From the Small Molecule Drug Discovery Group, Genetics Institute, Cambridge, Massachusetts 02140
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ABSTRACT |
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We report the cloning and characterization of a
novel membrane-bound, calcium-independent PLA2, named
cPLA2-. The sequence encodes a 541-amino acid protein
containing a domain with significant homology to the catalytic domain
of the 85-kDa cPLA2 (cPLA2-
). cPLA2-
does not contain the regulatory
calcium-dependent lipid binding (CaLB) domain found in
cPLA2-
. However, cPLA2-
does contain two
consensus motifs for lipid modification, a prenylation motif (
CCLA)
at the C terminus and a myristoylation site at the N terminus. We
present evidence that the isoprenoid precursor [3H]mevalonolactone is incorporated into the prenylation
motif of cPLA2-
. Interestingly, cPLA2-
demonstrates a preference for arachidonic acid at the sn-2
position of phosphatidylcholine as compared with palmitic acid.
cPLA2-
encodes a 3-kilobase message, which is highly
expressed in heart and skeletal muscle, suggesting a specific role in
these tissues. Identification of cPLA2-
reveals a newly
defined family of phospholipases A2 with homology to
cPLA2-
.
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INTRODUCTION |
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Phospholipases A2
(PLA2)1 are a
diverse group of enzymes that hydrolyze the sn-2 fatty acids
from phospholipids and play a role in a wide range of physiological
functions. Of particular interest is the role of these enzymes in the
production of factors involved in mediating the inflammatory response.
The phospholipases A2 family is large, and individual
members can be classified according to localization (extracellular
versus intracellular), sequence homology, and biochemical
characteristics (1). Known PLA2 members include the
secreted PLA2s and the cytosolic PLA2s. To date
only two cytosolic PLA2 sequences have been reported: the
calcium-dependent PLA2 (cPLA2-)
and the calcium-independent PLA2 (iPLA2)
(2-5). cPLA2-
has a predicted molecular mass of 85 kDa
and contains two domains, a calcium-dependent
lipid binding (CaLB) domain and a catalytic
domain (2, 6). The catalytic domain contains a lipase consensus
sequence and a novel catalytic triad that employs a serine, an
aspartate, and an arginine instead of the usual serine, aspartate, and
histidine found in many lipases and serine proteases (7-9).
cPLA2-
activity is regulated by the activation of the CaLB domain in response to increased intracellular calcium (6). The
activated CaLB domain translocates the enzyme to its substrate in the
nuclear envelope and endoplasmic reticulum (10). cPLA2-
activity is also increased by the phosphorylation of a MAP kinase consensus site, in response to stimulation of cells with cytokines such
as tumor necrosis factor and interleukin 1 (11, 12). These same
cytokines have also been found to increase the expression of
cPLA2-
(11, 12). Although there have been many studies that suggest the importance of cPLA2 in the generation of
prostaglandins and leukotrienes, the most convincing data have come
from studies using mice that are genetically deficient in
cPLA2 (13, 14). Studies demonstrate that
cPLA2-
is essential for both the calcium ionophore,
A23187, and lipopolysaccharide-induced prostaglandin E2 and leukotriene
B4 production in peritoneal monocytes (13, 14). The possible importance
of cPLA2 in asthma was also shown (13).
The 85-kDa calcium-independent iPLA2, purified by two
groups, shares no homology with cPLA2- except, like
other lipases, it contains the critical consensus sequence,
GXSXG (4, 5, 15). Interestingly,
iPLA2 contains a domain of eight ankyrin repeats, which may
be involved in protein-protein interactions (4). iPLA2
possesses no clear preference for a fatty acid at the sn-2
position, and it is thought to play a role in the remodeling of
phospholipids (16).
Although cPLA2- and iPLA2 are the only
intracellular PLA2s that have been cloned, many other
PLA2 activities, which presently seem to be distinct from
cPLA2-
and iPLA2, have been reported (17-19). The relationship of the enzymes responsible for these activities to the known PLA2 enzymes will be clear only
upon sequence determination.
