From the Department of Biochemistry and Molecular
Biology, Faculty of Medicine, The University of Tokyo, Core Research
for Evolutional Science and Technology of the Japan Science and
Technology Corporation, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan, the ¶ Faculty of Pharmaceutical Sciences, Nagoya City
University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-0027, Japan,
and the § Pharmaceutical Research Center, Meiji Seika
Kaisha. Ltd., 760 Morooka-cho, Kohoku-ku,
Yokohama 222-8567, Japan
Received for publication, November 27, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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To create the unique properties of a certain
cellular membrane, both the composition and the metabolism of membrane
phospholipids are key factors. Phospholipase A2
(PLA2), with hydrolytic enzyme activities at the
sn-2 position in glycerophospholipids, plays critical roles
in maintaining the phospholipid composition as well as producing
bioactive lipid mediators. In this study we examined the contribution
of a Ca2+-independent group IVC PLA2 isozyme
(cPLA2 Biological membranes contain a complicated mixture of
phospholipids differing from each other with respect to their
head-group structure, hydrocarbon chain length, and degree of
unsaturation of the acyl chains. The complexity of these phospholipid
structures results in their diverse roles in membrane dynamics, protein
regulation, signal transduction, and vesicular secretion. The assembly
of membranes requires the coordinate synthesis, catabolism, and
transport of phospholipids to create the unique properties of a certain cellular membrane. Considerable numbers of studies have identified many
enzymes involved in these multiple pathways (1-8).
Phospholipase A2
(PLA2)1 is a
superfamily of enzymes that hydrolyze the sn-2 ester bond in
glycerophospholipids, releasing free fatty acids and lysophospholipids
(9-11). To date, at least 19 enzymes have been identified in
mammals. Among them, cPLA2 In contrast, the information on cPLA2 Materials--
[1-14C]Arachidonic acid (2.1 GBq/mmol) was from Amersham Biosciences. Bovine serum albumin
(BSA, fatty acid-free) and catalase (Aspergillus
niger) were from Sigma. Herbimycin A, calphostin C, and
sodium orthovanadate were obtained from Calbiochem, and methyl
arachidonyl fluorophosphonate (MAFP) was from Cayman Chemical Co. (Ann
Arbor, MI). Hydrogen peroxide (H2O2) was from
Wako (Osaka, Japan). BODIPY Brefeldin A and MitoTracker Red CMXRos were
from Molecular Probes (Eugene, OR). Mammalian expression vector
pcDNA3.1/His was obtained from Invitrogen, and pEGFP-C1 was from Clontech.
Plasmid Constructions and Expression of Human
cPLA2 Cell Culture and Transfection--
HEK293 cells were cultured in
Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with
10% fetal calf serum (Sigma), 100 IU/ml penicillin, and 100 µg/ml
streptomycin. CHO cells were cultured in Nutrient Mixture F-12 HAM
(Sigma) supplemented with 10% fetal calf serum, 100 IU/ml penicillin,
and 100 µg/ml streptomycin. LipofectAMINE PLUS (Invitrogen) was used
for the transfection of HEK293 and CHO cells according to the
manufacturer's protocols. To obtain HEK293 cells stably expressing
cPLA2 Confocal Microscopy--
CHO cells were seeded on coverslips
(10-mm diameter) on glass-bottomed culture dishes (Matsunami, Osaka,
Japan) at a density of 1.5 × 104 cells/coverslip,
transiently transfected with 1 µg of the expression vector encoding a
GFP-cPLA2 Electrospray Ionization Mass Spectrometric (ESI-MS) Analysis of
Phospholipids--
About 1 × 107 cells were
collected, washed three times with phosphate-buffered saline (5 mM sodium phosphate buffer, pH 7.4, containing 150 mM NaCl), and then suspended in 1 ml of phosphate-buffered saline. The total phospholipids were extracted by the method of Bligh
and Dyer (22). The total lipid extract was dried under a gentle stream
of nitrogen, dissolved in 400 µl of chloroform/methanol (2:1, v/v),
and used for mass spectrometry analysis. Mass spectrometry analysis was
performed essentially as described previously (23). Lipid extracts from
the cells were analyzed using a Quattro II tandem quadrupole mass
spectrometer (Micromass, Manchester, UK) equipped with an electrospray
ion source. 5 µl of samples (500 pmol/µl) dissolved in
chloroform/methanol (2:1, v/v) were introduced by means of a flow
injector into the ESI chamber at a flow rate of 2 µl/min. The elution
solvent was acetonitrile/methanol/water (2:3:1, v/v/v) containing 0.1%
ammonium formate (pH 6.4). The mass spectrometer was operated in the
positive and negative ion scan modes. The nitrogen drying gas flow rate
was 10 liters/min, and its temperature was 80 °C. Essentially, the
capillary voltage was set at 3.7 kV, and the cone voltage was set at 30 V in both the positive and negative ion scan modes.
