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. Author manuscript; available in PMC: 2013 Apr 24.
Published in final edited form as: FEBS Lett. 2012 Mar 23;586(8):1135–1140. doi: 10.1016/j.febslet.2012.03.022

An autoregulatory feedback loop between Mdm2 and SHP that fine tunes Mdm2 and SHP stability

Zhihong Yang 1, Li Wang 1,*
PMCID: PMC3350641  NIHMSID: NIHMS366256  PMID: 22575647

Abstract

Mdm2 is a crucial negative regulator of the tumor suppressor function of p53. However, little is known about Mdm2 protein stability regulation by other tumor suppressors. Nuclear receptor small heterodimer partner (SHP, NROB2) functions as a tumor suppressor in liver cancer. We show here a surprising finding of a feedback regulatory loop between SHP and Mdm2. SHP stabilizes Mdm2 protein by abrogating Mdm2 self-ubiquitination, and Mdm2 in turn attenuates SHP protein levels under p53-deficient conditions. Such cross-regulation critically depends on the physical interaction of SHP with Mdm2 through the SHP K170 residue. The Mdm2-SHP interplay represents a novel component of Mdm2 signaling that is likely to dictate Mdm2 activity and function.

Keywords: nuclear receptor, small heterodimer partner, protein stability, Mdm2

1. Introduction

Small heterodimer partner (SHP, NROB2), a unique member of the nuclear receptor superfamily, is a pleiotropic transcriptional regulator of genes involved in various metabolic diseases [1–17]. Our recent studies also identified SHP as a critical regulator of microRNA expression and function [18–25]. Interestingly, we observed spontaneous hepatocellular carcinoma (HCC) formation in SHP−/− mice due to hepatocyte hyperproliferation [26] and decreased apoptosis [27]. SHP expression was also diminished in human HCC by epigenetic silencing of the SHP promoter [28], suggesting that SHP is a good prognostic factor in HCC [29]. Importantly, SHP functions as a transcriptional repressor of DNA methyltranferase Dnmt1 expression via ERRγ [30] and zinc/MTF-1 mediated regulation [31]. Despite these important findings, in particular the novel tumor suppressor function of SHP in liver cancer, it is poorly understood how SHP regulates carginogenesis through cross-interaction with other tumor suppressors or oncogenes.

Mdm2 is a well characterized negative regulator of the tumor suppressor p53 [32]. The direct binding of Mdm2 to p53 inhibits the transcriptional activation function of p53 [33]. Mdm2 targets p53 for proteasomal degradation through Mdm2 E3 ubiquitin ligase activity [34]. Phosphorylation of Mdm2 inhibits its E3 ligase activity and prevents the ability of Mdm2 to repress p53 [35, 36]. Mdm2 also suppresses p53 translation by interacting directly with p53 mRNA [37], or by targeting ribosomal protein RPL26 for degradation [38]. Mdm2 is overexpressed in a variety of human tumors, consistent with a role as an oncogene [39]. However, growing evidence suggests Mdm2 as a tumor suppressor under specific physiological conditions. Loss of Mdm2 in a p53 mutant mouse model enhanced tumor formation [40]. Mdm2 facilitates nuclear export of p53 [41], thereby activating the p53-mediated mitochondrial apoptotic pathway [42] and contributing to the tumor suppressor activity of p53 [43]. These observations suggest that Mdm2 may play as a double-edged sword in tumorigenesis.

Currently, little is known about Mdm2 protein stability regulation by other tumor suppressors and how this may contribute to its tumor suppressor function under certain cellular contexts. As a potential additional layer of regulation of protein stability, the present study explored the crosstalk between SHP and Mdm2. Demonstration of a feedback loop between SHP and Mdm2 reveals a fine tuned interplay of SHP and Mdm2 proteins. The findings provide the first evidence that Mdm2 protein stability is enhanced by a nuclear transcriptional repressor.

