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Molecular and Cellular Biology, September 2008, p. 5162-5171, Vol. 28, No. 17
0270-7306/08/$08.00+0     doi:10.1128/MCB.00387-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Anaphase-Promoting Complex/Cyclosome-Cdh1-Mediated Proteolysis of the Forkhead Box M1 Transcription Factor Is Critical for Regulated Entry into S Phase{triangledown}

Hyun Jung Park, Robert H. Costa, Lester F. Lau, Angela L. Tyner, and Pradip Raychaudhuri*

Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, College of Medicine, Chicago, Illinois 60607

Received 6 March 2008/ Returned for modification 9 April 2008/ Accepted 9 June 2008


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ABSTRACT
 
The forkhead box M1 (FoxM1) transcription factor is overexpressed in many cancers, and in mouse models it is required for tumor progression. FoxM1 activates expression of the cell cycle genes required for both S and M phase progression. Here we demonstrate that FoxM1 is degraded in late mitosis and early G1 phase by the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. FoxM1 interacts with the APC/C complex and its adaptor, Cdh1. Expression of Cdh1 stimulated degradation of the FoxM1 protein, and depletion of Cdh1 resulted in stabilization of the FoxM1 protein in late mitosis and in early G1 phase of the cell cycle. Cdh1 has been implicated in regulating S phase entry. We show that codepletion of FoxM1 inhibits early S phase entry observed in Cdh1-depleted cells. The N-terminal region of FoxM1 contains both destruction box (D box) and KEN box sequences that are required for targeting by Cdh1. Mutation of either the D box sequence or the KEN box sequence stabilized FoxM1 and blocked Cdh1-induced proteolysis. Cells expressing a nondegradable form of FoxM1 entered S phase rapidly following release from M phase arrest. Together, our observations show that FoxM1 is one of the targets of Cdh1 in late M or early G1 phase and that its proteolysis is important for regulated entry into S phase.


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INTRODUCTION
 
The forkhead box (Fox) family proteins comprise a large number of transcription factors, defined by a conserved winged-helix DNA binding domain (2, 7, 8). Transcription of the FoxM1 locus results in three differentially spliced mRNAs that are almost identical in sequence but differ by the addition of two small exons: the FoxM1b isoform (HFH-11B) contains no additional exons, while the Foxm1c (Trident, WIN, or MPP2) isoform contains one additional exon in the winged-helix DNA binding domain and the transcriptionally inactive Foxm1a (HFH-11A) isoform contains the exon in the DNA binding domain and an additional exon in the C terminus (12, 20, 39, 41). FoxM1 (or FoxM1b) has been shown to be expressed in all proliferating cell types examined, including many tumor-derived cell lines (12, 20, 39, 41). Accordingly, FoxM1 is ubiquitously expressed in the embryo but not in differentiated or resting cells in adult tissues. FoxM1 expression is confined to a few proliferating cell types in the adult, including the thymus, testis, and the crypts of the intestine and colon (12, 41). This expression pattern implicates FoxM1 in the transcription of the cell cycle regulatory genes in proliferating cells.

We previously used the albumin promoter/enhancer Cre recombinase transgene (Alb-Cre) to mediate hepatocyte-specific deletion of the Foxm1 fl/fl targeted allele. Liver regeneration studies demonstrated that Alb-Cre Foxm1–/– hepatocytes displayed an 80% reduction in DNA replication and a complete inhibition of mitosis (33). FoxM1 plays a pivotal role in mitotic progression by regulating transcription of the Cdc25B, cyclin B, polo-like kinase, Aurora B kinase, survivin, centromere protein A, and centromere protein B genes (4, 13, 15, 17, 31, 32, 37, 40). The cyclin-dependent kinase (CDK) inhibitor proteins p27Kip1 and p21Cip1 are phosphorylated by the Cdk2-cyclin E complex and are then recognized by the specificity subunits, i.e., S phase kinase-associated protein 2 (Skp2) and CDK subunit 1 (Cks1), of the Skp1-Cullin1-F box (SCF) ubiquitin ligase complex, which targets them for ubiquitin-mediated proteasome degradation (3, 6, 22, 28). FoxM1 participates in the G1-S transition by reducing nuclear levels of the CDK inhibitor proteins p27Kip1 and p21Cip1 through the transcriptional activation of the specificity subunits Skp2 and Cks1 of the SCF ubiquitin ligase complex (4, 31, 32).

In agreement with the important role of FoxM1 in cell cycle progression, several observations have implicated FoxM1 in tumor growth and progression. It has been shown that increased levels of FoxM1 protein in osteosarcoma U2OS cells stimulate anchorage-independent growth, as evidenced by an increased number and size of cell colonies in a soft agar assay (10). Moreover, the livers in which the Foxm1 fl/fl allele was conditionally deleted by Alb-Cre were highly resistant to development of hepatocellular carcinomas induced by a diethylnitrosamine/phenobarbital tumor induction protocol (10). In published studies, reduced expression of FoxM1 significantly diminished DNA replication and mitosis of tumor cells and reduced development of mouse tumors in response to carcinogens or oncogenes (9-11). Furthermore, the FoxM1 transcription factor is overexpressed in a number of aggressive human tumors (5, 19, 27, 29, 30, 37). These observations underscore the importance of determining the mechanism which regulates the level of FoxM1 during cell cycle progression. However, detailed molecular mechanisms that control the level of FoxM1 during cell cycle progression and tumorigenesis still remain elusive.

