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First published online 25 January 2005
doi: 10.1242/jcs.01657

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Raf/MEK/MAPK signaling stimulates the nuclear translocation and transactivating activity of FOXM1c

Department of Biochemistry, Faculty of Medicine, The University of Hong Kong, 3/F Laboratory Block, The Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, China

View larger version (61K): [in a new window]   Fig. 1. FOXM1 is phosphorylated and translocated to the nucleus before entry into the G2/M phase. (A) Flow diagrams of asynchronized and synchronized BJ1 cells. BJ1 cells were synchronized at the G1/S boundary by serum starvation/aphidicolin double block. Cells were released from arrest by removal of aphidicolin. At different time intervals after release, cells were harvested and stained with propidium iodide for DNA analysis using a flow cytometer. Synchronized cells gradually progressed through the cell cycle and G2/M cells could be detected at both 9 hours and 12 hours after release. (B) Cells at different time intervals after aphidicolin release were harvested for immunoblot analysis using anti-FOXM1 and anti-tubulin antibodies. Progressive mobility shifts (denoted by arrowheads) of the FOXM1 band were observed at 9 hours and 12 hours after release. (C) The cell lysate at 9 hours after release was treated with calf intestine phosphatase (for 1 hour) before immunoblot analysis. Such treatment abolished the mobility up-shift of the FOXM1 band. (D) BJ1 cells grown on coverslips were synchronized by serum starvation/aphidicolin double block and fixed at various time points after removal of aphidicolin. Cells were immunostained with anti-FOXM1 antibody and counterstained with propidium iodide to detect nuclear DNA. Merged images of FOXM1 and nuclear staining are also shown. FOXM1 was predominantly cytoplasmic (arrows) at 0 hour, 3 hours and 6 hours after release; FOXM1 became mainly nuclear (arrowheads) at 9 hours and 12 hours after release.

View larger version (41K): [in a new window]   Fig. 2. Activity of the Raf/MEK/MAPK pathway is required for nuclear translocation of FOXM1. (A) Immunoblot analysis of total ERK and phosphoERK expression to monitor the effect of U0126 and ATA treatment on Raf/MEK/MAPK activity. (B) Scheme for drug treatment to inhibit MEK1/2. Synchronized BJ1 cells were incubated with U0126 (10 µM) or the inactive analog U0124 (10 µM) from 7 hours to 8 hours after aphidicolin release. Cells were harvested 1 hour later (i.e. at 8 hours after release) and immunostained for FOXM1. (C) Without drug treatment, FOXM1 was predominantly cytoplasmic at 7 hours after release. (D) 1 hour later, FOXM1 became mainly nuclear. (E) Treatment with U0126 abolished FOXM1 nuclear translocation at 8 hours after release. (F) FOXM1 nuclear translocation was not affected by U0124 treatment. (G-K) ATA promotes nuclear translocation of FOXM1. (G) Scheme for ATA treatment and U0126/U0124 pre-treatment. (H) In cells treated with solvent (DMSO), FOXM1 was predominantly expressed in the cytoplasm. (I) After incubation with 200 µM ATA for 1 hour, FOXM1 became localized mainly to the nucleus. (J) ATA stimulation of FOXM1 nuclear translocation was abrogated by pre-incubation with 10 µM U0126 for 1 hour. (K) U0124 pre-treatment could not counteract the stimulatory effect of ATA.

View larger version (31K): [in a new window]   Fig. 3. Constitutively active MEK1 enhances the transactivating activity of FOXM1c. (A) Chromatin immunoprecipitation assays of asynchronized BJ1 cells were carried out as described in Methods section to detect FOXM1 binding to the cyclin B1 promoter. Cyclin B1 DNA was enriched more than 50-fold by the anti-FOXM1 antiserum. (B) Schematic diagrams of FOXM1c, FOXM1b and FOXM1cCter. Positions of the DNA binding domain (DBD), exon Va and transactivation domain (TAD) are shown. FOXM1b lacks exon Va. FOXM1cCter was generated by deletion of the last 71 amino acids of FOXM1. (C-E) Transient reporter assays. NIH3T3 cells were co-transfected with the various expression plasmids and cyclin B1 luciferase reporter. 48 hours after transfection, cells were harvested for luciferase assay. (C) 60 ng of FOXM1c, FOXM1b or the control vector pcDNA3 was co-transfected with the cyclin B1 reporter. Both FOXM1c and FOXM1b showed 2.5-fold stimulation of the cyclin B1 promoter when compared with the vector control. (D) caMEK1 enhances the transactivating activity of FOXM1c. Co-transfection of caMEK1 (30 ng) with an increasing amount of FOXM1 strongly enhanced the transactivating activity of FOXM1c, but not FOXM1b. (E) The caMEK1 enhancing effect requires the presence of functional FOXM1 protein. Various amounts of FOXM1 and MEK1 expression plasmids, and empty vectors (pcDNA3 and pSR), were co-transfected as indicated. Both caMEK1 and functional FOXM1c are required for the synergistic activation of cyclin B1 promoter. (F) Western blot to demonstrate the activating and inhibitory effect of caMEK1 and dnMEK1, respectively, on Raf/MEK/MAPK signaling.

