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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...

The death domain of the type 1 tumor necrosis factor receptor (TNFR1) mediates interactions with several proteins involved in signaling the downstream effects of TNF. We have used the yeast interaction trap to isolate a protein, MADD, that associates with the death domain of TNFR1 through its own C-terminal death domain. MADD interacts with TNFR1 residues that are critical for signal generation and coimmunoprecipitates with TNFR1, implicating MADD as a component of the TNFR1 signaling complex. Importantly, we have found that overexpression of MADD activates the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (ERK), and expression of the MADD death domain stimulates both the ERK and c-JUN N-terminal kinase MAP kinases and induces the phosphorylation of cytosolic phospholipase A 2 . These data indicate that MADD links TNFR1 with MAP kinase activation and arachidonic acid release and provide further insight into the mechanisms by which TNF exerts its pleiotropic effects. Tumor necrosis factor (TNF)1 is a central player in the regulation of immune responses. Produced mainly by activated macrophages, TNF promotes a wide variety of cellular activities, including the initiation of inflammation, the induction of antiviral responses, and apoptosis. Two receptors for TNF have been cloned, both of which belong to a superfamily of cell surface receptors that includes the Fas antigen and CD40 (1). The TNF receptors share 30% homology in their extracellular domains but are unrelated in their intracellular domains. The intracellular domains of the Fas antigen and the 55-kDa TNF receptor (TNFR1), both of which can trigger apoptosis, share approximately 28% identity in a region known as the death domain (2, 3). This region of TNFR1 has been shown to be necessary and sufficient for signaling cytotoxicity (3, 4).TNFR1 mediates most of the biological effects of TNF (3, 5-8). Engagement of this receptor activates a diverse group of intracellular signaling pathways. Among the early downstream effects of TNF are the activation of kinases, including members of the MAP kinase family (9 -14), and phospholipases, including cytosolic phospholipase A 2 (cPLA 2 ) (reviewed in Ref. 15). The activation of cPLA 2 results in the release of arachidonic acid, which is metabolized into the potent proinflammatory mediators prostaglandins and leukotrienes. TNF also activates several transcription factors, including NF-B and c-JUN/AP-1, leading to the up-regulation of a large number of genes involved in the inflammatory response (16). The signaling elements involved in initiating these pathways were not discovered until recently, when the yeast two-hybrid system was used to identify proteins that associate directly with TNFR1. Briefly, TRADD (17) is a death domain-containing protein that interacts directly with the death domain of TNFR1. TRADD is believed to act as an adaptor protein that recruits two other proteins, TRAF2 and RIP, to the receptor (18, 19). TRAF2 has been implicated in the pathway leading to the activation...

We have isolated a cDNA clone encoding the major protein-tyrosine-phosphatase (protein-tyrosine-phos- tTo whom reprint requests should be addressed. §The sequence reported in this paper has been deposited in the GenBank data base (accession no. M31724). 2735The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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