Inhibition of Cytosolic Phospholipase A2 by Annexin I

SPECIFIC INTERACTION MODEL AND MAPPING OF THE INTERACTION SITE*

Seung-Wook KimDagger §, Hae Jin RheeDagger , Jesang KoDagger , Yeo Jeong KimDagger , Hyung Gu Kim||, Jai Myung Yang||, Eung Chil Choi§**, and Doe Sun NaDagger **

From the Dagger  Department of Biochemistry, College of Medicine, University of Ulsan, 388-1 Poongnap-dong, Songpa-gu, Seoul 138-736, the § College of Pharmacy, Seoul National University, Sinrim-dong, Kwanak-gu, Seoul 151-742, and the || Department of Life Science, Sogang University, Shinsu-Dong, Mapo-gu, Seoul 121-742, South Korea

Received for publication, October 31, 2000, and in revised form, February 7, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Annexins (ANXs) display regulatory functions in diverse cellular processes, including inflammation, immune suppression, and membrane fusion. However, the exact biological functions of ANXs still remain obscure. Inhibition of phospholipase A2 (PLA2) by ANX-I, a 346-amino acid protein, has been observed in studies with various forms of PLA2. "Substrate depletion" and "specific interaction" have been proposed for the mechanism of PLA2 inhibition by ANX-I. Previously, we proposed a specific interaction model for inhibition of a 100-kDa porcine spleen cytosolic form of PLA2 (cPLA2) by ANX-I (Kim, K. M., Kim, D. K., Park, Y. M., and Na, D. S. (1994) FEBS Lett. 343, 251-255). Herein, we present an analysis of the inhibition mechanism of cPLA2 by ANX-I in detail using ANX-I and its deletion mutants. Deletion mutants were produced in Escherichia coli, and inhibition of cPLA2 activity was determined. The deletion mutant ANX-I-(1-274), containing the N terminus to amino acid 274, exhibited no cPLA2 inhibitory activity, whereas the deletion mutant ANX-I-(275-346), containing amino acid 275 to the C terminus, retained full activity. The protein-protein interaction between cPLA2 and ANX-I was examined using the deletion mutants by immunoprecipitation and mammalian two-hybrid methods. Full-length ANX-I and ANX-I-(275-346) interacted with the calcium-dependent lipid-binding domain of cPLA2. ANX-I-(1-274) did not interact with cPLA2. Immunoprecipitation of A549 cell lysate with anti-ANX-I antibody resulted in coprecipitation of cPLA2. These results are consistent with the specific interaction mechanism rather than the substrate depletion model. ANX-I may function as a negative regulator of cPLA2 in cellular signal transduction.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Annexins (ANXs)1 are structurally related, calcium-dependent, phospholipid-binding proteins that have been implicated in diverse cellular roles, including anti-inflammation, membrane fusion, differentiation, exocytosis, calcium channels, and interaction with cytoskeletal proteins (reviewed in Refs. 1 and 2). These proteins are defined structurally by a conserved core domain that contains either four or eight repeating units of ~70 amino acids each (3, 4). The conserved repeats account for the shared abilities of ANXs to bind phospholipids in a calcium-dependent manner, whereas the specific functions of each ANX are probably related to their type-specific N-terminal regions. Despite definitive structural characterization, the relationship between structure and function or precise biological function has not been well defined for any of the ANXs.

ANX-I, a 37-kDa member of the family, has been proposed as a mediator of the anti-inflammatory actions of glucocorticoids (5). These anti-inflammatory properties have been related to the ability of ANX-I to inhibit phospholipase A2 (PLA2) activity. PLA2 represents a growing family of enzymes with the common function of catalyzing the release of fatty acids from the sn-2-position of membrane phospholipids, thereby providing production of bioactive lipid metabolites and cytoprotective functions (6, 7). PLA2 enzymes can be subdivided into several groups based on their structure and enzymatic characteristics. Secretory PLA2 (sPLA2) enzymes are low molecular mass (14-18 kDa) enzymes with little fatty acid specificity that require a millimolar calcium concentration for catalysis. Types IIA and V sPLA2 isozymes are known to play a role in arachidonic acid release by certain stimuli (8). On the other hand, type VI Ca2+-independent PLA2 has been proposed to participate in fatty acid release associated with phospholipid remodeling (9, 10). In contrast, type IV cytosolic PLA2 (cPLA2) is a ubiquitously distributed 85-100-kDa enzyme, the activation of which has been shown to be tightly regulated by growth factors and pro-inflammatory cytokines. cPLA2 requires a submicromolar Ca2+ concentration for effective hydrolysis of its substrate, arachidonic acid-containing glycerophospholipids (11, 12). This requirement is associated with the C2 domain in the N terminus of cPLA2 that mediates calcium-dependent phospholipid binding and translocation of cPLA2 from the cytosol to membranes (13). In addition to the calcium-dependent translocation, cPLA2 is phosphorylated by kinases of the mitogen-activated protein kinase family (14), which is one of the important regulatory mechanisms for in vivo activation of cPLA2 (15, 16).

The mechanism by which ANX-I inhibits PLA2 is not fully understood. Most studies, which have been performed using a 14-kDa sPLA2, supported the "substrate depletion" model rather than the "specific interaction" model (17). In the presence of calcium, ANX-I tightly binds to negatively charged phospholipid substrates, which results in substrate depletion and apparent cPLA2 inhibition (18). To the contrary, our recent study using cPLA2 isolated from porcine spleen showed that ANX-I inhibited cPLA2 by specific interaction (19).

