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Cited by 262Cancer-related Molecular PathwaysDNA Repair MechanismsUbiquitin and proteasome pathways

ATM and Chk2-dependent phosphorylation of MDMX contribute to p53 activation after DNA damage

Open access
Chen, Lihong, Gilkes, Daniele M, Pan, Yu, Lane, William S, Chen, Jiandong·2005·The EMBO Journal
DOI 10.1038/sj.emboj.7600812PMC PMC1276172
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Abstract

The p53 tumor suppressor is activated after DNA damage to maintain genomic stability and prevent transformation. Rapid activation of p53 by ionizing radiation is dependent on signaling by the ATM kinase. MDM2 and MDMX are important p53 regulators and logical targets for stress signals. We found that DNA damage induces ATM-dependent phosphorylation and degradation of MDMX. Phosphorylated MDMX is selectively bound and degraded by MDM2 preceding p53 accumulation and activation. Reduction of MDMX level by RNAi enhances p53 response to DNA damage. Loss of ATM prevents MDMX degradation and p53 stabilization after DNA damage. Phosphorylation of MDMX on S342, S367, and S403 were detected by mass spectrometric analysis, with the first two sites confirmed by phosphopeptide-specific antibodies. Mutation of MDMX on S342, S367, and S403 each confers partial resistance to MDM2-mediated ubiquitination and degradation. Phosphorylation of S342 and S367 in vivo require the Chk2 kinase. Chk2 also stimulates MDMX ubiquitination and degradation by MDM2. Therefore, the E3 ligase activity of MDM2 is redirected to MDMX after DNA damage and contributes to p53 activation.

AI snapshot

Written from the paper’s full text.

TL;DR

DNA damage induces ATM- and Chk2-dependent phosphorylation of MDMX at C-terminal serines, promoting MDM2 binding, ubiquitination, and degradation of MDMX, which contributes to p53 activation. Reducing MDMX by RNAi enhances p53 response and cell-cycle arrest after ionizing radiation.

Key findings

  • DNA damage induces MDMX phosphorylation and degradation; phosphorylated MDMX is selectively bound and degraded by MDM2, and ATM-deficient fibroblasts fail to induce MDMX mobility shift/degradation after radiation.
  • Mass spectrometry mapped MDMX phosphorylation on S161 (post-IR), S342 (pre- and post-IR), S367 (pre- and post-IR), S391 (post-IR), and S403 (post-IR), with approximately 77% peptide coverage; S342 and S367 were confirmed by phosphopeptide-specific antibodies.
  • Mutation of S342, S367, and S403 each reduced MDM2-mediated polyubiquitination, with S367 having the most significant effect; the triple alanine mutant MDMX-3A was resistant to degradation after DNA damage and inhibited p53 activation more efficiently than wild-type MDMX.
  • Chk2 was required in vivo for S342 and S367 phosphorylation after irradiation; Chk2-null HCT116 cells showed less efficient MDMX degradation and reduced p53 stabilization, MDM2 induction, and p21 induction after 5 Gy. MDMX siRNA (200 nM) in U2OS cells increased p53 target induction after 10 Gy and made DNA synthesis inhibition after gamma irradiation significantly more efficient by BrdU ELISA (n=4).

Methods

  • Cell lines included MCF7, U2OS, H1299, Saos2, HCT116 wild-type/p53-null/Chk2-null, ATM-deficient Hs235.Sk, and normal human skin fibroblasts; experiments used MDMX point mutants, MDMX siRNA, retrovirus, ionizing radiation, MG132, caffeine, cycloheximide, and MDM2 antisense oligonucleotide.
  • Protein analysis used Western blot, gradient gel electrophoresis, immunoprecipitation, GST-MDM2 pull-down, calf intestinal phosphatase treatment, and BrdU incorporation ELISA; phosphorylation-specific antibodies against PS342 and PS367 were generated and validated.
  • Phosphorylation site mapping used FLAG-MDMX affinity purification, trypsin digestion, and LCQ DECA XP Plus ion trap MS/MS with SEQUEST/MUQUEST/FUZZYIONS; 32P metabolic labeling with 2-D phospho-amino-acid analysis and in vitro kinase reactions with ATM, Chk1, Chk2, Akt1, and CK1α were also performed.

Limitations

  • Stated in the paper: lack of a viable Chk1-null mammalian cell line and strong toxicity of Chk1 siRNA hampered investigation of Chk1’s role; lack of a PS403 antibody precluded testing direct ATM modification of S403; more rigorous testing of cooperative phosphorylation requires gene replacement to avoid MDMX overexpression; the Chk2-null result differed from a prior study, possibly due to experimental conditions.
  • Evident from the text: many conclusions rely on transfected/overexpressed MDMX and single versus triple mutants, with single mutants showing only minor differences in U2OS p53 activation; no animal models are reported, and the authors note Chk2 effects may be subtle and cell-line/damage-dose dependent.

