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J. Biol. Chem., Vol. 281, Issue 23, 15615-15624, June 9, 2006

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Highly Selective Hydrolysis of Fatty Acyl-CoAs by Calcium-independent Phospholipase A₂

ENZYME AUTOACYLATION AND ACYL-CoA-MEDIATED REVERSAL OF CALMODULIN INHIBITION OF PHOSPHOLIPASE A₂ ACTIVITY^*

Christopher M. Jenkins, Wei Yan, David J. Mancuso, and Richard W. Gross^¶1

From the Division of Bioorganic Chemistry and Molecular Pharmacology, Departments of Medicine and Molecular Biology & Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 and the ^¶Department of Chemistry, Washington University, St. Louis, Missouri 63130

Received for publication, October 26, 2005 , and in revised form, March 20, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Calcium-independent phospholipase A₂ (iPLA₂ ) participates in numerous diverse cellular processes, such as arachidonic acid release, insulin secretion, calcium signaling, and apoptosis. Herein, we demonstrate the highly selective iPLA₂-catalyzed hydrolysis of saturated long-chain fatty acyl-CoAs (palmitoyl-CoA myristoyl-CoA >> stearoyl-CoA >> oleoyl-CoA arachidonoyl-CoA) present either as monomers in solution or guests in host membrane bilayers. Site-directed mutagenesis of the iPLA₂ catalytic serine (S465A) completely abolished acyl-CoA thioesterase activity, demonstrating that Ser-465 catalyzes both phospholipid and acyl-CoA hydrolysis. Remarkably, incubation of iPLA₂ with oleoyl-CoA, but not other long-chain acyl-CoAs, resulted in robust stoichiometric covalent acylation of the enzyme. Moreover, S465A mutagenesis or pretreatment of wild-type iPLA₂ with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one unexpectedly increased acylation of the enzyme, indicating the presence of a second reactive nucleophilic residue that participates in the formation of the fatty acyl-iPLA₂ adduct. Radiolabeling of intact Sf9 cells expressing iPLA₂ with [³H]oleic acid demonstrated oleoylation of the membrane-associated enzyme. Partial trypsinolysis of oleoylated iPLA₂ and matrix-assisted laser desorption ionization mass spectrometry analysis localized the acylation site to a hydrophobic 25-kDa fragment (residues 400–600) spanning the active site to the calmodulin binding domain. Intriguingly, calmodulin-Ca²⁺ blocked acylation of iPLA₂ by oleoyl-CoA. Remarkably, the addition of low micromolar concentrations (5 µM) of oleoyl-CoA resulted in reversal of calmodulin-mediated inhibition of iPLA₂ phospholipase A₂ activity. These results collectively identify the molecular species-specific acyl-CoA thioesterase activity of iPLA₂ , demonstrate the presence of a second active site that mediates iPLA₂ autoacylation, and identify long-chain acyl-CoAs as potential candidates mediating calcium influx factor activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Phospholipases A₂ (PLA₂s)² catalyze the hydrolysis of ester-linked fatty acids from glycerophospholipids, thereby regulating cellular signaling pathways through the generation of lysophospholipids, free fatty acids (e.g. arachidonic acid), and their downstream metabolites. In eukaryotes, PLA₂s are broadly categorized into three families: secretory, cytosolic, and calcium-independent phospholipases A₂ (iPLA₂) (1). Secretory PLA₂s are low molecular mass (12–15 kDa) extracellular enzymes that require high micromolar to millimolar concentrations of Ca²⁺ for catalysis (2, 3). Six cytosolic phospholipases A₂ (, ,, , , and ) have been characterized at present, five of which (, , , , and ) contain C2 domains that require submicromolar Ca²⁺ for membrane association (4–7). Calcium-independent PLA₂s are intracellular, do not require calcium ion for membrane association or catalysis, and currently comprise seven family members (, , , , , , ) (8–11), all of which contain conserved nucleotide-binding (GXGXXG) and lipase (GXSXG) sequence motifs.

Studies of calcium-independent phospholipase A₂ have revealed its importance in agonist-induced arachidonic acid release (12, 13), apoptosis (14, 15), lymphocyte proliferation (16), fat cell differentiation (17), insulin secretion (18), and lysolipid production mediating capacitive calcium influx (19, 20). In addition, recent experiments utilizing transgenic mice selectively overexpressing iPLA₂ in myocardium have provided evidence that cardiac ischemia activates iPLA₂, precipitating ventricular tachyarrythmias, which can be suppressed by pretreatment with the mechanism-based iPLA₂ inhibitor, (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) (21). Previously, we have demonstrated that iPLA₂ activity is regulated through calmodulin-mediated inhibition of phospholipase A₂ activity in the presence of physiologic concentrations of calcium (200 nM) (22). Subsequent experiments identified the calmodulin binding domain of iPLA₂, containing "1-9-14" and IQ sequence motifs, located 160 and 240 amino acid residues, respectively, from the active site (23). During cellular stimulation and the depletion of intracellular Ca²⁺ stores, activation of iPLA₂ has been proposed to occur through disassociation of the iPLA₂·CaM complex (13, 20), potentially through the actions of a low molecular weight cellular component known as calcium influx factor (CIF) (20, 24). Although CIF was first described and partially characterized more than 10 years ago as a diffusible messenger released upon intracellular Ca²⁺ store depletion, which stimulated Ca²⁺ influx through the plasma membrane (25, 26), the precise molecular identity of CIF is unknown.

Multiple fatty acyl-CoA thioesterases have been purified from mammalian cytosol, peroxisomes, and mitochondria and have been cloned and characterized with respect to substrate selectivity, enzyme kinetics, and sensitivity to various inhibitors (27). In general, fatty acyl-CoA thioesterases have been classified as those that are induced by peroxisome proliferators (Type-I or Type-II thioesterases) and those that do not share significant sequence homology with these isoforms (27). Several other mammalian enzymes, such as lysophospholipases (28), secretory phospholipase A₂ (29), and palmitoyl-protein thioesterases (30–32), have also been shown to exhibit acyl-CoA hydrolase activity. Interestingly, hepatocyte nuclear factor-4 has been recently demonstrated to hydrolyze fatty acyl-CoAs, followed by binding of the fatty acid product to hepatocyte nuclear factor-4, thereby allowing cross-talk between the acyl-CoA and free fatty acid binding domains (33).

