Fas-induced Arachidonic Acid Release Is Mediated by Ca2+-independent Phospholipase A2 but Not Cytosolic Phospholipase A2, Which Undergoes Proteolytic Inactivation*

Gen-ichi Atsumi, Masae Tajima, Atsuyoshi Hadano, Yoshihito Nakatani, Makoto Murakami, and Ichiro KudoDagger

From the Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Fas-mediated apoptosis of human leukemic U937 cells was accompanied by increased arachidonic acid (AA) and oleic acid release from membrane glycerophospholipids, indicating phospholipase A2 (PLA2) activation. During apoptosis, type IV cytosolic PLA2 (cPLA2), a PLA2 isozyme with an apparent molecular mass of 110 kDa critical for stimulus-coupled AA release, was converted to a 78-kDa fragment with concomitant loss of catalytic activity. Cleavage of cPLA2 correlated with increased caspase-3-like protease activity in apoptotic cells and was abrogated by a caspase-3 inhibitor. A mutant cPLA2 protein in which Asp522 was replaced by Asn, which aligns with the consensus sequence of the caspase-3 cleavage site (DXXDdown-arrow X), was resistant to apo-ptosis-associated proteolysis. Moreover, a COOH-terminal deletion mutant of cPLA2 truncated at Asp522 comigrated with the 78-kDa fragment and exhibited no enzymatic activity. Thus, caspase-3-mediated cPLA2 cleavage eventually leads to destruction of a catalytic triad essential for cPLA2 activity, thereby terminating its AA-releasing function. In contrast, the activity of type VI Ca2+-independent PLA2 (iPLA2), a PLA2 isozyme implicated in phospholipid remodeling, remained intact during apoptosis. Inhibitors of iPLA2, but neither cPLA2 nor secretory PLA2 inhibitors, suppressed AA release markedly and, importantly, delayed cell death induced by Fas. Therefore, we conclude that iPLA2-mediated fatty acid release is facilitated in Fas-stimulated cells and plays a modifying although not essential role in the apoptotic cell death process.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Release of free fatty acids from glycerophospholipids, a major component of cell membranes, is crucial for various cellular responses, such as signal transduction and membrane remodeling and is tightly regulated by a diverse family of phospholipase A2 (PLA2)1 enzymes. Type IV cytosolic PLA2 (cPLA2), which exhibits strict substrate specificity for arachidonic acid (AA)-containing phospholipids in the presence of submicromolar concentrations of Ca2+, is a key enzyme that triggers stimulus-initiated AA metabolism, leading to production of bioactive lipid mediators in activated cells (1, 2). Various Ca2+-mobilizing agonists and proinflammatory cytokines induce cPLA2 activation for immediate and delayed eicosanoid biosynthesis, respectively, through mechanisms involving Ca2+-dependent translocation to the perinuclear and endoplasmic reticular membranes and phosphorylation by kinases belonging to the mitogen-activated protein kinase (MAPK) family (3-7). Evidence is also accumulating that certain phases of stimulus-initiated AA release are regulated by types IIA or V secretory PLA2 (sPLA2) isozymes, which require millimolar concentrations of Ca2+ for catalytic activity (8-12). In contrast, type VI Ca2+-independent PLA2 (iPLA2) has been proposed to participate in fatty acid release associated with phospholipid remodeling and to play a minimal role in signal transduction (13-15).

The programmed cell death pathway called apoptosis plays a fundamental role in tissue homeostasis, development, and host defense. The discovery of increasing numbers of death factors (e.g. Fas ligand and tumor necrosis factor (TNF) alpha ), their receptors, adapter molecules, and caspases have provided new insight into the molecular mechanisms leading to apoptosis that are highly conserved in eukaryotic cells (16, 17). Following ligation of these receptors by their cognate ligands, several cytoplasmic signal-transducing adapters are recruited to the receptors to form multimeric complexes, thereby leading to either apoptosis or cellular activation, depending on the adapters involved (18, 19). In the apoptotic pathway, these adapter molecules in turn recruit particular caspases (20-22), which trigger the protease cascade where multiple caspases are sequentially activated as a consequence of their proteolytic processing at a specific Asp residue, allowing them to become self-activated and activate one another. Activated caspases then cleave their respective substrates (23-30), which are key regulatory and structural proteins such as protein kinases and proteins involved in DNA repair and cytoskeleton integrity, thereby contributing to the demise of the cell.

The changes in glycerophospholipid metabolism, unlike those in protein and DNA levels, during apoptosis are rather obscure. Although the involvement of particular PLA2 enzymes in stimulus-coupled eicosanoid biosynthesis has been studied extensively as discussed above, relatively little is known about their roles in fatty acid release associated with apoptotic signaling. Recently, we and others showed that the plasma membrane phospholipids of apoptotic cells are the preferred substrates for type IIA sPLA2 (31, 32), which is induced by proinflammatory stimuli and has been implicated in the pathogenesis of inflammation (10, 33). It has been speculated that the AA thus liberated is taken up by surrounding live cells to be metabolized to eicosanoids, representing a particular transcellular pathway for AA metabolism that contributes to propagation of inflammation. cPLA2 has also been implicated in AA release during certain cell death processes, such as TNFalpha -induced apoptosis (34-36) and hydroperoxide-induced cytotoxicity (37). In these studies, the use of cPLA2 inhibitors, overexpression, and antisense technologies revealed significant correlations between the cPLA2 expression level, amounts of AA released, and cell death. However, the role of cPLA2 is uncertain in Fas-mediated apoptosis (38).

