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Department of Immunology and Oncology, Centro Nacional de Biotecnología, Campus de Cantoblanco, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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isoform of diacylglycerol
kinase. In this study, using the IL-2-dependent mouse T cell line
CTLL-2, we demonstrate that pharmacological inhibition of IL-2-induced
diacylglycerol kinase activation is found to block IL-2-induced late
G1 to S transition without affecting cell viability.
Herein, we demonstrate that diacylglycerol kinase inhibition has a
profound effect on the induction of the protooncogenes c-myc, c-fos,
and c-raf by IL-2, whereas expression of bcl-2 and bcl-xL
are not affected. When the IL-2-regulated cell cycle control
checkpoints are examined in detail, we demonstrate that inhibition of
diacylglycerol kinase activation prevents IL-2 induction of cyclin D3
without affecting p27 down-regulation. The strict control of cell
proliferation exerted by phosphatidic acid through activation of
diacylglycerol kinase is independent of other well-characterized
IL-2R-derived signals, such as the phosphatidylinositol-3 kinase/Akt
pathway, indicating the existence of a different and important
mechanism to control cell division. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(55
kDa), ß (75 kDa), and the common 
-chain (c; 64 kDa), which is
shared by several hematopoietic receptors (3). Signaling
by the IL-2R is mediated by ligand-induced heterodimerization of the
cytoplasmic domains of the IL-2Rß and c. The apparent function of
the -chain is to affect the affinity of the receptor, but not the
mechanism of IL-2 signaling. Heterodimerization of the IL-2R ß and
subunits allows activation of the src-family tyrosine kinases
(4) and the Janus kinases 1 and 3 (5) that,
in turn, create docking sites for the association/activation of
other signaling molecules, such as STAT3 and -5, Shc, and
phosphoinositide 3-kinase
(PI3K)3
(6). Several studies have addressed the signaling
mechanisms that regulate cell cycle entry following IL-2 binding, but,
to this date, the molecular events underlying IL-2-induced signal
transduction, particularly those involved in the IL-2-induced
proliferative response are not fully understood. For instance, it has
been shown in Ba/F3 cells transfected with the IL-2R that mitogenesis
can be fully supported by IL–2 receptors that lack the ability to
activate the Ras pathway (7), suggesting the existence of
other mechanisms by which IL-2R, and perhaps other cytokine receptors,
control cell proliferation.
In the search for novel mechanisms transducing IL-2 signals, we have
previously shown that IL-2 binding induces phosphatidic acid (PA)
formation by the rapid activation of the isoform of diacylglycerol
kinase (DGK) (8). Addition of IL-2 causes the rapid
translocation of this enzyme from its cytosolic location to the
perinuclear region of the cell. IL-2-induced activation of DGK can
be prevented by preincubation of the cells with the DGK inhibitor
R59949. Treatment of the cells with this inhibitor prevents
IL-2-induced proliferation, an effect similar to that seen with other
immunosuppressors, such as rapamycin or wortmannin. In the present
paper, we have extended our previous work by performing a detailed
study of the molecular events caused by DGK inhibition. The analysis of
IL-2-induced protooncogene expression demonstrates that inhibition of
IL-2-induced DGK activation prevents the induction of c-fos, c-myc,
and c-raf by this cytokine. In addition, inhibition of IL-2-induced DGK
activation prevents cyclin D3 expression and, consequently,
retinoblastoma tumor suppressor protein (Rb) hyperphosphorylation.
Inhibition of IL-2-induced DGK activation does not affect other
IL-2-induced signaling pathways known to be related to cell cycle
control, such as PI3K-dependent Akt activation. Our results confirm the
existence of a signal transduction pathway that relies on the
generation of PA and establishes a link between IL-2R-mediated PA
production and the cell cycle machinery. The relevance of this pathway
in IL-2-dependent responses and its relation with other IL-2-initiated
signaling pathways is further discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human IL-2 was generously provided by
Hoffman-LaRoche (Nutley, NJ). Recombinant murine IL-4 was from
Peprotech (Rocky Hill, NJ). DGK inhibitor II (R59949) was from
Calbiochem (San Diego, CA). Nonidet P-40, protease inhibitors
(leupeptin, aprotinin, and PMSF), and
sn-1,2-dioleoylglycerol were obtained from Sigma (Poole,
U.K.). Phosphatidylinositol and dioleoyl PA were from Avanti Polar
Lipids (Alabaster, AL). Histone H2B was from Boehringer Mannheim
(Mannheim, Germany). Anti-c-myc, anti-c-fos, anti-cyclin D3,
and anti-cRaf 1 Abs were from Santa Cruz Biotechnology (Santa Cruz,
CA). Anti-bcl-2, anti-bcl-xL, and anti-Rb
Abs were from PharMingen (San Diego, CA). Anti-Akt/protein kinase B Ab
was from Upstate Biotechnology (Lake Placid, NY). Anti-rabbit and
anti-mouse Ig HRP-linked whole Abs and the enhanced
chemiluminescence Western blotting detection system were from Amersham
(Aylesbury, U.K.). Silica Gel G-60 TLC plates were obtained from
Whatman (Clifton, NJ). [-32P]ATP were from
Amersham.
