Lukas C, Sorensen CS, Kramer E, Santoni-Rugiu E, Lindeneg C, Peters JM et al.. Accumulation of cyclin B1 requires E2F and cyclin-A-dependent rearrangement of the anaphase-promoting complex. Nature 401: 815-818
Institute of Cancer Biology, Danish Cancer Society, Copenhagen. Nature
(Impact Factor: 41.46).
11/1999; 401(6755):815-8. DOI: 10.1038/44611
In mammalian somatic-cell cycles, progression through the G1-phase restriction point and initiation of DNA replication are controlled by the ability of the retinoblastoma tumour-suppressor protein (pRb) family to regulate the E2F/DP transcription factors. Continuing transcription of E2F target genes beyond the G1/S transition is required for coordinating S-phase progression with cell division, a process driven by cyclin-B-dependent kinase and anaphase-promoting complex (APC)-mediated proteolysis. How E2F-dependent events at G1/S transition are orchestrated with cyclin B and APC activity remains unknown. Here, using an in vivo assay to measure protein stability in real time during the cell cycle, we show that repression of E2F activity or inhibition of cyclin-A-dependent kinase in S phase triggers the destruction of cyclin B1 through the re-assembly of APC, the ubiquitin ligase that is essential for mitotic cyclin proteolysis, with its activatory subunit Cdh1. Phosphorylation-deficient mutant Cdh1 or immunodepletion of cyclin A resulted in assembly of active Cdh1-APC even in S-phase cells. These results implicate an E2F-dependent, cyclin A/Cdk2-mediated phosphorylation of Cdh1 in the timely accumulation of cyclin B1 and the coordination of cell-cycle progression during the post-restriction point period.
Available from: Alessandro A Sartori
- "During S/G 2 phase, premature APC/C activation is at least in part prevented through CDK-mediated phosphorylation of Cdh1, which hinders association of Cdh1 with the APC/C (Lukas et al, 1999; Kramer et al, 2000; Miller et al, 2006). As a first indication of APC/C Cdh1 playing a role in the DDR, "
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ABSTRACT: Human cells have evolved elaborate mechanisms for responding to DNA damage to maintain genome stability and prevent carcinogenesis. For instance, the cell cycle can be arrested at different stages to allow time for DNA repair. The APC/CCdh1 ubiquitin ligase mainly regulates mitotic exit but is also implicated in the DNA damage-induced G2 arrest. However, it is currently unknown whether APC/CCdh1 also contributes to DNA repair. Here, we show that Cdh1 depletion causes increased levels of genomic instability and enhanced sensitivity to DNA-damaging agents. Using an integrated proteomics and bioinformatics approach, we identify CtIP, a DNA-end resection factor, as a novel APC/CCdh1 target. CtIP interacts with Cdh1 through a conserved KEN box, mutation of which impedes ubiquitylation and downregulation of CtIP both during G1 and after DNA damage in G2. Finally, we find that abrogating the CtIP–Cdh1 interaction results in delayed CtIP clearance from DNA damage foci, increased DNA-end resection, and reduced homologous recombination efficiency. Combined, our results highlight the impact of APC/CCdh1 on the maintenance of genome integrity and show that this is, at least partially, achieved by controlling CtIP stability in a cell cycle- and DNA damage-dependent manner.
Available from: Brett M Connolly
- "Phosphorylation of the CRS is required for nuclear trafficking of cyclin B1 . Lukas et al. has reported a fusion reporter of N terminus 119 amino acids of cyclin B1 linked to luciferase to measure Cdh1 ligase activity . In the present study, the N terminus of cyclin B1 includes the CRS, different from that reported by Lukas et al. , so the fusion protein is able to be transported into the cytoplasm for ubiquitination-dependent degradation. "
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Visualization of the cell cycle in living subjects has long been a big challenge. The present study aimed to noninvasively visualize mitotic arrest of the cell cycle with an optical reporter in living subjects.
An N-terminal cyclin B1–luciferase fusion construct (cyclin B-Luc) controlled by the cyclin B promoter, as a mitosis reporter, was generated. HeLa or HCT116 cells stably expressing cyclin B-Luc reporter were used to evaluate its cell cycle-dependent regulation and ubiquitination-mediated degradation. We also evaluated its feasibility to monitor the mitotic arrest caused by Taxotere both in vitro and in vivo.
We showed that the cyclin B-Luc fusion protein was regulated in a cell cycle-dependent manner and accumulated in the mitotic phase (M phase) in cellular assays. The regulation of cyclin B-Luc reporter was mediated by proteasome ubiquitination. In the present study, in vitro imaging showed that antimitotic reagents like Taxotere upregulated the reporter through cell cycle arrest in the M phase. Noninvasive longitudinal bioluminescence imaging further demonstrated an upregulation of the reporter consistent with mitotic arrest induced in tumor xenograft models. Induction of this reporter was also observed with a kinesin spindle protein inhibitor, which causes cell cycle blockage in the M phase.
Our results demonstrate that the cyclin B-Luc reporter can be used to image whether compounds are capable, in vivo, of causing an M phase arrest and/or altering cyclin B turnover. This reporter can also be potentially used in high-throughput screening efforts aimed at discovering novel molecules that will cause cell cycle arrest at the M phase in cultivated cell lines and animal models.
Available from: Arne Nedergaard Kousholt
- "The degradation of Cyclin B is regulated by the anaphase-promoting complex/cyclosome (APC/C) , an E3 ubiquitin ligase that targets specific proteins for proteasomal degradation. Cyclin B polyubiquitylation starts in metaphase, where it is critical for mitotic exit, and it continues until the early S phase where the polyubiquitylation is downregulated due to APC/C phosphorylation by Cyclin-CDK2 and binding of the APC/C inhibitor Emi1 [15,16]. "
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ABSTRACT: The maintenance of genome integrity is important for normal cellular functions, organism development and the prevention of diseases, such as cancer. Cellular pathways respond immediately to DNA breaks leading to the initiation of a multi-facetted DNA damage response, which leads to DNA repair and cell cycle arrest. Cell cycle checkpoints provide the cell time to complete replication and repair the DNA damage before it can continue to the next cell cycle phase. The G2/M checkpoint plays an especially important role in ensuring the propagation of error-free copies of the genome to each daughter cell. Here, we review recent progress in our understanding of DNA repair and checkpoint pathways in late S and G2 phases. This review will first describe the current understanding of normal cell cycle progression through G2 phase to mitosis. It will also discuss the DNA damage response including cell cycle checkpoint control and DNA double-strand break repair. Finally, we discuss the emerging concept that DNA repair pathways play a major role in the G2/M checkpoint pathway thereby blocking cell division as long as DNA lesions are present.
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