A Novel Calcium-independent Phospholipase A2, cPLA2-γ, That Is Prenylated and Contains Homology to cPLA2 *

We report the cloning and characterization of a novel membrane-bound, calcium-independent PLA2, named cPLA2-γ. The sequence encodes a 541-amino acid protein containing a domain with significant homology to the catalytic domain of the 85-kDa cPLA2 (cPLA2-α). cPLA2-γ does not contain the regulatory calcium-dependent lipid binding (CaLB) domain found in cPLA2-α. However, cPLA2-γ 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-γ. Interestingly, cPLA2-γ demonstrates a preference for arachidonic acid at the sn-2position of phosphatidylcholine as compared with palmitic acid. cPLA2-γ encodes a 3-kilobase message, which is highly expressed in heart and skeletal muscle, suggesting a specific role in these tissues. Identification of cPLA2-γ reveals a newly defined family of phospholipases A2 with homology to cPLA2-α.


From the Small Molecule Drug Discovery Group, Genetics Institute, Cambridge, Massachusetts 02140
We report the cloning and characterization of a novel membrane-bound, calcium-independent PLA 2 , named cPLA 2 -␥. The sequence encodes a 541-amino acid protein containing a domain with significant homology to the catalytic domain of the 85-kDa cPLA 2 (cPLA 2 -␣). cPLA 2 -␥ does not contain the regulatory calcium-dependent lipid binding (CaLB) domain found in cPLA 2-␣. However, cPLA 2 -␥ 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 [ 3 H]mevalonolactone is incorporated into the prenylation motif of cPLA 2 -␥. Interestingly, cPLA 2 -␥ demonstrates a preference for arachidonic acid at the sn-2 position of phosphatidylcholine as compared with palmitic acid. cPLA 2 -␥ encodes a 3-kilobase message, which is highly expressed in heart and skeletal muscle, suggesting a specific role in these tissues. Identification of cPLA 2 -␥ reveals a newly defined family of phospholipases A 2 with homology to cPLA 2 -␣.
Phospholipases A 2 (PLA 2 ) 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 A 2 family is large, and individual members can be classified according to localization (extracellular versus intracellular), sequence homology, and biochemical characteristics (1). Known PLA 2 members include the secreted PLA 2 s and the cytosolic PLA 2 s. To date only two cytosolic PLA 2 sequences have been reported: the calcium-dependent PLA 2 (cPLA 2 -␣) and the calcium-independent PLA 2 (iPLA 2 ) (2)(3)(4)(5). cPLA 2 -␣ 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)(8)(9). cPLA 2 -␣ 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). cPLA 2 -␣ 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 cPLA 2 -␣ (11,12). Although there have been many studies that suggest the importance of cPLA 2 in the generation of prostaglandins and leukotrienes, the most convincing data have come from studies using mice that are genetically deficient in cPLA 2 (13,14). Studies demonstrate that cPLA 2 -␣ 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 cPLA 2 in asthma was also shown (13).
The 85-kDa calcium-independent iPLA 2 , purified by two groups, shares no homology with cPLA 2 -␣ except, like other lipases, it contains the critical consensus sequence, GXSXG (4,5,15). Interestingly, iPLA 2 contains a domain of eight ankyrin repeats, which may be involved in protein-protein interactions (4). iPLA 2 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 cPLA 2 -␣ and iPLA 2 are the only intracellular PLA 2 s that have been cloned, many other PLA 2 activities, which presently seem to be distinct from cPLA 2 -␣ and iPLA 2 , have been reported (17)(18)(19). The relationship of the enzymes responsible for these activities to the known PLA 2 enzymes will be clear only upon sequence determination.
Our initial efforts to identify additional PLA 2 enzymes failed using low stringency cross-hybridization techniques with cPLA 2 -␣ sequences. 2 A search of the expressed sequence tag (EST) data base was quite successful, and two independent cPLA 2 -␣ related gene fragments were identified. Subsequent sequence analysis of the full-length clones revealed two novel homologs of cPLA 2 -␣, designated cPLA 2 -␤ and cPLA 2 -␥. The characterization of cPLA 2 -␤ will be described elsewhere. 3 Here, we report the sequence and characterization of a novel 60.9-kDa calcium-independent, membrane-associated cPLA 2, cPLA 2 -␥.