In addition to its regulation by calmodulin, iPLA₂ binds ATP through a conserved nucleotide binding motif (GXGXXG), resulting in both enzyme stabilization and activation (34, 35). From this perspective, we considered the possibility that iPLA₂ might also bind and hydrolyze long-chain acyl-CoAs, given the structural similarity of the 3'-phosphoadenosine moiety of CoA to ATP. In this paper, we demonstrate that iPLA₂ catalyzes the hydrolysis of saturated long-chain acyl-CoAs present as either monomers or as guests in host membrane vesicles at physiologic concentrations (1–5 mol %). Moreover, highly selective autoacylation of iPLA₂ by oleoyl-CoA occurred at a second nucleophilic site(s) within the catalytic domain, which is protected from oleoylation by Ca²⁺-activated calmodulin. Finally, oleoyl-CoA was found to attenuate calmodulin-mediated inhibition of iPLA₂ phospholipase A₂ activity. In summary, these results describe the previously unrecognized fatty acyl-CoA thioesterase activity and fatty acyl-CoA dependent covalent acylation of iPLA₂, thereby revealing additional levels of complexity in this multifunctional signaling enzyme.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—[1-¹⁴C]palmitoyl-CoA, [1-¹⁴C]oleoyl-CoA, and 1-palmitoyl-2-[1-¹⁴C]oleoyl-sn-glycero-3-phosphocholine were obtained from PerkinElmer Life Sciences. [1-¹⁴C]Arachidonoyl-CoA, [1-¹⁴C]myristoyl-CoA, [1-¹⁴C]stearoyl-CoA, and [methyl-¹⁴C]human albumin were obtained from American Radiolabeled Chemicals. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS) were purchased from Avanti%20Polar%20Lipids">Avanti Polar Lipids. Sf9 cell culture reagents and 2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3,4-diaza-s-indacene-3-propionyl)amino) undecyl)-sn-glycero-3-phosphocholine (BODIPY-PC) were purchased from Invitrogen. High purity bovine calmodulin was obtained from Calbiochem. Most other materials were obtained from either Sigma or Fisher. BEL was purchased from Cayman Chemical and separated into individual enantiomers as described previously (36).

Construction of Recombinant Baculoviruses Encoding His-tagged Wild-type and S465A Mutant iPLA₂ Proteins—cDNA encoding wild-type C. griseus (Chinese hamster) iPLA₂ (9) was utilized as template for PCR amplification of the sequence to introduce six in-frame His codons followed by a stop codon and XhoI restriction site (3' primer) for subcloning into pFASTBac1. The 5' primer contained a SalI site and Kozak sequence (GCCACC) 5' of the ATG start codon. The His-tagged S465A iPLA₂ construct was created by PCR amplification of the mutant cDNA (9) utilizing a 5' primer containing an EcoRI restriction site and a3' primer encoding a His₆ tag followed by a SalI site for subcloning into pFASTBac1. The 2.4-kb products were each inserted into a bacmid shuttle vector and sequenced in both directions to confirm the integrity of the constructs. S. frugiperda (Sf9) cells in 35-mm plates containing supplemented Grace's medium were transfected and incubated at 27 °C for 72 h to produce high titer baculoviral stocks for infection of Sf9 cells.

Expression and Affinity Purification of iPLA₂His₆ from Sf9 Cells—Sf9 cells were grown as 100 ml of suspended cultures (in 1-liter plastic bottles) in supplemented Grace's insect medium containing 10% heat-inactivated fetal bovine serum and 0.1% Pluronic F-68. Following infection of 3 x 100-ml cultures of Sf9 cells (1.5 x 10⁶ cells/ml) with baculovirus encoding iPLA₂His₆ for 48 h, cells were harvested by centrifugation (250 x g for 10 min), washed once with Grace's insect medium without serum, and resuspended in 30 ml of 25 mM sodium phosphate, pH 7.8, 20% glycerol, 2 mM -ME, 5 µg/ml aprotinin, 5 µg/ml leupeptin. After lysing the cells by sonication (30 1-s bursts), the homogenate was centrifuged at 100,000 x g for 1 h to obtain the cytosol, to which NaCl was added to a final concentration of 250 mM. The cytosol was then mixed by inversion with 3 ml of HIS-Select-Co²⁺ affinity resin (Sigma) for 1 h, and the cytosol-resin suspension was poured into an Amersham Biosciences 1 x 10-cm column. Following washing of the settled resin with 30 ml of Buffer A (25 mM sodium phosphate, pH 7.8, containing 500 mM NaCl, 20% glycerol, and 2 mM -ME), bound protein was eluted from the column at a flow rate of 0.25 ml/min utilizing a 250 mM imidazole gradient in Buffer A (50-ml total volume) generated using an Amersham Biosciences fast protein liquid chromatography system. Column fractions were assayed for iPLA₂ activity as described below, pooled, and dialyzed overnight against Buffer B (25 mM imidazole, pH 7.8, containing 20% glycerol, 1 mM dithiothreitol, and 1 mM EGTA). The dialyzed sample was applied to a 2.5-ml column of ATP-agarose equilibrated with Buffer B and washed with Buffer B containing 1 mM AMP and 50 mM NaCl. Bound iPLA₂His₆ was eluted with Buffer B containing 2 mM ATP and 100 mM NaCl, dialyzed against Buffer B (EGTA concentration was reduced to 0.1 mM) containing 100 mM NaCl, flash frozen in liquid nitrogen, and stored at –80 °C. Approximately 1 mg (65% yield) of iPLA₂His₆ with a specific activity of 0.5 µmol of oleic acid/min/mg, utilizing 5 µM [¹⁴C]POPC as substrate, was typically recovered from 300 ml of cultured Sf9 cells by this procedure.