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

Integrity signals

Registry

Crossref · 10.1038/sj.emboj.7600812

Cites

43 of 43 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 39 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    DNA damage induces degradation of MDMX.

    “We found that DNA damage induces ATMdependent phosphorylation and degradation of MDMX.”
    • Also stated in: § DNA damage induces degradation of MDMX · plain
      “MDMX was stable before irradiation but was rapidly degraded after DNA damage (Figure 1C).”
    Quote in the paperSubstantial claim
  • Findingown data

    Loss of ATM prevents p53 stabilization after DNA damage.

    “Loss of ATM prevents MDMX degradation and p53 stabilization after DNA damage.”
    Quote in the paperSubstantial claim
  • Findingown data

    Mutation of MDMX at S342, S367, and S403 each confers partial resistance to MDM2-mediated ubiquitination and degradation.

    “Mutation of MDMX on S342, S367, and S403 each confers partial resistance to MDM2-mediated ubiquitination and degradation.”
    • Also stated in: § Phosphorylation site mutations reduce ubiquitination and degradation by MDM2 · plain
      “The results showed that mutating S342, S367, and S403 each resulted in reduced poly-ubiquitination by MDM2, with S367 having the most significant effect (Figure 6C).”
    Quote in the paperSubstantial claim
  • Findingown data

    MDMX is phosphorylated on S342, S367, and S403 after DNA damage.

    “Phosphorylation of MDMX on S342, S367, and S403 were detected by mass spectrometric analysis, with the first two sites confirmed by phosphopeptide-specific antibodies.”
    • Also stated in: § Identification of phosphorylation sites on MDMX · plain
      “Analysis of the MS results led to detection of phosphorylation on S161 (post-IR), S342 (preand post-IR), S367 (pre-and post-IR), S391 (post-IR), and S403 (post-IR).”
    Quote in the paperSubstantial claim
  • Findingown data

    Phosphorylation of MDMX at S342 and S367 in vivo requires Chk2.

    “Phosphorylation of S342 and S367 in vivo require the Chk2 kinase.”
    • Also stated in: § Phosphorylation of MDMX by Chk2 · hedged
      “Loss of Chk2 abrogated the induction of S342 and S367 phosphorylation after irradiation (Figure 8C), suggesting that Chk2 is required for the phosphorylation of both sites in vivo.”
    Quote in the paperSubstantial claim
  • May combine two claims. 42
  • Classified as probably not a substantial claim. 17

  • May combine two claims.

    Findingown data

    Serine carries most of the phosphate on MDMX; threonine is modified less, and tyrosine modification was not detected.

    “In vivo metabolic labeling revealed that MDMX is predominantly phosphorylated on serine, with minor modifications on threonine and no detectable modification on tyrosine.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Chk1 modifies S342 and S367 in vitro, with strong preference for S342.

    “Western blot showed that Chk1 modified S342 and S367, but with strong preference for S342.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Endogenous MDMX phosphorylated on S342 or S367 is rapidly degraded after DNA damage and accumulates only after MG132 treatment.

    “Furthermore, endogenous MDMX phosphorylated on S342 or S367 were rapidly degraded after DNA damage and only accumulate after MG132 treatment.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ATM immunoprecipitated from transfected cells phosphorylated p53 S15 but did not show specific modification of MDMX on S342 and S367.

    “ATM immunoprecipitated from transfected cells phosphorylated S15 of p53 but did not show specific modification of MDMX on S342 and S367.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Caffeine partially inhibits MDMX mobility shift and degradation after radiation and blocks p53 stabilization and MDM2 and p21 induction.

    “This treatment partially inhibited the mobility shift and degradation of MDMX after radiation (Figure 3B).”
    • Also stated in: § ATM is required for stimulating MDMX phosphorylation · plain
      “As expected, caffeine also blocked stabilization of p53 and induction of MDM2 and p21 (Figure 3B and data not shown).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MDMX downregulation occurs in p53-deficient HCT116 cells and p53-mutant DLD1 and C33A cells without induction of MDM2.

    “However, further tests of a panel of cell lines showed that MDMX downregulation also occur in p53-deficient HCT116 cells and p53-mutant DLD1 and C33A cells without induction of MDM2 (Figure 1B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Deletion of the MDMX C-terminal RING domain causes complete loss of mobility shift after DNA damage, whereas deletion of the N-terminal p53-binding domain does not prevent phosphorylation.

