CALCIUM IONOPHORE-INDUCED SECRETION FROM MAST-CELLS CORRELATES WITH MYOSIN LIGHT-CHAIN PHOSPHORYLATION BY PROTEIN-KINASE-C

basophilic leukemia mast cells (RBL-2H3) secrete histamine when activa ted by Ag, This secretion correlates with increased phosphorylation of myosin light chain by protein kinase C (PKC), Calcium ionophores (A23 187) also elicit secretion, which is enhanced by PMA. To analyze the r oles of Ca2+ and PKC in the secretory process, A23187-induced myosin l ight chain phosphorylation was examined in the presence and absence of PMA, A23187-induced secretion correlated best with myosin tight chain phosphorylation by PKC, not with phosphorylation by myosin light chai n kinase (MLCK). A23187 induced the translocation to membranes of the alpha, beta, delta, and epsilon isozymes of PKC. PMA not only increase d the phosphorylation of myosin light chains at PKC-specific sites (Se r(1) and Ser(2)) but also at sites attributed to MLCK (Ser(19) and Thr (18) - Ser(19)). A23187 plus PMA induced higher levels of secretion co ncomitantly with increased myosin light chain phosphorylation at the P KC-specific sites. However, there was little correlation between the t ranslocation of specific PKC isozymes and the phosphorylation of myosi n light chains by PKC. Activation induced a novel triphosphorylated fo rm of myosin light chain with a higher level of phosphorylation at the diphosphorylated MLCK sites. Quantitation of A23187 plus PMA-induced myosin light chain phosphorylation revealed that phosphorylation at PK C sites increased from zero to 0.35 mol/mol, was little changed at the monophosphorylated MLCK site (0.30 mol/mol), and increased from zero to 0.06 mol/mol at the diphosphorylated MLCK sites. Therefore, Ca2+-in duced secretion correlates best with myosin light chain phosphorylatio n by PKC, and diphosphorylation by MLCK is unlikely to contribute to s ecretion.