An increasing number of reports have suggested that cPLA2 is a key enzyme responsible for signal transduction in inflammation, cytotoxicity, and mitogenesis (6, 7). ANX-I suppresses cPLA2 activity not only in vitro (21, 22), but also in cultured cells (23, 24). Thus, ANX-I may function as an endogenous negative regulator of cPLA2. Herein, we have studied the inhibition of cPLA2 by ANX-I in detail. Deletion mutants of ANX-I were constructed, and enzymatic studies were performed. Also, the protein-protein interaction between cPLA2 and ANX-I, a prerequisite for the specific interaction mechanism, was investigated.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- 1-Stearoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine (2-AA-PC; 56.0 mCi/mmol) was purchased from Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom) and used as the substrate. Unlabeled 2-AA-PC was purchased from Sigma. Scintillation fluid (Aquasol-2) was obtained from Molecular Probes, Inc. (Eugene, OR). Rabbit antiserum was raised against human ANX-I produced in Escherichia coli. Mouse anti-ANX-I antibody was purchased from Transduction Laboratories (Lexington, KY). Mouse anti-cPLA2 antibody was a product of Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Preparation of ANX-I Deletion Mutants-- Cloning of ANX-I cDNA and expression in E. coli have been described (3, 25). Briefly, ANX-I cDNA was selected by colony hybridization from a human placenta cDNA library. ANX-I cDNA was then cloned into plasmid pET-28a(+) (Novagen, Madison, WI) and expressed in E. coli. Full-length ANX-I and N-terminally deleted ANX-I were cloned into the NcoI and SalI sites of pET-28a by cloning procedures that do not involve PCR. The deletion mutants ANX-I-(1-274) and ANX-I-(1-196) were cloned by PCR amplification of the DNA fragment, followed by insertion into the NcoI and SalI sites of pET-28a. The DNA fragment encoding amino acids 1-274 was amplified using primers TTAccatggCAATGGTATCAG and TTAgtcgacTCATTTGCTTGTGGCGCA (lowercase letters represent the restriction enzyme sites). The DNA fragment encoding amino acids 1-196 was amplified using primers TTAccatggCAATGGTATCAG and TTAgtcgacTCATTCATTCACACCAAA. PCR-amplified clones were verified by nucleotide sequencing. ANX-I and the mutants were purified according to methods previously described (25).

Production of Glutathione S-Transferase (GST) Fusion Proteins-- Various deletion mutants of ANX-I were constructed as GST fusion proteins by inserting the full-length or deleted ANX-I cDNA into plasmid pGEX-5X-1 (Amersham Pharmacia Biotech). Portions of the ANX-I cDNA were isolated by PCR and subcloned into the BamHI and XhoI sites (full-length and C-terminal deletion mutants) or the EcoRI and XhoI sites (N-terminal deletion mutants) of pGEX-5X-1. The sequences of the PCR primers for GST-ANX-I-(1-346) were ATTggatccTTATGGCAATGGTATC (primer F1) and TTActcgagGTTTCCTCCACAAAG (primer R1). To clone the C-terminal deletion mutants, primer F1 was used as a common forward primer, and the reverse primers were as follows: GST-ANX-I-(1-274), TTActcgagTTTGCTTGTGGCGCA; GST-ANX-I-(1-196), TTActcgagTTCATTCACACCAAA; GST-ANX-I-(1-114), TTActcgagAGTTTTTAGCAGAGC; and GST-ANX-I-(1-33), TTActcgagTCCGGGACCACCTTT. To clone the N-terminal deletion mutants, primer R1 was used as a common reverse primer, and the forward primers were as follows: GST-ANX-I-(34-346), ATTgaattcTCAGCGGTGAGCCCCTAT; GST-ANX-I-(115-346), ATTgaattcCCAGCGCAATTTGATGCT; GST-ANX-I-(197-346), ATTgaattcGACTTGGCTGATTCAGAT; and GST-ANX-I-(275-346), ATTgaattcCCAGCTTTCTTTGCAGAG.

All PCR-amplified clones were verified by nucleotide sequencing. GST fusion proteins were expressed and purified according to the manufacturer's instructions. The E. coli lysate was bound to a glutathione-Sepharose 4B column (Amersham Pharmacia Biotech) and washed three times with phosphate-buffered saline. GST fusion proteins were eluted with 10 mM reduced glutathione in 50 mM Tris-HCl (pH 8.0) and dialyzed against Tris-buffered saline containing 10% glycerol. The protein concentration was determined by the Bradford method (26).

Preparation of PLA2 Enzymes-- Bee venom sPLA2 was purchased from Sigma. A 100-kDa cPLA2 was partially purified from porcine spleen according to previously described methods (27). Since purification of cPLA2 from porcine spleen is laborious and time-consuming, we cloned cPLA2 cDNA into the baculovirus vector pFastBacHTa (Life Technologies, Inc.) and produced cPLA2 in Sf9 cells. cPLA2 cDNA was cloned into plasmid pGEMT (Promega, Madison, WI) by reverse transcription-PCR from U937 cell mRNA using primers ATTgtcgacATGTCATTTATAGATCC and TAAaagcttCTATGCTTTGGGTTTACTTAG.

The nucleotide sequence was verified by DNA sequencing. Plasmid pGEMT-cPLA2 was digested with SalI and HindIII, and the insert was cloned into the SalI and HindIII sites of pFastBacHTa to produce pBac-cPLA2. To induce transposition between pBac-cPLA2 and Autographa californica nuclear polyhedrin virus DNA, pBac-cPLA2 was transformed into E. coli DH10Bac (maximum efficiency, Life Technologies, Inc.), which harbors A. californica nuclear polyhedrin virus bacmid. Transformed E. coli was incubated on LB plates containing 50 µg/ml kanamycin, 7 µg/ml gentamycin, 10 µg/ml tetracycline, 100 µg/ml 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside (X-gal), and 40 µg/ml isopropyl-beta -D-thiogalactopyranoside for 24 h. Recombinant bacmid PLA2-containing white colonies were then isolated. Bacmid cPLA2 was transfected into Sf9 cells using CellFectin (Life Technologies, Inc.) and cultured for 72 h, and recombinant virus cPLA2 production was confirmed by PCR. The titer of the recombinant virus cPLA2 in the culture medium was determined by a plaque assay. Fresh Sf9 cells were infected with this culture medium and cultured for 72 h. Cells from a 50-ml culture were lysed in 1 ml of buffer containing 20 mM Tris-HCl (pH 7.4), 10% glycerol, 1% Nonidet P-40, 0.1% BSA, 0.1 mM phenylmethylsulfonyl fluoride, CompleteTM EDTA-free protease inhibitor mixture (Roche Molecular Biochemicals), and 0.1 µM Ca2+ and used as the source of Sf9 cPLA2.