EXPERIMENTAL PROCEDURES
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 g/ml streptomycin, and 1 mM glutamine. Cells were incubated in a 37°C humidified atmosphere with 10% CO 2 . Chinese hamster ovary (CHO) cells were maintained in alpha medium (Life Technologies, Inc.) containing 50 units/ml penicillin, 50 g/ml streptomycin, 1 mM glutamine, 1 mg/ml G418 (Life Technologies, * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Clone Identification-The EST clone 258543 (GenBank accession N56796) was identified by searching the GenBank EST data base using the amino acid sequence of cPLA 2 -␣. The 900-base pair EcoRI-NotI fragment from clone 258543 was used to screen 10 6 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 32 Plabeled EcoRI-NotI fragment of cPLA2-␥. The blot was washed under high stringency conditions (0.2 ϫ SSC at 65°C). Each lane comprises approximately 2 g of poly(A) ϩ mRNA.
Construction of Expression Vectors-Two oligonucleotides, 5Ј-GT-TCACCTCATCCTCTCCTTCGAC-3Ј and 5Ј-TCGGGGTACCGAAT-TCGGGCCCTATGCCAAGCAGCAACTTCGGGCACT-3Ј, corresponding to the 3Ј-end of cPLA 2 -␥, were used to amplify the 3Ј-end coding region of cPLA 2 -␥ 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-cPLA 2 -␥WT, was sequenced to confirm the desired C-terminal coding sequence. Mutation of the C-terminal ϪCCLA was produced as above except using an oligonucleotide that introduced the mutation (C538S,C539S) to the 3Ј end of the sequence. The resulted clone, named pED⌬C-cPLA 2 -␥SSLA, was confirmed by sequencing to contain the desired C-terminal coding sequence.
Stable CHO Cell Lines Overexpressing cPLA 2 -␥ and cPLA 2 -␣-The plasmid of wild-type cPLA 2 ␥ was constructed as follows. The restriction fragment (EcoRI-EcoRI) containing the entire coding sequence was isolated from pED⌬C-cPLA 2 -␥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.
Stable CHO cell lines of cPLA 2 -␥ and cPLA 2 -␣ were generated by transfecting CHO cells that constitutively express tTA with pHTOP-cPLA 2 -␥ or pTOP6-cPLA 2 -␣. Transfection was performed using Lipofectin as recommended by the manufacturer (Life Technologies, Inc.). Transfectants were then selected in growth medium containing methotrexate at a concentration of 5-100 nM.
Prenylation Assay-70 -80% confluent COS cells (10-cm plate) were transfected with 8 g of pMev (obtained from ATCC (24), to facilitate uptake of mevalonolactone) and either 8 g of pED⌬C-cPLA 2 -␥WT, pED⌬C-cPLA 2 -␥SSLA, or pzflag (for vector control) or 4 g 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 M mevistatin (Biomol) for 1 h. The cells were then incubated in 3 ml of growth media containing 40 M mevistatin and 150 Ci of [ 3 H]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 l of lysis buffer (20 mM Tris-HCl, pH 7.5, 10% glycerol, 1% Triton X-100, 137 mM NaCl, 2 mM EDTA, 5 g/ml aprotinin, 10 g/ml leupeptin, and 2 mM phenylmethylsulfonyl fluoride for 10 min. A 20-l aliquot was removed and centrifuged, and the pellet was resuspended in 50 l of 2 ϫ 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 ϫ g, 4°C. The supernatant was immunoprecipitated with 10 l of anti-cPLA 2 -␥ 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 l of 2 ϫ 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 cPLA 2 -␥, aliquots of the samples were subjected to 4 -20% SDS-PAGE. Proteins were transferred to a nitrocellulose filter (Novex), immunoblotted for cPLA 2 -␥ with 44284 antibody, and detected by ECL (Amersham).

RESULTS
cPLA 2 -␥ Is a Novel cPLA 2 -The EST data base was searched to identify sequences similar to the human 85-kDa cPLA 2 gene (cPLA 2 -␣). This analysis led to the identification of two related genes, named cPLA 2 -␤ and cPLA 2 -␥. The cPLA 2 -␥ EST clone 258543 was shown to contain a partial cDNA insert with sequence similarity to the C terminus of cPLA 2 -␣. 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 cPLA 2 -␥ and cPLA 2 -␣ ( 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 cPLA 2 -␣ 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 cPLA 2 -␥ are the amino acids that make up the putative catalytic triad of cPLA 2 -␣ (8). These amino acids correspond to serine 82, aspartate 385, and arginine 54 in cPLA 2 -␥. cPLA 2 -␣ contains a CaLB or C-2 domain that has been shown to be important for calcium-dependent binding of the enzyme to membranes (6). cPLA 2 -␥ does not contain a CaLB domain and is therefore likely to be a novel calcium-independent phospholipase. Interestingly, a motif search of cPLA 2 -␥ reveals the presence of a C-terminal ϪCAAX box (ϪCCLA). This motif has been identified as a signal for prenylation, where C is the cysteine that becomes modified, A is an aliphatic amino acid, and X is any amino acid (27). The N terminus of cPLA 2 -␥ also contains a sequence that is a potential site for myristoylation (M-G-X-X-X-(S/small uncharged)-X) (Ref. 28 and the Prosite data base). Similar to other lipid-modified proteins, these putative lipid modifications may regulate the localization of cPLA 2 -␥ within the cell.
