Chen X Y, Lo T C
Department of Biochemistry, University of Western Ontario, London, Canada.
J Cell Physiol. 1993 Oct;157(1):145-57. doi: 10.1002/jcp.1041570119.
The present investigation examines the function and site(s) of involvement of an ecto-protein kinase and its substrate protein (a cell surface 112 kDa protein) in the in vitro myogenic pathway. The phosphorylated 112 kDa protein (p112) has recently been shown to be involved in myogenesis. Not much information is currently available on the role of the ecto-protein kinase and the 112 kDa protein in modulating the expression of the myogenic factors and various muscle-specific proteins. Five different p112-deficient rat myoblasts were used to examine the temporal order of the in vitro expression of the myogenic components; namely, L6 myoblasts treated with BrdUrd or phloretin, a conditional p112-defective mutant (clone D1), an ecto-protein kinase-deficient mutant (clone F72), and a mutant defective in the 112 kDa protein (clone D1/S4). All these p112-deficient myoblasts were also impaired in myogenesis. The absence of p112, ecto-protein kinase, and/or the 112 kDa protein was found to have no effect on the Myf-5 mRNA level. However, the expected increase in NCAM and Myf-4 mRNAs was not observed in any of the p112-deficient myoblasts examined. This suggests that the p112 site of action is probably located upstream of the Myf-4 and NCAM sites in the myogenic pathway. While 7-28 fold increases in the MLC, MHC, and TnT transcripts were observed during myogenesis, such increases were not observed in the p112-deficient myoblasts. However, when mutant D1/S4 was transfected with the myf-4 cDNA, expression of Myf-4 in the transfectant resulted in increased level of the MLC, MHC, and TnT mRNAs, and in myotube formation, even though the Myf-5 and NCAM mRNA levels and p112 were not altered. This suggests that p112 may function by activating transcription of Myf-4, which will subsequently promote the expression of muscle-specific proteins and myotube formation. In the absence of p112, Myf-5 cannot activate the expression of Myf-4, NCAM, MLC, MHC, TnT, and myotube formation. If all these components are involved in the same myogenic pathway, then p112 may be acting downstream from Myf-5, and upstream from NCAM and Myf-4.
本研究考察了一种胞外蛋白激酶及其底物蛋白（一种细胞表面112 kDa蛋白）在体外成肌途径中的功能及作用位点。最近研究表明，磷酸化的112 kDa蛋白（p112）参与了肌生成过程。目前关于胞外蛋白激酶和112 kDa蛋白在调节成肌因子及各种肌肉特异性蛋白表达方面的作用，所知信息不多。使用五种不同的p112缺陷型大鼠成肌细胞来研究成肌成分在体外表达的时间顺序；即，用BrdUrd或根皮素处理的L6成肌细胞、一种条件性p112缺陷突变体（克隆D1）、一种胞外蛋白激酶缺陷突变体（克隆F72）以及一种112 kDa蛋白缺陷的突变体（克隆D1/S4）。所有这些p112缺陷型成肌细胞在肌生成方面也存在缺陷。研究发现，p112、胞外蛋白激酶和/或112 kDa蛋白的缺失对Myf - 5 mRNA水平没有影响。然而，在所检测的任何p112缺陷型成肌细胞中，均未观察到预期的NCAM和Myf - 4 mRNA增加。这表明p112的作用位点可能位于成肌途径中Myf - 4和NCAM位点的上游。在肌生成过程中，观察到MLC、MHC和TnT转录本增加了7 - 28倍，但在p112缺陷型成肌细胞中未观察到这种增加。然而，当用myf - 4 cDNA转染突变体D1/S4时，转染体中Myf - 4的表达导致MLC、MHC和TnT mRNA水平升高，并形成了肌管，尽管Myf - 5和NCAM mRNA水平以及p112未发生改变。这表明p112可能通过激活Myf - 4的转录发挥作用，进而促进肌肉特异性蛋白的表达和肌管形成。在缺乏p112的情况下，Myf - 5无法激活Myf - 4、NCAM、MLC、MHC、TnT的表达以及肌管形成。如果所有这些成分都参与同一条成肌途径，那么p112可能作用于Myf - 5的下游，以及NCAM和Myf - 4的上游。
