From the Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Cantoblanco, 28049, Madrid, Spain and the § CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom
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ABSTRACT |
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Phosphatidic acid generation
through activation of diacylglycerol kinase Signal transduction by a wide variety of extracellular stimuli
involves phospholipid hydrolysis and/or the activation of lipid kinases
and phosphatases as a means to generate biologically active lipid
second messengers (LSMs).1
The second messengers, 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate are now well established as modulators of protein
kinase C (PKC) activity and intracellular calcium ion concentration,
respectively (1). In recent years, investigations have revealed that
phospholipids distinct from phosphatidylinositol 4,5-bisphosphate
(PIP2) can also act as substrates for activated phospholipases. One of these is phosphatidylcholine (PC), the principal
substrate for phospholipase D (PLD) which catalyzes its hydrolysis to
form phosphatidic acid (PA) and free choline (2). The other route for
the formation of PA is through the phosphorylation of DAG by DAG kinase
(DGK) (3). Structural analysis of precursor phospholipids and LSMs have
demonstrated that phospholipid hydrolysis by activated phospholipases
is not a random process, but rather is highly organized and selective for particular lipid classes leading to the formation of specific LSMs
(4-7). DAG and PA exist in biologically active and inactive forms
depending on their fatty acid compositions. Biologically active DAGs
are those which possess polyunsaturated fatty acids (i.e.
those containing three or more double bonds) (6, 7). Phosphorylation by
DGK attenuates their activity, generating polyunsaturated PAs which are
thought to be biologically inactive. In contrast, biologically active
PAs are those containing saturated, monounsaturated, and to a lesser
extent diunsaturated fatty acids (6, 8). Dephosphorylation by PA
phosphatase (PAP) switches off this PA signal by converting it to
inactive, primarily monounsaturated, DAG (7, 9). The conversion of
biologically active DAGs and PAs into their inactive counterparts is
important for the cessation of the incoming signal and for the
resynthesis of their precursor phospholipids to maintain membrane
integrity. As the interconversion between biologically active and
inactive DAGs and PAs is through DGK and PAP this implies their
generation through two distinct routes: via PLD for the generation of
biologically active PA and via phospholipase C (PLC) for the generation
of biologically active DAG. This model is supported by both the
kinetics of accumulation of species-specific DAGs and PAs and
additionally through the fatty acid analysis of their precursor
phospholipids which show a close match to DAG and PA (4). From the
information in the literature it has now become apparent that the
generation of LSMs is a highly ordered sequence of events that displays
a high degree of specificity (9).
Interleukin-2 (IL-2) is the cytokine responsible for T-cell
proliferation. After binding to its high affinity receptor on the cell
surface, the activated receptor initiates a variety of signal
transduction pathways of which those involving protein and lipid
kinases have been the best characterized. IL-2 neither causes the
hydrolysis of PIP2 nor provokes the generation of cyclic nucleotides as a means to generate second messenger molecules (10-12).
In contrast, a rapid elevation of PA levels is seen in T-lymphocytes
(13) due to the activation of DGK (14). Further investigations revealed
that this acute activation of DGK, specifically that of the DGK Our goal in this work was to identify the fatty acid composition of
both basal and IL-2-elevated PA in CTLL-2 cells to demonstrate that
despite the lack of IL-2-stimulatable PLD activity, IL-2 could generate
biologically active PA species via DGK Materials--
Human recombinant IL-2 was a generous gift from
Hoffman-LaRoche Inc. (Nutley, NJ). [ Cell Culture and Stimulations--
CTLL-2 cells were cultured as
published previously (16). Upon reaching a density of 1 × 106/ml they were washed twice in incomplete medium (RPMI
buffered with 10 mM HEPES pH 7.2, supplemented with 2 mM glutamine, 50 µM 2-mercaptoethanol, 100 units/ml penicillin, and 100 µg/ml streptomycin) before reincubating
for a starving period of 6 h in the presence of 0.3% BSA. At this
point experiments were initiated after washing the cells once with
incomplete medium. Cells were incubated ± 500 units/ml IL-2 for
various times, pelleted, and immediately frozen on dry ice.
PA Isolation--
Cellular phospholipids, together with 1 µg
of 17:0/17:0-PA internal standard, were extracted by the method of
Bligh and Dyer (17). After drying under a gentle stream of
N2, the total lipid extract was resuspended in 20 µl of
chloroform/methanol/NH4OH (50/48/2, v/v/v) before injection
onto a Kromasil 100-5SIL HPLC column (5 µm, 2.1 × 250 mm;
Hichrom Ltd., Reading, U.K.). PA was separated from all other neutral
and phospholipids using a linear gradient changing from 70% solvent A
(chloroform/methanol/NH4OH, 70/28/2, v/v/v) plus 30%
solvent B (chloroform/methanol/NH4OH, 50/48/2, v/v/v) to
100% solvent B over 50 min at 0.5 ml/min. Detection was with a Varex
MKIII evaporative light scattering detector (Alltech, Carnforth, U.K.).