Assay of PLA2 Activity-- PLA2 activity was assayed using sonicated liposomes prepared as described previously (19, 28). A stock solution of the substrate was prepared as follows. The substrate (10-20 nmol) was dried under nitrogen and then suspended in 0.5-1.0 ml of distilled water by sonication (3 × 10 s) in a bath-type sonicator (Ultrasonik 300, The J. M. Ney Company, Broomfield, CT). The standard reaction mixture (200 µl) for the PLA2 assay contained 0.33 nmol (1.65 µM) of radioactive substrate (~39,000 cpm), 200 µg of fatty acid-free BSA, and 10 ng (or an equivalent amount when partially purified enzyme was used) of PLA2 in 75 mM Tris-HCl (pH 7.5). Ten ng of purified cPLA2 yielded ~3000 cpm of the product under the standard conditions with 1 µM Ca2+. When partially purified porcine cPLA2 or the total cell lysate of Sf9 cPLA2 cells was used, the amount of cPLA2 was estimated from the activity. The reaction was started by addition of the enzyme to the reaction mixture. Assays were incubated at 37 °C for 1 h and then stopped by adding 1.25 ml of 2% NH2SO4, 20% n-heptane, and 78% isopropyl alcohol. Non-esterified fatty acid was extracted as follows. First, 0.55 ml of water was added, and the sample was Vortex-mixed and centrifuged at 5600 × g for 5 min. Then, 0.75 ml of the upper phase was transferred to a new tube, to which 100 mg of silica gel and 0.75 ml of n-heptane were added. The samples were Vortex-mixed and centrifuged again for 5 min. The supernatant was dried using a SpeedVac freeze drier, and the lipid was resuspended in chloroform/methanol (1:1, v/v) that contained unlabeled arachidonic acid (1 µg/µl) in methanol. Phospholipid and neutral lipid were separated by migration on layers of Silica Gel 60 F254 plates (Merck, Darmstadt, Germany) in petroleum ether/ethyl ether/acetic acid (80:20:1, v/v/v). After drying, the plates were subjected to iodine vapor, and lipids were identified by their comigration with unlabeled arachidonic acids. Products were quantified by scraping their corresponding spots into counting vials containing 2 ml of Aquasol-2. Radioactivity was determined using a Packard Tri-Carb scintillation spectrophotometer. For analysis in which the substrate concentration dependence was determined, unlabeled phospholipid was added to the labeled phospholipid to produce a designated final concentration. For accurate control of the Ca2+ concentration, a CaCl2/EGTA buffering system was used (29). In all analyses, samples were tested in triplicate and adjusted for nonspecific release by subtracting a control value in which preparation of the enzyme was omitted. For inhibition assays, 5-100 nM ANX-I was added to the reaction mixture.

Effect of ANX-I on PLA2 Activity-- All analyses were performed in triplicate and repeated at least three times. The effect of ANX-I was represented by the percentage of cPLA2 activity compared with the control value. All data shown are means ± S.E. The effect of ANX-I and its deletion mutants on cPLA2 activity was determined by the percentage of PLA2 activity using the following equation: % of PLA2 activity = (cpm test/cpm control) × 100.

Mammalian Two-hybrid Analyses-- Portions of cPLA2 and ANX-I cDNAs were cloned into the mammalian version of the bait and prey vectors, pM for GAL4 fusion and pVP16 for VP16 fusion (CLONTECH, Palo Alto, CA). To generate GAL4 fusion, ANX-I cDNA was subcloned into the BamHI and XbaI sites (full-length and C-terminal deletion mutant) or the EcoRI and XbaI sites (N-terminal deletion mutant) of pM. To generate VP16 fusion, cPLA2 cDNA was subcloned into the SalI and HindIII sites of pVP16. Portions of cPLA2 cDNA containing amino acids 1-80 or 81-793 were amplified with primers ATTgtcgacATGTCATTTATAGATCC and TAAaagcttATCCAAAATAAATTCAAA or, in the case of amino acids 81-793, primers ATTgtcgacCTTAATCAGGAAAATGTT and TAAaagcttTGCTTTGGGTTTACTTAG. The pG5CAT reporter was purchased from CLONTECH. Chloramphenicol acetyltransferase assays (Promega) were performed according to the manufacturer's instructions.

In Vitro Protein Binding of cPLA2-Delta C2 and ANX-I-- The 43 amino acids spanning amino acids 38-80 of the C2 domain of cPLA2 (Delta C2) were amplified by PCR using cPLA2 cDNA as a template and two oligonucleotide primers: ATAggatccATGCTTGATACTCCA and TAAaagcttATCCAAAATAAATTCAAA. After digestion with BamHI and HindIII, the amplified fragment was inserted into the BamHI and HindIII sites of pMAL-P2X, a maltose-biding protein (MBP) fusion vector (New England Biolabs Inc., Beverly, MA), to produce pMAL-Delta C2. The MBP fusion protein MBP-Delta C2 was expressed in E. coli and purified using amylose resin (New England Biolabs Inc.) (30) according to the manufacturer's instructions. The binding mixture contained 2 µg of either ANX-I or deletion mutant, 2 µg of MBP-Delta C2, and 20 µl of amylose resin in 300 µl of 20 mM Tris-HCl (pH 8.0), 30 mM NaCl, 1 mg/ml BSA, 0.1% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1 µM Ca2+. The mixture was incubated for 12 h at 4 °C and centrifuged at 14,00 × g for 15 s at 4 °C. The beads were washed three times with 1 ml of the binding buffer and subjected to 12% SDS-polyacrylamide gel electrophoresis for Western blotting as follows. Proteins were transferred to a nitrocellulose membrane (Schleicher & Schüll), probed with monoclonal antibody against ANX-I, and visualized using the ECL system (Amersham Pharmacia Biotech).