To determine the tissue distribution of cPLA 2 -␥, Northern blot analysis was performed. Hybridization of the EST clone with RNA from various human tissues indicates that cPLA 2 -␥ mRNA is approximately a 3-kilobase transcript. Strikingly, cPLA 2 -␥ is most abundant in skeletal muscle and heart, with lower levels in spleen, brain, placenta, and pancreas (Fig. 2). ]arachidonyl-PC, cPLA 2 -␥ seems to be as proficient at cleaving at the sn-1 site as the sn-2 site. This is dissimilar from cPLA 2 ␣ (Fig.  3B), which under the conditions used has no apparent sn-1 activity. Importantly, the PLA 1 and PLA 2 activity of cPLA 2 -␥ does not seem to be sequential, as the radiolabeled fatty acid released from 1-palmitoyl-2-[ 14 C]arachidonyl-PC and using 1-[ 14 C]palmitoyl-2-arachidonyl-PC showed similar kinetics. cPLA 2 -␥ Is Prenylated-cPLA 2 -␥ contains the C-terminal sequence ϪCCLA, 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 ϪAAX, followed by methylation of the cysteine carboxyl group (29). The sequence of cPLA 2 -␥ 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).
To investigate the utilization of the prenylation motif on cPLA 2 -␥, COS cells were transfected with either cPLA 2 -␥ or cPLA 2 -␥ with the C terminus mutated from CCLA to SSLA. A plasmid encoding Ras was transfected as a positive control for prenylation. Approximately 48 h post-transfection, the cells were incubated for 14 h with the isoprenoid precursor, [ 3 H]mevalonolactone. Cell lysates were prepared as described under "Experimental Procedures" and analyzed by SDS-polyacrylamide gel electrophoresis. Autoradiographic analysis reveals a band at 60 kDa in cells that were transfected with the wild-type cPLA 2 -␥ (Fig. 4A). Whereas cells transfected with plasmid encoding the mutant protein, vector only, or Ras show no bands at 60 kDa. Similar results were obtained when the cell lysate was immunoprecipitated with cPLA 2 -␥ antibody, 44282, confirming that the 60-kDa protein is cPLA 2 -␥ (data not shown). To determine if equal amounts of wild-type and mutant protein were expressed in the COS cells, Western blot analysis was performed on the samples and indicated equal expression of the two proteins (Fig. 4B). These data indicate that cPLA 2 -␥ is prenylated at its C terminus. cPLA 2 -␥ Is a Membrane-associated Protein-To determine the subcellular localization of cPLA 2 -␥, CHO cells stably transfected with cPLA 2 -␥ were lysed by nitrogen cavitation and centrifuged for 1 h at 100,000 ϫ g. cPLA 2 -␥ was then detected by Western analysis. As shown in Fig. 5, cPLA 2 -␥ 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 ϫ g results in the majority of the enzyme being present in the supernatant. This is unlike cPLA 2 -␣, 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 cPLA 2 -␥, as is the case for Ras. However, fractionation of cells transfected with cPLA 2 -␥ 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 cPLA 2 -␥ with the membrane. cPLA 2 -␥ Is Calcium-independent-Unlike the secreted PLA 2 s, which require millimolar concentrations of calcium for activity, the catalytic domain of cPLA 2 -␣ does not require calcium for activity (6). However, cPLA 2 -␣ does require micromolar concentrations of calcium for membrane binding through its CaLB domain. We were interested in determining the requirement of calcium for cPLA 2 -␥ activity. Cell lysates prepared from COS cells transfected with cPLA 2 -␥ or cPLA 2 -␣ were incubated with 1-palmitoyl-2-[ 14 C]arachidonyl-PC in the presence of 0 or 10 M or 10 mM calcium (Fig. 6). cPLA2-␥ activity is unaffected by calcium, unlike cPLA2-␣ activity, which increases 14-fold in the presence of 10 M calcium. This result demonstrates that cPLA 2 -␥ is a calcium-independent enzyme.
Substrate Specificity at the sn-2 Position-cPLA 2 -␣ selectively hydrolyzes arachidonic acid at the sn-2 position in several assay formats, whereas iPLA 2 selectivity is significantly more assay dependent. To determine if cPLA 2 -␥ 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 cPLA 2 -␥ or cPLA 2 -␣. cPLA 2 -␥ 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).

