Molecular cloning of two new human paralogs of 85-kDa cytosolic phospholipase A2

Two new cloned human cDNAs encode paralogs of the 85-kDa cytosolic phospholipase A2 (cPLA2). We propose to call these cPLA2beta (114 kDa) and cPLA2gamma (61 kDa), giving the name cPLA2alpha to the well known 85-kDa enzyme. cPLA2beta mRNA is expressed more highly in cerebellum and pancreas and cPLA2gamma more highly in cardiac and skeletal muscle. Sequence-tagged site mapping places cPLA2beta on chromosome 15 in a region near a phosphoinositol bisphosphate phosphatase. The mRNA for cPLA2beta is spliced only at a very low level, and Northern blots in 24 tissues show exclusively the unspliced form. cPLA2beta has much lower activity on 2-arachidonoyl-phosphatidylcholine liposomes than either of the other two enzymes. Its sequence contains a histidine motif characteristic of the catalytic center of caspase proteases of the apoptotic cascade but no region characteristic of the catalytic cysteine. Sequence-tagged site mapping places cPLA2gamma on chromosome 19 near calmodulin. cPLA2gamma lacks the C2 domain, which gives cPLA2alpha its Ca2+ sensitivity, and accordingly cPLA2gamma has no dependence upon calcium, although cPLA2beta does. cPLA2gamma contains a prenyl group-binding site motif and appears to be largely membrane-bound. cPLA2alpha residues activated by phosphorylation do not appear to be well conserved in either new enzyme. In contrast, all three previously known catalytic residues, as well as one additional essential arginine, Arg-566 in cPLA2alpha, are conserved in both new enzyme sequences. Mutagenesis shows strong dependence on these residues for catalytic activity of all three enzymes.     

Activation of cytosolic phospholipase A2 in permeabilized human neutrophils

Neutrophils (PMN) contain two types of phospholipase A₂ (PLA₂), a 14 kDa ‘secretory’ Type II PLA₂ (sPLA₂) and an 85 kDa ‘cytosolic’ PLA₂ (cPLA₂), that differ in a number of key characteristics: (1) cPLA₂ prefers arachidonate (AA) as a substrate but hydrolyzes all phospholipids; sPLA₂ is not AA specific but prefers ethanolamine containing phosphoacylglycerols. (2) cPLA₂ is active at nM calcium (Ca²⁺) concentrations; sPLA₂ requires μM Ca²⁺ levels. (3) cPLA₂ activity is regulated by phosphorylation; sPLA₂ lacks phosphorylation sites. (4) cPLA₂ is insensitive to reduction; sPLA₂ is inactivated by agents that reduce disulfide bonds. We utilized PMN permeabilized with Staphylococcus aureus α-toxin to determine whether one or both forms of PLA₂ were activated in porated cells under conditions designed to differentiate between the two enzymes. PMN were labeled with [³H]AA to measure release from phosphatidylcholine and phosphatidylinositol; gas chromatography-mass spectrometry was utilized to determine total AA release (mainly from phosphatidylethanolamine) and to asses oleate and linoleate mass. A combination of 500 nM Ca²⁺, a guanine nucleotide, and stimulation with n-formyl-met-leu-phe (FMLP) were necessary to induce maximal AA release in permeabilized PMN measured by either method; AA was preferentially released. [³H]AA and AA mass release occurred in parallel over time. A hydrolyzable form of ATP was necessary for maximum AA release and staurosporin inhibited PLA₂ activation. Dithiothreitol treatment had little affect on [³H]AA release and metabolism but inhibited AA mass release. Assay of cell supernatants after cofactor addition did not detect sPLA₂ activity and the cytosolic buffer utilized did not support activity of recombinant sPLA₂. These results strongly suggested that cPLA₂ was the enzyme activated in the permeabilized cell model and this is the first report which unambiguously demonstrates AA release in response to activation of a specific type of PLA₂ in PMN.     

A 38 kDa nuclear protein is involved in the retention of an antisense oligonucleotide directed against cytosolic phospholipase A2.

Recent studies suggest that antisense phosphorothioate oligonucleotides (APO) are useful tools not only to impair gene expression, but also to modify the splicing of pre-mRNA, as the classical view that they act by suppressing the translation of mature mRNA has been challenged by several examples showing their nuclear site of action. In this work we show that an APO directed against cytosolic phospholipase A2 (cPLA2) mRNA localises in the nucleus and interacts with a specific nuclear protein.     

Enzymatic properties of human cytosolic phospholipase A(2)gamma

The enzymatic properties of cytosolic phospholipase A(2)gamma (cPLA(2)gamma), an isoform of 85-kDa group IV cPLA(2)alpha (cPLA(2)alpha) were studied in vitro and when the enzyme was expressed in cells. cPLA(2)gamma expressed in Sf9 cells is associated with membrane. Membranes isolated from [(3)H]arachidonic acid-labeled Sf9 cells expressing cPLA(2)gamma, constitutively release [(3)H]arachidonic acid. The membrane-associated activity is inhibited by the group IV PLA(2) inhibitor methylarachidonyl fluorophosphonate, but not effectively by the group VI PLA(2) inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. cPLA(2)gamma has higher lysophospholipase activity than PLA(2) activity. Purified His-cPLA(2)gamma does not exhibit phospholipase A(1) activity, but sequentially hydrolyzes fatty acid from the sn-2 and sn-1 positions of phosphatidylcholine. cPLA(2)gamma overexpressed in HEK293 cells is constitutively active in isolated membranes, releasing large amounts of oleic, arachidonic, palmitic, and stearic acids; however, basal fatty acid release from intact cells is not increased. cPLA(2)gamma overexpressed in lung fibroblasts from cPLA(2)alpha-deficient mice is activated by mouse serum resulting in release of arachidonic, oleic, and palmitic acids, whereas overexpression of cPLA(2)alpha results primarily in arachidonic acid release.     

