Search

Mitochondria are involved in energy supply, signaling, cell death and cellular differentiation and have been implicated in several human diseases. Neks (NIMA-related kinases) represent a family of mammal protein kinases that play essential roles in cell-cycle progression, but other functions have recently been related. A yeast two-hybrid (Y2H) screen was performed to identify and characterize Nek5 interaction partners and the mitochondrial proteins Cox11, MTX-2 and BCLAF1 were retrieved. Apoptosis assay showed protective effects of stable hNek5 expression from Hek293-T's cell death after thapsigargin treatment (2 μM). Nek5 silenced cells as well as cells expressing a “kinase dead” version of Nek5, displayed an increase in ROS formation after 4 h of thapsigargin treatment. Mitochondrial respiratory chain activity was found decreased upon stable hNek5expression. Cells silenced for hNek5 on the other hand presented 1.7 fold increased basal rates of respiration, especially at the electrons transfer steps from TMPD to cytochrome c and at the complex II. In conclusion, our data suggest for the first time mitochondrial localization and functions for Nek5 and its participation in cell death and cell respiration regulation. Stable expression of h Nek5 in Hek293T cells resulted in enhanced cell viability, decreased cell death and drug resistance, while depletion of hNek5by shRNA overcame cancer cell drug resistance and induced apoptosis in vitro . Stable expression of hNek5 also inhibits thapsigargin promoted apoptosis and the respiratory chain complex IV in HEK293T cells.

NEKs ( N IMA-r e lated k inases) are a group of kinases that share high amino acid sequence identity to NIMA ( N ever i n m itosis 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.

A precise orchestration of the DNA damage response (DDR) is absolute essential to avoid genomic instability, which may lead to apoptosis or may initiate tumorigenesis. Recently, hNek1, 2, 4, 8, 9, ...

Aside from Polo and Aurora, a third but less studied kinase family involved in mitosis regulation is the never in mitosis-gene A (NIMA)-related kinases (Neks). The founding member of this family is the sole member NIMA of Aspergillus nidulans, which is crucial for the initiation of mitosis in that organism. All 11 human Neks have been functionally assigned to one of the three core functions established for this family in mammals: (1) centrioles/mitosis; (2) primary ciliary function/ciliopathies; and (3) DNA damage response (DDR). Recent findings, especially on Nek 1 and 8, showed however, that several Neks participate in parallel in at least two of these contexts: primary ciliary function and DDR. In the core section of this in-depth review, we report the current detailed functional knowledge on each of the 11 Neks. In the discussion, we return to the cross-connections among Neks and point out how our and other groups’ functional and interactomics studies revealed that most Neks interact with protein partners associated with two if not all three of the functional contexts. We then raise the hypothesis that Neks may be the connecting regulatory elements that allow the cell to fine tune and synchronize the cellular events associated with these three core functions. The new and exciting findings on the Nek family open new perspectives and should allow the Neks to finally claim the attention they deserve in the field of kinases and cell cycle biology.

Background The adaptor protein RACK1 (receptor of activated kinase 1) was originally identified as an anchoring protein for protein kinase C. RACK1 is a 36 kDa protein, and is composed of seven WD repeats which mediate its protein-protein interactions. RACK1 is ubiquitously expressed and has been implicated in diverse cellular processes involving: protein translation regulation, neuropathological processes, cellular stress, and tissue development. Results In this study we performed a biophysical analysis of human RACK1 with the aim of obtaining low resolution structural information. Small angle X-ray scattering (SAXS) experiments demonstrated that human RACK1 is globular and monomeric in solution and its low resolution structure is strikingly similar to that of an homology model previously calculated by us and to the crystallographic structure of RACK1 isoform A from Arabidopsis thaliana. Both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation techniques showed that RACK1 is predominantly a monomer of around 37 kDa in solution, but also presents small amounts of oligomeric species. Moreover, hydrodynamic data suggested that RACK1 has a slightly asymmetric shape. The interaction of RACK1 and Ki-1/57 was tested by sedimentation equilibrium. The results suggested that the association between RACK1 and Ki-1/57(122-413) follows a stoichiometry of 1:1. The binding constant (KB) observed for RACK1-Ki-1/57(122-413) interaction was of around (1.5 ± 0.2) × 106 M-1 and resulted in a dissociation constant (KD) of (0.7 ± 0.1) × 10-6 M. Moreover, the fluorescence data also suggests that the interaction may occur in a cooperative fashion. Conclusion Our SAXS and analytical ultracentrifugation experiments indicated that RACK1 is predominantly a monomer in solution. RACK1 and Ki-1/57(122-413) interact strongly under the tested conditions.