Phospholipase A₂ and Acyl-CoA Hydrolase Enzymatic Assays—Purified recombinant iPLA₂His₆ (0.1–1 µg) was incubated with radiolabeled phospholipid or acyl-CoA in 25 mM Tris-HCl, pH 7.2, containing 1 mM EGTA (200 µl final volume) for 1–2 min at 37 °C. In experiments using acyl-CoAs as guests in host phospholipid bilayers, radiolabeled acyl-CoAs were incorporated into POPC/DOPS (90:10 mol %) vesicles (100 µM) before addition to the reaction mix. Long-chain acyl-CoAs have been previously demonstrated to integrate into lipid bilayers within seconds (37). Reactions were terminated by extraction of the released radiolabeled fatty acids into 100 µl of butanol, separation of fatty acids from unreacted substrate by thin layer chromatography, and quantitation by scintillation spectroscopy as previous described (38). For experiments examining the effects of acyl-CoAs on calmodulin-mediated inhibition of iPLA₂, phospholipase A₂ activity was continuously measured utilizing a SPECTRAmax GEMINI XS Dual-Scanning Microplate Spectrofluorometer (Molecular Devices). BODIPY-PC substrate (1.17 mM in Me₂SO, 5 µM final concentration) was co-sonicated (10 min at 40% power, 50% duty cycle) with POPC (95 µM final concentration) in 25 mM HEPES, pH 7.2. Oleoyl-CoA and CaCl₂ were added at the indicated concentration to the lipid vesicles before addition to iPLA₂ with or without CaM (preincubated on ice for 10 min) present in individual wells of a black 96-well microtiter plate. Fluorescence readings were acquired at 15-s intervals for 2 min at 37 °C utilizing excitation/emission wavelengths of 495/515 nm, respectively.

Electrospray Ionization (ESI)/Mass Spectral (MS) Analyses—Purified iPLA₂ (0.25 µg) was incubated with POPC (95 µM) vesicles containing BODIPY-PC (5 µM) for 5 min at 37 °C as described above. The reactions were stopped by the addition of 4 ml of chloroform/methanol (1:1, v/v) containing internal standards (i.e. 14:1–14:1 PC and 17:0 LPC), and the lipid species were extracted into the chloroform layer as described previously (39). Extracted lipid samples were filtered with Millex-FG 0.20-µm filters (Millipore, Bedford, MA) and were routinely stored in 200 µl of chloroform/methanol (1/1, v/v) under nitrogen at –20 °C. ESI/MS analysis was performed using a Thermo Finnigan TSQ Quantum Plus spectrometer (San Jose, CA) equipped with an electrospray ion source as previously described in detail (40, 41). Samples were diluted with chloroform/methanol (1:1, v/v) (5 pmol/µl) prior to direct infusion into the ESI ion source at a flow rate of 4 µl/min with a 2-min period of signal averaging. Tandem mass scanning of neutral loss of 59 atomic mass units (loss of trimethylamine) was performed at a collision energy of 24 eV and a collision gas pressure of 1.0 millitorr.

Covalent Modification of iPLA₂ with ¹⁴C-labeled Long-chain Acyl-CoAs—Purified recombinant iPLA₂His₆ was incubated with POPC vesicles (90 µM) containing 10 mol % [1-¹⁴C]acyl-CoA for 1 h at 37 °C. In some experiments, iPLA₂His₆ was preincubated with BEL (3 min at 23 °C), N-ethylmaleimide (5 min at 30 °C) or iodoacetamide (5 min at 30 °C) prior to the addition of radiolabeled acyl-CoA. Chloroform/methanol precipitation of some samples was performed as described (42), utilizing 15 µg of bovine serum albumin as carrier. In experiments to examine the nature of the covalent linkage between oleic acid and iPLA₂, acid (HCl), base (NaOH), and hydroxylamine were added to the indicated concentrations, and the samples were incubated at 30 °C for 1 h. Bovine serum albumin (15 µg) and SDS-PAGE loading buffer were then added to each sample prior to dialysis against 50 mM Tris-HCl, pH 6.8, containing 10% glycerol and 1% SDS for 4 h. Samples were electrophoresed by SDS-PAGE, fixed (40% methanol containing 10% acetic acid), stained with Coomassie Blue R-250, incubated in Amplify fluorographic reagent, dried, and exposed to Eastman Kodak Co. Biomax MR film for 2–5 days at –80 °C.

Partial Trypsinolysis of Oleoylated iPLA₂—Purified iPLA₂His₆ (10 µM) was incubated with 50 µM [1-¹⁴C]oleoyl-CoA or unlabeled oleoyl-CoA in 25 mM imidazole, pH 7.8, containing 50 mM NaCl, 0.1 mM EGTA, 1 mM dithiothreitol, and 20% glycerol for 1 h at 37°C. Excess [1-¹⁴C]oleoyl-CoA was removed by using a Micro Bio-Spin (Bio-Rad) column equilibrated with the above buffer. Recovered iPLA₂ was partially digested with trypsin (1:25, w/w) for 1–30 min at 37 °C. Tryptic peptides were separated by SDS-PAGE, fixed in 40% methanol, 10% glacial acetic acid, stained with Coomassie Blue, and destained in the fixation solution. Gels containing the radiolabeled peptide fragments were soaked in Amplify fluorogenic reagent (Amersham Biosciences), dried, and exposed to film. In parallel samples utilizing unlabeled oleoyl-CoA, the band corresponding to the 25-kDa radiolabeled fragment was excised, cut into 1 x 1-mm pieces, and destained further by washing with 50% acetonitrile at 37 °C. The gel pieces were then dried in a SpeedVac, resuspended in 50 mM ammonium bicarbonate (100 µl) containing 0.5 µg of sequencing grade modified trypsin (Promega), and incubated for 12 h at 37 °C. After aliquoting the supernatant solution to a separate tube, residual peptides in the gel pieces were extracted into 50% acetonitrile, 20% isopropyl alcohol, 0.1% trifluoroacetic acid, combined with the supernatant solution, and concentrated utilizing a SpeedVac.

MALDI-TOF Analysis of iPLA₂ Tryptic Fragments—Concentrated peptide samples were diluted with 0.5% trifluoroacetic acid, absorbed to a C18 Zip-Tip (Millipore), and desorbed with a solution composed of 50% acetonitrile, 20% isopropyl alcohol, 0.1% trifluoroacetic acid, and containing in addition 5 mg/ml -cyano-4-hydroxycinnamic acid. Samples were applied to 192-spot sample plates (ABI) and allowed to air-dry. MS analyses were performed utilizing an Applied Biosystems 4700 Proteomics Analyzer (Framingham, MA), which possesses a 200-Hz Nd:YAG laser operating at 355 nm. The mass accuracy of the instrument was externally calibrated to the 4700 Proteomics analyzer calibration mixture of peptides. For MALDI-MS analysis, spectra were obtained by the accumulation of 2500 consecutive laser shots at a collision energy of 1 kV with air serving as the collision gas. Calculations of predicted peptide and peptide fragment masses were performed using programs developed at the University of California, San Francisco, Mass Spectrometry Facility (available on the World Wide Web at prospector.ucsf.edu).

