Functional studies have highlighted different roles of FFAR family in human health

. However, our study indicates PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19818229 that NEK2C is expressed at low levels in all cancer cells analysed, raising doubts on its contribution to the localization of NEK2 in primary tumours and in cell lines. Conversely, upregulation of NEK2A, but not NEK2B, is sufficient to induce its nuclear localization, suggesting that NEK2A is the prevalent isoform in the nucleus of cancer cells. Characterization of the subcellular distribution of NEK2 pointed out its co-fractionation with several splicing factors in the nuclear-insoluble material of cancer cells. Moreover, NEK2 co-localized with two SR proteins in nuclear splicing speckles. These inter-chromatin granules are particularly enriched in SR proteins and are supposed to function as nuclear storage sites for pre-mRNA processing regulators. Assembly of splicing speckles and active recruitment of SR proteins from these sites to the newly synthesized pre-mRNA is strictly regulated by reversible phosphorylation. Phosphorylation represents one of the main BAY-41-2272 mechanisms by which subtle regulation of the splicing process, and especially of AS, is achieved. These observations led us to hypothesize the existence of a functional interaction between NEK2 and splicing. Several results of our study support this hypothesis. First, NEK2 interacts with and phosphorylates SRSF1. Second, we found that the splicing activity of SRSF1 is modulated by NEK2. By contrast, our study suggests that NEK2 can modulate SRSF1 splicing activity similarly to SRPK1, the prototype member of the SRPK family of kinases that mediate phosphorylation of SR proteins. Overexpression of either NEK2 or SRPK1 induced a similar pattern of SR proteins phosphorylation and caused a similar modulation of E1A AS. Likewise, knockdown of NEK2 or SRPK1 similarly reduced the splicing activity of SRSF1 toward the BCL-X minigene. Although the effects of NEK2 might be indirect, three lines of evidence support a direct action. First, our in vitro kinase assays were performed using highly purified proteins, strongly indicating that NEK2 can directly phosphorylate SRSF1 and SRSF7. Second, although NEK2 was recently reported to induce activation of AKT in myeloma cells , a signalling kinase known to directly and indirectly modulate SR proteins phosphorylation, overexpression or knockdown of NEK2 did not alter the activity of AKT in HeLa cells. Third, the effect of NEK2 on BCL-X splicing was not affected by knockdown of SRPK1. Thus, these experiments suggest that NEK2 behaves as a bona fide splicing factor kinase in live cells. Our study implicates NEK2 in AS regulation of several SRSF1 target genes involved in cell viability. We found that knockdown of NEK2 mimicked that of SRSF1, or SRPK1, and induced expression of pro-apoptotic BCL-X, BIN1 and MKNK2 splice variants. Consistently, NEK2 depletion sensitized HeLa cells to spontaneous and stress-induced apoptosis, suggesting a pro-survival function for this kinase. Although protection from cell death may also involve other splicing-unrelated functions of NEK2, it is likely that enhanced splicing of the anti-apoptotic variants of BCL-X, BIN1 and MKNK2 contributes to this pro-survival effect. 3226 Nucleic Acids Research, 2014, Vol. 42, No. 5 As NEK2, SRPK1 is also overexpressed in human cancers, suggesting that these kinases may act in concert to modulate SRSF1 activity. In line with this hypothesis, we found that NEK2 affected BCL-X splicing independently of SRPK1. Thus, even if apparently redundant, SRPK1 and N