Identification of a new APC/C recognition domain, the A box, which is required for the Cdh1-dependent destruction of the kinase Aurora-A during mitotic exit
Identification of a new APC/C recognition domain, the A box, which is required for the Cdh1-dependent destruction of the kinase Aurora-A during mitotic exit
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DOI：
10.1101/gad.1007302
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发表时间：
2002-09-01
影响因子：
10.5
通讯作者：
Ruderman, JV
中科院分区：
文献类型：
--
作者：
Littlepage, LE;Ruderman, JV
有丝分裂激酶Aurora A（Aur-A）是有丝分裂纺锤体形成和染色体分离所必需的。在体细胞中，Aur-A蛋白和激酶活性水平在有丝分裂期间达到峰值，并且Aur-A在有丝分裂退出期间降解。在这里，我们研究了Aur-A蛋白和激酶活性水平的调节，利用非洲爪蟾卵和无细胞系统的快速同步细胞分裂周期。Aur-A激酶活性在早期胚胎细胞周期中振荡，就像在体细胞中一样，但Aur-A蛋白水平是恒定的，这表明受调节的激活和失活，而不是周期性的蛋白水解，是Aur-A调节在这些细胞周期中的主导模式。Cdh 1是一种APC/C激活剂，在体细胞有丝分裂晚期和G1期靶向许多有丝分裂蛋白进行泛素依赖性蛋白水解，但在非洲爪蟾卵和早期胚胎中缺失。我们发现，除了Cdh 1的鸡蛋提取物进行M期退出是足以诱导Aur-A的快速降解。Aur-A包含两种已知的APC/C识别信号，（1）C-末端D盒，类似于泛素依赖性破坏细胞周期蛋白B和其他几种有丝分裂蛋白所需的那些，和（2）N-末端KEN盒，类似于cdc 20上发现的，其响应APC/C-Cdh 1而被泛素化。Cdh 1诱导的Aur-A破坏需要D盒，但KEN盒不是。破坏还需要N端的一个短区域，其中包含一个新发现的识别信号，A盒。A盒在脊椎动物Aur-As中是保守的，并且含有丝氨酸53，其在M期被磷酸化。丝氨酸53突变为天冬氨酸，可以模拟磷酸化的作用，完全阻断Cdh 1依赖性的Aur-A破坏。这些结果表明，丝氨酸53在有丝分裂退出过程中的去磷酸化可以控制Aur-A破坏的时间，允许Cdh 1激活的APC/C识别A盒和D盒。
The mitotic kinase Aurora A (Aur-A) is required for formation of a bipolar mitotic spindle and accurate chromosome segregation. In somatic cells, Aur-A protein and kinase activity levels peak during mitosis, and Aur-A is degraded during mitotic exit. Here, we investigated how Aur-A protein and kinase activity levels are regulated, taking advantage of the rapid synchronous cell division cycles of Xenopus eggs and cell-free systems derived from them. Aur-A kinase activity oscillates in the early embryonic cell cycles, just as in somatic cells, but Aur-A protein levels are constant, indicating that regulated activation and inactivation, instead of periodic proteolysis, is the dominant mode of Aur-A regulation in these cell cycles. Cdh1, the APC/C activator that targets many mitotic proteins for ubiquitin-dependent proteolysis during late mitosis and G1 in somatic cells, is missing in Xenopus eggs and early embryos. We find that addition of Cdh1 to egg extracts undergoing M phase exit is sufficient to induce rapid degradation of Aur-A. Aur-A contains both of the two known APC/C recognition signals, (1) a C-terminal D box similar to those required for ubiquitin-dependent destruction of cyclin B and several other mitotic proteins, and (2) an N-terminal KEN box similar to that found on cdc20, which is ubiquitinated in response to APC/C-Cdh1. The D box is required for Cdh1-induced destruction of Aur-A but the KEN box is not. Destruction also requires a short region in the N terminus, which contains a newly identified recognition signal, the A box. The A box is conserved in vertebrate Aur-As and contains serine 53, which is phosphorylated during M phase. Mutation of serine 53 to aspartic acid, which can mimic the effect of phosphorylation, completely blocks Cdh1-dependent destruction of Aur-A. These results suggest that dephosphorylation of serine 53 during mitotic exit could control the timing of Aur-A destruction, allowing recognition of both the A box and D box by Cdh1-activated APC/C.