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is farnesylated and palmitoylated in mammalian cells
* Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206
Departments of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195
Departments of Pathology and Pharmacology, University of Colorado School of Medicine, Denver, CO 80206
Published, JLR Papers in Press, August 1, 2005. DOI 10.1194/jlr.M500230-JLR200
1 To whom correspondence should be addressed. e-mail: gelb{at}chem.washington.edu (M.H.G.); lesliec{at}njc.org (C.C.L.)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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(cPLA2) is a member of the group IV family of intracellular phospholipase A2 enzymes, but unlike the well-studied cPLA2
, it is constitutively bound to membrane and is calcium independent. cPLA2 contains a C-terminal CaaX sequence and is radiolabeled by mevalonic acid when expressed in cPLA2-deficient immortalized lung fibroblasts (IMLF–/–). The radiolabel associated with cPLA2 was identified as the farnesyl group. The protein farnesyltransferase inhibitor BMS-214662 prevented the incorporation of [3H]mevalonic acid into cPLA2 and partially suppressed serum-stimulated arachidonic acid release from IMLF–/– and undifferentiated human skeletal muscle (SkMc) cells overexpressing cPLA2, but not from cells overexpressing cPLA2. However, BMS-214662 did not alter the amount of cPLA2 associated with membrane.
These results were consistent in COS cells expressing the C538S cPLA2 prenylation mutant. cPLA2 also contains a classic myristoylation site and several potential palmitoylation sites and was found to be acylated with oleic and palmitic acids but not myristoylated. Immunofluorescence microscopy revealed that cPLA2 is associated with mitochondria in IMLF–/–, SkMc cells, and COS cells.
Abbreviations: cPLA2, cytosolic phospholipase A2; GFP, green fluorescent protein; GST, glutathione S-transferase; His, 6x histidine tag; IMLF, immortalized mouse lung fibroblasts; PLA2, phospholipase A2; Sf 9, Spodoptera frugiperda; SH, sulfhydryl; SkMc, skeletal muscle
Supplementary key words prenylation • fatty acylation • mitochondria
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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One member of the group IV PLA2 family, group IVA cPLA2 (cPLA2), has been well studied because of its specificity for releasing sn-2 arachidonic acid from membrane phospholipids for the production of eicosanoids (6, 8, 10). The oxygenated metabolites of arachidonic acid, prostaglandins, and leukotrienes, produced through the cyclooxygenase and lipoxygenase pathways, promote acute inflammatory responses and are also important in regulating many physiological processes (11). cPLA2 is subject to posttranslational regulatory controls via phosphorylation and Ca2+ (12–16). Calcium functions by binding to an N-terminal C2 domain and inducing the translocation of cPLA2 from the cytosol to the Golgi, endoplasmic reticulum, and nuclear envelope (17–20).
Two additional group IV enzymes have been identified. Group IVC cPLA2 (cPLA2) and group IVB cPLA2ß (cPLA2ß); both have 
30% homology to cPLA2 (21–23). The active site residues found in cPLA2 are conserved in these paralogs, suggesting that they have a similar catalytic mechanism (22). cPLA2ß contains an N-terminal C2 domain that confers calcium sensitivity and an additional N-terminal extension containing a JmjC domain, the function of which is unknown (21–24). In contrast, cPLA2 does not contain a C2 domain, consistent with its lack of regulation by Ca2+ (21, 22). The regulatory phosphorylation sites used by cPLA2 are not present in cPLA2, although it possesses multiple putative PKC phosphorylation sites, the use of which has yet to be investigated. Unlike cPLA2 and cPLA2ß, which are widely distributed in mammalian tissues, cPLA2 message is abundantly expressed in skeletal muscle (SkMc), brain, and heart (21, 22). In another significant departure from cPLA2, cPLA2 and cPLA2ß do not exhibit strong sn-2 acyl chain specificity (25, 26). cPLA2 exhibits relatively high lysophospholipase activity, as reported previously for cPLA2 (25, 27).
A unique property of cPLA2 is that it is constitutively bound to cell membrane and contains putative acylation sites and a C-terminal prenylation site that may regulate its membrane association (21). The C-terminal sequence CCLA on cPLA2 fits the consensus sequence of a CaaX box (a is usually, but not necessarily, an aliphatic residue), a motif that is recognized by protein prenyltransferases for the attachment of either a 15 carbon farnesyl or a 20 carbon geranylgeranyl to the cysteine sulfhydryl (SH) group (28, 29). Indeed, cPLA2 becomes radiolabeled when expressed in COS cells grown in the presence of [3H]mevalonic acid, the precursor of prenyl groups in mammalian cells (21). The structure of the prenylated C terminus cannot be inferred from the cPLA2 CaaX sequence. The CCLA sequence could be recognized by protein farnesyltransferase, resulting in the attachment of a farnesyl group to the N-terminal-most cysteine SH.
