Differentiation of U937 cells enables a phospholipase D-dependent pathway of cytosolic phospholipase A2 activation.

Treatment with dibutyryl cyclic AMP (dBcAMP) of the human, premonocytic U937 cell line results in differentiation toward a monocyte/granulocyte-like cell. This differentiation enables the cell to activate cytosolic phospholipase A2 (cPLA2) to release arachidonate upon stimulation. In contrast, undifferentiated cells are unable to release arachidonate even when stimulated with calcium ionophores. In the present research, a role for phospholipase D (PLD) in the regulation of cPLA2 was shown based on a number of observations. First, the ionomycin- and fMLP-stimulated production of arachidonate in differentiated cells was sensitive to ethanol (2% (v/v)). Ethanol acts as an alternate substrate in place of water for PLD producing phosphatidylethanol (PEt) instead of phosphatidic acid. Indeed, ionomycin stimulation of differentiated cells produced a 14-fold increase in PEt levels. Further evidence for the involvement of PLD in the regulation of cPLA2 came from the observation that the stimulated production of diacylglycerol (for which phosphatidic acid is a major source) was greatly diminished in undifferentiated cells as compared to differentiated cells. Moreover, the normally deficient activation of cPLA2 in undifferentiated cells could be stimulated to release arachidonate if the cells were electroporated in the presence of GTP[gamma]S and MgATP. This treatment stimulates phosphatidylinositol-4,5-bisphosphate (PIP2) production which appears to activate PLD and cPLA2 in subsequent steps. The phosphatidic acid (and diacylglycerol derived from phosphatidic acid) appears to greatly regulate the action of cPLA2 by an unknown mechanism, and undifferentiated cells lack the ability to stimulate PLD activity due to a dysfunction of PIP2 production.     

Molecular cloning and expression of human Ca(2+)-sensitive cytosolic phospholipase A2

Phospholipases A2 (PLA2s) play a key role in inflammatory processes through production of precursors of eicosanoids and platelet-activating factor. Recently, we described the purification of a novel approximately 100-kDa cytosolic PLA2 (cPLA2) from human monoblast U937 cells that is activated by physiological (intracellular) concentrations of Ca2+ (Kramer, R. M., Roberts, E. F., Manetta, J., and Putnam, J. E. (1991) J. Biol. Chem. 266, 5268-5272). Here we report the isolation of the complementary DNA encoding human cPLA2 and confirm its identity by expression in bacteria and in hamster cells. The predicted 749-amino acid cPLA2 protein has no similarity to the well known secretory PLA2s, but contains a structural element homologous to the C2 region of protein kinase C. The molecular cloning of cPLA2 will allow further studies defining the structure, function, and regulation of this novel PLA2.     

Cholesterol modulates PAF-stimulated Ca2(+)-mobilization in monocytic U937 cells

We investigated the effect of cellular cholesterol content on platelet activating factor (PAF)-stimulated Ca2+ mobilization in the human monocytic cell line U937. When cholesterol auxotroph U937 cells were depleted of cellular cholesterol by a 48-h incubation in delipidated medium, a 40% reduction in the PAF (100 nM)-stimulated increase in cytosolic Ca2+ concentration was seen. Ca2+ mobilization following stimulation with LTD4 (10 nM) or ATP (10 microM) was not affected. Addition of LDL (100 micrograms/ml, 24 h) to the delipidated medium completely recovered cellular cholesterol content and PAF-induced Ca2+ mobilization. These two LDL effects had very similar time- and dose-dependences. Partial recoveries of PAF-induced Ca2+ mobilization, seen after addition of pure cholesterol dissolved in ethanol (30 micrograms/ml, 24 h) or acetyl-LDL (100 micrograms/ml, 24 h), were associated with partial recoveries of cellular cholesterol content. Our results indicate that cellular cholesterol influences PAF-stimulated events in monocytic cells.     