Ubiquitin-mediated proteolysis controls the cellular abundance of a number of cell cycle regulatory proteins (23). The substrate specificity of degradation is largely conferred by the E3 ubiquitin ligases. There are several ubiquitin ligases involved in cell cycle regulation. For example, the SCF ligase regulates degradation of p27, facilitating cell cycle progression into S phase (23). The anaphase-promoting complex/cyclosome (APC/C) is responsible for targeting anaphase inhibitors and mitotic regulatory proteins for polyubiquitination and subsequent degradation in order for the cell to exit mitosis and for keeping the mitotic regulators low in G1 phase to prevent aberrant cell cycle progression into S phase (23). The substrate specificity of APC/C is governed by the coactivator Cdc20 and by Cdh1. Many APC/C substrates are identified by the presence of recognition sequences known as a destruction box (D box) sequence (38, 42) or a KEN box sequence, which is also found either alone or in combination with the D box in APC/C substrates (26).

In this study, we demonstrate that FoxM1 is one of the targets of the APC/C E3 ubiquitin ligase through recognition by the activating protein Cdh1. We show that FoxM1 contains both D box and KEN box sequences that are required for recognition by Cdh1 for ubiquitin-mediated degradation during late mitosis and early G1 phase of the cell cycle. Moreover, the APC/C-Cdh1-mediated degradation of FoxM1 is important for regulated entry into S phase following mitosis.


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MATERIALS AND METHODS
 
Cell culture and DNA transfection. HeLa and U2OS cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (Gibco). For transient transfection, cells were transfected with Fugene 6 reagent (Roche) or Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

Cell cycle arrest and flow cytometry. For double thymidine block, cells were arrested with 2 mM thymidine medium for 19 h. After cells were washed three times with phosphate-buffered saline (PBS) and released into fresh medium for 9 h, they were then treated with thymidine for an additional 16 h. To synchronize cells in G2/M phase, cells were treated with 40 ng/ml nocodazole (Sigma) for 16 h. To evaluate cell cycle distribution, cells were subjected to flow cytometry. For flow cytometry, cells were trypsinized and fixed in 70% ethanol for 16 h at 4°C. Cells were incubated with 40 µg/ml propidium iodide and 100 µg/ml RNase A (Sigma) in PBS for 1 h at 37°C. After being washed, cells were resuspended in PBS for further analysis. Data were acquired using a Beckman Coulter Epics Elite ESP apparatus (Hialeah, FL) and then analyzed using Multicycle AV (Phoenix Flow Systems, San Diego, CA). The flow cytometry and analysis were performed at the Research Resource Center at the University of Illinois at Chicago.

Plasmids, mutagenesis, and siRNAs. We cloned full-length FoxM1 and cytomegalovirus T7-FoxM1 as described previously (21). The D box deletion mutant was generated by PCR, using the primers 5'-GAAGATCTTCAGTGAAACATCAGAG-3' and 5'-CGGGATCCCTACTGTAGCTCAGG-3'. We generated FoxM1 KEN box point mutants with the QuikChange mutagenesis system (Stratagene). The small interfering RNA (siRNA) oligonucleotide sequences were 5'-GGACCACUUUCCCUACUUUUU-3' (FoxM1), 5'-AATGAGAAGTCTCCCAGTCAGdTdT-3' (Cdh1), and 5'-CGGCAGGACUCCGGGCCGAdTdT-3' (Cdc20).

IP and Western blot analysis. Cells were harvested and lysed in immunoprecipitation (IP) buffer on ice for 20 min. The IP buffer consisted of 50 mM Tris (pH 7.5), 100 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1% NP-40, 5% glycerol, freshly added 1x Complete Mini protease inhibitor cocktail (Roche), 2 mM phenylmethylsulfonyl fluoride, and 2 mM NaF and 2 mM NaVO4 as phosphatase inhibitors. Cell extracts were clarified by centrifugation at 10,000 rpm for 5 min at 4°C. Protein concentrations were determined by the Bradford method with the Bio-Rad protein assay reagent.

IP assays were performed as described previously (21). Briefly, 300 µg of protein was incubated with primary antibody overnight at 4°C, and protein A/G-Sepharose beads were added. The beads were washed three times with IP buffer and resuspended in sodium dodecyl sulfate sample buffer. Immunocomplexes were analyzed by immunoblotting. The following antibodies were used for this study: mouse anti-Aurora B kinase/AIM-1 (1:250) (BD Biosciences), mouse anti-β-actin (AC-15; 1:5,000) (Sigma), goat anti-cdc20 (1:500) (Santa Cruz Biotechnology, Inc.), mouse anti-Cdh1 (1:500) (Oncogene), mouse anti-Cdc27 (1:1,000) (Transduction Laboratories), and mouse anti-PLK1 (1:1,000) (Santa Cruz Biotechnology, Inc.).

BrdU staining. For bromodeoxyuridine (BrdU) staining, 10 µM of BrdU was added to the growth medium for 1 h. Cells were fixed with 4% paraformaldehyde for 15 min and washed with PBS three times. Fixed cells were then incubated with 2 M HCl for 1 h and neutralized with 0.1 M borate buffer (pH 8.5). Cells were stained with anti-BrdU antibody (Dako) and anti-mouse antibody-fluorescein isothiocyanate (Dako).

