Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2.
Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2.
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DOI：
10.1083/jcb.200102093
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发表时间：
2001-09-03
期刊：
影响因子：
--
通讯作者：
Yen TJ
中科院分区：
文献类型：
--
作者：
Sudakin V;Chan GK;Yen TJ
有丝分裂检查点通过抑制后期促进复合物/细胞周期体（APC/C）靶向关键蛋白进行遍在蛋白介导的蛋白质水解，防止染色体未对齐的细胞过早退出有丝分裂。我们已经研究了机制，其中检查点抑制APC/C的纯化APC/C抑制因子从HeLa细胞。我们称这种因子为有丝分裂检查点复合物（MCC），因为它由接近相等化学计量的hBUBR 1，hBUB 3，CDC 20和MAD 2检查点蛋白组成。MCC的抑制活性是重组MAD 2的3，000倍，重组MAD 2也显示出体外抑制APC/C。令人惊讶的是，MCC不是由动粒产生的，因为它也存在于间期细胞中并具有活性。然而，只有从有丝分裂细胞中分离的APC/C对MCC的抑制敏感。我们发现有丝分裂裂解物中的大多数APC/C与MCC相关，这可能有助于泛素连接酶活性的滞后。重要的是，染色体可以抑制APC/C的再激活。染色体不影响MCC的抑制活性或CDC 20的刺激活性。我们建议，预先形成的间期池MCC允许快速抑制APC/C细胞进入有丝分裂时。然后，未附着的动粒靶向APC/C，以通过MCC持续抑制。
The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from targeting key proteins for ubiquitin-mediated proteolysis. We have examined the mechanism by which the checkpoint inhibits the APC/C by purifying an APC/C inhibitory factor from HeLa cells. We call this factor the mitotic checkpoint complex (MCC) as it consists of hBUBR1, hBUB3, CDC20, and MAD2 checkpoint proteins in near equal stoichiometry. MCC inhibitory activity is 3,000-fold greater than that of recombinant MAD2, which has also been shown to inhibit APC/C in vitro. Surprisingly, MCC is not generated from kinetochores, as it is also present and active in interphase cells. However, only APC/C isolated from mitotic cells was sensitive to inhibition by MCC. We found that the majority of the APC/C in mitotic lysates is associated with the MCC, and this likely contributes to the lag in ubiquitin ligase activity. Importantly, chromosomes can suppress the reactivation of APC/C. Chromosomes did not affect the inhibitory activity of MCC or the stimulatory activity of CDC20. We propose that the preformed interphase pool of MCC allows for rapid inhibition of APC/C when cells enter mitosis. Unattached kinetochores then target the APC/C for sustained inhibition by the MCC.
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