In order to explore the role of particular PLA2 isozymes in fatty acid release and glycerophospholipid metabolism alterations associated with the apoptotic pathway, we chose the Fas system, which strongly promotes apoptosis but barely elicits cell activation signals. We now provide evidence that Fas-mediated apoptosis of human leukemic U937 cells is accompanied by gradual fatty acid release, which appears to be mediated by iPLA2 but not by cPLA2 or sPLA2. During apoptosis, cPLA2 is inactivated by caspase-3-dependent proteolytic cleavage at Asp522. Inhibition of iPLA2-evoked fatty acid release results in slight but significant retardation of cell death, suggesting that disturbed phospholipid turnover renders cells more susceptible to Fas-mediated apoptotic signaling.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Mouse cPLA2 cDNA and a rabbit antiserum against human cPLA2 were provided by Drs. M. Tsujimoto (RIKEN Institute) and J. D. Clark (Genetics Institute), respectively. The agonistic anti-Fas antibody (CH-11) (39) was purchased from MBL. The cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3) (40) was purchased from Calbiochem. Methyl arachidonylfluorophosphonate (MAFP), which inhibits both cPLA2 and iPLA2 (14), and the iPLA2 inhibitor bromoenol lactone (13-15) were purchased from Cayman Chemical. The type IIA sPLA2 inhibitor LY311727 (41) was donated by Dr. R. M. Kramer (Lilly Research). Two caspase inhibitors (Ac-YVAD-CHO and Ac-DEVD-CHO) and chymostatin were obtained from the Peptide Institute, and caspase substrates were obtained from Takara Biomedicals. LipofectAMINE PLUS reagent, Opti-MEM medium, and TRIzol reagent were obtained from Life Technologies. Etoposide, p-bromophenacyl bromide, 4',6'-diamidino-2-phenylindole (DAPI), leupeptin, antipain, pepstatin, cytochalasin B, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and Nalpha -benzoyl-arginine ethyl ester were purchased from Sigma. All the other reagents used were of analytical grade and purchased from Wako, unless stated otherwise. The human monocytic leukemia cell lines U937 and HL-60 and human embryonic kidney 293 cells (RIKEN Cell Bank) were maintained in RPMI 1640 medium (Nissui Pharmaceutical) supplemented with 10% (v/v) fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in humidified air with 5% CO2.

Fatty Acid Release from U937 Cells Undergoing Apoptosis-- In order to investigate AA and oleic acid release, U937 cells were preincubated with 0.1 and 0.5 µCi/ml [3H]AA and [3H]oleic acid (NEN Life Science Products), respectively, for 24 h. Then the cells were washed three times, resuspended in culture medium to produce 1 × 107 cells/ml, and incubated for various periods with or without the anti-Fas antibody. In the experiments to determine the effect of etoposide treatment, the cells were incubated with etoposide for 1 h, washed three times, and then incubated for a further 10 h in culture medium without etoposide. In some experiments, various PLA2 inhibitors were added to the cells during incubation. The process of cell death was monitored by observing the morphological changes, assessing the cell viability by trypan blue dye exclusion, and quantifying DNA fragmentation fluorometrically using DAPI as described previously (42). The free 3H-labeled fatty acids released were extracted by the method of Dole and Meinertz (43), and the associated radioactivity was counted using a liquid beta -scintillation counter (Aloka). The amount of each fatty acid released, expressed as a percentage, was calculated using the formula [S/(S + P)] × 100, where S and P are the radioactivities of equal portions of supernatant and cell pellet, respectively.

Immunoblotting-- Cells were washed with phosphate-buffered saline and then lysed in phosphate-buffered saline containing 100 µM p-4-(2-aminoethyl)-benzenesulfonyl fluoride, 5 µM iodoacetamide, 5 mM EDTA, 1 µM pepstatin, 1 mg/ml soybean trypsin inhibitor, and 100 µM leupeptin by sonication for 1 min with a Branson Sonifer (power 30, 50% pulse cycle). The samples (10 µg protein equivalents/lane) were subjected to 10% (w/v) SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions and electroblotted onto nitrocellulose membranes (Schleicher & Schuell), which were probed with the antibody against human cPLA2 and visualized with the ECL Western blot analysis system (Amersham Pharmacia Biotech), as described previously (8). The protein contents were quantified using a BCA protein assay kit (Pierce).

Assessment of PLA2 Activity-- In order to assess cPLA2 activity, cells were washed once with phosphate-buffered saline, suspended in buffer comprising 10 mM Tris-HCl (pH 7.4) and 150 mM NaCl (Tris-buffered saline), and lysed by sonication as described above. The resulting lysates were incubated in 250 µl of buffer comprising 100 mM Tris-HCl (pH 9.0), 4 mM CaCl2, and 2 µM 1-palmitoyl-2-[14C]arachidonyl-glycerophosphoethanolamine (NEN Life Science Products) as the substrate at 37 °C for 30 min.

In order to assess iPLA2 activity, the lysates, prepared in 10 mM HEPES (pH 7.5) containing 1 mM EDTA, 1 mM dithiothreitol, and 0.34 M sucrose, were incubated in 250 µl of buffer comprising 100 mM HEPES (pH 7.5), 5 mM EDTA, 0.4 mM Triton X-100, 0.1 mM ATP, and 10 µM 1-palmitoyl-2-[14C]arachidonyl-glycerophosphoethanolamine at 40 °C for 30 min (13, 15). The [14C]AA released was extracted by the method of Dole and Meinertz (43), and the associated radioactivity was counted.

Thin Layer Chromatography-- The total lipids were extracted from [3H]AA-labeled cells and their supernatants by the method of Bligh and Dyer (44) and developed by two-dimensional thin layer chromatography on silica 60 gel plates (Merck), according to a method described previously with a slight modification (31). The first solvent system consisted of chloroform/methanol/acetic acid/water (65/25/4/2, v/v/v), and the second comprised chloroform/methanol/formic acid (65/25/8.8, v/v/v). The zones on the silica gel corresponding to AA and phospholipids were identified by comparison with the mobilities of authentic standards and visualized with iodine vapor. Each zone was scraped into a vial, and its radioactivity was counted using a liquid beta -scintillation counter. To separate free fatty acids and other neutral lipids, including triacylglyceride, the neutral lipid fraction was developed further on fresh plates with a solvent system of hexane/ether/acetic acid (80/30/1, v/v/v).

Preparation of Mouse Recombinant cPLA2-- Sf9 cells (Invitrogen) were transfected with mouse cPLA2 cDNA that had been subcloned into the pVL1392 baculovirus transfection vector (Pharmingen), as described previously (8). The cells were harvested, suspended in a buffer comprising 250 mM sucrose, 10 mM Tris-HCl (pH 7.0), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Nalpha -benzoyl-arginine ethyl ester, 10 µg/ml leupeptin, and 10 µg/ml antipain, and disrupted by sonication for 1 min, as described above. The lysates were centrifuged at 100,000 × g for 1 h at 4 °C, and the resulting supernatants were applied to a DEAE-Sephacel column (Amersham Pharmacia Biotech) that had been pre-equilibrated with Tris-buffered saline. After washing the column with Tris-buffered saline, the bound proteins were eluted with a linear NaCl gradient from 0.15 to 0.5 M in 10 mM Tris-HCl (pH 7.4). The eluted fractions containing cPLA2 activity were pooled, applied to a phenyl-Superose column (Amersham Pharmacia Biotech) that had been pre-equilibrated with 10 mM Tris-HCl (pH 7.4) containing 1 M NaCl, and the bound cPLA2 was eluted stepwise with 10 mM Tris-HCl (pH 7.4).