Cells
CTLL-2 cells were maintained in basal medium (RPMI 1640, 2 mM L-glutamine, 50 µM 2-ME, buffered to pH 7.2 with 10 mM HEPES) supplemented with 10% (v/v) FCS and 20 U/ml recombinant human IL-2. To obtain maximal synchronization, cells were washed extensively and incubated for 8 h in basal medium. After this period of incubation, the majority of the cells were found in the G1 phase, and no apoptosis was observed.
Cell cycle and PI/annexin V analysis
CTLL-2 cells growing exponentially in IL-2-supplemented medium were washed free of IL-2 and FCS before resuspending in basal medium. After 8 h, the DGK inhibitor (R59949, 1 µM final) or vehicle (DMSO, 0.01% v/v final) was added 15 min before IL-2 (50 U/ml final) or IL-4 (10 ng/ml). Cells were harvested by centrifugation and washed in PBS. For cell cycle analysis, cells were resuspended in permeabilization solution (0.1% sodium citrate, 0.05% Nonidet P-40) before treatment with 50 µg/ml RNase A for 30 min at room temperature. Propidium iodide was then added yielding a final concentration of 20 mg/ml. After 20 min, the fluorescence of the propidium iodide-stained DNA was quantitated on a per cell basis with an EPICS-XL flow cytofluorometer (Coulter, Miami, FL). For PI/annexin V analysis, cells were incubated with FITC-labeled annexin V, a protein that binds to phosphatidylserine and propidium iodide (Annexin V FITC Kit; Immunotech, Marseille, France). Following 10 min of incubation, positive cells were analyzed by flow cytometry.
Preparation of cell lysates and immunoblot analysis
Exponentially growing CTLL-2 cells were washed twice and resuspended at a concentration of 2 x 105 cell/ml in IL-2- and serum-free medium. After 8 h of starvation, cells were restimulated with recombinant IL-2 (50 U/ml). When indicated, the DGK inhibitor R59949 (1 µM) was added 15 min before IL-2 addition. At the appropriate times, cells were collected by centrifugation at 4°C, washed twice with ice-cold PBS, followed by lysis of the cell pellet in radioimmunoprecipitation (RIPA) buffer. Protein concentrations were determined using the Bio-Rad (Richmond, CA) protein assay. Equal amounts of protein were resolved using SDS-PAGE, before transfer to nitrocellulose membranes (Bio-Rad) in 25 mM Tris (pH 8.3), 190 mM glycine, and 20% v/v methanol for 1 h at 200 mA. The membranes were then blocked by incubation with TBS (150 mM NaCl, 20 mM Tris-HCl (pH 7.4)) containing 0.5% w/v BSA, 0.05% v/v Tween, and 5% w/v non-fat dried milk. Proteins were detected with specific Abs and HRP-conjugated-anti-mouse or anti-rabbit Abs and visualized by enhanced chemiluminescence, according to the manufacturer’s recommendations.
Measurement of PI3K activity
CTLL-2 cells were washed free of serum and IL-2 before being placed in phosphate-free RPMI medium for 6 h. During the last 2 h, carrier-free orthophosphate (Amersham) was added at a radiochemical concentration of 400 µCi/ml. To determine the effect of the inhibitors, cells were preincubated with either DMSO (0.01% v/v (control)), R59949 (1 µM final concentration), or wortmannin (50 nM final concentration) for 15 min before IL-2 addition. Cells were stimulated with IL-2 for 10 min, after which the cells were pelleted, washed twice with ice-cold PBS, and immediately frozen on dry ice. Cellular lipids were extracted using CHCl3/MeOH/HCl followed by their deacylation as described previously (9). 3-phosphorylated phosphoinositides were separated by strong anion exchange HPLC as described (9).