A small tap before the detector enabled the fraction corresponding to
PA to be diverted into a glass vial for structural analysis. The fatty
acid content of PA was quantified by GC-MS relative to the 17:0/17:0-PA
internal standard (insignificant amounts of naturally occurring 17:0
fatty acid were found in the samples).
DRG Isolation and HPLC Molecular Species Analysis--
Total
cellular lipids plus 1 µg of 12:0/12:0-DAG internal standard were
extracted as above. On the same day, the DRG within the total lipid
extract was derivatized with excess 3,5-dinitrobenzoyl chloride as
described previously (4, 5). DRG-dinitrobenzoyl derivatives (DRG-DNBs)
were isolated by solid-phase extraction on tC18 Sep-Pak cartridges
(Waters Ltd., Watford, U.K.), then separated into DRG classes by normal
phase HPLC (Kromasil column, 5 µm, 2.1 × 250 mm, Hichrom Ltd.)
using a linear gradient of 100% cyclohexane, 2,2,4-trimethylpentane,
diethyl ether/propan-2-ol (49/49/2/0.1, by volume) changing to 100%
cyclohexane/diethyl ether/propan-2-ol (85/15/0.1, v/v/v) over 45 min at
0.4 ml/min with absorbance detection at 254 nm. Fractions corresponding
to the AAG-DNB + AEAG-DNB and DAG-DNB classes were collected. AAG-DNB and AEAG-DNB were separated from each other by TLC using hexane/diethyl ether (80/20, v/v). After lightly spraying the TLC plate with 0.01%
1,6-diphenyl-1,3,5-hexatriene in hexane and visualization under UV
light, the bands corresponding to AAG-DNB and AEAG-DNB were scraped off
and eluted from the silica with diethyl ether. After concentration,
separation of the isolated DRG-DNB classes into their component
molecular species was performed by reverse-phase HPLC (Spherisorb
S5ODS2 Excel column, 5 µm, 2.1 × 250 mm, Hichrom Ltd.)
employing a linear gradient of 100% acetonitrile/propan-2-ol (9/1,
v/v) changing to 100% acetonitrile/propan-2-ol (1/1, v/v) over 50 min
at 0.5 ml/min with absorbance detection at 254 nm. To verify the
identification of individual DRG species, HPLC peaks were collected
into glass vials, derivatized, and subjected to GC-MS.
Phospholipid Digests--
An exhaustive PLC digest of PC and PE
isolated from CTLL-2 cells was performed overnight at 25 °C in 300 µl of reaction buffer (100 mM sodium borate, pH 7.4, 1 mM CaCl2, 1 mM 2-mercaptoethanol, 0.5 mM ZnCl2) plus 300 µl of diethyl ether
using 10 units of B. cereus PC-PLC in order to generate a
mixture of AAG, AEAG, and DAG for use as standards. The DRG was
ether-extracted from the aqueous buffer four times, dried under
N2, then DNB-derivatized, as described earlier.
Structural Analysis by GC-MS--
The lipid was dried under a
stream of N2 then transmethylated with 3 M HCl
in dry methanol in a sealed vial for 2 h at 70 °C. After
cooling to room temperature, the contents of the vial were dried and
re-dissolved in 100 µl of hexane. Fatty acid methyl esters (FAMEs)
were identified by gas chromatography-mass spectrometry (GC-MS;
5890GC/5972MSD, Hewlett-Packard) using authentic FAME standards as
described previously (6). Transmethylation of 1-alk-1'-enyl-2-acyl
lipids generates FAMEs and dimethylacetyls (from hydrolysis of the
fatty vinyl alcohol at the sn1-position), the latter being
identified by their retention times relative to known standards and by
the appearance of a characteristic fragmentation ion at
m/z 75. For the 1-O-alkyl-2-acyl
lipids a two-step procedure was used; transmethylation of the fatty
acid at the sn2-position of the glycerol backbone followed
by trimethylsilylation of the free hydroxyl groups at the
sn2- and sn3-positions of the resultant 1-O-alkylglycerol using 100 µl of
bis(trimethylsilyl)trifluoroacetamide for 30 min at 60 °C. The FAMEs
and 1-O-alkyl-2,3-bis(trimethylsilyl (TMS)) glycerols
generated were identified by electron impact GC-MS using a DB-23 column
(0.25 mm × 30 m; J & W Scientific) with a temperature
program holding at 55 °C for 2 min, ramping to 140 at 70 °C/min
then to 240 at 2 °C/min, an injector temperature of 260 °C, a
detector temperature of 270 °C and a helium head pressure of 12 p.s.i. A characteristic fragmentation ion at m/z 205, corresponding to [TMS-O-CH = CH2-O-TMS]+ formed by cleavage of the C1-C2
bond within the glycerol backbone of 1-O-alkyl-2,3-bis-TMS glycerol,
was used for identification (18).