Immunoprecipitation Studies-- Coprecipitation of ANX-I in A549 cell lysate and purified MBP-Delta C2 was examined. A549 cells (2 × 107) were lysed in 200 µl of lysis buffer (20 mM Tris-HCl (pH 7.4), 10% glycerol, 1% Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 µM Ca2+, and CompleteTM EDTA-free protease inhibitor mixture). The binding mixture comprised 100 µl (~100 µg of protein) of A549 cell lysate, 2 µg of MBP-Delta C2, 2 µg of anti-MBP antibody, and 100 µl of binding buffer (75 mM Tris-HCl (pH 7.4), 1 mg/ml BSA, and 0.1 µM Ca2+). After a 4-h incubation at 4 °C, the immune complexes were precipitated with protein A-agarose (Santa Cruz Biotechnology Inc.). The pellet was boiled in the gel loading buffer, and proteins were analyzed by SDS-polyacrylamide gel electrophoresis, followed by Western blotting using anti-ANX-I antibody.

Coprecipitation of cPLA2 in the Sf9 cPLA2 cell lysate and purified GST-ANX-I was examined. Total cell lysate of Sf9 cPLA2 cells was prepared in the same way as described above for the preparation of A549 cell lysate. The binding mixture contained 100 µg of Sf9 cPLA2 cell lysate, 2 µg of ANX-I, 2 µg of anti-GST monoclonal antibody, and 100 µl of binding buffer. The immune complexes were analyzed by Western blotting using anti-cPLA2 antibody (31).

Immunoprecipitation of the cPLA2·ANX-I Complex from A549 Cell Lysate-- Existence of the cPLA2·ANX-I complex in cells was examined using A549 cell lysates. A549 cell lysate was prepared as described above. One-hundred µl of A549 cell lysate was precipitated with anti-ANX-I antibody and protein A-agarose. cPLA2 activity in the precipitate or supernatant was determined in a standard buffer containing 5 mM CaCl2. cPLA2 in the pellet was also analyzed by Western blotting.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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In the previous experiments of cPLA2 inhibition by ANX-I, we used cPLA2 purified from porcine spleen (19). Since purification of cPLA2 from porcine spleen is laborious and results are often inconsistent, human cPLA2 cDNA was cloned into a baculovirus vector and expressed in Sf9 cells. cPLA2 produced in Sf9 cPLA2 cells was characterized by the following methods using porcine spleen cPLA2 as a reference: 1) size determination by Western blot analysis using anti-cPLA2 antibody, 2) activity in the presence of dithiothreitol, and 3) Ca2+ concentration dependence of PLA2 activity. Although sPLA2 activity was sensitive to dithiothreitol, both cPLA2 enzymes from porcine spleen and Sf9 cells were essentially insensitive to the dithiothreitol concentration (data not shown). Sf9 cPLA2 was active at Ca2+ concentrations as low as 0.1 µM and showed nearly identical activity compared with porcine spleen cPLA2 at all Ca2+ concentrations. On the other hand, at least 0.1 mM Ca2+ was necessary for sPLA2 activity (data not shown). Therefore, Sf9 cPLA2 exhibited nearly identical activity compared with porcine spleen cPLA2.

Inhibition of Porcine Spleen cPLA2 by ANX-I and Its Deletion Mutants-- The effects of ANX-I and its deletion mutants on cPLA2 activity were determined. Full-length ANX-I (ANX-I-(1-346)) and the deletion mutants ANX-I-(33-346), ANX-I-(1-274), and ANX-I-(1-196) were cloned, expressed in E. coli, and purified to near homogeneity (Fig. 1A). The effects of ANX-I and its mutants on cPLA2 from porcine spleen were determined at various concentrations of ANX-I and Ca2+ using 2-AA-PC as a substrate. The reaction mixtures were incubated at 37 °C for 1 h, and radiolabeled arachidonic acid, produced by the hydrolyzing reaction of cPLA2, was measured. An incubation time of 1 h was chosen for the following reasons. First, measuring initial rates was prone to more errors due to the small counts/min of the product; and second, the ANX-I inhibition pattern was nearly identical at all time points until 2 h. The substrate concentration was 1.65 µM, which was significantly greater than the enzyme (0.5 nM) and ANX-I (5-100 nM) concentrations. Fig. 1B shows the results of experiments carried out at 1 µM Ca2+. The percentage of the remaining 2-AA-PC hydrolyzing activity in the presence of ANX-I was plotted against the ANX-I concentration. In the presence of either ANX-I-(1-346) or ANX-I-(33-346), PLA2 activity decreased, indicating inhibition of enzymatic activity. On the other hand, ANX-I-(1-274) and ANX-I-(1-196) had no effect on cPLA2 activity. The calcium concentration dependence of inhibition by 20 nM ANX-I was determined at 0.1, 1, 10, and 100 µM and 1 and 10 mM Ca2+. The inhibition by both ANX-I-(1-346) and ANX-I-(33-346) was greatest at 0.1 µM (and 1 µM) calcium and least at 10 mM calcium (Fig. 1C). Both ANX-I-(1-274) and ANX-I-(1-196) had no effect on cPLA2 activity at any calcium concentration. To rule out the possibility that the cPLA2 inhibition by ANX-I-(1-346) or ANX-I-(33-346) was due to substrate depletion, the substrate concentration was varied from 1.65 to 33 µM while holding the other components constant. As shown in Fig. 1D, the inhibition of cPLA2 by both ANX-I-(1-346) and ANX-I-(33-346) was essentially independent of the substrate concentration.


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Fig. 1.   Inhibition of porcine spleen cPLA2 by ANX-I and its deletion mutants. ANX-I-(1-346), ANX-I-(33-346), ANX-I-(1-274), and ANX-I-(1-196) were cloned into pET-28a(+), expressed in E. coli, and purified according to the methods described (25). Partially purified porcine spleen cPLA2 was used. A, ANX-I and its deletion mutants analyzed by 12% SDS-polyacrylamide gel electrophoresis. B, inhibition of cPLA2 by ANX-I and its deletion mutants. cPLA2 inhibition was determined using 2-AA-PC as a substrate. The reaction was carried out at 37 °C for 1 h in 75 mM Tris-HCl (pH 7.5) containing 0.5 nM cPLA2 (10 ng/200 µl), 1.65 µM 2-AA-PC, 1 µM Ca2+, and 1 mg/ml BSA. The concentration of ANX-I was varied from 5 to 100 nM. cPLA2 activity with or without ANX-I was determined, and the percentage of the remaining activity in the presence of ANX-I was calculated. C, calcium concentration dependence of cPLA2 inhibition. The reaction was performed as described for B, except that the substrate and ANX-I concentrations were maintained at 1.65 µM and 20 nM, respectively. The Ca2+ concentration was varied from 0.1 µM to 10 mM. D, substrate concentration dependence of cPLA2 inhibition by ANX-I. Assays were performed at 20 nM ANX and 1 µM calcium with various amounts of substrate. Data represent means ± S.E. of three independent experiments.

