A novel nuclear function of the centrosomal kinase Nek2 through modulation of alternative splicing in cancer cells

Protein kinases that regulate the centrosome cycle are often aberrantly regulated in neoplastic cells, thereby leading to cell cycle perturbations. The centrosomal kinase Nek2 is highly expressed and activated in neoplastic cells of testicular seminomas, as compared to normal testis or other testicular cancers. Interestingly, Nek2 staining was concentrated in the nucleus of seminoma cells. Furthermore, nuclear localization of Nek2 was found to be a common feature of cancers in which this kinase is upregulated, such as breast, prostate and colon cancers. Confocal microscopy revealed that in these cells Nek2 is enriched in nuclear speckles, where splicing regulators like SC35 and ASF/SF2 also accumulate. Cell fractionation experiments indicated that Nek2 is associated with nuclear matrix insoluble fractions, together with several splicing factors. To identify nuclear proteins that interact with Nek2, we performed affinity chromatography using, as bait, the C-terminal domain of the kinase, which is known to interact with substrates, fused to GST (GST-Nek2-Ct). We found that GST-Nek2-Ct selectively interacts with some splicing factors present in prostate cancer cell nuclear extracts, like ASF/SF2, hnRNP F/H and Sam68, but not others, like SRp20 or hnRNP C1/C2. We focused on Sam68 because this splicing factor is also upregulated in breast, prostate and testicular cancers like Nek2. In vitro kinase assays showed that Nek2 directly phosphorylates Sam68. Furthermore, upregulation of Nek2 in HEK293T cells induced phosphorylation of endogenous Sam68, whereas a kinase-dead version of the protein (Nek2KD) was ineffective. Sam68 regulates alternative splicing of target genes, including CD44. To investigate whether Nek2 modulates the splicing activity of Sam68, we used a minigene encoding the CD44 variable exon v5 flanked by constitutive exons as reporter system. Co-expression of Nek2, but not Nek2KD, increased Sam68-dependent exon v5 inclusion, whereas depletion of endogenous Sam68 abolished the effect. Finally, we observed that upregulation of Nek2 enhanced v5 inclusion in the endogenous CD44 pre-mRNA, whereas its depletion decreased it. Our observations indicate that upregulation and nuclear localization of the centrosomal protein kinase Nek2 is a relevant marker of human cancers and suggest that regulation of alternative splicing is a novel function of Nek2 in the nucleus of neoplastic cells.