Tang Zhanyun, Shu Hongjun, Oncel Dilhan, Chen She, Yu Hongtao
Department of Pharmacology, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
Mol Cell. 2004 Nov 5;16(3):387-97. doi: 10.1016/j.molcel.2004.09.031.
To ensure the fidelity of chromosome segregation, the spindle checkpoint blocks the ubiquitin ligase activity of APC/C(Cdc20) in response to a single chromatid not properly attached to the mitotic spindle. Here we show that HeLa cells depleted for Bub1 by RNA interference are defective in checkpoint signaling. Bub1 directly phosphorylates Cdc20 in vitro and inhibits the ubiquitin ligase activity of APC/C(Cdc20) catalytically. A Cdc20 mutant with all six Bub1 phosphorylation sites removed is refractory to Bub1-mediated phosphorylation and inhibition in vitro. Upon checkpoint activation, Bub1 itself is hyperphosphorylated and its kinase activity toward Cdc20 is stimulated. Ectopic expression of the nonphosphorylatable Cdc20 mutant allows HeLa cells to escape from mitosis in the presence of spindle damage. Therefore, Bub1-mediated phosphorylation of Cdc20 is required for proper checkpoint signaling. We speculate that inhibition of APC/C(Cdc20) by Bub1 in a catalytic fashion may partly account for the exquisite sensitivity of the spindle checkpoint.
为确保染色体分离的准确性，纺锤体检查点会响应单条染色单体未正确附着于有丝分裂纺锤体的情况，阻断后期促进复合物/细胞周期蛋白依赖性激酶20（APC/C(Cdc20)）的泛素连接酶活性。在此我们表明，通过RNA干扰使Bub1缺失的HeLa细胞在检查点信号传导方面存在缺陷。Bub1在体外直接磷酸化Cdc20，并催化性地抑制APC/C(Cdc20)的泛素连接酶活性。去除了所有六个Bub1磷酸化位点的Cdc20突变体在体外对Bub1介导的磷酸化和抑制具有抗性。在检查点激活后，Bub1自身会发生超磷酸化，并且其对Cdc20的激酶活性会被刺激。不可磷酸化的Cdc20突变体的异位表达使HeLa细胞在存在纺锤体损伤的情况下能够逃离有丝分裂。因此，Bub1介导的Cdc20磷酸化对于正确的检查点信号传导是必需的。我们推测，Bub1以催化方式对APC/C(Cdc20)的抑制可能部分解释了纺锤体检查点的高度敏感性。