Assessment of Caspase Activity-- Cells were washed with phosphate-buffered saline, resuspended (108 cells/ml) in an extraction buffer comprising 50 mM PIPES-NaOH (pH 7.0), 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM dithiothreitol, 20 µM cytochalasin B, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 50 µg/ml antipain, and 10 mg/ml chymostatin, and disrupted by freeze-thawing twice. Aliquots (10 µg of protein equivalents) were incubated with 1 µM Mca-YVADAPK(Dnp)-OH or Mca-DEVDAPK(Dnp)-OH, substrates for caspase-1 and caspase-3, respectively (45), in a buffer containing 100 mM HEPES (pH 7.5), 10% (w/v) sucrose, 0.1% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10 mM dithiothreitol, and 0.1 mg/ml ovalbumin, at 30 °C for 30 min. Then the fluorescence of each cleaved substrate was determined using a spectrofluorometer (Hitachi) at excitation and emission wavelengths of 325 and 392 nm, respectively.

Construction of Mutant cPLA2-- Substitution of Asp522 of mouse cPLA2 with Asn (cPLA2-D522N) was performed by altering the codon GTC to GTT using the QuikChangeTM site-directed mutagenesis kit (Stratagene) with two synthetic oligonucleotides for the sense and antisense strands: 5'-C CTT CGA TGA CGA GCT CAA CGC AGC GGT AGC AG-3' and 5'-C TGC TAC CGC TGC GTT GAG CTC GTC ATC GAA GG-3', respectively (altered codons are underlined). Briefly, mouse cPLA2 cDNA subcloned into pBK-CMV (Stratagene) at the EcoRI site was denatured and annealed with the two oligonucleotide primers containing the desired mutation. After incubation with Pfu DNA polymerase followed by treatment with DpnI, the resulting mutated plasmid was transfected into Escherichia coli XL1-Blue supercompetent cells (Stratagene). A cPLA2 deletion mutant, cPLA2(1-522), was constructed by polymerase chain reaction amplification of the cPLA2 coding sequence with ex Taq polymerase (Takara) using the oligonucleotide pair 5'-ATG TCA TTT ATA GAT CCT TAC-3' and 5'-TCA GTC GAG CTC GTC ATC GAA-3'. The polymerase chain reaction product was ligated into pCRTM3.1 (Invitrogen) and was transfected into Top10F' supercompetent cells (Invitrogen). Colonies were picked up, and the plasmids were isolated and sequenced using a Taq cycle sequencing kit (Takara) and an auto-fluorometric DNA sequencer (DSQ-1000L, Shimadzu) to confirm the mutation.

In Vitro Transcription and Translation of cPLA2-- [35S]Methionine-labeled cPLA2 and its mutants were synthesized using a PROTEINscriptTM kit (Ambion). Briefly, plasmids containing mouse cPLA2, cPLA2-D522N, or cPLA2(1-522) cDNA were transcribed using RNA polymerase and then incubated with [35S]methionine (NEN Life Science Products) and rabbit reticulocyte lysate. The products were subjected to SDS-PAGE and visualized autoradiographically.

Cleavage of cPLA2 Protein by Apoptotic Cell Lysates in Vitro-- Aliquots of [35S]cPLA2 or baculovirus-derived recombinant cPLA2 proteins prepared as described above were incubated with the cytosolic fraction obtained from U937 cells that had been treated for 12 h with or without the anti-Fas antibody in the presence or absence of a caspase inhibitor (Ac-YVAD-CHO or Ac-DEVD-CHO) (45). The reaction products were analyzed by SDS-PAGE followed by autoradiography and immunoblotting, respectively.

Transfection of 293 Cells with cDNAs for Native and Mutant cPLA2-- The cDNAs for mouse cPLA2 and its mutants cPLA2-D522N and cPLA2(1-522) that had been subcloned into pBK-CMV and PCR 3.1TM, respectively, were each transfected into 293 cells using LipofectAMINE PLUS reagent according to the manufacturer's instructions as described previously (46). The cells were harvested 48 h after transfection, lysed, and subjected to immunoblotting and PLA2 assay.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

AA Release from Anti-Fas Antibody-treated U937 Cells-- Human monocytic leukemia U937 cells were prelabeled with [3H]AA for 24 h, washed, and then exposed to various concentrations of the agonistic anti-Fas antibody for various periods to assess the changes in the free [3H]AA levels (Fig. 1A). AA release by cells cultured without the anti-Fas antibody increased minimally over 24 h, whereas that by replicate cells treated with the anti-Fas antibody increased significantly after culture for 3-24 h in a concentration-dependent manner. AA release by U937 cells treated with 10, 50, and 100 ng/ml anti-Fas antibody for 24 h was about 3.5, 5.0, and 6.5 times higher, respectively, than that by the cells before anti-Fas antibody addition (Fig. 1A). AA release was almost parallel to the reductions in cell viability and DNA fragmentation, assessed by trypan blue dye exclusion and fluorometric quantification with DAPI (33), respectively (Fig. 1, B and C), as well as DNA rudder formation, assessed by agarose gel electrophoresis (data not shown).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1.   AA release by U937 cells undergoing Fas-mediated apop-tosis. A, U937 cells prelabeled with [3H]AA were treated for the indicated periods with 0 (open circle ), 5 (bullet ), 10 (square ), 50 (black-square), and 100 (triangle ) ng/ml anti-Fas antibody. The free [3H]AA was extracted, and its radioactivity was counted as described under "Experimental Procedures." B and C, changes in cell viability, assessed by trypan blue staining (B) and DNA fragmentation quantified fluorometrically using DAPI (C) in cells cultured for various periods with 100 ng/ml anti-Fas antibody (left) and those cultured for 12 h with various concentrations of the antibody (right). Values are expressed as the means ± S.E. of three independent experiments; *, p < 0.05 versus untreated cells; **, p < 0.01 versus untreated cells.

After a 24-h preincubation of U937 cells with [3H]AA, nearly 95% of the radioactivity was incorporated into phospholipid pools, mainly into phosphatidylcholine and phosphatidylethanolamine, followed by phosphatidylinositol and phosphatidylserine (Fig. 2). Up to 5% of the radioactivity was incorporated into the neutral lipid fraction, mainly into triacylglycerol (Fig. 2, right panel). After incubation with 50 ng/ml anti-Fas antibody, the free [3H]AA level increased significantly, accompanied by reductions in the percentages of the total [3H]AA remaining in phosphatidylethanolamine and phosphatidylcholine without appreciable changes of those in other phospholipids and triacylglycerol (Fig. 2). In terms of the total counts, the combined decrements in these two phospholipids roughly matched the net increase in free [3H]AA.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   Phospholipid composition of U937 cells before and after anti-Fas antibody treatment. U937 cells prelabeled with [3H]AA were cultured in the presence or absence of 50 ng/ml anti-Fas antibody for 24 h. The lipids were extracted and separated into each component by thin layer chromatography as described under "Experimental Procedures." Inset, the neutral lipid fraction was further separated into free fatty acids and other neutral lipids (triacylglycerol) by thin layer chromatography. The values are expressed as the means ± S.E. of three independent experiments; *, p < 0.05 versus untreated cells. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PA, phosphatidic acid; SM, sphingomyelin; LPC, lysophosphatidylcholine; FFA, free fatty acid; NL, neutral lipid; TG, triacylglycerol; DG, diacylglycerol.