Inhibition of Sf9 cPLA2 by GST-ANX-I and Its Deletion Mutants-- The results shown in Fig. 1 demonstrate that the N-terminal 32 amino acids are not important for the cPLA2 inhibitory activity of ANX-I, whereas the C-terminal 72 amino acids are crucial for the inhibitory activity. To further investigate the important region of ANX-I, various deletion mutants were constructed. Attempts to produce ANX-I-(275-346) or ANX-I-(197-346) in E. coli failed due to very low expression levels. Therefore, use of the GST-ANX-I fusion protein was evaluated. The validity of Sf9 cPLA2 instead of porcine spleen cPLA2 was also evaluated. GST-ANX-I-(1-346) exhibited effects identical to those of ANX-I-(1-346) on the activity of cPLA2 from porcine spleen and Sf9 cPLA2 cells (data not shown). Therefore, various ANX-I mutants were constructed as GST fusion proteins and used for inhibition studies. Fig. 2A shows a schematic representation of various GST-ANX-I deletion mutants. All mutants were produced in E. coli, purified on a glutathione-Sepharose 4B column to near homogeneity, and used without cutting off the GST tail (Fig. 2B). Fig. 3 shows the effects of GST-ANX-I mutants on cPLA2 activity. No C-terminal deletion mutants exhibited any inhibitory activity (Fig. 3A), whereas all N-terminal deletion mutants exhibited full inhibitory activity (Fig. 3B). Therefore, the inhibitory activity is located in amino acids 275-346. Inhibition of cPLA2 by GST-ANX-I-(1-346) or GST-ANX-I-(275-346) was further characterized at various GST-ANX-I, substrate, and calcium concentrations. GST-ANX-I showed similar patterns with ANX-I (Fig. 1 versus Fig. 4). GST-ANX-I-(275-346) also showed similar patterns, except that the activity was independent of the calcium concentration (Fig. 4C).


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Fig. 2.   Production of GST-ANX-I and its deletion mutants in E. coli. Various ANX-I deletion mutants were constructed as GST fusion proteins using a GST fusion vector (pGEX-5X-1) as described under "Experimental Procedures." Each protein was produced in E. coli and purified on a glutathione-Sepharose 4B column. A, schematic representation of mutants. Arabic numbers represent the amino acid (aa) number of full-length ANX-I and indicate the positions of the loop regions between domains. N, N-terminal tail of ANX-I. Roman numerals represent the conserved repeat domains. B, 12% SDS-polyacrylamide gel electrophoresis of ANX-I and its deletion mutants. The arrows represent the molecular sizes of the mutants in kilodaltons.


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Fig. 3.   Inhibition of cPLA2 by GST-ANX-I and its deletion mutants. The cell lysate of Sf9 cPLA2 cells was used as a cPLA2 source. GST-ANX-I mutants (shown schematically in Fig. 2) were used. Other details are as described in the legend to Fig. 1. A, C-terminal deletion mutants; B, N-terminal deletion mutants.


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Fig. 4.   Inhibition of cPLA2 by GST-ANX-I-(1-346) and GST-ANX-I-(275-346). Assays were performed at various concentrations of GST-ANX-I (A), substrate (B), or calcium (C). Other details are as described in the legends to Figs. 1 and 3.

Interaction of cPLA2-(1-80) and ANX-I-(275-346) in a Mammalian Two-hybrid System-- The results presented in Figs. 1, 3, and 4 demonstrate that ANX-I inhibits cPLA2 by specific interaction and not by substrate depletion, which requires a protein-protein interaction. A mammalian two-hybrid assay was utilized to investigate the interaction between ANX-I and cPLA2. Deletion mutants of ANX-I and cPLA2 were cloned into a mammalian two-hybrid vector, and the interaction was examined as described under "Experimental Procedures." Since ANX-I-(275-346) inhibited cPLA2 as effectively as full-length ANX-I, the effects of ANX-I-(1-274), ANX-I-(275-346), and full-length ANX-I were examined. The C2 domain of cPLA2 (comprising amino acids 16-138), of which amino acids 38-80 (Delta C2) are highly conserved, is responsible for calcium-dependent phospholipid binding (11, 32). To evaluate the importance of this region, cPLA2-(1-80) and cPLA2-(81-793) were cloned. The mutants were transfected into A549 cells as well as into Rat2 cells, and protein-protein interaction was analyzed. ANX-I-(1-346) interacted with full-length cPLA2 as well as cPLA2-(1-80) in both A549 cells (Fig. 5A) and Rat2 cells (Fig. 5B). ANX-I-(275-346) was almost as effective as ANX-I-(1-346), whereas ANX-I-(1-274) was much less effective for the interaction with cPLA2. Therefore, it can be deduced that the C-terminal 72-amino acid region of ANX-I interacts with the N-terminal 80-amino acid region of cPLA2. This result is consistent with cPLA2 inhibition by ANX-I, shown in Figs. 3 and 4.


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Fig. 5.   Interaction of cPLA2-(1-80) and ANX-I-(275-346) in a mammalian two-hybrid system. The mammalian two-hybrid vectors pVP16 and pM were used to clone cPLA2 and ANX-I, respectively. Deletion mutants of cPLA2 and ANX-I cDNAs were also cloned into the same vector. A549 cells (A) or Rat2 cells (B) were transfected with different combinations of cPLA2 and ANX-I clones. Chloramphenicol acetyltransferase (CAT) assays were performed according to the manufacturer's instructions. C indicates untransfected cells (control); cPLA2 indicates full-length cPLA2 and ANX-I-(1-346); cPLA2-N indicates cPLA2-(1-80); and cPLA-C indicates cPLA2-(81-793).