Our initial efforts to identify additional PLA2
enzymes failed using low stringency cross-hybridization techniques with
cPLA2- sequences.2 A search of the
expressed sequence tag (EST) data base was quite successful, and two
independent cPLA2-
related gene fragments were
identified. Subsequent sequence analysis of the full-length clones
revealed two novel homologs of cPLA2-
, designated
cPLA2-
and cPLA2-
. The characterization
of cPLA2-
will be described elsewhere.3 Here, we report
the sequence and characterization of a novel 60.9-kDa
calcium-independent, membrane-associated cPLA2,
cPLA2-
.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Antibodies--
COS cells were maintained
in Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% fetal calf serum, 50 units/ml penicillin, 50 µg/ml streptomycin, and 1 mM glutamine. Cells were
incubated in a 37 °C humidified atmosphere with 10%
CO2. Chinese hamster ovary (CHO) cells were maintained in
alpha medium (Life Technologies, Inc.) containing 50 units/ml penicillin, 50 µg/ml streptomycin, 1 mM glutamine, 1 mg/ml G418 (Life Technologies, Inc.), and 10% dialyzed fetal calf
serum supplemented with 10 µg each of adenosine, deoxyadenosine, and
thymidine per ml for parental cells, 5 nM methotrexate for
cPLA2- overexpressing cells, and 20 nM
methotrexate for cPLA2-
overexpressing cells. Cells were
incubated in a 37 °C humidified atmosphere with 5% CO2.
Rabbit polyclonal antibodies 44282 and 44284, generated against peptides from cPLA2-
, amino acids 416-434, and amino
acids 476-495, were used for immunoprecipitation and immunoblotting.
Rabbit polyclonal anti-human Ras (Upstate Biotechnology) was used for
immunoprecipitation.
Clone Identification--
The EST clone 258543 (GenBank
accession N56796) was identified by searching the GenBank EST data base
using the amino acid sequence of cPLA2-. The 900-base
pair EcoRI-NotI fragment from clone 258543 was
used to screen 106 recombinates of the oligo(dT) primed
human skeletal muscle library (Stratagene). Clone 19A, which is a
phagemid DNA excised from the Lambda Uni-Zap XR phage vector, had its
DNA sequence determined.
Northern Blot Analysis--
Northern analysis was performed on
multiple human tissues blot (CLONTECH) using a
random-primed 32P-labeled EcoRI-NotI
fragment of cPLA2-. The blot was washed under high stringency
conditions (0.2 × SSC at 65 °C). Each lane comprises
approximately 2 µg of poly(A)+ mRNA.
Construction of Expression Vectors--
Two oligonucleotides,
5'-GTTCACCTCATCCTCTCCTTCGAC-3' and
5'-TCGGGGTACCGAATTCGGGCCCTATGCCAAGCAGCAACTTCGGGCACT-3', corresponding to the 3'-end of cPLA2-, were used to amplify the 3'-end
coding region of cPLA2-
by polymerase chain reaction,
using clone 19A DNA as a template. The polymerase chain reaction
product was digested with XbaI and EcoRI and
ligated with the XbaI-EcoRI fragment of clone 19A
and the EcoRI-digested vector pED
C. The resulted clone, named pED
C-cPLA2-
WT, was sequenced to confirm the
desired C-terminal coding sequence. Mutation of the C-terminal
CCLA
was produced as above except using an oligonucleotide that introduced
the mutation (C538S,C539S) to the 3' end of the sequence. The resulted
clone, named pED
C-cPLA2-
SSLA, was confirmed by
sequencing to contain the desired C-terminal coding sequence.