DISCUSSION
We have identified a novel 60.9-kDa calcium-independent phospholipase A 2 , which we termed cPLA 2 -␥. cPLA 2 -␥ contains 28.7% overall sequence identity with cPLA 2 -␣ 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). cPLA 2 -␥ contains the sequence GVS 82 GS, which is similar but slightly different from the consensus. However, this sequence aligns with the sequence in cPLA 2 -␣, and the change from glycine to serine also occurs in the corresponding region of cPLA 2 -␣, GLS 228 GS (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 cPLA 2 -␣, abrogated cPLA 2 -␣ 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 Cells were processed as stated under "Experimental Procedures." 1% Triton X-100 pellets were subjected to 4 -20% SDS-PAGE, soaked in entensify and exposed to Biomax MS film for 7 days (A). To examine the expression levels of cPLA2-␥, aliquots of samples were subjected to 4 -20% SDS-PAGE, followed by Western blot analysis using anti-cPLA2-␥ antibody, 44284 (B).

FIG. 5. Localization of cPLA2-␥ in CHO cells stably transfected
with cPLA2-␥. CHO cells overexpressing cPLA2-␥ were lysed by nitrogen cavitation (CL) and spun for 1 h at 100,000 ϫ g at 4°C. The supernatant was collected (S). The pellet (P) was resuspended in lysis buffer containing 1% Triton X-100 and centrifuged for 1 h at 100,000 ϫ g at 4°C. The supernatant and pellet fractions were collected. Samples were subjected to 4 -20% SDS-PAGE followed by Western blot analysis using anti-cPLA2-␥ antibody, 44282.  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. cPLA 2 -␣ 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 cPLA 2 -␣ are not conserved in the sequence of cPLA 2 -␥, suggesting a different regulatory mechanism. Interestingly, cPLA 2 -␥ 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 [ 3 H]mevalonolactone is readily incorporated into cPLA 2 -␥ 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 (ϪCCLA), we do not know which of these isoprenoids is modifying the protein.
One of the most striking differences between cPLA 2 -␣ and -␥ is the lack of a lipid binding CaLB domain in cPLA 2 -␥. The presence of lipidation motifs suggests that these regions may function as the CaLB domain in cPLA 2 -␣, localizing the enzyme to the membrane and being critical for activity. However, in a preliminary study, cPLA 2 -␥ 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 cPLA 2 -␥ 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 cPLA 2 -␥ in the cells. cPLA 2 -␥ will hydrolyze fatty acids at the sn-1 and sn-2 position of phosphatidylcholine. This suggests that cPLA 2 -␥ contains PLA 1 and PLA 2 activity. The evidence that cPLA 2 -␥ contains PLA 2 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 PLA 1 activity or if it is cleavage of the lysophospholipid. The kinetics of the reactions suggest that it is PLA 1 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 cPLA 2 -␥ is an enzyme with PLA 2 activity and a probable PLA 1 activity. cPLA 2 -␥ 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 cPLA 2 -␣ displays for arachidonic acid, 3.5-fold versus 24.5-fold, respectively. The substrate specificity of cPLA 2 -␥ 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. cPLA 2 -␥ 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, cPLA 2 -␣ activity is also regulated by MAP kinase phosphorylation of serine-505. This serine is not conserved in the sequence of cPLA 2 -␥; however, there are several potential protein kinase C phosphorylation sites, which may be utilized to regulate the enzyme. cPLA 2 -␥ 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)(18)(19). Hazen et al. (18) and McHowat and Creer (19) have identified membrane-bound, calcium-independent PLA 2 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 PLA 2 activity leads to the accumulation of lysophospholipids and subsequent injury to the heart tissue because of disruptions of the membrane. As the PLA 2 activity of this myocardial membrane-bound enzyme was shown to increase under hypoxic conditions, it was suggested that this calcium-independent PLA 2 is physiologically involved in ischemia (18). Because cPLA 2 -␥ 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 cPLA 2 -␥ 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 PLA 2 , cPLA 2 -␥. This enzyme shares identity with cPLA 2 -␣ and contains the potential critical amino acids for the catalytic site but is missing the key elements that regulate the activity of cPLA 2 -␣. This suggests that the mechanisms of regulation for cPLA 2 -␥ will be different from that of cPLA 2 -␣ and quite possibly employs the use of the lipid modification. Defining the mechanisms of regulation and the physiological substrate of cPLA 2 -␥ should shed some light on the physiological role of this newly identified PLA 2 .