An 100-kDa Ca2+-sensitive actin-fragmenting protein from unfertilized sea urchin egg

An actin-modulating protein was purified from unfertilized eggs of sea urchin, Hemicentrotus pulcherrimus, by means of DNase I affinity and DEAE-cellulose column chromatographies. This protein was a globular protein with a Stokes radius of 41-42 nm and consisted of a single polypeptide chain having an apparent molecular mass of 100 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Gel filtration chromatography revealed that one 100-kDa protein molecule binds two or three actin monomers in the presence of Ca2+, but such binding was not observed in the absence of Ca2+. The effect of the 100-kDa protein on the polymerization of actin was studied by viscometry, spectrophotometry and electron microscopy. The initial rate of actin polymerization was decreased at a very low molar ratio of 100-kDa protein/actin. Acceleration of the initial rate of polymerization occurred at a relatively high, but still substoichiometric, molar ratio of 100-kDa protein/actin. The 100-kDa protein produced fragmentation of muscle actin filaments at Ca2+ concentrations greater than 0.3 microM as revealed by viscometry and electron microscopy. Evidence was also presented that the 100-kDa protein binds to the barbed end of the actin filament.     

Calcium-independent phospholipase A2 mediates proliferation of human promonocytic U937 cells

We have investigated the possible involvement of two intracellular phospholipases A(2), namely group VIA calcium-independent phospholipase A(2) (iPLA(2)-VIA) and group IVA cytosolic phospholipase A(2) (cPLA(2)alpha), in the regulation of human promonocytic U937 cell proliferation. Inhibition of iPLA(2)-VIA activity by either pharmacological inhibitors such as bromoenol lactone or methyl arachidonyl fluorophosphonate or using specific antisense technology strongly blunted U937 cell proliferation. In contrast, inhibition of cPLA(2)alpha had no significant effect on U937 proliferation. Evaluation of iPLA(2)-VIA activity in cell cycle-synchronized cells revealed highest activity at G(2)/M and late S phases, and lowest at G(1). Phosphatidylcholine levels showed the opposite trend, peaking at G(1) and lowest at G(2)/M and late S phase. Reduction of U937 cell proliferation by inhibition of iPLA(2)-VIA activity was associated with arrest in G(2)/M and S phases. The iPLA(2)-VIA effects were found to be independent of the generation of free arachidonic acid or one of its oxygenated metabolites, and may work through regulation of the cellular level of phosphatidylcholine, a structural lipid that is required for cell growth/membrane expansion.     

HSP100, a 100-kDa heat shock protein, is a Ca2+-calmodulin-regulated actin-binding protein

The 100-kDa heat shock protein, HSP100, was purified from mouse lymphoma cells. Amino acid sequences of three peptide fragments which were obtained from the purified protein by lysylendopeptidase digestion were completely or nearly identical with those of a mouse endoplasmic reticulum protein, ERp99, of a hamster glucose-regulated protein, GRP94, and of a chicken heat shock protein, HSP108, all of which have been known to have strong homology with the 90-kDa heat shock protein, HSP90. HSP100 bound to actin filaments and an apparent Kd for the binding was determined to be 8 x 10(-7) M in 2 mM MgCl2 + 100 mM KCl. Calmodulin inhibited the binding in a Ca2+-dependent manner. Equilibrium gel filtration demonstrated that HSP100 has an ability to bind to calmodulin only in the presence of Ca2+. Moreover, HSP100 competed with HSP90 for binding to actin filaments. These results together with our previous findings that HSP90 and HSP100 have similar physicochemical properties (Koyasu, S., Nishida, E., Kadowaki, T., Matsuzaki, F., Iida, K., Harada, F., Kasuga, M., Sakai, H., and Yahara, I. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8054-8058) and HSP90 is a calmodulin-regulated actin-binding protein (Nishida, E., Koyasu, S., Sakai, H., and Yahara, I. (1986) J. Biol. Chem. 261, 16033-16036), strongly suggest that HSP100 is structurally and functionally related to HSP90.     

