Neks are serine-threonine kinases that are similar to NIMA, a protein found in Aspergillus nidulans which is essential for cell division. Eleven human Neks have been identified (1-11) and there is growing evidence pointing Neks role in diverse cellular process, such as cell cycle control, including cell division, mitotic spindle assembly, furrow ingression, primary cilium formation and maintenance, besides DNA damage response (DDR) and apoptosis control. Nek4 have already been associated with these process, although its role in these processes remains to be elucidated. In DNA damage response, its known that Nek4 interacts with proteins from Non-Homologous End Join (NHEJ) pathway and cells knockdown to Nek4 are etoposide-resistant. In an IP/MS assay performed by our group, several proteins related to DDR, mitochondrial metabolism and apoptosis were identified. The mitochondrial related function has already been described to Nek1 and Nek5, such as apoptosis and reactive oxygen species (ROS) levels regulation. Among the mitochondrial related proteins found in the Nek4 IP/MS assay, Mitofusin was identified. The GTPases Mitofusin 1 (Mfn1) and Mitofusin 2 (Mfn2) play a crucial role on the fusion process and the maintenance of the contacts between the mitochondria and endoplasmic reticulum. In this sense, Mfn2 plays a key role in the unfolded protein response (UPR) after endoplasmic reticulum stress. In the absence of Mfn2, the UPR is increased along with ROS and apoptosis activation deficiency. Our previous results have shown that both Nek4 isoforms interact with Mfn1 and Mfn2 as well as with the Mfn2 truncated version without the transmembrane domain, indicating that the interaction occurs through the N-terminus portion of Mfn2, the cytoplasmic region. We also verified that this interaction does not depend on the Nek4 kinase activity. Furthermore, surprisingly, we observe the presence of Nek4 in the mitochondria and endoplasmic reticulum contacting region, denominated MAMs (mitochondria-associated membranes), suggesting that Nek4 could be involved in UPR too. Corroborating with these results, we observed that Nek4 knockdown leads to alterations in the mitochondrial dynamics, indicated by the reduction in the ATP levels, increase in the ROS levels and alterations in the mitochondrial morphology. The involvement of Nek4 in DNA damage response has already been described, as well as the resistance to anticancer drugs in Nek4 depleted cells. Recently has been shown that Nek4 shows an anti-apoptotic role in TRAIL induced apoptosis in cancer cells. Considering this, the aim of this project is understanding the role of Nek4 in the mitochondria and verify if the Nek4/ Mfns interaction is component of nuclear mitochondria crosstalk after cellular stress. In this context we intend to investigate if Nek4, as a kinase, could regulate the Mfns activity. The cell cycle control and DDR, ROS levels regulation are process closely connected, and in this way, a better understanding of the crosstalk between nucleus-mitochondria signalling is essential to understanding cancer development.
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