Proteolysis of cPLA2 in Cells Undergoing Apoptosis-- Initially, we thought that the increased Fas-mediated AA release from apoptotic U937 cells was due to cPLA2 activation. Unexpectedly, however, the cPLA2 catalytic activity toward exogenous substrate gradually declined during the Fas-mediated apoptotic process: the cPLA2 activities of cells after treatment with 10 and 100 ng/ml anti-Fas antibody for 12 h were about one-half and one-ninth, respectively, of that of untreated cells (Fig. 3A). The cPLA2 inhibitor AACOCF3, at 1 µM, abolished the cPLA2 activity of U937 cells, whereas [3H]AA release by apoptotic cells incubated with and without AACOCF3 at 10 µM, the concentration at which it inhibits cPLA2 but not other PLA2s (Ref. 13 and data not shown), did not differ significantly (Fig. 3B). Moreover, net amounts of [3H]oleic acid comparable with those of [3H]AA were released by anti-Fas antibody-treated cells prelabeled with these fatty acids (Fig. 3C), arguing against the AA selective property of cPLA2.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Lack of involvement of cPLA2 in Fas-induced fatty acid release by apoptotic U937 cells. A, changes in cPLA2 activity in cells undergoing Fas-induced apoptosis. Cell lysates (105 cell equivalent) before and after anti-Fas antibody treatment were taken for cPLA2 assay. B, effects of AACOCF3 on [3H]AA release by apoptotic cells over 12 h (left) and cPLA2 activity in cell lysate (right). Representative results of three independent experiments are shown in A and B. C, release of [3H]AA and [3H]oleic acid from U937 cells treated for 12 h with or without 100 ng/ml anti-Fas antibody (mean ± S.E.; n = 3). *, p < 0.05.

In order to elucidate the mechanisms responsible for the decrease in cPLA2 activity in cells undergoing Fas-mediated apoptosis, we examined cPLA2 protein expression by immunoblotting. We found that treating U937 cells with the anti-Fas antibody resulted in time- (Fig. 4A) and dose-dependent (Fig. 4B) cleavage of intact cPLA2 with an apparent molecular mass of 110 kDa, which was present in untreated cells, to a proteolytic fragment of approximately 78 kDa. Proteolysis of cPLA2 was detectable within 3 h and complete after anti-Fas antibody treatment for 12 h (Fig. 4A), the time at which cPLA2 cleavage was dependent upon the anti-Fas antibody concentration (Fig. 4B), paralleling the onset of Fas-mediated apoptosis (Fig. 1, B and C). Nonspecific proteolysis could be discounted, because most of the other proteins were not degraded, as assessed by SDS-PAGE followed by silver staining, after anti-Fas antibody treatment for 12 h (data not shown).


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 4.   Cleavage of cPLA2 in U937 cells undergoing apoptosis. A, U937 cells were treated for the indicated periods with 100 ng/ml anti-Fas antibody. B, the cells were treated for 12 h with the indicated concentrations of anti-Fas antibody. C, the cells were treated with the indicated concentrations of etoposide for 1 h, washed, and cultured for a further 10 h in the absence of etoposide. Each cell lysate was subjected to immunoblotting using a rabbit anti-cPLA2 antiserum as described under "Experimental Procedures." Representative results of three to six independent experiments are shown.

Etoposide, a chemotherapeutic agent that directs to DNA topoisomerase II (47), induced apoptosis of U937 cells 10 h after treatment. Cleavage of cPLA2 was evident after incubation with as little as 1 µg/ml etoposide and, after exposure to 10 mg/ml, about half of the cPLA2 was converted to a 78-kDa fragment (Fig. 4C); the cell viability and DNA fragmentation after treatment with 10 µg/ml etoposide were about 40~50% (data not shown). Cleavage of cPLA2 was also observed when human leukemic HL-60 cells were exposed to the anti-Fas antibody (data not shown).

Cleavage of Recombinant cPLA2 by Apoptotic Cell Lysates in a Cell-free System-- In order to verify that apoptotic but not live U937 cells possessed protease activity that cleaved cPLA2 to produce a 78-kDa fragment, recombinant cPLA2, either prepared by in vitro transcription/translation or expressed by baculovirus-infected Sf9 insect cells, was incubated with lysates of U937 cells treated with or without the anti-Fas antibody for 12 h (Fig. 5). Detailed time course studies revealed that a reduction in the amount of 110-kDa [35S]cPLA2 translated in vitro was detected as early as 30 min after adding apoptotic U937 cell lysate, which correlated with the time when the 78-kDa fragment appeared (Fig. 5A). Cleavage of [35S] cPLA2 also depended on the amount of apoptotic cell lysate (Fig. 5B). Thus, all the 110-kDa [35S]cPLA2 was converted to the 78-kDa cleavage product by incubation with adequate apoptotic cell lysate within 4 h. In contrast, [35S]cPLA2 remained intact when incubated without the lysate (Fig. 5A) and with the lysate of nonapoptotic cells (Fig. 5B). Similar results were obtained in the experiment using baculovirus-derived recombinant cPLA2 (Fig. 5C). After mixing with the lysate prepared from cells treated with the anti-Fas antibody for 12 h, in which endogenous intact cPLA2 had been already degraded to a 78-kDa fragment (Fig. 4A), immunoblotting showed that recombinant cPLA2 disappeared gradually (Fig. 5C).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 5.   Cleavage of cPLA2 by apoptotic cell lysates in a cell-free system. A, 0.5 µg of the plasmid containing cPLA2 cDNA was subjected to in vitro transciption/translation in the presence of [35S]methionine. An aliquot (1/25 volume) of the in vitro translated [35S]cPLA2 was then incubated for the indicated periods with or without lysates prepared from U937 cells (105 cell equivalents) treated for 12 h with 100 ng/ml anti-Fas antibody. B, [35S]cPLA2 was incubated for 4 h with the indicated amounts of lysate prepared from U937 cells cultured for 12 h in the presence or the absence of 100 ng/ml anti-Fas antibody. C, baculovirus-derived recombinant cPLA2 was incubated for the indicated periods with the lysate of U937 cells treated for 12 h with 100 ng/ml anti-Fas antibody. The cPLA2 was visualized by autoradiography (A and B) and immunoblotting (C). Representative results of three independent experiments are shown.

