Choi O H, Adelstein R S, Beaven M A
Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1994 Jan 7;269(1):536-41.
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.
已知蛋白激酶C对肌球蛋白轻链和重链的磷酸化与RBL - 2H3细胞中颗粒的钙（Ca2+）依赖性分泌在时间上相关（Ludowyke, R. I., Peleg, I., Beaven, M. A., and Adelstein, R. S. (1989) J. Biol. Chem. 264, 12492 - 12501）。我们现在报告，在未受刺激的细胞中，肌球蛋白轻链主要由钙/钙调蛋白依赖性肌球蛋白轻链激酶在丝氨酸19处单磷酸化，而用抗原或其他刺激物刺激RBL - 2H3细胞会导致肌球蛋白轻链激酶在苏氨酸18处以及蛋白激酶C在丝氨酸1或丝氨酸2处对肌球蛋白轻链进行额外的磷酸化。肌球蛋白轻链激酶在丝氨酸19和苏氨酸18处的双磷酸化以及蛋白激酶C的单磷酸化与脱颗粒的速率和程度相关。只要抗原或低浓度的A23187（50 nM）和佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（20 nM）的组合刺激这两种酶的磷酸化，就会发生分泌。这些磷酸化似乎与RBL - 2H3细胞中的胞吐作用密切相关。因此，激酶抑制剂KT5926和ML - 7可以阻断磷酸化以及分泌。更具体地说，单独的佛波酯仅诱导蛋白激酶C对肌球蛋白轻链的磷酸化，但在伴有低浓度的A23187（激活肌球蛋白轻链激酶）之前不会诱导分泌。相反，在抗原刺激的细胞中用Ro31 - 7549选择性抑制蛋白激酶C的磷酸化会抑制脱颗粒，从而表明需要蛋白激酶C。