Stable CHO Cell Lines Overexpressing cPLA2- and
cPLA2-
--
The plasmid of wild-type
cPLA2
was constructed as follows. The restriction
fragment (EcoRI-EcoRI) containing the entire coding sequence was isolated from pED
C-cPLA2-
WT and
ligated to a controlled expression vector pHTOP. pHTOP was modified
from pED6 vector (20) essentially by inserting a 289-base pair
tetracycline operator sequence (21) at the XhoI site of
pED6. The expression vector of transactivator (tTA) was generated
similarly as described by Gossen and Bujard (21) using neomycin
transferase as selection marker.
Activity Assay--
Cell pellets (either from CHO cells
overexpressing cPLA2- or cPLA2-
or from
COS cells transiently transfected with cPLA2-
or
cPLA2-
) were resuspended in lysis buffer (10 mM HEPES, pH 7.5, 1 mM EDTA or 1 mM
EGTA, 0.1 mM dithiothreitol, 0.34 M sucrose, and 1 µg/ml leupeptin). Cells were lysed by nitrogen cavitation (750-1000 psi, 10 min) on ice. Approximately 5-15 µg of cell lysate (determined by Bradford assay, Bio-Rad) was used in the assay. 1-[14C]Palmitoyl-2-arachidonyl-phosphatidylcholine (PC)
(57 Ci/mmol), 1-palmitoyl-2-[14C]arachidonyl-PC (55-58
Ci/mmol), 1-palmitoyl-2-[14C]oleoyl-PC (58 Ci/mmol),
1-palmitoyl-2-[14C]linoleoyl-PC (58 Ci/mmol), and
1-O-hexadecyl-2 [3H]arachidonyl-PC (200 Ci/mmol) were obtained from DuPont NEN. Unlabeled
1-O-hexadecyl-2-arachidonyl-PC was obtained from Biomol. The
lipids were dried under N2 and sonicated with vesicle
buffer (50 mM HEPES, pH 7.5, 1 mM EGTA or 5 mM EGTA, and 7 mM CaCl2 for regiospecificity assay, 30% glycerol, 1 mg/ml fatty acid-free bovine
serum albumin, and 150 mM NaCl) adapted from Ghomashchi et al. (22). Aliquots of lysate were incubated with
substrate (20 µM) at 37 °C for the indicated amount of
time. Released fatty acid was measured as described (23).
Prenylation Assay--
70-80% confluent COS cells (10-cm
plate) were transfected with 8 µg of pMev (obtained from
ATCC (24), to facilitate uptake of mevalonolactone) and either 8 µg
of pEDC-cPLA2-
WT, pED
C-cPLA2-
SSLA, or pzflag (for vector control) or 4 µg of pCMV-Ras61L (obtained as a
generous gift from R. Davis) using lipofectamine according to the
manufacturer's instructions (Life Technologies, Inc.). Cells were
grown for 2 days and then incubated with 20 µM mevistatin (Biomol) for 1 h. The cells were then incubated in 3 ml of growth media containing 40 µM mevistatin and 150 µCi of
[3H]mevalonolactone (29 Ci/mmol) (DuPont NEN) for 14 h (25). Cells were washed twice in phosphate-buffered saline and
scraped into 3 ml of phosphate-buffered saline. The cell pellets were
lysed in 100 µl of lysis buffer (20 mM Tris-HCl, pH 7.5, 10% glycerol, 1% Triton X-100, 137 mM NaCl, 2 mM EDTA, 5 µg/ml aprotinin, 10 µg/ml leupeptin, and 2 mM phenylmethylsulfonyl fluoride for 10 min. A 20-µl
aliquot was removed and centrifuged, and the pellet was resuspended in
50 µl of 2 × Laemmli's sample buffer. SDS to 1% was added to
the remaining lysate and incubated for 10 min. Lysis buffer was added
to the lysate to 1 ml to reduce the SDS to 0.1%, incubated for another
30 min, and then centrifuged 1 h at 100,000 × g,
4 °C. The supernatant was immunoprecipitated with 10 µl of
anti-cPLA2-
antibody, 44282, or anti-Ras antibody (Upstate Biotechnology) for 3 h at 4 °C, then incubated with
protein A-Sepharose (Amersham Pharmacia Biotech) saturated with 10%
bovine serum albumin for 30 min. The beads were washed three times with lysis buffer containing 0.1% SDS and then resuspended in 50 µl of
2 × Laemmli's sample buffer. Samples were subjected to 4-20% SDS-PAGE (Novex), stained with Coomassie Blue, soaked in entensify (Amersham), and dried and exposed to Biomax MS film (Kodak) for 7 days.
