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Cited by 234Cancer-related Molecular PathwaysUbiquitin and proteasome pathwaysHeat shock proteins research

Mdmx stabilizes p53 and Mdm2 via two distinct mechanisms

Open access
Stad, Robert, Little, Natalie A, Xirodimas, Dimitris P, Frenk, Ruth, van der Eb, Alex J, Lane, David P, Saville, Mark K, Jochemsen, Aart G·2001·The EMBO Reports
DOI 10.1093/embo-reports/kve227PMC PMC1084126
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Abstract

The p53 protein maintains genomic integrity through its ability to induce cell cycle arrest or apoptosis in response to various forms of stress. Substantial regulation of p53 activity occurs at the level of protein stability, largely determined by the activity of the Mdm2 protein. Mdm2 targets both p53 and itself for ubiquitylation and subsequent proteasomal degradation by acting as an ubiquitin ligase, a function that needs an intact Mdm2 RING finger. For efficient degradation of p53 nuclear export appears to be required. The Mdmx protein, structurally homologous to Mdm2, does not target p53 for degradation, but even stabilizes both p53 and Mdm2, an activity most likely mediated by heterodimerization of the RING fingers of Mdm2 and Mdmx. Here we show that Mdmx expression leads to accumulation of ubiquitylated, nuclear p53 but does not significantly affect the Mdm2-mediated ubiquitylation of p53. In contrast, Mdmx stabilizes Mdm2 by inhibiting its self-ubiquitylation.

AI snapshot

Written from the paper’s full text.

TL;DR

Mdmx stabilizes p53 and Mdm2 through distinct mechanisms: it promotes accumulation of ubiquitylated nuclear p53 without substantially blocking Mdm2-mediated p53 ubiquitylation, while it stabilizes Mdm2 by inhibiting Mdm2 self-ubiquitylation. Mdmx does not stabilize cytoplasmic p53, but it does stabilize cytoplasmic Mdm2.

Key findings

  • Mdmx expression led to accumulation of ubiquitylated p53 and inhibited Mdm2-dependent p53 degradation, but did not detectably change the overall pattern of Mdm2-dependent p53 ubiquitylation in H1299 cells; in Saos-2 and p53/mdm2 double-knockout cells, Mdmx caused some relative reduction of slower-migrating ubiquitylated p53 forms.
  • Mdmx stabilized Mdm2 by inhibiting its self-ubiquitylation: co-expression of Mdmx significantly reduced poly-ubiquitylated Mdm2 despite increased total Mdm2 levels, and increased the fastest-migrating lower ubiquitylated forms of Mdm2.
  • In Saos-2 cells, mutant p53 alone was 49% exclusively nuclear and 50% strong nuclear with weak cytoplasmic staining; Mdm2 increased cytoplasmic p53, and additional Mdmx co-expression significantly reversed this shift, indicating inhibition of Mdm2-mediated nuclear export of p53.
  • Mdmx did not stabilize cytoplasmic p53(NLS−) in H1299 or Saos-2 cells, but it stabilized cytoplasmic Mdm2(NLS−) to the same extent as wild-type nuclear Mdm2; co-immunoprecipitation indicated Mdmx binds both wild-type Mdm2 and Mdm2(NLS−).

Methods

  • In vivo ubiquitylation assays were performed in H1299, p53/mdm2 double-knockout, and Saos-2 cells transfected with p53, Mdm2, Mdmx, and His6-tagged ubiquitin expression vectors; cells were untreated or treated with 20 µM MG132 for 4 h starting 40 h after transfection, then His-purified ubiquitylated proteins or total lysates were analyzed by western blot.
  • Immunofluorescence localization studies used Saos-2 cells transfected with p53, human mdm2, and human mdmx; in each experiment more than 100 cells were counted for p53 localization and categorized as only nuclear, strong nuclear/weak cytoplasmic, equal, or weak/no nuclear/strong cytoplasmic, with three independent experiments.
  • Co-immunoprecipitation was performed with anti-Mdmx serum p56; 80% of lysate was immunoprecipitated and 10% loaded directly, with Mdm2 detected by 4B2 antibody.

