From the Division of Basic Science, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206 and the Department of Pathology, University of Colorado, School of Medicine, Denver, Colorado 80262
Membrane phospholipid is an important reservoir
for the generation of bioactive mediators. Cellular phospholipases
hydrolyze membrane phospholipid in a structurally specific manner
releasing numerous lipid products that are responsible for transmitting diverse signals necessary for the induction of functional responses. Since this is a highly regulated process, the phospholipases are subject to complex mechanisms of activation and presumably
deactivation.
The phospholipase A2
(PLA2)1 enzymes hydrolyze fatty
acid from the sn-2 position of phospholipid with the
concomitant production of lysophospholipid. Mammalian cells contain
structurally diverse forms of PLA2 including secretory
PLA2 (sPLA2), calcium-independent PLA2, and the 85-kDa cytosolic PLA2
(cPLA2) (1-4). PLA2 enzymes function in the
digestion of dietary lipid, microbial degradation, and regulation of
phospholipid acyl turnover either in a housekeeping role for membrane
repair or for the production of inflammatory lipid mediators. The
presence of diverse PLA2 enzymes in mammalian cells
provides multiple, differentially regulated pathways for the important
process of fatty acid turnover. This review will focus on the
biochemical properties and regulation of cPLA2, which plays
an important role in mediating arachidonic acid release. cPLA2 shares no homology with other PLA2
enzymes and is the only well characterized PLA2 that
preferentially hydrolyzes sn-2 arachidonic acid (1, 5, 6).
It should be noted, however, that although sPLA2 and
calcium-independent PLA2 do not exhibit acyl chain
specificity they can also mediate arachidonic acid release depending on
the cell type and agonist involved (3, 4). Arachidonic acid is itself
an important regulator of specific cellular processes including
regulation of PKC and phospholipase C Distribution of cPLA2 and Regulation of Its
Synthesis cPLA2 is a widely distributed enzyme, and the
transcript is expressed at a fairly constant level in all human tissues
with somewhat elevated levels in lung and hippocampus (16, 17). At the
cellular level, cPLA2 is enriched in mononuclear phagocytes but has been found in most cells examined. One notable exception is
mature T and B lymphocytes that do not contain cPLA2,
whereas it is present in thymocytes and immature B cells (18). This suggests that expression of cPLA2 may be incompatible with
the function of mature lymphocytes. The ubiquitous expression of
cPLA2 is consistent with features of the 5 A variety of cytokines and mitogens such as interleukin-1, tumor
necrosis factor (TNF), colony-stimulating factor (CSF), epidermal growth factor, c-Kit ligand, and interferon Although cPLA2 and sPLA2 catalyze the same
reaction their catalytic mechanisms are different. cPLA2
has multiple enzymatic activities exhibiting in addition to
PLA2 activity high lysophospholipase activity and weak
transacylase activity (26-28). cPLA2 can also hydrolyze
fatty acid ester of 7-hydroxycoumarin, which has been used as a
substrate for continuously monitoring the activity of pure
cPLA2 (29). Although the transacylase activity may not have
physiological relevance, there is evidence that cPLA2 may function as a lysophospholipase in cells (30). The coordinate release
of sn-2 arachidonic acid followed by the sn-1
fatty acid of diacylphospholipids provides an efficient mechanism for
controlling the levels of potentially cytotoxic lysophospholipids. A
lysophospholipase with immunological identity to cPLA2 has
recently been purified from bovine brain and exhibits both high
activity and selectivity for arachidonoyl-substituted
lysophosphatidylcholine (31). It is suggested that the combined action
of brain phospholipase A1 and this lysophospholipase may
provide significant amounts of arachidonic acid in the brain. The
transacylase activity provides evidence that the cPLA2
reaction proceeds by formation of an acyl-enzyme intermediate, either
as an acyl-serine or acyl-cysteine. cPLA2, which contains 9 cysteine residues, is most stable in the presence of reducing agents
(32). This is in contrast to sPLA2 enzymes, which are
stabilized by disulfide linkages and inactivated by reducing agents.