To monitor the expression levels of cPLA2-
, aliquots of
the samples were subjected to 4-20% SDS-PAGE. Proteins were transferred to a nitrocellulose filter (Novex), immunoblotted for
cPLA2-
with 44284 antibody, and detected by ECL
(Amersham).
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RESULTS |
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cPLA2- Is a Novel cPLA2--
The EST
data base was searched to identify sequences similar to the human
85-kDa cPLA2 gene (cPLA2-
). This analysis
led to the identification of two related genes, named
cPLA2-
and cPLA2-
. The
cPLA2-
EST clone 258543 was shown to contain a partial
cDNA insert with sequence similarity to the C terminus of
cPLA2-
. A full-length clone was isolated from a human
skeletal muscle cDNA library. This clone contains a 541-amino acid
open reading frame with predicted molecular mass of 60.9 kDa.
Comparison of the amino acid sequences of cPLA2-
and
cPLA2-
(Fig. 1) reveals 28.7% identity. Within this putative catalytic domain there exists two
subdomains with greater sequence identity. Interestingly, the spacer
region separating these two domains corresponds to an area in
cPLA2-
considered to be an exposed hinge region
containing many protease-accessible sites, as well as the MAP kinase
activation site Ser-505 (6). cPLA2-
contains a sequence that is
similar to the lipase consensus sequence, GLSGS, in cPLA2-
, which
has been found to be critical for cPLA2-
activity (7). These
sequences are very similar to the lipase consensus sequence,
GXSXG, found in many lipases and serine proteases
(9, 26). Also conserved in cPLA2-
are the amino acids
that make up the putative catalytic triad of cPLA2-
(8).
These amino acids correspond to serine 82, aspartate 385, and arginine
54 in cPLA2-
.
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cPLA2- Encodes a Phospholipase
A2--
To determine the regioselectivity of
cPLA2-
, vesicle activity assays were performed using
1-[14C]palmitoyl-2-arachidonyl-PC,
1-palmitoyl-2-[14C]arachidonyl-PC, or
1-O-hexadecyl-2 [3H]arachidonyl-PC. Cell
lysates prepared from COS cells transfected with either vector,
cPLA2-
or cPLA2-
, were incubated with the various substrates for the indicated times (Fig.
3). Lysate from cPLA2-
transfected cells showed at least 4.4-fold more PLA2
activity than the lysate from vector transfected cells. Importantly,
cPLA2-
readily liberated arachidonate when
1-O-alkyl phospholipid (1-O-hexadecyl-2 [3H]arachidonyl-PC) was utilized as a substrate,
confirming its ability to cleave the sn-2 site. Comparing
the activity of cPLA2-
in the presence of the substrates
using 1-[14C]palmitoyl-2-arachidonyl-PC,
1-palmitoyl-2-[14C]arachidonyl-PC, or
1-O-hexadecyl-2 [3H]arachidonyl-PC,
cPLA2-
seems to be as proficient at cleaving at the
sn-1 site as the sn-2 site. This is dissimilar
from cPLA2
(Fig. 3B), which under the
conditions used has no apparent sn-1 activity. Importantly,
the PLA1 and PLA2 activity of
cPLA2-
does not seem to be sequential, as the
radiolabeled fatty acid released from
1-palmitoyl-2-[14C]arachidonyl-PC and using
1-[14C]palmitoyl-2-arachidonyl-PC showed similar
kinetics.
