Transitions through cell-cycle phases require the coordination of multiple events and are tightly regulated by protein kinases1,2,5
. In addition, transcriptional control of key cell-cycle regulators are important for cellcycle progression9,10
. Thus, defining the links between protein kinases and transcriptional networks is essential for an understanding of normal cell-cycle progression and how specific factors may contribute to the misregulation of this process in diseases.
In Saccharomyces cerevisiae
, the polo kinase Cdc5p coordinates cellcycle-dependent transcription at G2/M by directly phosphorylating and regulating the activity of a co-activator of the forkhead transcription family member Fkh2p (ref. 11
). The mammalian Polo-like kinase 1 (Plk1) is also essential for multiple events throughout M phase1,5
and several Plk1 targets involved in mitosis have been identified12–17
. So far, no direct link between Plk1 and the transcription factors controlling gene expression has been identified, although the conservation of functions between polo kinases in yeast and higher eukaryotes makes it tempting to speculate that Plk1 may have a role in mammalian cells similar to that recently demonstrated for Cdc5p in yeast11
The mammalian transcription factor Forkhead Box M1 (FoxM1) is important in regulating mitotic entry and subsequent execution of the mitotic programme by controlling the expression of a cluster of G2/M target genes6,8,18
. However, the mechanisms regulating the activation of FoxM1 at specific cell-cycle phases, and how this contributes to G2/M progression are largely unknown.
Here, using yeast two-hybrid screening, we identified FoxM1 as a direct binding partner of Plk1. Six of the twenty-four positive clones encoded various lengths of the FoxM1 C terminus, with the smallest clone encoding residues 463–748 of FoxM1. These initial results suggest that there is a link between Plk1 and a transcription factor controlling a G2/M transcriptional programme.
translated FoxM1 associated with GST-fused Plk1 (GST–Plk1), but not with GST (), indicating that Plk1 binds directly to FoxM1. In contrast, bacterially expressed FoxM1 failed to interact with in vitro
translated Plk1 (Supplementary Information, Fig. S1a
), suggesting that post-translational modification of FoxM1 may be required for this interaction. In addition, ectopically expressed FoxM1 formed a complex with endogenous Plk1 (), but not Plk2 or Plk3 (Supplementary Information, Fig. S1c
). Plk1 and FoxM1 show periodic expression patterns, with both proteins increasing markedly in G2/M cells19
(Supplementary Information, Fig. S1b
). Indeed, an endogenous Plk1–FoxM1 complex was readily observed in G2/M cells, whereas this complex was barely detectable in asynchronous cells ().
Figure 1 Plk1 interacts with FoxM1 in vitro and in vivo. (a) In vitro translated FoxM1 was used in a pulldown assay with either GST or GST–Plk1 immobilized on agarose beads. (b) 293T cells were transfected with constructs encoding Myc-tagged FoxM1 and (more ...)
A noticeable progressive decrease in FoxM1 mobility was observed when cells were released from G0/G1 arrest (Supplementary Information, Fig. S1b
), indicating that FoxM1 undergoes an initial phosphorylation in S phase followed by hyperphosphorylation during G2/M, a time at which Plk1 activity is maximal. Spatial and temporal coordination of Plk1 activity is mediated by the C-terminal Polo-box domain (PBD) of Plk1 (refs 20–22
). Furthermore, the PBD is required for recruitment of Plk1 to its substrates that have been ‘primed’ by phosphorylation at specific docking sites23
. Therefore, FoxM1 may undergo an initial priming phosphorylation, allowing it to be recruited and directly phosphorylated by Plk1.
Plk1 PBD recognizes a consensus sequence of S-pS/pT-P/X22,24
. FoxM1 sequence contains two potential PBD-binding sites (Thr 596 and Ser 678). Our yeast two-hybrid results indicated that a C-terminal domain of FoxM1 (residues 463–748) is essential for Plk1 binding. Therefore, we examined whether Thr 596 and Ser 678 of FoxM1 would mediate the interaction between these two proteins. Thr 596 and Ser 678 were mutated to Ala individually (FoxM1T596A
) or in combination (FoxM1TSAA
), and their ability to bind GST-tagged Plk1 PBD was analysed. FoxM1WT
associated with GST–PBD, but not with GST or the GST–PBD mutant (). Binding to the Plk1 PBD in the FoxM1T596A
single mutants was reduced; however, mutation of both residues abolished this interaction (). Thus, both Thr 596 and Ser 678 of FoxM1 seem to be key sites involved in a phosphorylation-dependent interaction between FoxM1 and Plk1 PBD.
