Human Group IVC Phospholipase A2 (cPLA2gamma )

ROLES IN THE MEMBRANE REMODELING AND ACTIVATION INDUCED BY OXIDATIVE STRESS*

Kenji AsaiDagger §, Tetsuya HirabayashiDagger , Toshiaki Houjou, Naonori UozumiDagger , Ryo Taguchi, and Takao ShimizuDagger ||

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (cPLA2gamma ), a paralogue of cytosolic PLA2alpha (cPLA2alpha ), 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 cPLA2gamma 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 cPLA2gamma -transfected human embryonic kidney 293 cells, possibly through the tyrosine phosphorylation pathway. Thus, we propose that cPLA2gamma is constitutively expressed in the endoplasmic reticulum and plays important roles in remodeling and maintaining membrane phospholipids under various conditions, including oxidative stress.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, cPLA2alpha 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).

In contrast, the information on cPLA2gamma , a Ca2+-independent paralogue of cPLA2alpha , is limited. It has been reported that cPLA2gamma 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 cPLA2gamma 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 cPLA2gamma , possibly through the tyrosine phosphorylation pathway.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 cPLA2gamma -- 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 cPLA2gamma were inserted into pcDNA3.1/His to obtain pcDNA3.1/His-cPLA2gamma . For confocal microscopy studies, cPLA2gamma was subcloned into pEGFP-C1, encoding a green fluorescence protein (GFP) at the N terminus between the BglII and SmaI sites to obtain pEGFP-cPLA2gamma .

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 cPLA2gamma , the cells were transfected with pcDNA3.1/His-cPLA2gamma , selected with 600 µg/ml G418 (Invitrogen), and maintained in the presence of 300 µg/ml G418. The expression of cPLA2gamma was confirmed by immunoblotting. HEK293 cells transfected with the vector alone were also kept in medium with G418 and used as a vector control.

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-cPLA2gamma fusion protein (pEGFP-cPLA2gamma ) 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-cPLA2gamma were incubated with 1 µM BODIPY Brefeldin A (lambda ex = 558 nm, lambda em = 568 nm) or 100 nM MitoTracker Red CMXRos (lambda ex = 579 nm, lambda 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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Subcellular Localization of cPLA2gamma -- To observe the subcellular localization of cPLA2gamma , CHO cells were transiently transfected with a vector coding for cPLA2gamma fused to GFP (GFP-cPLA2gamma ), and the distribution of the chimeric proteins was examined. GFP-cPLA2gamma 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-cPLA2gamma did not colocalize with a mitochondrial marker, MitoTracker Red (Fig. 1B). We also confirmed the absence of cPLA2gamma in the lysosome using a lysosomal marker (data not shown). Although we have previously shown different subcellular localization of GFP-cPLA2gamma 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 cPLA2gamma -overexpressing HEK293 cells confirmed the assignment of the localization of cPLA2gamma to the microsomal fraction (data not shown), in accordance with the results reported previously for cPLA2gamma expressed in CHO cells (19) and Sf9 (20). These results suggest that cPLA2gamma is localized predominantly in the ER and Golgi membranes.


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Fig. 1.   Subcellular localization of cPLA2gamma . A, CHO cells transiently transfected with GFP-cPLA2gamma were visualized under a fluorescence microscope at a wavelength of 488 nm for GFP. B, CHO cells transiently transfected with GFP-cPLA2gamma 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).

cPLA2gamma Changes the Fatty Acid Composition of PE in Transfected Cells-- Although the enzymatic properties of cPLA2gamma in vitro have previously been characterized (19-21), its roles at the cellular level have not been clarified. First, we hypothesized that cPLA2gamma 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 cPLA2gamma , we analyzed the molecular species of phospholipids in HEK293 cells stably expressing cPLA2gamma or control cells by ESI-MS.

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 cPLA2gamma -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 cPLA2gamma -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 cPLA2gamma -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 cPLA2gamma 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 cPLA2gamma 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.

cPLA2gamma Enhances H2O2-induced AA Release-- To search for stimuli which regulate cPLA2gamma 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 cPLA2gamma -dependent manner. Although the effect was also detected in control cells, much higher release was observed in cPLA2gamma -overexpressing cells. Addition of glucose and glucose oxidase to the medium also enhanced AA release from cells expressing cPLA2gamma (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 cPLA2gamma -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 cPLA2gamma 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.

To further address the involvement of cPLA2gamma in the H2O2-induced AA release, we tested the effect of MAFP, which inhibits both cPLA2alpha and Ca2+-independent PLA2 (iPLA2) and has also been reported to inhibit cPLA2gamma (21). Pretreatment with 100 µM MAFP exerted a significant inhibitory effect on the H2O2-induced AA release from cPLA2gamma -overexpressing cells. MAFP also inhibited H2O2-induced AA release from control cells, probably through inhibition of intrinsic cPLA2alpha or iPLA2.

