Cyclin D activates the Rb tumor suppressor by mono-phosphorylation.
Cyclin D activates the Rb tumor suppressor by mono-phosphorylation.
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DOI：
10.7554/elife.02872
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发表时间：
2014-06-04
期刊：
影响因子：
7.7
通讯作者：
Dowdy SF
中科院分区：
文献类型：
--
作者：
Narasimha AM;Kaulich M;Shapiro GS;Choi YJ;Sicinski P;Dowdy SF
广泛接受的G1细胞周期进程模型提出，在G1早期，cyclin D:Cdk4/6通过渐进式多磷酸化（称为低磷酸化）使Rb肿瘤抑制因子失活，从而释放E2F转录因子。然而，这一模型尚未得到生物化学上的证实，Rb的生物活性形式也尚不清楚。在本研究中，我们发现Rb在G1期早期被细胞周期蛋白D:Cdk4/6单磷酸化。单磷酸化Rb由14个独立的同工异构体组成，它们都是E1a癌蛋白的靶标，但表现出优先的E2F结合模式。在G1后期限制点，cyclin E:Cdk2通过量子超磷酸化使Rb失活。经历DNA损伤反应的细胞激活周期蛋白D:Cdk4/6来产生单磷酸化的Rb，从而调节全局转录，而正在分化的细胞则利用未磷酸化的Rb。这些观察结果从根本上改变了我们对G1细胞周期进程的理解，并表明由周期蛋白D:Cdk4/6产生的单磷酸化Rb是G1早期唯一的Rb亚型。DOI: http://dx.doi.org/10.7554/eLife.02872.001细胞在分裂之前要经历一个严格控制的多步骤过程。这种细胞分裂程序——细胞周期——是防止可能导致癌症的无节制细胞生长所必需的。细胞周期蛋白控制着细胞周期的每个阶段，不同的细胞周期蛋白在不同的阶段起作用。在细胞周期的G1期，细胞的体积增大并产生复制DNA所需的蛋白质。一旦细胞在G1期结束时通过了一个叫做“限制点”的检查点，它就会开始分裂。因此，控制G1阶段的事件尤为重要。视网膜母细胞瘤肿瘤抑制蛋白（Rb）在G1期的调控中起关键作用。Rb隔离对细胞周期进展至关重要的转录因子。以前，人们认为在G1期，一种称为周期蛋白D的复合物向Rb蛋白添加了越来越多的磷酸盐。这一过程预示着转录因子的缓慢释放，这些转录因子附着在DNA上并开始DNA复制过程。虽然许多研究提供了与该模型一致的数据，但缺乏这些事件的直接生化证据。Narasimha, Kaulich， Shapiro等人现在对Rb蛋白进行了生化分析，完全出乎意料地表明，在G1期，周期蛋白D复合体只向Rb添加了一个磷酸基团，尽管这个磷酸基团可以添加到14个不同的位点之一。由此产生的“单磷酸化”Rb品种可以隔离不同的转录因子并停止它们的工作。在限制点，更多的磷酸基团迅速加入，Rb蛋白被另一种细胞周期蛋白灭活。这种被称为周期蛋白e的周期蛋白会驱使细胞进入细胞周期的下一个阶段。确定周期蛋白E是如何被激活的是未来研究的重点。DOI: http://dx.doi.org/10.7554/eLife.02872.002
The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, to release E2F transcription factors. However, this model remains unproven biochemically and the biologically active form(s) of Rb remains unknown. In this study, we find that Rb is exclusively mono-phosphorylated in early G1 phase by cyclin D:Cdk4/6. Mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but show preferential E2F binding patterns. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by quantum hyper-phosphorylation. Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription, whereas cells undergoing differentiation utilize un-phosphorylated Rb. These observations fundamentally change our understanding of G1 cell cycle progression and show that mono-phosphorylated Rb, generated by cyclin D:Cdk4/6, is the only Rb isoform in early G1 phase. DOI: http://dx.doi.org/10.7554/eLife.02872.001 Cells go through a tightly controlled, multi-step procedure before they divide. This cell division program—the cell cycle—is necessary for preventing unrestrained cellular growth, which may lead to cancer. Proteins called cyclins control the progression through each of the phases of the cell cycle, with different cyclins working during different phases. During the G1 phase of the cell cycle, cells grow in size and produce the proteins that are required to copy DNA. Once a cell passes a checkpoint called the 'restriction point' at the end of the G1 phase, it is committed to dividing. It is therefore particularly important to keep events during G1 phase in check. The Retinoblastoma tumor suppresor protein (Rb) is a key player in regulating the G1 phase. Rb sequesters transcription factors that are essential for the cell cycle to progress. Previously, it was thought that a complex called cyclin D added more and more phosphates to the Rb protein during the G1 phase. This process predicted a slow release of transcription factors, which attach to DNA and start the process of DNA replication. While many studies have presented data that is consistent with this model, direct biochemical evidence of these events is lacking. Narasimha, Kaulich, Shapiro et al. now present biochemical analyses of Rb proteins that show—completely unexpectedly—that the cyclin D complex adds just one phosphate group to Rb during the G1 phase, although this group can be added to one of fourteen different sites. The resulting 'mono-phosphorylated' Rb varieties can each sequester different transcription factors and stop them working. At the restriction point, many more phosphate groups are then rapidly added, and the Rb protein is inactivated by a different cyclin. This cyclin—called Cyclin E—then drives cells into the next phase of the cell cycle. Establishing how cyclin E is activated is a priority for future research. DOI: http://dx.doi.org/10.7554/eLife.02872.002