Sulfhydryl-modifying reagents such as iodoacetamide or
dithiobisnitrobenzoic acid inactivate cPLA2; however, the
cysteine residues are not essential for catalytic activity based on
mutagenesis studies (32, 33). Cys331 is the residue that is
targeted by sulfhydryl-modifying reagents leading to a loss of
cPLA2 activity, which suggests that it is located in a
sensitive region of cPLA2, possibly near the active site
(34, 35) (Fig. 1).
Several lines of evidence have demonstrated that cPLA2
contains a serine nucleophile at the active site. NMR analysis of
cPLA2 complexed with an inhibitory trifluoromethylketone
analogue of arachidonic acid has revealed a similarity with
Regulation of cPLA2 by Calcium cPLA2 requires calcium for activity; however, unlike
sPLA2, calcium is necessary for binding cPLA2
to membrane or phospholipid vesicles rather than for catalysis.
cPLA2 contains an N-terminal calcium-dependent
phospholipid binding domain (CaLB) that shares homology with the C2
domains in the conventional isoforms of PKC, Unc-13, phospholipase
C The properties of cPLA2 are consistent with the enzyme
being soluble in resting cells at 50-100 nM calcium and
translocating to membrane when intracellular calcium levels increase.
It is well documented that arachidonic acid release is triggered in cells by many calcium-mobilizing agonists (1). In a rat mast cell line,
cPLA2 is cytosolic in resting cells but binds to nuclear membrane in response to calcium ionophore or IgE/antigen (44). The
extent of membrane binding correlates with the amount of arachidonic acid release, which is greater with A23187 than with IgE/antigen. In
Chinese hamster ovary cells overexpressing cPLA2, calcium
ionophore promotes translocation of the enzyme to endoplasmic reticulum and nuclear membrane, which is dependent on the CaLB domain (45). In
contrast, mutation of Ser505, the site of MAP kinase (MAPK)
phosphorylation, has no effect on cPLA2 translocation
although it abolishes arachidonic acid release. The subcellular
localization of cPLA2 can be dependent on cell density
(46). In confluent endothelial cells, cPLA2 is cytosolic,
but in subconfluent cells most of the cPLA2 is
intranuclear. Exposure of confluent and subconfluent cells to a variety
of agonists induces translocation of enzyme to a perinuclear region and
the nuclear envelope. In the confluent cells only, cPLA2
also localizes to intercellular junctions of the plasma membrane. This
is the first cell model in which cPLA2 has been shown to be
associated with the plasma membrane. In rat alveolar type II cells,
A23187-induced translocation of cPLA2 to nuclear membrane
results in preferential loss of arachidonic acid from this site
verifying functional activation of cPLA2 at the nuclear
membrane (47). Interestingly, 5-lipoxygenase, 5-lipoxygenase activating
protein, and COX 2 are also associated with nuclear membrane suggesting
that these enzymes form a functional complex for the production of
eicosanoids (48). It also infers that there may be a specific role for
eicosanoid production at the nuclear membrane. Preferential targeting
of cPLA2 to the nuclear membrane suggests that there may be
specific transport or binding proteins that mediate this process.
However, it should be kept in mind that cPLA2 is also
important in mediating eicosanoid production in platelets, which do not
contain a nucleus (49).
Regulation of cPLA2 by Phosphorylation In many cell types a stable increase in cPLA2 activity
occurs upon exposure to diverse agonists and can be reversed by
phosphatase treatment (1). Agonist-induced phosphorylation of
cPLA2 on serine residues has been verified, and the
increased cPLA2 activity in stimulated cells is attributed
to phosphorylation on Ser505 by MAPK (50-52).
cPLA2 has one consensus site for MAPK
(PLS505P), and phosphorylation of this site in
vitro by either p42 or p44 MAPKs increases cPLA2
activity and induces a characteristic retardation in its
electrophoretic mobility or gel shift (53, 54) (Fig. 1).
