Novel translocation responses of cytosolic phospholipase A2alpha fluorescent proteins

Cytosolic phospholipase A2 (cPLA2)alpha responds to the rise in cytosolic Ca2+ ([Ca2+]i) attending cell stimulation by moving to intracellular membranes, releasing arachidonic acid (AA) from these membranes, and thereby initiating the synthesis of various lipid mediators. Under some conditions, however, cPLA2alpha translocation occurs without any corresponding changes in [Ca2+]i. The signal for such responses has not been identified. Using confocal microscopy to track fluorescent proteins fused to cPLA2alpha or cPLA2alpha's C2 domain, we find that AA mimics Ca2+ ionophores in stimulating cPLA(2)alpha translocations to the perinuclear ER and to a novel site, the lipid body. Unlike the ionophores, AA acted independently of [Ca2+](i) rises and did not translocate the proteins to the Golgi. AA's action did not involve its metabolism to eicosanoids or acylation into cellular lipids. Receptor agonists also stimulated translocations targeting lipid bodies. We propose that AA is a signal for Ca2+-independent cPLA2alpha translocation and that lipid bodies are common targets of cPLA2alpha and contributors to stimulus-induced lipid mediator synthesis.     

Ser515 phosphorylation-independent regulation of cytosolic phospholipase A2alpha (cPLA2alpha) by calmodulin-dependent protein kinase: possible interaction with catalytic domain A of cPLA2alpha

Calmodulin (CaM)-dependent protein kinase (CaM kinase) is proposed to regulate the type alpha of cytosolic phospholipase A(2) (cPLA(2)alpha), which has a dominant role in the release of arachidonic acid (AA), via phosphorylation of Ser515 of the enzyme. However, the exact role of CaM kinase in the activation of cPLA(2)alpha has not been well established. We investigated the effects induced by transfection with mutant cPLA(2)alpha and inhibitors for CaM and CaM kinase on the Ca(2+)-stimulated release of AA and translocation of cPLA(2)alpha. The mutation of Ser515 to Ala (S515A) did not change cPLA(2)alpha activity, although S228A and S505A completely and partially decreased the activity, respectively. Stimulation with hydrogen peroxide (H(2)O(2), 1 mM) and A23187 (10 microM) markedly released AA in C12 cells expressing S515A and wild-type cPLA(2)alpha, but the responses in C12-S505A, C12-S727A, and C12-S505A/S515A/S727A (AAA) cells were reduced. In HEK293T cells expressing cPLA(2)alpha, A23187 caused the translocation of the wild-type, the every mutants, cPLA(2)alpha-C2 domain, and cPLA(2)alpha-Delta397-749 lacking proposed phosphorylation sites such as Ser505 and Ser515. Treatment with inhibitors of CaM (W-7) and CaM kinase (KN-93) at 10 microM significantly decreased the release of AA in C12-cPLA(2)alpha cells and C12-S515A cells. KN-93 inhibited the A23187-induced translocation of the wild-type, S515A, AAA and cPLA(2)alpha-Delta397-749, but not cPLA(2)alpha-C2 domain. Our findings show a possible effect of CaM kinase on cPLA(2)alpha in a catalytic domain A-dependent and Ser515-independent manner.     

1alpha,25(OH)2D3 and parathyroid hormone (PTH) signaling in rat intestinal cells: activation of cytosolic PLA2

In the current study, we have probed the role of cytosolic phospholipase A2 (cPLA2) activity in the cellular response to the calciotropic hormones, 1alpha,25,dihydroxy-vitamin D(3) [1alpha,25(OH)(2)D(3)] and PTH. Stimulation of rat enterocytes with either hormone, increased release of arachidonic acid (AA) 3H-AA] one-two fold in a concentration and time-dependent manner. The effect of either hormone on enterocytes was totally reduced by preincubation with the intracellular Ca(2+) chelator BAPTA-AM (5 microM), suggesting that the release of AA following cell exposure to the calciotropic hormones occurs mainly through a Ca(2+)-dependent mechanism involving activation of Ca(2+)-dependent cPLA2. Calciotropic homone stimulation of rat intestinal cells increases cPLA2 phosphorylation (three to four fold). This effect was decreased by PD 98059 (20 microM), a MAP kinase inhibitor, indicating that this action is, in part, mediated through activation of the MAP kinases ERK 1 and ERK2. Enterocytes exposure to 1alpha,25(OH)(2)D(3) (1nM) or PTH (10 nM) also resulted in P-cPLA2 translocation from cytosol to nuclei and membrane fractions, where phospholipase subtrates reside. Collectively, these data suggest that PTH and 1alpha,25(OH)(2)D(3) activate in duodenal cells, a Ca(2+)-dependent cytosolic PLA2 and attendant arachidonic acid release and that this activation requieres prior stimulation of intracellular ERK1/2. 1alpha,25(OH)(2)D(3) and PTH modulation of cPLA2 activity may change membrane fluidity and permeability and thereby affecting intestinal cell membrane function.     

