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 ∼40-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.

FIGURE 1.

Mapping the interaction between Dbf4 and the Cdc5 PBD. A, N-terminal Dbf4 deletion mutants were tested for a two-hybrid interaction with the PBD. 10-Fold serial dilutions of saturated cultures were spotted onto SCM−Trp−Leu plates to visualize total cells and SCM−Trp−Leu−His + 2 mm 3-aminotriazole (3-AT) plates to score the two-hybrid interaction. aa, amino acids. B, a schematic of the features in Dbf4 N terminus is shown including two potential destruction boxes (D-boxes), a conserved BRCT-like domain, and motifs N, M, and C along with a summary of the Dbf4-PBD two-hybrid data. C, two-hybrid results for various point mutants spanning Dbf4 residues 82–96 are summarized. Arg-83, Ile-85, Gly-87, and Ala-88 are critical for PBD binding. D, HA-Cdc7-Dbf4 complexes were immunoprecipitated (IP) from baculovirus-infected Sf9 cells and examined for co-immunoprecipitation of 3Myc-Cdc5. Cdc5 was co- immunoprecipitated by wild type Dbf4 but not by Dbf4-Δ82–88 and Dbf4-NΔ109 mutant proteins. WCE, whole cell lysates; WB, Western blot.

FIGURE 2.

A novel, non-consensus polo-box binding sequence in Dbf4. A, Dbf4 N-terminal deletion mutants were constructed to determine the minimal region (residues 78–96) required for a two-hybrid interaction with the PBD. B, a biotinylated Dbf4 peptide (residues 73–96, panel C) was tested for interaction with purified His₆-PBD using the AlphaScreen assay. Data represent the average of three independent experiments ± S.D. C, the Dbf4_(73–96)-PBD interaction was competed with the indicated non-biotinylated, wild type Dbf4 peptides (P1–P4), or mutant Dbf4 peptides as indicated in (D and E).

FIGURE 3.

Dbf4-RSIEGA mutants suppress the cdc5-1 temperature sensitivity. A, the indicated strains W303-1A, dbf4-Δ82–88 (M2805), cdc5-1 (M1614), cdc5-1 dbf4-Δ82–88 (M3112 and M3114), cdc5-1 dbf4-R83E (M3116, M3117), and cdc5-1 dbf4-NΔ109 (M2655, M2656) were spotted onto YPD plates and scored for growth at the indicated temperatures. B, various dbf4 deletions on an ARS CEN plasmid (pMW489) were introduced into M2600 (cdc5-1 dbf4Δ::kanMX6) and scored for growth by spotting serial dilutions on YPD media at the indicated temperatures. C, shown is a summary of dbf4 mutations, their effect on the Dbf4-PBD interaction, and suppression of the cdc5-1 ts. dbf4 mutants were scored for growth in the M2600 (cdc5-1 dbf4Δ::kanMX6) background by spotting serial dilutions on YPD media at increasing temperatures (supplemental Fig. S4). D, high copy plasmids expressing wild type DBF4, and the indicated mutants were transformed into M1614 (cdc5-1). Cultures were spotted onto SCM−Leu plates at 25 °C, indicating that high copy dbf4-NΔ65 lethality is alleviated by deleting residues 82–88. E, expression of the Dbf4 N terminus from the GAL1,10 promoter is lethal to cdc5-1 cells only if Dbf4 retains the ability to interact with Cdc5 as occurs in the WT, S84A, S84E, E86A, and E86K mutants.

FIGURE 4.

Dbf4 binds a surface on the PBD distinct from its phospho-protein binding site. A, a biotinylated Spc72 phospho-peptide (residues 223–242) bound the PBD in the AlphaScreen assay. B, the same (non-biotinylated) Spc72 phosphopeptide did not compete the Dbf4-PBD interaction. C, purified wild type PBD and PBD-HK proteins interact with Dbf4 in the AlphaScreen assay, but the PBD-HK mutant protein fails to interact with Spc72 phosphopeptide. D, two-hybrid Spc72_1–400 and Dbf4_66–227 interactions with the PBD were tested on the indicated plates. As in C, mutation of the PBD pincer residues H641A and K643M or W517F, H641A, and K643M had no affect on the Dbf4-PBD interaction but eliminated the Spc72-PBD interaction. 3-AT, 3-aminotriazole.

FIGURE 5.

The Cdc5 pincer residues are not required for yeast viability. A, the cdc5-HK and cdc5-WHK mutants complemented a cdc5Δ by plasmid shuffle into M1672 (cdc5Δ::kanMX6/pMW536[CDC5 URA3]) evidenced by growth on 5-fluoro-orotic acid plates and at various temperatures on YPD plates after loss of pMW536. B, growth curves of M1672 strains containing only the indicated CDC5 alleles in YPD at 30 °C are shown. C, the M1672-transformed strains in panel A were spotted onto YPD plates with/without benomyl (top). WT (M138), cdc5-HK (M3502), cdc5-5 (M1680), and cdc15-4 (M1999) strains containing integrated alleles were similarly spotted onto YPD with/without benomyl (bottom). D, representative tetrads from diploid strains of genotype cdc5-HK/CDC5 dbf2–1/DBF2, cdc5-HK/CDC5 cdc15-4/CDC15 TAB6-1/CDC14, or cdc5-HK/CDC5 cdc14-1/CDC14 that were sporulated and dissected onto YPD plates at 25 °C. Recombinant genotypes are indicated.

FIGURE 6.

Mutation of the Cdc5 pincer residues causes a G₂/M delay and alters spindle dynamics. A, flow cytometry profiles of asynchronous W303-1A (WT) and M3502 (cdc5-HK) strains are shown. B, average spindle length was quantitated in large-budded cells of the same strains, shown with the range that includes 25–75% of spindle lengths. The inset shows fraction of cells with short spindles, <2 μm. C, shown are flow cytometry profiles of W303-1A and M3502 arrested in G₂ with nocodazole for 3 h (t = 0) and after release at 30 °C. D, quantitation of spindle length at the indicated times after nocodazole release. S.E. were all less than 1%. E, shown is tubulin staining of representative photomicrographs of cells at 40 min after nocodazole release.