We have shown that antigen-stimulated exocytosis in a mast cell line, RBL- 2H3 cells is accompanied by an increased phosphorylation of the heavy (200 kDa) and light (20 kDa) chains of myosin, which can be attributed to protein kinase C (PKC). In case of 20 kDa myosin light chain, it has been shown that an additional site (serine- 18) was phosphorylated by myosin light chain kinase (MLCK) in unstimulated cells. By using a newly developed one-dimensional isoelectrofocusing technique, however, we found that in antigen stimulated cells, there is de novo phosphorylation of two sites, one by PKC (serine 1) and one by MLCK (threonine-19). The phosphorylation by MLCK, which is a calcium-regulated enzyme, and by PKC, may be significant because exocytosis in RBL-2H3 cells is totally dependent on a rise in [Ca2+]i and the activation of PKC. Consistent with this hypothesis these phosphorylations could be induced by Ca2+-ionophore A23187 and phorbol ester but only at concen- trations that were sufficient to induce secretion. Also selective suppression of MLCK (with EGTA) and PKC (with Ro31-7549) inhibited secretion and the phosphorylation by these enzymes. Therefore, it is plausible that the co-ordination between phosphorylation by MLCK and by PKC is necessary for exocytosis, because some kinase inhibitors (staurosporine, KT5926) suppress equally phosphorylation and secretion, whereas other inhibitors that do not inhibit secretion do not inhibit phosphorylation.
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