Dbf4 Regulates the Cdc5 Polo-like Kinase through a Distinct Non-canonical Binding Interaction

Abstract

Cdc7-Dbf4 is a conserved, two-subunit kinase required for initiating eukaryotic DNA replication. Recent studies have shown that Cdc7-Dbf4 also regulates the mitotic exit network (MEN) and monopolar homolog orientation in meiosis I (Matos, J., Lipp, J. J., Bogdanova, A., Guillot, S., Okaz, E., Junqueira, M., Shevchenko, A., and Zachariae, W. (2008) Cell 135, 662-678 and Miller, C. T., Gabrielse, C., Chen, Y. C., and Weinreich, M. (2009) PLoS Genet. 5, e1000498). Both activities likely involve a Cdc7-Dbf4 interaction with Cdc5, the single Polo-like kinase in budding yeast. We previously showed that Dbf4 binds the Cdc5 polo-box domain (PBD) via an ϳ40-residue N-terminal sequence, which lacks a PBD consensus binding site (S(pS/pT)(P/X)), and that Dbf4 inhibits Cdc5 function during mitosis. Here we identify a non-consensus PBD binding site within Dbf4 and demonstrate that the PBD-Dbf4 interaction occurs via a distinct PBD surface from that used to bind phosphoproteins. Genetic and biochemical analysis of multiple dbf4 mutants indicate that Dbf4 inhibits Cdc5 function through direct binding. Surprisingly, mutation of invariant Cdc5 residues required for binding phosphorylated substrates has little effect on yeast viability or growth rate. Instead, cdc5 mutants defective for binding phosphoproteins exhibit enhanced resistance to microtubule disruption and an increased rate of spindle elongation. This study, therefore, details the molecular nature of a new type of PBD binding and reveals that Cdc5 targeting to phosphorylated substrates likely regulates spindle dynamics.Cell cycle progression requires the highly accurate replication and segregation of chromosomes. Although these two events occur at different times, several cell cycle kinases regulate both DNA synthesis and chromosome segregation (3). In budding yeast the Cdc7-Dbf4 kinase (also called Dbf4-dependent kinase or DDK) 2 plays such a dual role in the cell cycle. The Dbf4 regulatory subunit binds to and activates Cdc7 kinase to initiate DNA replication (4, 5). DDK also promotes other aspects of chromosome biology including cohesin loading during early S-phase in Xenopus laevis (6), centromeric cohesion in Schizosaccharomyces pombe (6), and meiotic recombination (7, 8) and the Ndt80 (early meiotic) transcriptional program in Saccharomyces cerevisiae (9). Budding yeast DDK also promotes monopolar orientation of homologs in meiosis I and inhibits chromosome segregation in the mitotic cycle (1, 2, 10, 11). Both activities are likely mediated through an interaction with Cdc5, the single Polo-like kinase in S. cerevisiae. Polo-like kinases (Plks) regulate mitotic events and are also involved in the response to DNA damage and checkpoint adaptation (12-14). Genetic and physical interactions between Dbf4 and Cdc5 were described many years ago (15, 16) raising the possibility that DDK acted beyond S phase. The DDK-Cdc5 interaction raises interesting questions regarding how these distinct kinases interact and coordinate accurate cell cycle progression.The Polo gene was na...

Independent modulation of the kinase and polo-box activities of Cdc5 protein unravels unique roles in the maintenance of genome stability

“…These observations prompted us to create both moderate (cdc5-12: H641A, K643M) and severe (cdc5-16: W517F, H641A, K643M) alleles of Cdc5 PBD based on the observation that polo-like kinase 1 (Plk1) mutants corresponding to W517F or H641A-K643M have no detectable phosphopeptide-binding activity in vitro (9,25). Consistent with this observation, introducing these mutations in Cdc5 PBD prevented binding to all its known phospho-substrates (9,22,26,27). Heterozygous diploid yeast strains carrying one copy of cdc5-12 or cdc5-16 were sporulated, and the viability of the resulting spores was investigated.…”

Mitotic Exit and Separation of Mother and Daughter Cells

“…This is clearly the case for some Cdc5 substrates (Yoshida et al 2006). Intriguingly, however, recent analyses indicate that phosphopeptide binding by the Cdc5 PBD is not truly essential, and instead is required for only a subset of Cdc5-dependent processes (Chen and Weinreich 2010;Ratsima et al 2011).…”

Binding of Drosophila Polo kinase to its regulator Matrimony is noncanonical and involves two separate functional domains

“…Notably, the serine in R-S-I-E-G-A is not phosphorylated. Moreover, the PBD pincer mutant is still able to robustly interact with Dbf4 as assayed by Y2H analysis, suggesting that Dbf4 may interact with a distinct binding surface on the PBD (31).…”

“…Another example of a noncanonical Polo interaction is the binding of the yeast protein Dbf4 with the yeast homolog of Polo, Cdc5 (31). Dbf4 was shown to interact with the Cdc5 PBD through a noncanonical R-S-I-E-G-A protein motif in Dbf4.…”