ABSTRACT Mitosis is a dynamic and tightly regulated cell cycle phase that spans chromosome condensation, spindle formation, sister chromatid separation and segregation, and cytokinesis; the failure of cells to successfully navigate these diverse biological functions underlies many human diseases including birth defects and cancer. The orchestration of these functions is accomplished by a complex array of protein kinase signaling, including the understudied NIMA-related kinases Nek6, Nek7 and Nek9. Although gene depletion studies have implicated these Nek kinases in cell division and cytokinesis, the biochemical mechanisms by which they function remain unresolved. Thus, new research that uncovers the regulation and downstream effectors of Nek6, Nek7 and Nek9 functions has the potential to both improve our basic understanding of cell division biology as well as identify new entry points for therapeutic intervention in human disease. Here, we propose to develop and deploy targeted protein degradation coupled with mass spectrometry- based proteomics to discover new substrates and signaling networks that are regulated by Nek6, Nek7 and Nek9 kinase activities. Using a combination of CRISPR/Cas9-based targeting strategies, homology-directed repair and genetic engineering, we will knock-in a short degron tag at endogenous loci for these kinases in cell lines of diverse genetic and tissue origin. We will then employ quantitative proteomics methods to identify protein phosphorylation loci that exhibit differential occupancy upon rapid depletion of the respective Nek kinase. We expect these experiments will shed new light on the mechanisms that define cell division and will additionally serve as a roadmap for illuminating the rest of the understudied kinome.