Coprecipitation of MBP-Delta C2 and GST-ANX-I-- To further investigate the direct interaction between cPLA2 and ANX-I or the deletion mutants, co-immunoprecipitation of Delta C2 with GST-ANX-I was examined. The Delta C2 domain of cPLA2 was produced as a fusion protein with MBP. GST-ANX-I mutants (Fig. 2) and MBP-Delta C2 were produced in E. coli and purified by affinity column chromatography. GST-ANX-I and MBP-Delta C2 were mixed; the mixture was immunoprecipitated with anti-MBP antibody; and the precipitate was analyzed by Western blotting using anti-ANX-I antibody. As shown in Fig. 6A, the C-terminal deletion mutants did not interact with Delta C2, whereas the N-terminal deletion mutants did. These results are in agreement with the cPLA2 inhibition pattern shown in Figs. 3 and 4 and the results presented in Fig. 5. The interaction of cPLA2 and ANX-I was further investigated using purified proteins and cell lysates: MBP-Delta C2 and A549 cell lysate or GST-ANX-I and Sf9 cPLA2 cell lysate. In one experiment, MBP-Delta C2 was mixed with A549 cell lysate, and the mixture was immunoprecipitated with anti-MBP antibody, followed by Western blot analysis using anti-ANX-I antibody. In another experiment, GST-ANX-I-(1-346) or GST-ANX-I-(275-346) was mixed with the Sf9 cPLA2 cell lysate, and the mixture was immunoprecipitated with anti-GST antibody, followed by Western blotting using anti-cPLA2 antibody. As shown in Fig. 6B, ANX-I in the A549 cell lysate coprecipitated with MBP-Delta C2. Both GST-ANX-I-(1-346) and GST-ANX-I-(275-346) coprecipitated with cPLA2 in the Sf9 cPLA2 cell lysate (Fig. 6C).


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Fig. 6.   Coprecipitation of MBP-Delta C2 and GST-ANX-I. A, coprecipitation of GST-ANX-I mutants and MBP-Delta C2. The Delta C2 sequence spanning amino acids 38-80 of cPLA2 was cloned into an MBP fusion vector (pMAL-P2X) to produce the MBP-Delta C2 fusion protein in E. coli. Purified GST-ANX-I mutants and MBP-Delta C2 were mixed and incubated. The mixture was immunoprecipitated (IP) with anti-MBP antibody, and the pellet was analyzed by Western blotting using anti-ANX antibody (Ab). ANX was omitted in lane C (control). B, immunoprecipitation of MBP-Delta C2 and ANX-I in A549 cell lysate. One-hundred µg of A549 cell lysate was incubated with 2 µg of either MBP-Delta C2 or MBP as a control. The mixture was immunoprecipitated with anti-MBP antibody, and the pellet was analyzed by Western blotting using anti-ANX antibody. Lane C, untreated A549 cell lysate. C, immunoprecipitation of GST-ANX-I and cPLA2 in Sf9 cPLA2 cell lysate. One-hundred µg of Sf9 PLA2 cell lysate was incubated with 2 µg of GST-ANX-I-(1-346), GST-ANX-I-(275-346), or GST. The mixture was immunoprecipitated with anti-GST antibody, and the pellet was analyzed by Western blotting using anti-cPLA2 antibody. Lane C, untreated Sf9 PLA2 cell lysate.

Coprecipitation of cPLA2·ANX-I from A549 Cell Lysate-- The results shown in Fig. 6 demonstrate that ANX-I interacts with cPLA2. To determine whether the cPLA2·ANX-I complex exists in vivo, A549 cell lysate was precipitated with anti-ANX-I antibody, and the precipitate was analyzed by Western blotting using anti-cPLA2 antibody. To minimize the effect of Ca2+ during immunoprecipitation, cells were lysed in buffer containing 0.1 µM Ca2+. As shown in Fig. 7A, the cPLA2·ANX-I complex was coprecipitated by anti-ANX-I antibody. To further demonstrate precipitation of the cPLA2·ANX-I complex by anti-ANX-I antibody, cPLA2 activity in the pellet and supernatant was determined. This approach is based upon the observation that ANX-I inhibited cPLA2 at Ca2+ concentrations below 1 µM, but not at Ca2+ concentrations above 1 mM, under the assay conditions of this study (Fig. 1). Thus, cPLA2 precipitated with ANX-I at 0.1 µM Ca2+, but was able to display activity under the assay condition of 5 mM Ca2+, presumably due to its spontaneous dissociation from ANX-I. To verify the validity of this method, the interaction of GST-ANX-I with MBP-Delta C2 was examined at 0, 0.1, and 1 µM and 1 mM calcium. In the absence of phosphatidylcholine, the interaction was slightly influenced by calcium, whereas in its presence, this interaction depended on the calcium concentration. GST-ANX-I bound to MBP-Delta C2 at 0.1 and 1 µM calcium, but not at 5 mM calcium (data not shown). Fig. 7B shows the results of the coprecipitation experiments. cPLA2 activity was confined primarily to the pellet.


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Fig. 7.   Immunoprecipitation of the cPLA2·ANX-I complex from A549 cell lysate. A549 cells (2 × 107) were lysed in 200 µl of lysis buffer. One-hundred µg of cell lysate was incubated with anti-ANX-I antibody. The mixture was immunoprecipitated (IP) with anti-ANX-I or anti-cPLA2 antibody (Ab) as a control. A, the pellet was analyzed by Western blotting using anti-cPLA2 antibody. Lane C, untreated Sf9 cPLA2 cell lysate; lane 1, anti-cPLA2 antibody; lane 2, anti-ANX-I antibody; lane 3, anti-IgG antibody. B, the cPLA2 activity remaining in the supernatant or pellet was determined in standard buffer containing 5 mM CaCl2. IgG, precipitated with anti-IgG antibody; ANX Ab, precipitated with anti-ANX-I antibody.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mechanism by which ANX-I inhibits cPLA2 activity is still a controversial issue. In this study, we tried to address this issue by determining the effects of ANX-I and its deletion mutants I on cPLA2 activity. Enzymatic studies have revealed that 1) cPLA2 activity is specifically inhibited by ANX-I; 2) deletion from the N terminus to amino acid 274 has little effect on the inhibitory activity; 3) deletion of the C-terminal 72 amino acids abolishes the activity; and 4) inhibition is independent of the substrate concentration. Studies by immunoprecipitation and mammalian two-hybrid experiments have revealed that cPLA2 forms complexes with the C-terminal 72 amino acids of ANX-I, but not with its N-terminal 274 amino acids. These results are in agreement with the enzymatic studies. The previously proposed substrate depletion mechanism is based upon the observation that inhibition of sPLA2 by ANX-I is abolished with an increasing substrate concentration (17, 18). As shown in Figs. 1D and 4B, inhibition of cPLA2 by ANX-I was independent of the substrate concentration, which is consistent with the specific interaction mechanism.

