Abstract
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.