PHOSPHORYLATION AND ACTIVATION OF CA2+-SENSITIVE CYTOSOLIC PHOSPHOLIPASE-A(2) IN MCII MAST-CELLS MEDIATED BY HIGH-AFFINITY FC RECEPTOR FOR IGE

In the present study we examined the activation of Ca2+-sensitive cytosolic phospholipase A(2) (cPLA(2)) after aggregation of cell-surface high-affinity Fc receptors for IgE (Fc epsilon RI) on mast cells. MCII mast cells (a factor-dependent bone-marrow-derived murine mast cell line) produce significant amounts of leukotriene C-4 (LTC(4)) (70 ng/10(6) cells) on cross-linking of Fc epsilon RI. Using enzymic and immunochemical analysis we found that cPLA(2) is the predominant form of this enzyme in MCII mast cells (0.2 mu g/mg of total protein) and other forms (i.e. secretory PLA(2) or Ca2+-independent cytosolic PLA(2)) could not be detected. Therefore MCII mast cells represent an excellent cellular model for the study of the biochemical mechanism(s) responsible for Fc epsilon RI-induced activation of cPLA(2) and the involvement of cPLA(2) in Fc epsilon RI-mediated production of LTC(4). After activation of Fc epsilon RI by cross-linking, cPLA(2) in MCII mast cells exhibited a decreased electrophoretic mobility and its enzyme activity was increased 3-fold. Treatment with phosphatase reversed both the altered electrophoretic mobility and the enhanced enzyme activity demonstrating that they were the result of Fc epsilon RI-induced phosphorylation. On cross-linking of Fc epsilon RI, cPLA(2) was phosphorylated within 30 s and appeared to be an early substrate for Fc epsilon RI-activated protein kinases in MCII mast cells. Tyrosine phosphorylation may be a critical component in this process, as genistein, an inhibitor of protein tyrosine kinases, blocked the activation of cPLA(2). Using anti-phosphotyrosine antibodies we observed that the activating phosphorylation was not on tyrosine residues of cPLA(2), indicating that tyrosine kinases participate upstream in the signalling cascade that couples Fc epsilon RI to cPLA(2). We conclude that in MCII mast cells cPLA(2) is activated by kinase-dependent mechanisms and may be responsible for Fc epsilon RI-induced mobilization of arachidonic acid for the generation of LTC(4).