Metabolic Labeling of iPLA₂ in Intact Sf 9 Cells—Cultures (100 ml) of Sf9 cells (1.5 x 10⁶ cells/ml) were infected with control (pFB empty vector) or iPLA₂His₆-encoding baculovirus and incubated at 27 °C for 45 h. The cells were then pelleted by centrifugation (250 x g for 10 min), resuspended in 10 ml of Grace's insect medium (Sigma) containing 1% heat inactivated fetal bovine serum, 0.1% Pluronic F-68, and 1 mCi of [³H]oleic acid. Following incubation at 27 °C for 3 h, cells were harvested by centrifugation (250 x g for 10 min) and resuspended in 5 ml of 25 mM sodium phosphate, pH 7.8, containing 20% glycerol, 5 µg/ml aprotinin, and 5 µg/ml leupeptin. After sonication, cytosol and membrane fractions were separated by ultracentrifugation as described above.

Other Procedures—SDS-PAGE was performed according to Laemmli (43). ECL Western analyses for iPLA₂His₆ were performed utilizing a monoclonal anti-His antibody (BD Biosciences) in conjunction with an anti-mouse IgG-horseradish peroxidase conjugate. Silver staining of SDS-polyacrylamide gels was performed as described (44). Protein concentration was determined by a version of the Bradford protein assay (Bio-Rad) with bovine serum albumin as a standard.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
iPLA₂ Hydrolyzes Palmitoyl-CoA and Is Inhibited at Submicellar Concentrations of Substrate—Due to the structural similarity between ATP and the 3'-phosphoadenosine moiety of CoA, we hypothesized that iPLA₂ could bind to, and potentially hydrolyze, the thioester linkage of long-chain fatty acyl-CoAs. Accordingly, we overexpressed iPLA₂His₆ in Sf9 cells and purified the enzyme to apparent homogeneity (as determined by SDS-PAGE/silver staining) by sequential cobalt and ATP affinity chromatographies as described under "Experimental Procedures." Initial assays with iPLA₂ utilizing supramicellar concentrations of palmitoyl-CoA (100 µM) typically used for acyl-CoA thioesterases revealed very low rates of hydrolysis (Fig. 1A). Remarkably, robust palmitoyl-CoA thioesterase activity was demonstrated at low micromolar concentrations of substrate with a maximal rate of 250 nmol of palmitic acid/min/mg at 2.5 µM palmitoyl-CoA (Fig. 1A). Similar acyl-CoA-mediated substrate inhibition was observed in previous studies of a purified rabbit heart mitochondrial thioesterase (45) and peroxisomal acyl-CoA thioesterase 2 (46). It should be noted that significant inhibition of iPLA₂ by palmitoyl-CoA occurs below the critical micelle concentration (10–20 µM) (47) and implies the presence of a second acyl-CoA binding site on the enzyme (i.e. SES intermediate) (48).

iPLA₂ Catalyzes the Highly Selective Hydrolysis of Myristoyl-CoA and Palmitoyl-CoA Present as Guests in Host Phospholipid Bilayers—Since iPLA₂ would probably be expected to encounter acyl-CoAs in a membrane bilayer in vivo, we examined whether the enzyme could hydrolyze palmitoyl-CoA present as a guest (at a low mol %) in phospholipid host vesicles. Purified iPLA₂ effectively hydrolyzed palmitoyl-CoA at physiologically relevant concentrations of acyl-CoA (i.e. 1–5 mol %) present in POPC/DOPS vesicles (Fig. 1B). This was surprising, since POPC is an excellent substrate for iPLA₂ and would be expected to efficiently compete with the palmitoyl-CoA as substrate. To determine the acyl-CoA substrate selectivity of iPLA₂, incubations were performed with a series of different long-chain acyl-CoAs in host POPC/DOPS bilayers. A dramatic selectivity for myristoyl- and palmitoyl-CoA was observed (up to 20-fold) in comparison with stearoyl-, oleoyl-, and arachidonoyl-CoAs (Fig. 2). Thus, iPLA₂ displays a marked preference for hydrolysis of saturated acyl-CoA substrates (14–16 carbons in length) in comparison with longer unsaturated acyl-CoA molecular species (C18:1 and C20:4) in the presence of membrane bilayers.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Palmitoyl-CoA thioesterase activity of iPLA2. A, substrate inhibition of iPLA2 palmitoyl-CoA hydrolase activity. Purified iPLA2His6 was incubated with the indicated concentrations of [1-14C]palmitoyl-CoA for 1–2 min at 37 °C. Reactions were terminated by vortexing with butanol, and [1-14C]palmitic acid extracted into the butanol layer was resolved by TLC and quantified by liquid scintillation spectrometry as described under "Experimental Procedures." B, iPLA2 catalyzed hydrolysis of palmitoyl-CoA guest in host POPC/DOPS vesicles. The indicated mol % of [1-14C]palmitoyl-CoA was incorporated into POPC/DOPS (90:10) vesicles (100 µM final vesicle lipid concentration). Each data point represents the average ± S.E. for at least four separate determinations.

Site-directed mutagenesis of the lipase catalytic serine (GTS⁴⁶⁵TG) of iPLA₂ to alanine has been previously demonstrated to ablate PLA₂ activity (10). To determine if Ser-465 was equally crucial for iPLA₂ acyl-CoA thioesterase activity, we generated and purified the identical mutant (S465A) and compared the PLA₂ and palmitoyl-CoA hydrolase activities with those of its wild-type counterpart. Importantly, the S465A iPLA₂His₆ bound to ATP-agarose (as determined by Western analysis), indicating that the mutant protein was properly folded around the nucleotide binding site (⁴³¹GGGVKG⁴³⁶), which is 30 amino acid residues away from the lipase site. As expected, calcium-independent PLA₂ activity was abolished in the S465A mutant utilizing POPC as substrate (Fig. 3A). Hydrolysis of palmitoyl-CoA incorporated into POPC/DOPS vesicles also was virtually eliminated in the S465A mutant (Fig. 3A), indicating that the iPLA₂ active site serine hydroxyl probably serves as the primary nucleophile for both phospholipase A₂ and acyl-CoA thioesterase reactions catalyzed by iPLA₂.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. Substrate selectivity of iPLA2 long-chain acyl-CoA hydrolase activity in the presence of phospholipid vesicles. Purified iPLA2His6 was incubated with equal amounts of the indicated [1-14C]acyl-CoA guest (5 mol %) in POPC/DOPS (90:10) host vesicles for 2 min at 37 °C. Released [1-14C]fatty acid was extracted into butanol by vortexing, resolved by TLC, and quantified by liquid scintillation spectrometry as described under "Experimental Procedures." Each data point represents the average ± S.E. from eight separate determinations.