cPLA2 has been shown to be farnesylated when expressed in insect cells (30); however, the structure of the prenyl group on cPLA2 in mammalian cells has not been investigated. cPLA2 could also be a substrate for protein geranylgeranyltransferase type I, resulting in geranylgeranylation of the N-terminal-most cysteine SH. Finally, it may be noted that a subset of Rab GTPases contain the C-terminal sequence CCXX, and the enzyme protein geranylgeranyltransferase type II attaches a geranylgeranyl group to each of the two cysteine SH groups (31). Thus, cPLA2 could be a doubly geranylgeranylated protein in mammalian cells. cPLA2 also contains a putative myristoylation site as well as several potential fatty acylation sites.
Both myristoylation and palmitoylation are widespread fatty acid modifications on membrane-associated proteins. In addition to facilitating high-affinity membrane association on dually lipidated proteins, these modifications can aid in protein trafficking of enzymes to specific compartments or subdomains (32, 33). Palmitoylation has also been implicated in the regulation of enzymatic activity in proteins, notably multiple mitochondrial enzymes (34–36). In the present study, we have found that cPLA2 is farnesylated and acylated in mammalian cells. Prenylation of cPLA2 was also found to be important for the function of cPLA2 in intact cells. In addition, we have found that cPLA2 is localized to the mitochondria when expressed in mammalian cells.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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in Spodoptera frugiperda (Sf9) and mammalian cells, respectively, was carried out as described previously (25). The mitochondrion-specific monoclonal antibody to oxidative phosphorylation complex V, subunit b, and the secondary antibodies used for immunofluorescence were from Molecular Probes.
cPLA2 polyclonal antibody production and Western blot analysis
Human cPLA2 was cloned as described previously (25). To produce glutathione S-transferase (GST)-cPLA2, cPLA2 was cloned into the EcoRI/PstI sites of pAcGHLT baculovirus transfer vector, and baculovirus was generated as described previously (25). Sf 9 cells grown in suspension (500 ml) were infected with baculovirus for 48 h and lysed in 50 mM Hepes buffer, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 1 mM EDTA, and one complete protease inhibitor cocktail tablet per 50 ml (Roche). GST-cPLA2 was affinity-purified using glutathione agarose beads by standard protocols. GST-cPLA2 (50–100 µg) was added to an equal volume of complete Freund's adjuvant and injected subcutaneously into rabbits. Subsequent booster injections were carried out every 3 weeks using Freund's incomplete adjuvant. Antiserum was obtained 10 days after each injection and analyzed for reactivity to cPLA2 by Western blotting.
Cell homogenates for Western blot analysis of cytosol and membrane fractions were prepared by sonicating cells in 50 mM Hepes, pH 7.4, containing 0.34 M sucrose, 1 mM EGTA, 10% glycerol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Homogenates were centrifuged at 100,000 g for 45 min to obtain the cytosol and membrane fractions. The protein concentration of the cell fractions was determined using the bicinchoninic acid reagent. Lysates were diluted in Laemmli buffer, and proteins were separated on 10% polyacrylamide gels, transferred to nitrocellulose, and blocked for 1 h in Tris-buffered saline containing 0.25% Tween 20 and 5% nonfat dry milk. Nitrocellulose membranes were incubated overnight in a 1:1,000 dilution of anti-cPLA2 antiserum, and immunoreactive protein was detected using the Amersham Biosciences ECL system.
Cell culture, production of DNA constructs, and recombinant adenovirus
Immortalized mouse lung fibroblasts lacking group IVA cPLA2 (IMLF–/–) were isolated and immortalized with SV40, as described previously (38). The replication-deficient recombinant adenoviruses carrying the cDNA for untagged cPLA2 (Ad-cPLA2) and for GFP-tagged cPLA2 (Ad-GFPcPLA2) were generated using the AdEasy vector system (Qbiogene) and titered, and expression levels were determined as described previously (25). Freshly isolated human SkMc cells obtained from Cambrex were grown in complete human SkMc growth medium for two passages and then frozen. Cells were thawed and passaged once by trypsinization according to the manufacturer's protocol (Cambrex) before use in experiments. The C538 in the CaaX box of cPLA2 was mutated to a serine (C538S) using PCR-based site-directed mutagenesis (Stratagene) and the primer 5'-CTA TGC CAA GCA GCT ACT TCG GGC ACT-3'. The putative N-myristoylation site (G2) was mutated to alanine (G2A) by the same method. The primer used was 5'-TTC GGA CCG CAG TGC ACC ATG GCA AGC TCT GAA GTT-3'. The C-terminal truncated cPLA2 was created by mutating K490 into a TAG stop codon using the primer 5'-GAC ACA TAC GAC ACA TTC TAG CTT GCT GAC-3'. Wild-type and mutant cPLA2 were cloned into the pcDNA3.0 mammalian expression vector. All constructs were confirmed by sequencing. COS and HEK293 cells were transiently transfected using FuGENE transfection reagent according to the manufacturer's instructions (Roche Diagnostics, Indianapolis, IN). Expression of wild-type and mutant cPLA2 was confirmed by Western blotting. RT-PCR of endogenous cPLA2 in SkMc cells was conducted using 5'-GCT CAC ATT GCC TGC CTT GGG GTC CTG-3' and 5'-AGT GCC CGA AGT TGC TGC TTG GCA TAG-3' and RNA isolated with the RNeasy mini kit (Qiagen).