Phosphorylation of Ser505 is important in the activation of
cPLA2 in vivo since overexpression of mutant
cPLA2 (S505A) in Chinese hamster ovary cells fails to enhance agonist-induced arachidonic acid release as seen when wild type
enzyme is expressed (54). Other kinases such as PKC and protein kinase
A can phosphorylate cPLA2 in vitro, but this does not result in a significant increase in cPLA2 activity
or a gel shift. However, there is evidence for a role for PKC in the
activation of PLA2 and regulation of arachidonic acid
release. PKC activation can play a role by triggering a kinase cascade leading to MAPK activation (55). There is no evidence at this time that
PKC directly phosphorylates cPLA2 in vivo.
Agonist-induced phosphorylation (gel shift) and activation of
cPLA2 correlates with the activation of p42/p44 MAPKs in
many cell models (1). However, it has been shown that phosphorylation of cPLA2 in lipopolysaccharide- or TNF Although phosphorylation on Ser505 is important for
cPLA2 activation in certain cells it is not sufficient for
full activation leading to arachidonic acid release (1). In
macrophages, CSF1 is a strong activator of p42/p44 MAPKs and induces
phosphorylation and an increase in activity of cPLA2.
However, CSF1 does not induce arachidonic acid release, but it acts
synergistically with calcium-mobilizing agonists (63). This suggests
that an increase in intracellular calcium, necessary for
cPLA2 translocation, and cPLA2 phosphorylation, which enhances enzymatic activity, can act together to fully activate cPLA2. Interestingly, phosphorylation of cPLA2
must precede the increase in intracellular Ca2+ to fully
activate the enzyme for arachidonic acid release in HER14 cells, and
this suggests that cPLA2 may not be available for
phosphorylation if it is first translocated to membrane (64). However,
there is evidence suggesting alternative pathways for regulation of
cPLA2 activation and arachidonic acid release. In macrophages and neutrophils, PMA can induce an increase in
phosphorylation and catalytic activity of cPLA2 as well as
arachidonic acid release, and this occurs without an increase in
intracellular calcium (51, 65, 66). However, in other cell models PMA
treatment alone induces little arachidonic acid release despite
stoichiometric phosphorylation of cPLA2 and must be
combined with a calcium-mobilizing agonist (50). This suggests that
there are novel pathways for regulation of cPLA2 activation
and arachidonic acid release that are cell-specific. Okadaic acid also
induces arachidonic acid release in macrophages without increasing
intracellular calcium, and this correlates with phosphorylation of a
novel site on cPLA2 (66). Using cPLA2 expressed
in Sf9 insect cells with baculovirus, phosphorylation sites on
cPLA2 have been identified (52). cPLA2 is
expressed as an activated phosphoprotein in insect cells where it is
constitutively phosphorylated on Ser505 (52, 67). However,
Sf9 cells overexpressing cPLA2 exhibit little constitutive
release of arachidonic acid, but release can be greatly stimulated by
treatment with okadaic acid or calcium ionophore (52). The
phosphorylated residues on the expressed cPLA2 have been
identified as Ser437, Ser454,
Ser505, and Ser727 (52) (Fig. 1).
Ser437 and Ser505 are the major phosphorylation
sites on the enzyme from unstimulated Sf9 cells. In response to okadaic
acid, an increase in phosphorylation preferentially occurs on
Ser727. A23187 induces only a small increase in labeling of
all the sites. There is also evidence that Ser727 is
phosphorylated on cPLA2 in okadaic acid-treated human
monocytes and mouse macrophages. Since okadaic acid does not induce an
increase in intracellular calcium the results suggest alternative
mechanisms for cPLA2 activation perhaps involving
phosphorylation of Ser727. Ser727 and
Ser505 are conserved in cPLA2 from
evolutionarily distant species (human, murine, chicken, zebrafish)
consistent with important functional roles (1). The kinase that
phosphorylates Ser727 has not been identified, but it is
not a MAP kinase since Ser727 lies within a consensus
sequence for a basotrophic kinase (RXS).
