Polo kinases have crucial conserved functions in controlling the eukaryotic cell cycle through orchestrating several events during mitosis1,2. An essential element of cell cycle control is exerted by altering the expression of key regulators3. Here we show an important function for the polo kinase Cdc5p in controlling cell-cycle-dependent gene expression that is crucial for the execution of mitosis in the model eukaryote Saccharomyces cerevisiae. In particular, we find that Cdc5p is temporally recruited to promoters of the cell-cycle-regulated CLB2 gene cluster, where it targets the Mcm1p–Fkh2p–Ndd1p transcription factor complex, through direct phosphorylation of the coactivator protein Ndd1p. This phosphorylation event is required for the normal temporal expression of cell-cycle-regulated genes such as CLB2 and SWI5 in G2/M phases. Furthermore, severe defects in cell division occur in the absence of Cdc5p-mediated phosphorylation of Ndd1p. Thus, polo kinase is required for the production of key mitotic regulators, in addition to previously defined roles in controlling other mitotic events.
In S. cerevisiae the polo kinase Cdc5p has been implicated in several processes associated with mitosis, including mitotic entry, spindle assembly, cytokinesis and mitotic exit2. Although several direct targets for Cdc5p have been identified, including Swe1p, Scc1p and Bfa1p4,5,6,7, no direct links have been made between polo kinases and the transcription factor complexes that control cell-cycle-dependent transcription.
In S. cerevisiae transcriptional regulation of the CLB2 gene cluster in the G2 and M phases represents a key cell cycle control point. This gene cluster encodes several factors required for mitotic progression including the B-type cyclin Clb2p, transcriptional regulators such as Swi5p, and Cdc5p polo kinase3,8. A major determinant of this coordinated gene expression is a transcription factor complex that consists of Mcm1p, the forkhead transcription factor Fkh2p and the coactivator protein Ndd1p3,9,10,11,12,13. The regulation of the Mcm1p–Fkh2p–Ndd1p complex is linked to the cell cycle through the sequential phosphorylation of Fkh2p by the Clb5p–Cdc28p14 kinase complex, and Ndd1p by the Clb2p–Cdc28p15,16 kinase complex. Although these regulatory phosphorylation events promote Ndd1p recruitment and target gene activation, it is less clear whether they alone account for the complete regulation of the Mcm1p–Fkh2p–Ndd1p complex. We therefore investigated whether the polo kinase Cdc5p has a role in controlling the activity of the Mcm1p–Fkh2p–Ndd1p complex.
First we investigated whether we could detect Cdc5p at the promoters of genes within the CLB2 cluster by chromatin immunoprecipitation (ChIP) analysis. We used a strain overexpressing Cdc5p to permit the analysis of Cdc5p recruitment at times when endogenous Cdc5p is difficult to detect because of its cyclical expression17. Although little Cdc5p could be detected in cells arrested in S phase by treatment with hydroxyurea, on release from the hydroxyurea block, recruitment of Cdc5p to the promoters of all of the CLB2 cluster genes tested was observed, which was further enhanced when cells were arrested in M phase with nocodazole (Fig. 1a). Furthermore, after release from α-factor arrest in G1 phase, recruitment of Cdc5p was induced in a cell-cycle-dependent manner at both the CLB2 and SWI5 promoters, at a time consistent with the onset of transcription of these genes (Fig. 1b). However, Cdc5p recruitment to the promoter of a G1-phase-expressed cyclin-encoding gene, CLN2 (ref. 13), could not be observed (Fig. 1c). Glutathione S-transferase (GST)-conjugated Cdc5p was expressed at times when promoter recruitment was not observed, demonstrating that recruitment is a temporal event that is not dependent on the levels of Cdc5p (Supplementary Fig. S1). ChIP analysis of a control strain expressing GST alone showed little enrichment of the CLB2 promoter (Supplementary Fig. S2). Recruitment of Cdc5p expressed at endogenous levels could also be specifically detected at CLB2 cluster promoters (Fig. 1d). Thus, despite the catalogued localization of Cdc5p to specific subcellular structures such as the spindle pole bodies2 during mitosis, a portion of the cellular pool is associated with CLB2 cluster promoters.