In vivo ubiquitination assays. Cells were transfected with His-ubiquitin and T7-FoxM1, with either Cdh1 or empty vector. In case of the endogenous proteins, only the His-ubiquitin plasmid was transfected along with specific siRNAs. Cells were treated with MG132 (Calbiochem) for the last 6 h of transfection and lysed with ubiquitin assay buffer (6 M guanidine-HCl, 0.1 M Na2HPO4/Na2HPO4, 0.01 M Tris-HCl, 5 µM imidazole, 10 µM β-mercaptoethanol). His-ubiquitin-conjugated proteins were immunoprecipitated with Ni-nitrilotriacetic acid (Ni-NTA; Qiagen). Ubiquitinated proteins were eluted with elution buffer (200 µM imidazole, 0.15 M Tris-HCl, pH 6.7, 30% glycerol, 0.72 M β-mercaptoethanol, 5% sodium dodecyl sulfate). The presence of ubiquitinated FoxM1 was analyzed by immunoblotting with FoxM1 antibody.

Real-time PCR. U2OS cells were harvested 72 h following siRNA transfection for RNA preparation using RNA-STAT-60 (Tel-Test B Inc., Friendswood, TX). After DNase I treatment, cDNA was synthesized from 2 µg of total RNA, using a Bio-Rad cDNA synthesis kit. The following reaction mixture was used for all PCR samples: 1 µl IQ Sybr green supermix (Bio-Rad, Carlsbad, CA), 100 nM of each primer, and 1 µl of cDNA in a 25-µl total volume. Reactions were amplified and analyzed in triplicate, using a MyiQ single-color real-time PCR detection system (Bio-Rad, Carlsbad, CA). The following primers were used to amplify and measure FoxM1 mRNA: FoxM1-S, 5'-GGA GGA AAT GCC ACA CTT AGC G-3'; and FoxM1-AS, 5'-TAG GAC TTC TTG GGT CTT GGG GTG-3' (annealing temperature, 55.7°C). The real-time reverse transcription-PCR RNA levels were normalized to human cyclophilin mRNA levels, and the primers used were as follows: cyclophilin-S, 5'-GCA GAC AAG GTC CCA AAG ACA G-3'; and cyclophilin-AS, 5'-CAC CCT GAC ACA TAA ACC CTG G-3' (annealing temperature, 55.7°C).


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RESULTS
 
Regulation of FoxM1 during late mitosis and G1 phase of the cell cycle. In order to determine whether FoxM1 is regulated during cell cycle progression, we examined the levels of FoxM1 in synchronized cultures of HeLa cells. Cells were arrested in G1/S by double thymidine block, and then thymidine was removed to allow synchronous progression through the cell cycle. Protein extracts were prepared at different times following thymidine removal and analyzed by immunoblotting (Fig. 1A). Cell cycle distribution following release from double thymidine block was determined by flow cytometry (Fig. 1A, bottom panel). The level of FoxM1 increased during late DNA replication (S phase) and during the transition between G2 phase and mitosis (Fig. 1A). However, the FoxM1 protein level decreased significantly in late mitosis and during early G1 phase of the cell cycle (Fig. 1A). The decrease in FoxM1 level coincided with a reduction of the established transcriptional targets of FoxM1, such as Aurora B kinase and polo-like kinase 1 (Plk1) (Fig. 1A) (15, 31). Expression of the specificity subunit Cdc20 of the APC/C ubiquitin ligase complex was analyzed as a marker for M phase (Fig. 1A). Western blot analysis also revealed that the FoxM1 protein resolved as a doublet band for asynchronous cells and that the slower-migrating FoxM1 protein was present during the G2-M phase transition and late mitosis (Fig. 1A). We hypothesized that the slowly migrating FoxM1 band represented the phosphorylated form of the FoxM1 protein. To confirm that hypothesis, HeLa cells were blocked at early metaphase by treatment with nocodazole, an inhibitor of spindle microtubule formation. Extracts of the synchronized cells were incubated with or without phosphatase and then analyzed by Western blotting with an antibody specific to the FoxM1 protein. Only the slowly migrating FoxM1 protein band was found in the nocodazole-blocked cells, and it was similar to the slowly migrating FoxM1 band observed during the G2 and mitosis phases of the cell cycle. FoxM1 migrated faster after phosphatase treatment of nocodazole-treated cell extracts, suggesting that phosphorylation contributed to the change in migration of the FoxM1 protein band (Fig. 1B). We previously showed that activation of FoxM1 involved phosphorylation by cyclin B/Cdk1 (21), which is active in the G2/M phase. Therefore, it is likely that the slower-migrating band corresponded to cyclin B/Cdk1-phosphorylated FoxM1. In the current study, we focused on the downregulation of the FoxM1 protein in the late M and early G1 phases of the cell cycle.


Figure 1
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  		FIG. 1. Levels of FoxM1 protein decrease during late mitosis and early G1 phase of the cell cycle. (A) FoxM1 protein levels decrease in late mitosis and G1 phase of the cell cycle. HeLa cells were arrested at the G1-S boundary by double thymidine block, released into fresh medium, and harvested at the indicated times. Cell lysates were subjected to Western blot assays with antibodies specific to FoxM1, Aurora B kinase, Cdc20, and polo-like kinase 1 (Plk1) protein. β-Actin was used as a loading control. Cell cycle status at various time points following release from the double thymidine block was determined by flow cytometry. (B) FoxM1 is phosphorylated during the G2-M phase transition. Protein extracts were prepared from HeLa cells arrested in prometaphase by nocodazole treatment or in G1-S phase by double thymidine block. Extracts of nocodazole-arrested HeLa cells were treated with or without phosphatase and compared to double thymidine-arrested HeLa cell extracts. Migration of the FoxM1 protein in these extracts was determined by Western blotting with a FoxM1-specific antibody. (C) FoxM1 protein levels are reduced during late mitosis and G1 phase of the cell cycle. HeLa cells were released from double thymidine block (S phase) or nocodazole block (prometaphase) in the presence of cycloheximide (CHX) for the indicated times. To visualize FoxM1 protein levels, cell extracts were subjected to Western blotting with antibody specific to the FoxM1 protein.