View larger version (22K): [in a new window]   Fig. 4. FOXM1c is the predominant isoform expressed in BJ1, NIH 3T3 and various mouse tissues. (A) RT-PCR assays were designed using human and mouse primers, which flank exon Va, to assess the expression of FOXM1b and FOXM1c transcripts. FOXM1b and FOXM1c transcripts generate PCR fragments of 323 and 368 base pairs, respectively. (B-D) RT-PCR analysis. As positive controls, cDNAs encoding human FOXM1b and FOXM1c, and human and mouse testis cDNA libraries, were PCR-amplified to generate the FOXM1b and FOXM1c bands. GAPDH transcripts were also PCR-amplified in the mouse tissue samples to control for possible loading differences. mRNA of asynchronized and synchronized BJ1 cells [0 hour (G1), 4.5 hours (S), 9 hours (G2) after aphidicolin release] (B) and asynchronized, L-mimosine-arrested (G1/S) and etoposide-arrested (G2) NIH 3T3 cells (C) were extracted for RT-PCR analysis. Intestine (I), heart (H), liver (Li) and lung (Lu) of day 1 neonates, and pancreas (P) and thymus (Th) of adult mice, were harvested for RT-PCR analysis and compared to that of testis (Te) cDNA library (D).

View larger version (32K): [in a new window]   Fig. 5. Identification and functional analysis of the ERK1/2 target sites within FOXM1c. (A) Sequence analysis of FOXM1c identifies three putative ERK1/2 phosphorylation sites (PXS/TP) at amino acids 329-332, 618-621 and 702-705. DBD, DNA binding domain; TAD, transactivating domain. (B) The 331 and 704 motifs are conserved in the FOXM1c coding sequences from multiple species. (C) Activated Raf/MEK/MAPK signaling stimulates FOXM1 phosphorylation. Asynchronized BJ1 cells were incubated with 200 µM ATA or 200 nM TPA for 1 hour. Cells with or without drug treatment were lysed and endogenous FOXM1 immunoprecipitated with anti-FOXM1 antibody or control rabbit antiserum. The immunoprecipitates were immunoblotted with anti-FOXM1 and anti-phosphoserine antibodies. ATA and to a lesser extent TPA, enhanced the phosphorylation of FOXM1. Note that anti-phosphoserine antibody selectively detected the upper band of the FOXM1 doublet. (D) Both the 331 and 704 motifs are important for mediating the caMEK1 enhancing effect. The wild type (WT) and various substitutive mutants (S331A, S704A, SASA) were co-transfected with caMEK1 in transient reporter assays as described in Fig. 3. caMEK1 enhancement is shown as percentage of activation compared to the wild-type control (100%). Immunoblot analysis revealed expression of the various HA-tagged, mutant FOXM1c proteins.

View larger version (28K): [in a new window]   Fig. 6. Inhibition of Raf/MEK/MAPK signaling leads to G2/M delay and downregulation of FOXM1 target genes. U0126 (25 µM) was added to synchronized BJ1 cells at 5.5 hours after aphidicolin release (black arrows). Cells were harvested for flow cytometric (A), RT-PCR (B) and western blot (C) analyses. Data in A and B were quantified using Modfit and TotalLab software, respectively. (A) After U0126 treatment, slower progression through G2/M was revealed by the delayed re-entry of cells into the subsequent G1 phase. (B) RT-PCR analysis indicated that U0126 treatment attenuated the increase in cyclin B1 mRNA levels associated with cell cycle progression through G2/M. GAPDH mRNA levels were analyzed in parallel as loading control. (C) Cell lysates were immunoblotted with anti-ERK, anti-phosphoERK, anti-cyclin B1, anti-Cdc25B and anti-tubulin antibodies. U0126 suppressed the phosphorylation of ERK, and the expression of cyclin B1 and Cdc25B, but had no effect on the expression of ERK and tubulin.

View larger version (9K): [in a new window]   Fig. 7. A model to illustrate the cell cycle-dependent regulation of FOXM1c function. (A) Location of the PGS331P and PGS704P ERK1/2 phosphorylation sites (shown in red circles). The 331 motif within exon Va (yellow) is close to the DNA binding domain (DBD, red) and a putative NLS (sequence of the NLS is provided in Fig. S2 of the supplementary material). The 704 motif is in close proximity to the Cdk phosphorylation sites (green circles) identified within the transactivating domain (TAD, violet). (B) During late S phase, the Raf/MEK/MAPK pathway phosphorylates FOXM1 and stimulates its nuclear translocation and transactivating activity. Active cyclin-Cdk2 (Cyclin E/A-Cdk2) phosphorylates FOXM1 at the Cdk1/2 phosphorylation sites within the TAD. This promotes recruitment of p300/CBP and transcription of FOXM1 target genes like cyclin B1 and Cdc25B. Cdk1 becomes activated by binding of cyclin B1 and dephosphorylation by Cdc25B. Active cyclin B1-Cdk1 phosphorylates FOXM1 and further promotes transcription of cyclin B1 and Cdc25B. This positive-feedback loop produces a burst of cyclin B1-Cdk1 activity to drive the cell into mitosis.