The results shown in Figs. 5-7 demonstrate the specific interaction between cPLA2 and ANX-I, which further supports the specific interaction model. As shown in Figs. 1C and 4C, inhibition depended upon the calcium concentration and was greatest at 0.1-1 µM calcium and negligible above 1 mM calcium, which is consistent with the previous observation (19). These results are consistent with the following interpretation. At calcium concentrations less than 1 µM, binding of ANX-I to the substrate is little, and inhibition is primarily by specific interaction. The substrate binding of ANX-I increased with an increasing calcium concentration (data not shown); and at 1 mM calcium, inhibition by specific interaction was negligible (Fig. 1C), and if any inhibition is to occur, it would be by substrate depletion. Under the conditions for the substrate depletion model, apparent cPLA2 inhibition by ANX-I is observed only with an excess amount of ANX-I and a limiting amount of the substrate (17, 18). Since the inhibition assays in Figs. 1 and 4 were performed in the presence of a large excess of the substrate (5-100 nM ANX-I and 1.65 µM substrate), at 1 mM Ca2+, ANX-I binds mostly to the substrate and is unavailable for cPLA2 inhibition. This interpretation is supported by the observation that at 1 mM Ca2+, 300 nM ANX-I was required to inhibit cPLA2 in the presence of 1.65 µM substrate (data not shown). Studies with various phospholipids have indicated that ANX-I binds to anionic vesicles such as phosphatidylserine, but not to neutral vesicles such as phosphatidylcholine, at calcium concentrations up to 0.37 mM (33). In studies of the substrate depletion model, vesicles including anionic phospholipids were used as substrate (17, 18). The substrate depletion is due to binding of anionic substrate to a large excess of ANX-I. In contrast, since phosphatidylcholine was used in all assays in this study, the calcium dependence of cPLA2 inhibition by ANX-I (Figs. 3 and 4) is difficult to explain based on the previous result (33). We have performed binding assays using ANX-I and phosphatidylcholine under the assay conditions of the inhibition experiments and found that ANX-I bound to phosphatidylcholine at 5 mM calcium, but not at 0.1 and 1 µM calcium (data not shown). This result is in agreement with the inhibition data (Figs. 3 and 4).

As shown in Fig. 1, inhibition by ANX-I never exceeded 50%. Furthermore, an inhibitor/enzyme ratio of 40 (20:0.5 nM) was required to obtain the maximum inhibition. Reasons for both phenomena are not clear. In the in vivo experiments using cultured cells, deletion of the C2 domain abolishes translocation of cPLA2 to the membrane and agonist-induced arachidonic acid release (11, 32). The hydrophobic residues of the C2 domain in loops known as calcium-binding regions 1 and 3 are important for phospholipid binding, and ANX-I bound to the Delta C2 domain (Figs. 5 and 6), which lacks calcium-binding region 3. Thus, even if ANX-I binds to the Delta C2 domain, calcium-binding region 3 may still be available for weak calcium and phospholipid binding, resulting in partial inhibition of the phospholipid-binding property of cPLA2. Another point to be mentioned is that the inhibitor/enzyme ratio is 20 (10:0.5 nM) at 85% of the maximum inhibition and 40 (20:0.5 nM) at the maximum inhibition. It is unlikely that this number directly reflects the stoichiometry of ANX-I to cPLA2 in the inhibitor-enzyme complex. At present, it is not easy to explain the reason for the high inhibitor/enzyme ratio. As mentioned above, ANX-I has higher affinity for vesicles in which anionic phospholipids are present, such as natural membranes. In mammalian cells, both ANX-I and cPLA2 are ubiquitous; however, the expression level of ANX-I is far greater than that of cPLA2. The interplay among ANX-I, cPLA2, and phospholipids in cells seems to follow a complex rule.

It is notable that although cPLA2 inhibition by GST-ANX-I-(1-346) depended upon the calcium concentration, inhibition by GST-ANX-I-(275-346) was independent of the calcium concentration (Fig. 4C). This may be due to the number of the calcium-binding sites of ANX-I. ANX-I has six calcium-binding sites, and each is located in the loop region of the helix-loop-helix motif (3), whereas ANX-I-(275-346) has only one calcium-binding site and may bind to the substrate in a less calcium-dependent manner.

ANX-I consists of four domains, and each domain has five alpha -helix motifs (3). The four domains are highly conserved in all members of the ANX family, whereas the N terminus of each ANX varies. As shown in Fig. 3, the N-terminal region is not important for the cPLA2 inhibitory activity of ANX-I, and domain IV is the active domain. It is located at amino acids 275-346 (Fig. 3). This result is unexpected because region 275-346 lies within the core domain that is conserved in all ANXs. Even though the core domain sequences are highly conserved, there are apparently enough differences to differentiate the cPLA2-binding properties. This phenomenon is supported by the fact that the anti-ANX-I antibody derived from human ANX-I specifically recognizes all type I ANXs across the species from mold to human, whereas it does not recognize any other types of ANXs from any source, including human cells.2

Inhibition of cPLA2 by various members of the ANX family of proteins has been reported. However, most reported data are from experiments performed under conditions in which the substrate depletion mechanism dominates (34, 35). Even though there have been no detailed enzymatic studies on the mechanism of cPLA2 inhibition by ANXs other than ANX-I, most articles in the literature describe the mechanism as substrate depletion. There is only one other report (our previous research) that concludes that the inhibition mechanism is by specific interaction (19). If the mechanism is substrate depletion, all ANXs should display similar inhibition patterns because this mechanism is due to the ability of ANXs to bind to phospholipids. Inhibition studies using several ANXs have revealed that ANX-I, but neither ANX-II nor ANX-III, inhibits cPLA2 and that ANX-V causes much less effective inhibition (36). The differential effects of ANXs on cPLA2 activity provide evidence for the specific interaction model.

