MINIREVIEW:
Properties and Regulation of Cytosolic Phospholipase A2*

Christina C. Leslie Dagger

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

INTRODUCTION
Distribution of cPLA2 and Regulation of Its Synthesis
Catalytic Mechanism
Regulation of cPLA2 by Calcium
Regulation of cPLA2 by Phosphorylation
Role of cPLA2 in Cytotoxicity
Conclusion
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


INTRODUCTION

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 Cgamma 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.


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'-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).

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 gamma  (IFNgamma ) 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 IFNgamma 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 IFNgamma -activated sequence and IFNgamma 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.


Catalytic Mechanism

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).


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)]

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 alpha -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.


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 Cgamma , 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.

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 TNFalpha -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.

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 alpha , 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-alpha (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-alpha -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.


Role of cPLA2 in Cytotoxicity

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 TNFalpha -induced cell death has not been elucidated.


Conclusion

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.


FOOTNOTES

*   This minireview will be reprinted in the 1997 Minireview Compendium, which will be available in December, 1997. This is the fourth article of six in "A Thematic Series on Phospholipases." Work in the author's laboratory was supported by National Institutes of Health Grant HL 34303.
Dagger    To whom correspondence should be addressed: Dept. of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1214; Fax: 303-270-2155; E-mail: lesliec{at}njc.org.
1   The abbreviations used are: PLA2, phospholipase A2; sPLA2, secretory PLA2; cPLA2, 85-kDa cytosolic PLA2; PKC, protein kinase C; COX, cyclooxygenase; TNF, tumor necrosis factor; CSF, colony-stimulating factor; IFNgamma , interferon gamma ; PMA, phorbol myristate acetate; CaLB, calcium-dependent phospholipid binding domain; MAP, mitogen-activated protein; MAPK, MAP kinase.

ACKNOWLEDGEMENTS

I thank Dennis Voelker for critical review of the manuscript and preparation of the figure and Brenda Sebern for preparing the manuscript.


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