Polo-like kinase 1-mediated phosphorylation of Forkhead box protein M1b antagonizes its SUMOylation and facilitates its mitotic function

Abstract

Transcription factor Forkhead box protein M1b (FoxM1b) plays an important role during mitotic entry and progression. Our previous studies identified polo-like kinase 1 (PLK1) as a major regulator of FoxM1b. During G2/M transition, PLK1 directly interacts with and phosphorylates FoxM1b, resulting in full activation of the transactivation capacity of FoxM1b. Such a vital regulatory mechanism is essential for timely mitotic entry and progression. However, the molecular mechanism by which PLK1-mediated phosphorylation enhances the transcriptional activity of FoxM1b remains to be determined. We demonstrate that FoxM1b can be SUMOylated in vitro and in vivo, preferentially by SUMO-1. SUMOylation of FoxM1b was found to occur at multiple sites, leading to suppression of FoxM1b transcriptional activity. Such a posttranslational modification of FoxM1b was antagonized by PLK1-mediated phosphorylation. By immunofluorescence staining and subcellular fractionation, we demonstrate that SUMO conjugation promotes cytosolic translocation of FoxM1b. Moreover, SUMO modification of FoxM1b facilitates the ubiquitin-mediated proteasomal degradation of FoxM1b. PLK1-mediated phosphorylation of FoxM1b abrogates the inhibitory effect on FoxM1b by SUMO modification, thereby promoting its nuclear translocation and preventing its proteolytic degradation in the cytoplasm. Such an antagonistic regulatory mechanism is essential for the mitotic function of FoxM1b, ensuring timely mitotic entry and progression. Taken together, our studies have revealed a working mechanism by which PLK1 positively regulates the activity and level of FoxM1b, which would greatly facilitate therapeutic interventions that focus on targeting the PLK1-mediated and/or FoxM1-mediated signaling network.

Keywords: Cell Cycle; FoxM1b; Mitosis; Nuclear-Cytoplasmic Shuttling; PLK1; Phosphorylation; Protein Stability; Regulation; SUMOylation; Transcription Factor.

FIGURE 1.

FoxM1b is subjected to SUMO modification. A, ectopically expressed FoxM1b is subjected to SUMOylation in cells. U2OS cells were transfected with the indicated constructs. After 24 h, the cells were lysed, immunoprecipitated (IP) using anti-Myc antibody, and blotted with anti-Myc, anti-SUMO-1, and anti-SUMO-2/3 antibodies. B, FoxM1b is SUMOylated in vitro. In vitro SUMOylation assays were performed by incubating bacterially expressed FoxM1b with the SUMO conjugation machinery: Aos1/Uba2, Ubc9, and SUMO-1. A SUMO-1 mutant (MUT) has a deletion of the two C-terminal glycine residues required for its conjugation. The SUMOylation status of FoxM1b was examined by Western blotting. C, endogenous FoxM1b is SUMOylated by SUMO-1. U2OS cells transfected with SUMO-1 plasmid were lysed and immunoprecipitated using anti-FoxM1 antibody and blotted with anti-FoxM1 and anti-SUMO-1 antibodies. D, schematic of consensus SUMOylation sites in FoxM1b identified by the SUMOplot^TM analysis program. FKH, forkhead domain; TAD, transactivation domain. E, mapping SUMOylation sites on FoxM1b. U2OS cells were transfected with the indicated constructs. Cells were lysed and blotted with anti-Myc and anti-SUMO-1 antibodies.

FIGURE 2.

PLK1-mediated phosphorylation of FoxM1b modulates FoxM1b SUMOylation. A, PLK1 activity modulates FoxM1b SUMOylation. U2OS cells were transfected with Myc-tagged FoxM1b and FLAG-tagged WT, TD, or KD PLK1. The SUMOylation of FoxM1b was examined by Western blotting using anti-Myc and anti-SUMO-1 antibodies. FLAG-tagged PLK1 expression was monitored using anti-FLAG antibody. B, PLK1-dependent phosphorylation of FoxM1b interferes with its SUMO conjugation. U2OS cells were transfected with Myc-tagged FoxM1b WT or mutants in the presence or absence of SUMO-1. The SUMOylation of FoxM1b was determined by blotting with anti-Myc and anti-SUMO-1 antibodies. C and D, PLK1 directly interferes with FoxM1b SUMOylation. Bacterially expressed FoxM1b was subjected to an in vitro kinase assay using WT or KD PLK1 as kinase sources. C, PLK1-directed phosphorylation was determined by Western blot analysis with an anti-phospho-specific Ser(P)-724-FoxM1b antibody. D, following an in vitro kinase assay, those samples were analyzed using an in vitro SUMOylation assay. NS, non-specific band.

FIGURE 3.