2. Materials and Methods

2.1 Plasmids and reagents

Plasmids were obtained from Drs. Xiongbin Lu (HA-Mdm2) [44], Takeshi Imamura (HA-Ub) [45], Yang Shi (Myc-Ub) [46], and Moshe Oren (Mdm2ΔR, Mdm2ΔA) [38]. Other plasmids were from our laboratory [16] or purchased from Addgene. Antibodies against human MDM2, HA-tag, and β-actin were purchased from Sigma. M2 antibody, anti-FLAG M2 Magnetic Beads and anti-HA agarose were purchased from Sigma. Mouse monoclonal antibody against Myc-tag was purchased from Cell Signaling. MagneGST™ pull-down kit was obtained from Promega.

2.2 Cell culture and transfection

HepG2, Hela, HEK293T, and HCT116 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum. Transfection was performed by Lipofectamine 2000 according to the manufacturer’s instructions.

2.3 Co-immunoprecipitation

HEK293T cells were transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen), lysed in 500 μl lysis buffer and immunoprecipitated with anti-FLAG M2 Magnetic Beads (Sigma) for 4 hr at 4°C. The beads were washed four times with the lysis buffer. The bound proteins were separated by SDS-PAGE, followed by Western blotting with the indicated antibodies according to the standard procedures.

2.4 Ubiquitination assay

As described previously, FLAG-tagged SHP or HA-tagged Mdm2 was transfected with HA-ubi or myc-ubi into HEK293T cells for 24 hr, then treated with 5 mM MG132 (Cayman) for additional 6 hr. Cells were harvested and lysed in 500 ul of lysis buffer and immunoprecipitated with anti-FLAG M2 magnetic beads (Sigma) for 4 hr at 4°C. The beads were washed three times with the lysis buffer and analyzed with anti-HA, anti-FLAG or anti-myc antibody by Western blots.

2.5 Statistical analysis

Data are expressed as mean ± SEM. Statistical analyses were carried out using Student’s unpaired t test; p < 0.01 was considered statistically significant.

3. Results

SHP drastically increases endogenous Mdm2 protein expression

To identify a cell model that has abundant endogenous Mdm2 and SHP expression, we examined several cell lines and found HepG2 cells express high levels of Mdm2 (Fig. 1A). HepG2 cells also express abundant SHP, as reported in our earlier studies [28], thus were used for examining changes of endogenous Mdm2 by SHP overexpression and knockdown.

Fig. 1.

Fig. 1

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SHP drastically increases endogenous Mdm2 protein expression. (A) Western blots to determine both endogenously (Endo-Mdm2) and exogenously (HA-Mdm2) expressed Mdm2 protein in different cell lines using anti-Mdm2 antibodies. HepG2 cells showed the highest levels of Mdm2 protein than other cell lines. (B–C) Western blots to determine the effect of overexpession (B) or knockdown (C) SHP on endogenous Mdm2 protein expression in HepG2 cells. Ade, adenoviruses. Mdm2, SHP (both Flag-SHP and Endo-SHP) and p53 proteins were detected using anti-Mdm2, anti-SHP and anti-p53 antibodies, respectively.

Introducing a wild-type (WT) SHP expression plasmid in HepG2 cells markedly increased endogenous Mdm2 protein, as examined by a specific Mdm2 antibody (Fig. 1B). Due to the short exposure time, Mdm2 protein was not detected in SHP non-transfected cells. In contrast, knockdown of SHP using SHP siRNA adeniviruses reduced Mdm2 protein, which was accompanied by an induction of p53 protein (Fig. 1C). The results suggest that SHP positively regulates endogenous Mdm2 protein expression.

SHP markedly stabilizes exogenously expressed Mdm2 protein

We next tested whether SHP could affect exogenously expressed Mdm2 protein levels. Hela cells were used because of several advantages, including expressing low endogenous Mdm2, having higher transfection efficiency, and having been successfully expressed with a HA-tagged Mdm2 protein (Fig. 1A).

The SHPWT, as well as several naturally occurring human SHP mutations SHPK170N, SHPR38H, and SHPG171A [16], were examined. Overexpression of SHPWT, SHPK170N, SHPR38H, and SHPG171A increased the HA-Mdm2 protein in a dose-dependent fashion (Fig. 2A–2B). For a better comparison of the efficacy of individual SHP mutant, we conducted Western blots in a single gel. Interestingly, SHPK170N had the lowest expression due to its increased ubiquitination [16], however, it showed the strongest ability to increase Mdm2 protein, whereas SHPR38H and SHPG171A exhibited weaker effects than SHPWT (Fig. 2C).