    “The results indicated that deletion of the MDMX C-terminal RING domain caused a complete loss of mobility shift after DNA damage, whereas deletion of the N-terminal p53-binding domain did not prevent phosphory- lation (Figure 5B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MDMX is mainly phosphorylated on serine in vivo, with minor threonine and no detectable tyrosine phosphorylation.

    “Phospho-amino-acid analysis showed that MDMX was mainly phosphorylated on serine in vivo, with minor labeling of threonine and no detectable phosphorylation of tyrosine (Figure 5A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    MDMX ubiquitination and degradation by MDM2 can occur in both wild-type and Chk2-null HCT116 cells, indicating Chk2 is not absolutely necessary under these conditions.

    “In cotransfection experiments, MDMX ubiquitination and degradation by MDM2 can occur in both wild-type and Chk2-null HCT116 cells, suggesting that Chk2 was not absolutely necessary under these conditions.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    DNA-damaging agents induce MDMX phosphorylation, whereas taxol and nocodazole do not.

    “The results showed that agents that can induce DNA damage were all capable of inducing MDMX phosphorylation.Microtubule targeting agents taxol and nocodazole did not cause MDMX mobility shift (Figure 2B), suggesting that the effect was unique to DNA-damaging drugs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Chk2 modifies both S342 and S367 in vitro, with strong preference for S367.

    “Chk2 modified both S342 and S367, but with strong preference for S367 (Figure 8A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In HCT116-Chk2-/- cells after gamma irradiation, MDMX degradation is much less efficient than in wild-type, and MDM2 and p21 induction and p53 stabilization are reduced.

    “When the HCT116-Chk2À/À cells were compared to wildtype HCT116 after gamma irradiation (5 Gy), we reproducibly found that MDMX degradation was much less efficient (Figure 9C).MDM2 and p21 induction and p53 stabilization were also reduced compared to wild-type cells (Figure 9C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The triple alanine MDMX mutant shows significant resistance to degradation after DNA damage and inhibits p53 activation more efficiently than wild-type MDMX.

    “However, the triple alanine compound mutant (MDMX-3A) showed significant resistance to degradation after DNA damage, and inhibited p53 activation more efficiently than wild-type MDMX (Figure 6D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cotransfection of MDMX and Chk2 into HCT116-Chk2-/- cells restores phosphorylation of MDMX S367 after gamma irradiation, whereas kinase-dead Chk2-A347, activated myr-Akt1, and CK1a have no effect.

    “Cotransfection of MDMX and Chk2 plasmid into HCT116-Chk2À/À cells restored the phosphorylation of MDMX S367 after gamma irradiation, whereas kinase-dead Chk2-A347 mutant, activated myr-Akt1 and CK1a had no effect (Figure 9A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Expression of MDM2 stimulates MDMX ubiquitination more significantly in wild-type HCT116 compared to Chk2-null cells, consistent with Chk2 being a facilitator.

    “However, expression of MDM2 stimulated MDMX ubiquitination more significantly in wild-type HCT116 compared to the Chk2-null cells (Figure 9B), consistent with Chk2 being a facilitator.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Chk2 phosphorylates MDMX at S342 and S367 both in vitro and in vivo.

    “Of the three functionally relevant phosphorylation sites identified in this study, S342 and S367 are targets of Chk2 in vitro and in vivo.”
    • Also stated in: § Discussion · boosted
      “We show here that Chk2 is directly involved in the phosphorylation of MDMX on S342 and S367.”
    Quote in the paperSubstantial claim

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

    Findingown data

    Weak modification of S342 by Akt1 was detected in vitro, with unclear significance.

    “Weak modification of S342 by Akt1 was detected, the significance of this reaction is not clear at present.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Ionizing irradiation promotes MDMX nuclear translocation and degradation by proteasomes.

    “DNA damage induced by ionizing irradiation also promotes MDMX nuclear translocation and degradation by the proteasomes (Li et al, 2002;Pan and Chen, 2003).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Li et al, 2002

      Not checked in the free run.

    • cites Pan and Chen, 2003 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    MDMX cooperates with MDM2 to promote p53 degradation.

    “Recent evidence showed that MDMX cooperates with MDM2 to promote p53 degradation (Gu et al, 2002).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Gu et al, 2002

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    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).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Stad et al, 2001 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    MDMX-null mice die in utero in a p53-dependent manner, and this lethality is rescued by crossing into a p53-null background.

