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, 42 (12), 840-849

c-Cbl Acts as an E3 Ligase Against DDA3 for Spindle Dynamics and Centriole Duplication During Mitosis

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c-Cbl Acts as an E3 Ligase Against DDA3 for Spindle Dynamics and Centriole Duplication During Mitosis

Dasom Gwon et al. Mol Cells.

Abstract

The spatiotemporal mitotic processes are controlled qualitatively by phosphorylation and qualitatively by ubiquitination. Although the SKP1-CUL1-F-box protein (SCF) complex and the anaphase-promoting complex/cyclosome (APC/C) mainly mediate ubiquitin-dependent proteolysis of mitotic regulators, the E3 ligase for a large portion of mitotic proteins has yet to be identified. Here, we report c-Cbl as an E3 ligase that degrades DDA3, a protein involved in spindle dynamics. Depletion of c-Cbl led to increased DDA3 protein levels, resulting in increased recruitment of Kif2a to the mitotic spindle, a concomitant reduction in spindle formation, and chromosome alignment defects. Furthermore, c-Cbl depletion induced centrosome over-duplication and centriole amplification. Therefore, we concluded that c-Cbl controls spindle dynamics and centriole duplication through its E3 ligase activity against DDA3.

Keywords: DDA3; E3 ligase; c-Cbl; centriole; centrosome; spindle dynamics.

Conflict of interest statement

Disclosure

The authors have no potential conflicts of interest to disclose.

Figures

Fig. 1
Fig. 1. c-Cbl interacts with DDA3 and acts as a mitotic regulator
(A) The DDA3 complex was purified from mitotic cells and analyzed by mass spectrometry. A peptide of c-Cbl was identified three times. (B) Twenty-eight hours after transfection of the EV or HA-c-Cbl plasmid, HeLa cells were harvested and subjected to immunoprecipitation and western blotting with the indicated antibodies. p38MAPK served as a loading control. EV, empty vector. (C and D) HeLa cells were synchronized by a double thymidine block (C) or thymidine-nocodazole block (D), placed into fresh media, and harvested at the indicated times. Cell lysates were analyzed by western blotting with the indicated antibodies. AS, unsynchronized cells. (E) HeLa cells were transfected with control (siControl) or c-Cbl-specific siRNAs (siCbl-A and siCbl-B). Seventy-two hours after siRNA transfection, the transfected cells were harvested and lysed to measure protein levels by western blotting with the indicated antibodies. (F and G) Seventy-two hours after siRNA transfection, HeLa cells were fixed with MeOH and stained with antibodies as indicated. Images are maximum projections from Z-stacks of representative cells stained for c-Cbl (green), β-tubulin (red), and DNA (blue). The number of metaphase cells with unaligned chromosomes was quantified and plotted (G) (n = 300 metaphase cells from three independent experiments). (H) Seventy-two hours after siRNA transfection, HeLa cells expressing GFP-Histone H2B cells were imaged for GFP fluorescence by time lapse. Images were captured every 3 min to monitor mitotic progression. NEB, nuclear envelop breakdown. Unaligned, the initial formation of the metaphase plate. Data are represented as mean ± SEM. Scale bars = 5 μm. *P < 0.01.
Fig. 2
Fig. 2. c-Cbl modulates the levels of DDA3 and Kif2a at the mitotic spindle for spindle formation
(A) Forty-eight hours after siRNA transfection, HeLa cells were transfected with HA-Ubiquitin plasmid. Twenty-four hours after plasmid transfection, the cells were treated with 10 mM proteasome inhibitor, MG132, for 5 h and then harvested and subjected to immunoprecipitation and western blotting with the indicated antibodies. (B) Seventy-two hours after siRNA transfection, the cells were treated with nocodazole for 18 h. The mRNA levels of c-Cbl and DDA3 in mitotic cells were quantified by real-time RT-qPCR analysis (n = 3). (C and D) Seventy-two hours after siRNA transfection, HeLa cells were harvested to analyze protein levels with the indicated antibodies (C) or fixed with MeOH to analyze unaligned chromosomes in metaphase cells (D). Relative intensities of band were quantified by image processing software (C; Image Studio ver5.0). (E) Seventy-two hours after siRNA transfection, HeLa cells were fixed with MeOH and stained with antibodies as indicated. Images, which were acquired under a constant exposure time, are maximum projections from Z-stacks of representative cells. The intensity of DDA3 was quantified and plotted (n = 10 cells for each quantification). (F) HeLa cells were transfected with siRNA and prepared as in Figure 1E. For each quantification, the intensities of spindle and Kif2a were determined based on the immunofluorescence from 10 cells. (G) Forty-eight hours after siRNA transfection, HeLa cells were transfected with either the EV or HA-c-Cbl plasmid. The cells were fixed in MeOH and stained with β-tubulin antibody at 28 h after plasmid transfection. Images for β-tubulin were acquired under a constant exposure time and spindle intensity was quantified (n = 10 cells for each quantification). EV, empty vector; AU, arbitrary units. Data are represented as mean ± SEM. Scale bars = 5 μm. *P < 0.01.
Fig. 3
Fig. 3. c-Cbl controls spindle stability and spindle attachment to the kinetochore
(A and B) Seventy-two hours after siRNA transfection, HeLa cells were treated with 1 μg/ml nocodazole for 3 min, fixed in MeOH, and analyzed by immunofluorescence staining of β-tubulin. (C and D) After spindle disruption by treatment with 1 μg/ml nocodazole for 10 min, c-Cbl-depleted cells were placed in fresh media for 6 min and stained with β-tubulin antibody. The spindle intensity was quantified and plotted (n = 10 for each quantification). (E–H) Seventy-two hours after siRNA transfection, HeLa cells were stained with the indicated antibodies. The ratio of Mad2/Hec1 (F) or BubR1/CREST (H) ratios were quantified and plotted (n = 100 kinetochores from 10 cells for each quantification). AU, arbitrary units. Data are represented as mean ± SEM. Scale bars = 5 μm. *P < 0.01.
Fig. 4
Fig. 4. c-Cbl is involved in centriole duplication
(A–C) HeLa cells were transfected with a siControl or siCbl for 72 h. The cells were cotreated with a separase siRNA (siSeperase) for 72 h or 100 nM BI 2536, as a Plk1 inhibitor, for 2 h. Images are maximum projections from Z-stacks of representative cells stained for Centrin-1 (green), β-tubulin (red), and DNA (blue). The level of proteins were analyzed by western blotting using the indicated antibodies (B). Metaphase cells with multipolar spindles or pseudobipolar spindles were quantified and plotted (C; n = 100 cells for each quantification). (D) Seventy-two hours after siRNA transfection, HeLa cells were fixed in MeOH and stained for Centrin-1 (green), C-NAP1 (red), and DNA (blue). G2 cells with multiple or disengaged centrioles were quantified and plotted (n = 100 cells for each quantification). (E) HeLa cells were transfected with siRNAs and prepared as in Figure 2D. Metaphase cells with multipolar spindles were quantified and plotted (n = 100 cells for each quantification). (F and G) HeLa cells were transfected with a siControl or siCbl. The cells were fixed in MeOH and stained with antibodies as indicated. The Sas6, STIL, and PLK4 intensities were quantified and plotted (n = 100 centrioles for each quantification). Alternatively, the cells were harvested at 72 h post-transfection and lysates were analyzed by western blotting using the indicated antibodies (G). (H) Seventy-two hours after siRNA transfection, the mRNA levels of c-Cbl and DDA3 in nocodazole-treated mitotic cells were quantified by RT-qPCR analysis (n = 3). AU, arbitrary units. Data are represented as mean ± SEM. Scale bars = 5 μm. *P < 0.01.