Measurement of Akt activity
Anti-Akt immunoprecipitates were resuspended in 25 µl of
kinase buffer (50 mM Tris (pH 7.5), 10 mM MgCl2,
1 mM DTT) containing 2.5 µg of histone 2B (H2B). Reactions were
initiated by adding [-32P]ATP 50 µM (3
µCi) and incubated for 20 min at room temperature. H2B
phosphorylation was assessed by autoradiography of the SDS-PAGE. The
amount of Akt protein in each sample was determined by Western blot
analysis. For transfection experiments, CTLL-2 cells were
electroporated with 30 µg of the corresponding cDNAs.
Measurement of DGK activity
DGK activity in total cell lysates was measured as previously described (10). Briefly, 15 x 106 quiescent cells were stimulated with either buffer, 500 U/ml of recombinant IL-2, or 100 ng/ml of recombinant IL-4. When indicated, the inhibitor was added 15 min before IL-2. After the periods of time indicated in the figure legends, the cells were harvested, washed twice with ice-cold PBS, and frozen on dry ice. The cells were thawed and lysed by nitrogen cavitation (10 min at 500 p.s.i., 4°C) in a buffer containing 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 50 mM NaF, 2 mM Na3VO5, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The lysates were then centrifuged at 800 x g, and the supernatant was used to determinate DGK activity. At the end of the phosphorylation assay, lipids were extracted by the subsequent addition of 200 µl of CHCl3/MeOH (2:1 v/v), 50 µl of CHCl3, and 50 µl of 0.1 M HCl. After centrifugation at 500 x g, the organic layer was recovered, dried under a stream of nitrogen, dissolved in 20 µl of CHCl3/MeOH (2:1 v/v), and applied to silica gel G-60 plates along with dioleoyl-PA as a standard. Plates were developed with a solvent system consisting of CHCl3/MeOH/4 M NH4OH (9:7:2, v/v/v). Dried plates were subjected to autoradiography, and the bands corresponding to PA were quantified by scanning of the autoradiograms.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown, in the human T cell line YT, that the
DGK inhibitor R59949 prevents IL-2-induced activation of the DGK
isoform (8). Unfortunately, there are no specific Abs that
recognize the isoform of murine origin, and we cannot demonstrate
direct inhibition of this specific isoform following treatment with the
DGK inhibitor in CTLL-2 cells. However, since most of the DGK activity
stimulated by IL-2 in human T lymphocytes is that of the isoform,
we decided to evaluate the effect of this inhibitor on IL-2-stimulated
DGK activation in CTLL-2 cells. As can be seen in Fig. 1
, when R59949 was added before IL-2, the
DGK activation induced by the cytokine was impaired indicating that, as
previously shown for human T cells, inhibitor treatment prevented
IL-2-induced DGK activation.
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CTLL-2 cells can be arrested in G1 by
incubation for 8 h in medium free of both IL-2 and serum.
Following this period, the effect of IL-2 as both a survival and
proliferative factor can be assessed without any interference from
serum constituents. As can be seen in Fig. 2A, IL-2, in the absence of
serum, induced cell cycle entry of CTLL-2 cells in a synchronic fashion
(59% of the cells were found in S + G2/M 48
h after IL-2 addition). Due to the absence of serum in the culture
medium, there was a significant percentage of cells undergoing
apoptosis following IL-2 addition (15% at 36 h). However, when
IL-2 was not present in the culture medium, the percentage of apoptotic
cells was much higher and, by 48 h, 55% of the cells were found
in a sub-G1 population. When additional
experiments were performed under these established conditions, addition
of the DGK inhibitor R59949, 15 min before IL-2, prevented cell cycle
entry (26% of cells in S + G2/M at 48 h)
without a significant increase in cell death by apoptosis. To further
assess that DGK inhibition differentially affected proliferation rather
than cellular survival, the level of apoptotic cells in the various
conditions was examined by FACS analysis of annexin V-positive cells.
As shown in Fig. 2B, addition of IL-2 significantly reduced
the percentage of annexin V/PI-positive cells compared with that of
cells cultured in the absence of the cytokine. Again, addition of the
DGK inhibitor did not significantly increase the percentage of annexin
V-positive cells. These results demonstrate that, in CTLL-2
lymphocytes, inhibition of DGK activity prevents IL-2-induced cell
cycle entry without affecting the capacity of this cytokine as a
survival factor.
