title: The role of priming in the regulation of cytosolic Phospholipase A2
Creators: name: Stewart, Allison
Publisher: name: University of Glasgow
Year: 1996
Subjects: subject: Molecular biology.

description: Work carried out in this Thesis tried to elucidate the factors involved in the regulation and priming of cytosolic phospholipase A2 (cPLA2). The 85 kDa cPLA2 originally identified and purified from U937 cells (Clark et al., 1991) was shown to be present in Rat-1 Raf ER4 fibroblasts, Swiss 3T3 fibroblasts and HL60 cells. This enzyme was shown to be acutely primed by cytochalasin B in 5 day DMSO-differentiated HL60 cells and chronically primed by serum in Swiss 3T3 fibroblasts. These priming agents increased tyrosine phosphorylation, which did not result in arachidonate release, but enhanced subsequent agonist-stimulated arachidonate release. This increased tyrosine phosphorylation did not enhance the known regulatory moieties of cytosolic phospholipase A2, such as MAP kinase (Lin et al., 1993), calcium (Channon &amp; Leslie, 1990) and GTP-binding proteins (Cockcroft, 1992), therefore suggesting that an additional factor was involved in both the acute and chronic priming of cPLA2. However, all of the above had a role to play in the regulation of cPLA2. GTPS did not enhance agonist-stimulated arachidonate release; however, a GTP-binding protein was involved in both LPA- and bombesin-stimulated arachidonate release as GDP?S inhibited both agonist-medited responses. Raf-1-stimulated MAP kinase activity enhanced LPA-stimulated arachidonate release, but did not stimulate arachidonate release alone, demonstrating that an additional agonist-stimulated signal was required. LPA, bombesin and fMLP all stimulated an immediate increase in intracellular calcium concentrations in their appropriate cells. The calcium chelator BAPTA inhibited agonist- stimulated arachidonate release to varying degrees in the cells studied, suggesting that calcium is essential for maximal agonist-stimulated arachidonate release. Channon &amp; Leslie (1990) demonstrated that increased calcium concentrations resulted in a translocation of cPLA2 from the cytosol to a membrane fraction. Under all priming conditions, western blotting demonstrated that there was a translocation of phosphorylated cPLA2 from the cytosol to a membrane fraction, although there was no increased calcium concentrations. Therefore, calcium was not responsible for the translocation of the enzyme, but was essential for maximum arachidonate release. The membrane fraction to which cPLA2 translocated under both priming and agonist stimulation was shown to be in the nuclear area and not the plasma membrane as had been suggested. In the fibroblastic cell lines and HL60 cells, cPLA2 was expressed under all conditions studied. However, there was a differential expression of cPLA2 in B- lymphoid cell lines representative of distinct stages in B-lymphocyte development. Therefore, work presented within this Thesis will try to examine the regulation and priming of cytosolic phospholipase A2 in both the adherent and suspension cells studied. The involvement of the factors responsible will be discussed in relation to the nuclear location of the enzyme, compared to the predicted plasma membrane location.