Wu Yue, Erdodi Ferenc, Murányi Andrea, Nullmeyer Kevin D, Lynch Ronald M, Hartshorne David J
Muscle Biology Group, University of Arizona, Tucson, AZ 85721, USA.
J Muscle Res Cell Motil. 2003;24(8):499-511. doi: 10.1023/b:jure.0000009810.36038.53.
C2C12 cells offer a useful model to study the differentiation of non-muscle cells to skeletal muscle cells. Myosin phosphorylation and changes in related enzymes, with an emphasis on myosin phosphatase (MP) were analyzed over the first 6 days of C2C12 differentiation. There was a transition from myosin phosphatase target subunit 1 (MYPT1), predominant in the non-muscle cells to increased expression of MYPT2. Levels of MYPT1/2 were estimated, and both isoforms were higher in non- or partially differentiated cells compared to the concentrations in the differentiated isolated myotubes from day 6. A similar profile of expression was estimated for the type 1 protein phosphatase catalytic subunit, delta isoform (PP1c delta). Phosphatase activities, using phosphorylated smooth and skeletal muscle myosins, were estimated for total cell lysates and isolated myotubes. In general, smooth muscle myosin was the preferred substrate. Although the expression of MYPT1/2 and PP1c delta was considerably reduced in isolated myotubes the phosphatase activities were not reduced to corresponding levels. Most of the MP activity was due to PP1c, as indicated by okadaic acid. In spite of relatively high expression of MYPT1/2 and PP1c delta, marked phosphorylation of non-muscle myosin (over 50% of total myosin) was observed at day 2 (onset of expression of muscle-specific proteins) and both mono- and diphosphorylated light chains were observed. Partial inhibition of MLCK by 1-(5-chloronaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine HCl (ML-9) or by a construct designed from the autoinhibitory domain of MLCK, resulted in an increase in small myotubes (3-5 nuclei) after 3 days of differentiation and a decrease in larger myotubes (compared to control). The effect of ML-9 was not due to a reduction in intracellular Ca2+ levels. These results suggest that phosphorylation of non-muscle myosin is important in growth of myotubes, either in the fusion process to form larger myotubes or indirectly, by its role in sarcomere organization.
C2C12细胞为研究非肌肉细胞向骨骼肌细胞的分化提供了一个有用的模型。在C2C12分化的前6天，分析了肌球蛋白磷酸化及相关酶的变化，重点是肌球蛋白磷酸酶（MP）。存在从非肌肉细胞中占主导的肌球蛋白磷酸酶靶向亚基1（MYPT1）到MYPT2表达增加的转变。对MYPT1/2的水平进行了评估，与第6天分化的分离肌管中的浓度相比，非分化或部分分化细胞中这两种亚型的水平更高。对1型蛋白磷酸酶催化亚基δ亚型（PP1cδ）的表达情况进行了类似的评估。使用磷酸化的平滑肌和骨骼肌肌球蛋白对总细胞裂解物和分离的肌管进行了磷酸酶活性测定。总体而言，平滑肌肌球蛋白是首选底物。尽管在分离的肌管中MYPT1/2和PP1cδ的表达大幅降低，但磷酸酶活性并未降至相应水平。如冈田酸所示，大部分MP活性归因于PP1c。尽管MYPT1/2和PP1cδ表达相对较高，但在第2天（肌肉特异性蛋白开始表达时）观察到非肌肉肌球蛋白有明显磷酸化（超过总肌球蛋白的50%），并且观察到单磷酸化和双磷酸化轻链。用1-（5-氯萘-1-磺酰基）-1H-六氢-1,4-二氮杂卓盐酸盐（ML-9）或由肌球蛋白轻链激酶（MLCK）的自抑制结构域设计的构建体对MLCK进行部分抑制，导致分化3天后小肌管（3-5个核）数量增加，大肌管数量减少（与对照相比）。ML-9的作用并非由于细胞内Ca2+水平降低。这些结果表明，非肌肉肌球蛋白的磷酸化在肌管生长中很重要，无论是在形成更大肌管的融合过程中，还是通过其在肌节组织中的作用间接发挥作用。