Effects of Various Inhibitors on H2O2-induced AA Release-- We investigated H2O2-initiated signaling pathways, because we could not detect the activation of cPLA2gamma 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, cPLA2gamma enhanced H2O2-induced AA release through a tyrosine phosphorylation pathway. We could not detect the direct tyrosine phosphorylation of cPLA2gamma 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 cPLA2gamma . A and B, HEK293 cells expressing cPLA2gamma 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 cPLA2gamma 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The major findings of the present study are as follows. 1) cPLA2gamma is localized in the ER and Golgi, where membrane phospholipids are abundantly produced. 2) expression of cPLA2gamma changes the fatty acid composition in PE but not in PC, as determined by ESI-MS. 3) cPLA2gamma is involved in H2O2-induced AA release.

ESI-MS analysis showing that the expression of cPLA2gamma changed the fatty acid composition in PE (Fig. 2) suggests that cPLA2gamma has a substrate preference for PE in intact cells. In an in vitro assay using cell lysates derived from HEK293 cells overexpressing cPLA2gamma , 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 cPLA2gamma -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 cPLA2gamma , and the derived lysoPE may be reacylated with PUFA by other enzymes with acyltransferase activities. Alternatively, cPLA2gamma may have transacylase or acyltransferase activity in itself, as in the case of other PLA2s (28, 29). Lysophospholipase activity of cPLA2gamma (21) may also contribute to the remodeling of the phospholipids.

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 cPLA2gamma . 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. cPLA2gamma 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 cPLA2gamma by the overexpression system, we have to await definite localization in native cells until a good quality antibody is available.

H2O2-induced AA release by cPLA2gamma suggests that ROS regulate the activity of cPLA2gamma at the cellular level. To determine whether the activity of cPLA2gamma 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 cPLA2alpha (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, cPLA2gamma 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 cPLA2gamma 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).

Regarding the mechanism by which H2O2 activates cPLA2gamma , 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, cPLA2alpha is reported to be activated by H2O2 through the activation of extracellular signal-regulated kinase and p38 MAP kinase in mesangial cells (46). Unlike cPLA2alpha , where MAP kinase phosphorylation sites exist (53-55), cPLA2gamma is postulated to possess consensus sequence for PKC and PTK phosphorylation. In this work we showed that cPLA2gamma 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 cPLA2gamma by immunoprecipitation and Western blotting (data not shown). These results suggest that there may be associated molecules that regulate the cPLA2gamma activity in response to the oxidative stress. Other possibilities remain to be explored.

In conclusion, we have provided several new insights into the properties of human cPLA2gamma , 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 cPLA2gamma 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 cPLA2gamma knockout mice.

    ACKNOWLEDGEMENTS

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.

    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.