Involvement of Caspase-3-like Protease(s) in the Site-specific Cleavage of cPLA2-- Because the cleavage of cPLA2 and onset of apoptosis occurred in parallel, we reasoned that caspases might cleave cPLA2 proteolytically. In order to explore this, we monitored the changes in the caspase activities in U937 cells after anti-Fas antibody treatment. As shown in Fig. 6A, the caspase-3-like activity increased during culture with the anti-Fas antibody for 3-12 h, showing good correlations with the apoptotic process (Fig. 1, B and C), AA release (Fig. 1A), and cPLA2 cleavage (Fig. 4A). The caspase-1-like activity increased transiently during Fas stimulation for 1-6 h and was much weaker than the caspase-3-like activity throughout (data not shown). When [35S]cPLA2 was incubated with apoptotic cell lysate in the presence of the caspase-1 and caspase-3 inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO, respectively, Ac-DEVD-CHO, at a concentration as low as 0.1 µM, inhibited cleavage of [35S]cPLA2 markedly, whereas Ac-YVAD-CHO was at least 100-fold weaker (Fig. 6B).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6.   Involvement of caspase-3-like protease(s) in cPLA2 cleavage. A, changes in the activities of caspase-3 toward its substrate Mca-DEVDAPK(Dnp)-OH in lysates of U937 cells cultured for the indicated periods with 100 ng/ml anti-Fas antibody. Values are expressed as the means ± S.E. of three independent experiments. *, p < 0.05 versus values at 0 h. B, effects of caspase inhibitors on proteolysis of [35S]cPLA2. The indicated concentrations of the caspase-1 and caspase-3 inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO, respectively, were added to the incubation mixture comprising [35S]cPLA2 and lysates of U937 cells cultured for 12 h with 100 ng/ml anti-Fas antibody. The cPLA2 was visualized by SDS-PAGE followed by autoradiography.

A potential caspase-3 cleavage site, DXXDdown-arrow X (48), is present in cPLA2 around Asp522 (2). If cPLA2 is cleaved at this site (DELD522down-arrow A), the predicted size of the resulting fragment, comprising the amino terminus of the protein, is consistent with the size of the cleavage fragment observed in this study. Indeed, a cPLA2 mutant, D522N, in which Asp522 was replaced by Asn, was not cleaved by the apoptotic cell lysate (Fig. 7A). Another cPLA2 mutant truncated at Asp522, cPLA2(1-522), comigrated with the 78-kDa fragment derived from apoptotic cleavage of cPLA2 on SDS-PAGE (Fig. 7B). These results suggest that caspase-3 or a related protease(s) is responsible for the proteolysis of cPLA2 at Asp522 in apoptotic cells.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 7.   Asp522 is the site of cPLA2 cleavage by caspase-3. A, 35S-labeled cPLA2 and its mutant (D522N) were incubated with apoptotic cell lysate for the indicated periods and subjected to SDS-PAGE. B, the electrophoretic (SDS-PAGE) mobility of [35S]cPLA2(1-522) was compared with that of the 78-kDa fragment produced after proteolytic cleavage of [35S]cPLA2 by apoptotic U937 cell lysate. Lane 1, cPLA2(1-522); lane 2, [35S]cPLA2; lane 3, [35S]cPLA2 incubated for 4 h with U937 cell lysate treated for 12 h with 100 ng/ml anti-Fas antibody. The cPLA2 was visualized by autoradiography. C, expression of native and mutant cPLA2 proteins assessed by immunoblotting (inset) and associated cPLA2 activities in lysates of 293 cells 2 days after transfection with the respective cDNAs.

Native cPLA2, cPLA2-D522N, and cPLA2(1-522) were overexpressed in human embryonic kidney 293 cells to assess their enzymatic activities. Cells were harvested 48 h after transfection and lysed by sonication, and the cPLA2 activity in each lysate was measured. As shown in Fig. 7C, cPLA2 expression, as assessed by immunoblotting, increased dramatically in cells transfected with native cPLA2, with a concomitant increase in the cPLA2 activity. cPLA2-D522N exhibited enzymatic activity comparable with that of the native enzyme. On the other hand, no PLA2 activity was associated with cPLA2(1-522), confirming that this fragment has no enzymatic function.

Possible Participation of iPLA2 in Fas-mediated AA Release and Apoptosis-- The results shown above support the hypothesis that cPLA2 is not involved in fatty acid release from cells undergoing Fas-mediated apoptosis. In an attempt to establish which PLA2 isozymes are involved in Fas-mediated AA release, the effects of various PLA2 inhibitors were tested. Because both AA and oleic acid were released from Fas-stimulated cells in parallel (Fig. 3C), we focused on sPLA2 and iPLA2, which exhibit no fatty acid preference in in vitro PLA2 assays (10, 14). No appreciable inhibition of [3H]AA release from anti-Fas antibody-treated U937 cells was observed with LY311727, a sPLA2-selective inhibitor (Fig. 8A), or p-bromophenacyl bromide, which inactivates all known sPLA2s by binding covalently to the catalytic center histidine (33) (data not shown). Consistent with these findings, sPLA2 expression was not detected in U937 cells (data not shown). In contrast, Fas-mediated [3H]AA release was significantly suppressed by MAFP, which inhibits both cPLA2 and iPLA2 (14), as well as by bromoenol lactone, a fairly selective iPLA2 inhibitor (13-15) (Fig. 8A). MAFP blocked [3H]AA release from phosphatidylethanolamine in apoptotic cells (Fig. 8B), indicating that this agent indeed blocked the PLA2-mediated reaction. The iPLA2 activities in lysates of U937 cells treated for 12 h with increasing concentrations of anti-Fas antibody were almost equal to that of control cells (Fig. 8C) and were suppressed by 85% by 1 µM MAFP. Verification that MAFP inhibits iPLA2-dependent [3H]AA release at the in vivo level is provided by the observation that increased spontaneous [3H]AA release by iPLA2-overexpressing 293 cells was attenuated by MAFP.2 These results imply that iPLA2 or a related MAFP-sensitive PLA2 is responsible for Fas-mediated AA release.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 8.   Involvement of iPLA2 in Fas-mediated AA release and apoptosis. A and B, effects of sPLA2 and iPLA2 inhibitors on AA release by cells undergoing Fas-mediated apoptosis. [3H]AA-prelabeled U937 cells were cultured for 24 h with 100 ng/ml anti-Fas antibody in the presence of the indicated concentrations of each inhibitor, and the [3H]AA liberated (A) and associated with phosphatidylethanolamine (B) was quantified. C, iPLA2 activities in lysates of U937 cells cultured for 12 h with the indicated concentrations of anti-Fas antibody. D, effect of MAFP on cell viability. U937 cells were cultured for the indicated periods with or without 100 ng/ml anti-Fas antibody in the presence or absence of 10 µM MAFP, and cell viablity was determined by trypan blue dye exclusion. Values are the means ± S.E. of three independent experiments; *, p < 0.05 versus untreated cells. BEL, bromoenol lactone.