Calcium-independent phospholipase A2 is required for lysozyme secretion in U937 promonocytes

As a part of their surveillance functions in the immune system, monocytes/macrophages secrete large amounts of the bactericidal enzyme lysozyme to the extracellular medium. We report here that lysozyme secretion in activated U937 promonocytes depends on a functional calcium-independent phospholipase A(2) (iPLA(2)). Inhibition of the enzyme by bromoenol lactone or by treatment with a specific antisense oligonucleotide results in a diminished capacity of the cells to secrete lysozyme to the extracellular medium. Calcium-independent PLA(2) is largely responsible for the maintenance of the steady state of lysophosphatidylcholine (lysoPC) levels within the cells, as manifested by the marked decrease in the levels of this metabolite in cells deficient in iPLA(2) activity. Reconstitution experiments reveal that lysoPC efficiently restores lysozyme secretion in iPLA(2)-deficient cells, whereas other lysophospholipids, including lysophosphatidic acid, lysophosphatidylserine, and lysophosphatidylethanolamine, are without effect. Arachidonic acid mobilization in activated U937 cells is under control of cytosolic phospholipase A(2) (cPLA(2)). Selective inhibition of cPLA(2) results in a complete abrogation of the arachidonate mobilization response, but has no effect on lysozyme secretion. These results identify iPLA(2)-mediated lysoPC production as a necessary component of the molecular machinery leading to lysozyme secretion in U937 cells and rule out a role for cPLA(2) in the response. Collectively, the results demonstrate distinct roles in inflammatory cell signaling for these two intracellular phospholipases.     

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.     

A novel 21-kDa cytochrome c-releasing factor is generated upon treatment of human leukemia U937 cells with geranylgeraniol

Geranylgeraniol (GGO) induces apoptosis in various lines of human tumor cells through a mitochondrion-dependent pathway. The present study describes identification of a 21-kDa cytochrome c-releasing factor that appears in the cytosolic fraction after treatment of human leukemia U937 cells with GGO. Incubation of isolated mitochondria with a lysate of U937 cells that had been treated with GGO resulted in the release of cytochrome c from the mitochondria. Utilizing this cell-free system, we purified a 21-kDa protein that induced the release of cytochrome c from mitochondria and appeared to be involved in the apoptosis that is induced in U937 cells by GGO. We designated this protein cytochrome c-releasing factor 21 (CRF21). Overexpression of CRF21 in HeLa cells induced the release of cytochrome c from mitochondria, with subsequent apoptosis. Our results suggest that CRF21 might play an important role in the induction of apoptosis by GGO in leukemia U937 cells.     

Purification of a phospholipase A2 from human monocytic leukemic U937 cells. Calcium-dependent activation and membrane association

The existence of an intracellular phospholipase A2 (PLA2) involved in the production of 1-O-alkyl-sn-glycero-3-phosphocholine and free arachidonic acid has been repeatedly postulated. Using 1-O-hexadecyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine as a substrate and a series of conventional and high-pressure liquid chromatographic techniques, we have purified a PLA2 from the soluble fraction of differentiated human monocytic U937 cells. The enzyme has been purified nearly 2000-fold to homogeneity. The purified enzyme has a molecular mass of 56 kDa, under reducing conditions, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The enzyme activity has a pH optimum of 8.0 and is calcium concentration-dependent. The EC50 for the activation of the enzyme activity by calcium is 300 nM. When the cells were homogenized in the presence of the calcium chelator EGTA (0.2 mM), the enzyme was found to be soluble (more than 90% of the activity in the 100,000 x g supernatant). However, when Ca2+ concentration was controlled from 10 nM to 100 microM in Ca2(+)-EGTA buffers, increasing amounts of the activity were found in the particulate fraction (100,000 x g pellet). This suggests that membrane translocation and activation of the soluble PLA2 may be regulated by physiological intracellular levels of Ca2+. The purified enzyme hydrolyzed different phosphatidylcholine substrates presented in either vesicular or Triton X-100 mix micellar forms. In both situations, the enzyme showed a high degree of specificity for arachidonic acid on the sn-2 position of the substrate. Substitution of palmitic or oleic on the sn-2 position substantially reduced the hydrolytic activity of the enzyme. When vesicles of arachidonic acid-containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol were presented to the purified enzyme, all of them were hydrolyzed with comparable efficiency. However, only phosphatidylcholine and phosphatidylinositol were hydrolyzed when presented in Triton X-100 mixed micelles.     