    ABBREVIATIONS

The abbreviations used are: PLA2, phospholipase A2; AA, arachidonic acid; BSA, bovine serum albumin; CHO, Chinese hamster ovary; cPLA2alpha , cytosolic PLA2alpha (group IVA phospholipase A2); cPLA2gamma , 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.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Dircks, L., and Sul, H. S. (1999) Prog. Lipid Res. 38, 461-479[CrossRef][Medline] [Order article via Infotrieve]
2. Farooqui, A. A., Horrocks, L. A., and Farooqui, T. (2000) Chem. Phys. Lipids 106, 1-29[CrossRef][Medline] [Order article via Infotrieve]
3. Lands, W. E. (2000) Biochim. Biophys. Acta 1483, 1-14[Medline] [Order article via Infotrieve]
4. MacDonald, J. I., and Sprecher, H. (1991) Biochim. Biophys. Acta 1084, 105-121[Medline] [Order article via Infotrieve]
5. Ohvo-Rekila, H., Ramstedt, B., Leppimaki, P., and Slotte, J. P. (2002) Prog. Lipid Res. 41, 66-97[CrossRef][Medline] [Order article via Infotrieve]
6. Tabas, I. (2000) Biochim. Biophys. Acta 1529, 164-174[Medline] [Order article via Infotrieve]
7. Yamashita, A., Sugiura, T., and Waku, K. (1997) J. Biochem. (Tokyo) 122, 1-16[Abstract]
8. Chilton, F. H., Fonteh, A. N., Surette, M. E., Triggiani, M., and Winkler, J. D. (1996) Biochim. Biophys. Acta 1299, 1-15[Medline] [Order article via Infotrieve]
9. Six, D. A., and Dennis, E. A. (2000) Biochim. Biophys. Acta 1488, 1-19[Medline] [Order article via Infotrieve]
10. Murakami, M., and Kudo, I. (2002) J. Biochem. (Tokyo) 131, 285-292[Abstract]
11. Cho, W. (2000) Biochim. Biophys. Acta 1488, 48-58[Medline] [Order article via Infotrieve]
12. Dessen, A., Tang, J., Schmidt, H., Stahl, M., Clark, J. D., Seehra, J., and Somers, W. S. (1999) Cell 97, 349-360[Medline] [Order article via Infotrieve]
13. Glover, S., de Carvalho, M. S., Bayburt, T., Jonas, M., Chi, E., Leslie, C. C., and Gelb, M. H. (1995) J. Biol. Chem. 270, 15359-15367[Abstract/Free Full Text]
14. Schievella, A. R., Regier, M. K., Smith, W. L., and Lin, L. L. (1995) J. Biol. Chem. 270, 30749-30754[Abstract/Free Full Text]
15. Hirabayashi, T., Kume, K., Hirose, K., Yokomizo, T., Iino, M., Itoh, H., and Shimizu, T. (1999) J. Biol. Chem. 274, 5163-5169[Abstract/Free Full Text]
16. Uozumi, N., Kume, K., Nagase, T., Nakatani, N., Ishii, S., Tashiro, F., Komagata, Y., Maki, K., Ikuta, K., Ouchi, Y., Miyazaki, J., and Shimizu, T. (1997) Nature 390, 618-622[CrossRef][Medline] [Order article via Infotrieve]
17. Sapirstein, A., and Bonventre, J. V. (2000) Biochim. Biophys. Acta 1488, 139-148[Medline] [Order article via Infotrieve]
18. Bonventre, J. V., Huang, Z., Taheri, M. R., O'Leary, E., Li, E., Moskowitz, M. A., and Sapirstein, A. (1997) Nature 390, 622-625[CrossRef][Medline] [Order article via Infotrieve]
19. Pickard, R. T., Strifler, B. A., Kramer, R. M., and Sharp, J. D. (1999) J. Biol. Chem. 274, 8823-8831[Abstract/Free Full Text]
20. Underwood, K. W., Song, C., Kriz, R. W., Chang, X. J., Knopf, J. L., and Lin, L. L. (1998) J. Biol. Chem. 273, 21926-21932[Abstract/Free Full Text]
21. Stewart, A., Ghosh, M., Spencer, D. M., and Leslie, C. C. (2002) J. Biol. Chem. 277, 29526-29536[Abstract/Free Full Text]
22. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-918
23. Taguchi, R., Hayakawa, J., Takeuchi, Y., and Ishida, M. (2000) J. Mass Spectrom. 35, 953-966[CrossRef][Medline] [Order article via Infotrieve]
24. Deng, Y., Bennink, J. R., Kang, H. C., Haugland, R. P., and Yewdell, J. W. (1995) J. Histochem. Cytochem. 43, 907-915[Abstract/Free Full Text]
25. Jin, N., Hatton, N. D., Harrington, M. A., Xia, X., Larsen, S. H., and Rhoades, R. A. (2000) Free Radic. Biol. Med. 29, 736-746[CrossRef][Medline] [Order article via Infotrieve]
26. Konishi, H., Tanaka, M., Takemura, Y., Matsuzaki, H., Ono, Y., Kikkawa, U., and Nishizuka, Y. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11233-11237[Abstract/Free Full Text]
27. Guyton, K. Z., Liu, Y., Gorospe, M., Xu, Q., and Holbrook, N. J. (1996) J. Biol. Chem. 271, 4138-4142[Abstract/Free Full Text]
28. Reynolds, L. J., Hughes, L. L., Louis, A. I., Kramer, R. M., and Dennis, E. A. (1993) Biochim. Biophys. Acta 1167, 272-280[Medline] [Order article via Infotrieve]