To investigate the mechanism that regulates the level of FoxM1 protein during the M and G1 phases of the cell cycle, we considered proteolysis and measured the decay rate of FoxM1. HeLa cells were synchronized in S phase by double thymidine block or in early M phase by nocodazole treatment, as described in Materials and Methods. The cells were then released from the block by use of fresh medium. To analyze the decay of FoxM1 following release, cycloheximide was included in the medium. Additional sets of cultures were also incubated in medium containing MG132 to prevent degradation by the 26S proteasome. Two, 4, and 6 h following release in cycloheximide-containing medium, cells were harvested and extracts were subjected to Western blot analysis using FoxM1-specific antibody. As can be seen in Fig. 1C, cells released from S phase block exhibited stable FoxM1 and very little decay during the time frame of our analysis. The cells released from M phase arrest, on the other hand, exhibited significant decay of FoxM1 by 2 h, suggesting that FoxM1 is specifically unstable in the M and early G1 phases of the cell cycle. Moreover, the addition of MG132 prevented the decay of FoxM1 following release from M phase block, suggesting that the decay of FoxM1 in the M and early G1 phases involves degradation by the 26S proteasome.

FoxM1 is targeted by APC/C-Cdh1 in the M and G1 phases of the cell cycle. To further investigate the mechanism of FoxM1 proteolysis, we considered involvement of the APC/C, an E3 ubiquitin ligase that participates in the proteolysis of several cell cycle regulatory proteins in mitosis. Notably, the expression pattern of the FoxM1 protein shows similarity to those of other cell cycle proteins, such as Aurora B kinase, polo-like kinase, and cdc20, all of which are degraded by APC/C E3 ubiquitin ligases (Fig. 1A). We therefore decided to test whether the FoxM1 protein interacted with the APC/C complex. Protein extracts were prepared from HeLa cells released from the double thymidine arrest, and co-IP assays were performed with an antibody against the APC/C core subunit Cdc27, followed by Western blot analysis with either Cdc27 or FoxM1 antibody. Although FoxM1 and Cdc27 accumulated in G2 and peaked in mitosis, the co-IP assay showed that interaction between FoxM1 and the Cdc27 APC/C core subunit occurred only at the time point coinciding with the disappearance of the FoxM1 protein during late mitosis and early G1 phase (Fig. 2A). The association between FoxM1 and Cdc27 coincided with FoxM1 degradation, suggesting that the APC/C complex plays an important role in the destruction of the FoxM1 protein.


Figure 2
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  		FIG. 2. FoxM1 interacts with the APC/C-Cdh1 complex. (A) FoxM1 interacts with the Cdc27 subunit of the ubiquitin-APC/C complex during late mitosis and early G1 phase of the cell cycle. HeLa cells were synchronized at G1/S by double thymidine block, and protein extracts were prepared at the indicated hours after release from the double thymidine block. Cdc27 was immunoprecipitated with anti-Cdc27, and the presence of FoxM1 and Cdc27 proteins in the immunocomplexes was analyzed by Western blot assays with either the Cdc27 or FoxM1 antibody. (B) FoxM1 interacts with Cdh1 in co-IP assays. To determine the interaction between FoxM1 and the APC/C coactivator, i.e., Cdc20 or Cdh1, U2OS cells were transfected with T7 epitope-tagged FoxM1 (T7-FoxM1) and Myc epitope-tagged Cdh1 (Myc-Cdh1) or Cdc20 (Myc-Cdc20) expression vector. Cells were lysed after 24 h of transfection and subjected to co-IP assays using either antibody against the Myc epitope tag (anti-Myc) or control mouse immunoglobulin G, and immunoprecipitates were analyzed by Western blotting with antibodies specific to either FoxM1 or the Myc epitope tag.

We next examined whether FoxM1 could associate with the substrate specificity subunit of APC/C, i.e., Cdc20 or Cdh1. We transiently transfected U2OS cells with T7-FoxM1 and either Myc-Cdc20 or Myc-Cdh1 expression vectors. Extracts were prepared 24 h after transfection. The extracts were immunoprecipitated with antibody against the c-Myc epitope tag and then analyzed by Western blot analysis with either FoxM1 or c-Myc epitope tag antibody. The co-IP assays provided evidence for a specific interaction between Cdh1 and the FoxM1 protein, further supporting the notion that FoxM1 is a target of APC/C-Cdh1 (Fig. 2B).

To investigate whether FoxM1 is a target of APC/C-Cdh1, we ectopically expressed Cdh1 or Cdc20 in conjunction with FoxM1. Expression of Cdh1 resulted in significant reduction in the FoxM1 protein level, whereas expression of Cdc20 had no effect on it (Fig. 3A). To further confirm a role of Cdh1 in the proteolysis of FoxM1, we performed in vivo ubiquitination assays. U2OS cells were transfected with His-ubiquitin and T7-FoxM1 expression vectors along with either empty vector or Myc-Cdh1. Twenty-four hours following transfection, cells were treated with MG132 for 6 h, and protein extracts were bound to Ni-NTA to bind the ubiquitinated proteins. The levels of ubiquitinated FoxM1 protein were analyzed by Western blot analysis using FoxM1 antibody. As shown in Fig. 3B, expression of Cdh1 caused a significant increase in the ubiquitination of FoxM1. To analyze the endogenous proteins, cells were depleted of Cdh1 or Cdc20 by siRNA transfection. To monitor ubiquitination, the cells were also transfected with the His-ubiquitin expression plasmid. The cells were treated with MG132 for 6 h before being harvested. The protein extracts were bound to Ni-NTA beads, and the bound proteins were analyzed by Western blotting using FoxM1 antibody. Clearly, depletion of Cdh1 but not Cdc20 caused a severe inhibition of the ubiquitination of FoxM1 (Fig. 3C).


