Identification of biological pathways and processes regulated by NEK5 in breast epithelial cells via an integrated proteomic approach

Abstract

Specific members of the Nima-Related Kinase (NEK) family have been linked to cancer development and progression, and a role for NEK5, one of the least studied members, in breast cancer has recently been proposed. However, while NEK5 is known to regulate centrosome separation and mitotic spindle assembly, NEK5 signalling mechanisms and function in this malignancy require further characterization. To this end, we established a model system featuring overexpression of NEK5 in the immortalized breast epithelial cell line MCF-10A. MCF-10A cells overexpressing NEK5 exhibited an increase in clonogenicity under monolayer conditions and enhanced acinar size and abnormal morphology in 3D Matrigel culture. Interestingly, they also exhibited a marked reduction in Src activation and downstream signalling. To interrogate NEK5 signalling and function in an unbiased manner, we applied a variety of MS-based proteomic approaches. Determination of the NEK5 interactome by Bio-ID identified a variety of protein classes including the kinesins KIF2C and KIF22, the mitochondrial proteins TFAM, TFB2M and MFN2, RhoH effectors and the negative regulator of Src, CSK. Characterization of proteins and phosphosites modulated upon NEK5 overexpression by global MS-based (phospho)proteomic profiling revealed impact on the cell cycle, DNA synthesis and repair, Rho GTPase signalling, the microtubule cytoskeleton and hemidesmosome assembly. Overall, the study indicates that NEK5 impacts diverse pathways and processes in breast epithelial cells, and likely plays a multifaceted role in breast cancer development and progression.

Mitochondria transcription and cancer

“…Additionally, Cyclophilin-D (CypD) can directly interact with TFB2M to inhibit mtDNA transcription [ 113 ]. Ferezinwe and colleagues employed various MS-based proteomic approaches to investigate the interactome of Nima Related Kinases 5 (NEK5) [ 114 ]. Their study uncovered the association between NEK5 and mitochondrial proteins, such as TFAM, TFB2M, and MFN2, suggesting their potential involvement in mitochondrial maintenance, transcription, and repair processes [ 114 ].…”

The Mitochondrial Connection: The Nek Kinases’ New Functional Axis in Mitochondrial Homeostasis

“…Nek5 was shown to interact with COX11, an intrinsic mitochondrial membrane protein essential for the assembly of an active cytochrome c oxidase complex (complex IV) in a yeast two-hybrid screening [ 16 ]. In 2022, an analysis of Nek5-overexpressing MCF10A cells proteome using BioID revealed interesting mitochondrial partners such as HIGD1A, TFAM, C2orf47, SLC25A12, GSR, TIGAR, IARS2, IDH3B, CLPX, TFB2M, MFN2, PPA2, MRPS11, and MT-ATP6 (from the highest to the lowest logFC, p -value < 0.05) [ 18 ]. The interactome enrichment pathway analysis of NEK5 interactors showed that mitochondrial organization and mitochondrial gene expression are among the top five enriched pathways in MCF10A Nek5-overexpressing cells, reinforcing our hypothesis of Nek5 participation in mitochondrial mtDNA homeostasis and mitochondrial functions ( Figure 1 and Figure 3 ).…”

“…Indeed, participation in DNA damage response, alternative splicing, and cilia formation has already been described for several members of the Nek family (for review, see [ 9 , 10 , 11 ]) [ 12 , 13 ]. A mitochondrion-related function was first shown for Nek1 [ 14 , 15 ], and from that, it was extended to other members: Nek5 [ 16 , 17 , 18 ], Nek10 [ 19 ], Nek4 [ 13 , 20 ], and Nek6 [ 20 , 21 ], so far.…”

“…Mitochondrial functions are regulated by mitochondrial morphology, protein composition, and mtDNA content and integrity. Our interactome studies on Nek1 [ 21 ], Nek4 [ 13 ], Nek5 [ 16 ], Nek10 [ 18 ], and Nek6 [ 22 ] revealed several mitochondrial or mitochondrion-related proteins. Based on that, we suggest five Nek family members that participate in the regulation of distinct mitochondrion-related functions, such as mitochondrion composition/structure, metabolism, mtDNA maintenance, cell stress response, and autophagy ( Figure 1 ).…”

The pseudokinase NRBP1 activates Rac1/Cdc42 via P-Rex1 to drive oncogenic signalling in triple-negative breast cancer

“…MS-based proteomic analyses were undertaken on control and shRNA#1-mediated NRBP1 knockdown MDA-MB-231 cells. NRBP1 knockdown was induced by 1 µg/ml doxycycline for 48 h. These analyses were performed in triplicate on independent biological replicates according to previously-published protocols [21,46].…”