Structure and mechanism of human cytosolic phospholipase A(2)

cPLA(2) is an 85-kDa enzyme whose primary function, the release of arachidonic acid from phospholipid membranes, is a crucial reaction in the metabolism of lipid mediators of inflammation. cPLA(2) consists of two domains: an N-terminal, C2-type unit analogous to those present in other membrane-targeting molecules, and a catalytic domain harboring an active site dyad at the bottom of a deep, mostly hydrophobic catalytic funnel. The absence of a third active site residue in the cPLA(2) cleft, as observed in other lipases, suggests that the enzyme proceeds through a novel catalytic mechanism. Crystallographic and biochemical studies of cPLA(2) will provide essential information for the development of small molecule inhibitors which may be employed in the control of inflammatory and other highly regulated processes.     

Involvement of cytosolic phospholipase A2 and secretory phospholipase A2 in arachidonic acid release from human neutrophils

The purpose of this study was to define the role of secretory phospholipase A2 (sPLA2), calcium-independent PLA2, and cytosolic PLA2 (cPLA2) in arachidonic acid (AA) release from fMLP-stimulated human neutrophils. While fMLP induced the release of extracellular sPLA2 activity and AA, 70% of sPLA2 activity remained associated with the cell. Treatment with the cell-impermeable sPLA2 inhibitors DTT or LY311-727, or the anti-sPLA2 Ab 3F10 all inactivated extracellular sPLA2 activity, but had minimal effect on neutrophil AA mass release. In contrast, coincubation of streptolysin-O toxin-permeabilized neutrophils with DTT, LY311-727, or 3F10 all decreased [3H8]AA release from [3H8]AA-labeled, fMLP-stimulated cells. Exposure to fMLP resulted in a decrease in the electrophoretic mobility of cPLA2, a finding consistent with cPLA2 phosphorylation, and stimulated the translocation of cPLA2 from cytosolic to microsomal and nuclear compartments. The role of cPLA2 was further evaluated with the cPLA2 inhibitor methyl arachidonyl fluorophosphonate, which attenuated cPLA2 activity in vitro and decreased fMLP-stimulated AA mass release by intact neutrophils, but had no effect on neutrophil sPLA2 activity. Inhibition of calcium-independent PLA2 with haloenol lactone suicide substrate had no effect on neutrophil cPLA2 activity or AA mass release. These results indicate a role for cPLA2 and an intracellular or cell-associated sPLA2 in the release of AA from fMLP-stimulated human neutrophils.     

Characterization of the human interleukin-2 receptor beta-chain gene promoter: regulation of promoter activity by ets gene products.

The interleukin-2 receptor (IL-2R) beta chain (IL-2R beta) is an essential signaling component of high- and intermediate-affinity IL-2Rs. Our laboratory previously reported that a DNA fragment containing 857 bp of 5'-flanking sequence of the human IL-2R beta gene exhibited promoter activity. We have now further characterized the promoter and delineated cis-acting regulatory regions. The region downstream of -363 is critical for basal and phorbol myristate acetate-inducible IL-2R beta promoter activity and contains at least three enhancer-like regions. Among them, the -56 to -34 enhancer was the most potent and had high-level activity in two T-cell lines but not in nonlymphoid HeLaS3 and MG63 cells. This enhancer contains a GGAA Ets binding site which bound two Ets family proteins, Ets-1 and GA-binding protein in vitro. Mutation of the Ets motif strongly diminished both promoter and enhancer activities. We conclude that this Ets binding site plays a key role in regulating basal and phorbol myristate acetate-inducible IL-2R beta promoter activity and may also contribute to tissue-specific expression of the IL-2R beta gene.     

Glucocorticoids inhibit TNF alpha-induced cytosolic phospholipase A2 activity.

The cytosolic phospholipase A2 (PLA2) was characterized in the human epithelial carcinoma cell line HEp-2 by its apparent molecular mass (about 80 kDa); its in vitro activation by micromolar concentrations of calcium; and its calcium-dependent association with cellular membranes. The activity of this enzyme was induced by an overnight incubation with tumor necrosis factor alpha (TNF alpha). Glucocorticoids only moderately reduced PLA2 activity in control cells, but completely inhibited the TNF alpha-induced increase in the activity of the high-molecular-weight cytosolic PLA2.     

Interferon-alpha-induced phosphorylation and activation of cytosolic phospholipase A2 is required for the formation of interferon-stimulated gene factor three.

Treatment of cells with interferon (IFN)-alpha caused phosphorylation and activation of cytosolic phospholipase A2 (cPLA2). The protein tyrosine kinase Jak1 was found to be necessary for the activation of cPLA2. Jak1 could be co-immunoprecipitated with cPLA2 from cell extracts, indicating that a close physical interaction occurs between these two proteins. The induction of IFN-stimulated gene factor three (ISGF3) by IFN-alpha, is blocked by cPLA2 inhibitors in cell cultures and in cell-free reconstituted systems. However, these inhibitors do not block IFN-alpha or gamma-induced binding of STAT1 to the inverted repeat (IR) element of the IFN regulatory factor 1 (IRF-1) gene. Thus, cPLA2 activations occurs as an early event in the IFN-alpha response and is selectively involved in ISGF3-dependent gene activation.