Identification of Specific Autoacylation of iPLA₂ by Oleoyl-CoA—Since prior work has demonstrated that various proteins, such as rhodopsin (51), G-protein subunits (52, 53), and protein kinase C (54) are auto-acylated in the presence of palmitoyl-CoA, we sought to determine if iPLA₂ could become similarly acylated in the presence of saturated and unsaturated long-chain acyl-CoA substrates. Remarkably, although incubations with [1-¹⁴C]myristoyl-CoA, [1-¹⁴C]palmitoyl-CoA, and [1-¹⁴C]stearoyl-CoA displayed either no observable or only diminutive acylation of iPLA₂ following SDS-PAGE, those containing [1-¹⁴C]oleoyl-CoA resulted in 10–100-fold greater radiolabeling of iPLA₂ in comparison (Fig. 4A). Furthermore, incubations with [1-¹⁴C]arachidonoyl-CoA resulted in 10-fold-less signal intensity than with [1-¹⁴C]oleoyl-CoA, but iPLA₂ was still arachidonoylated under these conditions (Fig. 4A, top). To our surprise, similar experiments with S465A iPLA₂ displayed a shift in the selectivity of acylation (i.e. autoacylation was greatest with stearoyl-CoA, labeling with palmitoyl-CoA became clearly detectable, and the enzyme exhibited reduced reactivity with oleoyl-CoA in comparison with wild-type iPLA₂) (Fig. 4A, middle). Similar acylation experiments with BEL-pretreated iPLA₂ revealed marked increases in acylation with palmitoyl-CoA and stearoyl-CoA that were not observed with the wild-type protein (Fig. 4A, bottom). Thus, inactivation of the catalytic site through either site-directed mutagenesis or pretreatment with BEL does not abolish iPLA₂ acylation, supporting the existence of a second active site, which catalyzes autoacylation.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Elimination of iPLA2 palmitoyl-CoA hydrolase activity by site-directed mutagenesis of Ser-465 and inhibition of iPLA2 palmitoyl-CoA hydrolase activity by (R)- and (S)-BEL. A, equivalent amounts of purified iPLA2His6 (WT) or mutant iPLA2His6 (S465A) were incubated with either 1-palmitoyl-2-[1-14C]oleoyl-sn-glycero-3-phosphocholine (5 µM) or[1-14C]palmitoyl-CoA (5 µM) for 2 min at 37 °C. Radiolabeled fatty acids from the reaction were extracted into butanol, resolved by TLC, and quantified by liquid scintillation spectrometry as described under "Experimental Procedures." Results represent the average ± S.E. of four separate determinations. B, purified iPLA2His6 was preincubated with the indicated concentrations of either enantiomer of BEL or ethanol vehicle for 3 min at 23 °C in 100 mM Tris-HCl, pH 7.2, containing 1 mM EGTA. [1-14C]palmitoyl-CoA guest (5 mol %) in POPC (100 µM) host vesicles was added to the samples, which were incubated for 2 min at 37 °C and processed as described above. Results are representative of three separate experiments performed in duplicate.

View larger version (62K): [in this window] [in a new window]   FIGURE 4. Selective and stoichiometric acylation of wild-type iPLA2 by oleoyl-CoA, effects of chemical treatments on oleoylated iPLA2, and oleoylation of iPLA2 in intact cells. A, equivalent amounts of the indicated [1-14C]acyl-CoA guests (5 mol %) were incorporated into host POPC (100 µM) vesicles and incubated for 1 h at 37°C with equal amounts of either wild-type (WT) iPLA2His6, S465A iPLA2His6, or BEL-pretreated wild-type iPLA2His6 enzyme as described under "Experimental Procedures." Samples were resolved by SDS-PAGE (10% gel), fixed, and dried before visualization by autoradiography. B, purified iPLA2His6 (2 µM) was incubated with the indicated concentrations of [1-14C]oleoyl-CoA present as guest in host POPC (100 µM) vesicles for 1 h at 37 °C. Samples were electrophoresed in parallel with standard amounts (0.5–10 nCi) of [methyl-14C]bovine serum albumin of known activity (not shown). The fixed and dried gel was exposed to film, and the resultant signals from the [methyl-14C]bovine serum albumin were quantified utilizing an Eastman Kodak Co. Imagestation (1D software) to generate a standard curve to determine the incorporation of [1-14C]oleate into iPLA2. C, purified iPLA2His6 was incubated with [1-14C]oleoyl CoA (10 mol %) incorporated as guest in host POPC vesicles (100 µM final vesicle lipid concentration) or with 10 µM [14C]POPC (in the presence or absence of 1 mM CoASH) for 1 h at 37°C. Samples containing N-ethylmaleimide and iodoacetamide were pretreated with 5 mM of either reagent for 5 min at 30 °C before the addition of [1-14C]oleoyl-CoA. Oleoylated iPLA2 samples were subsequently incubated with 1 N HCl, 1 N NaOH, or 2 N neutral hydroxylamine for 1 h at 30 °C, as indicated. All samples, except lane 1, were precipitated with CHCl3/MeOH and washed with 70% acetone prior to SDS-PAGE and autoradiography. D, iPLA2 (4 µM) was preincubated with 20 µM oleoyl-CoA as a guest in POPC vesicles (80 µM) for 60 min at 37 °C. An identical amount (20 µM) of [1-14C]oleoyl-CoA (55 µmol/µCi) in the presence of POPC vesicles (80 µM) was then added, and aliquots of radiolabeled enzyme were removed at the indicted time points, subjected to SDS-PAGE, and visualized by autoradiography.