Limitations

  • The paper states that the mechanism by which Mdmx prevents relocalization of p53 into the cytoplasm remains unclear.
  • The text makes evident that effects on p53 ubiquitylation vary by cell line: Mdmx did not detectably change the ubiquitylation pattern in H1299 cells but partly reduced slower-migrating ubiquitylated p53 forms in Saos-2 and double-knockout cells, and this partial inhibition was not sufficient to prevent degradation of cytoplasmic p53.
  • The text makes evident reliance on transfected overexpression systems and notes some supporting observations as “not shown” or “data not shown” (e.g., mouse embryo fibroblasts, U2OS cells, Saos-2 Mdm2 ubiquitylation).

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1093/embo-reports/kve227

Cites

16 of 16 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 23 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    In Saos-2 cells, mutant p53 V143A transfected alone is predominantly nuclear (49% exclusively nuclear, 50% strong nuclear/weak cytoplasmic).

    “When mutant p53 was transfected alone into Saos-2 cells, 49% of the p53 positive cells showed exclusively nuclear and 50% strong nuclear and weak cytoplasmic staining (Figure 2B, left).”
    Quote in the paperSubstantial claim
  • Findingown data

    Mdmx stabilizes Mdm2 by inhibiting its self-ubiquitylation.

    “In contrast, Mdmx stabilizes Mdm2 by inhibiting its self-ubiquitylation.”
    Quote in the paperSubstantial claim
  • Findingown data

    In DKO cells, Mdmx causes some reduction in slower-migrating ubiquitylated forms of p53 relative to faster-migrating forms.

    “However, in contrast to H1299 cells, in these cells this effect was accompanied by some reduction in the amount of slow migrating ubiquitylated forms of p53 relative to the faster migrating forms”
    Quote in the paperNot a substantial claim
  • Findingown data

    In H1299 cells, Mdmx does not significantly alter the ubiquitylation pattern of p53(NLS-) upon proteasome inhibition.

    “Upon inhibition of the proteasome, Mdmx expression does not significantly alter the ubiquitylation pattern of p53(NLS -).”
    Quote in the paperSubstantial claim
  • Findingown data

    Mdmx expression does not significantly affect Mdm2-mediated ubiquitylation of p53.

    “Here we show that Mdmx expression leads to accumulation of ubiquitylated, nuclear p53 but does not significantly affect the Mdm2-mediated ubiquitylation of p53.”
    Quote in the paperSubstantial claim
  • May combine two claims. 20
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 3

  • May combine two claims.

    Findingown data

    In p53/mdm2 double knockout cells, Mdmx does not cause accumulation of ubiquitylated p53, indicating Mdmx lacks intrinsic ubiquitin ligase activity toward p53.

    “In the absence of endogenous Mdm2, Mdmx did not result in the accumulation of ubiquitylated p53 (Figure 1B, His purified, lanes 7, 8), indicating that Mdmx by itself does not have ubiquitin ligase activity towards p53.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Mdmx has similar effects on Hdm2 and in Saos-2 cells.

    “A similar effect was observed with Hdm2, although the pattern of ubiquitylated Mdm2 and Hdm2 is somewhat different (Figure 1D, lanes 5-8).”
    • Also stated in: § 2001; see Methods). · plain
      “In addition, the same effect was observed in Saos-2 cells (not shown).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Across cell lines, Mdmx either does not (H1299) or only partly (Saos-2, DKO) prevent Mdm2-mediated polyubiquitylation of p53.

    “Together, the data in Figure 1A-C show that, depending on the cell line tested, Mdmx does not (H1299) or partly (Saos-2, DKO) prevent Mdm2-mediated (poly-)ubiquitylation of p53.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In all tested cases, ubiquitylated p53 levels with both Mdm2 and Mdmx are higher than with p53 alone, indicating stabilization of ubiquitylated p53.

    “In all cases, the amount of ubiquitylated p53 in the presence of both Mdm2 and Mdmx is higher than after transfection of only p53, indicating a stabilization of ubiquitylated p53.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    With wild-type p53, Mdm2 has only a very limited effect on p53 localization, but Mdmx increases nuclear localization of p53.

