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 cyto solic phospholipase A(2) (cPLA(2)) after aggregation of cell-surface h igh-affinity Fc receptors for IgE (Fc epsilon RI) on mast cells. MCII mast cells (a factor-dependent bone-marrow-derived murine mast cell li ne) produce significant amounts of leukotriene C-4 (LTC(4)) (70 ng/10( 6) cells) on cross-linking of Fc epsilon RI. Using enzymic and immunoc hemical 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 fo rms (i.e. secretory PLA(2) or Ca2+-independent cytosolic PLA(2)) could not be detected. Therefore MCII mast cells represent an excellent cel lular 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 activat ion of Fc epsilon RI by cross-linking, cPLA(2) in MCII mast cells exhi bited a decreased electrophoretic mobility and its enzyme activity was increased 3-fold. Treatment with phosphatase reversed both the altere d electrophoretic mobility and the enhanced enzyme activity demonstrat ing 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-activate d protein kinases in MCII mast cells. Tyrosine phosphorylation may be a critical component in this process, as genistein, an inhibitor of pr otein tyrosine kinases, blocked the activation of cPLA(2). Using anti- phosphotyrosine antibodies we observed that the activating phosphoryla tion was not on tyrosine residues of cPLA(2), indicating that tyrosine kinases participate upstream in the signalling cascade that couples F c epsilon RI to cPLA(2). We conclude that in MCII mast cells cPLA(2) i s activated by kinase-dependent mechanisms and may be responsible for Fc epsilon RI-induced mobilization of arachidonic acid for the generat ion of LTC(4).