In this study, we aimed to gain more insight into how FoxM1 activity is regulated during an ongoing cell cycle. Our data point to the existence of a cyclin A-dependent mechanism that controls transactivation by FoxM1, allowing a tight restriction of FoxM1 transactivation to the G2 phase of the cell cycle.
We provide evidence that although FoxM1 can already be expressed in late G1
and in S phase and can bind to its endogenous promoters, its transcriptional activity is kept low until entry into G2
. Our data indicate that cyclin A, but not cyclin B, plays an important role in the regulation of FoxM1 transcriptional activity during G2
. Ectopic expression of cyclin A greatly increases FoxM1 transcriptional activity, while removal of cyclin A through RNAi-mediated depletion leads to a strong reduction in FoxM1 activity, similar to what is seen after expression of dominant negative versions of cdk1 and cdk2. More importantly, cyclin A depletion results in a defect in G2
/M progression and a reduction in FoxM1 target gene expression, similar to what is seen in FoxM1-deficient cells. Previously, cyclin B/cdk2 was shown to phosphorylate FoxM1 (8
), but the exact contribution of this phosphorylation to FoxM1 activation remains to be determined. Cyclin E/cdk2 was also shown to phosphorylate and activate FoxM1 at G1
). A similar observation was reported by Major and coworkers, who showed that the cyclin E/cdk2 complex binds and phosphorylates the C-terminal region of FoxM1 in order to recruit the transcriptional coactivator p300 (8
). Accordingly, a minor shifted form of FoxM1 is seen in thymidine-blocked cells (Fig. ), suggesting that phosphorylation of FoxM1 is a multistep process that starts as early as late G1
. However, because cyclin E/cdk2 activity is limited to the G1
/S transition of the cell cycle, it may not be sufficient for full activation of FoxM1 but could prime FoxM1 for eventual activation by cyclin A/cdk complexes in G2
. Indeed, the major cyclin A-dependent shift in FoxM1 mobility that is observed as cells progress from S to G2
suggests that cyclin A plays a major role in promoting FoxM1 phosphorylation. Accordingly, we also found that FoxM1 is in vitro phosphorylated by cyclin A/cdk2 complexes and that the endogenous protein is in vivo phosphorylated during G2
in a cyclin A-dependent manner. On the basis of our observations, it is difficult to discriminate which cyclin A/cdk complex is most crucial for FoxM1 activation during G2
phase, as both cdk1 and cdk2 complexes could act redundantly.
We found that a truncated mutant of FoxM1 that lacks the N-terminal domain is a hyperactive transcriptional regulator, consistent with recent results from others (9
). Here, we show that transcriptional activation of this mutant no longer depends on cyclin A/cdk activity, as neither overexpression of dominant negative forms of cdk's nor depletion of cyclin A could inhibit the activity of this mutant. These data imply that the N-terminal repressor domain is regulated in a cell cycle-dependent fashion, requiring cyclin A/cdk-dependent inactivation in G2
. We found that phosphorylation of the C-terminal TAD (T600 and T611 residues) is required for G2
-specific activation of FoxM1. Substitution of these residues to alanine clearly reduced the level of FoxM1 phosphorylation by cyclin A/cdk complexes and prevented activation of full-length FoxM1 in G2
, while it did not affect transcriptional activity of ΔN-FoxM1. These data strongly suggest that cyclin A/cdk-mediated phosphorylation of the C-terminal TAD is required for relief of the inhibitory function of the N terminus.
Recently, it was shown that the N- and C-terminal domains of FoxM1 can form a direct complex (14
), suggesting that inhibition by the N terminus occurs through direct interaction of this domain with the C-terminal transactivation domain, thereby preventing transcriptional activation of FoxM1. Importantly, our data indicate that this molecular interaction within FoxM1 can be disrupted by active cyclin A/cdk complexes, allowing for full activation of FoxM1. Moreover, point mutations in two conserved RXL/LXL sites in the TAD of FoxM1 (R716/L718 and L722/724) strongly reduced the interaction with the N-terminal repressor domain of FoxM1 and behaved as hyperactive mutants. Similar to the N-terminal deletion mutant, mutation of the RXL/LXL motifs eliminates the requirement of cyclin A/cdk for FoxM1 activation, indicating that these RXL/LXL motifs mediate the binding between the N terminus and the TAD of FoxM1. In contrast, mutation of another LXL motif present in the TAD of FoxM1 (L656A), which has been recently shown to be required for FoxM1B activation (9
), did not show any significant effect on FoxM1C transactivation (data not shown), suggesting that the different FoxM1 isoforms require different modes of regulation.
Taken together, our findings uncover a crucial role for cyclin A/cdk complexes in optimal activation of FoxM1 during the G2 phase of the cell cycle. These data are most consistent with a model in which FoxM1 activity is kept low due to repression by its own N-terminal domain through direct binding to the C-terminal TAD. This binding might also allow docking of cellular components, yet unknown, that further suppress FoxM1 activity. Through constitutive cyclin A/cdk action, and once sufficient levels of phosphorylated FoxM1 protein are achieved, repression by the N terminus is relieved, allowing full activation of FoxM1.