AA Release Assay--
Cells were seeded onto 12-well culture
plates at a density of 8 × 104 cells/well in DMEM.
After 24 h of incubation the medium was removed, and the cells
were labeled by incubation for 24 h in 1 ml of DMEM containing
0.05 µCi of [14C]AA and 0.2% (w/v) fatty acid-free
BSA. The cells were then washed three times with DMEM containing 0.2%
(w/v) fatty acid-free BSA (the control medium) and stimulated with
ligands in the same buffer at 37 °C. The radioactivity of the
supernatants and cell lysates (in 1% Triton X-100) was measured using
a liquid scintillation counter. The amount of radioactivity released
into the supernatant was expressed as a percentage of the total
incorporated radioactivity.
Subcellular Localization of
cPLA2 cPLA2
Examination of the PE molecular species in the negative-ion mode
demonstrated a relative abundance of polyunsaturated fatty acid (PUFA)
species such as 1-alkyl or alkenyl-2-acyl 16:1-20:4 (m/z 722.6) and diacyl 18:0-20:4
(m/z 766.7) in
cPLA2 cPLA2
To further address the involvement of cPLA2 Effects of Various Inhibitors on
H2O2-induced AA Release--
We investigated
H2O2-initiated signaling pathways, because we
could not detect the activation of cPLA2 The major findings of the present study are as follows. 1)
cPLA2 ESI-MS analysis showing that the expression of cPLA2 ER is a major organelle wherein the biosynthesis of various kinds of
phospholipids, including PE, occurs (30-33). Due to the fact that PE
has a unique property in that it possesses high proportion of AA or
docosahexaenoic acid (22:6) at the sn-2 position, the fatty
acid composition in PE may be rearranged to the proper proportion in
the ER by cPLA2 H2O2-induced AA release by cPLA2 Regarding the mechanism by which H2O2 activates
cPLA2 In conclusion, we have provided several new insights into the
properties of human cPLA2), a paralogue of cytosolic PLA2
(cPLA2
), to phospholipid remodeling. The enzyme was
localized in the endoplasmic reticulum and Golgi apparatus, as seen
using green fluorescence fusion proteins. Electrospray ionization mass spectrometric analysis of membrane extracts revealed that
overexpression of cPLA2
increased the proportion of
polyunsaturated fatty acids in phosphatidylethanolamine, suggesting
that the enzyme modulates the phospholipid composition. We also found
that H2O2 and other hydroperoxides induced
arachidonic acid release in cPLA2
-transfected human
embryonic kidney 293 cells, possibly through the tyrosine phosphorylation pathway. Thus, we propose that cPLA2
is
constitutively expressed in the endoplasmic reticulum and plays
important roles in remodeling and maintaining membrane phospholipids
under various conditions, including oxidative stress.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is the only PLA2
enzyme that shows significant selectivity toward phospholipids
containing arachidonic acid (AA) at the sn-2 position. A
number of studies have clarified its structure (12), localization (13-15), and pathophysiological roles in vivo (16-18).