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References

    1. Arquint C., Nigg E.A. The PLK4-STIL-SAS-6 module at the core of centriole duplication. Biochem Soc Trans. 2016;44:1253–1263. doi: 10.1042/BST20160116. - DOI - PMC - PubMed
    1. Castro A., Bernis C., Vigneron S., Labbe J.C., Lorca T. The anaphase-promoting complex: a key factor in the regulation of cell cycle. Oncogene. 2005;24:314–325. doi: 10.1038/sj.onc.1207973. - DOI - PubMed
    1. Cheeseman I.M., Niessen S., Anderson S., Hyndman F., Yates J.R., 3rd, Oegema K., Desai A. A conserved protein network controls assembly of the outer kinetochore and its ability to sustain tension. Genes Dev. 2004;18:2255–2268. doi: 10.1101/gad.1234104. - DOI - PMC - PubMed
    1. Choi Y.H., Han Y., Lee S.H., Jin Y.H., Bahn M., Hur K.C., Yeo C.Y., Lee K.Y. Cbl-b and c-Cbl negatively regulate osteoblast differentiation by enhancing ubiquitination and degradation of Osterix. Bone. 2015;75:201–209. doi: 10.1016/j.bone.2015.02.026. - DOI - PubMed
    1. Dulic V., Lees E., Reed S.I. Association of human cyclin E with a periodic G1-S phase protein kinase. Science. 1992;257:1958–1961. doi: 10.1126/science.1329201. - DOI - PubMed
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