Figure 3
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  		FIG. 3. FoxM1 degradation is stimulated by Cdh1. (A) The FoxM1 protein is preferentially degraded by overexpression of the Cdh1 subunit of the ubiquitin-APC/C complex. U2OS cells were transfected with the T7-FoxM1 expression vector together with an expression vector for either hemagglutinin-Cdh1 (HA-Cdh1) or HA-Cdc20. The steady-state level of the FoxM1 protein was analyzed by Western blot assays with the FoxM1 antibody. (B) Cdh1-stimulated ubiquitination of the FoxM1 protein in vivo. U2OS cells were transfected with T7-FoxM1 along with Myc-Cdh1 expression vector or empty vector. Cells were lysed, and the lysates were bound to Ni-NTA beads. The bound fractions containing the ubiquitinated proteins were analyzed for polyubiquitination of the FoxM1 protein by Western blot assays with FoxM1 antibody. (C) Cdh1 is required for ubiquitination of endogenous FoxM1. U2OS cells were transfected with siRNA against Cdh1 or Cdc20 or with control siRNA. The cells were also transfected with a plasmid expressing His-ubiquitin (His-ubi). Lysates of the transfected cells were bound to Ni-NTA beads, and the bound fractions were analyzed with FoxM1 antibody in Western blot assays.

To further investigate the requirement of Cdh1 in the degradation of the FoxM1 protein, we reduced the activity of APC/Cdh1 by transfecting cells with Cdh1 siRNA. Partial depletion of Cdh1 levels in U2OS cells by siRNA transfection increased the steady-state level of the FoxM1 protein without increasing the level of FoxM1 mRNA in asynchronous cells (Fig. 4A). Depletion of Cdc20 did not alter the level of FoxM1 significantly. To investigate the role of Cdh1 in the cell cycle proteolysis of FoxM1, we performed siRNA transfection experiments with synchronized HeLa cells. Cells were synchronized in G1/S phase by a double thymidine block. Following the first thymidine block (as indicated in Fig. 4B), the cells were transfected with Cdh1 siRNA or a control siRNA. The transfected cells were subjected to a second thymidine block. The cells were then released for 14 h, a time point corresponding to early G1 phase, and the extracts of the synchronized cells at G1/S phase and early G1 phase were compared for the levels of FoxM1. In the control siRNA-transfected set, there was a clear reduction in the level of FoxM1 in G1 cells. Transfection of Cdh1 siRNA, on the other hand, prevented the loss of FoxM1 in the early G1 phase cells (Fig. 4B), suggesting a role of Cdh1 in the degradation of FoxM1 in the late M and early G1 phases of the cell cycle.


Figure 4
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  		FIG. 4. Depletion of Cdh1 increases FoxM1 levels. (A) Depletion of Cdh1 stabilizes FoxM1. HeLa cells were transfected with control siRNA or siRNA duplexes specific to Cdh1, Cdc20, or FoxM1. Seventy-two hours after siRNA transfection, levels of FoxM1 were analyzed by Western blot analysis with FoxM1 antibody. The levels of Cdh1 and Cdc20 were also analyzed by Western blotting, and β-actin was used as a loading control. FoxM1 mRNA levels were measured by quantitative reverse transcription-PCR as described in Materials and Methods. Data from a flow cytometric analysis of the siRNA-transfected cells are shown. (B) Depletion of Cdh1 in HeLa cells by siRNA transfection prevents reduction of FoxM1 protein levels in G1 phase of the cell cycle. The experimental scheme indicates the sequence of the double thymidine G1/S phase synchronization and Cdh1 siRNA transfection experiments with HeLa cells (upper panel). Cell extracts were prepared from the G1/S phase cells and from the cells at G1 phase. The extracts were subjected to Western blot analysis with FoxM1 antibody.

Codepletion of FoxM1 blocks early entry into S phase of cells depleted of Cdh1. Interestingly, it has been shown that APC/C-Cdh1 participates in the proteolysis of the Skp2 subunit of the SCF complex in early G1 phase (1, 35). Also, it has been suggested that the proteolysis of Skp2 is important for regulated entry into S phase because that allows for accumulation of p27 (28). Consistent with that notion, Cdh1 depletion by Cdh1 siRNA in HeLa cells caused accelerated entry into S phase after nocodazole block (1). Because FoxM1 is a transcriptional activator of Skp2 and is a target of APC/C-Cdh1, we sought to investigate whether targeting of FoxM1 is critical for APC/C-Cdh1 in the G1-S phase transition. First, we confirmed the role of Cdh1 in the early G1 proteolysis of FoxM1 following release from nocodazole arrest. We transfected HeLa cells with Cdh1 siRNA, Cdc20 siRNA, or control siRNA. The cells were then arrested in M phase by treatments with nocodazole. At the indicated time points following release from the nocodazole arrest, the cells were harvested, and the extracts were assayed for FoxM1 (Fig. 5A). As expected, the cells expressing control siRNA or Cdc20 siRNA exhibited reductions in the level of FoxM1 in early G1 phase. The cells expressing Cdh1 siRNA, on the other hand, did not exhibit a reduction of FoxM1 following release from the M phase block (Fig. 5A).


