scholarly journals Translocation of cytosolic phospholipase A2 to the nuclear envelope elicits topographically localized phospholipid hydrolysis

1996 ◽  
Vol 318 (3) ◽  
pp. 797-803 ◽  
Author(s):  
Marc PETERS-GOLDEN ◽  
Keli SONG ◽  
Teresa MARSHALL ◽  
Thomas BROCK
Keyword(s):  
Nuclear Envelope ◽  
Phospholipase A2 ◽  
Nuclear Membrane ◽  
Microscopic Analysis ◽  
Cytosolic Phospholipase A2 ◽  
Alveolar Epithelial Cells ◽  
Ionophore A23187 ◽  
Membrane Phospholipids ◽  
Phospholipid Hydrolysis ◽  
Cytosolic Phospholipase

Cytosolic phospholipase A2 (cPLA2) is a good candidate for mediating the agonist-stimulated release of arachidonic acid (AA) from membrane phospholipids. This enzyme undergoes a Ca2+-dependent translocation from the cytosol to a membrane site in a variety of cell types, and this site has recently been identified as the nuclear envelope in leucocytes. The functional correlate of this finding has not yet been established. The present study was therefore undertaken to determine whether translocation of cPLA2 to the nuclear envelope was associated with localized phospholipid hydrolysis at this site. Rat alveolar epithelial cells, previously shown to contain cPLA2, were prelabelled with [3H]AA and stimulated with the model agonist, ionophore A23187. Ionophore-induced AA release exhibited characteristics typical of a cPLA2-mediated response, in that it was Ca2+-dependent, sn-2 AA-selective, and inhibited by arachidonyl trifluoromethyl ketone. As determined by indirect immunofluorescence microscopic analysis as well as subcellular fractionation with immunoblotting, ionophore treatment resulted in a translocation of cPLA2 protein from the cytoplasm to the nuclear envelope. To determine whether the nuclear membrane was indeed the source of released AA, prelabelled cells were incubated in the presence or absence of A23187, after which the phospholipid radioactivity was quantified in nuclear and non-nuclear membrane fractions. [3H]AA was distributed in both nuclear and non-nuclear membrane phospholipids. Following A23187 stimulation, the loss of [3H]AA from nuclear membrane phospholipids accounted for 88.1±5.8% of the total loss from phospholipids and for 92.9±2.3% of the total [3H]AA released into the medium. These results demonstrate for the first time that agonist-stimulated translocation of cPLA2 to the nuclear envelope is associated with phospholipid hydrolysis which is preferentially localized to that site.

scholarly journals Role of Phosphorylation Sites and the C2 Domain in Regulation of Cytosolic Phospholipase A2

1999 ◽  
Vol 145 (6) ◽  
pp. 1219-1232 ◽  
Author(s):  
Miguel A. Gijón ◽  
Diane M. Spencer ◽  
Alan L. Kaiser ◽  
Christina C. Leslie
Keyword(s):  
Arachidonic Acid ◽  
Nuclear Envelope ◽  
Phospholipase A2 ◽  
Okadaic Acid ◽  
Cytosolic Phospholipase A2 ◽  
Phosphorylation Sites ◽  
C2 Domain ◽  
Arachidonic Acid Release ◽  
Cytosolic Phospholipase ◽  
Acid Release

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.

scholarly journals Stress stimuli increase calcium-induced arachidonic acid release through phosphorylation of cytosolic phospholipase A2

1999 ◽  
Vol 344 (2) ◽  
pp. 359-366 ◽  
Author(s):  
Marcus BUSCHBECK ◽  
Farideh GHOMASHCHI ◽  
Michael H. GELB ◽  
Steve P. WATSON ◽  
Angelika G. BÖRSCH-HAUBOLD
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Cytosolic Phospholipase A2 ◽  
Ionophore A23187 ◽  
Arachidonic Acid Release ◽  
High Osmolarity ◽  
Cytosolic Phospholipase ◽  
Acid Release ◽  
Sb 203580

Stress stimuli such as free radicals, high osmolarity or arsenite activate stress-activated protein kinases (SAPKs) in a wide variety of cells. In the present study, we have investigated the ability of several stress stimuli to activate SAPKs in platelets and to induce phosphorylation of their substrates. Treatment of human platelets with H2O2 stimulated SAPK2a and its downstream target mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP-K2). Kinase activity reached a maximum after 2-5 min and declined towards basal levels after 15 min. Arsenite caused a steady increase of MAPKAP-K2 activity up to 15 min. The level of maximal kinase activation by H2O2 and arsenite was comparable with the effect caused by the physiological platelet stimulus thrombin. A high osmolarity solution of sorbitol induced comparatively small activation of SAPK2a and MAPKAP-K2. The 42-kDa extracellular signal-regulated kinase (ERK) 2 was not activated by H2O2, sorbitol or arsenite. None of these stimuli triggered significant arachidonic acid release on their own. However, H2O2 and sorbitol enhanced the release of arachidonic acid induced by the calcium ionophore A23187. This effect was reversed by the inhibitor of SAPK2a, 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl) imidazole (SB 203580), but not by the inhibitor of the ERK2-activating pathway, 2-(2-amino-3-methoxyphenyl)-oxanaphthalen-4-one (PD 98059). Both H2O2 and sorbitol increased phosphorylation of cytosolic phospholipase A2 (cPLA2) and its intrinsic activity; both responses were blocked by SB 203580. Phosphorylation of cPLA2 by H2O2 occurred on Ser-505, a reaction that is known to increase the intrinsic lipase activity of the enzyme. Our results demonstrate that activation of SAPKs by stress stimuli primes cPLA2 activation through phosphorylation. In vivo, this mechanism would lead to the sensitization of platelet activation and may be an important risk factor in thrombotic disease.

