Importin β-dependent nuclear import of TopBP1 in ATR-Chk1 checkpoint in Xenopus egg extracts.
Importin β-dependent nuclear import of TopBP1 in ATR-Chk1 checkpoint in Xenopus egg extracts.
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DOI：
10.1016/j.cellsig.2014.01.006
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发表时间：
2014-05
影响因子：
4.8
通讯作者：
Yan S
中科院分区：
文献类型：
--
作者：
Bai L;Michael WM;Yan S
TopBP 1是一种含有多个BRCT的蛋白质，在DNA复制、DNA损伤反应和转录调控等DNA代谢过程中发挥着重要作用。临床研究发现TopBP 1的胞浆定位与乳腺癌易感性相关，提示TopBP 1的亚细胞定位具有生物学意义。然而，在正常或应激条件下，TopBP 1如何穿梭进入细胞核并募集到染色质中仍然是难以捉摸的。利用非洲爪蟾卵提取物，我们鉴定出Importin β是TopBP 1 C端的一个新的相互作用蛋白。我们通过GST pulldown和免疫共沉淀试验验证了TopBP 1-Importin β的结合。然后，我们证明了含有推定的NLS（核定位信号）的TopBP 1的C-末端基序（命名为CTM，23个氨基酸）是Importin β相互作用所需的，并且Importin β的CT 100（Importin β的末端C-末端的100个氨基酸）是TopBP 1相互作用所需的。进一步的结构-功能分析表明，CTM的TopBP 1是必不可少的TopBP 1的核输入和随后的染色质招募，从而发挥重要作用的DNA复制和丝裂霉素C（MMC）诱导的Chk 1磷酸化。此外，Importin β特异性抑制剂importazole抑制TopBP 1的核输入和MMC诱导的Chk 1磷酸化。随着DNA复制的进行，TopBP 1的Importin β依赖性核输入确实是MMC诱导的Chk 1磷酸化所必需的。我们的数据还表明，检查点激活需要比DNA复制更多的TopBP 1。在无核AT 70系统中可以绕过TopBP 1的CTM基序对ATR-Chk 1检查点的要求。综上所述，我们的研究结果表明CTM基序介导的TopBP 1通过Importin β穿梭进入细胞核在非洲爪蟾卵提取物中的ATR-Chk 1检查点信号传导中起重要作用。
TopBP1, a multiple-BRCT-containing protein, plays diverse functions in DNA metabolism including DNA replication, DNA damage response and transcriptional regulation. The cytoplasmic localization of TopBP1 has been found associated with breast cancer susceptibility in clinical studies, suggesting the biological significance of TopBP1’s sub-cellular localization. However, it remains elusive how TopBP1 is shuttled into nucleus and recruited to chromatin under normal or stressful conditions. Taking advantage of Xenopus egg extract, we identified Importin β as a new interacting protein of the TopBP1 C-terminus. We verified the TopBP1-Importin β association via GST pulldown and coimmunoprecipitation assays. We then demonstrated that TopBP1’s C-terminal motif (designated as CTM, 23 amino acids) containing a putative NLS (Nuclear Localization Signal) was required for Importin β interaction and that CT100 of Importin β (100 amino acids of extreme C-terminus of Importin β) was required for TopBP1 interaction. Further structure-function analysis reveals that the CTM of TopBP1 is essential for TopBP1’s nuclear import and subsequent chromatin recruitment, thereby playing important roles in DNA replication and mitomycin C (MMC)-induced Chk1 phosphorylation. In addition, Importin β-specific inhibitor importazole inhibits TopBP1’s nuclear import and the MMC-induced Chk1 phosphorylation. With ongoing DNA replication, the Importin β-dependent nuclear import of TopBP1 was indeed required for the MMC-induced Chk1 phosphorylation. Our data also suggest that checkpoint activation requires more TopBP1 than DNA replication does. The requirement of TopBP1’s CTM motif for ATR-Chk1 checkpoint can be bypassed in a nucleus-free AT70 system. Taken together, our findings suggest the CTM motif-mediated TopBP1 shuttling into nucleus via Importin β plays an important role in the ATR-Chk1 checkpoint signaling in Xenopus egg extracts.
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影响因子：
64.8
作者：
Bartkova, J;Horejsi, Z;Bartek, J
通讯作者：
Bartek, J
影响因子：
16
作者：
Gong, Zihua;Kim, Ja-Eun;Leung, Charles Chung Yun;Glover, J. N. Mark;Chen, Junjie
通讯作者：
Chen, Junjie
影响因子：
2.8
作者：
Forma E;Krzeslak A;Bernaciak M;Romanowicz-Makowska H;Brys M
通讯作者：
Brys M
DOI：
10.1016/j.bbamcr.2010.10.014
发表时间：
2011-09
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Chook YM;Süel KE
通讯作者：
Süel KE
影响因子：
10.5
作者：
Guo, ZJ;Kumagai, A;Dunphy, WG
通讯作者：
Dunphy, WG