    “With the use of a wild-type p53 expression vector, the effect of Mdm2 on p53 localization is only very limited in our hands, but still in this case co-expression of Mdmx leads to an increase in nuclear localization of p53 (Figure 2B, right).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mdmx localization largely depends on Mdm2 localization, and Mdmx binds both wild-type and NLS-mutant Mdm2.

    “since Mdmx localization was largely dependent on the localization of Mdm2 (Figure 3A panels 9, 10 and 20, 21) and co-immunoprecipitation studies indicate that Mdmx binds to both wild-type Mdm2 and the Mdm2(NLS -) mutant (Figure 3D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In H1299 cells, Mdmx causes a general increase in ubiquitylated p53 but does not detectably change the overall pattern of Mdm2-dependent p53 ubiquitylation.

    “While Mdmx caused a general increase in the level of ubiquitylated p53 it did not detectably change the overall pattern of Mdm2-dependent ubiquitylation of p53 (Figure 1A, His purified, lanes 5, 6 compared to lanes 3, 4)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mdmx can block Mdm2-dependent p53 degradation without significantly affecting the pattern of p53 ubiquitylation.

    “In this report we show that Mdmx can block Mdm2-dependent degradation of p53 without significantly affecting the pattern of p53 ubiquitylation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mdmx significantly reduces polyubiquitylated Mdm2 while total Mdm2 levels increase.

    “Co-expression of Mdmx significantly reduced the amount of poly-ubiquitylated Mdm2 (Figure 1D, His purified, lane 3 and 4), despite the total levels of Mdm2 being increased (Figure 1D, total lysate, compare lanes 1, 2 with 3, 4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Mdmx can modestly affect p53 ubiquitylation in the absence of co-transfected Mdm2 only when endogenous Mdm2 is expressed, likely because Mdmx stabilizes endogenous Mdm2.

    “Mdmx does not have intrinsic ubiquitin ligase activity and Mdmx can modestly affect p53 ubiquitylation in the absence of co-transfected Mdm2 only when endogenous Mdm2 is expressed, which is probably stabilized by the transfected Mdmx.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Efficient degradation of p53 requires its nuclear export.

    “Several reports suggest that nuclear export of p53, mediated through the nuclear export signal (NES) of Mdm2 and/or through the NES of p53 itself, is required for its efficient degradation (Roth et al., 1998;Tao and Levine, 1999;Boyd et al., 2000;Geyer et al., 2000).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Roth et al., 1998 source

      Not checked in the free run.

    • cites Tao and Levine, 1999 source

      Not checked in the free run.

    • cites Boyd et al., 2000 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Mdmx stabilizes Mdm2 independent of its localization, most likely by repressing its auto-ubiquitylation.

    “These data are consistent with the previous results showing that Mdmx prevents Mdm2-dependent degradation of p53 by stabilization of nuclear, ubiquitylated p53, whereas Mdmx stabilizes Mdm2 independent of its localization, most likely by repressing its auto-ubiquitylation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    In cells co-transfected with Mdm2 and Mdmx, MG132 does not further increase ubiquitylated p53, indicating Mdmx blocks proteasome-mediated degradation of ubiquitylated p53.

    “In cells co-transfected with Mdm2 and Mdmx, MG132 had no further effect on the level of ubiquitylated p53, indicating that Mdmx blocks proteasome-mediated degradation of ubiquitylated p53.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Mdmx does not target p53 for degradation but instead stabilizes p53 and Mdm2.

    “However, Mdmx does not target p53 for degradation but even prevents Mdm2-mediated degradation of p53 and also stabilizes Mdm2 (Sharp et al., 1999;Jackson and Berberich, 2000;Stad et al., 2000).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Sharp et al., 1999 source

      Not checked in the free run.

    • cites Jackson and Berberich, 2000 source

      Not checked in the free run.

    • cites Stad et al., 2000 source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Mdm2 is a RING-type E3 ubiquitin ligase that ubiquitylates itself and p53, leading to their proteasomal degradation.