Elevated cytosolic Ca2+ activates phospholipase D in human platelets

We have examined the activation of phospholipase D in human platelets treated with alpha-thrombin. When incubated with 1-O-[9,10-3H2]hexadecyl-2-lysophosphatidylcholine (PtdCho) and 1-alkyl-[32P]lysoPtdCho for 2 h, platelets formed 3H/32P-labeled PtdCho in a ratio of 11:1. After incubation of such labeled platelets with alpha-thrombin for 5 min, increased accumulation of 3H/32P-labeled phosphatidic acid (PtdOH) was detected in the same ratio, indicating the action of phospholipase D. The Ca2+ ionophore A23187 and alpha-thrombin each stimulated the formation of labeled PtdOH as above in a time- and concentration-dependent manner, with only minor changes in labeled diglyceride. A23187 was able to cause increases in labeled PtdOH comparable to those observed with alpha-thrombin. beta-Phorbol 12,13-dibutyrate, an activator of protein kinase C, only slightly stimulated the accumulation of labeled PtOH. The protein kinase C inhibitor, staurosporine, totally blocked these changes but only slightly inhibited the increases in labeled PtdOH promoted by alpha-thrombin. These results suggest that an increase in intracellular Ca2+, rather than protein kinase C activity, is a major factor regulating phospholipase D in platelets exposed to alpha-thrombin. We have also examined the relative contributions of phospholipase D and diglyceride kinase (following phospholipase C action) to PtdOH accumulation in [32P]Pi-labeled platelets by comparing the 32P-specific radioactivities of PtdOH, PtdCho, and metabolic gamma-ATP in control and alpha-thrombin-exposed platelets. Based on these determinations, we conclude that 13 and 87% of incremental PtdOH in human platelets exposed to alpha-thrombin arises via phospholipase D acting on PtdCho and phospholipase C/diglyceride kinase, respectively.     

Translocation of protein kinase C in human polymorphonuclear neutrophils. Regulation by cytosolic Ca2(+)-independent and Ca2(+)-dependent mechanisms

[3H]Phorbol dibutyrate [( 3H]PDB) rapidly and reversibly binds to human polymorphonuclear neutrophils (PMN). Ca2+/diacylglycerol/phospholipid-dependent protein kinase C appeared to be the receptor for this binding because: a diacylglycerol, dioctanoylglycerol, competed with [3H]PDB for PMN binding sites; a blocker of protein kinase C-phospholipid interactions, sphinganine, inhibited PMN binding of [3H]PDB; and changes in cytosolic Ca2+ apparently regulated PMN binding of the label. Relevant to the last point, disrupted PMN contained 9 X 10(5) phorbol diester receptors/cell, whereas intact PMN had only 1.6 X 10(5) such receptors that were accessed by the ligand. This number fell to 1.0 X 10(5) in Ca2(+)-depleted PMN and rose to 2.5 X 10(5) in cells stimulated with the Ca2+ ionophore, ionomycin. This ionomycin effect lasted for greater than 16 min, correlated temporally with changes in cytosolic Ca2+, did not occur in Ca2(+)-depleted PMN, and was blocked by sphinganine. A second ionophore, A23187, likewise induced Ca2(+)-dependent rises in [3H]PDB binding. These results fit the standard model, wherein rises in cytosolic Ca2+ cause protein kinase C to translocate from cytosol to plasmalemma and thereby become more available to [3H]PDB. In contrast, two humoral agonists, N-formyl-Met-Leu-Phe (fMLP) and leukotriene (LT)B4, had actions that did not fit this model. They stimulated PMN to increase the availability of PDB binding sites by a sphinganine-sensitive mechanism, but their actions differed from those of ionophores. They induced biphasic (t = 15 and 60 s) increases in [3H]PDB binding while eliciting monophasic (t = 15 s), short-lived (t less than 1 min) rises in cytosolic Ca2+. In Ca2(+)-depleted PMN, moreover, fMLP and LTB4 stimulated slow (t greater than or equal to 30 s), monophasic, prominent rises in [3H]PDB binding and binding site number without appreciably altering cytosolic Ca2+. We suggest, therefore, that fMLP and LTB4 translocate protein kinase C using two sequential mechanisms. The first involves Ca2+ transients and thus produces abrupt (t = 15 s), rapidly reversing responses. The second mechanism uses an unrelated signal to effect a more slowly evolving (t = 60 s) movement of protein kinase C to plasmalemma. Hence, the standard model does not explain all instances of protein kinase C translocation, and a cytosolic Ca2(+)-independent signal contributes to the regulation of protein kinase C as well as those responses elicited by the effector enzyme.     

