在大约5%的非小细胞肺癌患者中发现EML4-ALK致癌融合蛋白。存在不同的EML4-ALK融合变体,变体3(V3)与明显高于其他常见变体的风险相关。如V1。V3患者对靶向ALK抑制剂的反应较差,有加速的转移率和较差的总体生存率。已经描述了EML4-ALKV3下游的途径,其不依赖于ALK催化活性,但依赖于NEK9和NEK7激酶。已经提出EML4-ALKV3:NEK9:NEK7复合物在微管上的组装导致细胞形成间充质样形态并表现出增强的迀移。然而,该复合体的下游目标仍有待确定。这里,我们证明了基于微管的驱动蛋白运动蛋白,响应于EML4-ALKV3的表达,将Eg5募集至相间微管,而Eg5的化学抑制逆转细胞的间充质形态。此外,我们显示,NEK7的耗竭干扰Eg5募集到微管中表达EML4-ALKV3的细胞,并导致细胞长度减少,但这是通过在一个位点共表达Eg5的磷模拟突变体而逆转的,S1033,被NEK7磷酸化。有趣的是,我们还发现Eg5-S1033D的表达导致表达EML4-ALKV1的细胞采用更多的间充质样形态。一起来看,我们提出Eg5在表达EML4-ALKV3的细胞中充当NEK7的底物,并且Eg5磷酸化促进这些细胞典型的形态改变。
Echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) oncogenic fusion proteins are found in approximately 5% of non-small cell lung cancers. Different EML4-ALK fusion variants exist with variant 3 (V3) being associated with a significantly higher risk than other common variants, such as variant 1 (V1). Patients with V3 respond less well to targeted ALK inhibitors, have accelerated rates of metastasis, and have poorer overall survival. A pathway has been described downstream of EML4-ALK V3 that is independent of ALK catalytic activity but dependent on the NEK9 and
NEK7 kinases. It has been proposed that assembly of an EML4-ALK V3-NEK9-
NEK7 complex on microtubules leads to cells developing a mesenchymal-like morphology and exhibiting enhanced migration. However, downstream targets of this complex remain unknown. Here, we show that the microtubule-based kinesin, Eg5, is recruited to interphase microtubules in cells expressing EML4-ALK V3, whereas chemical inhibition of Eg5 reverses the mesenchymal morphology of cells. Furthermore, we show that depletion of
NEK7 interferes with Eg5 recruitment to microtubules in cells expressing EML4-ALK V3 and cell length is reduced, but this is reversed by coexpression of a phosphomimetic mutant of Eg5, in a site, S1033, phosphorylated by
NEK7. Intriguingly, we also found that expression of Eg5-S1033D led to cells expressing EML4-ALK V1 adopting a more mesenchymal-like morphology. Together, we propose that Eg5 acts as a substrate of
NEK7 in cells expressing EML4-ALK V3 and Eg5 phosphorylation promotes the mesenchymal morphology typical of these cells.