    “Several studies showed that MDMX-null mice also die in utero in a p53-dependent fashion, which can be rescued by crossing into the p53-null background (Finch et al, 2002;Migliorini et al, 2002;Parant et al, 2002).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Finch et al, 2002

      Not checked in the free run.

    • cites Migliorini et al, 2002 source

      Not checked in the free run.

    • cites Parant et al, 2002 source

      Not checked in the free run.

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

    Premisecitation

    MDM2 binds p53 and acts as an ubiquitin E3 ligase to promote p53 degradation by proteasomes.

    “P53 turnover is regulated by MDM2, which binds p53 and functions as an ubiquitin E3 ligase to promote p53 degradation by the proteasomes (Zhang and Xiong, 2001).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Zhang and Xiong, 2001

      Not checked in the free run.

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

    Premisecitation

    ATM phosphorylates p53 on serine 15, enhancing its transcriptional activity.

    “ATM phosphorylates p53 on serine 15, enhancing its transcriptional activity (Banin et al, 1998).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Banin et al, 1998 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    After DNA damage, MDM2 E3 ligase activity is redirected to MDMX and contributes to p53 activation.

    “Therefore, the E3 ligase activity of MDM2 is redirected to MDMX after DNA damage and contributes to p53 activation.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    DNA damage induces p53 accumulation by phosphorylation.

    “Stress signals such as DNA damage induce p53 accumulation by phosphorylation (Prives and Hall, 1999).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Prives and Hall, 1999 source

      Not checked in the free run.

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

    Premisecitation

    ATM activates Chk2, which phosphorylates p53 on serine 20 and inhibits MDM2 binding.

    “ATM also activates Chk2, which in turn phosphorylates p53 on serine 20 and inhibits MDM2 binding (Shieh et al, 1997;Banin et al, 1998;Chehab et al, 2000;Shieh et al, 2000).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Shieh et al, 1997 source

      Not checked in the free run.

    • cites Banin et al, 1998 source

      Not checked in the free run.

    • cites Chehab et al, 2000 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    DNA damage induces MDMX degradation in p53-deficient cells without induction of MDM2.

    “A recent study showed that DNA damage also induces MDMX degradation in p53-deficient cells, without induction of MDM2 (Kawai et al, 2003).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Kawai et al, 2003 source

      Not checked in the free run.

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

    Premisecitation

    ATM functions as the primary signal transducer of DNA double-strand breaks.

    “ATM functions as the primary signal transducer of DNA double-strand breaks (Shiloh, 2003).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Shiloh, 2003 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    ATM is important for rapid p53 activation after ionizing radiation, as shown by delayed p53 accumulation in ATM-deficient cells.

    “The importance of ATM in rapid p53 activation after ionizing radiation (IR) was revealed by the delay in p53 accumulation in ATM-deficient cells (Kastan et al, 1993).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Kastan et al, 1993 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    MDMX can be ubiquitinated and degraded by MDM2.

    “Several reports showed that MDMX can be ubiquitinated and degraded by MDM2 (De Graaf et al, 2003;Kawai et al, 2003;Pan and Chen, 2003).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites De Graaf et al, 2003 source

      Not checked in the free run.

    • cites Kawai et al, 2003 source

      Not checked in the free run.

    • cites Pan and Chen, 2003 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    MDMX and MDM2 form heterodimers through their C-terminal RING domains, and the MDM2 RING domain alone is sufficient to degrade MDMX.

    “MDMX and MDM2 form heterodimers through the C-terminal RING domains and the MDM2 RING domain alone is sufficient to degrade MDMX (Sharp et al, 1999;Tanimura et al, 1999;Pan and Chen, 2003).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Sharp et al, 1999 source

      Not checked in the free run.

    • cites Tanimura et al, 1999 source

      Not checked in the free run.

    • cites Pan and Chen, 2003 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Phosphorylated MDMX binds MDM2 more efficiently, which may cause its selective degradation by MDM2.

    “These results showed that phosphorylated MDMX bound to MDM2 more efficiently, which may cause its selective degradation by MDM2.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    DNA damage activates ATM and then Chk2.

    “The current results suggest that DNA damage activates ATM and subsequently Chk2.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    A population of MDMX is phosphorylated and then rapidly degraded after ionizing irradiation.

    “This suggested that a population of MDMX was phosphorylated and then rapidly degraded after ionizing irradiation.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    The role of ATM in p53 response is well established, but the significance of Chk2 is debated.

    “Although a role of ATM in p53 response is well established, the significance of Chk2 is still a matter of debate.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Zhang et al, 1998 source

      Not checked in the free run.

    • cites Chehab et al, 2000 source

      Not checked in the free run.