In Vitro DGK Assays--
250 µg of
1-O-hexadecyl-2-oleoyl-PC and 1-palmitoyl-2-oleoyl-PC were
separately hydrolyzed with PLC as described above. The extent of PC
hydrolysis was checked by TLC (using chloroform/acetone/methanol/acetic acid/water, 50/20/10/10/5, by volume) of a chloroform/methanol extract
of the post-incubation reaction buffer. Using iodine vapor, no PC was
evident indicating that the PLC-mediated hydrolysis was essentially
100% (detection limit approximately 2 µg of lipid). The AAG and DAG
products (1-O-hexadecyl-2-oleoylglycerol and
1-palmitoyl-2-oleoylglycerol, respectively) generated were stored at
In Vivo Metabolic Labeling--
During the last 24 h of
exponential growth, CTLL-2 cells were labeled with 1 µCi/ml of
1-O-[3H]octadecyl lysophosphatidylcholine.
Thereafter, the cells were washed twice with incomplete medium before
reincubating for a starving period of 6 h in the presence of 0.3%
BSA. At this point experiments were initiated after washing the cells
once with incomplete medium. Cells (2 × 106) were
incubated ± 500 units/ml IL-2 for various times, pelleted and
immediately frozen on dry ice. Total lipids were extracted, (17),
followed by the separation of
[3H]1-O-alkyl-2-acyl-PC (AAG-PC) and
[3H]1-O-alkyl-2-acyl-PE (AAG-PE) by TLC using
a solvent system consisting of propan-1-ol/propionic
acid/chloroform/water (60/40/40/20, by volume). Radioactivity within
the AAG-PC and AAG-PE fractions of CTLL-2 cells (comigrating with
authentic phospholipid standards) was determined after scraping the
silica into vials followed by liquid scintillation counting.
Incorporation of D-[U-14C]Glucose and
[9,10-3H]Oleic Acid into Glycerolipids--
CTLL-2 cells
starved of both serum and IL-2 for 6 h were washed twice with
Krebs-HEPES buffer (118.46 mM NaCl, 4.74 mM
KCl, 2.54 mM CaCl2, 1.18 mM
KH2PO4, 1.18 mM MgSO4,
24.88 mM NaHCO3, and 10 mM HEPES
buffered to pH 7.35) containing 0.1% (v/v) BSA before incubation in
the latter for 1 h. Cells (5 × 106) were
incubated ± 500 units/ml IL-2 for 10 min followed by a 4-min
incubation with 5 µCi of D-[U-14C]glucose.
Cell incubations were terminated by rapidly pelleting the cells and
their immediate freezing on dry ice. Total lipids were extracted, (17),
followed by their separation using two-dimensional TLC (19).
Radioactivity within the lipid fractions of interest (DRG, PA,
triacylglycerol, PC, and PE, identified using authentic lipid
standards) was determined as described above. The incorporation of
oleic acid into the DRG fraction of CTLL-2 cells involved incubating IL-2- and serum-starved CTLL-2 cells (1 × 106) with 1 µCi of [9,10-3H]oleic acid for various periods of time
(from 30 s to 2 h) in the presence or absence of 500 units/ml
IL-2. Cell incubations were terminated by the rapid addition of 9 volumes of ice-cold phosphate-buffered saline, containing 1% BSA
followed by cell centrifugation and freezing of the pellets on dry ice.
After total lipid extraction (17), the DRG was separated from all other neutral lipids by TLC using the solvent system composed of petroleum ether/diethyl ether/glacial acetic acid (70/30/2, v/v/v). Radioactivity within the DRG fraction of the CTLL-2 cells (comigrating with authentic
1,2-dioleoylglycerol standard) was determined as described above.
In T-lymphocytes, IL-2 has been reported to both stimulate DGK has been implicated in
interleukin-2-dependent T-lymphocyte proliferation. To
investigate this lipid signaling in more detail, we characterized the
molecular structures of the diradylglycerols and phosphatidic acids in
the murine CTLL-2 T-cell line under both basal and stimulated
conditions. In resting cells, 1,2-diacylglycerol and
1-O-alkyl-2-acylglycerol subtypes represented 44 and 55%
of total diradylglycerol, respectively, and both showed a highly
saturated profile containing primarily 16:0 and 18:1 fatty acids.