Structural analysis of the cPLA2 prenyl group
IMLF–/– were plated at 1 x 106 cells/100 mm dish and incubated for 10 h in DMEM containing 2% FBS. The cells were washed and incubated in 4 ml of serum-free DMEM containing 0.1% BSA and Ad-cPLA2 as described previously (25). After incubation for 90 min, additional DMEM containing 0.1% BSA, 1 mCi of [3H]mevalonic acid, and 10 µM simvastatin was added to the cells to prevent the mevalonate from becoming metabolized to cholesterol. For some experiments, the medium also included 1 µM of the farnesyltransferase inhibitor BMS-214662 or Me-BMS-214662. After incubation for 26 h, cells were rinsed with PBS and solubilized in ice-cold lysis buffer (50 mM Hepes, pH 7.4, 150 mM sodium chloride, 1% Nonidet P-40, 200 µM sodium orthovanadate, 10 mM tetrasodium pyrophosphate, 100 mM sodium fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 100 µM phenylmethylsulfonyl fluoride, and 300 nM p-nitrophenyl phosphate). The lysate was incubated at 0°C for 15 min, then centrifuged for 10 min at 4°C at full speed in a microfuge. Total protein in the supernatant was quantified using the bicinchoninic acid method. Immunoprecipitation of cPLA2 was carried out by incubating the lysates at 4°C with protein A-Sepharose beads and anti-cPLA2 antiserum (1:25) overnight while rotating. The beads were washed five times in lysis buffer and then boiled in Laemmli buffer for 10 min. One-third of the labeled proteins was separated by SDS-PAGE, and the gel was fixed in isopropanol-water-acetic acid (25:65:10) for 10 min. The gel was incubated for 30 min in Amplify at room temperature before drying and then exposed to film for 8 days for visualization of the bands.
For samples to be extracted for HPLC prenylation analysis, the remaining two-thirds of the immunoprecipitate was electrophoresed and dried without fixation or Amplify treatment. The pieces of dried gel corresponding to the location of radiolabeled cPLA2 (61 kDa) were excised. Protein in the gel slices was extracted in the presence of 100 mg of BSA as carrier (39), and half of the extract was further processed. Extracted protein was precipitated with trichloroacetic acid, and the precipitate was washed with cold acetone as described (39). The dried protein was dissolved in guanidine-HCl/sodium phosphate buffer, N-acetyl-cysteine(S-farnesyl) and N-acetyl-cysteine(S-geranylgeranyl) (20 mg each; Bachem, Inc.) were added, and the sample was treated with Raney-nickel as described (39). The pentane extract was concentrated and submitted to HPLC analysis, and radioactivity in the column fractions was determined by scintillation counting (39).
Analysis of cPLA2 fatty acylation
Adenoviral infection and immunoprecipitation were modified from the protocol for the farnesylation experiments described above. IMLF–/– cells were cultured in serum-free DMEM containing 0.1% fatty acid-free BSA for 22 h and then incubated for 4 h in the same medium containing either 0.3 mCi/ml [3H]palmitate or 0.1 mCi/ml [3H]myristate. After immunoprecipitation of cPLA2 (as described above), proteins were separated by SDS-PAGE and the gels were either submitted to fluorography or incubated briefly in water and dried. For analysis of fatty acylation, bands corresponding to cPLA2 were excised. The gel slice was mixed in 1 ml of 50% aqueous methanol at room temperature in a polypropylene tube. After 2–3 h, the liquid was removed and the gel slice was washed again as above. The gel slice was dried with a Speed-Vac (Savant Instruments), and 0.7 ml of 1.5 M aqueous NaOH was added to the tube. After incubation for 2 h at 30°C, the solution was brought to pH 2 by the addition of 6 M HCl (monitored with pH paper). The gel slice and solution were transferred to a glass tube, and 2.5 ml of Dole solvent (24 ml of isopropanol, 0.6 ml of 0.5 M H2SO4, and 6 ml of n-heptane) was added (using a portion to rinse the polypropylene tube). The sample was vortexed, 1 ml of water and 1.5 ml of n-heptane were added, and the sample was vortexed again.
