Involvement of Myc activity in a G1/S-promoting mechanism parallel to the pRb/E2F pathway
Involvement of Myc activity in a G1/S-promoting mechanism parallel to the pRb/E2F pathway
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DOI：
10.1128/mcb.20.10.3497-3509.2000
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发表时间：
2000-05-01
影响因子：
5.3
通讯作者：
Lukas, J
中科院分区：
文献类型：
--
作者：
Santoni-Rugiu, E;Falck, J;Lukas, J
视网膜母细胞瘤蛋白（pRb）/E2 F途径调节哺乳动物细胞复制DNA的承诺。另一方面，有丝分裂原刺激的细胞剥夺E2 F活性仍然可以保持生理相关水平的细胞周期蛋白E依赖性激酶活性，并逐渐进入S期，这表明存在的DNA合成诱导机制平行于pRb/E2 F轴。在这里，我们表明，可调节的异位表达的细胞周期蛋白E或转录活性Myc可以迅速诱导DNA合成的U2 OS衍生的细胞系，其E2 F活性被阻断的组成型活性pRb（pRb三角洲cdk）突变体。Myc的作用与Cdc 25 A磷酸酶和细胞周期蛋白E-CDK 2激酶激活相关，并通过用显性阴性（dn）MadMyc嵌合体拮抗Myc活性而消除。此外，虽然内源性E2 F或Myc活性的消除仅延迟和降低同步化U2 OS细胞或大鼠二倍体成纤维细胞中的DNA合成，但同时两者的中和作用使其消除，而异位MSc和E2 F1挽救了由pRb Delta cdk引起的G（1）/S延迟（或dnDP 1）和MadMyc，细胞周期蛋白E或Cdc 25 A可以恢复DNA复制，甚至在同时暴露于pRb Delta cdk和MadMyc的细胞中。然而，dnCDK 2的共表达中和了所有这些拯救作用。最后，细胞周期蛋白E和Cdc 25 A在G（1）/S转换时的适当转录需要Myc和E2 F活性，并且亚阈值水平的异位细胞周期蛋白E和Cdc 25 A协同恢复具有沉默的Myc和E2 F活性的细胞中的DNA合成。这些结果表明，Myc控制G（1）/S-促进机制调节细胞周期蛋白E-CDK 2平行于“经典”pRb/E2 F途径。
The retinoblastoma protein (pRb)/E2F pathway regulates commitment of mammalian cells to replicate DNA. On the other hand, mitogen-stimulated cells deprived of E2F activity can still maintain physiologically relevant levels of cyclin E-dependent kinase activity and gradually enter S phase, suggesting the existence of a DNA synthesis-inducing mechanism parallel to the pRb/E2F axis. Here we show that regulatable ectopic expression of cyclin E or transcriptionally active Myc can rapidly induce DNA synthesis in U2OS-derived cell lines whose E2F activity is blocked by a constitutively active pRb (pRb Delta cdk) mutant. The effect of Myc is associated with Cdc25A phosphatase and cyclin E-CDK2 kinase activation and abolished by antagonizing Myc activity with the dominant-negative (dn) MadMyc chimera. Moreover, while abrogation of either endogenous E2F or Myc activity only delays and lowers DNA synthesis in synchronized U2OS cells or rat diploid fibroblasts, concomitant neutralization of both abolishes it. Whereas ectopic MSc and E2F1 rescue the G(1)/S delay caused by pRb Delta cdk (or dnDP1) and MadMyc, respectively, cyclin E or Cdc25A can restore DNA replication even in cells concomitantly exposed to pRb Delta cdk and MadMyc. However, coexpression of dnCDK2 neutralizes all of these rescuing effects. Finally, proper transcription of cyclin E and Cdc25A at the G(1)/S transition requires both Myc and E2F activities, and subthreshold levels of ectopic cyclin E and Cdc25A synergistically restore DNA synthesis in cells with silenced Myc and E2F activities. These results suggest that Myc controls a G(1)/S-promoting mechanism regulating cyclin E-CDK2 in parallel to the "classical" pRb/E2F pathway.