Fig. 2.

Fig. 2

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SHP markedly stabilizes exogenously expressed Mdm2 protein. (A–C) Western blots to determine the effects of SHPWT (wild type), SHPK170N, SHPR38H, or SHPG171A on exogenously expressed HA-Mdm2 protein expression in Hela cells. Mdm2 and SHP proteins were detected using anti-HA and anti-Flag antibodies, respectively. (D) Western blots to determine the effect of SHP on Mdm2 protein half-life in the presence of protein synthesis inhibitor cycloheximide (CHX).

To address the question that the induction of Mdm2 protein by SHP is not due to increased Mdm2 synthesis, we treated the cells with protein synthesis inhibitor cycloheximide (CHX). The half-life of Mdm2 protein was significantly increased in SHP co-expressed cells compared with cells without SHP expression (Fig. 2D), the later showed barely detectable Mdm2 protein because of short exposure time.

Overall, the results in Fig. 2 suggest that SHP stabilizes exogenously expressed Mdm2 protein, likely through slowing down Mdm2 degradation.

SHP and Mdm2 interaction via SHPK170 is important to protect Mdm2 from self-ubiquitination

Because Mdm2 undergoes self-ubiquitination and degradation [32, 36], it is postulated that SHP may affect Mdm2 protein stability by modulating its ubiquitination. In vitro ubiquitination assays were performed in 293T cells that produce suitable amounts of ectopically expressed proteins for Co-IP experiments.

In the presence of SHP co-expression, the ubiquitinated Mdm2 was markedly reduced (Fig. 3A). Because SHPK170N exhibited the highest potency to increase Mdm2 protein (Fig. 2C), we mutated K170 to a positively charged Arg (K170R) to further examine the effect of SHP on Mdm2 protein stability. Interestingly, SHPK170R failed to inhibit Mdm2 ubiquitination (Fig. 3B), which correlated with its lack of effect to increase Mdm2 protein expression (Fig. 3C).

Fig. 3.

Fig. 3

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Overexpression of SHP protects Mdm2 from self-ubiquitination. (A–B) In vitro ubiquitination assays to determine the effect of SHPWT (A) or SHPK170R (B) on Mdm2 protein ubiquitination (indicated by a solid line) in 293T cells. (B) Western blots to determine the effect of SHP170R on HA-Mdm2 protein expression. Mdm2 and SHP proteins were detected using anti-HA and anti-Flag antibodies, respectively. (C) Immunoprecipitation and Western blots to determine the association of SHPWT, SHPK170N and SHPK170R proteins with the Mdm2 protein. Anti-HA antibody was used to IP Mdm2, and anti-Flag or anti-HA antibodies were used to detect SHP or Mdm2 in Western blots, respectively.

Co-IP and Western blots revealed a weak binding between SHPWT and Mdm2, which was markedly enhanced by the K170N mutation, but abolished by the K170R mutation (Fig. 3D). The results suggest that the interaction of SHP and Mdm2 is important for SHP regulation of Mdm2 self-ubiquitination and that the SHP K170 residue plays a critical role for SHP interaction with Mdm2.

The in vivo interaction of SHP with Mdm2 is independent of p53

Next, HepG2 cells were used to determine whether SHP and Mdm2 proteins were directly associated with each other in vivo in cells using anti-Mdm2 and anti-SHP antibodies. The endogenous SHP protein was immunoprecipitated with Mdm2 protein in HepG2 cells (Fig. 4A). Interestingly, we also found SHP was immunoprecipitated with the p53 protein. To further define the nature of the protein-protein interaction between SHP and Mdm2, we used p53 deficient cells. SHP exhibited a similar ability to interact with Mdm2 in HCT116p53−/− cells as compared to the HCT116p53+/+ cells (Fig. 4B). Our data demonstrate that the SHP/Mdm2 interaction is independent of the presence of p53.

Fig. 4.