The upper organic phase was transferred to a glass tube, and solvent was removed from the organic and aqueous phases with a Speed-Vac. To the tube containing the residue from the aqueous phase and gel slice was added 0.7 ml of 6 M HCl, the top of the tube was sealed with a glassblower torch, and the tube was heated in an incubator for 4 h at 100°C. After cooling, the tube was opened with the aid of a scoring file, and solvent was removed with a Speed-Vac. Water was added to the residue (0.7 ml), and the sample was extracted by the addition of Dole solvent, water, and n-heptane as described above. The upper organic phase was transferred to a glass tube, and the solvent was removed from both tubes with a Speed-Vac. Methanol (1 ml) was added to each tube, and the radioactivity in 0.1 ml aliquots was determined by scintillation counting.
Methanol in the tube containing the organic extract of the NaOH-treated and HCl-acidified sample (see above) was transferred to a 1.5 ml vial with a Teflon-septum screw cap, 1 mmol each of myristic acid, palmitic acid, and oleic acid were added (from 50 mM stock solutions in methanol), and solvent was removed with a Speed-Vac. Diisopropylethylamine (0.5 ml of 10%, v/v; Aldrich) and 1% (v/v) pentafluorobenzyl bromide (Pierce) in CH3CN was added to the residue, and the capped vial was heated at 60°C for 15 min. An overnight Speed-Vac treatment removed the solvent and excess reagents. The residue was dissolved in CH3CN and injected onto a C18 reverse-phase HPLC column (1 x 25 cm; Vydac 218TP1010) equilibrated previously with 70% CH3CN in water at a flow rate of 1.5 ml/min. The column was developed with a solvent gradient of 70% CH3CN in water to 100% CH3CN over 20 min and then held at 100% CH3CN for 60 min. Absorbance at 254 nm was monitored, and 2 min fractions were collected into scintillation vials. Solvent was removed with a Speed-Vac, and the residues were dissolved in scintillation fluid and counted.
Arachidonic acid release assays
Cells were plated at 1.25 x 104 cells/cm2 on 24-well plates in DMEM containing 2% FBS for IMLF–/– or in complete SkMc medium for growing SkMc cells and incubated overnight. COS cells were plated at the same density and transfected as described above. The cells were washed and then incubated in 150 µl of DMEM containing 0.1% BSA with Ad-cPLA2 or Ad-GFP control virus. After incubation for 90 min, DMEM (500 µl) containing 0.1% BSA and 0.2 µCi/ml [3H]arachidonic acid was added. In some experiments 1 µM BMS-214662 or Me-BMS-214662 was added. After a 26 h incubation, cells were washed two times in medium containing 0.1% BSA to remove unincorporated arachidonic acid. Cells in this medium were then stimulated with 10% mouse serum for 3 h (25). The amount of radioactivity released into the medium was determined and expressed as a percentage of the total counts incorporated into the cells.
Immunofluorescence microscopy
IMLF–/– or SkMc cells were plated on 35 mm glass-bottomed MatTek plates at 1.25 x 104 cells/cm2 and infected with Ad-cPLA2 as described above. The cells were rinsed once with PBS and incubated for 15 min in ice-cold fixative containing 3.2% paraformaldehyde and 3% sucrose in PBS. After fixation, cells were rinsed five times with cold PBS and incubated for 15 min in 0.1% Triton X-100 in PBS. Cells were then rinsed with PBS and blocked for 1 h in PBS containing 10% FBS. Fixed cells were incubated with anti-cPLA2 polyclonal antiserum (1:50) overnight, followed by a 2 h incubation with goat anti-rabbit Texas Red-conjugated secondary antibody (1:100). Cells were costained with mitochondrion-specific anti-oxidative phosphorylation complex V monoclonal antibody (1:20) and an AlexaFluor 488-conjugated anti-mouse secondary antibody (1:100). Control experiments with Texas Red anti-rabbit secondary antibody only and with both primary antibodies and both secondary antibodies were included to identify any nonspecific reactions. After incubation with each antibody, the cells were washed five times in PBS containing 10% FBS. All antibodies were diluted into blocking solution and centrifuged at full speed in a microfuge before use. Controls using secondary antibodies alone were included and revealed only a low level of background fluorescence (data not shown). IMLF–/– were visualized using a Nikon diaphot inverted microscope with a 60x, 1.4 numerical aperture oil-immersion lens and a Photometrics charge-coupled device camera using FITC and Tetramethyl Rhodamine Iso-Thiocyanate filters. Images were acquired with IP Labs software (Scanalytics, Inc.). Immunofluorescence microscopy of SkMc cells was carried out using an Olympus inverted microscope with a 60x, 1.25 numerical aperture oil-immersion objective, and images were collected with a charge-coupled device camera using Chroma dichroic mirrors fitted with emission filters for FITC and Texas Red detection. Image acquisition and analysis were performed using TILL visION software (TILL Photonics).