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cPLA2- Is Prenylated--
cPLA2-
contains the C-terminal sequence
CCLA, which is a motif for
prenylation. Protein prenylation is mediated by the addition of either
farnesyl (C-15) or geranylgeranyl (C-20) to the cysteine of the
CAAX motif (29). The process is initiated by cleavage of the
three most C-terminal amino acid residues
AAX, followed by
methylation of the cysteine carboxyl group (29). The sequence of
cPLA2-
also resembles the sequence CCXX,
which is another motif for prenylation (27). This motif signals the
addition of geranylgeranyl to the protein and occurs via mechanisms
that differ from the CAAX modification. This motif is mostly
found on the Rab family of proteins (29).
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cPLA2- Is a Membrane-associated Protein--
To
determine the subcellular localization of cPLA2-
, CHO
cells stably transfected with cPLA2-
were lysed by
nitrogen cavitation and centrifuged for 1 h at 100,000 × g. cPLA2-
was then detected by Western
analysis. As shown in Fig. 5,
cPLA2-
is found to localize to the particulate (pellet)
fraction. Treatment of the particulate fraction with 1% Triton X-100
followed by centrifugation at 100,000 × g results in
the majority of the enzyme being present in the supernatant. This is
unlike cPLA2-
, which is found in the supernatant in the
absence of calcium and in the pellet fraction following the addition of
calcium (6, 10). Lipid modification may be responsible for the membrane
association of cPLA2-
, as is the case for Ras. However,
fractionation of cells transfected with cPLA2-
mutated
at both the N- and C termini, to disrupt possible lipidation sites,
revealed that this mutated protein remains in the membrane fraction
(data not shown). This result indicates that there is another component
that is involved in the association of cPLA2-
with the
membrane.
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cPLA2- Is Calcium-independent--
Unlike the
secreted PLA2s, which require millimolar concentrations of
calcium for activity, the catalytic domain of cPLA2-
does not require calcium for activity (6). However,
cPLA2-
does require micromolar concentrations of calcium
for membrane binding through its CaLB domain. We were interested in
determining the requirement of calcium for cPLA2-
activity. Cell lysates prepared from COS cells transfected with
cPLA2-
or cPLA2-
were incubated with
1-palmitoyl-2-[14C]arachidonyl-PC in the presence of 0 or
10 µM or 10 mM calcium (Fig.
6). cPLA2-
activity is unaffected by
calcium, unlike cPLA2-
activity, which increases 14-fold in the
presence of 10 µM calcium. This result demonstrates that
cPLA2-
is a calcium-independent enzyme.
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Substrate Specificity at the sn-2
Position--
cPLA2- selectively hydrolyzes arachidonic
acid at the sn-2 position in several assay formats, whereas
iPLA2 selectivity is significantly more assay dependent. To
determine if cPLA2-
has a preference for the fatty acid
at the sn-2 position, vesicle assays were performed using
phosphatidylcholine that contains palmitoyl in the sn-1
position and radiolabeled arachidonyl, oleyl, linoleyl, or palmitoyl in
the sn-2 position. The substrates were incubated for 15 min
with lysates from CHO cells stably transfected with either
cPLA2-
or cPLA2-
. cPLA2-
seems to prefer lipids that are unsaturated at the sn-2
position, and it does seem to prefer arachidonic acid approximately
3.5-fold over palmitic acid (Table
I).
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DISCUSSION |
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We have identified a novel 60.9-kDa calcium-independent
phospholipase A2, which we termed cPLA2-.
cPLA2-
contains 28.7% overall sequence identity with
cPLA2-
and was identified by searching the EST data base
for related proteins.
A common motif found in many lipases is the consensus sequence,
GXSXG, which is essential for enzymatic activity
(9, 26). cPLA2- contains the sequence
GVS82GS, which is similar but slightly different from the
consensus. However, this sequence aligns with the sequence in
cPLA2-
, and the change from glycine to serine also
occurs in the corresponding region of cPLA2-
,
GLS228GS (7). Mutation of serine-228 and aspartate-549 was
shown to abolish cPLA2-
activity, consistent with their role in the putative catalytic triad (7, 8). Catalytic triads of lipases and serine
proteases also frequently contain critical histidines; however,
mutation of these residues in the catalytic domain of cPLA2-
had no
affect on activity (8, 9, 26). Surprisingly, mutation of arginine-200,
in what is thought to be the novel catalytic triad of
cPLA2-
, abrogated cPLA2-
activity (8).