, a
Ca2+-independent paralogue of cPLA2
, is
limited. It has been reported that cPLA2
is
predominantly expressed in the brain, heart, and skeletal muscle in
humans (19, 20) and that the enzyme is activated in vivo by
serum (21). However, the fatty acid selectivity is controversial, and
its subcellular localization, cellular roles, and regulation of
activity have not been documented. In this study we demonstrated that
cPLA2
is localized in the endoplasmic reticulum (ER) and Golgi and is involved in the mobilization of fatty acids in
phosphatidylethanolamine (PE). During the search for regulators of the
enzymatic activity at the cellular level, we found that reactive oxygen
species (ROS) activate the activity of cPLA2
, possibly
through the tyrosine phosphorylation pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
The mammalian expression vector
pcDNA3.1/His was used to express proteins fused with the N-terminal
Xpress epitope. The cDNA fragments encoding full-length human
cPLA2
were inserted into pcDNA3.1/His to obtain
pcDNA3.1/His-cPLA2
. For confocal microscopy studies,
cPLA2
was subcloned into pEGFP-C1, encoding a green fluorescence protein (GFP) at the N terminus between the
BglII and SmaI sites to obtain
pEGFP-cPLA2
.
, the cells were transfected with
pcDNA3.1/His-cPLA2
, selected with 600 µg/ml G418
(Invitrogen), and maintained in the presence of 300 µg/ml G418. The
expression of cPLA2
was confirmed by immunoblotting. HEK293 cells transfected with the vector alone were also kept in medium
with G418 and used as a vector control.
fusion protein (pEGFP-cPLA2
) or a control vector, pEGFP-C1, with LipofectAMINE PLUS according to the
manufacturer's protocol, and used for experiments 48 h after
transfection. For double staining, cells transiently expressing GFP-cPLA2
were incubated with 1 µM BODIPY
Brefeldin A (
ex = 558 nm,
em = 568 nm) or 100 nM MitoTracker Red CMXRos
(
ex = 579 nm,
em = 599 nm) in
Hanks' balanced salt solution containing 10 mM HEPES and
0.1% BSA for 20 min at 37 °C. The fluorescent signal was observed
with an AX-80 analytical microscope system (Olympus, Tokyo, Japan) or a
Zeiss LSM510 Laser Scanning Microscope using a Zeiss 100 × 1.3 NA
oil immersion lens.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
To observe the subcellular localization of
cPLA2
, CHO cells were transiently transfected with a
vector coding for cPLA2
fused to GFP
(GFP-cPLA2
), and the distribution of the chimeric proteins was examined. GFP-cPLA2
was located in the
nuclear envelope and in an extensive reticular pattern that was typical
for ER localization (Fig. 1A).
These structures were identified to be ER and Golgi by loading the
cells with BODIPY Brefeldin A, which recognizes both ER and Golgi (24)
(Fig. 1B). GFP-cPLA2
did not colocalize with
a mitochondrial marker, MitoTracker Red (Fig. 1B). We also
confirmed the absence of cPLA2
in the lysosome using a
lysosomal marker (data not shown). Although we have previously shown
different subcellular localization of GFP-cPLA2
in a
preliminary experiment (56), the localization turned out to be caused
by a peroxisomal targeting signal generated incidentally in the linker region of the chimeric proteins. Western blotting analysis of protein
extracts from cPLA2
-overexpressing HEK293 cells
confirmed the assignment of the localization of cPLA2
to
the microsomal fraction (data not shown), in accordance with the
results reported previously for cPLA2
expressed in CHO
cells (19) and Sf9 (20). These results suggest that
cPLA2
is localized predominantly in the ER and Golgi
membranes.
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Fig. 1.
Subcellular localization of
cPLA2 . A, CHO cells
transiently transfected with GFP-cPLA2
were visualized
under a fluorescence microscope at a wavelength of 488 nm for GFP.
B, CHO cells transiently transfected with
GFP-cPLA2
were incubated with 1 µM BODIPY
Brefeldin A or 100 nM MitoTracker Red CMXRos in Hanks'
balanced salt solution containing 10 mM HEPES and 0.1% BSA
for 20 min at 37 °C. The fields shown were visualized under a
confocal microscope at appropriate wavelengths for BODIPY Brefeldin A,
MitoTracker, and GFP, and the two images were overlaid
(Merge).
