The centrosomal kinase Nek2 is a novel regulator of alternative splicing in cancer cells

Nek2 is a serine/threonine kinase highly enriched in the centrosome, wherein it promotes splitting and separation in the S/G2 phase of the cell cycle. Nek2 is frequently up-regulated in cancer cells, where it contributes to formation of supernumerary centrosomes and genomic instability. Surprisingly, examination of a panel of cancer tissues and cell lines revealed that Nek2 is concentrated in the nucleus of neoplastic cells. Confocal microscopy revealed that Nek2 is enriched in nuclear SC35- and ASF/SF2-containing speckles. Pull-down assays showed that Nek2 selectively interacts with ASF/SF2, hnRNP F/H and Sam68, but not with SRp20 or hnRNP C1/C2. Furthermore, in vitro and in vivo analyses indicated that Nek2 can directly phosphorylate Sam68 and ASF/SF2, two splicing factors with pathological implications in human cancers. To test the physiological role of Nek2-dependent phosphorylation of splicing factors, we studied the alternative splicing of CD44, a cancer-relevant target of Sam68. Up-regulation of Nek2, but not a kinase-dead mutant, increased Sam68-dependent exon v5 inclusion in a CD44 minigene, whereas depletion of endogenous Sam68 abolished this effect. Moreover, UV crosslink experiments showed that over-expression of Nek2 enhances binding of Sam68 to the endogenous CD44 exon v5 in vivo, suggesting that serine/threonine phosphorylation facilitates recruitment of Sam68 to the pre-mRNA. Lastly, we found that inclusion of variable exon V5 is modulated during the S and G2 phases of the cell cycle, concomitantly with the reported activity of Nek2. Importantly, depletion of endogenous Nek2 strongly reduced exon v5 splicing in S and G2 phases, but not in G1 when the kinase activity is minimal. Our study identifies Nek2 as a novel regulator of alternative splicing in cancer cells, and links its activity during cell cycle progression with alternative processing of target pre-mRNAs.