Coefficients of variation
were less than 10% of mean values. IL-2, closed circles; IL-2
+ R59022, closed triangles; IL-2 + R59949, closed squares.
This study shows what our understanding is the first evidence
of agonist activation of DGK. Following three different
approaches: immunoprecipitation, inhibition studies, and confocal
microscopy analysis, the data presented in this study indicate for the
first time that IL-2 activates
DGK. Indirect evidence, such as the
lack of selectivity for different DG substrates as well as
phosphatidylserine-mediated activation of this DGK, also indicates that
DGK is the isoform activated by IL-2 (data not shown). Moreover,
our studies indicate that IL-2 activation of this isoform of DGK is the
main source of PA production through this mechanism. Finally, another
important observation derived from the present work is the finding
that, in the IL-2 signaling system, PA and not DG is the lipid second
messenger with mitogenic activity; PA generation is a key event for the
correct proliferative function of this cytokine.
The results
presented here demonstrate that the formation of
[P]PA and thus DGK activation takes place over a
similar time course after IL-2 addition in both total cell lysates and
specific immunoprecipitates of
DGK. While DGK activity is
completely abolished when R59022 is added to the immunoprecipitates,
there is some activity still present if the assay is performed on cell
lysates. These data suggest that another DGK isoforms could also be
activated by IL-2. In this regard we have previously reported the
presence of DGK activity in the membrane fraction that is enhanced in
response to IL-2 (11) . This subtype of DGK could be similar to
the arachidonoyl-DGK identified by Glomset and others as a
membrane-bound DGK(23) .
Initial studies demonstrated that
DGK is mostly present in the cytosolic fraction, being the content
in the particulate fraction very scarce(24) . By analogy with
other DG-binding proteins, translocation from the cytosol to the plasma
membrane has been postulated. To determine if IL-2 induces
translocation of
DGK, confocal studies were performed. The
confocal immunofluorescence assays indicate that, in resting cells,
DGK is present not only in the cytosol but also in the nucleus and
is perhaps partly associated with the cytoskeleton. This is not
surprising since DGK activity has been previously described in both the
nuclei (25) and associated to the
cytoskeleton(26, 27, 28) . Our results here
show that, upon IL-2 stimulation of the cells, the enzyme does not
translocate to the plasma membrane and is instead accumulated mostly in
the perinuclear space. In view of these results, it must be postulated
that at least some if not most of the PA being generated in response to
IL-2 binding is produced in inner compartments and not in the plasma
membrane.
Previous studies by Sakane and co-workers (29) suggested that the translocation of DGK to membranes
occurs in a Ca
-dependent manner. Furthermore, the
existence of EF-hand motifs in the
DGK sequence suggested that the
enzyme was functionally linked to Ca
signals(30) . IL-2 fails to induce Ca
mobilization, but we have shown here that
DGK is
successfully translocated in an IL-2-dependent manner. In this regard
other groups have described the Ca
-independent
activation and translocation of this DGK
isoform(31, 32) . Interestingly, Kanoh and co-workers (33) have reported that an
DGK mutant lacking EF-hands
lost Ca
binding activity, but could be fully
activated by phosphatidylserine or deoxycholate in the absence of
Ca
. Recently another DGK isoform containing EF-hands
in its sequence has been cloned, and its activity resulted to be
Ca
-independent(33) . All these experiments
suggest that a Ca
-independent mechanism may be
responsible for the IL-2-induced
DGK translocation, and therefore
some other unidentified roles could be attributed to the EF-hand motif.
As for other possible mechanisms responsible for the enzyme
translocation, Besterman and co-workers (32) have reported that
the presence of DG and phosphatidylserine is enough to induce
translocation of the DGK enzyme from cytosol to membranes. The same
mechanism where substrate concentration is responsible for the
translocation has been also described for protein kinase C-depleted 3T3
cells when cell-permeable dioctanoylglycerol is added(34) .
