Kiess M, Gill R M, Hamel P A
Department of Cellular and Molecular Pathology, University of Toronto, Ontario, Canada.
Cell Growth Differ. 1995 Oct;6(10):1287-98.
The activity of the E2 F-family of transcription factors is tightly linked to control of the cell cycle. p107 and p130, two closely related members of the retinoblastoma protein-family of negative cell cycle regulators, modulate the activity of the E2f-family proteins by direct interaction with these factors. To understand the role of p107 and p130 in progression through or exit from the cell cycle, we have characterized the expression, phosphorylation state, cyclin-binding, and E2f-binding activity of p107 and p130 during terminal differentiation of rat myoblast cells into immature skeletal muscle (myotubes). In exponentially growing L6 myoblasts, p107 is phosphorylated in a cell cycle-dependent manner, and E2f-site binding complexes containing p107 is phosphorylated in a cell cycle-dependent manner, and E2f-site binding complexes containing p107 can be observed throughout the cell cycle. During differentiation of L6 cells, p107 levels are reduced, while p130 protein levels are increased 8-fold. Despite both p107 and p130 becoming hypophosphorylated during myogenesis, the E2F-site DNA-binding complexes containing p107 observed in exponentially growing myoblasts are quantitatively replaced in myotubes with complexes containing only p130. In myotubes, p107 is not associated with E2f-family proteins that are capable of binding DNA. The failure to observe p107-containing complexes in myotubes appears to be due to the differentiation-specific induction of both p130 and cyclin D3, p107 is found in complexes with cyclin D3 in myotubes, and the addition of exogenous cyclin D3 or p130 to lysates from undifferentiated L6 cells was able to disrupt p107-containing E2F-site binding complexes. In myotubes, p130 also forms complexes with cyclin D3 as well as cyclin E, cdk2, and cdk4. We are able to copurify cyclin D3 with cyclin E from myotubes, indicating the presence of a macromolecular complex containing both cyclin E and cyclin D3 simultaneously bound to p130. Thus, in myoblasts, p107 is normally involved in regulation of E2f-family proteins during cell cycle progression, while p130 is a differentiation-specific regulator of E2f activity. Our results also provide evidence that the apparent positive regulator of cell cycle progression, cyclin D3, has a function in terminally differentiated muscle cells.
转录因子E2F家族的活性与细胞周期的调控紧密相关。p107和p130是细胞周期负调控因子视网膜母细胞瘤蛋白家族中两个密切相关的成员，它们通过与这些因子直接相互作用来调节E2f家族蛋白的活性。为了了解p107和p130在细胞周期进程或退出过程中的作用，我们对大鼠成肌细胞向未成熟骨骼肌（肌管）终末分化过程中p107和p130的表达、磷酸化状态、细胞周期蛋白结合以及E2f结合活性进行了表征。在指数生长的L6成肌细胞中，p107以细胞周期依赖性方式磷酸化，并且在整个细胞周期中都能观察到含有p107的E2f位点结合复合物以细胞周期依赖性方式磷酸化。在L6细胞分化过程中，p107水平降低，而p130蛋白水平增加8倍。尽管在肌生成过程中p107和p130都发生了去磷酸化，但在指数生长的成肌细胞中观察到的含有p107的E2F位点DNA结合复合物在肌管中被仅含有p130的复合物定量取代。在肌管中，p107不与能够结合DNA的E2f家族蛋白相关联。在肌管中未观察到含p107复合物似乎是由于p130和细胞周期蛋白D3的分化特异性诱导，在肌管中发现p107与细胞周期蛋白D3形成复合物，并且向未分化的L6细胞裂解物中添加外源细胞周期蛋白D3或p130能够破坏含p107的E2F位点结合复合物。在肌管中，p130还与细胞周期蛋白D3以及细胞周期蛋白E、细胞周期蛋白依赖性激酶2（cdk2）和细胞周期蛋白依赖性激酶4（cdk4）形成复合物。我们能够从肌管中共同纯化细胞周期蛋白D3和细胞周期蛋白E，这表明存在一种同时含有细胞周期蛋白E和细胞周期蛋白D3并与p130结合的大分子复合物。因此，在成肌细胞中，p107通常在细胞周期进程中参与E2f家族蛋白的调节，而p130是E2f活性的分化特异性调节因子。我们的结果还提供了证据，表明细胞周期进程的明显正调节因子细胞周期蛋白D3在终末分化的肌肉细胞中具有功能。
