Recognition of nuclear export signals by CRM1 carrying the oncogenic E571K mutation.
Recognition of nuclear export signals by CRM1 carrying the oncogenic E571K mutation.
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DOI：
10.1091/mbc.e20-04-0233
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发表时间：
2020-08-01
影响因子：
3.3
通讯作者：
Chook YM
中科院分区：
文献类型：
--
作者：
Baumhardt JM;Walker JS;Lee Y;Shakya B;Brautigam CA;Lapalombella R;Grishin N;Chook YM
CRM1的E571K突变在一些癌症中非常普遍，但其肿瘤发生机制尚不清楚。 CRM1 的 Glu571 位于其核输出信号 (NES) 结合沟中，表明所选 NES 的结合可能会改变。我们使用 CRISPR/Cas9 生成了具有单等位基因 CRM1WT/E571K 或双等位基因 CRM1E571K/E571K 的 HEK 293 细胞。我们还将野生型和 E571K CRM1 的 27 种不同 NES 的结合亲和力和结构分析与基于结构的生物信息学相结合。虽然大多数 NES 与两个 CRM1 的结合相似，但来自 Mek1、eIF4E 转运蛋白和 RPS2 的 NES 显示出 > 10 倍的亲和力差异。这些 NES 具有多个靠近 CRM1 位置 571 的带电侧链，但仅此特征不足以预测与 CRM1(E571K) 的不同结合。与 eIF4E 转运蛋白 NES 与 CRM1(E571K) 的结合较弱一致，eIF4E 转运蛋白在携带 CRM1(E571K) 的肿瘤细胞中错误定位。这作为概念证明，了解 CRM1(E571K) 如何影响 NES 结合提供了一个平台，用于识别因 CRM1 突变而在癌症中错误定位的货物。最后，我们表明，一些 NES 肽（Mek1 和 RPS2）所观察到的大亲和力变化并不总是转化为全长货物，这表明当前 NES 预测方法的局限性。因此，像我们这样的综合研究势在必行，以确定具有真正致病潜力的 CRM1 货物。
The E571K mutation of CRM1 is highly prevalent in some cancers, but its mechanism of tumorigenesis is unclear. Glu571 of CRM1 is located in its nuclear export signal (NES)-binding groove, suggesting that binding of select NESs may be altered. We generated HEK 293 cells with either monoallelic CRM1WT/E571K or biallelic CRM1E571K/E571K using CRISPR/Cas9. We also combined analysis of binding affinities and structures of 27 diverse NESs for wild-type and E571K CRM1 with structure-based bioinformatics. While most NESs bind the two CRM1 similarly, NESs from Mek1, eIF4E-transporter, and RPS2 showed >10-fold affinity differences. These NESs have multiple charged side chains binding close to CRM1 position 571, but this feature alone was not sufficient to predict different binding to CRM1(E571K). Consistent with eIF4E-transporter NES binding weaker to CRM1(E571K), eIF4E-transporter was mislocalized in tumor cells carrying CRM1(E571K). This serves as proof of concept that understanding how CRM1(E571K) affects NES binding provides a platform for identifying cargoes that are mislocalized in cancer upon CRM1 mutation. Finally, we showed that large affinity changes seen with some NES peptides (of Mek1 and RPS2) do not always translate to the full-length cargoes, suggesting limitations with current NES prediction methods. Therefore, comprehensive studies like ours are imperative to identify CRM1 cargoes with real pathogenic potential.