Involvement of calcium-independent phospholipase A2 in hydrogen peroxide-induced accumulation of free fatty acids in human U937 cells

Previous studies have demonstrated that U937 cells are able to mobilize arachidonic acid (AA) and synthesize prostaglandins in response to receptor-directed and soluble stimuli by a mechanism that involves the activation of Group IV cytosolic phospholipase A(2)alpha. In this paper we show that these cells also mobilize AA in response to an oxidative stress induced by H(2)O(2) through a mechanism that appears not to be mediated by cytosolic phospholipase A(2)alpha but by the calcium-independent Group VI phospholipase A(2) (iPLA(2)). This is supported by the following lines of evidence: (i) the response is essentially calcium-independent, (ii) it is inhibited by bromoenol lactone, and (iii) it is inhibited by an iPLA(2) antisense oligonucleotide. Enzyme assays conducted under a variety of conditions reveal that the specific activity of the iPLA(2) does not change as a result of H(2)O(2) exposure, which argues against the activation of a specific signaling cascade ending in the iPLA(2). Rather, the oxidant acts to perturb membrane homeostasis in a way that the enzyme susceptibility/accessibility to its substrate increases, and this results in altered fatty acid release. In support of this view, not only AA, but also other fatty acids, were found to be liberated in an iPLA(2)-dependent manner in the H(2)O(2)-treated cells. Collectively, these studies underscore the importance of the iPLA(2) in modulating homeostatic fatty acid deacylation reactions and document a potentially important route under pathophysiological conditions for increasing free fatty acid levels during oxidative stress.     

Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2

To test thehypothesis that intracellular Ca²⁺activation of large-conductanceCa²⁺-activatedK⁺ (BK) channels involves thecytosolic form of phospholipase A₂ (cPLA₂), we first inhibited theexpression of cPLA₂ by treating GH₃ cells with antisenseoligonucleotides directed at the two possible translation start siteson cPLA₂. Western blot analysis and a biochemical assay of cPLA₂activity showed marked inhibition of the expression ofcPLA₂ in antisense-treated cells.We then examined the effects of intracellularCa²⁺ concentration([Ca²⁺]_i)on single BK channels from these cells. Open channel probability (P_o) for thecells exposed to cPLA₂ antisenseoligonucleotides in 0.1 µM intracellularCa²⁺ was significantly lower thanin untreated or sense oligonucleotide-treated cells, but the voltagesensitivity did not change (measured as the slope of theP_o-voltagerelationship). In fact, a 1,000-fold increase in[Ca²⁺]_ifrom 0.1 to 100 µM did not significantly increaseP_oin these cells, whereas BK channels from cells in the other treatmentgroups showed a normalP_o-[Ca²⁺]_iresponse. Finally, we examined the effect of exogenous arachidonic acidon theP_oof BK channels from antisense-treated cells. Although arachidonic aciddid significantly increaseP_o,it did so without restoring the[Ca²⁺]_isensitivity observed in untreated cells. We conclude that although [Ca²⁺]_idoes impart some basal activity to BK channels inGH₃ cells, the steepP_o-[Ca²⁺]_irelationship that is characteristic of these channels involves cPLA₂.

     

Activation and possible involvement of calpain, a calcium-activated cysteine protease, in down-regulation of apoptosis of human monoblast U937 cells

An active form of calpain mu, a low-Ca(2+)-requiring intracellular cysteine protease, was detected using a cleavage site-directed antibody in apoptotic human monoblast U937 cells treated with tumor necrosis factor-alpha and interferon-gamma. Membrane-permeable calpain inhibitors accelerated apoptosis of U937 cells thus induced and suppressed the activation of procalpain. These findings suggest that calpain down-regulates apoptosis by shutting off the intracellular signals for cell death.     