Fig. 4

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The in vivo interaction of SHP with Mdm2 is independent of p53. (A) Immunoprecipitation and Western blots to determine the in vivo association of SHP with Mdm2 or p53 in HepG2 cells. Anti-SHP antibody was used to IP SHP, and anti-Mdm2, anti-SHP or anti-p53 antibodies were used to detect Mdm2, SHP or p53 in Western blots, respectively. (B) Immunoprecipitation and Western blots to determine the in vivo association of SHP with Mdm2 or p53 in HCT116p53+/+ and HCT116p53−/− cells. Anti-SHP antibody was used to IP SHP, and anti-Mdm2, anti-p53 or anti-SHP antibodies were used to detect Mdm2, p53 or SHP in Western blots, respectively.

p53-deficiency augments SHP degradation by Mdm2

Because Mdm2 has intrinsic E3 ligase activity and regulates the turnover of numerous signaling proteins, we characterized the cross-regulation between Mdm2 and SHP in HCT116p53+/+ and HCT116p53−/− cells using anti-Mdm2 and anti-SHP antibodies. Mdm2ΔR (C-terminal RING domain deletion) and Mdm2ΔA (acidic domain deletion) [38] were used for comparison. Mdm2ΔR lacks intrinsic E3 ligase activity and self-ubiquitination, and thus was expressed at much higher levels than Mdm2ΔA and Mdm2 in HCT116p53−/− cells (Fig. 5A, row 1, lane 2 vs. 1&3) and HCT116p53+/+ cells (row 1, lane 2′ vs. 1′&3′). Co-expression of SHPWT with Mdm2 significantly elevated Mdm2 to similar levels as Mdm2ΔR in both HCT116p53−/− (row 1, lane 5 vs. 6) and HCT116p53+/+ cells (row 1, lane 5′ vs. 6′). The observation confirms that SHP-mediated Mdm2 protein stability is associated with Mdm2 ubiquitinaton and is also independent of p53.

Fig. 5.

Fig. 5

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The reduction of SHP protein by Mdm2 is enhanced by p53-deficiency. (A) Western blots to determine the effects of Mdm2, Mdm2ΔR or Mdm2ΔA on SHP protein expression in HCT116p53−/− and HCT116p53+/+ cells. The Mdm2, SHP and p53 proteins were detected using anti-Mdm2, anti-SHP or anti-p53 antibodies, respectively. (B) Western blots to determine the effect of Mdm2 on exogenously expressed SHP protein expression in Hela cells. The SHP and Mdm2 proteins were detected using anti-Flag or anti-HA antibodies, respectively. (C) Western blots to determine the effect of Mdm2 on SHP protein half-life in the presence of protein synthesis inhibitor cycloheximide (CHX).

To our surprise, SHP levels were markedly downregulated by Mdm2 co-expression in p53-deficient HCT116p53−/− cells (row 2, lane 5 vs. 4), which was not obvious in HCT116p53+/+ cells (row 2, lane 5′ vs. 4′). Mdm2ΔR and Mdm2ΔA affected SHP turnover to the same extent as its wild-type counterpart in both cells (row 2, lanes 6&7 vs. 5, lane 6′&7′ vs. 5′).

To confirm SHP downregulation by Mdm2, Hela cells, which have no functional p53, were used as an additional cell model. Overexpressing Mdm2 reduced SHP protein levels by about 40% ~ 50% (Fig. 5B). In addition, SHP protein showed a short half-life (Fig. 5C), consistent with early observations [16, 47]. The half-life of SHP protein was shortened from 30 min to 15 min upon Mdm2 overexpression (Fig. 5C). Overall, the results suggest that in the absence of p53, Mdm2 is able to cause rapid SHP degradation.

Mdm2 and p53 bind preferentially to each other rather than to SHP

Thus far, we observed that SHP interacted with both Mdm2 and p53. Mdm2 is known to interact strongly with p53. The next question is whether Mdm2 and p53 binding could be disrupted by SHP. 293T cells were used for Co-IP experiments. The amount of immunoprecipitated Mdm2 associated with SHP was attenuated with increasing amounts of p53 expression, likely due to the enhanced Mdm2 and p53 interaction (Fig. 6A). In addition, the association of SHP with p53 was also decreased by co-expression of Mdm2 due to increased Mdm2 binding to p53 (Fig. 6B). The data suggest that Mdm2 competes for binding between SHP and p53, and p53 competes for binding between SHP and Mdm2. Thus, the binding between SHP and Mdm2 or SHP and p53 is weaker than the binding between Mdm2 and p53.