Many docking sites on Plk1-binding proteins are initially phosphorylated by Cdk1/cyclin B. FoxM1 preferentially binds to the Cdk1/cyclin B complex in G2, and Thr 596 of FoxM1 was previously identified as a Cdk1 phosphorylation site7
. We determined whether phosphorylation of FoxM1 by Cdk1 is responsible for the formation of Plk1–FoxM1 complex. We performed pulldown assays using bacterially expressed GST–Plk1 PBD and MBP–FoxM1 fusion proteins, with or without Cdk1/ cyclin B. Phosphorylation of MBP–FoxM1 by Cdk1 promoted a strong interaction between FoxM1 and Plk1 PBD (Supplementary Information, Fig. S2a
). Inhibition of Cdk1, but not Plk1 (ref. 25
), markedly reduced the binding of endogenous FoxM1 to GST–Plk1 PBD (Supplementary Information, Fig. S2b, e
). Thus, Cdk1-dependent phosphorylation of FoxM1 is required for its interaction with Plk1.
FoxM1 is hyperphosphorylated during G2/M phase, which correlates with its transcriptional activity being cell-cycle-restricted. Therefore, we hypothesized that Plk1 phosphorylates and activates FoxM1 during G2/M transition. We found that FoxM1 was phosphorylated by the constitutively active Plk1 (Plk1TD) in vitro, but not by the catalytically inactive Plk1KD mutant ().
Figure 2 Plk1 phosphorylates FoxM1 in vitro and in vivo. (a) Bacterially expressed FoxM1 was subjected to in vitro kinase assays. Kinases used were constitutively active human Plk1 (TD) or a kinase-defective (KD) mutant of Plk1 purified from insect cells. Loading (more ...)
We next sought to map the major Plk1 phosphorylation sites on FoxM1. A series of FoxM1deletion-mutants were generated and tested for their capacity to be phosphorylated by Plk1 in vitro
. Although the amino-terminal segments of FoxM1 were weakly phosphorylated by Plk1, C-terminal segments, especially those containing the transcriptional activation domain (TAD, residues 617–748; Supplementary Information, Fig. S3a, b
), showed much higher levels of phosphorylation.
Plk1 substrates do not in general contain a strict consensus phosphorylation motif26,27
. Therefore, we used mass spectrometry analysis for the identification of Plk1-dependpent FoxM1 phosphorylation sites. Using recombinant FoxM1 protein purified from bacteria and phosphorylated in vitro
by Plk1, we identified four Ser residues (Ser 680, Ser 702, Ser 715 and Ser 724) located within the TAD of FoxM1 as potential Plk1 phosphorylation sites. Single- and double-site mutants were generated and their capacity to be phosphorylated by Plk1 was determined in vitro
. Single mutation of Ser 715 or Ser 724 reduced Plk1-dependent phosphorylation of FoxM1 (Supplementary Information, Fig. S3c
), whereas phosphorylation was almost abolished when the S715A;S724A double mutant was used (). Thus, Ser 715 and Ser 724 seem to be the two major Plk1-dependent phosphorylation sites within the TAD region of FoxM1. Importantly, these two Ser residues, as well as the surrounding amino acids, are highly conserved among vertebrates (), indicating that the phosphorylation of these two sites may have an evolutionarily conserved role in regulating FoxM1 activity.