Figure 5
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  		FIG. 5. FoxM1 is a critical target of Cdh1 to inhibit premature S phase entry. (A) Diminished expression of Cdh1 in HeLa cells by siRNA transfection inhibits reduction of FoxM1 protein levels in late mitosis and G1 phase of the cell cycle. Control siRNA-, Cdc20 siRNA-, or Cdh1 siRNA-transfected HeLa cells were arrested in prometaphase by nocodazole, released, and then harvested at the indicated times following removal of nocodazole. Cell extracts were subjected to Western blot assays with anti-FoxM1, with β-actin as a loading control. (B) Codepletion of FoxM1 prevents aberrant S phase entry after Cdh1 depletion. HeLa cells were transfected with control siRNA, Cdh1 or FoxM1 siRNA, or a combination of FoxM1 siRNA and Cdh1 siRNA. After 72 h, cells were arrested in G2/M phase by nocodazole for 16 h and then released for the indicated times. BrdU (10 µM) was added to the culture medium 1 h before cells were harvested. BrdU-positive cells were determined by immunostaining as described in Materials and Methods. For each time point, the average percentage of BrdU-positive cells from three sets is plotted.

To analyze S phase entry following release from nocodazole arrest, cells were transfected with FoxM1 siRNA along with Cdh1 siRNA or with the individual siRNAs. The siRNA-transfected cells were arrested in early M phase by nocodazole treatment. At the indicated time points following release from arrest, the cells were treated with BrdU (10 µM) for 1 h. At the end of BrdU treatment, the cells were fixed and subjected to immunostaining using anti-BrdU antibody. The cells were also stained with DAPI (4',6'-diamidino-2-phenylindole) for quantification purposes. The percentages of BrdU-positive cells at different time points were plotted (Fig. 5B). As reported in a previous study (1), Cdh1 siRNA-transfected cells entered S phase earlier than control siRNA-transfected cells did after release from nocodazole block. Interestingly, however, we observed that cells transfected with both FoxM1 siRNA and Cdh1 siRNA showed significantly delayed S phase entry. Similar results were obtained with serum-starved cells. To study serum-induced entry into S phase, we employed NIH 3T3 cells because these cells can easily be synchronized to G0/G1 phase by serum starvation. Serum starvation caused a reduction in the level of FoxM1 (Fig. 6A), but cells depleted of Cdh1 retained a much higher level of FoxM1 following serum starvation (Fig. 6B). We measured BrdU incorporation following serum addition to cells that were transfected with siRNAs and serum starved following the same general approach as in the nocodazole experiment shown in Fig. 5. As expected, the Cdh1 siRNA-transfected cells exhibited more BrdU incorporation at earlier time points than did the control siRNA-transfected cells. But cotransfection of the FoxM1 siRNA caused a clear delay in BrdU incorporation by cells transfected with Cdh1 siRNA (Fig. 6C). These results are consistent with the notion that FoxM1 is a target of Cdh1 in early G1 phase. However, some caveats exist for these experiments. For example, it would appear from our results (Fig. 5B and 6C) that Cdh1 siRNA stimulates S phase mainly by stabilizing FoxM1. That is unlikely to be true, though, because Cdh1 also targets Skp2 (1, 35).


Figure 6
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  		FIG. 6. Codepletion of FoxM1 prevents premature S phase entry induced by Cdh1 depletion after serum induction. (A) FoxM1 levels are decreased in serum-starved cells. NIH 3T3 cells were serum starved for 24 h. For one set, during the last 6 h, MG132 was added to inhibit 26S proteasome-dependent proteolysis. (B) Depletion of Cdh1 prevents the decrease in FoxM1 protein levels in serum-depleted cells. Cdh1 siRNA was transfected into NIH 3T3 cells, and then the cells were serum starved for the indicated times. FoxM1 protein levels were determined by Western blot analysis. Data for flow cytometric analysis of the cells 0 h and 48 h after serum starvation are shown. (C) NIH 3T3 cells were transfected with the indicated siRNA. After 48 h of serum starvation, the siRNA-transfected cells were incubated with medium containing 10% fetal bovine serum for the indicated times. BrdU (10 µM) was added for 1 h before the cells were harvested. BrdU-positive cells were stained with anti-BrdU antibody and counted as described in Materials and Methods. For each point, the average percentage of BrdU-positive cells from three sets is plotted.

Degradation of FoxM1 protein requires D box and KEN box sequences. Most of the substrates of the APC/C complex contain a KEN box sequence and/or a D box sequence (26, 38, 42). Analysis of the amino-terminal region of the FoxM1 protein sequence shows two consecutive D box sequences (Fig. 7A) (amino acids 1 to 25) and one KEN box sequence (Fig. 7A) (amino acids 203 to 216), which is adjacent to the winged-helix DNA binding domain (Fig. 7A, DBD). To examine whether the D box and/or KEN box sequence is responsible for FoxM1 degradation by the APC/C-Cdh1 complex, we constructed vectors that expressed FoxM1 mutants that contained either a deleted D box sequence ({Delta}D) or site-directed mutagenesis of the KEN box sequence (KA). Cotransfection of the Cdh1 expression vector into U2OS cells reduced expression of the wild-type (WT) Myc-FoxM1 protein, whereas Cdh1 did not influence expression of the Myc-FoxM1 D box deletion mutant ({Delta}D) protein or the KEN box mutant (Fig. 7B). A Myc-tagged WT FoxM1 protein or a {Delta}D/KEN double mutant was used to generate a stable cell line, using U2OS cells. We synchronized the cells in prometaphase with nocodazole. At different times after release from the mitotic block, cells were harvested and subjected to immunoblotting with FoxM1 antibody. As expected, the double mutant was stabilized during cell cycle progression, whereas the WT FoxM1 protein was degraded in late mitosis and G1 phase (Fig. 7C). Taken together, these results suggest that both the D box and the KEN box are critical for the degradation of FoxM1 by APC/C-Cdh1.