Likely candidates for recruiting Cdc5p to CLB2 cluster promoters are components of the Mcm1p–Fkh2p–Ndd1p transcription factor complex. Indeed, Fkh2p, but not Ndd1p, formed a complex with Cdc5p in vitro (Fig. 1e). Similarly, GST–Cdc5p could co-precipitate Fkh2p when co-expressed in vivo (data not shown). The observed recruitment of Cdc5p to CLB2 cluster promoters indicated that Cdc5p might directly phosphorylate one or more components of the Mcm1p–Fkh2p–Ndd1p complex. In contrast with Fkh2p, which was only poorly phosphorylated by Cdc5p, Ndd1p was phosphorylated as efficiently as the known Cdc5p substrate Bfa1p (Fig. 1f, and Supplementary Fig. S3)4,6. A catalytically inactive version, Cdc5-KD, had a much lower ability to phosphorylate Ndd1p (Fig. 1g).
Ndd1p contains a perfect match at Ser 85 to the consensus phosphoacceptor motif for the mammalian polo kinase, Plk1 (ref. 18) (Fig. 2a). We therefore replaced Ser 85 of Ndd1p with an alanine residue (S85A) and analysed the phosphorylation of the mutant protein by Cdc5p. In vitro kinase assays revealed a decrease in total phosphorylation of Ndd1p(S85A) by Cdc5p (Fig. 2b); moreover, western blotting with an anti-phospho-Ser85 antibody showed that phosphorylation at Ser 85 was abolished in Ndd1p(S85A) (Fig. 2b). In contrast, the S85A mutation had little effect on Ndd1p phosphorylation by Clb2p–Cdc28p complexes and phosphorylation at Ser 85 was not observed (Fig. 2b).
To probe the potential role of Cdc5p in the phosphorylation of Ndd1p in vivo, we analysed the phosphorylation of wild-type and mutant (S85A) versions of Ndd1p in yeast strains containing a temperature-sensitive allele of CDC5 (cdc5ts). Significantly, phosphorylation of Ser 85 could be detected in the wild-type, but not the mutant, Ndd1p at the permissive temperature (Fig. 2c). Furthermore, phosphorylation of Ser 85 in wild-type Ndd1p was attenuated on inactivation of Cdc5p at 37 °C (Fig. 2c).
Clb2p–Cdc28p-mediated phosphorylation of Thr 319 in Ndd1p is important in Ndd1p-mediated activation of the CLB2 cluster16. However, phosphorylation of Thr 319 is not required for Cdc5p-dependent phosphorylation of Ndd1p (Supplementary Fig. S4).
Fkh2p is important for the recruitment of Ndd1p to promoters of genes in the CLB2 cluster9. As Cdc5p is also recruited to these promoters, and binds to Fkh2p in vitro, we tested whether Fkh2p is required for Cdc5p recruitment in vivo. Haemagglutinin (HA)-epitope-tagged Cdc5p was expressed in FKH2 (wild-type control) and fkh2Δ strains, and promoter recruitment was monitored by ChIP analysis. Cell-cycle-dependent temporal recruitment of Cdc5p to CLB2 cluster promoters was observed in wild-type cells but was largely lost in fkh2Δ cells (Fig. 2d, and Supplementary Fig. S5). Using the same approach, we also determined whether Fkh2p was required for Ndd1p phosphorylation in vivo. Ndd1p was phosphorylated at Ser 85 in wild-type and fkh1Δ cells, whereas phosphorylation of Ser 85 was severely diminished in fkh2Δ cells (Fig. 2e). These results therefore suggest that Fkh2p has a function in nucleating the formation of a Fkh2p–Ndd1p–Cdc5p complex on CLB2 cluster promoters, and the subsequent Cdc5p-dependent phosphorylation of Ndd1p at Ser 85.
