Characterization of hNek10 and hNek5 kinases in DNA damage response

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NEK5 interacts with topoisomerase II beta and is involved in the DNA damage response induced by etoposide

Cells are daily submitted to high levels of DNA lesions that trigger complex pathways and cellular responses by cell cycle arrest, apoptosis, alterations in transcriptional response, and the onset of DNA repair. Members of the NIMA-related kinase (NEK) family have been related to DNA damage response and repair and the first insight about NEK5 in this context is related to its role in centrosome separation resulting in defects in chromosome integrity. Here we investigate the potential correlation between NEK5 and the DNA damage repair index. The effect of NEK5 in double-strand breaks caused by etoposide was accessed by alkaline comet assay and revealed that NEK5-silenced cells are more sensitive to etoposide treatment. Topoisomerase II beta (TOPII beta) is a target of etoposide that leads to the production of DNA breaks. We demonstrate that NEK5 interacts with TOPII beta, and the dynamics of this interaction is evaluated by proximity ligation assay. The complex NEK5/TOPII beta is formed immediately after etoposide treatment. Taken together, the results of our study reveal that NEK5 depletion increases DNA damage and impairs proper DNA damage response, pointing out NEK5 as a potential kinase contributor to genomic stability120101685316866CAPES - Coordenação de Aperfeiçoamento de Pessoal e Nível SuperiorCNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESP – Fundação de Amparo à Pesquisa Do Estado De São PauloSem informação308868/2018‐8; 302534/ 2017‐2010/15262‐2; 2010/51730‐0; 2010/16831‐0; 2015/06458‐4; 2017/03489‐1; 2014/15982‐

Identification of NEK3 interacting proteins and functional characterization of its signaling mechanisms

NEKs (NIMA-related kinases) are a group of kinases that share high amino acid sequence identity to NIMA (Never in mitosis gene A), which exists as a single member in the fungi Aspergillus nidulans and is functionally involved in the initiation of mitosis. NEK3 is a 506 amino acid serine/threonine kinase, localizes both to the nucleus and cytoplasm, and its gene localizes to 13q14.2 chromosome. It has an N-terminal catalytic domain and a C-terminal regulatory domain, which contains Thr475 in its PEST domain, which gets phosphorylated upon activation. Expression of mutants without Thr475 or PEST domain causes changes in cellular morphology and polarity of neuronal cells. NEK3 is also involved in cell motility and invasiveness of breast cancer tumor cells through interaction with regulators of the Rho GTPases Rac1 and RhoA, mediated by prolactin induced association of NEK3 to the human Prolactin Receptor (PRLR). Using the Matchmaker Gold Yeast Two-Hybrid system, a screening for interaction partners wasperformed and 65 clones were obtained, which cDNAs encode 27 different proteins. The identified candidate interacting proteins are functionally involved in sumoylation, ubiquitinylation, transcriptional regulation, DNA repair, RNA processing, and the regulation of cell proliferation, invasiveness and metastasis.Some interaction partners for NEK3 are located in the nucleus and plasma membrane but most of them localize to the cytoplasm. One of the cytoplasmatic interactors for NEK3, RhoGDI2, is a regulator of RhoGTPases and inhibits Rac1 and RhoA activation levels. In our pull down assay, NEK3 overexpression increases Rac1-GTP while concomittant overexpression of RhoGDI2 reducesit to non-detectable levels. Our data suggests that NEK3 interaction with RhoGDI2 is a important new regulatory elements in Rac1 signaling pathways.

Fasciculation and elongation zeta-1 protein (FEZ1) interacts with the retinoic acid receptor and participates in transcriptional regulation of the Hoxb4 gene

Fasciculation and elongation zeta-1 (FEZ1) protein is involved in axon outgrowth and is highly expressed in the brain. It has multiple interaction partners, with functions varying from the regulation of neuronal development and intracellular transport mechanisms to transcription regulation. One of its interactors is retinoic acid receptor (RAR), which is activated by retinoic acid and controls many target genes and physiological process. Based on previous evidence suggesting a possible nuclear role for FEZ1, we wanted to deepen our understanding of this function by addressing the FEZ1-RAR interaction. We performed in vitro binding experiments and assessed the interface of interaction between both proteins. We found that FEZ1-RAR interacted with a similar magnitude as RAR to its responsive element DR5 and that the interaction occurred in the coiled-coil region of FEZ1 and in the ligand-binding domain of RAR. Furthermore, cellular experiments were performed in order to confirm the interaction and screen for induced target genes from an 86-gene panel. The analysis of gene expression showed that only in the presence of retinoic acid did FEZ1 induce hoxb4 gene expression. This finding is consistent with data from the literature showing the hoxb4 gene functionally involved in development and acute myeloid leukemia, as is FEZ1

Insights into the full-length SRPK2 structure and its hydrodynamic behavior

The serine/arginine-rich protein kinase 2 (SRPK2) has been reported as upregulated in several cancer types, with roles in hallmarks such as cell migration, growth, and apoptosis. These findings have indicated that SRPK2 is a promising emerging target in drug discovery initiatives. Although high-resolution models are available for SRPK2 (PDB 2X7G), they have been obtained with a heavily truncated recombinant protein version (-50% of the primary structure), due to the presence of long intrinsically unstructured regions. In the present work, we sought to characterize the structure of a full-length recombinant version of SRPK2 in solution. Low-resolution Small-Angle X-ray Scattering data were obtained for both versions of SRPK2. The truncated Delta N Delta S-SRPK2 presented a propensity to dimerize at higher concentrations whereas the full-length SRPK2 was mainly found as dimers. The hydrodynamic behavior of the full-length SRPK2 was further investigated by analytical size exclusion chromatography and sedimentation velocity analytical ultracentrifugation experiments. SRPK2 behaved as a monomer-dimer equilibrium and both forms have an elongated shape in solution, pointing to a stretched-to closed tendency among the conformational plasticity observed. Taken together, these findings allowed us to define unique structural features of the SRPK2 within SRPK family, characterized by its flexible regions outside the bipartite kinase domain137205214CAPES - Coordenação de Aperfeiçoamento de Pessoal e Nível SuperiorCNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPEMIG - Fundação de Amparo à Pesquisa Do Estado De Minas GeraisFAPESP – Fundação de Amparo à Pesquisa Do Estado De São PauloSem informação485011/2012-3; 420648/2016-0; 471415/2013-8; 303129/2015-8CBB-01637-13; CBB-APQ02556-15; RED-00140-162011/23110-0; 2012/50161-8; 2014/07206-6; 2017/07335-9; 2012/00195-3; 13/50724-5; 2017/03489-