Distinct surfaces on Cdc5/PLK Polo-box domain orchestrate combinatorial substrate recognition during cell division

Abstract

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...

Exo1 recruits Cdc5 polo kinase to MutLγ to ensure efficient meiotic crossover formation

“…We found that Exo1 interacts with Cdc5 through the same mode as the only example described so far, Dbf4. Besides the importance for the meiotic recombination context, our study therefore adds another case where the recently described interaction surface on Cdc5, distinct from the phosphopeptide recognition surface, promotes interaction with a protein relevant for a biological process (48). This surface is conserved in human PLK, and our work should open new avenues to search for other partners of Cdc5 or human PLKs with similar interacting motifs, and to inhibitors that may specifically target this new interacting surface.…”

“…Importantly, mutation of the key residue of this surface (S630Q mutation) abolished interaction with Exo1, as it did for Dbf4, but kept intact the interaction with Spc72, a canonical interacting substrate of Cdc5 (Figs. 5D and S7D) (48). Conversely, mutation of the WHK motif of Cdc5 involved in phosphopeptide recognition (44) did not alter interaction with Exo1 or Dbf4, but reduced interaction with Spc72 (Figs.…”

“…The Cdc5 PBD structure has recently been solved, and shows a specific binding surface for the Dbf4 motif, opposite from the canonical phosphopeptide binding site (Fig. 5Ci) (48). Interestingly, the Exo1 RSIEGA-like motif could be modeled interacting exactly with the same Cdc5 surface as Dbf4 (Fig.…”

“…Conversely, mutation of the WHK motif of Cdc5 involved in phosphopeptide recognition (44) did not alter interaction with Exo1 or Dbf4, but reduced interaction with Spc72 (Figs. 5D and S7D) (47,48). Furthermore, mutation of the equivalent of the key residues of Dbf4 of this motif in Exo1 (R570E I572D G574A, hereafter called exo1-cid, Cdc5 interaction-deficient) abolished the two-hybrid interaction between Exo1 and Cdc5 (Figs.…”

“…The RSIEGA motif of Exo1 was modeled based the structure of Cdc5 bound to a Dbf4-derived peptide encompassing the RSIEGA motif of Dbf4 (PDB ID: 6MF6; (48). The sequence was edited in Coot (79) and the rotamers of each residue chosen to prevent clashes between Cdc5 and the modeled peptide.…”