To understand the functional consequences of Cdc5p-mediated phosphorylation of Ndd1p at Ser 85, we analysed the cellular phenotypes associated with mutations of this site. In contrast with wild-type control cells (more than 90% normal morphology), more than 90% of the ndd1Δ cells expressing Ndd1p(S85A) had aberrant morphologies, such as elongated daughter cells, hyphal-like growth and cells with problems in separating (Fig. 3a, and Supplementary Fig. S6). Moreover, these cells exhibited multiple different severe defects in nuclear morphology and mitotic spindle structure (Fig. 3a; see Supplementary Information for details). In addition, cytokinesis was defective, as demonstrated by the lack of formation of normal septa (Fig. 3b). Many of the defects observed in Ndd1p(S85A)-expressing cells resemble those observed in cdc5 mutant cells, including defects in septum formation and cytokinesis19. This indicates that at least some of the cdc5-dependent defects might be primarily due to deficiencies in Ndd1p-mediated gene regulation rather than problems in other functions of Cdc5p.
Next we examined whether phosphorylation of Ser 85 is involved in the recruitment of Ndd1p to promoters in the CLB2 cluster. HA-epitope-tagged versions of Ndd1p were expressed from plasmids in a strain expressing Tandem Affinity Purification (TAP)-tagged wild-type Ndd1p from the normal chromosomal locus, to allow the cell cycle to proceed normally. Under these conditions, plasmid-expressed HA-tagged wild-type Ndd1p is more efficiently recruited to the SWI5 promoter than HA-tagged Ndd1p(S85A) in the presence of TAP-tagged wild-type Ndd1p (Fig. 4a). In contrast, TAP-tagged wild-type Ndd1p is preferentially recruited only in the presence of plasmid-expressed HA-tagged Ndd1p(S85A) (Fig. 4a). These data indicated that wild-type Ndd1p might be more efficiently recruited to CLB2 gene cluster promoters than Ndd1p(S85A). Indeed, consistent with this hypothesis was our observation of delayed recruitment of mutant Ndd1p(S85A) to several CLB2 gene cluster promoters in synchronized cells co-expressing wild-type Ndd1p (Fig. 4b, and Supplementary Fig. S7). Moreover, fusion proteins containing Ndd1p and the DNA-binding domain of Gal4p (Gal4p-DBD) demonstrated equal recruitment of wild-type and mutant Ndd1p to a Gal4p-driven promoter but decreased recruitment of Ndd1p mutated at Ser 85 to the CLB2 promoter (Supplementary Fig. S8b). Taken together, these data therefore suggest that Cdc5p-mediated phosphorylation of Ndd1p at Ser 85 is important in controlling the temporal recruitment of Ndd1p at promoters of CLB2 cluster genes.
As a result of the severe defects observed in strains expressing only Ndd1p(S85A) (see Fig. 3), we were unable to analyse cell-cycle-dependent expression of the CLB2 gene cluster in these cells. Therefore, to examine the potential role of Ser 85 on CLB2 cluster gene expression, we developed a system in which we could rapidly switch between endogenous and plasmid-borne expression of Ndd1p (Supplementary Fig. S9). The expression of CLB2 cluster genes was then compared in cells expressing either wild-type Ndd1p or Ndd1p(S85A) after release from an α-factor block (Supplementary Fig. S10). Significantly, periodic gene expression that occurs early in the cell cycle in G1 phase (CLN2) and S phase (HHT2 and HTB1) was similar in cells expressing either wild-type Ndd1p or Ndd1p(S85A) (Fig. 4c). In contrast, cells expressing Ndd1p(S85A) had decreased expression of the CLB2 cluster genes CLB2, SWI5 and CDC5 (Fig. 4c). To investigate further the role of Cdc5p in controlling late cell-cycle-dependent gene expression, cdc5ts cells were arrested in G1 phase, then released at the permissive (25 °C) or non-permissive (37 °C) temperature. In concordance with the effects observed in Fig. 4c, cell-cycle-dependent induction of CLB2 was found to be severely attenuated on inactivation of Cdc5p (Supplementary Fig. S11). An additional role for phosphorylation of Ser 85 in regulating the intrinsic transactivation function of Ndd1p was revealed with a GAL1pr–LacZ reporter gene assay because Gal4p-DBD–Ndd1p(S85A) had a decreased ability to activate transcription (Supplementary Fig. S8a).