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The binding of IL-2 to its high-affinity receptor induces
expression of the protooncogenes c-myc, c-fos,
bcl-xL, and bcl-2 (11). While
expression of bcl-2 and bcl-xL have been
implicated in the prevention of apoptosis, IL-2-induced expression of
c-myc has been shown to be essential to induce proliferation
(12). To determine the effect of DGK inhibition on
protooncogene induction by IL-2, the expression of these proteins was
analyzed by Western blotting. Fig. 3
indicates that inhibition of IL-2-induced DGK activation by R59949 had
a dramatic effect on the induction of c-fos and c-myc by IL-2.
Furthermore, inhibition of IL-2-induced DGK also reduced induction of
c-raf-1 by this cytokine.
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Effect of DGK inhibition on Rb hyperphosphorylation and the cellular levels of p27 and cyclin D3
The retinoblastoma tumor suppressor protein, Rb, plays a central
role in the control of the G1 to S transition.
The association of Rb with transcription factors, such as E2F, is
thought to be responsible for Rb’s ability to block the passage of
cells through the G1 checkpoint. The
hyperphosphorylation of Rb renders this protein incapable of
associating with these factors, causing their release and, as a
consequence, allows progression of cells into S phase
(14). We have previously demonstrated, using CTLL-2 cells,
that IL-2 deprivation induces the rapid dephosphorylation of Rb
(15). To investigate the effect of DGK inhibition on Rb
hyperphosphorylation, CTLL-2 cells were pretreated with R59949 before
IL-2 addition. Rb phosphorylation was then assessed by Western
blotting. As shown in Fig. 4A,
inhibition of IL-2-induced DGK activation prevented Rb phosphorylation,
which correlated with the inhibition of S phase entry.
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B, treatment with the DGK inhibitor did
not affect the down-regulation of p27 levels induced by IL-2 treatment
of the cells. However, when the same membrane was analyzed for cyclin
D3 expression, following DGK inhibition, expression of cyclin D3
induced by IL-2 was dramatically reduced (Fig. 4C). Inhibition of DGK activation has no effect on IL-2-induced PI3K activation
One of the earliest signaling events following IL-2 binding to its
high-affinity receptor is the activation of PI3K (9, 21, 22). It has been recently shown that the activation of this
enzyme has a very important effect on some of the events regulating S
phase entry (23). At least one isoform of DGK (DGK 
) is
known to control the level of araquidonoyl-stearoyl-PA, an intermediate
in the PI cycle. Therefore, we reasoned that inhibition of DGK activity
could be related with the inhibition of the generation of
3-phosphorylated lipids. We measured the generation of such lipids in
vivo following the addition of IL-2 with or without DGK inhibitor. As
shown in Fig. 5, treatment of the cells
with the DGK inhibitor did not affect the in vivo generation of either
PI3,4P2 or PI3,4,5P3, the
two principal products of PI3K activity. As a positive control for PI3K
inhibition, we used wortmannin, a well-recognized PI3K inhibitor, to
demonstrate inhibition of stimulated PI3,4P2 and
PI3,4,5P3 generation.
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To further confirm that cell cycle arrest following DGK inhibition
was not related to PI3K-derived signaling pathways, we measured the
effect of DGK inhibition on IL-2-stimulated activation of Akt, the
kinase that is directly activated by 3-phosphorylated phosphoinositides
(24). As can be seen in Fig. 6, Akt was activated following IL-2
treatment of CTLL-2 cells. This activation was prevented following
treatment of the cells with wortmannin or by cell transfection with
p85, the regulatory subunit of PI3K, which acts as a dominant negative
form of this enzyme. However, treatment of CTLL-2 cells with R59949 had
no effect on IL-2-stimulated Akt activation, indicating that the
formation of PA in response to IL-2 was not related to the generation
of 3-phosphorylated lipids.