We next determined whether FoxM1 is phosphorylated by Plk1 in vivo
. We generated phospho-specific anti-FoxM1 antibodies. As shown in , Both the anti-p715 and anti-p724 antibodies immunoreacted with wild-type FoxM1; however, only the anti-p715 antibody recognized the FoxM1S724A
mutant and vice versa
. Furthermore, neither antibody detected λ-phosphotase-treated wild-type FoxM1 (Supplementary Information, Fig. S3d
). Thus, the anti-p715 and anti-p724 antibodies are highly specific and only recognize their corresponding targets. Using these phospho-specific antibodies, we showed that phosphorylation of endogenous FoxM1 could be readily detected in G2/M but not in asynchronous HeLa cells (), indicating that FoxM1 is phosphorylated at Ser 715 and Ser 724 sites at G2/M. To determine whether Plk1 is required for these phosphorylation events, we depleted endogenous Plk1 by siRNA and observed a marked reduction of FoxM1 phosphorylation at Ser 715 and Ser 724 sites (), supporting the suggestion that Plk1 is the kinase that phosphorylates FoxM1 in vivo
We directly examined the possibility that Cdk1 could function as a priming kinase of FoxM1. To test this, we performed in vitro
kinase assays using Cdk1, Plk1, or both as kinase sources. Consistent with earlier studies7
, Cdk1 could phosphorylate FoxM1 to some extent (Supplementary Information, Fig. S2c
). Interestingly, in the presence of Cdk1 the phosphorylation of FoxM1 by Plk1 was markedly elevated when compared with Plk1 alone. Furthermore, Cdk1-mediated phosphorylation was initiated as early as S/G2 phase in vivo
, whereas Plk1-mediated phosphorylation occurred later, mainly at G2/M phases (Supplementary Information, Fig. S2d
), suggesting that FoxM1 undergoes an initial priming phosphorylation by Cdk1, which allows its subsequent phosphorylation by Plk1.
To better understand the functional significance of Plk1-dependent phosphorylation of FoxM1, we tested whether Plk1 regulates FoxM1 transcriptional activity, assayed using the 6× FoxM1
–TATA–luciferase reporter plasmid7
. Wild-type Plk1, but not the kinase-inactive mutant of Plk1, significantly upregulated FoxM1 transcriptional activity in a concentration-dependent manner (), suggesting a direct regulation of FoxM1 by Plk1-dependent phosphorylation.
Figure 3 Plk1 activates FoxM1 transcriptional activity. (a) U2OS cells were transiently transfected with the reporter plasmid 6×FoxM1 TATA–luciferase plasmid encoding wild-type FoxM1, and increasing amounts of plasmids encoding for either wild-type (more ...)
We next investigated whether the association between Plk1 and FoxM1 is necessary for Plk1-dependent activation of FoxM1. Mutation of one of the two residues involved in the docking of Plk1 to FoxM1 resulted in only a partial decrease in Plk1-enhanced FoxM1 transcriptional activity (T596A, 55%; S678A, 30%). In contrast, mutation of both residueled to a marked reduction (>90% decrease) in Plk1-dependent FoxM1 transcriptional activity (). These results are consistent with those shown in and indicate that phosphorylation of either Thr 596 or Ser 678 of FoxM1 can independently mediate the binding to Plk1 and this binding is essential for Plk1-dependent activation of FoxM1.
To test the hypothesis that Plk1-mediated phosphorylation of FoxM1 enhances its transcriptional activity, we mutated the two major Plk1 phosphorylation sites of FoxM1 (Ser 715 and Ser 724) to Ala (FoxM1AA) or to glutamic acid (FoxM1EE) and examined the effects on FoxM1 transcriptional activity. We included a FoxM1 mutant lacking the TAD (FoxM11–616) as a negative control in these experiments. Indeed, the phospho-mutant of FoxM1 was not activated by Plk1 (), whereas FoxM1EE showed robust transcriptional activity with or without Plk1 (). Taken together, these results indicate that after an initial priming phosphorylation of FoxM1 by Cdk1, Plk1 is recruited to and directly phosphorylates FoxM1, thereby enhancing FoxM1 transcriptional activity.
To address the functional relevance of Plk1-dependent FoxM1 phosphorylation in vivo
, we developed a rescue assay using U2OS cells stably expressing various wild-type or mutant forms of FoxM1. Endogenous FoxM1 levels were depleted using siRNA and rescue of the FoxM1
knock down phenotype was analysed in cells stably expressing Myc-tagged wild-type FoxM1 or siRNA-resistant Myc-tagged wild-type (WT-r) or S715A;S724A mutant (AA-r) FoxM1. As controls, U2OS cells stably transfected with empty vector were treated with siRNAs targeting FoxM1 or the firefly luciferase gene. Endogenous FoxM1 and Myc-tagged wild-type FoxM1 were significantly depleted but WT-r and AA-r remained abundant (, Supplementary Information, Fig. S4a
), confirming that the siRNA-resistant FoxM1 constructs were effective.
Figure 4 Plk1-dependent phosphorylation of FoxM1 is required for expression of the G2/M transcriptional programme and orderly mitotic progression in vivo. U2OS cells stably expressing empty vector (EV), or Myc-tagged FoxM1WT, siRNA-resistant wild-type FoxM1 (WT-r) (more ...)