Figure 7
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  		FIG. 7. FoxM1 degradation requires retention of both the D box and KEN box sequences. (A) Schematic representation of the FoxM1 protein depicting two D box sequences, a KEN box sequence, and the amino acid positions of these sequences. Also indicated on the FoxM1 protein are the winged-helix DNA binding domain (DBD), the C-terminal transcriptional activation domain (TAD), and the amino acid positions of these domains. (B) Deletion of the D box sequence or mutation in the KEN box of the FoxM1 protein prevents degradation by Cdh1. U2OS cells were transfected with either a WT Myc-FoxM1, Myc-FoxM1 D box deletion mutant ({Delta}D), or Myc-FoxM1 KEN box mutant (KEN) expression vector, with or without Myc-Cdh1 expression plasmid, and protein extracts were evaluated for the FoxM1 protein level by Western blot analysis with FoxM1 antibody. (C) The {Delta}D/KEN double mutant protein exhibits enhanced stability in G1 phase. Cells expressing WT or mutant FoxM1 were synchronized by nocodazole and then released for the indicated times in the presence of cycloheximide (100 µg/ml). The extracts were evaluated for FoxM1 levels by Western blot assays with the FoxM1 antibody. (D) Cells expressing a nondegradable mutant ({Delta}D/KEN) enter S phase earlier than do WT FoxM1-expressing cells. Cells expressing the WT or the nondegradable form ({Delta}D/KEN) of the FoxM1 protein were arrested at prometaphase with nocodazole and then released for the indicated times. BrdU (10 µM) was added for 1 hour before fixation and staining. BrdU-positive cells were analyzed by immunostaining with anti-BrdU and anti-mouse-fluorescein isothiocyanate antibodies, as described in Materials and Methods. (E) Cdh1 depletion further accelerates S phase entry of cells expressing the nondegradable form of FoxM1. Cells expressing the nondegradable form ({Delta}D/KEN) of FoxM1 or the parental line were transfected with control or Cdh1 siRNA. After 72 h of transfection, cells were arrested at G2/M phase by nocodazole treatment for 16 h and then released into fresh medium for the indicated times. One hour prior to fixation, cells were incubated with BrdU as described for panel D. For each point, the average percentage of BrdU-positive cells from three sets is plotted.

We employed a nondegradable mutant (harboring mutations in both the D and KEN boxes) of FoxM1 to further confirm the significance of FoxM1 proteolysis in the late M and early G1 phases. We generated stable U2OS cell lines expressing the WT or the nondegradable form of FoxM1. To study S phase entry following mitosis, we synchronized the cells to M phase by treating them with nocodazole for 16 h. The nocodazole-containing medium was then removed, and the cells were cultured in medium without drug to allow progression through the G1 and S phases. To determine the kinetics of S phase entry, the cells were treated with BrdU (10 µM) for 1 h at different time points following nocodazole removal. At the end of BrdU treatment, the cells were fixed and subjected to immunostaining using anti-BrdU antibody. The cells were also stained with DAPI for quantification purposes. The percentages of BrdU-positive cells at different time points were plotted (Fig. 7D). Clearly, the cells expressing the nondegradable form of FoxM1 exhibited a higher rate of BrdU incorporation at earlier time points following release from M phase arrest than did cells expressing the degradable form of FoxM1, providing further evidence that the degradation of FoxM1 is important for the regulated entry into S phase.

The nondegradable mutant of FoxM1 allowed us to investigate whether FoxM1 is the main target of Cdh1, through which it regulates S phase entry. We compared the kinetics of S phase entry in cells expressing Cdh1 siRNA with that in cells expressing the nondegradable mutant of FoxM1. The cells were synchronized to M phase by being treated with nocodazole for 16 h and then were released for progression through G1 and S phases. The cells were also treated with BrdU to measure S phase entry, as in the previous experiment. We observed that the knockdown of Cdh1 caused a greater stimulation of S phase entry than that in cells expressing a nondegradable form of FoxM1 (Fig. 7E). Moreover, knockdown by Cdh1 siRNA in cells expressing the nondegradable form of FoxM1 exhibited a greater acceleration than that in cells expressing the nondegradable form of FoxM1 alone (Fig. 7E). These results are consistent with the notion that there are other important targets of Cdh1 that regulate S phase entry.


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DISCUSSION
 
The work presented here is significant in several ways. First, we show that the cell cycle transcription factor FoxM1 is regulated by proteolysis at the late M and early G1 phases of the cell cycle. Second, we identify APC/C-Cdh1 as the E3 ubiquitin ligase that targets FoxM1, involving the D box and the KEN box motifs found in the N-terminal region of FoxM1. Finally, we provide evidence that the cell cycle proteolysis of FoxM1 is important for regulated entry into S phase. Together, these observations provide new insights into the cell cycle regulation of the transcription factor FoxM1.

It has been demonstrated that FoxM1 plays a pivotal role in cell cycle progression by regulating the expression of important cell cycle regulators, such as Cdc25B, cyclin B, polo-like kinase, and Aurora B kinase (13, 15, 17, 31). In agreement with the role of FoxM1 in cell cycle progression, several studies have implicated FoxM1 in tumor growth and progression. In published studies, reduced expression of FoxM1 significantly diminishes DNA replication and mitosis of tumor cells and inhibits development of mouse tumors in response to carcinogens or oncogenes (9-11). Moreover, enforced expression of FoxM1 in osteosarcoma U2OS cells dramatically increases anchorage-independent growth, as evidenced by stimulation of colony formation in soft agar assays (10). It also has been reported that FoxM1 plays an important role in cell invasion and angiogenesis (34). Furthermore, a growing body of evidence indicates that expression of FoxM1 is significantly elevated in various human malignancies and positively correlated with tumor grades (5, 27, 29, 30, 37). These published studies underscore the importance of understanding the mechanisms that control FoxM1.