ANX-I is a major substrate for the epidermal growth factor receptor kinase, which has been implicated in membrane-related events along the endocytic pathway, in particular the sorting of internalized epidermal growth factor receptors occurring in the multivesicular body (MVB) (37). Truncation of the N-terminal 26 residues of ANX-I altered its intracellular distribution, shifting it from early to late and multivesicular endosomes, indicating the regulatory importance of the N-terminal domain and the involvement of ANX-I in early endocytic processes (38). ANX-I is associated with both the plasma membrane and MVB in a calcium-dependent manner, but can be phosphorylated only in MVBs. ANX-I and ANX-II are localized differently in human epidermal keratinocytes (39). ANX-I translocates from the cytosol to the nucleus by epidermal growth factor or stress signals through the N-terminal region (40). These evidences indicate that the variable N-terminal residue of ANXs is important for localization of ANXs and is involved in the interaction with MVBs, rather than with the plasma membrane. This interaction may be important for signal-induced phosphorylation and endocytic processing. On the other hand, the C-terminal domain of ANX-I may be involved in plasma membrane binding and in regulation of cPLA2. On the contrary, involvement of ANX-I domain I in the regulation of the cPLA2 signal has been observed (24). Since the study was designed to observe phorbol myristate acetate-induced c-fos activation, it is likely that several factors other than cPLA2 also participate in this process. The importance of the N-terminal region in mediation of the anti-inflammatory function of glucocorticoids and inflammatory signal transduction has been reported by Flower and co-workers (41-43). In the study of neutrophil migration, a mechanism has been proposed that the N-terminus of ANX-I binds to a receptor-like molecule on the surface of the neutrophils (44). Therefore, it is reasonable to assume that the N terminus is involved in the interaction with the MVB and the receptor-like molecule and that the C terminus is involved in the interaction with cPLA2.

cPLA2 binds to phospholipids by hydrophobic interaction. The hydrophobic residues of cPLA2 have a significant function in membrane binding of this domain. The C2 domain of cPLA2 preferentially binds to phospholipids with hydrophobic head groups, such as phosphatidylcholine. However, the C2 domain of protein kinase C binds to the membrane by electrostatic force (45). Whether specific interaction between cPLA2 and ANX-I is driven by a hydrophobic or electrostatic force is not known. The C-terminal domain of ANX-I may bind to the phospholipid-binding site of cPLA2 by hydrophobic interaction, thereby interfering with the binding of cPLA2 to membranes. The C-terminal domain of ANX-I has five alpha -helices. Each helix may form a hydrophobic patch that binds to the Delta C2 domain. However, ANX-I-(275-346) binding to the regulatory site of cPLA2 by electrostatic interaction cannot be ruled out. We are conducting mutation studies of the hydrophobic region of ANX-I-(275-346) to determine the interaction mechanism between ANX-I and cPLA2.

The existence of the cPLA2·ANX-I complex in the A549 cell lysate strongly suggests that this complex exists in vivo and that ANX-I regulates cPLA2 activity in cellular signal transduction. Inhibition of cPLA2 by ANX-I in cellular models supports this hypothesis (23, 24). Upon activation of cells, intracellular calcium concentration is elevated from 0.1 to 1 µM, and cPLA2 is phosphorylated and translocated from the cytosol to the membrane (11). ANX-I is also translocated upon activation of cells (46, 47). Since the degree of inhibition due to ANX-1 is similar at calcium concentrations of 0.1 and 1 µM (Figs. 1 and 4), it is unlikely that elevation of calcium concentration is the sole mechanism of cPLA2 regulation by ANX-I. It is probable that phosphorylation of ANX-I affects its protein-protein binding property. Considering that both cPLA2 and ANX-I bind to phospholipids, it is reasonable to assume that their binding properties in the cytosol or near the membrane are different. Whatever the mechanism, its specific interaction with cPLA2 and phosphorylation following various extracellular signals strongly suggest that ANX-I has an essential role in cell regulation, probably through the inhibition of cPLA2 activity.

In conclusion, the mechanism of cPLA2 inhibition by ANX-I is consistent with the specific interaction model, and the interaction between cPLA2 inhibition and ANX-I supports this interpretation. cPLA2 inhibition is a specific function of ANX-I and is not a general function of all ANXs. The C-terminal region is important for cPLA2 inhibition. The results presented here are important since ANX-I specifically inhibits cPLA2 at intracellular Ca2+ concentrations. ANX-I may regulate several biological processes through regulation of cPLA2 activity.

    FOOTNOTES

* This work was supported in part by Ministry of Health and Welfare Grant HMP-98-B-2-0007 and Ministry of Science and Technology Grant 00-G-08-02-A-100 (to D. S. N.).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.

Present address: Div. of Hematology-Oncology, Dept. of Medicine, Harold Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75390-8594.

** To whom correspondence should be addressed. Tel.: 82-2-880-7874; Fax: 82-2-866-5802; E-mail: ecchoi@snu.ac.kr (E. C. C.). Tel.: 82-2-224-4275; Fax: 82-2-477-9715; E-mail: dsna@www.amc.seoul.kr (D. S. N.).

Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M009905200

2 S.-W. Kim, H. J. Rhee, J. Ko, Y. J. Kim, and D. S. Na, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: ANXs, annexins; PLA2, phospholipase A2; sPLA2, secretory phospholipase A2; cPLA2, cytosolic phospholipase A2; 2-AA-PC, 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine; PCR, polymerase chain reaction; GST, glutathione S-transferase; BSA, bovine serum albumin; MBP, maltose-biding protein; MVB, multivesicular body.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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