Changes the Fatty Acid Composition of PE in
Transfected Cells--
Although the enzymatic properties of
cPLA2
in vitro have previously been
characterized (19-21), its roles at the cellular level have not been
clarified. First, we hypothesized that cPLA2
might
change the phospholipid composition in the membrane, because the enzyme
is membrane-bound, possesses Ca2+-independent
PLA2 activity (19-21), and is localized in the ER (Fig. 1)
where phospholipid biosynthesis occurs. To test this possibility and
also to determine intracellular substrates for cPLA2
, we
analyzed the molecular species of phospholipids in HEK293 cells stably
expressing cPLA2
or control cells by ESI-MS.
-overexpressing HEK293 cells. In contrast,
saturated or monounsaturated fatty acid species such as diacyl
16:0-16:1 (m/z 688.6), diacyl 16:0-18:1 (m/z 716.6) and diacyl 18:0-18:0
(m/z 746.7) were unchanged or rather decreased in
cPLA2
-expressing HEK293 cells (Fig.
2A). On the other hand,
analysis of the phosphatidylcholine (PC) molecular species in the
positive ion mode demonstrated no major difference between the two cell
lines (Fig. 2B).
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Fig. 2.
ESI-MS analysis of phospholipid species in
cPLA2 -overexpressing and control
HEK293 cells. Total lipids were extracted from both cells and then
subjected to lipid analysis using ESI-MS. A, the ESI-MS
spectra of PE from both cells in the negative ion scan mode are shown.
The values representing 100% of the y-axis for
cPLA2
and the control are 1.03 and 1.08 × 104 eV, respectively. * indicates the major differences
between the cells. B, the ESI-MS spectra of
phosphatidylcholine (PC) from both cells in the positive ion
scan mode are shown. The values representing 100% of the
y-axis for cPLA2
and the control are 6.04 and
6.45 × 106 eV, respectively. alk denotes
either alkenyl or alkyl in panel A and alkyl in
panel B.
Enhances H2O2-induced
AA Release--
To search for stimuli which regulate
cPLA2
activity at the cellular level, we examined the
effect of various compounds on AA release and found that
H2O2 enhanced AA release. As shown in Fig.
3A, 1 mM
H2O2 enhanced AA release from cells in a
cPLA2
-dependent manner. Although the effect
was also detected in control cells, much higher release was observed in
cPLA2
-overexpressing cells. Addition of glucose and
glucose oxidase to the medium also enhanced AA release from cells
expressing cPLA2
(data not shown). These effects were
completely inhibited by adding catalase to the reaction medium. Other
ROS, such as cumene hydroperoxide, enhanced the H2O2-induced AA release in a
cPLA2
-dependent manner (data not shown).
4-Hydroxy 2-nonenal, a major oxidized product of fatty acids or hydroxy
radicals produced by the combination of Fe2+ and
H2O2, did not induce AA release (data not
shown). The effect of H2O2 on AA release was
evident at 5 min and lasted for at least 30 min (data not shown). Under
the assay conditions, no significant loss or floating of cells from the
plates were seen (data not shown). Similar results were obtained in
three independent stable clones.
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Fig. 3.
H2O2-induced AA
release. A, HEK293 cells expressing
cPLA2 or the control vector were labeled with
[14C]AA, washed, and incubated for 30 min with control
medium (see "Experimental Procedures"), 1 mM
H2O2, or 1 mM
H2O2 and catalase (1000 units/ml) in the
control medium. B, cells labeled with [14C]AA
were preincubated for 2 h with or without 100 µM
MAFP, washed, and incubated for 30 min with the control medium only or
with 1 mM H2O2 in the control
medium. The amounts of [14C]AA released into the
supernatants were assayed as described under "Experimental
Procedures." The results are the means ± S.E. of three
experiments.
in the
H2O2-induced AA release, we tested the effect
of MAFP, which inhibits both cPLA2
and
Ca2+-independent PLA2 (iPLA2) and
has also been reported to inhibit cPLA2
(21).