These amino acids may serve as a catalytic triad, providing the active
site for hydrolysis, or it is possible that the arginine may function in another but unknown critical role, such as in transition state stabilization (8). We have also shown that serine, aspartate, and
arginine are conserved in cPLA2-
, providing further evidence that
these amino acids are important and may indeed be part of a novel
catalytic triad.
cPLA2- is regulated by at least two post-translational
mechanisms: 1) calcium-induced membrane association through its CaLB domain and 2) phosphorylation of serine-505 by a MAP kinase (6, 10,
30). The phosphorylation site and the CaLB domain of
cPLA2-
are not conserved in the sequence of
cPLA2-
, suggesting a different regulatory mechanism.
Interestingly, cPLA2-
contains a potential prenylation
motif at its C terminus and a putative signal for myristoylation at its
N terminus. Initial studies have failed to indicate that the
myristoylation site is utilized, whereas the prenylation site is indeed
utilized. The isoprenoid precursor [3H]mevalonolactone is
readily incorporated into cPLA2-
expressed in COS cells.
We do not know, however, if the modifying isoprenoid is a farnesyl or a
geranylgeranyl. Generally, in the consensus sequence CAAX,
when the C-terminal (X) amino acid is a methionine, serine,
glutamine, or alanine, this signals that the lipid will be farnesyl
(27), whereas a leucine signals that the modifying lipid is a
geranylgeranyl. However, there is also a motif XXCC, CXC, or CCXX found on the Rab family of proteins
that modifies the two cysteines with geranylgeranyl (31). Because both
of these motifs match the cPLA2-
sequence (
CCLA), we do not know which of these isoprenoids is modifying the protein.
One of the most striking differences between cPLA2- and
-
is the lack of a lipid binding CaLB domain in
cPLA2-
. The presence of lipidation motifs suggests that
these regions may function as the CaLB domain in cPLA2-
,
localizing the enzyme to the membrane and being critical for activity.
However, in a preliminary study, cPLA2-
mutant protein
that disrupts the possible N- and C-terminal lipid modification sites
did not affect its activity in a phopholipid vesicle
assay.4 Moreover, this mutant
protein fails to alter its association with the membrane fraction.
However, it remains possible that lipid modification may be important
in the subcellular localization of cPLA2-
and/or its
ability to associate with other proteins. Interestingly, Ras shows
increased affinity toward other proteins when it is prenylated compared
with its nonprenylated form, and it has been shown that oncogenic forms
of Ras need to be modified to transform cells (29). Therefore, it is
possible that lipid modifications may play a role in regulating the
activity of cPLA2-
in the cells.
cPLA2- will hydrolyze fatty acids at the sn-1
and sn-2 position of phosphatidylcholine. This suggests that
cPLA2-
contains PLA1 and PLA2
activity. The evidence that cPLA2-
contains
PLA2 activity is also confirmed by its ability to cleave
1-O-hexadecyl-2-arachindonyl-phosphatidylcholine. However,
we do not know whether the sn-1 cleavage is PLA1
activity or if it is cleavage of the lysophospholipid. The kinetics of the reactions suggest that it is PLA1 activity, as the
hydrolysis of sn-1 would show a time-dependent
lag as compared with sn-2 hydrolysis if sequential cleavage
were taking place, unless cleavage of sn-1 from
lysophospholipid occurs rapidly. Taken together, all of these data
provide evidence that cPLA2-
is an enzyme with PLA2 activity and a probable PLA1 activity.
cPLA2- prefers arachidonic acid to palmitic acid in the
sn-2 position of phosphatidylcholine. However, this
preference is modest in comparison to the strong preference that
cPLA2-
displays for arachidonic acid, 3.5-fold
versus 24.5-fold, respectively. The substrate specificity of
cPLA2-
should be considered cautiously, however, because
of the artificial nature of the substrate presentation. The selectivity
of the enzyme using a natural membrane as a substrate may be a more
relevant method to determine the preferred physiological substrate for
this enzyme.
