Calcium-mediated Translocation of Cytosolic Phospholipase A2 to the Nuclear Envelope and Endoplasmic Reticulum

Abstract

Cytosolic phospholipase A 2 (cPLA 2 ) is activated by a wide variety of stimuli to release arachidonic acid, the precursor of the potent inflammatory mediators prostaglandin and leukotriene. Specifically, cPLA 2 releases arachidonic acid in response to agents that increase intracellular Ca 2؉. In vitro data have suggested that these agents induce a translocation of cPLA 2 from the cytosol to the cell membrane, where its substrate is localized. Here, we use immunofluorescence to visualize the translocation of cPLA 2 to distinct cellular membranes. In Chinese hamster ovary cells that stably overexpress cPLA 2 , this enzyme translocates to the nuclear envelope upon stimulation with the calcium ionophore A23187. The pattern of staining observed in the cytoplasm suggests that cPLA 2 also translocates to the endoplasmic reticulum. We find no evidence for cPLA 2 localization to the plasma membrane. Translocation of cPLA 2 is dependent on the calcium-dependent phospholipid binding domain, as a calcium-dependent phospholipid binding deletion mutant of cPLA 2 (⌬CII) fails to translocate in response to Ca 2؉ . In contrast, cPLA 2 mutated at Ser-505, the site of mitogen-activated protein kinase phosphorylation, translocates normally. This observation, combined with the observed phosphorylation of ⌬CII, establishes that translocation and phosphorylation function independently to regulate cPLA 2 . The effect of these mutations on cPLA 2 translocation was confirmed by subcellular fractionation. Each of these mutations abolished the ability of cPLA 2 to release arachidonic acid, establishing that cPLA 2 -mediated arachidonic acid release is strongly dependent on both phosphorylation and translocation. These data help to clarify the mechanisms by which cPLA 2 is regulated in intact cells and establish the nuclear envelope and endoplasmic reticulum as primary sites for the liberation of arachidonic acid in the cell.The 85-kDa cytosolic phospholipase A 2 (cPLA 2 ), 1 which selectively releases arachidonic acid from the sn-2 position of membrane phospholipids, is crucial to the initiation of the inflammatory response. cPLA 2 activity is stimulated by a wide variety of agents, including the proinflammatory cytokines interleukin 1 (1, 2) and tumor necrosis factor (3), macrophage colony-stimulating factor (4), thrombin (5, 6), ATP (5), mitogens (7-10), and endothelin (11). The release of arachidonic acid is the rate-limiting step in the generation of prostaglandins and leukotrienes, the proinflammatory eicosanoids. Cleavage of arachidonoyl-containing phospholipids also results in the release of lysophospholipid, the precursor of the inflammatory mediator platelet-activating factor (12).cPLA 2 is expressed in many cell types. Many of these are associated with the inflammatory response, such as monocytes (4), neutrophils (13), and synovial fibroblasts (14). However, cPLA 2 is also expressed in kidney, spleen, heart, lung, liver, testis, and hippocampus (15). This diverse pattern of expression is consistent with accumulating evidence tha...

Intraarticular Osteoid Osteoma Associated with Synovitis: A Possible Role of Cyclooxygenase-2 Expression by Osteoblasts in the Nidus

“…Cytosolic PLA2 is present in almost all cells and mediates the release of arachidonic acid from the lipid bi-layer of the cell membrane (30,31). Inflammatory cytokines IL-1␤ and TNF␣ are stimulants for the activation of the arachidonic acid metabolic pathway.…”

The bovine papillomavirus type 1 E6 oncoprotein sensitizes cells to tumor necrosis factor alpha-induced apoptosis

“…It is possible that E6BP associates with and activates endogenous PLA 2 of mammalian origin. Interestingly, immediately after cell activation, cPLA 2 was shown to translocate from the cytosol to the perinuclear envelope and the ER (Schievella et al, 1995), a location where E6BP was found. Moreover, the enzymatic activity of cPLA 2 is calcium dependent (Qiu et al (1998) and references therein).…”

Inhibition of human cytosolic phospholipase A2 by human annexin V

“…Recent work demonstrates that, on activation, cPLA # translocates to the nuclear envelope, where binding to specific regions must occur [11,12]. It is of interest that it has now been shown that annexin V also translocates to this nuclear envelope on cell activation [13] and hence a modulatory role of this annexin on cPLA # cannot be discounted.…”

“…Annexin V has a high intracellular concentration, where it can form up to 2 % of total cell protein in certain tissues [10]. Therefore, considering that both annexin V and cPLA # are found within the cytosol and have both been demonstrated to translocate to the nuclear membrane on increases in intracellular Ca# + levels [11][12][13], it is of importance to investigate mechanisms by which annexin V could modulate cPLA # catalytic activity and, in particular, whether a substrate depletion mechanism could operate successfully in the cell.…”