FBXW7 is involved in Aurora B degradation

Abstract

FBXW7, a component of E3 ubiquitin ligase, plays an important role in mitotic checkpoint, but its role remains unclear. Aurora B is a mitotic checkpoint kinase that plays a pivotal role in mitosis by ensuring correct chromosome segregation and normal progression through mitosis. Whether Aurora B and FBXW7 are coordinately regulated during mitosis is not known. Here, we show that FBXW7 is a negative regulator for Aurora B. Ectopic expression of FBXW7 can suppress the expression of Aurora B. Accordingly, FBXW7 deficiency leads to Aurora B elevation. Mechanistic studies show that all FBXW7 isoforms are negative regulators of Aurora B expression through ubiquitination-mediated protein degradation. Aurora B interacts with R465 and R505 residues of WD 40 domain of FBXW7. Significantly, inverse correlation between FBXW7 and Aurora B elevation is translated into the deregulation of mitosis. FBWX7 expression mitigates Aurora B-mediated cell growth and mitotic deregulation. In addition, FBXW7 reduces the percentage of multinucleated cells caused by Aurora B overexpression. These data suggest that FBXW7 is an important negative regulator of Aurora B, and that the loss or mutation of FBXW7 as seen in many types of cancer could lead to an abnormal elevation of Aurora B and result in deregulated mitosis, which accelerates cancer cell growth.

Figure 1. FBXW7 regulates Aurora B stability. (A) FBXW7 negatively regulates the steady-state expression of Aurora B. 293T cells were co-transfected with the indicated plasmids and increasing amounts of FBXW7. Equal amounts of cell lysates were immunoblotted with the indicated antibodies. (B) FBXW7-mediated degradation of Aurora B is proteasome-dependent. 293T cells were co-transfected with the indicated plasmids. Cells were treated with MG132 for 6 h before harvesting. Equal amounts of cell lysates were immunoblotted with the indicated antibodies. (C) Aurora B turnover rate is increased in FBXW7 expressing cells. 293T cells transfected with the indicated plasmids were treated with cycloheximide (CHX) (100 µg/ml) for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. Integrated OD values of GFP-Aurora B bands at each time point were measured using a densitometer. Levels of GFP-Aurora B at time 0 were set at 100%. Remaining GFP-Aurora B is indicated graphically (right).

Figure 2. FBXW7 isoforms increase Aurora B turnover via increasing Aurora B ubiquitination. (A) Different FBXW7 isoforms regulate the steady-state expression of Aurora B in a dose-dependent manner. Lysates of 293T cells transfected with increasing amounts of the indicated FBXW7 isoforms and GFP-Aurora B were immunoblotted with the indicated antibodies. (B) FBXW7 downregulates Aurora B by increasing Aurora B ubiquitination. 293T cells were transfected with the indicated plasmids. The proteasome inhibitor MG132 was added 6 h prior to cell harvesting. The amount of ubiquitinated Aurora B was analyzed by immunoprecipitated (IP) with anti-Aurora B followed by immunoblotting (IB) with anti-His. (C) FBXW7α dereases the turnover of Aurora B. Indicated 293T cells were transfected with GFP-Aurora B with or without FBXW7 and then treated with cycloheximide (CHX) (100 µg/ml) for the indicated times. Signals of GFP-Aurora B from cells were observed under microscope.

Figure 3. FBXW7 deficiency leads to Aurora B stabilization. (A) FBXW7 deficiency increases the steady-state expression of Aurora B. Indicated wt and FBXW7−/− HCT116 cells were immunoblotted with anti-Aurora B or actin antibodies. (B) Aurora B stability regulation is proteasome-dependent. Indicated wt and FBXW7−/− HCT116 cells were treated with MG132 6 h before harvesting. Equal amounts of cell lysates were immunoblotted with anti-Aurora B or Actin antibodies. (C) Aurora B turnover rate is reduced in FBXW7 deficient cells. Indicated wt and FBXW7−/− HCT116 cells were treated with cycloheximide (CHX) (100 µg/ml) for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. Integrated OD values of bands at each time point were measured using a densitometer. Levels of Aurora B at time 0 were set at 100%. Remaining Aurora B is indicated graphically (right).