To further establish that the modification of iPLA₂ with oleoyl-CoA was covalent, [1-¹⁴C]oleoyl-iPLA₂ was precipitated with CHCl₃/CH₃OH and extensively washed with 70% acetone before SDS-PAGE (Fig. 4C). This treatment did not result in an appreciable decrease in signal intensity, indicating that the [1-¹⁴C]oleate is covalently bound to iPLA₂. Since esterification of fatty acids to proteins has been shown to occur through either amide, oxyester, or thioester bonds that can be distinguished through chemical treatment with strong acid (HCl), strong base (NaOH), or neutral hydroxylamine, additional experiments were performed to determine the nature of the covalent linkage. In the case of [1-¹⁴C]oleoyl-iPLA₂, the addition of either 1 N HCl or 1–2 N neutral hydroxylamine did not result in a significant decrease in radiolabeling, whereas the addition of N 1 NaOH completely eliminated the majority of covalently bound [1-¹⁴C]oleate (Fig. 4C). The insensitivity of [1-¹⁴C]oleoyl-iPLA₂ to hydroxylamine and HCl would indicate the absence of thioester and oxyester linkages, respectively, whereas the disappearance of radiolabeling in the presence of NaOH is consistent with an amide linkage. Pretreatment of iPLA₂ with N-ethylmaleimide or iodoacetamide decreased [1-¹⁴C]oleoylation (Fig. 4C), indicating that free thiol (cysteine) groups are important for either the formation of oleoyl-iPLA₂ intermediate(s) or for subsequent transfer to the terminal acceptor residue(s). Interestingly, acylation of iPLA₂ was not detectable utilizing 1-palmitoyl-2-[1-¹⁴C]oleoyl-sn-glycero-3-phosphocholine (with or without CoASH) (Fig. 4C). This suggests that the acyl-enzyme intermediate formed with POPC, which is readily hydrolyzed by water, is fundamentally distinct from that which leads to the relatively stable iPLA₂ acyl adduct(s) formed with oleoyl-CoA.

View larger version (75K): [in this window] [in a new window]   FIGURE 5. Oleoylation of iPLA2 in intact Sf9 cells. 1.5 x 108 Sf9 cells (100-ml cultures) were infected with either control (pFB) or recombinant (iPLA2) baculovirus for 45 h at 27 °C, pelleted by centrifugation, and resuspended in 10 ml of Grace's medium containing 1% heat-inactivated fetal bovine serum and 1 mCi of [3H]oleic acid. Following incubation at 27 °C for 3 h, cells were harvested by centrifugation, resuspended in homogenization buffer, and lysed by sonication, and cellular membranes were isolated by ultracentrifugation as described under "Experimental Procedures." Membrane proteins were resolved by SDS-PAGE and analyzed by autoradiography (left) and ECL Western blotting (right) against iPLA2His6 (indicated by an arrow).

Oleoylation of iPLA₂ in Sf 9 Cells—Although iPLA₂ was robustly acylated by oleoyl-CoA in vitro, it remained to be determined if the enzyme could be oleoylated in an intact cell. Accordingly, we incubated control and iPLA₂His₆-containing Sf9 cells with [³H]oleic acid for 3 h and analyzed the cytosolic and membrane fractions by SDS-PAGE followed by autoradiography. A unique radiolabeled protein band was observed in the membrane fraction of cells expressing iPLA₂ but not in the membranes of control cells (Fig. 5, left). A corresponding radiolabeled protein was not observed in the cytosol of cells containing iPLA₂ (data not shown), indicating that the majority of the acylated protein is localized to cellular membranes. To confirm the identity of the radiolabeled band, Western blotting for iPLA₂ was performed. A robust immunoreactive band representing iPLA₂ was detected in Sf9 cell membranes at the same molecular weight as that of the unique radiolabeled band (Fig. 5, right).

View larger version (91K): [in this window] [in a new window]   FIGURE 6. Partial trypsinolysis of [1-14C]Oleoyl-iPLA2. [1-14C]oleoyl-iPLA2His6 was prepared by incubation of the unmodified enzyme (10 µM) with 50 µM [1-14C]oleoyl-CoA for 1 h at 37°C. Trypsin (1:25, w/w) was added and incubated with [1-14C]oleoyl-iPLA2His6 for the indicated times. Following termination of proteolysis by addition of loading buffer, tryptic peptides were resolved by SDS-PAGE, and radiolabeled fragments were visualized by autoradiography as described under "Experimental Procedures."

View this table: [in this window] [in a new window]   TABLE 1 25-kDa iPLA2His6 fragment tryptic peptides identified by MALDI-MS analysis Unlabeled oleoylated iPLA2His6 was prepared and partially trypsinized for 10 min as described under "Experimental Procedures." The band corresponding to the 25-kDa radiolabeled fragment in Fig. 8 was excised and in-gel trypsinized, and the resultant peptides were identified by comparison of mass peaks obtained by MALDI-TOF with predicted iPLA2His6 tryptic peptide masses. Letters in parentheses indicate the neighboring amino acid residues in the primary sequence before and after the tryptic cleavage sites.

Calmodulin-mediated Protection of iPLA₂ against Oleoylation by Oleoyl-CoA—The proximity of the oleoylated iPLA₂ 25-kDa tryptic fragment to calmodulin binding domain next led us to investigate whether Ca²⁺-CaM could protect the enzyme against covalent acylation by oleoyl-CoA. Although the addition of either Ca²⁺ or CaM in the presence of EGTA alone did not alter the extent of oleoylation of iPLA₂ (Fig. 7B), the combination of Ca²⁺ and CaM significantly decreased autoacylation of the enzyme. From these results, acyl-CoA-mediated acylation would be predicted to primarily occur after dissociation of the iPLA₂·CaM complex.