There is suggestive evidence that cPLA2 is also
phosphorylated on tyrosine residues. In HEL-30 keratinocytes treated
with transforming growth factor cPLA2 plays a role in mediating several forms of cell
injury. In some models the mechanism involved has not been defined
whereas in other models, arachidonic acid metabolites are implicated. UV light has been shown to induce activation and increased synthesis of
cPLA2 in keratinocytes and human skin (70). UVB is a potent inducer of inflammation, and this is thought to be important in its
ability to act as a tumor promoter. The production of prostaglandin E2 after UVB exposure plays an important role in mediating
UV erythema, and cPLA2 is the enzyme responsible for
UV-induced prostaglandin E2 production in human
keratinocytes (70). In human skin, UV-induced erythema correlates with
increased synthesis of cPLA2, which is mediated by
formation of free radicals. UVB irradiation is known to induce a potent
oxidative response. UV-induced cPLA2 synthesis is inhibited
by antioxidants, and oxidizing agents can increase cPLA2
synthesis (71). These results indicate that cPLA2 is
regulated by UV exposure through generation of free radicals and plays
an important role in UV-induced injury.
In other models, cellular oxidants have also been shown to activate
cPLA2. In vascular smooth muscle cells, hydrogen peroxide (H2O2) induces serine phosphorylation and
activation of cPLA2 (72). In this system, cPLA2
activation and arachidonic acid production are implicated in the
induction of c-fos and c-jun and the mitogenic
response induced by H2O2. In contrast,
treatment of kidney epithelial cells with H2O2
has been shown to activate cPLA2, which contributes to
oxidant-induced cytotoxicity (73). In kidney cells overexpressing
either cPLA2 or sPLA2, only cells expressing
cPLA2 are susceptible to
H2O2-induced cell injury. Of interest,
chelating extracellular calcium protects against H2O2-induced injury and is consistent with the
known role of calcium in cPLA2 activation. However, simply
raising intracellular calcium with A23187 to activate cPLA2
does not induce injury, suggesting that other cellular changes induced
by H2O2, perhaps membrane lipid peroxidation,
together with cPLA2 activation contribute to the toxic
response. The observations that exogenous arachidonic acid does not
enhance H2O2-induced injury and that the kidney cells produce few eicosanoids suggest that these PLA2
products may not contribute to H2O2-induced
cell injury.
cPLA2 is also involved in TNF-induced cell death. Ceramide
is an important second messenger that mediates the response to TNF
(74). TNF induces cPLA2 activation, and the released
arachidonic acid is implicated as a regulator of sphingomyelinase
activation leading to ceramide production. Importantly, it has been
shown that a cPLA2-deficient L929 cell line is resistant to
TNF-induced toxicity, but this response is restored when
cPLA2 is expressed (75). However, Fas-induced apoptosis is
independent of cPLA2 since cross-linking this receptor
induces cell death equally well in deficient or
cPLA2-expressing L929 cells (76). Thus the Fas and TNF type
I receptors act through different pathways for inducing apoptosis. Many
cell types are not killed by TNF, but some can be rendered susceptible
to TNF in conjunction with inhibitors of transcription or translation,
and this susceptibility also correlates with the level of
cPLA2 present (77). The role that cPLA2 plays
in mediating TNF In conclusion, cPLA2 is subject to diverse mechanisms
of regulation that we are only beginning to understand.
cPLA2 can be quickly activated by post-translational
mechanisms to rapidly mobilize arachidonic acid for eicosanoid
production. It is also subject to increased synthesis and long term
activation leading to prolonged production of lipid mediators. These
different mechanisms of regulation are important in determining the
diverse functional roles of cPLA2. Depending on the tissue
or cell type, cPLA2 has now been implicated to function in
various cellular responses such as mitogenesis, differentiation,
inflammation, and cytotoxicity. cPLA2 can play a functional
role through its ability to trigger arachidonic acid release and
eicosanoid production. However, in many cases the mechanisms have not
been defined, and it is possible that cPLA2 may act by
affecting local changes in membrane structure or by modulating
protein/protein interactions important for signaling mechanisms.
and modulation of
Ca2+ transients (7-11). Arachidonic acid can also be
converted to potent inflammatory lipid mediators, the eicosanoids. This
can occur enzymatically through the lipoxygenase or cyclooxygenase (COX) pathways for the production of leukotrienes, lipoxins,
thromboxanes, or prostaglandins (12, 13). Arachidonic acid is also
subject to non-enzymatic, free radical oxidation to bioactive
isoprostanes and isoleukotrienes (14, 15). The important role of
arachidonic acid in cellular activation ensures that its levels are
tightly controlled. cPLA2 plays a role in maintaining
arachidonate levels and is subject to complex mechanisms of regulation
at both the transcriptional and post-translational levels. The
involvement of cPLA2 in lipid mediator production makes it
a potentially important pharmacological target for anti-inflammatory
drugs.
