Secretion from rat basophilic RBL-2H3 cells is associated with diphosphorylation of myosin light chains by myosin light chain kinase as well as phosphorylation by protein kinase C.

The phosphorylation of myosin light chains and heavy chains by protein kinase C is known to be temporally correlated with Ca(2+)-dependent secretion of granules from RBL-2H3 cells (Ludowyke, R. I., Peleg, I., Beaven, M. A., and Adelstein, R. S. (1989) J. Biol. Chem. 264, 12492-12501). We now report that whereas myosin light chains are predominantly monophosphorylated by the Ca2+/calmodulin-dependent myosin light chain kinase at serine 19 in unstimulated cells, stimulation of RBL-2H3 cells with antigen or other stimulants causes additional phosphorylation of myosin light chains by myosin light chain kinase at threonine 18, as well as by protein kinase C at serine 1 or serine 2. This diphosphorylation at serine 19 and threonine 18 by myosin light chain kinase and the monophosphorylation by protein kinase C is correlated with the rate and extent of degranulation. Secretion occurs whenever phosphorylation by both enzymes is stimulated by antigen or by the combination of low concentrations of A23187 (50 nM) and phorbol 12-myristate 13-acetate (20 nM). These phosphorylations appear to be closely associated with exocytosis in RBL-2H3 cells. Thus, phosphorylation, as well as secretion, can be blocked by the kinase inhibitors KT5926 and ML-7. More specifically, phorbol ester alone induces phosphorylation of myosin light chains by protein kinase C exclusively, but fails to induce secretion until accompanied by low concentrations of A23187, which activates myosin light chain kinase. Conversely, selective suppression of phosphorylation by protein kinase C (with Ro31-7549 in antigen-stimulated cells) suppresses degranulation, thereby indicating a requirement for protein kinase C.