Ligation of the alpha2M* signaling receptor regulates synthesis of cytosolic phospholipase A2

We have studied the regulation of cytosolic phospholipase A2 (cPLA2) synthesis in macrophages stimulated with receptor-recognized forms of alpha2-macroglobulin (alpha2M*). [35S]methionine-labeled cells were stimulated with alpha2M* and [35S]cPLA2 was immunoprecipitated with a monoclonal antibody directed against cPLA2. The precipitates were electrophoresed, immunoblotted, cPLA2 detected by Enhanced Chemifluorescence, and its radioactivity determined. Stimulation of cells with alpha2M* caused a two- to threefold increase in cPLA2 synthesis compared to buffer-treated cells which was consistently maximal at 200 pM of alpha2M*. Actinomycin D or cycloheximide treatment of cells drastically reduced alpha2M*-induced cPLA2 synthesis. Likewise, inhibition of protein kinase C with chelerythrin, farnesyl transferase with manumycin A, MEK kinase with U0126, Erk1/2 kinases with PD98059, p38MAPK with SB203580, PI 3-kinase with wortmannin or LY294002, p70s6k with rapamycin, or depletion of [Ca2+]i with either BAPTA/AM or EGTA drastically reduced alpha2M* induction of cPLA2. Inhibition of NFKB activation with BAY11-7182 or PGA1 also abolished alpha2M* induction of cPLA2. We conclude that alpha2M*-induced cPLA2 synthesis is controlled by [Ca2+]i levels, tyrosine kinase activity, the p21ras-dependent MAPK and PI 3-kinase downstream signaling pathways, and regulation of NFkappaB.     

Acceleration by ceramide of calcium-dependent translocation of phospholipase A2 from cytosol to membranes in platelets

The effect of ceramide on Ca2+-dependent translocation of cytosolic phospholipase A2 (cPLA2) to membranes was studied. Pretreatment of platelets with sphingomyelinase or C6-ceramide (N-hexanoylsphingosine) led to apparent enhancement of Ca2+-ionophore A23187-stimulated arachidonic acid release but did not affect the cytosolic phospholipase A2 (cPLA2) activity. Under these conditions, the cPLA2 proteins in membranes increased significantly, compared with those by A23187 alone. Sphingomyelinase and C6-ceramide, but not C6-dihydroceramide, a control analog of C6-ceramide, also facilitated the Ca2+-dependent increase in the cPLA2 protein, as well as the activity, in membranes induced by addition of Ca2+ into platelet lysate. Protein kinase Calpha, which possesses a Ca2+-dependent lipid binding domain, was increased in membranes in a Ca2+-dependent manner, but the increase was not accelerated by sphingomyelinase or C6-ceramide. These findings suggest that ceramide in membranes potentiates Ca2+-dependent cPLA2 translocation from cytosol to membranes, probably through modification of membrane phospholipid organization.     

fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis?2-aminophenoxy?thane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.     

Protein kinase C: subcellular redistribution by increased Ca2+ influx. Evidence that Ca2+-dependent subcellular redistribution of protein kinase C is involved in potentiation of beta-adrenergic stimulation of pineal cAMP and cGMP by K+ and A23187