scholarly journals Role of cytosolic phospholipase A2 in arachidonic acid release of rat-liver macrophages: regulation by Ca2+ and phosphorylation

1995 ◽  
Vol 311 (1) ◽  
pp. 189-195 ◽  
Author(s):  
P Ambs ◽  
M Baccarini ◽  
E Fitzke ◽  
P Dieter
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Map Kinase ◽  
Rat Liver ◽  
Kinase Inhibitor ◽  
Cytosolic Phospholipase A2 ◽  
Ionophore A23187 ◽  
Liver Macrophages ◽  
Cytosolic Phospholipase ◽  
Stimulation Of

In this study we have verified the existence of a cytosolic phospholipase A2 (cPLA2) in rat-liver macrophages. Stimulation of these cells with phorbol 12-myristate 13-acetate (PMA), zymosan and lipopolysaccharide (LPS), but not with the Ca(2+)-ionophore A23187, leads to phosphorylation of cPLA2 and activation of mitogen-activated protein (MAP) kinase, supporting the hypothesis that MAP kinase is involved in cPLA2 phosphorylation. We show furthermore, that the tyrosine kinase inhibitor genistein prevents the LPS- but not the PMA- or zymosan-induced phosphorylation of cPLA2 and activation of MAP kinase, indicating that tyrosine kinases participate in LPS- but not in PMA- and zymosan-induced cPLA2 phosphorylation and MAP kinase activation. Phosphorylation of cPLA2 does not strongly correlate with stimulation of the arachidonic acid (AA) cascade: (1) A23187, a potent stimulator of AA release, fails to induce cPLA2 phosphorylation; (2) withdrawal of extracellular Ca2+, which inhibits PMA-stimulated AA release (Dieter, Schulze-Specking and Decker (1988) Eur. J. Biochem. 177, 61-67), has no effect on PMA-induced phosphorylation of cPLA2; (3) LPS induces cPLA2 phosphorylation within minutes, whereas increased AA release upon treatment with LPS is detectable for the first time after 4 h; and (4) genistein, which prevents LPS-induced cPLA2 phosphorylation, does not inhibit AA release in response to LPS. From these data we suggest that a rise in intracellular Ca2+, but not phosphorylation of cPLA2, is essential for activation of the AA cascade in rat-liver macrophages.

scholarly journals The properties of a cloned human high-molecular-mass cytosolic phospholipase A2 investigated using a continuous fluorescence displacement assay: evidence for enzyme clustering on phospholipid vesicles

1995 ◽  
Vol 306 (3) ◽  
pp. 857-864 ◽  
Author(s):  
A Creaney ◽  
D J Masters ◽  
M B Needham ◽  
R D Gordon ◽  
R Mott ◽  
...  
Keyword(s):  
Phospholipase A2 ◽  
Cytosolic Phospholipase A2 ◽  
Kinetic Properties ◽  
Complete Hydrolysis ◽  
Phospholipid Hydrolysis ◽  
Cytosolic Phospholipase ◽  
Displacement Assay ◽  
Burst Phase ◽  
Hydrolysis Of ◽  
Outer Monolayer

The 85 kDa human cytosolic phospholipase A2 has been cloned and expressed in insect Sf21 cells. The pure enzyme has been investigated using a fluorescence displacement assay that provides a continuous record of phospholipid hydrolysis [Wilton (1990) Biochem. J. 266, 435-439]. The unusual kinetic properties of this enzyme, previously described using radioactive assays, were readily demonstrated using the continuous fluorescence assay and were examined in detail. It is proposed that the enzyme clusters on the surface of a fixed number of substrate vesicles during the initial stages of catalysis and that the characteristic burst phase of hydrolysis represents the hydrolysis of these vesicles. This clustering produced a molar ratio of total phospholipid substrate to enzyme of about 450:1 at vesicle saturation with enzyme. Under limiting substrate conditions, the lower secondary rate that is observed results eventually in almost complete hydrolysis of the phospholipid; this was confirmed using radioactive substrate. Evidence is presented that during the initial burst phase, equivalent to hydrolysis of the outer monolayer of the vesicle, the enzyme remains tightly bound but is released as the reaction proceeds towards complete hydrolysis of the phospholipid substrate. In the presence of excess substrate, about 370 mol of fatty acid are released per mol of enzyme during the burst phase and it is calculated that this value also approximates to hydrolysis of the outer monolayer of the vesicle. It is proposed that the formation of a stable enzyme-vesicle complex during the burst phase of phospholipid hydrolysis may be due, at least in part, to protein-protein interactions between adjacent enzyme molecules in order to account for the clustering phenomenon.

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