29. Lio, Y. C., and Dennis, E. A. (1998) Biochim. Biophys. Acta 1392, 320-332[Medline] [Order article via Infotrieve]
30. Lykidis, A., Baburina, I., and Jackowski, S. (1999) J. Biol. Chem. 274, 26992-27001[Abstract/Free Full Text]
31. Spencer, A. G., Woods, J. W., Arakawa, T., Singer, I. I, and Smith, W. L. (1998) J. Biol. Chem. 273, 9886-9893[Abstract/Free Full Text]
32. Rusinol, A. E., Cui, Z., Chen, M. H., and Vance, J. E. (1994) J. Biol. Chem. 269, 27494-27502[Abstract/Free Full Text]
33. Henneberry, A. L., Wright, M. M., and McMaster, C. R. (2002) Mol. Biol. Cell 13, 3148-3161[Abstract/Free Full Text]
34. de Figueiredo, P., Drecktrah, D., Katzenellenbogen, J. A., Strang, M., and Brown, W. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8642-8647[Abstract/Free Full Text]
35. Choukroun, G. J., Marshansky, V., Gustafson, C. E., McKee, M., Hajjar, R. J., Rosenzweig, A., Brown, D., and Bonventre, J. V. (2000) J. Clin. Invest. 106, 983-993[Abstract/Free Full Text]
36. Imai, K., Aimoto, T., Shima, T., Nakashima, T., Sato, M., and Kimura, R. (2000) Biol. Pharm. Bull. 23, 415-419[Medline] [Order article via Infotrieve]
37. Rapoport, S. M., and Schewe, T. (1986) Biochim. Biophys. Acta 864, 471-495[Medline] [Order article via Infotrieve]
38. Uchida, K., Shiraishi, M., Naito, Y., Torii, Y., Nakamura, Y., and Osawa, T. (1999) J. Biol. Chem. 274, 2234-2242[Abstract/Free Full Text]
39. Birbes, H., Gothie, E., Pageaux, J. F., Lagarde, M., and Laugier, C. (2000) Biochem. Biophys. Res. Commun. 276, 613-618[CrossRef][Medline] [Order article via Infotrieve]
40. Martinez, J., and Moreno, J. J. (2001) Arch Biochem. Biophys. 392, 257-262[CrossRef][Medline] [Order article via Infotrieve]
41. Buschbeck, M., Ghomashchi, F., Gelb, M. H., Watson, S. P., and Borsch-Haubold, A. G. (1999) Biochem. J. 344, 359-366[CrossRef][Medline] [Order article via Infotrieve]
42. Mori, A., Yasuda, Y., Murayama, T., and Nomura, Y. (2001) Eur. J. Pharmacol. 417, 19-25[CrossRef][Medline] [Order article via Infotrieve]
43. McHowat, J., Swift, L. M., Arutunyan, A., and Sarvazyan, N. (2001) Cancer Res. 61, 4024-4029[Abstract/Free Full Text]
44. Cummings, B. S., McHowat, J., and Schnellmann, R. G. (2002) Am. J. Physiol. Renal Physiol 283, F492-498[Abstract/Free Full Text]
45. Rao, G. N., Runge, M. S., and Alexander, R. W. (1995) Biochim Biophys Acta 1265, 67-72[Medline] [Order article via Infotrieve]
46. Hayama, M., Inoue, R., Akiba, S., and Sato, T. (2002) Am. J. Physiol. Renal Physiol 282, F485-F491[Abstract/Free Full Text]
47. Nigam, S., and Schewe, T. (2000) Biochim. Biophys. Acta 1488, 167-181[Medline] [Order article via Infotrieve]
48. Chaitidis, P., Schewe, T., Sutherland, M., Kuhn, H., and Nigam, S. (1998) FEBS Lett. 434, 437-441[CrossRef][Medline] [Order article via Infotrieve]
49. Rashba-Step, J., Tatoyan, A., Duncan, R., Ann, D., Pushpa-Rehka, T. R., and Sevanian, A. (1997) Arch. Biochem. Biophys. 343, 44-54[CrossRef][Medline] [Order article via Infotrieve]
50. Stafforini, D. M., Rollins, E. N., Prescott, S. M., and McIntyre, T. M. (1993) J. Biol. Chem. 268, 3857-3865[Abstract/Free Full Text]
51. Waku, K., Uda, Y., and Nakazawa, Y. (1971) J. Biochem. (Tokyo) 69, 483-491[Medline] [Order article via Infotrieve]
52. Okano, G., Matsuzaka, H., and Shimojo, T. (1980) Biochim. Biophys. Acta 619, 167-175[Medline] [Order article via Infotrieve]
53. Borsch-Haubold, A. G., Bartoli, F., Asselin, J., Dudler, T., Kramer, R. M., Apitz-Castro, R., Watson, S. P., and Gelb, M. H. (1998) J. Biol. Chem. 273, 4449-4458[Abstract/Free Full Text]
54. Lin, L. L., Wartmann, M., Lin, A. Y., Knopf, J. L., Seth, A., and Davis, R. J. (1993) Cell 72, 269-278[Medline] [Order article via Infotrieve]
55. Nemenoff, R. A., Winitz, S., Qian, N. X., Van Putten, V., Johnson, G. L., and Heasley, L. E. (1993) J. Biol. Chem. 268, 1960-1964[Abstract/Free Full Text]
56. Hirabayashi, T., and Shimizu, T. (2000) Biochim. Biophys. Acta 1488, 124-138[Medline] [Order article via Infotrieve]


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