Abstract
To create the unique properties of a certain cellular membrane, both the composition and the metabolism of membrane phospholipids are key factors. Phospholipase A(2) (PLA(2)), with hydrolytic enzyme activities at the sn-2 position in glycerophospholipids, plays critical roles in maintaining the phospholipid composition as well as producing bioactive lipid mediators. In this study we examined the contribution of a Ca(2+)-independent group IVC PLA(2) isozyme (cPLA(2)gamma), a paralogue of cytosolic PLA(2)alpha (cPLA(2)alpha), to phospholipid remodeling. The enzyme was localized in the endoplasmic reticulum and Golgi apparatus, as seen using green fluorescence fusion proteins. Electrospray ionization mass spectrometric analysis of membrane extracts revealed that overexpression of cPLA(2)gamma increased the proportion of polyunsaturated fatty acids in phosphatidylethanolamine, suggesting that the enzyme modulates the phospholipid composition. We also found that H(2)O(2) and other hydroperoxides induced arachidonic acid release in cPLA(2)gamma-transfected human embryonic kidney 293 cells, possibly through the tyrosine phosphorylation pathway. Thus, we propose that cPLA(2)gamma is constitutively expressed in the endoplasmic reticulum and plays important roles in remodeling and maintaining membrane phospholipids under various conditions, including oxidative stress.
Highlights
To create the unique properties of a certain cellular membrane, both the composition and the metabolism of membrane phospholipids are key factors
Subcellular Localization of cPLA2␥—To observe the subcellular localization of cPLA2␥, CHO cells were transiently transfected with a vector coding for cPLA2␥ fused to green fluorescence protein (GFP) (GFPcPLA2␥), and the distribution of the chimeric proteins was examined
Western blotting analysis of protein extracts from cPLA2␥-overexpressing HEK293 cells confirmed the assignment of the localization of cPLA2␥ to the microsomal fraction, in accordance with the results reported previously for cPLA2␥ expressed in CHO cells [19] and Sf9 [20]
Summary
Phospholipase A2 (PLA2), with hydrolytic enzyme activities at the sn-2 position in glycerophospholipids, plays critical roles in maintaining the phospholipid composition as well as producing bioactive lipid mediators. Biological membranes contain a complicated mixture of phospholipids differing from each other with respect to their head-group structure, hydrocarbon chain length, and degree of unsaturation of the acyl chains. The complexity of these phospholipid structures results in their diverse roles in membrane dynamics, protein regulation, signal transduction, and vesicular secretion. In this study we demonstrated that cPLA2␥ is localized in the endoplasmic reticulum (ER) and Golgi and is involved in the mobilization of fatty acids in phosphatidylethanolamine (PE). During the search for regulators of the enzymatic activity at the cellular level, we found that reactive oxygen species (ROS) activate the activity of cPLA2␥, possibly through the tyrosine phosphorylation pathway
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