1-O-Alk-1'-enyl-2-acylglycerol represented 1-2% of total
diradylglycerol. Interleukin-2 stimulation did not alter the molecular
species profiles, however, it did selectively reduce total
1-O-alkyl-2-acylglycerol by over 50% at 15 min while only causing a 10% drop in 1,2-diacylglycerol. When radiolabeled CTLL-2 cells were challenged with interleukin-2, no change in the cellular content of phosphatidylcholine nor phosphatidylethanolamine was observed thereby ruling out phospholipase C activity as the source of
diradylglycerol. In addition, interleukin-2 failed to stimulate de novo synthesis of diradylglycerol. Structural analysis
revealed approximately equal amounts of 1,2-diacyl phosphatidic acid
and 1-O-alkyl-2-acyl phosphatidic acid under resting
conditions, both containing only saturated and monounsaturated fatty
acids. After acute (2 and 15 min) interleukin-2 stimulation the total
phosphatidic acid mass increased, almost entirely through the formation
of 1-O-alkyl-2-acyl species. In vitro assays
revealed that both 1,2-diacylglycerol and
1-O-alkyl-2-acylglycerol were substrates for
1,2-diacylglycerol kinase
, the major isoform in CTLL-2 cells, and
that the lipid kinase activity was almost totally inhibited by R59949.
In conclusion, this investigation shows that, in CTLL-2 cells,
1,2-diacylglycerol kinase
specifically phosphorylates a
pre-existing pool of 1-O-alkyl-2-acylglycerol to form the
intracellular messenger 1-O-alkyl-2-acyl phosphatidic acid.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
isoform, was essential for subsequent cell cycle progression and in
addition, DGK
was rapidly translocated to the perinuclear region of
T-cells (15). The increase in PA is thought to be entirely due to the
activation of DGK
as no PLD activity at equivalent times is detected
in IL-2-stimulated cells (16). These observations underline the
importance of DGK
activation for the generation of biologically
active PA species during T-cell proliferation and distinguish it from
other signal transduction mechanisms.
instead. In addition, the
molecular species analysis of the diradylglycerols (DRGs) (consisting
of DAG, 1-O-alkyl-2-acylglycerol (AAG) and 1-O-alk-1'-enyl-2-acylglycerol (AEAG)) was determined in
order to define the substrates for the IL-2-stimulated DGK
in CTLL-2 cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (specific
activity 3000 Ci/mmol), 1-O-[3H]octadecyl
lysophosphatidylcholine (specific activity 110-210 Ci/mmol),
D-[U-14C]glucose (specific activity 230-370
mCi/mmol), and [9,10-3H]oleic acid (specific activity
2-10 Ci/mmol) were purchased from Amersham (Amersham, United Kingdom).
Fetal calf serum and all cell culture media were bought from Life
Technologies, Inc. (Paisley, U.K.). Silica gel (60 Å) TLC plates were
from Whatman (Clifton, NJ) and Merck (Darmstadt, Germany).
1-O-Hexadecyl-2-oleoyl-PC (1-O-16:0/18:1n-9-PC),
1-palmitoyl-2-oleoyl-PC (16:0/18:1n-9-PC), diheptadecanoyl-PA
(17:0/17:0-PA), PA (egg yolk), 1,2-dilauroylglycerol (12:0/12:0-DAG),
1,2-dioleoylglycerol, PC (egg yolk), phosphatidylethanolamine (PE) (egg
yolk), triacylglycerol, 1,6-diphenyl-1,3,5-hexatriene, bovine serum
albumin (BSA, fraction V), and PC-specific phospholipase C (PC-PLC)
(Bacillus cereus, type XI) were from Sigma (Poole, U.K.).
Anti-HA antibody was purchased from Berkeley Antibody Co. (Richmond,
CA). R59949 was obtained from Calbiochem (Nottingham, U.K.).
3,5-Dinitrobenzoylchloride and all HPLC grade/analytical grade solvents
were from Fisher Scientific U.K. Ltd. (Loughborough, U.K.).
70 °C under argon and used within a few days to minimize both
carbon-carbon double bond oxidation and acyl migration. COS cells
(routinely cultured in Dulbecco's modified Eagle's medium buffered
with 10 mM HEPES pH 7.2 supplemented with 10% (v/v) fetal
calf serum, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin) were transfected by electroporation with a
plasmid (pcDNA3.1, Invitrogen, Leek, Holland) containing HA-tagged
murine DGK
(GenBank accession number: AF085219)2 or empty plasmid
as a control. After 48 h, the cells were collected by
trypsinization, lysed, and HA-tagged murine DGK
was
immunoprecipitated with anti-HA antibodies. 10% of the
immunoprecipitate was subjected to SDS-polyacrylamide gel
electrophoresis followed by Western blotting (first antibody, anti-HA;
second antibody, anti-horseradish peroxidase) to check for the presence
of HA-tagged murine DGK
. DGK assays using the immunoprecipitates
were performed with 15 µg of either 1-O-hexadecyl-2-oleoyl
glycerol or 1-palmitoyl-2-oleoylglycerol as substrate as described
previously (15). Where indicated, immunoprecipitates were preincubated
on ice with 1 µM R59949 (DGK inhibitor type II) for 15 min before the addition of lipid substrate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
activity (14, 15) and raise PA levels (13) at early time points (less
than 30 min). To investigate the molecular species composition of this
PA we initially analyzed its fatty acid content in resting CTLL-2
cells. Fatty acids identified as 14:0, 15:0, 16:0, 16:1n-7, 16:1n-5,
18:0, 18:1n-9, and 18:1n-7 were present in varying quantities (Fig.