-flanking region
of the human gene, which has certain characteristics of a housekeeping
promoter in that it has no TATA box (16, 19, 20). However, unlike many
housekeeping promoters, it does not contain a GC-rich region or SP1
sites. The 5
-flanking region of the cPLA2 gene contains a
27-base pair region with a polypyrimidine sequence that is responsible for the basal expression of cPLA2 (16). In addition, a
48-base purine/pyrimidine repeat (CA repeat) appears to confer an
inhibitory effect on cPLA2 gene transcription (20). By
genetic analysis, the human cPLA2 gene has been localized
to chromosome 1q31-41 between markers F13B and D1S74 (16). However, by
structural analysis it has been mapped to chromosome 1q25 (21). Of
interest, the COX 2 gene has also been mapped to the 1q25 region
(22).
(IFN
) have been shown to induce activation and increase synthesis of cPLA2
in diverse cell models (1). In WI-38 cells, the increase in
cPLA2 synthesis induced by interleukin-1 correlates with
PGE2 production, and both effects are suppressed by
glucocorticoids (23). In some models, there is coordinate up-regulation
of both cPLA2 and COX 2 (1). The induction of
cPLA2 gene expression by IFN
in an epithelial cell line
is proposed to occur at the transcriptional level (24). Consistent with
this, the promoter for human cPLA2 contains a putative
IFN
-activated sequence and IFN
response elements (20). In
addition, DNA sequence analysis has revealed two glucocorticoid
response elements suggesting that steroids may act to suppress
cPLA2 synthesis at the transcriptional level. There is also
evidence for post-transcriptional regulation of cPLA2
synthesis. In rat mesangial cells, phorbol myristate acetate (PMA),
platelet-derived growth factor, and serum and epidermal growth factor
increase the half-life of cPLA2 mRNA (25). An adenosine-uridine-rich sequence in the 3
-untranslated region of
cPLA2 is thought to be responsible for the instability of
the cPLA2 transcript. Stimulation of cPLA2
synthesis occurs over hours and results in the prolonged release of
arachidonic acid and eicosanoid production.
Fig. 1.
Schematic representation of the primary
structure of cPLA2. The N-terminal CaLB domain
(yellow) mediates calcium-dependent membrane
binding. Sites phosphorylated (green) on recombinant cPLA2 expressed in insect cells are indicated.
S505 is the site phosphorylated by MAP kinases. Residues
essential for catalytic activity (orange) are shown.
S228 is the active site nucleophile. Thiol modification of
C331 (pink) leads to loss of cPLA2
activity.
[View Larger Version of this Image (21K GIF file)]
-chymotrypsin complexed with a peptidyltrifluoromethyl ketone
inhibitor (36). This suggests that the cPLA2 complex exists
as a hemiketal and implicates an active site serine. cPLA2
shares some sequence homology with phospholipase B from
Penicillium notatum, which exhibits both PLA2
and lysophospholipase activity. Mutation of a conserved serine
(Ser228) in cPLA2 that aligns with a lipase
consensus sequence of phospholipase B (Gly-Leu-Ser-Gly-Gly) abolishes
both PLA2 and lysophospholipase activities suggesting a
role for Ser228 in catalysis (33) (Fig. 1). The role of
Ser228 as the active site nucleophile has recently been
confirmed (37). When COS cells are cotransfected with COX 1 and mutant
forms of cPLA2 in which Ser228 is replaced with
Ala, Cys, or Thr, they cannot provide arachidonic acid for
prostaglandin production in response to A23187 unlike cells transfected
with wild type cPLA2. Although the S228C mutant cPLA2 is inactive on phospholipid substrates, it exhibits
measurable activity against 7-hydroxycoumarin esters unlike the S228A
mutant. These results support an acyl-enzyme intermediate and show that Ser228 can be replaced by cysteine as the active site
nucleophile albeit with reduced catalytic activity. Ser228
is located in the sequence GLS228GS
(GXSXS) that is proposed to be an active site
consensus sequence for a second group of lipolytic serine esterases
(38). Arg200 and Asp549 are also essential for
cPLA2 activity (35) (Fig. 1). The Asp549 is
comparable with the aspartic acid residue in the catalytic site of the
subtilisin proteases. Surprisingly, a histidine residue does not appear
to play a role in cPLA2 action. The mechanism for the
involvement of Arg200 is not known. Thus cPLA2
contains a novel catalytic center that is responsible for its multiple
catalytic activities.