Taken together, our data show that Cdc5p-mediated phosphorylation of Ndd1p is important in controlling CLB2 cluster gene expression and normal cell cycle progression. Because CDC5 itself is part of the CLB2 gene cluster, the phenotypic defects associated with mutation of Ser 85 of Ndd1p are likely to be due in part to downstream defects caused by lower Cdc5p levels. Moreover, our data extend a previous model in which Clb2p-mediated phosphorylation of Ndd1p helps to establish a positive feedback loop that in turn leads to further increases in CLB2 transcription and entry into M phase3,15,16. Here Cdc5p is identified as another cell cycle regulator that can generate a positive feedback loop, because the CDC5 gene is also part of the CLB2 gene cluster, which is a target of the Mcm1p–Fkh2p–Ndd1p complex10,12,20,21,22. Hence, Cdc5p and Clb2p–Cdc28p together converge on Ndd1p to activate one of the key switches that controls cell cycle progression (Fig. 4d). This is reminiscent of the convergence of Cdc5p and Clb2p–Cdc28p on the regulation of Swe1p kinase and the subsequent promotion of mitotic entry23. Thus, the combinatorial action of these kinases seems to be a common mechanism to control late cell cycle events precisely.
Forkhead transcription factors are also important for late cell-cycle-dependent gene expression in the evolutionarily divergent yeast Schizosaccharomyces pombe24,25,26,27 and in mammalian cells28,29. Similarly, some of the previously described functions of polo kinases are also conserved between yeast and higher eukaryotes2, and genetic studies link the Cdc5p homologue Plo1p to the regulation of M/G1-phase-specific transcription in S. pombe30. Given these parallels, it is quite possible that a function for polo kinases in regulating cell-cycle-dependent gene expression in conjunction with forkhead transcription factor complexes will eventually be uncovered in mammalian systems.
More detailed methods are supplied in Supplementary Information.
Plasmid constructs and yeast strains
Details of plasmid construction and yeast strains used in this study can be found in Supplementary Information.
RNA analysis and chromatin immunoprecipitations
RNA levels were analysed by northern blotting11 or by real-time polymerase chain reaction with reverse transcription (RT–PCR; for further details see Supplementary Information). ChIP assays were performed as described previously14.
Protein production, pulldown assays and western blotting
GST pulldown assays with proteins translated in vitro were performed as described previously14. Fusion proteins with maltose-binding protein (MBP) were prepared by maltose-affinity chromatography (New England Biolabs). Western blots were performed with antibodies recognizing epitope tags or with an antibody raised against a phosphorylated peptide (NSSSNESS[P]LVENS; Eurogentec).
Protein kinase production and kinase assays
Protein kinase assays using Flag-tagged Clb2p-kinase complexes purified from yeast cells, bacterially expressed and purified Clb2p and Cdc28p15 or GST–Cdc5p-WT and GST–Cdc5p-KD6 with bacterially expressed and purified substrates were performed as described previously15.
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We thank A. Clancy for technical assistance; I. Hagan, P. March, M. Jackson and G. Pereira for advice; A. Whitmarsh, S.-H. Yang and members of our laboratories for comments on the manuscript and helpful discussions; and M. Walberg, U. Surana, G. Pereira and D. Lydall for reagents. This work was supported by Cancer Research UK, the BBSRC, the MRC and the Wellcome Trust.
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