The auto-ubiquitylation of E3 ubiquitin-protein ligase Chfr at G2 phase is required for accumulation of polo-like kinase 1 and mitotic entry in mammalian cells

Abstract

The E3 ubiquitin-protein ligase Chfr is a mitotic stress checkpoint protein that delays mitotic entry in response to microtubule damage; however, the molecular mechanism by which Chfr accomplishes this remains elusive. Here, we show that Chfr levels are elevated in response to microtubule-damaging stress. Moreover, G(2)/M transition is associated with cell cycle-dependent turnover of Chfr accompanied by high autoubiquitylation activity, suggesting that regulation of Chfr levels and auto-ubiquitylation activity are functionally significant. To test this, we generated Chfr mutants Chfr-K2A and Chfr-K5A in which putative lysine target sites of auto-ubiquitylation were replaced with alanine. Chfr-K2A did not undergo cell cycle-dependent degradation, and its levels remained high during G(2)/M phase. The elevated levels of Chfr-K2A caused a significant reduction in phosphohistone H3 levels and cyclinB1/Cdk1 kinase activities, leading to mitotic entry delay. Notably, polo-like kinase 1 levels at G(2) phase, but not at S phase, were ∼2-3-fold lower in cells expressing Chfr-K2A than in wild-type Chfr-expressing cells. Consistent with this, ubiquitylation of Plk1 at G(2) phase was accelerated in Chfr-K2A-expressing cells. In contrast, Aurora A levels remained constant, indicating that Plk1 is a major target of Chfr in controlling the timing of mitotic entry. Indeed, overexpression of Plk1 in Chfr-K2A-expressing cells restored cyclin B1/Cdk1 kinase activity and promoted mitotic entry. Collectively, these data indicate that Chfr auto-ubiquitylation is required to allow Plk1 to accumulate to levels necessary for activation of cyclin B1/Cdk1 kinase and mitotic entry. Our results provide the first evidence that Chfr auto-ubiquitylation and degradation are important for the G(2)/M transition.

FIGURE 1.

Chfr levels are elevated in response to mitotic stress in mammalian cells. A, DLD1 cells were transiently transfected with 3xFLAG-CMV 7.1 (Con) or FLAG-Chfr (WT). DLD1 cells were harvested after treatment of nocodazole (Noc, 100 ng/ml) for the indicated times and analyzed by immunoblotting with anti-FLAG and anti-phospho H3 antibodies. B, T24 and HeLa cells were transfected with FLAG-Chfr and treated with different concentrations of nocodazole for 12 h. Expression levels of Chfr were determined by immunoblotting. C, to obtain G₂ and mitotic phase cells, FLAG- or FLAG-Chfr-transfected HeLa cells were treated with nocodazole (100 ng/ml) for 12 h and floating mitotic cells and residual adherent cells (G₂) were separately collected by mechanical shake-off. Expression profiles of Chfr were analyzed by densitometry (Student's t test; *, p < 0.05; **, p < 0.005).

FIGURE 2.

Cell cycle-dependent degradation of Chfr protein by auto-ubiquitylation. A, HeLa cells were synchronized at the G₁/S boundary by the DTB method. After first thymidine treatment, cells were transfected with the FLAG-Chfr (WT) followed by treatment of second thymidine for 14 h. Asynchronous (Asy) and synchronized cells at the indicated time points after the release from DTB were harvested and analyzed by immunoblotting. B, HeLa cells were synchronized at S or G₂ phase by treatment with hydroxyurea (HU, 2 mm) for 18 h and roscovitine (Ros, 100 μm) for 5 h at R7 (7 h after DTB release). The Chfr expression levels were determined by immunoblotting. C, HeLa cells were transfected with FLAG-Chfr ΔRF or FLAG-Chfr^I306A. For collecting cells at each time points, cells were synchronized at G₁/S boundary using DTB method and released from DTB. Lysates were subjected to immunoblotting with anti-phosphohistone H3, anti-FLAG (Chfr), and anti-actin antibodies. D, HeLa cells co-transfected with FLAG-Chfr and His-ubiquitin (Ub) were synchronized at each phase by treatment with hydroxyurea and roscovitine as described above and followed by treatment with MG132 (2 μm) for 6 h before harvest. The ubiquitylation of Chfr was evaluated by anti-Ub antibody after lysates were immunoprecipitated (IP) with anti-FLAG antibody. E, HeLa cells were co-transfected with FLAG-Chfr and synchronized as described in Fig. 2B. The immunoprecipitates obtained with anti-FLAG antibody were conjugated with ubiquitin in the presence of E1, UbcH5b, ubiquitin, and ATP for 1 h. The ubiquitylation patterns were determined by immunoblotting. F, the immunoprecipitates obtained with anti-FLAG antibody were incubated with 60 units of CIP for 1 h and subjected to in vitro ubiquitylation as described in E.

