Turboid reveals NEK2a's semi-dynamic cell cycle interactions and Nusap1 as a novel partner

Nek2A is a cell cycle regulated kinase, which is involved in several cellular processes and overexpressed in numerous cancer types. It has been associated with chromosome instability, increased cell proliferation and drug resistance in cancers. Our study primarily aims to identify the specific partners that interact with Nek2A during the cell cycle, deepening our understanding of Nek2's role in cancer. To meet this objective, we employed the TurboID proximity labeling technique on synchronized cell groups, exploring the dynamic interactions of Nek2A as the cell cycle advances. We synchronized the cells using a double thymidine block and harvested them at designated release time points, focusing on G1/S, late S, and G2/M cell cycle phases. Through mass spectrometry, we identified biotinylated proteins, which we further analyzed using MaxQuant, Cassiopeia, and Amica tools. Our experiments reliably identified previously recognized Nek2A partners like the Anaphase Promoting Complex (APC) and Kif24. We confirmed proteins closely interacting with Nek2A during the G1/S, late S, and G2/M stages using western blotting. Co-immunoprecipitation unveiled direct interactions of Nek2A with proteins such as Nusap1, Kif2c, Mapre3, and Mpg. We also confirmed the colocalization of these proteins with Nek2A using fluorescence microscopy. We took a special interest in Nusap1 due to its unique role, distinct from other Nek2A partners. Nusap1 acts as a stabilizer for DNA damage proteins, and its depletion can lead to DNA strand breaks, negatively impacting cell survival. Our analysis highlighted a prevalent occurrence of ubiquitin-driven proteolysis during the cell cycle's progression. We initially studied Nusap1's expression in cells with either diminished or amplified Nek2A activity. Remarkably, there was an increase in Nusap1 levels post-siRNA treatment and in two distinct Nek2 null (KO) cell variants. Conversely, overexpression of Nek2A led to a short-lived reduction in Nusap1 amounts, lasting just 6 hours, before other cell mechanisms presumably balanced it. Using the well- known proteasome inhibitor, MG132, we were able to counteract this reduction. Moreover, we observed a notable reduction in Nusap1 ubiquitination in Nek2A KO cells, pointing to the possible role of proteasomal breakdown processes. A broad protein analysis in Nek2A deficient cells confirmed many proteins, including Nusap1, showed decreased expression. In essence, our findings indicate Nusap1 as a new Nek2A partner, with Nek2A possibly affecting Nusap1's degradation through ubiquitination. The exact nature of this interaction and the clinical significance require further exploration.