The preferred substrate for an enzyme provides a clue to its
physiological role, as can its distribution within tissues.
cPLA2- is highly expressed in heart and skeletal muscle.
The calcium independence of this enzyme may be important for its high
expression in muscle, where contractions cause large fluxes in calcium
concentrations. Therefore, it may be necessary in this environment to
regulate a phospholipase in a calcium-independent manner, such as
phosphorylation. As previously stated, cPLA2-
activity
is also regulated by MAP kinase phosphorylation of serine-505. This
serine is not conserved in the sequence of cPLA2-
;
however, there are several potential protein kinase C phosphorylation
sites, which may be utilized to regulate the enzyme.
cPLA2- may be highly expressed in these muscles because
heart and skeletal muscle encounter physical stress upon increased load. It may be necessary to regulate the remodeling of the
phospholipid bilayer when cells undergo stress. This speculation is
substantiated by the reports of several calcium-independent
phospholipases expressed in heart (17-19). Hazen et al.
(18) and McHowat and Creer (19) have identified membrane-bound,
calcium-independent PLA2 activity that prefers the
myocardia-abundant lipid, plasmologen, as a substrate. They have shown
increased hydrolysis of plasmologen under hypoxic conditions, such as
in ischemia. It is believed that in ischemia, a PLA2
activity leads to the accumulation of lysophospholipids and subsequent
injury to the heart tissue because of disruptions of the membrane. As
the PLA2 activity of this myocardial membrane-bound enzyme
was shown to increase under hypoxic conditions, it was suggested that
this calcium-independent PLA2 is physiologically involved
in ischemia (18). Because cPLA2-
is abundantly expressed in heart, and its properties (including calcium independence and membrane localization) are similar to that reported in heart muscle, it
may be that cPLA2-
is involved in ischemia-induced
injury to heart muscle.
In summary, we have described the molecular cloning and initial
characterization of a novel 60.9-kDa membrane-associated, calcium-independent PLA2, cPLA2-. This
enzyme shares identity with cPLA2-
and contains the
potential critical amino acids for the catalytic site but is missing
the key elements that regulate the activity of cPLA2-
.
This suggests that the mechanisms of regulation for
cPLA2-
will be different from that of
cPLA2-
and quite possibly employs the use of the lipid
modification. Defining the mechanisms of regulation and the
physiological substrate of cPLA2-
should shed some light
on the physiological role of this newly identified
PLA2.
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ACKNOWLEDGEMENTS |
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We thank Roger Davis for pCMV-RAS61L; Dianne
Sako for the cPLA2-:CHO cell line; Kevin Bean, Kerry
Kelleher, and Heather Finnerty for DNA sequencing; and Mark Proia for
screening the cDNA library. We are especially grateful to James
Clark, Simon Jones, and Thomas Noland for many helpful discussions.
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FOOTNOTES |
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* 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.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed: Genetics Institute, 87 Cambridge Park Dr., Cambridge, MA 02140. Tel.: 617-498-8934; Fax: 617-498-8993.
The abbreviations used are: cPLA2, cytosolic phospholipase A2CaLB, calcium-dependent lipid bindingiPLA2, cytosolic calcium-independent PLA2MAP, mitogen-activated proteinEST, expressed sequence tagPC, phosphatidylcholineCHO, Chinese hamster ovaryPAGE, polyacrylamide gel electrophoresis.
2 R. Kriz, unpublished results.
3 C. Song, X. J. Chang, K. Bean, M. Proia, J. L. Knopf, and R. W. Kriz, manuscript in preparation.
4 K. Underwood, unpublished data.
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REFERENCES |
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