Next, we examined the effect of MAFP on Fas-mediated apoptotic cell death. The viabilities of U937 cells treated for 24 h with anti-Fas antibody in the presence and absence of MAFP were reduced to similar extents (Fig. 8D). However, MAFP treatment slightly but consistently reduced the proportions of dead cells after incubation with the anti-Fas antibody for 6-12 h (Fig. 8D). These results indicate that fatty acid release by iPLA2 may play a role in the relatively early phase of Fas-induced cell death, although it is not essential.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In the present study, we attempted to establish the roles of particular PLA2 enzymes in AA release and changes in glycerophospholipid metabolism in cells stimulated with Fas, a receptor that transduces death signal. We found that treatment of U937 cells with an agonistic anti-Fas antibody enhanced delayed AA release, which occurred in parallel with the hallmarks of apoptosis. Unexpectedly, however, we found that cPLA2 lost its catalytic activity during apoptosis. Furthermore, the cPLA2 inhibitor AACOCF3 failed to suppress AA release, and oleic acid was also released in comparable amounts to AA, suggesting that Fas-induced AA release is largely cPLA2-independent. Detailed analysis revealed that intact cPLA2 disappeared as a result of proteolytic cleavage in apoptotic cells. Pharmacological studies suggest that iPLA2 is the most likely PLA2 isozyme responsible for liberation of fatty acids from apoptotic cell membranes.

Activation of cPLA2 by post-translational modifications, such as Ca2+-dependent translocation and MAPK-dependent phosphorylation, is essential for the initiation of stimulus-initiated AA release (1-7). The results of this study throw light on a novel post-translational modification of cPLA2, proteolytic cleavage by caspase-3, which leads to inactivation of cPLA2. We found that Fas-induced apoptosis of human leukemic cells led to cPLA2 cleavage, producing a catalytically inactive fragment with an apparent molecular mass of 78 kDa. Several lines of evidence indicate that this cPLA2 cleavage is mediated by a caspase-3-like protease(s): (i) cPLA2 cleavage in vivo and increased caspase-3-like activity occurred in parallel; (ii) apoptosis induced by the anti-cancer agent etoposide, which activates caspase-3 (49), was also accompanied by cPLA2 cleavage; (iii) cPLA2 cleavage in vitro was suppressed by the caspase-3 inhibitor Ac-DEVD-CHO; (iv) a mutant cPLA2 protein in which Asp522 was replaced by Asn, which is located within the putative consensus sequence of the caspase-3 cleavage site (DELD522down-arrow A), was not cleaved by the apoptotic cell lysate; and (v) another cPLA2 mutant truncated at Asp522, cPLA2(1-522), had the same apparent molecular mass, 78 kDa, as the catalytically inactive fragment. Thus, cPLA2 can be categorized as a physiological target of the death effector proteases. Further support for this mechanism of cPLA2 inactivation is provided by the finding that this cleavage process destroyed a catalytic triad (Arg200, Ser228, and Arg549) that is essential for cPLA2 function (50).

Several workers have suggested that cPLA2 is involved in apoptosis or cytotoxicity. Sapirstein et al. (37) reported that cPLA2-overexpressing LLC-PK1 renal epithelial cells were more susceptible to oxidant damage than their parental cells. Voelkel-Johnson et al. (34, 51) showed, in studies employing cPLA2 overexpression and cPLA2 antisense introduction, that cPLA2 was both necessary for and rate-limiting in TNFalpha -induced apoptosis of various melanoma cells. Hayakawa et al. (35) showed that the TNFalpha -resistant subline of L929 cells, which showed reduced cPLA2 expression compared with the parental line, regained a TNFalpha -sensitive phenotype after overexpression of transfected cPLA2. Subsequently, cPLA2 was shown to participate in TNFalpha -induced apoptosis of this cell line through the ceramide-dependent pathway (52). However, our present results suggest that AA release by cPLA2 is not necessary for the apoptotic pathway and that the involvement of cPLA2 in apoptosis observed in previous studies (34-37, 51, 52) may reflect secondary or cell type- or stimulus-specific events. While this study was underway, Wissing et al. (36) reported that cPLA2 was cleaved by caspase-3, presumably at the same site we identified, during TNFalpha -induced apoptosis of MCF-7S1 breast carcinoma cells and stated that this proteolytic process led to cPLA2 activation based on the observations that AA release from TNFalpha -treated cells was inhibited by AACOCF3. In their study, however, they did not measure cPLA2 activity directly and, even though cPLA2 cleavage occurred to some extent, the major portion of cPLA2 remained uncleaved. In view of our findings that both caspase-3-cleaved cPLA2 and recombinant cPLA2(1-522) were catalytically inactive, it is more likely that the TNFalpha -induced AA release Wissing et al. observed was the result of the activation of intact, not cleaved, cPLA2, which may undergo some other post-translational modifications, such as phosphorylation by MAPK family protein kinases downstream of the TNF-specific signal transduction pathway.

Nonetheless, our results reinforce the hypothesis that TNFalpha - and Fas-mediated signaling pathways are segregated; cPLA2 is involved in AA release by the former but not the latter. This proposal is consistent with previous reports that the cPLA2 expression level correlated with TNFalpha -dependent but not Fas-induced apoptosis of L929 cells (35, 38). In this respect, the JNK and p38 subfamilies of MAPKs, which are capable of phosphorylating cPLA2 (53, 54), are known to be strongly activated by type 1 TNF receptors but only minimally activated by Fas (27). Alternatively, a unique death domain-containing adapter molecule, MADD, which links type 1 TNF receptors but not Fas to the ERK subfamily MAPKs and cPLA2 activation (55), may facilitate the linkage of cPLA2 to the TNF-specific apoptotic pathway through an as yet unidentified mechanism. These TNF-specific signalings may be coupled with cPLA2 activation, which modulates apoptosis under certain conditions.