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CTLL-2 cells are IL-2-dependent, but they can also grow in
response to IL-4. We decided to test the effect of the DGK inhibitor on
IL-4-induced CTLL-2 cell proliferation. As shown in Fig. 7, treatment of CTLL-2 cells with IL-4
increased DGK activity, and this activation was prevented by addition
of R59949. Cell cycle analysis of IL-4-stimulated CTLL-2 cells
demonstrated that, as is the case for IL-2, inhibition of DGK
activation prevented IL-4-induced proliferation. Following 36 h of
IL-4 addition, 54% of the cells were found in the S +
G2/M phases of the cell cycle. Addition of the
DGK inhibitor significantly reduced the percentage of cycling cells
(36% in S + G2/M). As previously shown for IL-2,
DGK inhibition did not significantly increase the percentage of
apoptotic cells determined either by propidium iodide analysis (14% vs
20%) or by FACS analysis of annexin V-positive cells (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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isoform of DGK, since this isoform is primarily expressed in
oligodendrocytes and lymphocytes (27). By using specific
Abs against the isoform of human origin, we have previously
demonstrated that IL-2 binding to its high-affinity receptor induces
DGK activation, and that this activation could be prevented by using
the potent DGK inhibitor R59949 (8). However, our previous
observations were performed in YT cells, which do not require IL-2 for
growth. We have now examined the effect of this same inhibitor in the
IL-2-dependent murine T cell line CTLL-2 to further investigate the
molecular mechanisms underlying the inhibition of cell proliferation
caused by prevention of DGK activation. The reason that we chose this
cell line as our experimental model was that, in CTLL-2 cells, the
effect of IL-2 as both an inducer of survival and as a mitogen could be
demonstrated in the absence of any added serum. This is very important
to properly ascertain that the signal is due only to the effect of the
cytokine and not to additional growth factors present in the serum. As
we have demonstrated, addition of IL-2 in the absence of the survival
factors contained in the serum promoted cell cycle entry, although, as
expected, a significantly higher proportion of cells underwent
apoptosis. However, the population of apoptotic cells, measured as
annexin V-positive cells, did not significantly increase in the
presence of the DGK inhibitor. Together, these experiments demonstrate
that PA production following IL-2 addition is essential for
cytokine-induced cell proliferation. Our results, presented here, demonstrate that DGK inhibition prevents IL-2 induction of c-fos, c-myc, and c-raf-1, three of the protooncogenes up-regulated by this cytokine during T cell expansion. Reciprocally, prevention of DGK activation does not affect the IL-2-regulated expression of bcl-x protein, indicating again that PA-derived signals are directly related to the proliferation machinery but not to the IL-2-regulated survival mechanisms. The involvement of both c-raf-1 and c-myc in mitogenic control is well established, although the exact mechanism underlying the regulated expression of these proteins by IL-2 has not been fully defined. Studies in freshly isolated T cells have demonstrated that the promotion of G1 progression by IL-2 was associated with an increase in c-raf protein expression (28). It has been previously suggested that c-raf-1 is constitutively expressed in CTLL-2 cells, but our experiments demonstrate that this is mostly due to the presence of serum. If CTLL-2 cells are deprived of both serum and IL-2, c-raf-1 protein expression is reduced. Readdition of IL-2 restores the levels of this protooncogene, an effect similar to that described in activated lymphocytes. Under these circumstances, inhibition of DGK activation prevents c-raf-1 induction, demonstrating for the first time that generation of PA is implicated in the control of c-raf-1 expression. Previous experiments by ourselves and others have demonstrated that addition of exogenous PA induces c-myc expression in T cells (10, 29), indicating the existence of a pathway governed by PA that is responsible for the induction of this gene. The present demonstration that DGK inhibition reduces c-myc expression further confirms that PA generation through DGK activation is essential for the induction of this protooncogene.
The binding of IL-2 to its high-affinity receptor regulates the transit
of T cells through the G1 phase to reach a
restriction point, after which cell proliferation is no longer
controlled by growth factor receptor ligation. The transition through S
phase is largely regulated by Rb hyperphosphorylation, which is in turn
controlled by a complex network of events. It has been previously
demonstrated that IL-2 regulates cyclin expression as well as p27
degradation. Our results here indicate that DGK inhibition prevents
IL-2-induced up-regulation of cyclin D3 and does not affect the
down-regulation of p27 that follows IL-2 addition. In this regard, the
cell arrest induced by DGK inhibition differs from the effect caused by
other immunosuppressors, such as rapamycin, since rapamycin treatment
of T cells prevents the IL-2-induced decrease in the protein levels of
p27 (19). Additionally, it has recently been demonstrated,
using the PI3K inhibitor LY29002, that PI3K/Akt signals were required
for IL-2-induced hyperphosphorylation of Rb in peripheral blood-derived
T lymphoblasts and Kit225 cells (23). The same authors
demonstrated that PI3K-derived signals were required for IL-2
up-regulation of cyclin D3, as well as down-regulation of p27. Our
results indicate that inhibition of DGK does not interfere with either
the generation of 3-phosphorylated lipids or the activation of Akt by
IL-2. Moreover, G1 progression is prevented
without effects on p27 down-regulation, indicating a different level of
control by PA than PI3K/Akt-derived signals in IL-2-induced cell cycle
progression. Previous studies with IL-2R mutants have indicated that
IL-2-induced DNA synthesis required the integration of a network of
signals generated by the IL-2Rß and subunits (11).