Pretreatment with 100 µM MAFP exerted a significant inhibitory effect on the H2O2-induced AA
release from cPLA2
-overexpressing cells. MAFP also
inhibited H2O2-induced AA release from control cells, probably through inhibition of intrinsic cPLA2
or
iPLA2.
by
H2O2 in vitro (data not shown). ROS
cause protein phosphorylation through the activation of protein
tyrosine kinase (PTK) and protein kinase C (PKC) in different cell
types (25-27), including vascular smooth muscle and cultured
endothelial cells. We then tested various inhibitors that regulate the
tyrosine or serine/threonine phosphorylation. As shown in Fig.
4, calphostin C, an inhibitor of PKC, did
not have any inhibitory effects on H2O2-induced
AA release but rather a slightly up-regulatory effect. On the other
hand, herbimycin A, a PTK inhibitor, inhibited
H2O2-induced AA release, whereas orthovanadate
(Na3VO4), a protein tyrosine phosphatase
inhibitor, enhanced H2O2-induced AA release. We
could not detect any effect induced by addition of
Na3VO4 by itself (data not shown). Thus, cPLA2
enhanced H2O2-induced AA
release through a tyrosine phosphorylation pathway. We could not detect
the direct tyrosine phosphorylation of cPLA2
by
immunoprecipitation and Western blotting (data not shown). The
mechanisms involved in this activation process remain to be
elucidated.
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Fig. 4.
Effects of various inhibitors on
H2O2-induced AA release by
cPLA2 . A and
B, HEK293 cells expressing cPLA2
or the
control vector were labeled with [14C]AA, preincubated
for 2 h with or without 500 nM calphostin C
(A) or 1 µM herbimycin A (B),
washed, and incubated for 30 min with medium only or with 1 mM H2O2. C, HEK293 cells
expressing cPLA2
or the control vector were labeled with
[14C]AA, washed, and incubated for 30 min with the
control medium, 1 mM H2O2, or 1 mM H2O2 and 100 µM
orthovanadate. The amounts of [14C]AA released into the
supernatants were assayed as described under "Experimental
Procedures." The results are the means ± S.E. of three
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is localized in the ER and Golgi, where membrane
phospholipids are abundantly produced. 2) expression of
cPLA2
changes the fatty acid composition in PE but not
in PC, as determined by ESI-MS. 3) cPLA2
is involved in
H2O2-induced AA release.
changed the fatty acid composition in PE (Fig. 2) suggests that
cPLA2
has a substrate preference for PE in intact cells.
In an in vitro assay using cell lysates derived from HEK293
cells overexpressing cPLA2
, the enzyme preferred
PE over PC or
phosphatidylinositol.2 The
precise mechanism by which a relative abundance of PUFA at the
sn-2 position of PE was produced in
cPLA2
-overexpressing cells (Fig. 2A) remains
uncertain. Saturated or monounsaturated fatty acids at the
sn-2 position of PE may be preferentially hydrolyzed by the
PLA2 activity of cPLA2
, and the derived
lysoPE may be reacylated with PUFA by other enzymes with
acyltransferase activities. Alternatively, cPLA2
may
have transacylase or acyltransferase activity in itself, as in the case
of other PLA2s (28, 29). Lysophospholipase activity of
cPLA2
(21) may also contribute to the remodeling of the phospholipids.
. The membranes of the ER and
Golgi, especially those of the smooth ER, constantly participate in
protein and lipid mass transport by local vesicle formation and fusion.
cPLA2
may be involved in the local vesicle formation and
fusion as suggested for other PLA2s (34, 35). As we only
determined the subcellular localization of cPLA2
by the
overexpression system, we have to await definite localization in native
cells until a good quality antibody is available.
suggests that ROS regulate the activity of cPLA2
at the
cellular level. To determine whether the activity of
cPLA2
can be regulated at the cellular level, we
searched for stimuli that enhanced the AA release and found H2O2 to be a candidate.
