Nagita Mana, Inoue Hiroki, Nakamura Norihiro, Kanazawa Hiroshi
Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043.
J Biochem. 2003 Dec;134(6):919-25. doi: 10.1093/jb/mvg223.
We previously showed that calcineurin B homologous protein 1 (CHP1) interacts with nuclear apoptosis-inducing protein kinase DRAK2, and that overexpression of DRAK2 induces the nuclear accumulation of CHP1, although CHP1 usually resides in the cytoplasm [Matsumoto et al. (2001) J. Biochem. 130, 217-225]. Here we show that CHP1 has two functional nuclear export signal (NES) sequences in its carboxyl-terminal region. Treatment of several cell lines with leptomycin B, a specific inhibitor of CRM1-dependent nuclear export, induces the nuclear accumulation of CHP1. Moreover, CHP1-GFP fusion proteins with deletions or point mutations affecting the two putative NES sequences accumulate in the nucleus to a greater extent than wild-type CHP1-GFP. Tagging glutathione S-transferase-GFP fusion protein with each NES sequence caused a shift in their intracellular localization from all over the cells to the cytoplasm. These results suggest that after CHP1 has entered the nucleus, it is exported to the cytoplasm in an NES-dependent manner.
我们之前发现，钙调神经磷酸酶B同源蛋白1（CHP1）与核凋亡诱导蛋白激酶DRAK2相互作用，并且DRAK2的过表达会诱导CHP1在细胞核内积累，尽管CHP1通常位于细胞质中[松本等人（2001年）《生物化学杂志》130卷，217 - 225页]。在此我们表明，CHP1在其羧基末端区域有两个功能性核输出信号（NES）序列。用莱普霉素B（一种依赖CRM1的核输出特异性抑制剂）处理几种细胞系，会诱导CHP1在细胞核内积累。此外，与野生型CHP1 - GFP相比，影响两个假定NES序列的缺失或点突变的CHP1 - GFP融合蛋白在细胞核内积累的程度更大。用每个NES序列标记谷胱甘肽S - 转移酶 - GFP融合蛋白会导致它们的细胞内定位从遍布细胞转变为细胞质。这些结果表明，CHP1进入细胞核后，会以依赖NES的方式输出到细胞质中。
