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MDM2 mediates p73 ubiquitination: a new molecular mechanism for suppression of p73 function - PubMed

  • ️Thu Jan 01 2015

MDM2 mediates p73 ubiquitination: a new molecular mechanism for suppression of p73 function

Hong Wu et al. Oncotarget. 2015.

Abstract

The protein p73, a homologue of the tumor suppressor protein p53, is capable of inducing apoptosis and cell cycle arrest. MDM2 is transcriptionally activated by p73 and represses the functions of p73, including p73-dependent transactivation and growth suppression. However, the molecular mechanism of this repression is unknown. In this study, we show that MDM2 mediates p73 ubiquitination. MDM2 mainly utilizes K11, K29 and K63-linked chains to mediate p73 ubiquitination in vivo and in vitro. However, MDM2 is unable to promote p73 degradation in most tested cell lines. Surprisingly, we observe that overexpression of Mdm2 promotes p73 degradation mainly through Itch in Mdm2-null MEFs. We further find that Itch interacts with the transfected Mdm2 in Mdm2-null cells. Moreover, our findings reveal that the E3 ligase activity of MDM2 is required to repress p73-dependent apoptosis and cell cycle arrest but not p73-dependent transcriptional activity. Furthermore, the data suggest a link between p73 ubiquitination/MDM2 E3 ligase activity and p73 biological functions.

Keywords: oncogene; protein degradation; tumor suppressor; ubiquitin E3 ligase; ubiquitination.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. MDM2 promotes p73 ubiquitination in vivo

A. Saos-2 cells were cotransfected with plasmids expressing Flag-p73α and pCMV-Bam-MDM2, MDM2ΔRING, ITCH, or ITCH mutant (C830A) as well as Myc-Ubwt or Myc-Ub4KR as indicated. p73 was immunoprecipitated with a Flag-specific antibody (M5) and analyzed by immunoblotting with a Myc-specific antibody (upper image) and ER-15 for p73 (second image). Direct western blots for MDM2 and ITCH are shown in the lower panels. B. The same procedure as (A) was used, except that Saos-2 cells were cotransfected with a Flag-p73β-expressing plasmid instead of Flag-p73α. C. The same procedure as (A) was used, except that Saos-2 cells were cotransfected with plasmids encoding p73α or p73β and MDM2 along with a number of ubiquitin mutants as indicated. Asterisk (*) indicates the migration position of p73-Ub conjugates. D. Saos-2 cells were transfected with plasmids expressing His-ubiquitin, p73α or p73β, and MDM2 or ITCH. His-ubiquitinated proteins were isolated from denatured whole extract extracts, and analyzed by western blot with a p73 specific antibody (ER-15). Direct western blots for MDM2 and ITCH are shown in the lower panels.

Figure 2
Figure 2. MDM2 is required for p73 ubiquitination in vivo

A. HEK293 cells were transfected with MDM2-siRNA or control-siRNA constructs. Forty hours later, the cells were further transfected with a plasmid expressing HA-Ub. Lysates were immunoprecipitated with a p73specific antibody (ER-15) and analyzed by western blotting with an HA-specific antibody to detect ubiquitinated p73. The western blots for p73, MDM2, and actin are shown in the lower panel. B. The same procedure as (A) was used, except that after the second transfection with the HA-Ub expression plasmid, the cells were treated with the proteasome inhibitor MG132 (20 μM) 6 hr prior to harvest. C. Similar to (A) except that the cell extracts were obtained from the wild-type mouse embryonic fibroblasts (MEFs) and Mdm2 null MEFs. In addition, Mdm2 expression plasmid was reintroduced into Mdm2 null MEFs (lane 3). D. HEK293 cells were immunoprecipitated with anti-p73 (ER-15) or anti-MDM2 (SMP14) as indicated, and immunoblotted with antip73 (upper image, ER-15) and anti-MDM2 antibodies (lower image).

Figure 3
Figure 3. MDM2 is an E3 ligase for p73 in vitro

A. and B. GST-MDM2 was evaluated for its capacity to ubiquitinate purified His-p73α and His-p73β using immunoblotting with anti-Ub to reveal ubiquitinated products (upper image) and an antibody directed to p73 (ER-15) to reveal ubiquitinated p73 species. C. MDM2 preferentially utilizes Ub with Lys residues at Lys11 and Lys29 for p73 ubiquitination in vitro. Affinity-purified GST-MDM2, His-p73α, or His-p73β was added to bacterial extracts containing recombinant E1 and E2 (UbcH5b), Ubwt or ubiquitin mutants. Immunoblotting was performed with anti-Ub to reveal ubiquitinated products (upper image) and anti-p73 (ER-15) to reveal ubiquitinated p73 species (lower image). D. As in (C) but after the ubiquitination reaction, the samples were immunoprecipitated with RIPA buffer and anti-p73 (ER-15) antibody and analyzed by immunoblotting with anti-Ub (upper image), anti-p73 (ER-15, middle image), polyubiquitin-specific FK-1 (third image), and anti-ubiquitin, Lys63-specific and Lys48-specific antibodies (lower image) as indicated. E. Western blot analysis of p73α or p73β ubiquitination was performed with a p73-specific antibody (ER-15, upper image), a polyubiquitinspecific antibody FK-1(lower image), and varying amounts of GST-MDM2 (0.1X: 0.3 ng; 1X: 3 ng; 10X: 30 ng) in the presence of Ubwt or UbKO. E1 and E2 were included in all reactions. Asterisk (*) indicates the migration position of p73-Ub conjugates. F. GST-ITCH, GST-ITCHC830A and GST-ITCHΔHECT were evaluated for E3 activity in the presence of recombinant E1, E2 (UbcH5b), ubiquitin and p73α protein. Following the ubiquitination reaction, the samples were analyzed by western blotting with ubiquitin-specific and p73-specific (ER-15) antibodies.