A 32-kDa protein associated with phospholipase A2-inhibitory activity from human placenta

Two monomeric 32-kDa proteins, termed 32K-I (pI 5.8) and 32K-II (pI 5.1), were isolated from human placenta, which was solubilized by a Ca2+-chelator. Only 32K-I was associated with PLA2-inhibitory activity. CNBr peptide mapping indicated that 32K-I was distinct from 32K-II and two 36-kDa proteins, called calpactin I and II or lipocortin II and I, which have been shown to possess PLA2-inhibitory activity. 32K-I bound to PS in a Ca2+-dependent manner. 32K-I was detected in many tissues except brain, cardiac and skeletal muscle.     

Overexpression of cytosolic group IVA phospholipase A2 protects cells from Ca2+-dependent death

The calcium ionophore ionomycin induces apoptosis-like events in the human embryonic kidney cell line at early times. Plasma membrane blebbing, mitochondrial depolarization, externalization of phosphatidylserine, and nuclear permeability changes can all be observed within 15 min of treatment. However, there is no activation of caspases or chromatin condensation. Expression of a fusion protein containing the enhanced green fluorescent protein (EGFP) and human cytosolic Group IVA phospholipase A(2)alpha (EGFP-cPLA(2)alpha) in these cells prevents ionomycin-induced phosphatidylserine externalization and death. Cells expressing the cPLA(2)alpha mutant D43N, which does not bind calcium, retain their susceptibility to ionomycin-induced cell death. Both nonexpressing and EGFP-D43N-cPLA(2)alpha-expressing human embryonic kidney cells can be spared from ionomycin-induced cell death by pretreating them with exogenous arachidonic acid. Moreover, during calcium overload, mitochondrial depolarization is significantly lower in the EGFP-cPLA(2)alpha-expressing cells than in cells expressing normal amounts of cPLA(2)alpha. These results suggest that early cell death events promoted by an overload of calcium can be prevented by the presence of high levels of arachidonic acid.     

Ca2+ binding effects on the C2 domain conformation of human cytosolic phospholipase A2

It has been reported that the cooperative binding of calcium ions indicated a local conformational change of the human cytosolic phospholipase A2 (cPLA2) C2 domain (Nalefski et al., (1997) Biochemistry 36, 12011-12018). However its structural evidence is less known (Malmberg et al., (2003) Biochemistry 42, 13227-13240). In this letter, life-time decay and fluorescence quenching techniques were employed to compare the calcium-induced conformational changes. The life-time decay parameters and fluorescence quenching constant changes were small between the apo- and holo-C2 domains when tryptophan residue was excited at 295 nm. In contrast, the quenching constant change was large, from 0.52 M(-1) for the apo-C2 to 8.8 M(-1) for the holo-C2 domain, when tyrosine residues were excited at 284 nm. Our results provide new information on amino acid side chain orientation change at calcium binding loop 3, which is necessary for Ca2+ binding regulated membrane targeting of human cytosolic phospholipase A2.     

Group IVC cytosolic phospholipase A2gamma is farnesylated and palmitoylated in mammalian cells

Cytosolic phospholipase A(2)gamma (cPLA(2)gamma) is a member of the group IV family of intracellular phospholipase A(2) enzymes, but unlike the well-studied cPLA(2)alpha, it is constitutively bound to membrane and is calcium independent. cPLA(2)gamma contains a C-terminal CaaX sequence and is radiolabeled by mevalonic acid when expressed in cPLA(2)alpha-deficient immortalized lung fibroblasts (IMLF(-/-)). The radiolabel associated with cPLA(2)gamma was identified as the farnesyl group. The protein farnesyltransferase inhibitor BMS-214662 prevented the incorporation of [(3)H]mevalonic acid into cPLA(2)gamma and partially suppressed serum-stimulated arachidonic acid release from IMLF(-/-) and undifferentiated human skeletal muscle (SkMc) cells overexpressing cPLA(2)gamma, but not from cells overexpressing cPLA(2)alpha. However, BMS-214662 did not alter the amount of cPLA(2)gamma associated with membrane. These results were consistent in COS cells expressing the C538S cPLA(2)gamma prenylation mutant. cPLA(2)gamma 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 cPLA(2)gamma is associated with mitochondria in IMLF(-/-), SkMc cells, and COS cells.