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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is farnesylated in mammalian cells were grown in the presence of [3H]mevalonic acid followed by immunoprecipitation of cPLA2. One-third of the total volume of the immunoprecipitate was resolved by SDS-PAGE, and the gel was submitted to fluorography. As shown in Fig. 1A
, a major radioactive band was seen with an apparent molecular mass of 60 kDa, which is the predicted molecular mass of cPLA2, demonstrating that cPLA2 is labeled by mevalonic acid, consistent with previous results (21). The remaining two-thirds of the immunoprecipitate was resolved by SDS-PAGE but not submitted to fluorography, and a gel piece corresponding to the cPLA2 region was excised. Protein was eluted from the gel slice and treated with Raney-nickel, which cleaves the carbon-sulfur bond of protein prenyl groups (31, 39). HPLC analysis of the released radiolabeled material clearly shows that it comigrates with the 15 carbon trimethyl dodecatriene (fraction 32) and that no 20 carbon material was detected (Fig. 1B). The results demonstrate that cPLA2 is a farnesylated protein in mammalian cells.
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expressed in IMLF–/–. cPLA2 was immunoprecipitated from both untreated cells and cells exposed to 1 µM BMS-214662. A radiolabeled band at 60 kDa was present in untreated cells. As shown in Fig. 2
, incorporation of [3H]mevalonic acid into the 60 kDa cPLA2 band was almost completely blocked by the farnesyltransferase inhibitor BMS-214662. The inhibitor was added to the cells at the time of adenovirus infection to prevent the prenylation of cPLA2 as it was being expressed. Prenylation is thought to be an irreversible modification; therefore, the inhibitor would not cause the loss of the prenyl group from an existing pool of cPLA2 protein. BMS-214662 did not affect the levels of cPLA2 expression (see Fig. 5).
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function, the effect of BMS-214662 on [3H]arachidonic acid release from IMLF–/– expressing cPLA2 was determined. We have previously reported that cPLA2 expressed in IMLF–/– is activated by serum, resulting in the release of arachidonic acid and other fatty acids (25). IMLF–/– were infected with Ad-cPLA2, and with Ad-GFP as a control, and the effect of BMS-214662 on serum-induced arachidonic acid release was determined. As shown in Fig. 3
(upper panel), there was increased arachidonic acid release from IMLF–/– expressing cPLA2 compared with control cells infected with Ad-GFP, and BMS-214662 inhibited cPLA2-mediated [3H]arachidonic acid release by 60%. A recent study has shown that mutation of the CaaX sequence also suppresses A23187-stimulated arachidonic acid release in HEK293 cells overexpressing cPLA2 (40).
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-mediated arachidonic acid release was tested (Fig. 3, lower panel). Serum stimulation of IMLF–/– expressing cPLA2 resulted in a large increase in arachidonic acid release compared with control cells, as we reported previously, and this response was unaffected by the protein farnesyltransferase inhibitor. Similar experiments were carried out in primary cultures of SkMc cells to determine whether prenylation of cPLA2 plays a functional role (Fig. 4)
. SkMc cells overexpressing cPLA2 released more arachidonic acid in response to mouse serum than control cells infected with Ad-GFP, and cPLA2-mediated arachidonic acid release was inhibited to near basal levels by BMS-214662. Another inhibitor, Me-BMS-214662, which inhibits mammalian protein farnesyltransferase with a similar potency as BMS-214662 (M. Gelb, unpublished observation), similarly suppressed arachidonic acid release from SkMc cells expressing cPLA2. Arachidonic acid release experiments were also conducted using transiently transfected COS cells overexpressing both the wild type and the cPLA2 C538S prenylation mutant. Consistent with the inhibitor experiments, the mutation partially prevented arachidonic acid release by cPLA2 in COS cells (Fig. 4B).