Phenylephrine is known to stimulate translocation of protein kinase C in rat pinealocytes (Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P., and Anderson, W. B. (1985) Nature 314, 359-361). In the present study, the receptor mediating this effect was found to belong to the alpha 1-adrenoceptor subclass. Activation of this receptor is also known to produce a sustained increase in [Ca2+]i by increasing net influx (Sugden, A. L., Sugden, D., and Klein, D. C. (1985) J. Biol. Chem. 261, 11608-11612), which points to the possible importance of Ca2+ influx in the subcellular redistribution (activation) of protein kinase C in intact cells. This possibility was investigated by reducing extracellular Ca2+ ((Ca2+]o) with EGTA or by inhibiting Ca2+ influx with inorganic Ca2+ blockers. These treatments reduced alpha 1-adrenoceptor-mediated translocation of protein kinase C. This suggested that elevation of Ca2+ influx alone triggers activation of protein kinase C. In support of this, it was found that treatments which elevate Ca2+ influx, including increased extracellular K+ and addition of the Ca2+ ionophore A23187, cause redistribution of protein kinase C. The effect of K+ was blocked by nifedipine and that of A23187 by EGTA, indicating that effects of these agents are Ca2+-dependent. The possible role of phospholipase C activation in these effects was examined by measuring the formation of [3H]diacylglycerol by cells labeled with [3H]arachidonic acid. Although [3H]diacylglycerol formation was easily detected in the presence or absence of an effective concentration of an inhibitor of diacylglycerol kinase, none of the agents which cause rapid translocation of protein kinase C were found to cause a rapid increase in the generation of [3H]diacylglycerol. These findings establish that an increase in Ca2+ influx is sufficient to trigger translocation of protein kinase C. In addition, we found that a very close correlation exists between translocation of protein kinase C by phenylephrine, K+, and A23187 and their ability to potentiate beta-adrenergic stimulation of cAMP and cGMP accumulation. This provides strong support to the proposal that translocation of protein kinase C is required for potentiation of beta-adrenergic stimulation of pinealocyte cAMP and cGMP accumulation.     

Phosphatidylinositol 4,5-bisphosphate anchors cytosolic group IVA phospholipase A2 to perinuclear membranes and decreases its calcium requirement for translocation in live cells

The eicosanoids are centrally involved in the onset and resolution of inflammatory processes. A key enzyme in eicosanoid biosynthesis during inflammation is group IVA phospholipase A2 (also known as cytosolic phospholipase A2alpha, cPLA2alpha). This enzyme is responsible for generating free arachidonic acid from membrane phospholipids. cPLA2alpha translocates to perinuclear membranes shortly after cell activation, in a process that is governed by the increased availability of intracellular Ca2+. However, cPLA2alpha also catalyzes membrane phospholipid hydrolysis in response to agonists that do not mobilize intracellular Ca2+. How cPLA2alpha interacts with membranes under these conditions is a major, still unresolved issue. Here, we report that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] promotes translocation of cPLA2alpha to perinuclear membranes of intact cells in a manner that is independent of rises in the intracellular Ca2+ concentration. PtdIns(4,5)P2 anchors the enzyme to perinuclear membranes and allows for a proper interaction with its phospholipid substrate to release arachidonic acid.     

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.     

Arachidonic acid released by phospholipase A(2) activation triggers Ca(2+)-dependent apoptosis through the mitochondrial pathway

We studied the effects of the divalent cation ionophore A23187 on apoptotic signaling in MH1C1 cells. Addition of A23187 caused a fast rise of cytosolic Ca(2+) ([Ca(2+)](c)), which returned close to the resting level within about 40 s. The [Ca(2+)](c) rise was immediately followed by phospholipid hydrolysis, which could be inhibited by aristolochic acid or by pretreatment with thapsigargin in Ca(2+)-free medium, indicating that the Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) was involved. These early events were followed by opening of the mitochondrial permeability transition pore (PTP) and by apoptosis in about 30% of the cell population. In keeping with a cause-effect relationship between addition of A23187, activation of cPLA(2), PTP opening, and cell death, all events but the [Ca(2+)](c) rise were prevented by aristolochic acid. The number of cells killed by A23187 was doubled by treatment with 0.5 microm MK886 and 5 microm indomethacin, which inhibit arachidonic acid metabolism through the 5-lipoxygenase and cyclooxygenase pathway, respectively. Consistent with the key role of free arachidonic acid, its levels increased within minutes of treatment with A23187; the increase being more pronounced in the presence of MK886 plus indomethacin. Cell death was preceded by cytochrome c release and cleavage of caspase 9 and 3, but not of caspase 8. All these events were prevented by aristolochic acid and by the PTP inhibitor cyclosporin A. Thus, A23187 triggers the apoptotic cascade through the release of arachidonic acid by cPLA(2) in a process that is amplified when transformation of arachidonic acid into prostaglandins and leukotrienes is inhibited. These findings identify arachidonic acid as the causal link between A23187-dependent perturbation of Ca(2+) homeostasis and the effector mechanisms of cell death.     

