Institute of Molecular Biology and Pathology (IBPM), CNR Consiglio Nazionale delle Ricerche, 00185, Rome, Italy.
Institut Jacques Monod, UMR7592 CNRS-Université Paris Diderot, Sorbonne Paris Cité, F-75205, Paris, France.
Oncogene. 2020 Jan;39(2):454-468. doi: 10.1038/s41388-019-0989-x. Epub 2019 Sep 6.
The nuclear transport receptor importin-β/karyopherin-β1 is overexpressed in cancers that display genomic instability. It is regarded as a promising cancer target and inhibitors are being developed. In addition to its role in nucleo-cytoplasmic transport, importin-β regulates mitosis, but the programmes and pathways in which it operates are defined only in part. To unravel importin-β's mitotic functions we have developed cell lines expressing either wild-type or a mutant importin-β form in characterised residues required for nucleoporin binding. Both forms similarly disrupted spindle pole organisation, while only wild-type importin-β affected microtubule plus-end function and microtubule stability. A proteome-wide search for differential interactors identified a set of spindle regulators sensitive to mutations in the nucleoporin-binding region. Among those, HURP (hepatoma up-regulated protein) is an importin-β interactor and a microtubule-stabilising factor. We found that induction of wild type, but not mutant importin-β, under the same conditions that destabilise mitotic microtubules, delocalised HURP, indicating that the spatial distribution of HURP along the spindle requires importin-β's nucleoporin-binding residues. Concomitantly, importin-β overexpression sensitises cells to taxanes and synergistically increases mitotic cell death. Thus, the nucleoporin-binding domain is dispensable for importin-β function in spindle pole organisation, but regulates microtubule stability, at least in part via HURP, and renders cells vulnerable to certain microtubule-targeting drugs.
核转运受体 importin-β/importin-β1 在显示基因组不稳定的癌症中过表达。它被认为是一个有前途的癌症靶点，正在开发抑制剂。除了在核质转运中的作用外，importin-β 还调节有丝分裂，但它所作用的程序和途径仅部分定义。为了解开 importin-β 的有丝分裂功能，我们开发了表达野生型或突变型 importin-β 的细胞系，这些细胞系在核孔蛋白结合所需的特征残基中表达。这两种形式都同样破坏了纺锤体极组织，而只有野生型 importin-β 影响微管末端功能和微管稳定性。对差异相互作用蛋白的全蛋白质组搜索鉴定了一组对核孔蛋白结合区突变敏感的纺锤体调节剂。其中，HURP（肝癌上调蛋白）是 importin-β 的相互作用蛋白和微管稳定因子。我们发现，在同样破坏有丝分裂微管的条件下，诱导野生型 importin-β，但不是突变型 importin-β，会使 HURP 去定位，表明 HURP 在纺锤体上的空间分布需要 importin-β 的核孔蛋白结合残基。同时，importin-β 的过表达使细胞对紫杉烷类药物敏感，并协同增加有丝分裂细胞死亡。因此，核孔蛋白结合结构域对于 importin-β 在纺锤体极组织中的功能是可有可无的，但至少部分通过 HURP 调节微管稳定性，并使细胞易受某些微管靶向药物的影响。
