Overexpression of Cytosolic Group IVA Phospholipase A2 Protects Cells from Ca2+-dependent Death

Abstract

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 ␣ (EGFPcPLA 2 ␣) in these cells prevents ionomycin-induced phosphatidylserine externalization and death. Cells expressing the cPLA 2 ␣ mutant D43N, which does not bind calcium, retain their susceptibility to ionomycin-induced cell death. Both nonexpressing and EGFP-D43N-cPLA 2 ␣-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 ␣-expressing cells than in cells expressing normal amounts of cPLA 2 ␣. 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.

JNK and Ceramide Kinase Govern the Biogenesis of Lipid Droplets through Activation of Group IVA Phospholipase A2

“…[ 3 H]Arachidonic Release-Stimulated [ 3 H]AA release from cells, which is an estimate of cPLA 2 ␣ activity, was measured as described (7,10,27 4. After 1 h, cells were washed and coverslips mounted in a static chamber on an inverted Nikon TE2000U microscope with a conventional epifluorescence system.…”

“…Asp-43 resides in the Ca 2ϩ -binding loop 1 at the membrane binding face of the C2 domain of the enzyme and participates in the coordination of the two Ca 2ϩ ions that drive membrane binding (11). Substitution of this residue for Asn increases 100-fold the Ca 2ϩ requirement for activity in vitro (31), and it prevents translocation of the enzyme to membranes in response to Ca 2ϩ ionophores (15,27). In agreement with this, EGFP-D43N-cPLA 2 ␣ did not relocate in CHO-K1 cells stimulated with 5 M ionomycin, unlike the wild type enzyme, which translocated to perinuclear membranes and structures resembling the Golgi apparatus (Fig.…”

Predicting Outcome in Follicular Lymphoma by Using Interactive Gene Pairs

“…In addition, the expression of PLA2G3, part of the extracellular signalregulated kinase/mitogen-activated protein kinase signaling pathway, has been shown to reliably distinguish diffuse large B cell lymphoma (DLBCL) from FL cases (20), and seems to have a role in stimulating both tumor cell growth (47) and angiogenesis (48), and protecting against apoptosis (49).…”

The cationic cluster of group IVA phospholipase A2 (Lys488/Lys541/Lys543/Lys544) is involved in translocation of the enzyme to phagosomes in human macrophages

“…The enhanced green fl uorescent protein (EGFP)-PLC ␦ 1 -PH construct with the pleckstrin domain from the PLC ␦ 1 was kindly provided by Dr. Tobias Meyer (Stanford University Medical Center, Stanford, CA) ( 24 ). The EGFP-cPLA 2 ␣ plasmid, the mutant for the PIP 2 binding site EGFP-4KE/A-cPLA 2 ␣ , the iPLA 2 -VIA-enhanced yellow fl uorescent protein (EYFP) plasmid, and the sPLA 2 -V-EGFP plasmid have been described elsewhere ( 17,25,26 ). pDsRed-Monomer-C1 was obtained from Clontech (Mountain View, CA).…”