Polo-like kinases (Plks) are key cell cycle regulators. They contain a kinase domain followed by a polobox domain that recognizes phosphorylated substrates and enhances their phosphorylation. The regulatory subunit of the Dbf4-dependent kinase complex interacts with the polo-box domain of Cdc5 (the sole plk in Saccharomyces cerevisiae) in a phosphorylation-independent manner. We have solved the crystal structures of the polo-box domain of Cdc5 on its own and in the presence of peptides derived from Dbf4 and a canonical phosphorylated substrate. The structure bound to the Dbf4-peptide reveals an additional density on the surface opposite to the phospho-peptide binding site that allowed us to propose a model for the interaction. We found that the two peptides can bind simultaneously and non-competitively to the polo-box domain in solution. Furthermore, point mutations on the surface opposite to the phosphopeptide binding site of the polo-box domain disrupt the interaction with the Dbf4 peptide in solution and cause an early anaphase arrest phenotype distinct from the mitotic exit defect typically observed in cdc5 mutants. Collectively, our data illustrates the importance of noncanonical interactions mediated by the polo-box domain and provide key mechanistic insights into the combinatorial recognition of substrates by Polo-like kinases. Processes that drive mitotic progression are under strict cellular regulation to ensure the faithful propagation of newly replicated genetic material. Cellular defects that arise during mitosis-such as sister chromatid mis-alignment or spindle pole mis-positioning-can lead to chromosome segregation defects and give rise to polyploid and aneuploid daughter cells 1-3. It is during these events that the cell activates signaling cascades known as checkpoints to inhibit mitotic processes 4. Misregulation of mitotic checkpoints gives cells a proliferative advantage-one of the hallmarks of carcinogenesis 3,5. A conserved family of kinases known as Polo-like kinases (Plks) control mitotic events 6. Plks function in mitotic entry, spindle pole dynamics, chromosome condensation 7 , sister-chromatid segregation 8 , and cytokinesis 2,6,9. Plk1 is upregulated in multiple human tumors and, therefore, has become an attractive anti-cancer target 10,11. The structural similarity with other cellular kinases, however, has limited the potential of inhibiting Plk1 due to off-target effects. Some of the most selective drugs against Plk1 also inhibit Plk2 and Plk3 with similar potency 12,13. Therefore, targeting interactions that regulate kinase activity provide a promising approach to specifically target Plk1. Much of our understanding of Plk1 comes from Cdc5, the sole Plk in budding yeast 4,14,15. Cdc5 promotes the release of Cdc14 from the nucleolus and regulates the mitotic exit network (MEN) 9,16-18 as well as cytokinesis 19-21. Full release of Cdc14 leads to the downstream activation of the anaphase promoting complex, cyclin destruction, mitotic spindle disassembly, and displacement of the septin ri...

Centrosomes are best known as the microtubule organizing centers (MTOCs) of eukaryotic cells. In addition to their classic role in chromosome segregation, centrosomes play diverse roles unrelated to their MTOC activity during cell proliferation and quiescence. Metazoan centrosomes and their functional doppelgängers from lower eukaryotes, the spindle pole bodies (SPBs), act as important structural platforms that orchestrate signaling events essential for cell cycle progression, cellular responses to DNA damage, sensory reception and cell homeostasis. Here, we provide a critical overview of the unconventional and often overlooked roles of centrosomes/SPBs in the life cycle of eukaryotic cells.