Dissociation of cPLA2 from Fas-mediated AA release prompted us to examine the effects of various inhibitors for other PLA2 isoforms. Of the PLA2 inhibitors examined so far, only iPLA2 inhibitors inhibited Fas-mediated AA release, suggesting that iPLA2 (or related enzymes), an 85-kDa cytosolic protein that exists as a multimeric complex of ~300 kDa, that exhibits no fatty acid selectivity, and that contains eight ankyrin motifs (56), is involved at least in part in apoptosis-associated fatty acid release from membrane phospholipids. Ongoing studies by Dennis and his colleagues (14, 15) have so far provided support for the notion that iPLA2 is involved in phospholipid remodeling, further support for which was provided by our recent overexpression studies (10). Because iPLA2 activity in vitro remained unchanged during Fas-mediated apoptosis, we surmise that apoptotic signals may render membrane phospholipids more susceptible to iPLA2 or that some regulatory proteins in the iPLA2 complex may be modified in vivo, leading to increased fatty acid release. Interestingly, treating the cells with an iPLA2 inhibitor retarded cell death significantly. Chilton and his colleagues (57) demonstrated that uneven AA redistribution between glycerophospholipid subclasses, which is caused by inhibition of CoA-independent transacylase, correlated with apoptosis. Thus, we speculate that perturbed phospholipid remodeling may affect the structures, dynamics, integrity, or asymmetry of bilayer membranes, thereby influencing apoptotic changes of the cell.

    ACKNOWLEDGEMENTS

We thank Dr. J. D. Clark and M. Tsujimoto for providing antiserum and cDNA for cPLA2, respectively. We thank Dr. R. M. Kramer for providing the sPLA2 inhibitor LY311727.

    FOOTNOTES

* This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 81-3-3784-8196; Fax: 81-3-3784-8245; E-mail: kudo{at}pharm.showa-u.ac.jp.

1 The abbreviations used are: PLA2, phospholipase A2; cPLA2, cytosolic phospholipase A2; sPLA2, secretory PLA2; iPLA2, Ca2+-independent PLA2; TNF, tumor necrosis factor; AA, arachidonic acid; AACOCF3, arachidonoyl trifluoromethyl ketone; MAFP, methyl arachidonylfluorophosphonate; PAGE, polyacrylamide gel electrophoresis; MAPK, mitogen-activated protein kinase; DAPI, 4',6'-diamidino-2-phenylindole; PIPES, 1,4-piperazinediethanesulfonic acid.

