Possible role of phosphorylation in the function of chicken MyoD1.
Possible role of phosphorylation in the function of chicken MyoD1.
复制标题
磷酸化在鸡 MyoD1 功能中的可能作用。
DOI：
10.1016/s0021-9258(19)50252-6
复制
发表时间：
1993
期刊：
影响因子：
--
通讯作者：
S. Nakamura
中科院分区：
文献类型：
--
作者：
S. Nakamura
鸡MyoD 1（CMD 1），相当于小鼠MyoD 1，在鸡骨骼肌中表达（Lin，Z.是的，德谢讷角一、Eldridge，J.，和Paterson，B. M. 03 The Dog of the Woman（1989）3，986-996），并纯化至几乎同质。该CMD 1通过32 P标记实验直接证明是磷蛋白。磷酸化氨基酸分析表明，只有丝氨酸残基被磷酸化。鸡胚11天胸肌原代培养物中CMD 1的磷酸化氨基酸也是丝氨酸。磷酸化CMD 1在Sf 9细胞中产生的SDS-聚丙烯酰胺凝胶电泳上的电泳迁移率和从原代培养的肌肉中获得的电泳迁移率是不可区分的。凝胶阻滞和甲基化干扰试验表明，纯化的CMD 1结合特异性的小鼠肌肉肌酸激酶增强子在体外翻译的E12的组合。CMD 1单独对靶DNA几乎没有亲和力。当用小牛肠磷酸酶处理纯化的CMD 1时，其与E12组合的靶DNA的亲和力降低约5倍。通过小麦胚芽提取物中含有的激酶对CMD 1的可能的再磷酸化，至少部分地恢复了亲和力。这些结果表明，CMD 1的磷酸化可能参与了肌肉分化的调节。
Chicken MyoD1 (CMD1), an equivalent to the mouse MyoD1, expressed in chicken skeletal muscle (Lin, Z.-Y., Dechesne, C. A., Eldridge, J., and Paterson, B. M. (1989) Genes & Dev. 3, 986-996), was produced in Spodoptera frugiperda (Sf9) cells by Baculovirus expression vector and purified to almost homogeneity. This CMD1 was directly demonstrated to be a phosphoprotein by a 32P-labeling experiment. Phosphoamino acid analysis revealed that only serine residue was phosphorylated. Phosphoamino acid of CMD1 from chick primary culture of 11-day embryonic breast muscle was also serine. Electrophoretic mobility on SDS-polyacrylamide gel electrophoresis of the phosphorylated CMD1 produced in Sf9 cells and that obtained from primary culture of muscle were indistinguishable. Gel retardation and methylation interference assays showed that purified CMD1 bound specifically to the mouse muscle creatine kinase enhancer in combination with the in vitro translated E12. CMD1 alone had almost no affinity to the target DNA. When purified CMD1 was treated with calf intestinal phosphatase, its affinity to the target DNA in combination with E12 was reduced by approximately 5-fold. The affinity recovered at least partially by possible rephosphorylation of CMD1 by kinase(s) contained in wheat germ extract. These results suggested that phosphorylation of CMD1 could be involved in the regulation of muscle differentiation.