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. 2011 Apr 15;10(8):1322-30.
doi: 10.4161/cc.10.8.15372. Epub 2011 Apr 15.

βTrCP regulates BMI1 protein turnover via ubiquitination and degradation

Anagh A Sahasrabuddhe  1 , Manjari Dimri, Prashant V Bommi, Goberdhan P Dimri
Affiliations

βTrCP regulates BMI1 protein turnover via ubiquitination and degradation

Anagh A Sahasrabuddhe et al. Cell Cycle. .

Abstract

The polycomb group protein BMI1 has been linked to proliferation, senescence, cancer progression and stem cell phenotype. At present, very little is known about its regulation. Here, we report that BMI1 contains a functional recognition motif for the F box protein βTrCP, which regulates ubiquitination and proteasome-mediated degradation of various proteins. We show that overexpression of wild-type βTrCP but not the ΔF mutant of it promotes BMI1 ubiquitination and degradation, and knockdown of βTrCP results in increased expression of BMI1. Furthermore, a mutant of BMI1 with an altered βTrCP recognition motif is much more stable than wild-type BMI1. We also show that wild-type BMI1 but not the mutant BMI1 interacts with βTrCP. Accordingly, compared to wild-type BMI1, mutant protein exhibited increased pro-oncogenic activity. In summary, our findings suggest that βTrCP regulates turnover of BMI1 and its function relevant to oncogenesis, cellular senescence and aging.