    “Mdm2 is a RING-type E3 ubiquitin ligase (Honda et al., 1997;Fang et al., 2000;Honda and Yasuda, 2000), which directs the ubiquitylation of itself and p53.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Honda et al., 1997 source

      Not checked in the free run.

    • cites Fang et al., 2000 source

      Not checked in the free run.

    • cites Honda and Yasuda, 2000 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Partial inhibition of Mdm2-mediated p53 ubiquitylation by Mdmx in Saos-2 is not sufficient to prevent p53 degradation; Mdmx stabilization of p53 requires nuclear localization.

    “These results strongly indicate that the partial inhibition of Mdm2-mediated p53 ubiquitylation by Mdmx in Saos-2 is not sufficient to prevent the degradation of p53, but that stabilization of p53 by Mdmx is dependent on a nuclear localization.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Co-expression of Mdm2 with p53 in Saos-2 or U2OS cells enhances nuclear export of p53, which is required for efficient degradation.

    “Two recent reports suggest that co-expression of Mdm2 with p53 in Saos-2 or U2OS cells results in enhanced nuclear export of p53, required for its efficient degradation (Boyd et al., 2000;Geyer et al., 2000).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Boyd et al., 2000 source

      Not checked in the free run.

    • cites Geyer et al., 2000 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Inhibition of p53 ubiquitylation by Mdmx in some cell lines is neither necessary nor sufficient for p53 stabilization.

    “Although some inhibition of p53 ubiquitylation is observed in some cell lines, this effect appears neither necessary nor sufficient for the stabilization by Mdmx.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Swapping Mdm2's RING finger with Praja 1's RING finger did not affect Mdm2 ubiquitylation but prevented p53 ubiquitylation.

    “This switch did not affect Mdm2 ubiquitylation but prevented the ubiquitylation of p53 (Fang et al., 2000).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Fang et al., 2000 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    p14ARF stabilizes Mdm2 and blocks Mdm2-mediated p53 degradation, but its effects on p53 and Mdm2 ubiquitylation are opposite to Mdmx.

    “While p14ARF also stabilizes Mdm2 and blocks Mdm2-mediated degradation of p53, its effects on p53 and Mdm2 ubiquitylation are the opposite of Mdmx.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Xirodimas et al., 2001 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    p14ARF inhibits polyubiquitylation of p53 while Mdm2 ubiquitylation is unaffected.

    “p14ARF inhibits the poly-ubiquitylation of p53 while the ubiquitylation of Mdm2 is unaffected (Xirodimas et al., 2001).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Xirodimas et al., 2001 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Mdm2 S395D mutant is impaired in p53 degradation and strongly inhibited in stimulating p53 nuclear export, with preliminary data suggesting ubiquitin ligase activity not decreased.

    “This effect was explained by the observation that the Mdm2 S395D mutant was strongly inhibited in its ability to stimulate nuclear export of p53, while preliminary data suggested that the ubiquitin ligase activity was not decreased.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Maya et al., 2001 source

      Not checked in the free run.

22 of 22

  • The paper relies on this without support.

    Poly-ubiquitylated proteins, rather than mono- or di-ubiquitylated proteins, are efficiently recognized and degraded by the proteasome.

    “It has been shown that poly-ubiquitylated rather than mono-or di-ubiquitylated proteins are efficiently recognized and degraded by the proteasome.”

  • The paper relies on this without support.

    Cytoplasmic p53 is efficiently ubiquitylated and degraded by cytoplasmic Mdm2.

    “Cytoplasmic p53 is efficiently ubiquitylated and degraded by cytoplasmic Mdm2.”

  • The paper relies on this without support.

    Heterodimerization of the RING fingers of Mdmx and Mdm2 is required for Mdmx's effects.

    “Heterodimerization of the RING fingers of Mdmx and Mdm2 appears to be required for these effects, but the exact mechanism by which Mdmx operates has not been determined.”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Mdm4 contributes to Mdm2 stability by disrupting self-ubiquitination of Mdm2.

    “Mdm4 further contributes to Mdm2 stability by disrupting self-ubiquitination of the latter (Stad et al., 2001), while also stimulating Mdm2-mediated polyubiquitination of p53 (Linares et al., 2003).”