1). 16:0 represented over 80% of the total fatty acid found within the PA fraction, whereas 15:0 was the
most minor component (less than 1% of total). No di- or more unsaturated fatty acids were found in CTLL-2 cell PA. IL-2-stimulation for 2 and 15 min caused the total amount of fatty acid in the PA
fraction to rise to 141 and 204% of control, respectively, indicative
of a rise in total PA mass. The monounsaturated fatty acids were found
to increase above their respective control levels to a proportionately
greater extent as compared with their saturated counterparts, although
in total mass terms the saturates remained predominant (Fig. 1). The
increase at 2 min was to 134% of control for total saturated fatty
acids and 187% of control for total monounsaturated fatty acids, while
at 15 min it was 177 and 364%, respectively. No new fatty acids
appeared in the PA fraction after any IL-2 stimulation conditions.
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Fig. 1.
IL-2 stimulates acute accumulation of
saturated and monounsaturated PA. Arrested CTLL-2 cells were
incubated ± IL-2 (500 units/ml) for the times indicated. PA was
isolated by HPLC, transmethylated, and the resultant FAMEs identified
and quantified by GC-MS. The data are represented as mean ± S.D.
(n = 4).
Since the IL-2 stimulated increase in PA at short times is thought to
be entirely through the action of DGK (15, 16), yet the fatty acid
composition of saturated and monounsaturated species was very similar
to that seen in lysophosphophatidic acid-stimulated porcine aortic
endothelial cells where the PA was generated by a PLD (6), we
determined the DRG (DAG, AAG, and AEAG) composition of CTLL-2 cells.
DAG represented 44% of the total DRG, AAG represented 55%, while AEAG
accounted for the remaining 1-2%, under resting conditions. Molecular
species analysis by reverse-phase HPLC revealed 16 major DAG peaks
corresponding to distinct molecular species (Fig.
2A and Table
I). 16:0/18:1n-9 (peak 11) was the most
abundant species. The most abundant polyunsaturated species,
18:0/20:4n-6 (peak 8), was present as only 1% of total DAG.
Reverse-phase HPLC of AAG demonstrated 10 major peaks (Fig. 2a), the
most abundant being identified as 1-O-hexadecyl-2-oleoyl
glycerol (1-O-16:0/18:1n-9; peak 8). Other AAG molecular
species detected are listed in Table II.
No polyunsaturated AAGs were detected. Although AEAG mass was too low
for a full species analysis, transmethylation of total cellular DRG
followed by GC-MS detected only 16 and 18 carbon-saturated dimethylacetyls derived from the corresponding alk-1'-enyl groups.
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IL-2 stimulation caused no obvious changes in the DRG molecular species profiles, however, at 15 min, it did cause a small drop in total DAG mass (from 2498 ± 46 to 2245 ± 248 pmol/107 cells) and a halving of total AAG mass (from 3059 ± 26 to 1416 ± 70 pmol/107 cells). Preincubation with the DGK inhibitor, R59949, blocked this IL-2-induced DRG response by approximately 90% (data not shown).
The results of the molecular species analyses of DAG and AAG prompted
us to reinvestigate the short-term IL-2-stimulated changes in PA. Since
AAG represented approximately half of the total DRG in CTLL-2 cells, we
envisaged that an alkyl, acyl structure could also be present in the PA
fraction. By initially transmethylating PA and then trimethylsilylating
the products we were able to identify, using GC-MS, both the component
fatty acids and any alkyl ethers present. Fig.
3A shows the electron impact
mass spectrum for 1-O-16:0 monoalkylglycerol following
trimethylsilylation to 1-O-16:0, 2,3-bis-TMS glycerol. The
C1-C2 bond within the 1-O-16:0, 2,3-bis-TMS glycerol is
susceptible to cleavage and generates a major ion fragment at
m/z 205 corresponding to [TMS-O-CH2 = CH-O-TMS]+. The ions at m/z 73, 103, 117, and 147 correspond to various fragmentation and
re-arrangement products tentatively identified as [TMS]+
(or [(CH3)3Si]+), [TMS-O = CH2]+, [TMS-O-CH2 = CH2]+, and
[(CH3)2Si = O-Si(CH3)3]+, respectively. No
molecular ion could be detected. As the ion at
m/z 205 could only arise from the decomposition
of the 1-O-alkyl-2,3-bis-TMS glycerol we used this to
quantify the alkyl, acyl content of PA.