Oleoyl-CoA-mediated Reversal of the Inhibition of iPLA₂ by Calmodulin—Depletion of intracellular calcium stores has been previously shown to initiate the influx of extracellular calcium through a poorly understood process, potentially involving an unknown cellular metabolite referred to as calcium influx factor (CIF) (25, 26, 60). Recent studies have demonstrated that CIF activates iPLA₂ by reversal of Ca²⁺-calmodulin inhibition of the enzyme (19, 20, 24). To determine if acyl-CoA could mitigate the inhibition of iPLA₂ by CaM, we utilized a real time fluorescence assay employing the PLA₂ substrate, BODIPY-PC, to measure the kinetic effects of oleoyl-CoA (guest in POPC (95 mol %)/BODIPY-PC (5 mol %) host vesicles) on CaM inhibition of iPLA₂ phospholipase A₂ activity. In the absence of calmodulin, iPLA₂ efficiently hydrolyzes BODIPY-PC present at 5 mol % in a POPC background as demonstrated by a robust time-dependent increase in fluorescence intensity (Fig. 8A). The presence of calcium ion did not appreciably affect the phospholipase A₂ activity of iPLA₂ under these conditions (data not shown). In contrast, the presence of Ca²⁺-bound CaM inhibited iPLA₂-catalyzed hydrolysis of BODIPY-PC by 70–80% (Fig. 8A). Remarkably, the addition of 1 mol % oleoyl-CoA could activate CaM-inhibited iPLA₂ (40% of initial activity) (Fig. 8B), and the presence of 2.5–5 mol % oleoyl-CoA completely eliminated CaM-mediated inhibition of iPLA₂ (Fig. 8, C and D) under these conditions. To confirm that iPLA₂ was in fact hydrolyzing BODIPY-PC and that the increase in fluorescence observed was not due to either proteinfluorophore or acyl-CoA-fluorophore interactions, the reaction substrates and products were extracted into chloroform/methanol in the presence of internal standards and subsequently quantified by shotgun lipidomics (61). As anticipated, the production of lyso-BODIPY-PC, 16:0-LPC, and 18:1-LPC was dependent upon the presence of iPLA₂, and the amount of each product was diminished (80%) by the presence of Ca²⁺-bound CaM (Fig. 8, E–G). Importantly, the addition of 5 mol % of oleoyl-CoA to the POPC/BODIPY-PC vesicles in the presence of Ca²⁺/CaM/iPLA₂ completely reversed the inhibition of iPLA₂ by Ca²⁺/CaM as evidenced by the recovery of similar amounts of 16:0-LPC, 18:1-LPC, and lyso-BODIPY-PC to that observed with iPLA₂ alone (Fig. 8H). Finally, to establish that the effects of oleoyl-CoA on Ca²⁺/CaM-mediated inhibition of iPLA₂ were not dependent on the presence of BODIPY-PC, similar experiments were performed with 1-palmitoyl-2-[1-¹⁴C]oleoyl-sn-glycero-3-phosphocholine as substrate. Similar to the real time fluorescence assays, CaM inhibited iPLA₂ activity by 70%, and the presence of oleoyl-CoA alone caused moderate inhibition (20%) of iPLA₂ activity (Fig. 8I), presumably due to interactions with iPLA₂ at or near the substrate binding site. As anticipated, the presence of oleoyl-CoA significantly attenuated the CaM-mediated inhibition of iPLA₂, resulting in 75% of the activity observed with oleoyl-CoA alone (Fig. 8I). Collectively, these results demonstrate the rescue of the calmodulin-inhibited iPLA₂ activity by oleoyl-CoA by three independent methods and identify fatty acyl-CoAs as potential candidates for calcium influx factor.

View larger version (33K): [in this window] [in a new window]   FIGURE 7. Ca2+-CaM blocks covalent acylation of iPLA2 by oleoyl-CoA but does not inhibit palmitoyl-CoA thioesterase activity. A, iPLA2His6 (0.5 µg) was preincubated in the presence of Ca2+ (1 mM) or Ca2+-CaM (6 µg) on ice before addition to 95 µM POPC containing either 5 mol % [1-14C]POPC or [1-14C]palmitoyl-CoA in 100 mM Tris-HCl, pH 7.2, containing 0.25 mM CaCl2. After incubation at 37 °C for 3 min, reactions were terminated by vortexing in the presence of butanol, and released radiolabeled fatty acids were resolved by TLC and quantitated by liquid scintillation spectrometry. Results are presented as the average ± S.E. of four separate determinations. B, purified iPLA2His6 was incubated with [1-14C]oleoyl CoA (10 mol %) incorporated as guest in host POPC (100 µM final lipid concentration) vesicles in the presence of EGTA (5 mM), Ca2+ (1 mM), CaM (3 µg), or Ca2+-CaM for 1 h at 37°C. Samples were resolved by SDS-PAGE and exposed to film as described under "Experimental Procedures."

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (36K): [in this window] [in a new window]   FIGURE 8. Reversal of Ca2+/CaM-mediated inhibition of iPLA2 activity by oleoyl-CoA. A–D, purified iPLA2His6 in the presence (filled circles) or absence (open squares) of Ca2+/CaM was incubated with POPC/BODIPY-PC (95:5 mol %, 100 µM phospholipid) host vesicles for 2 min at 37 °C with the indicated concentrations of guest oleoyl-CoA. Relative fluorescence was recorded at 15-s intervals utilizing 495-nm excitation and 515-nm emission wavelengths as described under "Experimental Procedures." Results are representative of four independent experiments performed in duplicate. E–H, ESI/MS analysis of phosphatidylcholine molecular species from the reactions described in A and D. Lipid species were extracted into chloroform in the presence of internal standards (I.S.; 14:1–14:1 PC and 17:0 LPC for phosphatidylcholine and lysophosphatidylcholine species, respectively) and were subjected to ESI tandem mass analysis as described under "Experimental Procedures." Spectra were acquired by scanning for the neutral loss of 59 atomic mass units in the positive ion mode at a collision energy of 24 eV and a collision gas pressure of 1.0 millitorr. The relative abundance of lipid species in each spectrum was normalized to that of the LPC internal standard (I.S., 17:0 LPC). I, purified iPLA2His6 in the presence or absence of Ca2+/CaM was incubated with 1-palmitoyl-2-[1-14C]oleoyl-sn-glycero-3-phosphocholine (100 µM) host vesicles with or without guest oleoyl-CoA (10 µM) for 3 min at 37 °C. Radiolabeled fatty acid was extracted into butanol, resolved by TLC, and quantified by scintillation spectrometry as described under "Experimental Procedures." Results represent the average ± S.E. of four separate determinations.

