The centrosomal kinase Nek2 phosphorylates Sam68 and enhances the inclusion of the CD44 variable exon v5

Protein kinases that regulate the centrosome cycle are often aberrantly regulated in neoplastic cells. Changes in their expression or activity can lead to cell cycle perturbations. We have previously described that the centrosomal kinase Nek2 is highly expressed and activated in the neoplastic cells of testicular seminomas as compared to normal testis. Remarkably, up-regulation of Nek2 was not observed in other types of testicular cancer, such as teratomas and yolk sac tumours, indicating that Nek2 represents a marker for seminoma lesions. Interestingly, Nek2 staining was concentrated in the nucleus of neoplastic cells. The same nuclear localization was observed in the seminoma cell line Tcam-2. Given its nuclear localization in neoplastic germ cells, we have focused our attention on the potential targets of this kinase in the nucleus. Nek2 is known to interact with substrates and activators through the C-terminal regulatory region. We have used purified GST-Nek2-C terminal protein as bait in affinity chromatography to identify interacting proteins in nuclear extracts of HEK293T cells and T-cam2 seminoma cells. Pull down assays demonstrated that that the RNA-binding protein Sam68 was specifically associated with GST-Nek2-C. In vitro kinase assays using Nek2 immunoprecipitated from HEK293T cells showed that this kinase is able to phosphorylate Sam68 in vitro. Nek2 phosphorylated both the C-terminal region and, albeit to a lesser extent, the N-terminal region of Sam68, but not the GSG RNA-binding domain. A well characterized function of Sam68 is regulation of alternative splicing, and regulation of its activity by serine/thronine or tyrosine phosphorylation has been previously reported. Thus, we investigated whether Nek2 could modulate the splicing activity of Sam68. As a reporter system, we used a minigene encoding the CD44 variable exon v5 flanked by constitutive exons. Co-expression of Nek2 increased Sam68-dependent exon v5 inclusion in a dose-dependent manner. The effect of Nek2 was likely mediated by phosphorylation, because a kinase-dead mutant did not affect Sam68 splicing activity. Finally, we found that Sam68 is strongly expressed in male germ cells and its expression is further increased in human seminomas, but not other types of testicular tumors, like Nek2, suggesting that Sam68 is a relevant target of Nek2 activity in neoplastic germ cells. Our observations indicate that up-regulation and nuclear localization of the protein kinase Nek2 is an important marker of human testicular seminomas and suggest that regulation of alternative splicing is a novel function of Nek2 in the nucleus of neoplastic cells.