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

Author(s)

Ferezin, Camila de Castro ; Sian, Terry C. C. Lim Kam ; Wu, Yunjian ; Ma, Xiuquan ; Chueeh, Anderly C. ; Huang, Cheng ; Schittenhelm, Ralf B. ; Kobarg, Jorg ; Daly, Roger J.

• Document type: Journal article
• Source: CELL COMMUNICATION AND SIGNALING; v. 20, n. 1, p. 15-pg., 2022-12-22.

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. (AU)

• FAPESP's process: 16/10530-5 - The funtion of the protein kinase Nek5 in normal and cancer cells: from the identification of physiological substrates to inhibitor design
• Grantee: Camila de Castro Ferezin
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 19/11435-4 - NEK5 phosphoproteomic profiling: identification of phospho-substrates in cancer cells and validation of known targets
• Grantee: Camila de Castro Ferezin
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• FAPESP's process: 17/03489-1 - From functional studies to searching for new inhibitors for cancer: exploring kinases that regulate the cell cycle of the human NEK family
• Grantee: Jörg Kobarg
• Support Opportunities: Research Projects - Thematic Grants