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In resting cells, 1-O-alkyl-2-acyl-PA (AAG-PA) represented
over 50% of total PA and consisted of a mixture of
1-O-14:0, 1-O-16:0, 1-O-18:0, and
1-O-18:1 alkyl structures based on retention times relative
to an authentic standard (data not shown). 1-O-16:0 was the
predominant form, representing approximately one-third of the total
AAG-PA. IL-2 stimulation for 2 and 15 min increased the level of AAG-PA
by 2- and 4-fold, respectively (Fig. 3B). At 15 min almost
all the PA is of the AAG-PA form since there is approximately a 1:1
ratio of total fatty acid to alkyl groups at this time point (Figs. 1
and 3B). The increases in AAG-PA are similar to those for
the monounsaturated fatty acids (Fig. 1) suggesting that DGK may
preferentially phosphorylate AAG containing unsaturated acyl chains
when available.
When the CTLL-2 PC molecular profile was analyzed following PLC
hydrolysis and DNB derivatization, 16:0/18:1n-9 PC and 1-O-16:0/18:1n-9 PC were found to be the most abundant species (data not shown). The
whole profile was similar to that for DRG, suggesting PC as a possible
source of DRG. We decided to investigate this possibility by
radiolabeling CTLL-2 cells with
1-O-[3H]octadecyl lysophosphatidylcholine
which is rapidly and specifically incorporated into the AAG-PC and
AAG-PE phospholipid fractions (87 and 12% of total radioactivity
incorporated, respectively). In addition, we found a minor proportion
(approximately 1%) of the radiolabel associated with the AAG fraction
of CTLL-2 cells. Over a time course of 0-30 min, IL-2 did not affect
the radioactivity associated with AAG-PC, AAG-PE, nor AAG (Fig.
4 and data not shown), suggesting that
hydrolysis of AAG-PC and AAG-PE was not taking place in order to
generate AAG. Another route for the generation of DRG is that through
de novo synthesis. To assess if IL-2 increased the de
novo synthesis of DRG, we measured the incorporation of radioactivity from [9,10-3H]oleic acid into the DRG
fraction in CTLL-2 cells. IL-2 treatment of CTLL-2 cells did not cause
an increase in the incorporation of oleic acid (over a period of
30 s to 2 h) compared with control cell treatment (data not
shown). We also measured the incorporation of
D-[U-14C]glucose into DRG, triacylglycerol,
PA, PC, and PE in control- and IL-2-stimulated CTLL-2 cells. When
radioactivity from a glucose tracer was determined in the glycerol
backbone of DRG (which incorporated by far the greatest proportion of
radioactivity at the end of a 4-min period of linear
D-[U-14C]glucose uptake) from cells acutely
treated with IL-2, there was no increase with respect to
control-treated cells confirming that IL-2 did not affect de
novo synthesis (Table III). In
addition, we found that IL-2 did not increase de novo
synthesis of triacylglycerol nor of PE. Very small changes (less than
10%) in the de novo synthesis of PC and PA were observed
after treatment of the cells with IL-2 compared with control medium.
However, these were not sufficient to alter the value of the estimated
flux (the sum of DRG, triacylglycerol, PC, and PE (19)) through PAP, an
enzyme involved in glycerolipid synthesis, shown in Table III.
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We have previously shown IL-2-dependent short-term
activation of DGK leading to PA generation (14, 15). In order to
complement the results of the in vivo AAG-PA generation, we
tested the ability of DGK
to phosphorylate DAG and AAG in
vitro. COS cells were either transfected with an empty plasmid or
a plasmid containing HA-tagged murine DGK
. The DGK
was
immunoprecipitated through its tag. Only those cells transfected with
the HA-tagged DGK
expressed the protein of approximately 80 kDa as
determined by Western blotting (Fig.
5A). DGK
immunoprecipitates
were preincubated with or without R59949 before incubation with the
substrates, 1-O-16:0/18:1n-9 (AAG) or 16:0/18:1n-9 (DAG), in
the presence of [
-32P]ATP. Fig. 5B shows
that the HA-tagged murine DGK
was able to phosphorylate both the AAG
and DAG substrates to the same extent. This enzymatic activity was
inhibited by over 90% by preincubation of the immunoprecipitates with
the DGK inhibitor II, R59949.
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DISCUSSION |
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It is becoming increasingly important when examining
mitogen-stimulated cells not only to measure the mass generation of
LSMs, such as DRG and PA, but to define the molecular species of each LSM generated. By reference to the molecular structure of precursor phospholipids (such as PC and PIP2), the DRG and PA species
formed may be mapped to their appropriate sources thereby fully
characterizing a signaling pathway. At present, there exists a limited
amount of information on this subject, but together it forms a model describing the formation and clearance of the LSMs DRG and PA through
the tight regulation of at least the four enzymes: PLC, PLD, DGK, and
PAP. These appear to function in many, but not all, signal transduction
events delivered by mitogens (9). One of the exceptions to this general
rule is IL-2, the T-lymphocyte growth factor, which deploys
nonconventional signal transduction pathways (10-16). It therefore
offers a unique opportunity to study novel LSM generation following
cytokine receptor stimulation in T-lymphocytes which up to now has
received little attention. To this end we investigated the effect of
IL-2 on PA and DRG metabolism in mouse CTLL-2 cells and show that
in vivo stimulated DGK preferentially utilizes a putative
PKC inhibitor, AAG, to form AAG-PA with putative second messenger functions.