As previously indicated, activation of the PI3K/Akt pathway has been
shown to be essential for E2F transactivation (23). In
addition, there is considerable evidence indicating that the activation
of STATs is required for the proliferative response of lymphoid cells
(30). We demonstrate that DGK inhibition has no effect on
IL-2-stimulated PI3K/Akt activation, nor does it reduce IL-2R
surface expression (data not shown), indicating that STAT5 activation
is not affected. The elucidation of the integration of the signals
derived from DGK activation with other IL-2R-induced signals should
clarify the mechanisms regulating cytokine-induced cell
proliferation.
PA generation has been shown to have an important role in mitogenesis in response to different mitogens in a variety of cell systems. Our results now indicate that not only IL-2, but also IL-4, activates DGK in CTLL-2 cells and that inhibition of IL-4-induced DGK activation also prevents IL-4-induced cell proliferation. Previous studies have shown that IL-4 activates several proliferative pathways on its own and also primes cells for a response to IL-2-triggered proliferative signals (31). The molecular mechanism underlying these effects is the effective activation by IL-2 in IL-4-cultured cells of several molecules that play an important role in promoting proliferation, including the induction of expression of c-myc, c-fos, and cyclin D3. Interestingly, the same genes the authors described in the above-mentioned study, up-regulated by both IL-4 and IL-2, were impaired in our studies following DGK inhibition, confirming a role for this enzyme in mitogenic control. Although the importance of PA generation in proliferation is broadly accepted, to this date the exact role of this lipid is not well established. The profound effect that DGK inhibition exerts on IL-2- and IL-4-regulated proliferation indicates that the cellular levels of the mitogenic lipid PA must regulate the activity, intracellular localization, or protein-protein interactions of certain key cell components. Therefore, the intracellular concentration of PA in the cell could constitute one of the factors that the cell checks before making the decision to enter mitosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Isabel Merida, Department of Immunology and Oncology, Centro Nacional de Biotecnologia, Campus de Cantoblanco, Madrid 28049, Spain. E-mail address:
3 Abbreviations used in this paper: PI3K, phosphoinositide 3-kinase; DGK, diacylglycerol kinase; PA, phosphatidic acid; Rb, retinoblastoma tumor suppressor protein; cdk, cyclin-dependent kinase; cdki, inhibitor of cdk; H2B, histone 2B.
Received for publication January 21, 1999. Accepted for publication May 3, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain cytoplasmic domain are required for signaling. Nature 369:333.[Medline]
DGK activation is an essential step in IL-2-mediated lymphocyte proliferation. J. Biol. Chem. 271:10334.This article has been cited by other articles:
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  T. Matsubara, Y. Shirai, K. Miyasaka, T. Murakami, Y. Yamaguchi, T. Ueyama, M. Kai, F. Sakane, H. Kanoh, T. Hashimoto, et al. Nuclear Transportation of Diacylglycerol Kinase {gamma} and Its Possible Function in the Nucleus J. Biol. Chem., March 10, 2006; 281(10): 6152 - 6164. [Abstract] [Full Text] [PDF]
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A. Avila-Flores, T. Santos, E. Rincon, and I. Merida Modulation of the Mammalian Target of Rapamycin Pathway by Diacylglycerol Kinase-produced Phosphatidic Acid J. Biol. Chem., March 18, 2005; 280(11): 10091 - 10099. [Abstract] [Full Text] [PDF]
|
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A. Palicz, T. R. Foubert, A. J. Jesaitis, L. Marodi, and L. C. McPhail Phosphatidic Acid and Diacylglycerol Directly Activate NADPH Oxidase by Interacting with Enzyme Components J. Biol. Chem., January 26, 2001; 276(5): 3090 - 3097. [Abstract] [Full Text] [PDF]
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L. Bregoli, J. J. Baldassare, and D. M. Raben Nuclear Diacylglycerol Kinase-theta Is Activated in Response to alpha -Thrombin J. Biol. Chem., June 22, 2001; 276(26): 23288 - 23295. [Abstract] [Full Text] [PDF]
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