During cell division, the levels of many cell cycle regulatory proteins are largely controlled by orderly synthesis and degradation. Thus, we hypothesized that the level of FoxM1 also fluctuates during the cell cycle. In this study, we provide evidence that FoxM1 is ubiquitinated and degraded via the ubiquitin-proteasome pathway and that rapid degradation of FoxM1 during mitotic exit is dependent on the activity of APC/C-Cdh1. The APC/C-Cdh1 complex recognizes target proteins containing specific motifs, i.e., the D box and KEN box. Both of these motifs are found in the N-terminal region of FoxM1. Moreover, these motifs in FoxM1 are critical for targeting by Cdh1 (Fig. 7). The level of the FoxM1 protein rapidly decreases when cells exit the cell cycle, induced by serum depletion and differentiation (data not shown). A mutant of FoxM1 lacking the Cdh1 target sequences (D/KEN) was very stable during cell cycle exit, suggesting that downregulation of the FoxM1 protein during cell cycle exit is dependent on APC/C.

Recently, three independent studies indicated that the transcriptional activity of FoxM1 is lower at G1/S phase than at G2/M phase (14, 25, 36). Wierstra and Alves (36) first reported that the N-terminal region of FoxM1 contains a regulatory domain that negatively regulates the transcriptional activity of FoxM1. This observation was confirmed by Laoukili et al. (14) and by Park et al. (25). It was shown that cyclin-Cdk-mediated phosphorylation of FoxM1 could overcome the inhibitory function of the N-terminal regulatory domain. Cyclin-Cdk-mediated phosphorylation of FoxM1 in G2/M phase (Fig. 1) activates the transcriptional activity of FoxM1 (14, 25, 36). The APC/C-Cdh1-mediated proteolysis of FoxM1 in early G1 phase, as described in this study, is distinct from the transcriptional regulation of FoxM1 in the late G1 and early S phases. In fact, the FoxM1 protein is relatively more stable in the late G1 and S phases than in early G1 phase (Fig. 1C). The transcriptional inhibitory motif in the N-terminal region has not been identified. It will be interesting to see whether it overlaps with the D box and KEN box motifs and whether the interaction with Cdh1 is also involved in the inhibition of the transactivation domain of FoxM1. On the other hand, it is possible that other proteins associate with the N-terminal region of FoxM1 to block targeting by Cdh1 in the late G1 and S phases. Further analyses of the N-terminal region and studies on the timing of the Cdh1 interaction will be valuable in understanding the cell cycle regulation of FoxM1 activity.

It is noteworthy that Skp2, Aurora B, and PLK1, which are transcriptionally activated by FoxM1, are also degraded, in a pathway involving APC/C-Cdh1, in a cell cycle-dependent manner (1, 18, 24, 35). Therefore, it is intriguing that APC/C-Cdh1 also targets FoxM1 and degrades it in the late M and early G1 phases of the cell cycle. We think that the proteolysis of FoxM1 serves as a more effective mechanism to control the levels of Skp2, Aurora B, and PLK1 during the cell cycle.

Previously, we reported that enforced expression of FoxM1 in mouse hepatocytes induces premature S phase entry following carbon tetrachloride injury in the liver (32). Furthermore, we observed that S phase entry is significantly reduced in FoxM1-depleted mouse embryonic fibroblasts, indicating that FoxM1 plays a critical role in G1/S phase progression. Therefore, we examined whether expression of a degradation-resistant FoxM1 mutant ({Delta}D/KEN) could accelerate S phase entry. As expected, U2OS cells expressing a nondegradable form of FoxM1 showed rapid S phase entry after release from nocodazole block. A more rapid entry into S phase by expression of the nondegradable FoxM1 form than that by degradable WT FoxM1 is consistent with the notion that FoxM1 proteolysis in early G1 phase is important for regulated entry into S phase.

Although no critical mutations have been found in either the APC/C machinery or its target genes in cancers, recent studies indicate that many APC/C substrates are deregulated in various human malignancies, implying that APC/C-dependent proteolysis is somehow impaired in a number of malignant tumors (16). Therefore, we speculate that deregulated APC/C activity in tumors may explain why FoxM1 is significantly overexpressed in various human cancers, leading to a poor prognosis.


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ACKNOWLEDGMENTS
 
We dedicate this work to the memory of Robert H. Costa.

We thank M. Pagano (NYU) for Cdh1 and Cdc20 plasmids.

This work was supported by U.S. Public Health Service grants CA 124488 and CA 100035 to P.R., AG021842 to L.F.L., and DK 44525 and DK068503 to A.L.T.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Genetics (M/C 669), University of Illinois at Chicago, College of Medicine, 900 S. Ashland Ave., MBRB Rm. 2302, Chicago, IL 60607-7170. Phone: (312) 413-0255. Fax: (312) 355-3847. E-mail: pradip{at}uic.edu Back

{triangledown} Published ahead of print on 23 June 2008. Back


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Molecular and Cellular Biology, September 2008, p. 5162-5171, Vol. 28, No. 17
0270-7306/08/$08.00+0     doi:10.1128/MCB.00387-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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