Figure 4
Figure 4. Overexpression of Mdm2 promotes p73 degradation in Mdm2-null MEFs

A. Mdm2 null MEFs were transfected with plasmids expressing p73α or an empty vector with Myc-tagged Itch, along with a GFP expressing plasmid (pEGFP) and analyzed by western blotting with a p73-specific antibody (H-79), a Myc-specific antibody for Myc-Itch, a GFP-specific antibody (B-2), and actin as a loading control. B. The same procedure as (A) was used, except that Mdm2 null MEFs were cotransfected with plasmids expressing GFP, p73α along with Itch and Mdm2. C. and D. Mdm2-null MEFs were transfected with an Itch-specific siRNA or a control siRNA. Thirty hours later, the cells were transfected with plasmids expressing GFP, Mdm2 and analyzed by western blot using anti-p73, anti-Itch, anti-p53 (Pab421), anti-GFP (B-2), and anti-Mdm2 (MD-219) antibodies as indicated. E. Mdm2-null MEFs were transfected with plasmids expressing GFP and increased amounts of Mdm2 and analyzed by western blotting with p73-specific (H-79), GFP-specific (B-2) and Mdm2-specific (MD-219) antibodies.

Figure 5
Figure 5. Mdm2 interacts with Itch and regulates the stability of p73 in Mdm2-null MEFs

A. Mdm2 interacts with Itch in vivo. Wild type MEFs and Mdm2-null MEFs were transfected with a plasmid expressing Mdm2. The cells were treated with the proteasome inhibitor MG132 (20 μM) for 6 hours prior to harvest. The cell extracts were immunoprecipitated with anti-Mdm2 or anti-Itch antibodies and analyzed by western blot with indicated antibodies. B. Wild type MEFs and Mdm2 null MEFs cells were treated with cycloheximide (CHX) (20 μg/ml) as indicated. Endogenous p73 levels were determined by immunoblotting with a p73-specific antibody (H-79). An antibody against β-actin was used as a loading control. C. Expression levels were determined by densitometry of the immunoblots in (B) Errors bars indicate the SEM (n = 3). D. Mdm2 null MEFs were transfected with plasmids expressing HA-Ub and p73α or Myc-Itch. Cell extracts were immunoprecipitated with a p73-specific antibody (H-79) and analyzed by western blotting with HA-specific, Myc-specific (for Itch), and p73-specific antibodies. E. HEK293 cells were transfected with ITCH-siRNA or control-siRNA. Two days later, the cells were further transfected with plasmids expressing HA-Ub and an MDM2 expression plasmid or empty vector as indicated. Cell extracts were immunoprecipitated with a p73-specific antibody (ER-15) and analyzed by western blotting with HA-specific, p73-specific, Lys63-specific and Lys48-specific antibodies (lower image) as indicated. Direct western blots for p73, ITCH, MDM2 and actin are shown in the lower panel. F. Mdm2 null MEFs were transfected with plasmids expressing His-Ub, p73α or p73β, and Mdm2 or Itch. His-ubiquitinated proteins were isolated from denatured whole extract extracts, and analyzed by western blot with a p73 specific antibody (H-79). Direct western blots for Mdm2 and Itch are shown in the lower panels.

Figure 6
Figure 6. MDM2 inhibits p73-dependent apoptosis and cell cycle arrest but not p73-dependent transcriptional activity

A. H1299 cells were cotransfected with a p21-Luc reporter plasmid and the p73α or p73β expression construct in combination with the MDM2, MDM2ΔRING, ITCH, or ITCH C830A expression constructs or empty vector (pcDNA3.1). The transcriptional activity of p73 is shown, and error bars indicate the SEM (n = 3). p < 0.01 (2-tailed Student t test). B. Western blots of p73α, p73β, ITCH, ITCHC830A, MDM2 and MDM2ΔRING with p73-specific (ER15), Flag-specific for ITCH and ITCHC830A and MDM2-specific antibodies were indicated. C. HEK293 cells were transfected with MDM2-siRNA (siMDM2), ITCH-siRNA (siITCH), control-siRNA, or MDM2-siRNA and ITCH-siRNA. Two day later, the cells were transfected with a p21 Luc reporter plasmid and a p73α expression plasmid. The transcriptional activity of p73 is shown, and error bars indicate the SEM (n = 3). p < 0.01 (2-tailed Student t test). D. H1299 cells were cotransfected with a CD20 expression construct, pcDNA3-p73α, or p73β (3 μg) and pcDNA3-MDM2 (15 μg), pcDNA3-MDM2ΔRING (15 μg), or pcDNA3-MDM2Δ222–303 as indicated. The inhibitory effect of MDM2 on p73-dependent apoptosis was determined by annexin V staining of CD20-positive cells and flow cytometry. Error bars indicate the SEM. (n = 3). p < 0.01 (2-tailed Student t test). E. H1299 cells were transfected with MDM2-siRNA or control-siRNA for 30 h. The cells were then transfected with the p53, p73α, or p73β expression constructs and CD20 expression plasmids. The number of surviving CD20-positive cells was measured by flow cytometry 24 hr after transfection. p < 0.01 (2-tailed Student t test). F. Human bladder carcinoma EJ cells were transfected with p73α alone or with MDM2, MDM2ΔRING, or empty vector (pcDNA3). The cell cycle profile was determined by propidium iodide staining and flow cytometry. The results represent the average of triplicate experiments.

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