Previous studies have shown that cells lacking FoxM1 have multiple cellcycle defects, including a delay in G2 and reduced expression of G2/M FoxM1 target genes. Although some FoxM1-depleted cells eventually enter mitosis, additional mitotic defects may occur, resulting in the formation of polyploid and aneuploid cells6,8
. Indeed, FACS, morphological analyses and time-lapse microscopy showed an increase in 4N cells and an accumulation of polyploid (>4N) cells (; Supplementary Information, Fig. S4a, c
) after FoxM1 depletion. Numerous mitotic defects, including misaligned and/or mis-segregated chromosomes, aberrant furrow formation, failed cytokinesis and prolonged mitotic progression, were also observed, confirming that FoxM1 is required for G2/M transition and proper mitotic progression. A delay of cells in G2/M transition and an increase in the number of binucleated cells (10.4%, Supplementary Information, Fig. S4b
) seems to account for the accumulation of cells with 4N DNA content in the absence of FoxM1. Expression of wild-type and siRNA-resistant wild-type FoxM1 partially or completely restored normal cell-cycle progression (). However, the siRNA-resistant FoxM1 S715A;S724A mutant was not able to rescue these phenotypes, suggesting that Plk1-dependent phosphorylation and activation of FoxM1 is crucial for FoxM1 function at G2/M.
To provide an in vivo
correlation, we next examined the expression of FoxM1 target genes, in the context of the rescue assay described above. Indeed, in FoxM1-depleted cells, we observed downregulation of FoxM1 target genes, including Plk1
, Cyclin B1
, and Aurora B
, all of which are required for mitotic progression; expression of siRNA-resistant wild-type FoxM1 rescued this downregulation (). In contrast, Plk1
, Cyclin B1
and Aurora B
levels remained low in cells expressing the siRNA-resistant FoxM1 S715A;S724A mutant cells (). Next, we blocked Plk1 activity using a chemical inhibitor25
. Although inhibition of Plk1 activity reduced the expression of downstream targets of FoxM1, no effect was observed in cells expressing the constitutively active FoxM1EE
mutant (). Moreover, time-lapse imaging analysis revealed that inhibition of Plk1 activity leads to mitotic defects, such as prolonged early mitotic phases and prometaphase arrest in wild-type cells. These defects were partially rescued in cells expressing FoxM1EE
(; Supplementary Information, Movies 1, 2
). Together, these data indicate that Plk1 regulates FoxM1 transcriptional activity by direct phosphorylation and thereby controls execution of a transcriptional programme required for mitotic progression.
We have developed a model to describe the role of Plk1-mediated regulation of FoxM1 at G2/M transition (). This suggests that at late S and G2 phase, FoxM1 is initially phosphorylated by Cdk1 kinases, creating docking sites for the PBD of Plk1. Subsequently, Plk1 binds and directly phosphorylates FoxM1. This activates FoxM1 transcriptional activity, resulting in the enhanced expression of key mitotic regulators. As Plk1
is a target gene of FoxM1, this mode of regulation can generate a positive-feedback loop, leading to a further increase in Plk1 levels and FoxM1 activity. Furthermore, as the major transcription factor during G2/M transition, full activation of FoxM1 ensures the coordinated expression of transcriptional networks essential for timely entry into M phase. This mode of regulation is reminiscent of the Rb-E2F pathway during G1/S transition, where Rb is hyperphosphorylated and releases E2F, allowing for transactivation of target genes that promote S phase entry28
The polo kinase Cdc5p also regulates a G2/M transcriptional programme in S.cerevisiae11
. Here we have extended these observations and revealed a link between Plk1 and regulation of a G2/M transcriptional network in mammalian cells. In contrast to yeast Cdc5p-mediated phosphorylation of a co-activator Ndd1p, Plk1 directly phosphorylates and thereby activates FoxM1 transcriptional activity. Our data do not exclude the possibility that other transcription factor(s) may collaborate with FoxM1 to coordinate the appropriate expression of transcriptional networks. As various cell-cycle kinases are upregulated in many cancers, the results presented here should help to provide further insights into the link between aberrant expression of these kinases and alterations in cell proliferation as well as genomic integrity, potentially through misregulation of transcriptional programmes.