2 M. Murakami, T. Kambe, S. Shimbara, and I. Kudo, unpublished observation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Leslie, C. C. (1997) J. Biol. Chem. 272, 16709-16712[Free Full Text]
  2. Clark, J. D., Lin, L.-L., Kriz, R. W., Ramesha, C. S., Sultzman, L. A., Lin, A.-Y., Milona, N., and Knopf, J. L. (1991) Cell 65, 1043-1051[Medline] [Order article via Infotrieve]
  3. Schievella, A. R., Regier, M. K., Smith, W. L., and Lin, L.-L. (1995) J. Biol. Chem. 270, 30749-30754[Abstract/Free Full Text]
  4. Lin, L-L., Wartmann, M., Lin, A.-Y., Knopf, J. L., Seth, A., and Davis, R. J. (1993) Cell 72, 269-278[Medline] [Order article via Infotrieve]
  5. Glover, S., de Carvalho, M. S., Bayburt, T., Jonas, M., Chi, E., Leslie, C. C., and Gelb, M. H. (1995) J. Biol. Chem. 270, 15359-15367[Abstract/Free Full Text]
  6. Roshak, A., Sathe, G., and Marshall, L. A. (1994) J. Biol. Chem. 269, 25999-26005[Abstract/Free Full Text]
  7. Murakami, M., Kuwata, H., Amakasu, Y., Shimbara, S., Nakatani, Y., Atsumi, G., and Kudo, I. (1997) J. Biol. Chem. 272, 19891-19897[Abstract/Free Full Text]
  8. Murakami, M., Nakatani, Y., and Kudo, I. (1996) J. Biol. Chem. 271, 30041-30051[Abstract/Free Full Text]
  9. Kuwata, H., Nakatani, Y., Murakami, M., and Kudo, I. (1998) J. Biol. Chem. 273, 1733-1740[Abstract/Free Full Text]
  10. Murakami, M., Shimbara, S., Kambe, T., Kuwata, H., Winstead, M. V., Tischfield, J. A., and Kudo, I. (1998) J. Biol. Chem. 273, in press
  11. Balboa, M. A., Balsinde, J., Winstead, M. V., Tischfield, J. A., and Dennis, E. A. (1996) J. Biol. Chem. 271, 32381-32384[Abstract/Free Full Text]
  12. Reddy, S. T., Winstead, M. V., Tischfield, J. A., and Herschman, H. R. (1997) J. Biol. Chem. 272, 13591-13596[Abstract/Free Full Text]
  13. Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. (1995) J. Biol. Chem. 270, 445-450[Abstract/Free Full Text]
  14. Balsinde, J., and Dennis, E. A. (1997) J. Biol. Chem. 272, 16069-16072[Free Full Text]
  15. Balsinde, J., Balboa, M. A., and Dennis, E. A. (1997) J. Biol. Chem. 272, 29317-29321[Abstract/Free Full Text]
  16. Cleveland, J. L., and Ihle, J. N. (1995) Cell 81, 479-482[Medline] [Order article via Infotrieve]
  17. Liu, Z.-G., Hsu, H., Goeddel, D. V., and Karin, M. (1996) Cell 87, 565-576[Medline] [Order article via Infotrieve]
  18. Hsu, H., Shu, H.-B., Pan, M.-G., and Goeddel, D. V. (1996) Cell 84, 299-308[Medline] [Order article via Infotrieve]
  19. Hsu, H., Xiong, J., and Goeddel, D. V. (1995) Cell 81, 495-504[Medline] [Order article via Infotrieve]
  20. Boldin, M. P., Goncharav, T. M., Goltsev, Y. V., and Wallach, D. (1996) Cell 85, 803-815[Medline] [Order article via Infotrieve]
  21. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996) Cell 85, 817-827[Medline] [Order article via Infotrieve]
  22. Duan, H., and Dixit, V. M. (1997) Nature 385, 86-89[CrossRef][Medline] [Order article via Infotrieve]
  23. Enari, M., Talanian, R. V., Wong, W. W., and Nagata, S. (1996) Nature 380, 723-726[CrossRef][Medline] [Order article via Infotrieve]
  24. Fraser, A., and Evan, G. (1996) Cell 85, 781-784[Medline] [Order article via Infotrieve]
  25. Chinnaiyan, A. M., O'Rourke, K., Tewari, M., and Dixit, V. M. (1995) Cell 81, 505-512[Medline] [Order article via Infotrieve]
  26. Martins, L. M., and Earnshaw, W. C. (1997) Trends Cell Biol. 7, 111-114[CrossRef]
  27. Nagata, S. (1997) Cell 88, 355-365[Medline] [Order article via Infotrieve]
  28. Martin, S. J., and Green, D. R. (1995) Cell 82, 349-352[Medline] [Order article via Infotrieve]
  29. Liu, X., Zou, H., Slaughter, C., and Wang, X. (1997) Cell 89, 175-184[Medline] [Order article via Infotrieve]
  30. Rudel, T., and Bokoch, G. M. (1997) Science 276, 1571-1574[Abstract/Free Full Text]
  31. Atsumi, G., Murakami, M., Tajima, M., Shimbara, S., Hara, N., and Kudo, I. (1997) Biochim. Biophys. Acta 1349, 43-54[Medline] [Order article via Infotrieve]
  32. Wang, H., Harrison-Shostak, D. C., Lemasters, J. J., and Herman, B. (1996) FASEB J. 10, 1318-1325
  33. Kudo, I., Murakami, M., Hara, S., and Inoue, K. (1993) Biochim. Biophys. Acta 1170, 217-231[Medline] [Order article via Infotrieve]
  34. Voelkel-Johnson, C., Thorne, T., and Laster, S. M. (1996) J. Immunol. 156, 201-207[Abstract]
  35. Hayakawa, M., Ishida, N., Takeuchi, K., Shibamoto, S., Hori, T., Oku, N., Ito, F., and Tsujimoto, M. (1993) J. Biol. Chem. 268, 11290-11295[Abstract/Free Full Text]
  36. Wissing, D., Mouritzen, H., Egeblad, M., Poirier, G. G., and Jaattela, M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 5073-5077[Abstract/Free Full Text]
  37. Sapirstein, A., Spech, R. A., Witzgall, R., and Bonventre, J. V. (1996) J. Biol. Chem. 271, 21505-21513[Abstract/Free Full Text]
  38. Enari, M., Hug, H., Hayakawa, M., Ito, F., Nishimura, Y., and Nagata, S. (1996) Eur. J. Biochem. 236, 533-538[Abstract]
  39. Yonehara, S., Ishii, A., and Yonehara, M. (1989) J. Exp. Med. 169, 1747-1756[Abstract]
  40. Street, I. P., Lin, H.-K., Laliberte, F., Ghomashchi, F., Wang, Z., Perrier, H., Tremblay, N. M., Huang, Z., Weech, P. K., and Gelb, M. H. (1993) Biochemistry 32, 5935-5940[Medline] [Order article via Infotrieve]
  41. Schevitz, R. W., Bach, N. J., Carlson, D. G., Chirgadze, N. Y., Clawson, D. K., Dillard, R. D., Draheim, S. E., Hartley, L. W., Jones, N. D., Mihelich, E. D., Olkowski, J. L., Snyder, D. W., Sommers, C., and Wery, J.-P. (1995) Nat. Struct. Biol. 2, 458-465[Medline] [Order article via Infotrieve]
  42. Kizaki, H., Tadakuma, T., Odaka, C., Muramatsu, J., and Ishimura, Y. (1989) J. Immunol. 143, 1790-1794[Abstract/Free Full Text]
  43. Dole, V. P., and Meinertz, H. (1960) J. Biol. Chem. 235, 2595-2599[Medline] [Order article via Infotrieve]
  44. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-918
  45. Enari, M., Talanian, R. V., Wong, W. W., and Nagata, S. (1996) Nature 380, 723-726[CrossRef][Medline] [Order article via Infotrieve]
  46. Naraba, H., Murakami, M., Matsumoto, H., Shimbara, S., Ueno, A., Kudo, I., and Oh-ishi, S. (1998) J. Immunol. 160, 2974-2982[Abstract/Free Full Text]
  47. Mochly-Rosen, D. (1995) Science 268, 247-251[Medline] [Order article via Infotrieve]
  48. Cohen, G. M. (1997) Biochem. J. 326, 1-16[Medline] [Order article via Infotrieve]
  49. Martins, L. M., Kottke, T., Mesner, P. W., Basi, G. S., Sinha, S., Frigon, N., Jr., Tatar, E., Tung, J. S., Bryant, K., Takahashi, A., Svingen, P. A., Madden, B. J., McCormick, D. J., Earnshaw, W. C., and Kaufmann, S. H. (1997) J. Biol. Chem. 272, 7421-7430[Abstract/Free Full Text]
  50. Pickard, R. T., Chiou, X. G., Strifler, B. A., DeFelippis, M. R., Hyslop, P. A., Tebbe, A. L., Yee, Y. K., Reynolds, L. J., Dennis, E. A., Kramer, R. M., and Sharp, J. D. (1996) J. Biol. Chem. 271, 19225-19231[Abstract/Free Full Text]
  51. Voelkel-Johnson, C., Entingh, A. J., Wold, W. S. M., Gooding, L. R., and Laster, S. M. (1995) J. Immunol. 154, 1707-1716[Abstract/Free Full Text]
  52. Jayadev, S., Hayter, H. L., Andrieu, N., Gamard, C. J., Liu, B., Balu, R., Hayakawa, M., Ito, F., and Hannun, Y. A. (1997) J. Biol. Chem. 272, 17196-17203[Abstract/Free Full Text]
  53. Kramer, R. M., Roberts, E. F., Um, S. L., Borsch-Haubold, A. G., Watson, S. P., Fisher, M. J., and Jakubowski, J. A. (1996) J. Biol. Chem. 271, 27723-27729[Abstract/Free Full Text]
  54. Hernandez, M., Bayon, Y., Crespo, M. S., and Nieto, M. L. (1997) Biochem. J. 328, 263-269[Medline] [Order article via Infotrieve]
  55. Schievella, A. R., Chen, J. H., Graham, J. R., and Lin, L.-L. (1997) J. Biol. Chem. 272, 12069-12075[Abstract/Free Full Text]
  56. Tang, J., Kriz, R. W., Wolfman, N., Shaffer, M., Seehra, J., and Jones, S. S. (1997) J. Biol. Chem. 272, 8567-8575[Abstract/Free Full Text]
  57. Surette, M. E., Winkler, J. D., Fonteh, A. N., and Chilton, F. H. (1996) Biochemistry 35, 9187-9196[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.