Dual signaling by the alpha(v)beta(3)-integrin activates cytosolic PLA(2) in bovine pulmonary artery endothelial cells

Vitronectin, which ligates the alpha(v)beta(3)-integrin, increases both lung capillary permeability and lung endothelial Ca(2+). In stable monolayers of bovine pulmonary artery endothelial cells (BPAECs) viewed with confocal microscopy, multimeric vitronectin aggregated the apically located alpha(v)beta(3)-integrin. This caused arachidonate release that was inhibited by pretreating the monolayers with the anti-alpha(v)beta(3) monoclonal antibody (MAb) LM609. No inhibition occurred in the presence of the isotypic MAb PIF6, which recognizes the integrin alpha(v)beta(5). Vitronectin also caused membrane translocation and phosphorylation of cytosolic phospholipase A(2) (cPLA(2)) as well as tyrosine phosphorylation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) 2. The cPLA(2) inhibitor arachidonyl trifluoromethylketone, the tyrosine kinase inhibitor genistein, and the MAPK kinase inhibitor PD-98059 all blocked the induced arachidonate release. PD-98059 did not inhibit the increase of cytosolic Ca(2+) or cPLA(2) translocation, although it blocked tyrosine phosphorylation of ERK2. Moreover, although the intracellular Ca(2+) chelator MAPTAM also inhibited arachidonate release, it did not inhibit tyrosine phosphorylation of ERK2. These findings indicate that ligation of apical alpha(v)beta(3) in BPAECs caused ERK2 activation and an increase of intracellular Ca(2+), both conjointly required for cPLA(2) activation and arachidonate release. This is the first instance of a tyrosine phosphorylation-initiated "two-hit" signaling pathway that regulates an integrin-induced proinflammatory response.     

Epidermal growth factor (EGF), tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium and stimulate arachidonic acid release and PGE2/6-keto PGF1 alpha production in cultured porcine thyroid cells

Epidermal growth factor (EGF), 12-O-tetradecanoylphorbol 13-acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium ([Ca2+]i) and stimulate arachidonic acid release and production of PGE2 and 6-keto PGF1 alpha, an end metabolite of PGI2, in cultured porcine thyroid cells. Addition of EGF, TPA or A23187 to the cells loaded with fura-2, a fluorescent Ca2+ indicator, causes an immediate increase in [Ca2+]i, which is the earliest event after mitogen stimulation. This [Ca2+]i response occurs immediately, reaching a maximum within several seconds. EGF, TPA and A23187 stimulate arachidonic acid release and PGE2 and 6-keto PGF1 alpha production; the maximum effects are obtained after 2-4 h incubation. EGF, TPA and A23187 increase [Ca2+]i and then stimulate arachidonic acid release and PG production.     

Function, activity, and membrane targeting of cytosolic phospholipase A(2)zeta in mouse lung fibroblasts

Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) initiates eicosanoid production; however, this pathway is not completely ablated in cPLA(2)alpha(-/-) lung fibroblasts stimulated with A23187 or serum. cPLA(2)alpha(+/+) fibroblasts preferentially released arachidonic acid, but A23187-stimulated cPLA(2)alpha(-/-) fibroblasts nonspecifically released multiple fatty acids. Arachidonic acid release from cPLA(2) alpha(-/-) fibroblasts was inhibited by the cPLA(2)alpha inhibitors pyrrolidine-2 (IC(50), 0.03 microM) and Wyeth-1 (IC(50), 0.1 microM), implicating another C2 domain-containing group IV PLA(2). cPLA(2) alpha(-/-) fibroblasts contain cPLA(2)beta and cPLA(2)zeta but not cPLA(2)epsilon or cPLA(2)delta. Purified cPLA(2)zeta exhibited much higher lysophospholipase and PLA(2) activity than cPLA(2)beta and was potently inhibited by pyrrolidine-2 and Wyeth-1, which did not inhibit cPLA(2)beta. In contrast to cPLA(2)beta, cPLA(2)zeta expressed in Sf9 cells mediated A23187-induced arachidonic acid release, which was inhibited by pyrrolidine-2 and Wyeth-1. cPLA(2)zeta exhibits specific activity, inhibitor sensitivity, and low micromolar calcium dependence similar to cPLA(2)alpha and has been identified as the PLA(2) responsible for calcium-induced fatty acid release and prostaglandin E(2) production from cPLA(2) alpha(-/-) lung fibroblasts. In response to ionomycin, EGFP-cPLA(2)zeta translocated to ruffles and dynamic vesicular structures, whereas EGFP-cPLA(2)alpha translocated to the Golgi and endoplasmic reticulum, suggesting distinct mechanisms of regulation for the two enzymes.     

