Figure 4. USP28 is catalytically required for p53 stabilization during centrosome loss-induced G1 arrest.
(A) Organization of the conserved catalytic domains in USP28. UCH-1 (Cys box, amino acids 162–196) and UCH-2 (His box, amino acids 580–649). (B) Catalytic-inactive USP28CI cannot rescue the G1 arrest in PLK4as; USP28-/- cells after centrosome removal. Wild type UPS8 or USP28CI was mildly expressed under the tetracycline inducible promoter in stable, clonal, centrosomal PLK4as; USP28-/- cells (see Materials and methods) during which centrosome loss was induced by 3MBPP1 addition. BrdU was added on day six after 3MBPP1 addition and cells were harvested 24 hr later for BrdU incorporation assay (3MBPP1 treatment for seven days in total). Data are means ± SD. n>150, N = 3. (C) Representative immunofluorescence images of cells in (B) stained with the indicated antibodies seven days after 3MBPP1 treatment. USP28 was stained with anti-HA antibody. Scale bar, 5 μm. (D) USP28 deubiquitinates p53 in vitro. Immunoblot of ubiquitinated p53 incubated with or without USP28 in an in vitro deubiquitination assay (see Materials and methods). Note the reduction in the polyubiquitinated form of p53 in the presence of USP28. (E) High levels of USP28 can ectopically stabilize nuclear p53 in the absence of mitotic stress. Immunofluorescence images of cells stained with the indicated antibodies. Expression of wild type USP28 or USP28CI was induced in PLK4as; USP28-/- cells with 10 ng/μl (low level expression) or 100 ng/μl (overexpression) of doxycycline for two days before cell fixing and staining. The percentage in the merged panel indicates the proportion of cells with p53 nuclear accumulation. Scale bar, 5 μm. (F) Nuclear p53 accumulation caused by USP28WT overexpression is not due to increased p53 transcription. Quantification of p53 mRNA levels relative to GAPDH in (E) by qRT-PCR. Data are means ± SD. n = 6 from two independent experiments. (G) USP28 ectopically stabilizes nuclear p53 independently of 53BP1. Immunofluorescence images of cells stained with the indicated antibodies. Wild type USP28 was induced in PLK4as; 53BP1-/- cells with 10 ng/μl (low level expression) or 100 ng/μl (overexpression) of doxycycline for two days. The percentage in the merged panel indicates the proportion of cells with p53 nuclear accumulation. Scale bar, 5 μm. (H) Overexpression of 53BP1 can ectopically stabilize nuclear p53 in the absence of mitotic stress. Immunofluorescence images of cells stained with the indicated antibodies. Expression of wild type 53BP1 or 53BP1ΔBRCT was induced in PLK4as; 53BP1-/- cells with 10 ng/μl (low level expression) or 1 μg/μl (overexpression) of doxycycline for two days before cell fixing and staining. The percentage in the merged panel indicates the proportion of cells with p53 nuclear accumulation. Scale bar, 5 μm. (I) Ectopic stabilization of nuclear p53 by 53BP1 requires USP28. Immunofluorescence images of cells stained with the indicated antibodies. Wild type 53BP1 was induced in PLK4as; USP28-/- cells with 10 ng/μl (low level expression) or 1 μg/μl (overexpression) of doxycycline for two days. The percentage in the merged panel indicates the proportion of cells with p53 nuclear accumulation. Scale bar, 5 μm.
Figure 4—figure supplement 1. USP28WT and USP28CI are exogenously expressed to similar levels in PLK4as; USP28-/- cells.
Figure 4—figure supplement 2. Schematic outlining the timeline of drug treatments used.
