M. Petersgolden et al., TRANSLOCATION OF CYTOSOLIC PHOSPHOLIPASE A(2) TO THE NUCLEAR-ENVELOPEELICITS TOPOGRAPHICALLY LOCALIZED PHOSPHOLIPID HYDROLYSIS, Biochemical journal, 318, 1996, pp. 797-803
Cytosolic phospholipase A(2) (cPLA(2)) is a good candidate for mediati
ng the agonist-stimulated release of arachidonic acid (AA) from membra
ne phospholipids. This enzyme undergoes a Ca2+-dependent translocation
from the cytosol to a membrane site in a variety of cell types, and t
his site has recently been identified as the nuclear envelope in leuco
cytes. The functional correlate of this finding has not yet been estab
lished. The present study was therefore undertaken to determine whethe
r translocation of cPLA(2) to the nuclear envelope was associated with
localized phospholipid hydrolysis at this site. Rat alveolar epitheli
al cells, previously shown to contain cPLA(2), were prelabelled with [
H-3]AA and stimulated with the model agonist, ionophore A23187. Ionoph
ore-induced AA release exhibited characteristics typical of a cPLA(2)-
mediated response, in that it was Ca2+-dependent, sn-2 AA-selective, a
nd inhibited by arachidonyl trifluoromethyl ketone. As determined by i
ndirect immunofluorescence microscopic analysis as well as subcellular
fractionation with immunoblotting, ionophore treatment resulted in a
translocation of cPLA(2) protein from the cytoplasm to the nuclear env
elope. To determine whether the nuclear membrane was indeed the source
of released AA, prelabelled cells were incubated in the presence or a
bsence of A23187, after which the phospholipid radioactivity was quant
ified in nuclear and nonnuclear membrane fractions. [H-3]AA was distri
buted in both nuclear and non-nuclear membrane phospholipids. Followin
g A23187 stimulation, the loss of [H-3]AA from nuclear membrane phosph
olipids accounted for 88.1 +/- 5.8% of the total loss from phospholipi
ds and for 92.9 +/- 2.3% of the total [H-3]AA released into the medium
. These results demonstrate for the first time that agonist-stimulated
translocation of cPLA(3) to the nuclear envelope is associated with p
hospholipid hydrolysis which is preferentially localized to that site.