Sensitizing effect of lysophosphatidic acid on Ca2+ response to hypotonic stress in cultured lens epithelial cells.

The effects of lysophosphatidic acid (LPA), a bioactive phospholipid, on the response of the cytosolic free Ca2+ concentration ([Ca2+]i) to hypotonic stress were studied in cultured bovine lens epithelial cells, to test whether LPA affects cellular swelling-mediated increase in [Ca2+]i, which may relate to formation of sugar cataracts. Exposure of the cells to a 30% hypotonic stress caused only a slight increase in [Ca2+]i. Pretreatment with LPA (10 microM) significantly augmented the hypotonic stress-induced [Ca2+]i response, whereas addition of LPA to the cells did not affect [Ca2+]i. The hypotonic stress-induced increase in [Ca2+]i in the presence of LPA was inhibited by Gd3+, a blocker of mechanosensitive cation channels, but not by nicardipine, a L-type Ca2+ channel blocker, or thapsigargin, an inhibitor of endoplasmic reticulum-ATPase pump. These results show that LPA sensitizes the response to hypotonic stress via increase in Ca2+ influx through Gd3+-sensitive stretch-activated ion channels, and not via Ca2+ release from intracellular stores. On the other hand, LPA did not affect the [Ca2+]i response to ATP, a Ca2+ mobilizing agonist. Therefore, LPA sensitizes the hypotonic stress-induced [Ca2+]i response in lens epithelial cells, suggesting that LPA potentiates the development of cataracts induced by cellular swelling such as sugar cataract.     

Cytosolic phospholipase A(2) activity associated with nuclei is not inhibited by arachidonyl trifluoromethyl ketone in macrophages stimulated with receptor-recognized forms of alpha(2)-macroglobulin

We have studied the translocation of cytosolic phospholipase A(2) (cPLA(2)) to nuclei in macrophages stimulated with receptor-recognized forms of alpha(2)-macroglobulin (alpha(2)M*). Translocation of phosphorylated cPLA(2) to nuclei was determined by immunoprecipitation of cPLA(2) in (32)P(i)-labeled cells. The identity of cPLA(2) was established by comparing its mobility on gels with an authentic cPLA(2) standard. cPLA(2) activity was quantified by measuring the release of [(14)C]arachidonic acid from the substrate 1-palmitoyl-2-[1-(14)C]arachidonyl-sn-glycerophosphatidylcholine. alpha(2)M* caused a two- to threefold increase in cPLA(2) phosphorylation and its translocation to nuclei. The p38 MAPK inhibitor SB203580, PKC inhibitor chelerythrin, or depletion of intracellular Ca(2+) profoundly decreased cPLA(2) activity in nuclei isolated from agonist-stimulated cells. The requirement for Ca(2+), PKC, and p38 MAPK activation appears to be of major importance for nuclear cPLA(2) activity. In contrast to cellular cPLA(2) activity, nuclear cPLA(2) activity was not inhibited by arachidonyl trifluoromethyl ketone (AACOCF(3)) in agonist-stimulated cells. It is concluded that the association of cPLA(2) with nuclear membranes in agonist-stimulated cells modifies the activity and the sensitivity of the enzyme to inhibition by AACOCF(3) in this phospholipid environment.     

Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.     

The role of the cytosolic free Ca2+ transient for fMet-Leu-Phe induced actin polymerization in human neutrophils

We have addressed the important question as to if and how the cytosolic free Ca2+ concentration, [Ca2+]i, is involved in fMet-Leu-Phe induced actin polymerization in human neutrophils. Stimulation of human neutrophils with the chemotactic peptide (10(-7) M), known to result in a prompt rise of the [Ca2+]i to above 500 nM, also induced a rapid decrease of monomeric actin, G-actin, content (to 35% of basal) and increase of filamentous actin, F-actin, content (to 320% of basal). A reduction of the fMet-Leu-Phe induced [Ca2+]i transient to about 250 nM, resulted in a less pronounced decrease of G-actin content (to 80% of basal) and increase of F-actin content (to 235% of basal). A total abolishment of the chemotactic peptide induced [Ca2+]i rise, still led to a decrease of the G-actin content (to 85% of basal) and increase of F-actin (to 200% of basal). These results indicate that the [Ca2+]i rise is not an absolute requirement, but has a modulating role for the fMet-Leu-Phe induced actin polymerization. Another possible intracellular candidate for fMet-Leu-Phe induced actin polymerization is protein kinase C. However, direct activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) only resulted in a minor increase of F-actin content. The recent hypothesis that a metabolite of the polyphosphoinositide cycle, independently of [Ca2+]i and protein kinase C, is responsible for actin polymerization agrees well with these results and by the fact that preexposure to pertussis toxin totally abolished a subsequent increase of F-actin content induced by fMet-Leu-Phe.     