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is necessary for its constitutive association with membrane, the effects of BMS-214662 and the C538S prenylation mutant on the relative amounts of cPLA2 in the cytosol and on the membrane were investigated. IMLF–/– and SkMc cells were infected with Ad-cPLA2 in the presence and absence of 1 µM BMS-214662, and the relative levels of cPLA2 in the 100,000 g soluble and particulate fractions were determined by Western blotting. Infection of both cell types with Ad-cPLA2 resulted in increased levels of cPLA2 that were predominantly associated with the membrane, although a lower level was observed in the soluble fraction, as reported previously in CHO and Sf 9 cells overexpressing cPLA2 (Fig. 5)
(21, 25). Inhibition of prenylation, by treatment of the cells with BMS-214662 (Fig. 5A) or expression of the C538S prenylation mutant (Fig. 5B), had no effect on the distribution of cPLA2, which remained primarily membrane-associated. This is consistent with previous findings showing that mutation of the CaaX sequence and the putative myristoylation site does not affect the membrane binding of cPLA2 expressed in CHO cells (21). In contrast, a recent study has suggested that mutation of the CaaX sequence partially decreases the affinity of cPLA2 for membrane in HEK293 cells based on enzymatic assays, although the relative amount of cPLA2 present in the membrane by Western blot analysis was not determined (40). In our experiments, transfection of C538S mutant cPLA2 into HEK293 cells did not result in a change in membrane distribution of the cPLA2 according to Western blotting (data not shown). In addition, when membranes (100,000 g pellet) from COS cells expressing cPLA2 mutated at the prenylation site (C538S) were treated with 1.0 M NaCl, cPLA2 remained associated with the membrane (data not shown).
To determine whether the hydrophobic and basic regions in the C terminus of cPLA2 play a role in the membrane association of the enzyme, the C terminus of cPLA2 was truncated. The addition of a stop codon replacing a lysine (K490) allowed the expression of an 55 kDa truncated version of cPLA2. The truncation also removed the CaaX sequence, thus preventing farnesylation of the enzyme, in addition to removing the hydrophobic and basic regions of the enzyme. Overexpression of the truncated cPLA2 and separation of the membrane fraction (100,000 g) in HEK293 cells indicated that without these regions, cPLA2 remained associated with the membrane (Fig. 5C).
Fatty acylation of cPLA2
IMLF–/– infected with Ad-cPLA2 were incubated in medium containing [3H]palmitate or [3H]myristate for 4 h, followed by immunoprecipitation of cPLA2, separation by SDS-PAGE, and fluorography to visualize tritiated bands. As shown in Fig. 6A
, major radioactive bands were seen with an apparent molecular mass of 60 kDa, the predicted molecular mass of cPLA2, demonstrating that cPLA2 is labeled by [3H]fatty acids. Samples from separate experiments were treated identically but not submitted to fluorography, and a gel piece corresponding to the cPLA2 region was excised. When the gel slice containing immunoprecipitated cPLA2 obtained from [3H]myristate- or [3H]palmitate-labeled cells was washed three times with 50% aqueous methanol, <1% of the total radioactivity in the gel slice was obtained in the combined washes. This shows that all of the radiolabel is protein bound (i.e., that there are no free radiolabeled fatty acids in the gel slice). The washed gel slice was treated with 1.5 M aqueous NaOH at 30°C for 2 h, conditions known to hydrolyze thiolester- or oxyester-linked fatty acid groups from proteins (41). It was found that 35% and 38% of the total radioactivity in the gel slice was obtained in the organic phase of the Dole solvent extract from [3H]myristate- and [3H]palmitate-labeled cells, respectively.
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are ester-linked rather than amide-linked. The radioactivity remaining in the water layer after hydrolysis in 6 M HCl is presumably attributable to radiolabeled amino acids that resulted from metabolic ß-oxidation of the radiolabeled fatty acids into acetyl-CoA and then into amino acids in the fibroblasts, but we did not further investigate this water-soluble radiolabeled material.
The material resulting from Dole extraction of the aqueous NaOH-treated sample was treated with pentafluorobenzyl bromide in the presence of the carrier nonradiolabeled fatty acids myristic, palmitic, and oleic acids, and the sample was analyzed by reverse-phase HPLC using ultraviolet absorbance at 254 nm to monitor the carrier fatty acid esters and by scintillation counting to monitor the radiolabel. For [3H]palmitate-labeled cells, two peaks of radioactivity coeluted precisely with the nonradiolabeled oleic acid and palmitic acid ester standards, and no radioactivity eluted with the myristic acid ester standard (Fig. 6B). The HPLC retention times for the myristate and oleate esters are reproducible to within 10 s, a time much shorter than the difference in retention times for the two esters. The radioactivity clearly comigrates with the 18:1 ester without a discernible shoulder at a shorter retention time. These results show that cPLA2 contains ester-linked oleoyl and palmitoyl groups. The same HPLC pattern was seen for [3H]myristate-labeled cells (data not shown), indicating that the 14 carbon fatty acid was converted to the 16 and 18 carbon fatty acids in fibroblasts and that cPLA2 is not myristoylated. The formation of the cPLA2-linked radiolabeled oleoyl group is presumably the result of elongation of the radiolabeled fatty acyl-CoA to stearoyl-CoA followed by the action of stearoyl-CoA desaturase to form oleoyl-CoAs.