The fatty acid composition of PA in resting CTLL-2 cells revealed only
saturated and monounsaturated fatty acids (Fig. 1), similar to that we
have previously reported for porcine aortic endothelial and Swiss 3T3
cells (6). The fatty acids found in PA did not change following IL-2
stimulation although their relative abundance did alter, with an
increase in monounsaturated species as compared with that seen in
resting cells. Since IL-2 stimulates acute PA generation through the
activation of DGK (14, 15), rather than through breakdown of PC by
PLD (16), then the PA must be derived from DRG already present in the
CTLL-2 cells.
Using DNB derivatization followed by HPLC, we were able to separate
DAG, AAG, and AEAG families of DRGs. AAG represented approximately 55%
of the total DRG in resting CTLL-2 cells which is rather more than that
seen in some other cell types (4, 6). Molecular species profiling of
DAG (Fig. 2) showed similarities to that of 3T3 cells (4), but was
distinct from that of porcine aortic endothelial cells (6) where
polyunsaturated forms (containing 20:3n-9, 20:4n-6, or 20:5n-3 at the
sn2-position) represented over 25% of the total DAG. This
was over 20 times higher than that seen in CTLL-2 cells where
polyunsaturated species are very minor components which do not change
significantly following IL-2 treatment. Previous work has failed to
detect any PIP2-PLC activation by IL-2 in these cells,
indicating that this cytokine does not signal via the classical
polyunsaturated DAG/inositol 1,4,5-trisphosphate pathways (10).
Furthermore, in PKC-deficient and PKC down-regulated T-lymphocytes,
IL-2 is still a mitogenic agent (20, 21). These results together
demonstrate that IL-2-driven cell proliferation is independent of the
involvement of classical isoforms of PKC. It is possible that atypical
PKC isoforms, including PKC, are important in IL-2-stimulated CTLL-2
cells since this subfamily does not display a requirement for DAG (22,
23) and interestingly they have been reported to be activated by PA
(24, 25). In another murine T-lymphocyte cell line, TS1
, IL-2
has been shown to regulate actin organization through the activation of
Rho and PKC
(26) and our own work with porcine aortic endothelial
cells has shown a requirement for PA in the Rho-mediated formation of actin stress fibers (27) although PKC
involvement was not examined.
It is interesting to note that the major AAG species,
1-O-16:0/18:1n-9 in CTLL-2 cells corresponds to that of the
most abundant DAG species, 16:0/18:1n-9, which is also the most
abundant PC species, which could imply a close relationship between the
synthesis of these DRGs and PC. In the absence of evidence for a PC-PLC or a PE-PLC (Fig. 4), for PLD (16) or for IL-2-stimulated
phosphocholine exchange between PC and ceramide (producing
sphingomyelin and DRG) in CTLL-2
cells,3 the source of the DRG
appeared to be through de novo synthesis, producing a
reservoir both for PC synthesis when required (hence the profile
similarities) and PA generation by IL-2-stimulated DGK. By measuring
the incorporation of radioactivity into DRG from
[9,10-3H]oleic acid and
D-[U-14C]glucose, de novo
synthesis was not up-regulated by treatment of the cells with IL-2
indicating that the pre-existing pool of DRG was sufficient for its
subsequent conversion into AAG-PA and DAG-PA by IL-2-activated DGK
.
Furthermore, the calculated flux through PAP (the sum of DRG,
triacylglycerol, PC, and PE (19)) was rapid (due to the low level of
radioactivity found within PA) and was unchanged by IL-2 at equivalent
times, confirming that IL-2 does not affect the activities of the
enzymes involved in de novo lipid synthesis.
AEAG represented a minor component of total DRG in CTLL-2 cells which is similar to that found in 3T3 fibroblasts (4, 7). Unfortunately these low levels made a comprehensive analysis impractical, although very small amounts of saturated 16 and 18 carbon alk-1'-enyl structures were detected. Stimulated increases in AAG and AEAG have been observed in other cell types. In response to interleukin-1 (28, 29) and choleocystokinin (30) in mesangial and pancreatic cells, respectively, a high proportion of AAG and/or AEAG arising from PE and PC hydrolysis, is generated as part of the total DRG production. In contrast to DAGs, the function of both AAGs and AEAGs is poorly understood and early reports concerning their properties compared with those known about DAGs were rather confusing (31-33), however, the current consensus is that if they have any PKC modulating function then it is as inhibitors rather than activators (29, 34).