A combination of partial trypsinolysis and MALDI-MS was utilized to localize the region of acylation to amino acid residues 400–600 (adjacent to the calmodulin binding domain), which includes the nucleotide and lipase consensus sequence motifs. Thus, acylation by oleoylCoA occurs near the catalytic domain of iPLA₂, although it does not appear to inhibit or block substrate (i.e. palmitoyl-CoA or POPC) access to the active site serine (Ser-465) for catalysis. Interestingly, Ca²⁺-bound CaM blocked covalent acylation of iPLA₂ by oleoyl-CoA. In contrast, Ca²⁺-CaM significantly inhibited only the phospholipase A₂ activity of iPLA₂, whereas the acyl-CoA thioesterase activity of the enzyme was unaffected under similar conditions. This result suggests that the less sterically bulky hydrophobic portion of the acyl-CoA substrate may have greater access to the iPLA₂ active site (Ser-465) than the bulkier diacyl phospholipid substrate in the presence of Ca²⁺-bound CaM.

To our knowledge, iPLA₂ is the only intracellular phospholipase A₂ to exhibit substantial amounts of long-chain acyl-CoA thioesterase activity and represents the first acyl-CoA thioesterase identified at the molecular level shown to efficiently hydrolyze membrane-associated acyl-CoAs. In contrast, in vitro assays with purified recombinant cytosolic PLA₂ (76) and iPLA₂³ in our hands did not detect appreciable long-chain acyl-CoA hydrolase activities.³ A 54-kDa acyl-CoA hydrolase from rat intestinal microsomes was found to cleave long-chain acyl-CoAs in the presence of phosphatidylcholine vesicles, although the sequence identity of this enzyme has not been described since its original purification (77).

Myocardial ischemia is known to be accompanied by activation of iPLA₂ (21), although the mechanism(s) contributing to such activation have been the subject of much debate. It is known that iPLA₂ largely exists as an inhibited complex in transgenic mice, since vast overexpression does not result in substantive increases in phospholipolysis (21). However, induction of myocardial ischemia results in the dramatic activation of the enzyme demonstrated by fatty acid release and lysolipid accumulation. The present results identify a likely mechanism contributing to the activation of iPLA₂, since acyl-CoA is known to dramatically increase during myocardial ischemia due to the blockade of mitochondrial fatty acid oxidation (78). Accordingly, these results potentially link the activation of iPLA₂ in hearts during ischemia with that which occurs during the influx of extracellular calcium (see below) in other tissues.

Depletion of intracellular calcium stores in smooth muscle cells has been previously demonstrated to activate iPLA₂ through a mechanism hypothesized to involve the dissociation of CaM from the enzyme (13, 36). Store-operated calcium (cation) channels in the plasma membrane are then activated in response to agonist-stimulated intracellular calcium pool depletion for the purpose of replenishing the emptied calcium stores. Recent work by Bolotina and co-workers has provided additional details of this process by showing that iPLA₂ is required for activation of store-operated calcium (cation) channels through generation of lysophospholipids (20). Furthermore, the inhibitory complex between CaM and iPLA₂ could be disrupted by a partially purified preparation of CIF (20). Although attempts to elucidate the molecular identity of CIF over the past 10 years have not been successful, these studies have determined various chemical properties of calcium influx factor. In general, CIF is believed to be a nonprotein, diffusible, phosphorylated "sugar nucleotide," which is resistant to heat, alkaline pH, and protease treatment and is retained on a C18 reverse phase matrix (79). In this work, we demonstrate that oleoyl-CoA is able to mimic the properties of CIF by restoring the phospholipase A₂ activity of Ca²⁺/CaM-inhibited iPLA₂. The acute production of acyl-CoAs due to fatty acid influx or release (e.g. from phospholipids or triacylglycerol) and subsequent thioesterification in specific membrane microenvironments containing complexes of Ca²⁺/CaM-inhibited iPLA₂ would probably be sufficient to mediate iPLA₂ activation through displacement of calmodulin in a temporally and spatially specific manner. Thus, this process would be predicted to initiate an amplification cascade by a subset of activated iPLA₂, which initially releases fatty acids (from surrounding phospholipids) for thioesterification to CoA, thereby activating adjacent iPLA₂·Ca²⁺/CaM complexes. We specifically point out that many other CIF-like cellular constituents capable of reversing Ca²⁺/CaM-iPLA₂ inhibition may exist and that other membrane components and conditions that occur in vivo (accessory proteins, membrane surface charge and curvature, membrane electrochemical potential, etc.) may facilitate this process.

Multiple acyl-CoA thioesterases have been cloned from mammalian sources and are classified on the basis of their subcellular localization (cytosolic, mitochondrial, or peroxisomal), sequence similarity, and ability to be induced by peroxisome proliferators. The majority of these thioesterases, as well as all known intracellular phospholipases A₂, contain the canonical GXSXG lipase (esterase) consensus sequence motif. Amino acid sequence alignments of iPLA₂ with the known mammalian acyl-CoA thioesterases did not reveal any significant sequence homology outside of the GXSXG consensus motif. This is not completely unexpected, given the diversity among the different classes of acyl-CoA thioesterases. Some of the established acyl-CoA thioesterase family members (e.g. mitochondrial acyl-CoA thioesterases and cytosolic acyl-CoA thioesterases) possess conserved putative nucleotide binding sequences (GXGXXG). Interestingly, calcium-independent phospholipase A₂ displays an acyl-CoA substrate selectivity (C14-C20) similar to the cytosolic Type-I thioesterase (27). In addition, iPLA₂, like this cytosolic acyl-CoA thioesterase, is not inhibited by high concentrations of CoASH³, indicating that these enzymes are probably not involved in "sensing" and regeneration of free CoASH through acyl-CoA hydrolysis, as has been ascribed to peroxisomal acyl-CoA thioesterase-2 (46). Calcium-independent phospholipase A₂ and other acyl-CoA thioesterases probably serve multiple pleotropic roles in metabolism and cellular signaling.

In this study, we demonstrate that purified recombinant iPLA₂ possesses robust palmitoyl-CoA hydrolase activity in addition to its previously well characterized lysophospholipase and phospholipase A₂ activities. Moreover, iPLA₂ is covalently autoacylated in a highly substrate-specific fashion (by oleoyl- but not palmitoyl-CoA), which occurs at a second active site distinct from the hydrolytic lipase site (GXSXG). Thus, iPLA₂ could potentially have multiple effects on the production of lipid metabolites (arachidonic acid and lysolipid