Myosin phosphatase and myosin phosphorylation in differentiating C2C12 cells.
Myosin phosphatase and myosin phosphorylation in differentiating C2C12 cells.
复制标题
分化 C2C12 细胞中的肌球蛋白磷酸酶和肌球蛋白磷酸化。
DOI：
10.1023/b:jure.0000009810.36038.53
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发表时间：
2003
影响因子：
2.7
通讯作者：
Hartshorne,DavidJ
中科院分区：
文献类型：
--
作者：
Wu,Yue;Erdodi,Ferenc;Murányi,Andrea;Nullmeyer,KevinD;Lynch,RonaldM;Hartshorne,DavidJ
C2 C12细胞为研究非肌细胞向骨骼肌细胞的分化提供了一个有用的模型。肌球蛋白磷酸化和相关酶的变化，重点是肌球蛋白磷酸酶（MP）在C2 C12分化的前6天进行了分析。从肌球蛋白磷酸酶靶亚基1（MYPT 1）（主要在非肌细胞中）到MYPT 2表达增加的转变。估计MYPT 1/2的水平，并且与第6天分化的分离的肌管中的浓度相比，两种亚型在未分化或部分分化的细胞中更高。1型蛋白磷酸酶催化亚基δ亚型（PP 1c δ）的表达谱相似。磷酸酶活性，使用磷酸化平滑肌和骨骼肌肌球蛋白，估计总细胞裂解物和分离的肌管。一般来说，平滑肌肌球蛋白是首选底物。虽然MYPT 1/2和PP 1c δ的表达在分离的肌管中显著降低，但磷酸酶活性没有降低到相应的水平。大部分MP活性是由于PP 1c，如冈田酸所示。尽管MYPT 1/2和PP 1c δ的表达相对较高，但在第2天（肌肉特异性蛋白表达开始）观察到非肌肉肌球蛋白的显著磷酸化（超过总肌球蛋白的50%），并观察到单磷酸化和二磷酸化轻链。通过1-（5-氯萘-1-磺酰基）-1H-六氢-1，4-二氮杂环庚三烯盐酸盐（ML-9）或通过由MLCK的自抑制结构域设计的构建体对MLCK的部分抑制导致分化3天后小肌管（3-5个核）的增加和较大肌管的减少（与对照相比）。ML-9的作用不是由于细胞内Ca 2+水平的降低。这些结果表明，非肌肉肌球蛋白的磷酸化在肌管的生长中是重要的，无论是在融合过程中形成更大的肌管，或间接地，通过其在肌节组织中的作用。
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, δ isoform (PP1cδ). 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 PP1cδ 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 PP1cδ, 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 l-(5-chloronaphthalene-l-sulphonyl)-1H-hexahydro-l,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.