Cyclin E-induced S phase without activation of the pRb/E2F pathway
Cyclin E-induced S phase without activation of the pRb/E2F pathway
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DOI：
10.1101/gad.11.11.1479
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发表时间：
1997-06-01
影响因子：
10.5
通讯作者：
Bartek, J
中科院分区：
文献类型：
--
作者：
Lukas, J;Herzinger, T;Bartek, J
在高等真核细胞中，依赖于细胞周期蛋白D的CDK4和CDK6，以及可能依赖于细胞周期蛋白E的CDK2，通过磷酸化视网膜母细胞瘤蛋白，从而释放控制S期基因的E2F转录因子，正向调节从G(1)到S的转变。在此，我们用大鼠R12成纤维细胞、条件过表达细胞周期蛋白D1或E的衍生物和人U-2-OS细胞进行显微注射和基因转染实验，以探讨G(1)细胞周期蛋白的作用以及E2F和细胞周期蛋白E在S期诱导中的关系。我们证明，细胞周期蛋白E的异位表达，而不是细胞周期蛋白D1的异位表达，可以超越针对CDK4和CDK6的p16(INK4a)CDK抑制剂或一种新的磷酸化缺陷突变体pRb对G(1)的抑制作用。几种评估E2F激活的互补方法，包括活细胞中的定量报告分析，表明在没有E2F介导的反式激活的情况下，细胞周期蛋白E诱导的S期和细胞分裂周期的完成可以发生。再加上Cyclin E克服了显性-负性突变体DP-1(E2FS的异二聚体伙伴)表达诱导的G(1)期阻滞，这些结果为Cyclin E控制的S控制的体细胞中与pRb磷酸化下游或平行的、不依赖于E2F激活的阶段促进事件提供了证据。他们进一步表明，缺乏E2F介导的反式激活可以通过过度激活这种周期蛋白E控制的事件来弥补。
In cells of higher eukaryotes, cyclin D-dependent kinases Cdk4 and Cdk6 and, possibly, cyclin E-dependent Cdk2 positively regulate the G(1)- to S-phase transition, by phosphorylating the retinoblastoma protein (pRb), thereby releasing E2F transcription factors that control S-phase genes. Here we performed microinjection and transfection experiments using rat R12 fibroblasts, their derivatives conditionally overexpressing cyclins D1 or E, and human U-2-OS cells, to explore the action of G(1) cyclins and the relationship of E2F and cyclin E in S-phase induction. We demonstrate that ectopic expression of cyclin E, but not cyclin D1, can override G(1) arrest imposed by either the p16(INK4a) Cdk inhibitor specific for Cdk4 and Cdk6 or a novel phosphorylation-deficient mutant pRb. Several complementary approaches to assess E2F activation, including quantitative reporter assays in live cells, showed that the cyclin E-induced S phase and completion of the cell division cycle can occur in the absence of E2F-mediated transactivation. Together with the ability of cyclin E to overcome a G(1) block induced by expression of dominant-negative mutant DP-1, a heterodimeric partner of E2Fs, these results provide evidence for a cyclin E-controlled S phase-promoting event in somatic cells downstream of or parallel to phosphorylation of pRb and independent of E2F activation. They furthermore indicate that a lack of E2F-mediated transactivation can be compensated by hyperactivation of this cyclin E-controlled event.