    From USP7 Is a Master Regulator of Genome Stability (2020) · cites this paper as Stad et al., 2001

  • Mdmx is overexpressed in many human malignancies and, together with Mdm2, negatively regulates the p53 tumor suppressor.

    “The oncogene Mdmx is overexpressed in many human malignancies, and together with Mdm2, negatively regulates the p53 tumor suppressor.”

    From Mdmx promotes genomic instability independent of p53 and Mdm2 (2015) · cites this paper as [24]

  • Mdmx negatively regulates p53 by inhibiting p53 transcriptional activity rather than by promoting p53 degradation through ubiquitination.

    “While Mdmx negatively regulates p53, it does so through inhibiting p53 transcriptional activity rather than promoting p53 degradation through ubiquitination, as is the case for Mdm2.24,25”

    From Mdmx promotes genomic instability independent of p53 and Mdm2 (2015) · cites this paper as [24]

  • Through their RING domains, Mdmx binds Mdm2 and enhances Mdm2's ability to regulate p53.

    “Additionally, it has been shown that through their RING domains, Mdmx binds Mdm2 and enhances the ability of Mdm2 to regulate p53.23,24,26–28”

    From Mdmx promotes genomic instability independent of p53 and Mdm2 (2015) · cites this paper as [24]

  • MDMX binds p53 and inhibits its transcriptional function, but alone does not promote p53 ubiquitination or degradation in vivo.

    “MDMX shares strong homology to MDM2 at the amino-acid sequence level and can bind to p53 and inhibit its transcriptional function.However, MDMX alone does not promote p53 ubiquitination or degradation in vivo (Stad et al, 2001).”

    From ATM and Chk2-dependent phosphorylation of MDMX contribute to p53 activation after DNA damage (2005) · cites this paper as Stad et al, 2001

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 2A panel 3 is assigned opposite p53 localization patterns

    Minor

    The Figure 2 caption identifies panel 3 as both strong nuclear with weak cytoplasmic staining and weak or no nuclear with strong cytoplasmic staining.

1 of 1

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Authors
Stad, R., Little, N. A., Xirodimas, D. P., Frenk, R., van der Eb, A. J., Lane, D. P., Saville, M. K., & Jochemsen, A. G.
Affiliations
  • Leiden University Medical Center (NL)
  • University of Dundee (GB)
ORCID
Venue
The EMBO Reports
Publisher
Springer Science and Business Media LLC
Published
2001-11-01
Volume
2
Issue
11
Pages
1029-1034
ISSN
  • 1469-221X (ISSN-L)
  • 1469-3178
DOI
10.1093/embo-reports/kve227
PMC
PMC1084126
OpenAlex
W1982545718
Semantic Scholar
9a1a4f2eec163e17e139e410aef986cb5dffe4d8
Cited by
234
References
16
Citations by year
2025: 5 · 2024: 3 · 2023: 7 · 2022: 4 · 2021: 9 · 2020: 7 · 2019: 5 · 2018: 5 · 2017: 8 · 2016: 6 · 2015: 2 · 2014: 17 · 2013: 5 · 2012: 13
FWCI
4.71
Type
journal article
Language
en
Open access
Licence
Locations
  • EMBO Reports (publishedVersion)
  • PubMed Central (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed (publishedVersion)
  • HAL (submittedVersion)
Funders
KWF Kankerbestrijding
Topics
  • Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heat shock proteins research
Keywords
MDMX, p53 stabilization, MDM2, protein stability, ubiquitination, proteasomal degradation, ubiquitin ligase, tumor suppressor regulation
MeSH terms
Animals, Cell Cycle, Cell Line, Cell Nucleus, Cytoplasm, Humans, Ligases, Microscopy, Fluorescence, Mutation, Nuclear Proteins, Plasmids, Protein Binding, Proto-Oncogene Proteins, Transfection, Tumor Suppressor Protein p53, Apoptosis, Protein Structure, Tertiary, Mice, Knockout, Dimerization, Active Transport, Cell Nucleus, Ubiquitin, Ubiquitin-Protein Ligases, Mice, Proto-Oncogene Proteins c-mdm2

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