An integrated analysis of the protein-protein interaction network of the conserved mitotic kinase, Polo

Abstract

Polo kinase, first identified in Drosophila over 30 years ago, is a highly conserved enzyme that functions pleiotropically during multiple stages of cell division. Members of this protein family have crucial roles in cell cycle progression, centriole duplication, mitosis, cytokinesis and the DNA damage response. Although some Polo substrates and regulatory mechanisms have been identified, we still lack complete understanding of the cellular and molecular roles of this kinase. Previous work in the Wakefield lab identified 40 proteins that physically interact with Polo in Drosophila embryos, but the functional significance of these components remains unknown. As genetic interaction screening can identify functional relationships between genes, I performed a highly sensitive assay called Variable Dose Analysis (VDA) in Drosophila S2R+ cells to determine which of the physical interactors also have genetic interactions with Polo. Inhibiting Polo using the selective small-molecule inhibitor BI-2536 and transfecting shRNA against the genes of interest allowed to easily screen cells based on their viability phenotype. Known Polo genetic interactors, Map205 and mtrm, were identified by the VDA screen, validating its robustness and utility in identifying genetic interactors. Fourteen genes were selected as hits, with components of the ubiquitination system enriched among them, particularly all member proteins of the Skp, Cullin, F-box containing complex (SCF complex). Six candidate polo interactors, SkpA, Cul1, slmb, Ck1α, Klp61F, and cher were selected for validation and further characterization of the interactions in vivo. Inhibition of Ck1α and slmb via RNAi resulted in larval lethality. Live imaging of SkpA RNAi and Cul1 RNAi larvae showed an increase in the cortical localization of poloGFP during late anaphase/telophase. Together, these results suggest that polo may be degraded in an SCF-dependent manner in Drosophila. Further follow up work is needed to gain deeper insight into the relationship between the ubiquitination system and polo.