Fig. 6.

Fig. 6

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Mdm2 and p53 bind preferentially to each other rather than to SHP. (A) Immunoprecipitation and Western blots to determine the effect of p53 on SHP-Mdm2 interaction. Anti-HA antibody was used to IP Mdm2, and anti-Flag, anti-Myc or anti-HA antibodies were used to detect SHP, p53 or Mdm2 proteins in Western blots, respectively. (B) Immunoprecipitation and Western blots to determine the effect of Mdm2 on SHP-p53 interaction. Anti-Myc antibody was used to IP p53, and anti-Flag, anti-HA or anti-Myc antibodies were used to detect SHP, Mdm2 or p53 proteins in Western blots, respectively.

Discussion

In this study, we show that SHP markedly increases Mdm2 protein stability by preventing Mdm2 self-ubiquitination. Mdm2 also causes rapid SHP degradation under p53-deficient conditions, thus creating an autoregulatory loop with SHP. Our study reveals a fine tuned interplay between SHP and Mdm2, which provides new insight into the mechanisms of Mdm2 activation mediated by a nuclear transcriptional repressor.

The recent discovery of certain natural occurring SHP variants provided key insights into the structural basis of the SHP-Mdm2 interaction. Despite its lower stability SHPK170N shows the highest ability to induce Mdm2 protein, whereas the more stable SHPK170R is unable to induce Mdm2 protein or decrease its degradation. This likely reflects the enhanced binding of Mdm2 to SHPK170N and its decreased binding to SHPK170R. Therefore, K170 appears to be a critical lysine residue through which SHP exerts its activity to regulate Mdm2 protein stability. On the other hand, the Mdm2 and SHP interaction is independent of p53, and Mdm2 does not require the presence of p53 to decrease SHP. The Mdm2-mediated SHP decrease is not affected by deletion of its RING or acidic domain, confirming that the interaction between SHP and Mdm2 through K170 is critical for their cross-regulation.

The autoregulatory feedback loop between SHP and Mdm2 is somewhat similar, although not identical, to the regulatory relationship between p53 and Mdm2. Accordingly, p53 activates Mdm2 gene transcription [48] and Mdm2 in turn targets p53 protein for proteasomal degradation [34]. A recent study reported that SHP was ubiquitinated at K122 and K123, and mutation of these sites to arginine (R) increased SHP stability by reducing ubiquitination [47]. However, the SHPK122/123R protein was still rapidly degraded by Mdm2 (not shown), suggesting that K122 and K123 may not be the essential residues for Mdm2-mediated SHP degradation in the cellular context of the present study.

We are aware of one prior report of crosstalk among TR3 (Nur77), p53, and Mdm2 [49]. There are fundamental differences with regard to the crosstalk between SHP or TR3 with Mdm2. SHP induces Mdm2, but TR3 represses Mdm2. SHP interacts with both Mdm2 and p53, yet TR3 interacts with p53 but not Mdm2. Mdm2 and p53 bind to each other with higher affinity than to SHP, whereas TR3 has a higher p53 binding affinity and can sequestrate p53 from Mdm2. It is apparent that SHP and TR3 play unique roles in regulating Mdm2 pathway which results in distinct outcomes. It remains to be determined whether the interaction between SHP and TR3 [50] affects the crosstalk between SHP and Mdm2.

In summary, our results establish a critical role for SHP in keeping the protein level of Mdm2 in check. SHP itself is also tightly regulated by Mdm2 in a negative feedback fashion. Future studies will be focused on elucidating how their cross-regulation affects the function of Mdm2 and SHP in carcinogenesis.

Highlights.

  • SHP drastically stabilizes Mdm2 protein by abrogating Mdm2 self-ubiquitination

  • Mdm2 causes SHP degradation under p53-deficient conditions

  • SHP K170 residue is critical for SHP and Mdm2 interaction and cross-regulation

Acknowledgments

We are very grateful for the generous plasmids from Drs. Xiongbin Lu, Takeshi Imamura, Yang Shi, and Moshe Oren. This work is supported in part by NIH Grant DK080440 to L.W.

Footnotes

Conflict of Interest

The authors declare no conflict of interest.

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