FIGURE 3.

Substitution of alanine for putative auto-ubiquitylation target lysine residues. A, shown is a schematic representation of Chfr domain structures (FHA, forkhead-associated domain; RF, ring finger domain; CR, cysteine-rich region), and substitution of putative auto-ubiquitylation target Lys sites into Ala (K2A, K3A, and K5A) are indicated in red. B, HeLa cells were transfected with the indicated FLAG-Chfr plasmids (WT, K2A, K5A, ΔRF), and Chfr expression levels of proteins were determined by immunoblotting. C, subcellular localization of Chfr expressing different mutant constructs was determined by immunofluorescence analyses. Chfr is shown in red (FLAG), and nuclei are shown in blue (DAPI). Images were analyzed by confocal microscopy.

FIGURE 4.

Stabilization of Chfr in vivo by mutation of putative auto-ubiquitylation target sites. A, FLAG-Chfr- or FLAG-Chfr K2A-expressing cells were synchronized by the DTB method, and expression profiles of Chfr in DLD1, T24, and HeLa cells are shown and analyzed by densitometry (Student's t test; *, p < 0.01). B, HeLa cells were transfected with FLAG-Chfr or FLAG-Chfr K2A and along with His-Ub expressing cells were synchronized at each time points after DTB. For evaluating the level of ubiquitylation of Chfr, the lysates were immunoprecipitated (IP) with anti-FLAG antibody followed by analyzed immunoblotting with anti-Ub antibody.

FIGURE 5.

Delay of mitotic entry by abrogation of cell cycle-dependent degradation of Chfr. A, HeLa cells were synchronized at the G₁/S boundary by the DTB method. During the DTB, the cells were transfected with the FLAG-Chfr K2A in two different doses (2 and 4 μg). At the indicated time points after the release from DTB, cells were harvested for immunoblotting. B, HeLa cells were harvested as described in A, and DNA contents were analyzed by flow cytometry. C, Mitotic indices of synchronized HeLa cells transfected with FLAG, FLAG-Chfr, and FLAG-Chfr K2A were determined by aceto-orcein staining. D, cell lysates were obtained at the indicated times after the DTB release. The lysates were immunoprecipitated anti-cyclin B₁ antibody and used to assess cyclin B/Cdk1 kinase activity in in vitro kinase assays. The phosphorylated substrates (histone H1) were detected by autoradiography, and immunoblotting for cyclin B₁ levels were shown in the lower panels.

FIGURE 6.

Plk1 acts as a downstream mediator of Chfr controlling timing of mitotic entry in mammalian cells. A, HeLa cells were transfected with control (pCDNA3.1) or FLAG-Chfr (WT, K2A, K5A, and ΔRF). Cells were synchronized at S phase with treatment of 2 mm hydroxyurea (HU) for 20 h or at G₂ phase with 100 μm roscovitine (Ros) for 4 h at R8. Expression profiles of Plk1 and Aurora A protein were determined and analyzed by densitometry (Student's t test; *, p < 0.01). B, HeLa cells were co-transfected with His-Ub and FLAG-Chfr (WT, K2A, K5A, and ΔRF), and cells were synchronized as in A to obtain S and G₂ phase-arrested cells. Cells were treated with MG132 (2 μm) for 8 h before harvest. IP, immunoprecipitate. C, shown is the mitotic index of synchronized HeLa cells transiently expressing FLAG-Chfr K2A with or without Myc-Plk1 after release from DTB. Cells were stained with aceto-orcein, and the mitotic index was determined by counting cells with condensed chromatin under optical microscopy. D, cyclin B₁/Cdk1 kinase activity of synchronized HeLa cells is shown. Cells were transfected with FLAG-Chfr K2A with or without Myc-Plk1 and synchronized by the DTB method. The phosphorylation profiles of histone H1 during G₂/M progression were determined and analyzed by densitometry (right panel). Bars represent the means of two independent experiments.