From the 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
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ABSTRACT |
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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.
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.
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 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- Immunoprecipitation Studies--
Coprecipitation of ANX-I in
A549 cell lysate and purified MBP-
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.
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.
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).
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 ( Coprecipitation of MBP- 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- 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 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 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 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (X-gal),
and 40 µg/ml isopropyl-
-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.
C2 and
ANX-I--
The 43 amino acids spanning amino acids 38-80 of the C2
domain of cPLA2 (
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-
C2. The MBP fusion protein MBP-
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-
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).
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-
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.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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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.
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).
C2 and GST-ANX-I--
To further
investigate the direct interaction between cPLA2 and ANX-I
or the deletion mutants, co-immunoprecipitation of
C2 with GST-ANX-I
was examined. The
C2 domain of cPLA2 was produced as a
fusion protein with MBP. GST-ANX-I mutants (Fig. 2) and MBP-
C2 were
produced in E. coli and purified by affinity column
chromatography. GST-ANX-I and MBP-
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
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-
C2 and A549 cell lysate or GST-ANX-I and Sf9
cPLA2 cell lysate. In one experiment, MBP-
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-
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- C2 and GST-ANX-I. A,
coprecipitation of GST-ANX-I mutants and MBP-
C2. The
C2 sequence
spanning amino acids 38-80 of cPLA2 was cloned into an MBP
fusion vector (pMAL-P2X) to produce the MBP-
C2 fusion protein in
E. coli. Purified GST-ANX-I mutants and MBP-
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-
C2 and
ANX-I in A549 cell lysate. One-hundred µg of A549 cell lysate was
incubated with 2 µg of either MBP-
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.
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-
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
C2 domain (Figs. 5 and 6), which
lacks calcium-binding region 3. Thus, even if ANX-I binds to the
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.
-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
-helices. Each helix may form a hydrophobic patch
that binds to the
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.
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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.
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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.
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