SUMOylation inhibits the transcriptional activity of FoxM1b, which is antagonized by PLK1-mediated phosphorylation. A, SUMO-1 suppressed the transcriptional activity of FoxM1b in a dose-dependent manner. U2OS cells were transfected with a FoxM1b reporter plasmid, Myc-tagged FoxM1b, and increasing amounts of SUMO-1 plasmids (100, 200, 300, 400, and 500 ng). Luciferase activity was measured and normalized to Renilla luciferase activity. The data are expressed as a percentage of WT FoxM1b activity (mean ± S.D. of three separate experiments in triplicate). Exogenous FoxM1b protein levels were measured by immunoblotting (IB) using anti-Myc antibodies. *, p < 0.05 versus control. EV, empty vector. B, cotransfection of Ubc9 along with SUMO-1 further reduced the transactivation capacity of FoxM1b. U2OS cells were transfected with a FoxM1b reporter plasmid, Myc-tagged FoxM1b, and SUMO-1 or Ubc9 or both plasmids. Luciferase activity was measured. *, p < 0.05 versus control (FoxM1 only). C–E, PLK1-mediated phosphorylation antagonized SUMOylation-induced suppression of FoxM1b transcriptional activity. U2OS cells were transfected with a FoxM1 reporter plasmid and either Myc-tagged FoxM1 WT or a mutant (6KR, SUMO-1-WT, AA, EE, or AA/6KR). These cells were left unsynchronized (C and D) or synchronized at G₁, S, or G₂/M phase (E). The data are expressed as a percentage of WT FoxM1b activity. *, p < 0.05 versus WT FoxM1. EV, empty vector.

FIGURE 4.

PLK1-mediated phosphorylation promotes FoxM1b nuclear translocation by interfering with its SUMO conjugation. A, U2OS cells were transfected with Myc-tagged FoxM1b WT or a mutant (6KR, SUMO-1-WT, AA, EE, or AA/6KR). The cellular localization of ectopically expressed WT or mutant FoxM1b was examined by immunofluorescence staining using anti-Myc antibody. Quantification of a representative experiment is shown in the right panels. Similar results were obtained from three independent experiments. N, nucleus; C, cytoplasm. Scale bar = 10 μm. B, an additional set of samples was subjected to subcellular fractionation. The levels of exogenous FoxM1b in nuclear and cytoplasmic fractions were determined by immunoblotting with anti-Myc antibody. The relative purity of the nuclear and cytoplasmic fractions was confirmed by sequential probing for the nuclear marker Lamin A/C and the cytoplasmic marker α-tubulin.

FIGURE 5.

SUMOylation of FoxM1b promotes ubiquitin-mediated proteasome degradation of FoxM1b. A, SUMOylation of FoxM1b affects its protein stability. U2OS cells were transfected with Myc-tagged FoxM1b WT or a mutant (6KR, SUMO-1-WT, AA, EE, or AA/6KR). 48 h post-transfection, cells were either left untreated (0 h) or treated with cycloheximide (CHX, 20 μg/ml) for 2, 4, 6, or 8 h. The protein levels of FoxM1b were determined by Western blot analysis (left panel). Quantification of the exogenous FoxM1b protein levels relative to β-actin is shown in the right panel. Error bars show mean ± S.D. B, SUMOylation of FoxM1b facilitates its ubiquitination. U2OS cells were cotransfected with Myc-tagged FoxM1b WT or a mutant (6KR, SUMO-1-WT, AA, EE, or AA/6KR) with or without His-ubiquitin. After 48 h, cells were treated with vehicle (dimethyl sulfoxide) or 5 μm MG132 for 6 h. Cells were then lysed, and cell lysates were bound to nickel-nitrilotriacetic acid (Ni-NTA) beads. The bound fractions containing the ubiquitinated proteins were separated by SDS-PAGE, and ubiquitination of FoxM1b was detected by anti-FoxM1 antibody.

FIGURE 6.

SUMOylation of FoxM1b occurs primarily during late mitosis. HeLa cells stably expressing His₆-tagged SUMO-1 were synchronized at prometaphase (Pro), metaphase (Meta), anaphase (Ana), and telophase (Telo). A, a portion of the synchronized cells was stained for α-tubulin (green) and DNA (blue) by FITC-conjugated anti-α-tubulin and DAPI, respectively. Scale bar = 10 μm. B, the remaining synchronized cells were lysed and immunoprecipitated (IP) using anti-FoxM1 antibody and blotted with anti-FoxM1 and anti-SUMO-1 antibodies. The protein levels of FoxM1 showed significant differences during mitotic progression. To compare the level of SUMOylation during different mitotic phases, the input was adjusted to have a similar level of total FoxM1b from the different mitotic phases.

FIGURE 7.

PLK1-dependent regulation of FoxM1b SUMOylation is required for timely entry into and progress through M phase. U2OS cells stably expressing empty vector (EV), Myc-tagged FoxM1 WT, or a mutant (6KR, SUMO-1-WT, AA, EE, or AA/6KR) were infected with lentivirus encoding FoxM1b shRNA (targeting the 3′-UTR) to knock down endogenous FoxM1b. A, cell cycle distributions were determined by flow cytometry analysis 72 h after infection. PI, propidium iodide. B, the percentage of binucleated cells was determined on the basis of DAPI and α-tubulin staining 72 h post-infection. C, cell lysates were collected 72 h after infection and analyzed by immunoblotting. Blotting for α-tubulin was used as a loading control (Ctrl). D, total RNA was extracted 48 h after transfection and subjected to RT-PCR analysis. RT-PCR for GAPDH was used as an internal control.

FIGURE 8.

Model for PLK1-mediated regulation of FoxM1 during late cell cycle phases.