Figure 4. FBXW7 interacts with Aurora B through WD 40 repeat. (A) Endogenous assciation between FBXW7 and Aurora B. 293T cells were immunoprecipitated with indicated antibodies or IgG and immunoblotted with anti-Aurora B in the presence or absence of MG132. (B) Interaction of FBXW7 isoforms with Aurora B. Equal amounts of 293T cell lysates transfected with indicated FBXW7 isoforms were immunoprecipitated with anti-Aurora B and immunoblotted with anti-Flag. (C) Mapping of Aurora B binding domains on FBXW7. Indicated Flag-FBXW7α or C terminus FBXW7 (DN-FBXW7α) was cotransfected with Aurora B into 293T cells. Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-Aurora B. (D) Mapping of Aurora B binding sites on FBXW7α. Indicated Flag-DN FBXW7α or DN FBXW7α mutants were cotransfected with GFP-Aurora B into 293T cells. Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with the indicated antibodies. Schematic representation of the mutants is shown.

Figure 5. FBXW7 mitigates Aurora B-mediated cell proliferation and reduces multi-nuclei formation. (A) Aurora B promotes cell proliferation. Cells transfected with Aurora B were estimated by MTT assay every day for a total of 4 d. Results are expressed as an OD570. (B) FBXW7 inhibits Aurora B-mediated cell proliferation. Cell cotransfected with Aurora B and FBXW7 were estimated by MTT assay every day for a total of 4 d. Results are expressed as an OD570. (C) FBXW7 inhibits Aurora B-mediated increased DNA content during cell cycle. Cells cotransfected with Aurora B and FBXW7 were analyzed by FCCS. DNA histogram was investigated by FLOWJO program. PE-A indicates the DNA content. % of max is the % of cell analyzed. (D) FBXW7 antagonizes Aurora B-induced multinucleate cell formation. 293T cells were transfected with GFP-Aurora B or GFP-Aurora B + FBXW7 (in a plasmid ratio of 1:3) for 48 h. Phase-contrast images and the corresponding fluorescent micrographs are shown. DNA was stained with DAPI and was pseudocolored red. GFP-Aurora B-expressing cells are circled in the color-merged micrographs. Note that GFP-Aurora B cells cotransfected with FBXW7 exhibited less multinucleation than GFP-Aurora B cells. Random areas of the green cells were counted, and % multinucleate cells were plotted. Error bars: 95% confidence.

Figure 6. FBXW7 deficiency leads to polyploidy. Indicated wt and FBXW7−/− HCT116 cells were synchronized to the G₂→M phase by nocodazole. Cell samples, at labeled time points after release from nocodazole block, were stained with propidium iodide (PI) and analyzed by FACS for DNA content. DNA content histograms are shown for the time points as labeled.

Figure 7. FBXW7-Aurora B axis is involved in the regulation of p53 stability and mitotic progression. (A) Aurora B decreased the steady-state protein level of p53. H1299 were transfected with the indicated plasmids. Cell lysates were immunoblotted with the indicated antibodies. (B) Enrichment score graph and Ranked list metric graph showing the upregulation of p53-suppressed target genes in Aurora B-highly expressing patients (Gene Set Enrichment Analysis, Broad Institute, MIT). Cohort: GSE-20194 consisting of 255 untreated stage I–stage III breast cancer patients at MD Anderson Cancer Center. (C) Heat map from transcriptomics analysis showing that p53-suppressed target genes were upregulated in Aurora B-highly expressing breast cancer patients. Cohort: GSE-20194 consisting of 255 untreated stage I–stage III breast cancer patients at MD Anderson Cancer Center. (D) Aurora B inhibition leads to multi-nucleated cells. MCF7 cells were treated with AZD1152-HQPA for 24 h and stained with DAPI. (E) Model of the FBXW7-Aurora B axis in regulating p53 and mitosis.