If IL-2 induces translocation of the enzyme, could the increases on
activity be due to an increase on the level of protein? In the
immunoprecipitation studies, the content of DGK in the
immunoprecipitates is the same in resting and activated cells. This
implies that the increase in the DGK activity is not related to an
increase in the
DGK protein level and suggests that the activation
occurs independently and previous to translocation. In fact, maximum
translocation measured by microscopy confocal analysis is obtained
about 10 min after IL-2 addition, several minutes after the maximum
activation is obtained in the in vitro studies. It could be
hypothesized that a covalent modification occurs. In this context,
Schaap and co-workers (35) have reported that
DGK can be
phosphorylated by Ser/Thr kinases and in Tyr by epidermal growth factor
receptor. However, we have not been able to demonstrate any increase on
tyrosine phosphorylation of the enzyme in response to IL-2 treatment
(data not shown). Experiments are currently under way to study if any
other covalent modification of the
DGK takes place in response to
IL-2.
The main biological function of DGK has largely been
considered to modulate the levels of DG that, in turn, regulate the
activation of classical and new PKCs. Our experiments support a new
role for this enzymatic activity in the generation of PA, a lipid with
a potential central role in IL-2-induced proliferation. When DGK is
inhibited by using specific inhibitors in IL-2-stimulated cells,
IL-2-induced proliferation is impaired. Cell cycle analysis
demonstrates that, upon DGK inhibition, the cells become arrested in
the late G phase. These results indicate that, in contrast
to what has been described for other systems, it is not DG but the
DGK product PA that is the effector molecule in this system. We
must therefore consider the novel hypothesis where accumulation of
certain
DGK-derived PA species following IL-2 stimulation
constitutes an essential step for the T cells to reach the restriction
point and enter S-phase, progressing afterward through the cell cycle.
Several observations have previously suggested that PA may act as a
second messenger. Specific species of PA that contain polyunsaturated
fatty acids have been shown to be increased in ras-transformed
fibroblasts(36) . PA has also been shown to induce invasion of
tumor cells in vitro(37) . When added exogenously to
cells, PA exhibits growth factor-like activity (11, 16, 38, 39, 40) and
can induce actin polymerization(41) . Moreover, intracellular
accumulation of PA, rather than an increase in DG, correlates well with
mitogenesis in growth factor-stimulated fibroblasts(42) ,
similarly to what has been described here. Therefore, it appears that
specific PA species may be associated with proliferation and a high
level of this bioactive lipid could be present in tumor cells. Cellular
targets of PA action have not been identified to date, although
activation of cellular kinases has been suggested (43) .
Limatola and co-workers(44) , using a cell-free assay system,
have demonstrated a strong PA-dependent activation of the
DG-insensitive isotype of PKC. Interestingly, PA-induced
activation of PKC
is inhibited by Ca
. This would
imply that, in intact stimulated cells, PKC
can only be activated
by PA when Ca
remains at basal levels as it happens
upon IL-2 binding. On the other hand, Gomez and co-workers (45) have recently reported, using antisense techniques, that
the
isotype of PKC could be implicated in the control of IL-2
mediated proliferation. Nevertheless, further studies will be necessary
to establish the exact role of
PKC on IL-2 signaling processes and
its activation mechanism by PA. Future work must also contemplate the
identification of other possible targets of the PA action as well as
the characterization of the fatty acid composition of the effector PA
species generated in response to IL-2.
In the last few years, a great number of studies have emerged describing the role of the sphingomyelin cycle in signal transduction (46) . Ceramide, the product of this pathway, has been implicated as a mediator of programmed cell death(47, 48, 49) , cell cycle arrest(50) , and differentiation(51) . All of these mechanisms imply a stop in cell cycle progression. Thus, ceramide could be considered an antimitogenic lipid. Interestingly, ceramide inhibits the activity of a DGK isoform (52) . It also inhibits the activation of phosphatidylcholine-phospholipase D by several agonists(53) . Therefore, the generation of ceramide would inhibit the production of PA either by DGK or phosphatidylcholine-phospholipase D activation. Furthermore, it has been reported that an inverse relationship exists between the cellular concentrations of ceramide and the proliferative capacity of human T-lymphocytes after IL-2 stimulation(47) . Thus, we can envisage the exciting hypothesis that PA (a mitogenic lipid) and ceramide (an antimitogenic lipid) act as biosensors of the cellular state determining, together with other factors, if the cells are going to die, proliferate, or remain quiescent.