Subcellular localization of cPLA2
The localization of expressed untagged cPLA2 in fixed IMLF–/–, SkMc cells, and COS cells was carried out using a rabbit polyclonal antibody generated against full-length cPLA2. A comparison of cPLA2 localization and a variety of organelle markers demonstrated that cPLA2 localized primarily to mitochondria. The morphology of mitochondria is heterogeneous in cells, but they often appear as small ovoid structures or branched "threads" (42). Mitochondria often concentrate around the cell nucleus, where they are closely apposed to the endoplasmic reticulum. However, mitochondria can be clearly resolved in the cell periphery, where they are not as closely associated with the endoplasmic reticulum (42).
In IMLF–/–, the localization of cPLA2 was concentrated in the central, thickest region of the cell around the nucleus, where little structural detail could be observed (Fig. 7)
. However, farther out in the cell extensions, cPLA2 was clearly localized to branched, thread-like structures that stained with antibodies to the mitochondrial marker oxidative phosphorylation complex V, subunit b. The mitochondrial localization of cPLA2 was also clearly apparent when it was expressed in SkMc cells (Fig. 8)
. cPLA2 localized on branched, tubular structures that wrapped around the nucleus and extended toward the periphery of the cell, and these structures costained with antibodies to the mitochondrial marker oxidative phosphorylation complex V. Although overexpressed cPLA2 predominately costained with the mitochondrial markers, there was a small amount of cPLA2 in these cells that did not colocalize with the endoplasmic reticulum, lysosomes, nucleus, or mitochondria (data not shown). Control experiments with secondary antibody alone (Fig. 7F) as well as each secondary antibody with the opposite primary antibody were negative (Fig. 7G). Additional experiments demonstrated that BMS-214662 had no effect on the localization of cPLA2 on the mitochondria in SkMc cells (data not shown).
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localizes to the endoplasmic reticulum when expressed in HEK293 cells, based on a cytoplasmic reticular staining pattern, although this was not confirmed with organelle markers (40). In HEK293 cells, FLAG-tagged cPLA2 was found enriched in the perinuclear region, but no signal was seen on the nuclear envelope, which is an extension of the endoplasmic reticulum (40). In another study, it was reported that cPLA2 with an N-terminal GFP tag localized at the Golgi, endoplasmic reticulum, and nuclear envelope when expressed in CHO cells but did not colocalize with a lysosomal or mitochondrial marker (26).
The reports suggesting that N-terminal-tagged cPLA2 primarily localizes to the endoplasmic reticulum and that mutation of the prenylation site affects the localization of FLAG-tagged cPLA2 are distinct from our results with untagged cPLA2 (26, 40). Although it is possible that the localization is cell type-dependent, it is also possible that expression of cPLA2 with an N-terminal tag has an effect on its localization and conformation on the membrane. We have found that N-terminal GFP and 6x histidine (His) tags negatively affect the enzymatic activity of cPLA2, resulting in 80% and 60% less activity than in the wild-type enzyme, respectively (25). The N-terminal tags also suppressed the amount of arachidonic acid released by cPLA2 when expressed in Sf 9 cells (Fig. 9)
. We reported previously that Sf 9 cells can be used as a model to study the function of cPLA2, which is activated in Sf 9 cells by A23187, resulting in the release of arachidonic acid (19, 43). As shown in Fig. 9, A23187 also induced a large increase in arachidonic acid release from Sf 9 cells expressing cPLA2 compared with vector control cells. In contrast to cells expressing untagged cPLA2, A23187-induced arachidonic acid release from Sf 9 cells expressing GFP- or His-tagged cPLA2 was dramatically reduced to near basal levels, suggesting that the N terminus of the enzyme is critical for the function of cPLA2. The N-terminal-tagged cPLA2 enzymes were expressed at similar levels as the wild-type enzyme in Sf 9 cells and were constitutively bound to membrane (25). It is important to note that the ability of A23187 to activate cPLA2 is cell type-specific and is also observed when cPLA2 is overexpressed in HEK293 cells (40), but A23187 does not stimulate arachidonic acid release from IMLF–/– or SkMc cells overexpressing cPLA2. The ability of A23187 to activate cPLA2 is unexpected, because cPLA2 is calcium-independent; however, an increase in intracellular calcium may trigger unique signals in some cells that play a role in regulating cPLA2. There is a pronounced lag phase preceding cPLA2-mediated arachidonic acid release in Sf 9 cells treated with A23187, consistent with an indirect mechanism. In contrast, arachidonic acid release from Sf 9 cells expressing cPLA2 occurs rapidly in response to A23187, as reported previously, as a result of the direct effect of Ca2+ binding to the C2 domain of cPLA2 and inducing translocation to the membrane (19, 43). We have also observed similar differences in the time course of serum-induced arachidonic acid release from IMLF–/– expressing cPLA2 and cPLA2 (25).
