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Title: The Role of Calcium and Phosphorylation in the Activation of cPLA2 During TNF-induced Apoptosis
Authors: Draper, David William
Advisors: Scott M Laster, Committee Chair
Keywords: phosphorylation
Issue Date: 12-Apr-2004
Degree: PhD
Discipline: Microbiology
Abstract: Tumor necrosis factor (TNF) is a pleotropic cytokine that mediates many inflammatory and innate immune responses. TNF also causes apoptosis in certain transformed cell lines and cells that are infected with certain viruses or intracellular bacteria. Cytosolic phospholipase A2 (cPLA2) is an inflammatory enzyme that mediates its activities, by specifically catalyzing the release of arachidonic acid leading to the generation of eicosanoids. The activity of cPLA2 is necessary during TNF-induced apoptosis and the goal of this study was to identify signals that mediate the activation of cPLA2 during cell death. Intracellular calcium and phosphorylation are well documented to activate cPLA2 under many inflammatory conditions. Therefore, I examined the ability of these signals to regulate cPLA2 during TNF-induced apoptosis. I first examined calcium levels during the TNF-induced apoptosis of C3HA fibroblasts and determined that an influx of extracellular calcium occurs early during cell death. This influx, as well as the release [3H]arachidonic acid, was blocked by verapamil indicating that the calcium response is necessary for the activation of cPLA2 during this process. To analyze the effects that phosphorylation has during TNF-induced apoptosis, cPLA2 proteins, containing serine phosphorylation site mutations, were stably overexpressed in WM793 melanoma cells. Although PMA was able to enhance the release of [3H]arachidonic acid from cells that overexpressed cPLA2, the treatment of the same cells with TNF and cycloheximide had no effect. However, subsequent experiments using PMA demonstrated novel roles for the phosphorylation of Ser-437 and -727. One was an activation role for Ser-437 as its phosphorylation enhanced [3H]arachidonic acid release. The other was an inhibitory role for the phosphorylation of Ser-727 as its mutation suppressed the release in response to PMA. In conclusion, though the activation of cPLA2, by calcium responses, during apoptotic and non-apoptotic systems is consistent, the regulation of cPLA2 by phosphorylation may involve both positive and negative regulatory signals.
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