Translocation of phospholipase A2 to membranes by oxidized LDL and hydroxyoctadecadienoic acid to contribute to cholesteryl ester formation

We examined the mechanisms underlying the activation of group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) contributing to the supply of fatty acids required for the formation of cholesteryl ester in oxidized low-density lipoprotein (oxLDL)-stimulated macrophages. The possible involvement of oxidized lipids was also examined. In [(3)H]arachidonic acid-labeled mouse peritoneal macrophages, oxLDL stimulated the release of arachidonic acid, which was suppressed by methyl arachidonyl fluorophosphonate (MAFP), a cPLA(2)alpha inhibitor. oxLDL induced an increase in PLA(2)alpha levels in the membrane fraction without affecting those in whole cells or the activity in the lysate. Among 13-hydroxyoctadecadienoic acid (13-HODE), 7-ketocholesterol, and 25-hydroxycholesterol, oxidized lipids present in oxLDL particles, only 13-HODE induced the release of arachidonic acid, which was also sensitive to MAFP. Under conditions where addition of Ca(2+) to the cell lysate induced an increase in cPLA(2)alpha protein in the membrane fraction, preincubation with 13-HODE facilitated the Ca(2+)-dependent translocation of cPLA(2)alpha. Furthermore, 13-HODE increased cholesteryl ester formation in the presence of [(3)H]cholesterol. These results suggest that 13-HODE mediates the oxLDL-induced activation of cPLA(2)alpha through an increase in cPLA(2)alpha protein in the membranes, thus contributing, in part, to the supply of fatty acids required for the esterification of cholesterol in macrophages.     

N-terminal and C-terminal plasma membrane anchoring modulate differently agonist-induced activation of cytosolic phospholipase A2.

The 85 kDa cytosolic phospholipase A2 (cPLA2) plays a key role in liberating arachidonic acid from the sn-2 position of membrane phospholipids. When activated by extracellular stimuli, cPLA2 undergoes calcium-dependent translocation from cytosol to membrane sites which are still a matter of debate. In order to evaluate the effect of plasma membrane association on cPLA2 activation, we constructed chimeras of cPLA2 constitutively targeted to the plasma membrane by the N-terminal targeting sequence of the protein tyrosine kinase Lck (Lck-cPLA2) or the C-terminal targeting signal of K-Ras4B (cPLA2-Ras). Constitutive expression of these chimeras in Chinese hamster ovary cells overproducing the alpha2B adrenergic receptor (CHO-2B cells) did not affect the basal release of [3H]arachidonic acid, indicating that constitutive association of cPLA2 with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck-cPLA2 increased [3H]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA2-Ras inhibited it, compared with parental CHO-2B cells and CHO-2B cells producing comparable amounts of recombinant wild-type cPLA2. The lack of stimulation of cPLA2-Ras was not due to a decreased enzymatic activity as measured using an exogenous substrate, or to a decreased phosphorylation of the protein. These results show that the plasma membrane is a suitable site for cPLA2 activation when orientated correctly.     

Involvement of different protein kinases and phospholipases A2 in phorbol ester (TPA)-induced arachidonic acid liberation in bovine platelets

The effect of various phospholipase A2 and protein kinase inhibitors on the arachidonic acid liberation in bovine platelets induced by the protein kinase activator 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied. TPA stimulates arachidonic acid release mainly by activating group IV cytosolic PLA2 (cPLA2), since inhibitors of this enzyme markedly inhibited arachidonic acid formation. However, group VI Ca2+-independent PLA2 (iPLA2) seems to contribute to the arachidonic acid liberation too, since the relatively specific iPLA2 inhibitor bromoenol lactone (BEL) decreased arachidonic acid generation in part. The pronounced inhibition of the TPA-induced arachidonic acid release by the protein kinase C (PKC) inhibitors GF 109203X and Ro 31-82220, respectively, and by the p38 MAP kinase inhibitor SB 202190 suggests that the activation of the PLA2s by TPA is mediated via PKC and p38 MAP kinase.