H2O2 is one of the highly important ROS because
of its ability to penetrate cellular membranes; as a precursor of the
hydroxy radical, a powerful free radical, it can cause severe oxidative
damage to membrane phospholipids, especially by oxidizing
polyunsaturated fatty acids at the sn-2 position. Any
oxidative modification of membrane phospholipids is a deleterious process, altering membrane fluidity, protein structure and cell signaling (36-38). The best way for repairing the phospholipids is the
selective cleavage of the peroxidized fatty acid residues and their
subsequent replacement by native fatty acids. Although several studies
have shown that ROS cause AA release from cells through
PLA2, such as iPLA2 or cPLA2
(39-49), capabilities in these enzymes for selective cleavage of the
peroxidized fatty acid residues have not been addressed to date. So
far, the platelet-activating factor acetylhydrolases of type II, which
cleave preferentially peroxidized or lipoxygenated phospholipids, are
proposed to be competent for the repair (47, 50). Taken together,
cPLA2
may release AA in response to oxidative stress and
increase the AA level to repair the oxidized fatty acids in
phospholipids. Furthermore, considering the major tissue distribution
of cPLA2
in skeletal muscle and heart, it may contribute
to the repair of the oxidized phospholipids in these distinct tissues
in which oxidative stress on the unique phospholipid compositions were noted (51, 52).
, at least three possibilities exist: 1)
modification of the enzyme itself or associated molecules; 2) changes
in the phospholipids environment; and 3) existence of a recognition
mechanism for the oxidized product or oxidative state. Several signal
transduction pathways in cultured mammalian cells have been activated
by the application of H2O2, including PTKs,
mitogen-activated protein (MAP) kinases, PKC isoforms, and the
epidermal growth factor receptor (25-27). For example,
cPLA2
is reported to be activated by
H2O2 through the activation of extracellular
signal-regulated kinase and p38 MAP kinase in mesangial cells (46).
Unlike cPLA2
, where MAP kinase phosphorylation sites
exist (53-55), cPLA2
is postulated to possess consensus
sequence for PKC and PTK phosphorylation. In this work we showed that
cPLA2
is activated by H2O2
through the tyrosine phosphorylation pathway, as demonstrated by the
inhibitor assays. In our preliminary experiments we could not detect
direct tyrosine phosphorylation of cPLA2
by
immunoprecipitation and Western blotting (data not shown). These
results suggest that there may be associated molecules that regulate
the cPLA2
activity in response to the oxidative stress.
Other possibilities remain to be explored.
, including PE preference
in vivo, localization in the ER and Golgi, and involvement
of H2O2-induced AA release. Herein we have
elucidated the possible roles of cPLA2
in mammalian
cells, i.e. membrane remodeling and oxidative stress-induced AA release. The physiological and pathological functions of the enzyme
would be clarified by analyzing cPLA2
knockout mice.
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ACKNOWLEDGEMENTS |
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We thank Drs. M. Murakami and I. Kudo at Showa University for invaluable suggestions and Drs. S. Hoshiko, F. Osawa, and Y. Akamatsu at the Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd. for the encouragement they gave us.
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FOOTNOTES |
---|
* This work was supported in part by Grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology and by the Human Frontier Special Program.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.
To whom correspondence should be addressed. Tel.:
81-3-5802-2925; Fax: 81-3-813-8732; E-mail:
tshimizu@m.u-tokyo.ac.jp.
Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.M212117200
2 K. Asai, T. Hirabayashi, N. Uozumi, and T. Shimizu, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
PLA2, phospholipase A2;
AA, arachidonic acid;
BSA, bovine
serum albumin;
CHO, Chinese hamster ovary;
cPLA2, cytosolic PLA2
(group IVA phospholipase A2);
cPLA2
, group IVC phospholipase A2;
DMEM, Dulbecco's modified Eagle's medium;
ER, endoplasmic reticulum;
ESI-MS, electrospray ionization mass spectrometry;
GFP, green
fluorescence protein;
HEK293, human embryonic kidney 293;
iPLA2, Ca2+-independent phospholipase
A2;
MAFP, methyl arachidonyl fluorophosphonate;
MAP, mitogen-activated protein;
PC, phosphatidylcholine;
PE, phosphatidylethanolamine;
PKC, protein kinase C;
PTK, protein tyrosine
kinase;
PUFA, polyunsaturated fatty acid;
ROS, reactive oxygen
species.
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