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is farnesylated in mammalian cells and document that prenylation plays a role in cPLA2-induced fatty acid hydrolysis. The C-terminal residue of cPLA2 is alanine, and it may be noted that yeast -factor also has a CaaX box with an X residue of alanine and is farnesylated. It is currently thought that the most preferred substrates for protein geranylgeranyltransferase type I have a CaaX box in which X is leucine or phenylalanine (28). Our studies with cPLA2 show that not all proteins with a CCXX sequence are doubly geranylgeranylated. Rab proteins have been shown to contain sequences in addition to the double cysteine motif that are recognized by the Rab escort protein that delivers the Rab protein to protein geranylgeranyltransferase type II (44).
The mechanism involved in regulating cPLA2 function by farnesylation is not known, but our results suggest that it alone is not responsible for membrane binding. For membrane targeting of other farnesylated proteins such as Ras, the farnesylated C terminus is not sufficient but requires either a polybasic domain or protein fatty acylation (45). In addition to membrane anchoring, prenylation can play a role in the heterodimeric protein interaction that is proposed to involve a two-site recognition (46). cPLA2 also has a cluster of basic residues adjacent to a hydrophobic region in the C terminus that may be significant in its association with the membrane (30). Because cPLA2 mutated at the prenylation site does not separate to the soluble fraction in the presence of 1.0 M NaCl, electrostatic interactions and prenylation alone are unlikely to determine the membrane binding of cPLA2. To investigate the role that the hydrophobic and basic regions in the cPLA2 C terminus may play in membrane binding more thoroughly, a truncated cPLA2, missing these regions, was created. However, our results indicate no change in membrane association of cPLA2 attributable to this mutation, suggesting that these regions do not play a predominant role in membrane association. In addition to experiments to determine the possible mechanism of farnesylation, fatty acid labeling experiments were conducted using cells expressing wild-type cPLA2 and the C538S mutant. Farnesylation could be required for the fatty acylation of cPLA2. However, our data indicate that this is not the case for cPLA2, because the fatty acid labeling is not significantly changed when the prenylation site is mutated (data not shown).
cPLA2 contains a putative N-myristoylation site; however, our results demonstrate that cPLA2 is acylated with palmitic and oleic acids, but we saw no evidence that myristoylation of the enzyme occurs. Consistent with this observation, we found that cPLA2 mutated at both the N-terminal glycine (to block possible myristoylation) and the prenylation site remains bound to the membrane (data not shown). Although cPLA2 is radiolabeled with [3H]myristic acid, it is first metabolized into [3H]palmitic and [3H]oleic acids, which subsequently associate with cPLA2. Thus, although our data show that N-terminal tags on cPLA2 negatively affect function, this is not attributable to blocking myristoylation but may have an affect on enzyme conformation, acylation, or membrane trafficking. Although these results indicate that cPLA2 is fatty acylated, the locations of the oleic and palmitic acyl groups on the protein chain remain to be determined. It is possible that both types of fatty acyl groups could be linked to the same residue or that the enzyme could be acylated on multiple residues. Investigation of mutations of the individual cysteines is under way. However, the more rare palmitoylation of serine or threonine residues via oxyester bonds can also occur and must be considered.
The observation that cPLA2 primarily associates with mitochondria suggests that it may play a role in the function of this organelle. Although cPLA2 message is abundant in human SkMc, to date, primarily immortalized cells have been used to study cPLA2 (21, 23, 25, 26, 40). By RT-PCR, we have confirmed that cPLA2 message is produced in both myotubes and myoblasts of SkMc cells (data not shown). However, despite the presence of message, only very low levels of endogenous cPLA2 protein were detectable in SkMc cells, making the direct study of endogenous cPLA2 difficult. The regulation of membrane binding and localization of cPLA2 to mitochondria are very different from the known properties of cPLA2, which has not been shown to associate with mitochondria. Recently, an increase in the group IVC cPLA2 in apoptotic macrophage cells was observed (47). This observation suggests a potential role in the mitochondrial induction of apoptosis for cPLA2. Both calcium-dependent and -independent PLA2 activities have been found associated with isolated mitochondria, including the group VI calcium-independent PLA2 and the group IIA sPLA2 (48–51). The presence of diverse PLA2 enzymes in mitochondria suggests that there are multiple, independently regulated pathways in this organelle for the hydrolysis of membrane phospholipid.
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ACKNOWLEDGMENTS |
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Manuscript received June 7, 2005 and in revised form July 22, 2005.
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