, synaptotagmin, rabphilin, and p120GAP to name a few (1)
(Fig. 1). An increasing number of proteins containing C2 domains are
being identified, and many play a role in signal transduction and
membrane trafficking (39). The first C2 domain of synaptotagmin I has
been the most extensively characterized and contains 5 aspartate
residues that coordinate two calcium ions (40, 41). The C2 domain of
cPLA2 spans from amino acid residue 18 to 141 (39). Four of
the five acidic groups that are thought to bind Ca2+ are
present in the CaLB domain of cPLA2 (Asp37,
Asp43, Asp93, and Glu100). The 5th
residue in cPLA2 (Asn95) is a conservative
substitution (39). Calcium at 0.3-1.0 µM promotes
binding of cPLA2 to membrane and correlates with the concentration of calcium necessary to stimulate catalytic activity in vitro using phospholipid vesicles as substrate (6, 42). A
fragment of cPLA2 containing the CaLB domain binds membrane at the same concentrations of calcium required for the intact enzyme,
whereas a C-terminal fragment lacking the C2 domain fails to bind
membrane but retains catalytic activity against monomeric phospholipid
substrates (43). This work clearly delineates two distinct structural
and functional domains of cPLA2.
-treated
neutrophils and thrombin-stimulated platelets occurs independent of p42
or p44 MAPK activation (56-58). Since cPLA2 exhibits a gel
shift and increased activity in these models, it suggests that the
enzyme is phosphorylated by a proline-directed kinase on
Ser505, and p38 kinase has recently been implicated (59,
60). The MAPK homologues p38 and c-Jun N-terminal kinases (JNK) can
directly phosphorylate recombinant cPLA2 in
vitro and induce a cPLA2 gel shift implicating
phosphorylation on Ser505 (61). However, it has recently
been shown in platelets using an inhibitor of p38 kinase that
proline-directed phosphorylation of cPLA2 is not required
for mobilization of arachidonic acid in response to thrombin (62). This
suggests that alternative mechanisms are involved in functional
activation of cPLA2 in platelets or that an increase in
intracellular calcium alone is sufficient.
, cPLA2 is detected in
antiphosphotyrosine immunoprecipitates (68). In HeLa S3 cells,
immunoprecipitated cPLA2 is tyrosine-phosphorylated in
unstimulated cells, and this increases after treatment with IFN-
(69). Interestingly, the tyrosine kinase Jak1 co-immunoprecipitates
with cPLA2 in these cells, and this association is not
dependent on IFN treatment. IFN induces arachidonic acid release in
these cells, and Jak has been shown to be required for this response.
In HeLa S3 cells, cPLA2 is required for IFN-
-induced
assembly of interferon-stimulated gene factor 3 (69). The mechanism
whereby cPLA2 mediates this response is not known but is
postulated not to involve an arachidonic acid metabolite. The results
showing tyrosine phosphorylation of cPLA2 are intriguing
and await confirmation by phosphoamino acid analysis. In several models
in which serine phosphorylation of cPLA2 has been confirmed
by phosphoamino acid analysis, no tyrosine phosphorylation of
cPLA2 has been evident. This suggests that the tyrosine
phosphorylation is a cell type- and stimulus-specific event.
-induced cell death has not been elucidated.
I thank Dennis Voelker for critical review of the manuscript and preparation of the figure and Brenda Sebern for preparing the manuscript.