Kim H S, Lee I H, Chung C H, Kang M S, Ha D B
Department of Molecular Biology, College of Natural Sciences, Seoul National University, Korea.
Dev Biol. 1992 Apr;150(2):223-30. doi: 10.1016/0012-1606(92)90237-b.
The pattern of protein phosphorylation was found to change in differentiating chick embryonic myoblasts in culture. The extent of phosphorylation of 42-, 50-, and 100-kDa proteins increased while that of a 63-kDa protein declined in extracts of myoblasts that had been cultured for increasing periods. Of these, the increase in phosphorylation of the 100-kDa protein occurred most dramatically in extracts of myoblasts in an early stage of differentiation and was specifically inhibited by trifluoperazine (TFP) and other calmodulin (CaM) antagonists including chlorpromazine and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7). Treatment of increasing concentrations of TFP to culture medium also decreased the phosphorylation state of the 100-kDa protein and the degree of myoblast fusion in parallel. In addition, levels of both the kinase activity and the 100-kDa protein but not of CaM appeared to rise in the cells cultured for longer periods. These results suggest that (1) a Ca2+/CaM-dependent protein kinase is responsible for phosphorylation of the 100-kDa protein, (2) the TFP-mediated myoblast fusion block may be associated with the inhibitory effect of the drug against the kinase activity, and (3) the increase in phosphorylation state of the 100-kDa protein during myogenic differentiation is due to the rise in levels of the kinase and its substrate.
研究发现，体外培养的分化期鸡胚成肌细胞中蛋白质磷酸化模式发生了变化。在培养时间不断延长的成肌细胞提取物中，42 kDa、50 kDa和100 kDa蛋白质的磷酸化程度增加，而63 kDa蛋白质的磷酸化程度下降。其中，100 kDa蛋白质磷酸化的增加在分化早期的成肌细胞提取物中最为显著，并且受到三氟拉嗪（TFP）以及包括氯丙嗪和N-（6-氨基己基）-5-氯-1-萘磺酰胺（W-7）在内的其他钙调蛋白（CaM）拮抗剂的特异性抑制。向培养基中添加浓度不断增加的TFP进行处理，也会使100 kDa蛋白质的磷酸化状态以及成肌细胞融合程度同时下降。此外，在培养时间较长的细胞中，激酶活性和100 kDa蛋白质的水平均有所升高，但CaM的水平未升高。这些结果表明：（1）一种Ca2+/CaM依赖性蛋白激酶负责100 kDa蛋白质的磷酸化；（2）TFP介导的成肌细胞融合阻滞可能与该药物对激酶活性的抑制作用有关；（3）在成肌分化过程中100 kDa蛋白质磷酸化状态的增加是由于激酶及其底物水平的升高。