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Figures

Figure 1
Figure 1
BMI1 is postranslationally regulated by ubiquitin proteasome system. (A) MCF10A-BMI1 cells were treated with CHX for the indicated time points and the immunoblot (IB) of BMI1 and β-actin (loading control) was performed (left part). The densitometric quantification of BMI1 normalized to β-actin was plotted against various time points to determine its half life (right parts). For densitometric analysis the light exposure of the blot was chosen in each case. The half life of BMI1 was also determined after further treating CHX-treated extracts with CIP, and performing western blot analysis and densitometry of BMI1 signal present at each time point (bottom part). (B) 26S-Proteasomal machinery orchestrates BMI1 proteolysis. Asynchronously growing MCF10A-B0 and MCF10A-BMI1 cells were treated with MG132 (10 µM), and lysosomal inhibitor Chloroquine (25 µM) as indicated. BMI1 was detected by western blot analysis. The relative expression (Rel. exp.) of BMI1 was determined by densitometric quantification of BMI1 bands normalized to α-tubulin levels. (C) SCF complex targets BMI1 for degradation. U2OS cells transiently transfected with ΔCul 1 FLAG plasmid as well as GFP-tubulin as transfection control. Cell lysates were analyzed after 48 hr. Densitometric values (relative expression) were obtained after BMI1 levels were normalized with the GFP-tubulin levels.
Figure 2
Figure 2
βTrCP, an F box component of SCF complex regulates BMI1 proteolysis and BMI1-mediated H2A K119Ub activity of PRC1. (A) Sequence alignment of putative βTrCP degron site of BMI1 with known βTrCP degron sites of CDC25A, MCL1, Snail and β catenin (left part), and sequence alignment of βTrCP degron site of BMI1 from various mammalian species (right part). (B) Knockdown of βTrCP stabilizes BMI1. U2OS cells stably overexpressing HA tagged βTrCP were transiently transfected with shRNAs against βTrCP. After 48 hr, cells were harvested and βTrCP, BMI1 and α-tubulin were analyzed by western blot analysis. The quantitative densitometric values were obtained after BMI1 and TrCP HA levels were normalized to α-tubulin levels. (C) MCF10A cells were stably transfected with Vector (B0), HA-tagged TrCP full length protein (TrCP WT-HA) and F box deletion mutant of the TrCP (TrCPΔF-HA). After stable selection of cells, TrCP (HA), BMI1 and β-actin levels were analyzed by western blot analysis. The relative levels of BMI1 were determined by densitometric analysis. (D) The levels of BMI1 and H2A K119Ub were examined in vector control and TrCPΔF-HA expressing MCF10A cells (as indicated) by western blot analysis (left part). Similarly, the expression of BMI1, and H2A K119Ub levels were determined in control (Conti) and βTrCPi expressing 293T cells. 293T cells were transiently transfected with either 8 µg of conti-1 plasmid or with 4 µg each of TrCPi-1 and TrCPi-2 plasmids together with 100 ng of GFP-tubulin as a transfection efficiency control. Total cell lysates or total histones were prepared and analyzed by western blot analysis using respective antibodies.
Figure 3
Figure 3
βTrCP promotes BMI1 ubiquitination and βTrCP degron mutant of BMI1 is expressed at higher levels. (A) Whole cell extracts (WCEs) from MCF10A-derived B0, TrCPWT-HA and TrCPΔF-HA cells were subjected to immunoprecipitation (IP) with anti-BMI1 antibody and analyzed by immunoblotting for the indicated proteins. (B) Expression levels of wild-type and mutant (βTrCP degron site mutant) BMI1 proteins in U2OS and MCF10A cells were determined by the western blot analysis. Relative expression of wild-type and mutant BMI1 were determined by densitometry and normalized to control α-tubulin levels. The expression level of wild-type BMI1 was considered 1. The expression level of endogenous BMI1 was not relevant and thus not determined (ND). (C) Whole cell extracts (WCEs) from MCF10A derived BMI1 WT-FLAG and BMI1 Mut-FLAG cells were subjected to IP with anti-FLAG antibody in the presence of solvent control or MG132 and analyzed by immunoblotting for ubiquitinated BMI1 and FLAG (input). (D) U2OS cells stably overexpressing wild-type BMI1 and either TrCPWT HA or TrCPΔF HA were treated with 10 µM MG132 for 5 hr. WCEs were subjected to IP with anti-HA and anti-BMI1 antibodies and analyzed by immunoblotting for the indicated proteins. (E) U2OS cells stably overexpressing TrCPWT-HA and BMI1 WT-FLAG or BMI1 Mut-FLAG were treated with 10 µM MG132 for 5 hr. WCEs were subjected to IP with anti-HA and anti-Flag antibodies and analyzed by immunoblotting for the indicated proteins.
Figure 4
Figure 4
Mutant BMI1 exhibits increased pro-oncogenic activities. (A) MCF10A cells overexpressing BMI1 WT and BMI1 Mut along with B0 control were growth factor deprived using D3 medium for 72 hr and stimulated for 30 min by addition of D medium containing 12.5 ng/ml epidermal growth factor (EGF). Western blot analysis of phospho-AKT, total AKT (AKT1 and AKT2), phospho-ERK, total ERK, phospho-GSK and total GSK in MCF10A B0 and MCF10A-derived cells (as indicated) was done and densitometric quantifications were done for each phosphorylated signaling intermediate after normalizing it with the levels of their respective total proteins. (B) Short term proliferation assay of MCF10A cells stably overexpressing BMI1 WT and BMI1 Mut along with B0 (vector) control. (C) Invasive potential of MCF 10A-derived B0 (vector), BMI1 WT and BMI1 Mut cells towards EGF was determined as described in Materials and Methods. Columns indicate mean of triplicates and bars indicate ±SD. (D) The migration potential of MCF10A-derived B0, BMI1 WT and BMI1 Mut was determined by wound-healing assay as described in the Materials and Methods. The values were plotted as indicated (right part).
Figure 5
Figure 5
Mutant BMI1 promotes proliferation and bypasses senescence more efficiently than the wild-type BMI1. (A) IMR90-derived B0, BMI1 WT and BMI1 Mut cells were analyzed for the expression of H2A K119Ub and total H2A by western blot analysis. (B) ChIP analysis of BMI1 binding to p16INK4a promoter. Left part shows ethidium bromide stained agarose gel picture showing the amplified product of p16INK4a after ChIP by anti-BMI1 antibody or control IgG as indicated. The values obtained from qPCR performed in triplicates were plotted as indicated (right part). (C) Relative mRNA levels of p16INK4a were analyzed by qRT PCR from IMR90-derived B0, BMI1 WT and BMI1 Mut cells as indicated. (D) IMR90-derived B0, BMI1 WT and BMI1 Mut cells were analyzed by western blot analysis for expression of BMI1, p21, p53, p16, pRb and β-actin (loading control). The densitometric values were obtained after normalizing the value of the indicated proteins with β-actin. *For determining the relative expression of wild-type and mutant BMI1, only two lower bands of BMI1 were considered as the mutant BMI1 lacks slow migrating top band(s). The expression level of wild-type BMI1 was considered 1. (E) Short term proliferation rate of IMR90 cells stably overexpressing BMI1 WT and BMI1 Mut along with B0 (vector) control was determined in triplicates as described in the Materials and Methods. (F) SA-β-gal assay was performed on IMR90-derived B0, BMI1 WT and BMI1 Mut cells. The left part shows the staining pictures taken at 10× magnification. The % senescence (fraction of SA-β-gal positive cells) was plotted as indicated (right part).
See this image and copyright information in PMC

Comment in

  • BMI1: a target of βTrCP.
    Raychaudhuri P. Raychaudhuri P. Cell Cycle. 2011 Jun 15;10(12):1893-4. doi: 10.4161/cc.10.12.15691. Epub 2011 Jun 15. Cell Cycle. 2011. PMID: 21577051 No abstract available.
  • BMI1 suffers a degrading experience.
    Hoenerhoff M, Chu IM, Green JE. Hoenerhoff M, et al. Cell Cycle. 2011 Jun 15;10(12):1894-5. doi: 10.4161/cc.10.12.15688. Epub 2011 Jun 15. Cell Cycle. 2011. PMID: 21593587 Free PMC article. No abstract available.

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