As IL-2 is known to signal through the activation of DGK and the
finding that AAG represented a high proportion of the total DRG, it was
necessary to assess if within the PA fraction there were AAG-PA
species. Using the electron impact-mediated decomposition properties of
the 1-O-alkyl-2,3-bis-TMS derivatives of AAG-PA, we were
able to identify structures containing 1-O-14:0,
1-O-16:0, 1-O-18:0, and 1-O-18:1 alkyl
groups. Quantification revealed that while approximately half of the PA
was of the AAG-PA form in resting cells, this increased to almost 100%
following acute stimulation. This fits with the observations that acute
IL-2 stimulation caused a large drop in AAG levels but had little
effect on DAG and that these changes could be blocked by R59949. Thus
IL-2-activated DGK
(14, 15) preferentially uses AAG as a substrate
in CTLL-2 cells.
In order to show that the IL-2-induced formation of AAG-PA could be
attributable to DGK, we transfected murine DGK
into COS cells
which do not normally express this isoform (3). This provided us with
an essentially pure enzyme preparation, after immunoprecipitation, for
studies on its substrate specificity. In vitro DGK
assays
revealed that it phosphorylated both AAG and DAG to the same extent
(Fig. 4). Furthermore, the DGK
activity was blocked by R59949, a
recognized inhibitor of this enzyme (3, 35), confirming our previously
published observations (15, 16). The proportionately greater
utilization of AAG for stimulated PA synthesis by DGK
in
vivo as compared with that seen in vitro suggests that
other factors effect selectivity within the cell such as restrictions
on substrate accessibility.
Thus in CTLL-2 cells IL-2 delivers a mitogenic signal which provokes
saturated/monounsaturated PA generation, an event previously described
as being absolutely essential for cell cycle entry and ultimately cell
proliferation (15). PA is known to exhibit numerous biological
properties (36), however, its intracellular targets remain to be
identified. Now with the finding that in addition to DAG-PA there
exists AAG-PA in T-lymphocytes and the fact that these species are
positively regulated by IL-2, there exists the possibility that the
biological properties of PAs are perhaps broader than first thought and
it is likely that the PA-binding proteins show selectivity for certain
classes and/or molecular species, similar to that seen with the DRGs.
Future work revealing the biological properties of the IL-2-stimulated
PA species, by examining the activation and inhibition of various
enzymes, will further extend our knowledge concerning downstream
elements of IL-2 signaling. This work has shown for the first time that
a member of the DGK family is able to phosphorylate AAG to the same extent as that of DAG in vitro. In vivo, the physiological
relevance of IL-2-stimulated DGK activation could be that of the
clearance of AAG to form AAG-PA thereby reducing the intracellular
level of the former which is considered to have senescent and growth inhibitory properties (34). For that reason the commonly used term
"diacylglycerol kinase" should be used with caution in order to
both remove previous concepts of DAG being its unique substrate and at
the same time not to ignore AAGs as valid substrates. The term
"diradylglycerol kinase" would be a more appropriate substitute for
previous DGK nomenclature.
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ACKNOWLEDGEMENT |
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We thank Prof. Carlos Martínez-A. for valuable criticisms and discussions during the preparation of this manuscript.
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FOOTNOTES |
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* This work was supported in part by Association for International Cancer Research Grant 97-15 and Dirección General de Enseñanza Superior e Investigación Científica Grant PM97-0132 (to I. M.). Work in the host laboratory (Birmingham, U. K.) was funded by The Wellcome Trust. The Department of Immunology and Oncology was founded and is supported by the Consejo Superior de Investigaciones Científicas and Pharmacia and Upjohn.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a short term European Molecular Biology Organization fellowship.
¶ To whom correspondence should be addressed. Tel.: 34-91-585-4665; Fax: 34-91-372-0493; E-mail: imerida{at}cnb.uam.es.
2 M. A. Sanjuán and I. Mérida, unpublished results.
3 D. R. Jones and I. Mérida, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: LSM, lipid second messenger; DAG, 1,2-diacylglycerol; AAG, 1-O-alkyl-2-acylglycerol; AEAG, 1-O-alk-1'-enyl-2-acylglycerol; PA, phosphatidic acid; IL-2, interleukin-2; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PIP2, phosphatidylinositol 4,5-bisphosphate; DAG-PA, 1,2-diacyl-PA; AAG-PA, 1-O-alkyl-2-acyl-PA; AAG-PC, 1-O-alkyl-2-acyl-PC; AAG-PE, 1-O-alkyl-2-acyl-PE; FAME, fatty acid methyl ester; PLC, phospholipase C; PLD, phospholipase D; DGK, diacylglycerol kinase; PAP, PA phosphatase; DNB, dinitrobenzoyl derivative; PKC, protein kinase C; DRG, diradylglycerol; HPLC, high performance liquid chromatograpy; GC-MS, gas chromatography-mass spectrometry; BSA, bovine serum albumin; HA, hemagglutinin.
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REFERENCES |
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