E1A can provoke G1 exit that is refractory to p21 and independent of activating Cdk2
E1A can provoke G1 exit that is refractory to p21 and independent of activating Cdk2
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DOI：
10.1161/01.res.85.4.319
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发表时间：
1999-08-20
影响因子：
20.1
通讯作者：
Schneider, MD
中科院分区：
文献类型：
--
作者：
Akli, S;Zhan, S;Schneider, MD
E1a可以通过取代肿瘤抑制蛋白中的E2F转录因子，以及通过一条特征较少的p300依赖的途径，在心肌细胞和其他类型的细胞中唤起G1退出。绕过Pocket蛋白(通过E2F-1的过度表达)可以复制Pocket蛋白的失活效果(通过E1a结合)；然而，Pocket蛋白除了与E2F结合外，还与许多分子靶标结合。因此，E1a结合的口袋蛋白可能参与了E2F-1以外的细胞周期重入机制。为了验证这一假设，我们使用腺病毒基因转移在新生大鼠心肌细胞中表达各种E2F-1和E1a蛋白，这些心肌细胞已经对有丝分裂血清不敏感，但没有或存在几种互补的细胞周期抑制物-p16、p21或显性阴性的细胞周期蛋白依赖性激酶-2(CDK2)。E2F-1与Rb的结合既不是G1退出的必要条件，也不是充分条件，而DNA结合是必需的；因此，外源E2F-1不仅仅通过竞争Rb的“口袋”来发挥作用。“通用”的CDK抑制剂p21可阻断E2F-1诱导的G1期退出，但不能被CDK4/6的特异性抑制剂p16阻断；p21对E2F-1诱导的细胞周期蛋白E和A有抑制作用，但可阻止它们对CDK2激酶活性的刺激。此外，E2F-1诱导的G1期退出被显性负性CDK2阻断。强迫表达Cyclin E可诱导内源性CDK2活性，但不能诱导G1期退出。因此，E2F-1诱导的CDK2功能是必要的，但不是充分的，以触发心肌细胞的DNA合成。相反，口袋蛋白结合形式的E1a诱导G1退出，而通过E1Ap300途径的G1退出对p21抑制敏感。两个E1A途径-通过口袋蛋白和通过p300-上调周期蛋白E和A以及CDK2的活性，与CDK2在E1A诱导的G1退出中的作用一致。然而，p21同样阻断了两条E1a通路诱导的CDK2激酶活性。因此，如果口袋蛋白结合域完整，E1a可以在不增加CDK2活性的情况下导致G1退出。在U2OS细胞中，如果口袋蛋白结合域是完整的，E1a也覆盖了p21；因此，E1a的这一新功能并不是心肌细胞所独有的。综上所述，E1a与Pocket蛋白的结合具有超越E2F-1单独产生的影响，并且可以驱动S进入对p21具有抗性的阶段，而不依赖于CDK2功能的增加。这表明其他内源性Rb结合蛋白或替代的E1a靶点可能参与了这一过程。
E1A can evoke G1 exit in cardiac myocytes and other cell types by displacing E2F transcription factors from tumor suppressor "pocket" proteins and by a less well-characterized p300-dependent pathway. Bypassing pocket proteins (through overexpression of E2F-1) reproduces the effect of inactivating pocket proteins (through E1A binding); however, pocket proteins associate with a number of molecular targets apart from E2F. Hence, pocket protein binding by E1A might engage mechanisms for cell cycle reentry beyond those induced by E2F-1. To test this hypothesis, we used adenoviral gene transfer to express various E2F-1 and E1A proteins in neonatal rat cardiac myocytes that are already refractory to mitogenic serum, in the absence or presence of several complementary cell cycle inhibitors-p16, p21, or dominant-negative cyclin-dependent kinase-2 (Cdk2). Rb binding by E2F-1 was neither necessary nor sufficient for G1 exit, whereas DNA binding was required; thus, exogenous E2F-1 did not merely function by competing for the Rb "pocket." E2F-1-induced G1 exit was blocked by the "universal" Cdk inhibitor p21 but not by p16, a specific inhibitor of Cdk4/6; p21 was permissive for E2F-1 induction of cyclins E and A, but prevented their stimulation of Cdk2 kinase activity. In addition, E2F-1-induced G1 exit was blocked by dominant-negative Cdk2. Forced expression of cyclin E induced endogenous Cdk2 activity but not G1 exit. Thus, E2F-1-induced Cdk2 function was necessary, although not sufficient, to trigger DNA synthesis in cardiac muscle cells. In contrast, pocket protein-binding forms of E1A induced G1 exit that was resistant to inhibition by p21, whereas G1 exit via the E1A p300 pathway was sensitive to inhibition by p21. Both E1A pathways-via pocket proteins and via p300-upregulated cyclins E and A and Cdk2 activity, consistent with a role for Cdk2 in G1 exit induced by E1A. However, p21 blocked Cdk2 kinase activity induced by both E1A pathways equally. Thus, E1A can cause G1 exit without an increase in Cdk2 activity, if the pocket protein-binding domain is intact. E1A also overrides p21 in U2OS cells, provided the pocket protein-binding domain is intact; thus, this novel function of E1A is not exclusive to cardiac muscle cells. In summary, E1A binding to pocket proteins has effects beyond those produced by E2F-1 alone and can drive S-phase entry that is resistant to p21 and independent of an increase in Cdk2 function. This suggests the potential involvement of other endogenous Rb-binding proteins or of alternative E1A targets.