    • cites Jallepalli et al, 2003 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Chk2 plays a role in regulating the p53 response to gamma irradiation, possibly through promoting degradation of MDMX.

    “These results showed that Chk2 plays a role in regulating p53 response to gamma irradiation, possibly through promoting degradation of MDMX.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Phosphorylation of S342, S367, and S403 may each contribute to stimulating poly-ubiquitination and degradation by MDM2, with S367 having the most dominant role.

    “Therefore, phosphorylation of S342, S367, and S403 may each contribute to stimulating poly-ubiquitination and degradation by MDM2, with S367 having the most dominant role.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Degradation of MDMX after DNA damage facilitates p53 activation and cell cycle arrest.

    “These results suggested that degradation of MDMX after DNA damage facilitates p53 activation and cell cycle arrest.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    MDMX phosphorylation occurs before p53 stabilization after ionizing irradiation, suggesting that degradation of MDMX contributes to p53 activation.

    “Therefore, MDMX phosphorylation occurs before p53 stabilization after ionizing irradiation, suggesting that degradation of MDMX contributes to p53 activation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Multiple phosphorylation in the MDMX C-terminal region may cause conformational changes in the RING domain, increasing MDM2 binding and leading to MDMX ubiquitination and degradation.

    “Multiple phosphorylation of MDMX C-terminal region may lead to conformational changes in the MDMX RING domain, increasing the affinity for MDM2 binding, and leading to ubiquitination and degradation of MDMX.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    A previous study using the same HCT116-Chk2-/- cell line reported that Chk2 knockout did not affect p53 response to higher doses of ionizing irradiation.

    “The results in Figure 9C were unexpected since a previous study using the same HCT116-Chk2À/À cell line reported that Chk2 knockout did not affect p53 response to higher doses (12 Gy) of ionizing irradiation (Jallepalli et al, 2003).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Jallepalli et al, 2003 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    ATM is required for activation of Chk2, which mediates S342 and S367 phosphorylation.

    “This is consistent with the interpretation that ATM is required for activation of Chk2, which mediates S342 and S367 phosphorylation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Phosphorylation of S367 is not necessary for MDM2 binding but may further stimulate MDM2 binding or induce a conformational change favoring degradation.

    “Therefore, phosphorylation of S367 was not necessary for MDM2 binding, but may further stimulate MDM2 binding or induce a conformational change that favor degradation by MDM2.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Phosphorylation of p53 S15 and S20 by ATM and Chk2 stimulates p53 transcription activation and reduces MDM2 binding.

    “Previous studies showed that phosphorylation of S15 and S20 of p53 by ATM and Chk2 stimulate p53 transcription activation function and reduce MDM2 binding.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    DNA damage also accelerates MDM2 degradation through a mechanism requiring its own RING domain.

    “A recent study showed that DNA damage also accelerates MDM2 degradation through a mechanism that requires its own RING domain (Stommel and Wahl, 2004).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Stommel and Wahl, 2004 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Removing MDMX would help activate p53 by freeing p53 from MDMX and by lowering MDM2's E3 ligase activity.

    “Elimination of MDMX would facilitate p53 activation by relieving p53 from MDMX binding, and reducing the E3 ligase activity of MDM2.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    Protein expression levels were confirmed by analyzing a matching set of 8C6 immunoprecipitates with MDMX Western blot.

    “An identical set of 8C6 IP samples was analyzed by MDMX Western blot to confirm protein expression level.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    14-3-3 copurifies with MDMX.

    “We have recently found that 14-3-3 copurifies with MDMX (Chen et al, 2005).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Chen et al, 2005 source

      Not checked in the free run.

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

    Premisecitation

    The phosphorylated form of MDMX S367 may serve as a 14-3-3 binding site.

    “It is noteworthy that phosphorylated S367 of MDMX is a potential 14-3-3 binding site (Tzivion et al, 2001).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Tzivion et al, 2001 source

      Not checked in the free run.

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

    Premisecitation

    c-Abl phosphorylation of MDM2 Y394 and ATM phosphorylation of S395 lower MDM2's capacity to promote p53 degradation.

    “Phosphorylation of MDM2 Y394 by c-Abl and S395 by ATM reduce its ability to promote p53 degradation.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    E. coli-expressed His6-MDMX served as a control to test antibody crossreactivity with unphosphorylated MDMX.

    “His6-MDMX expressed in E. coli was used as a control for crossreactivity to nonphosphorylated MDMX.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Stress signals can redirect MDM2's E3 ligase activity toward either p53 or MDMX, so MDM2 has both negative and positive effects on p53 function.

    “The ubiquitin E3 ligase activity of MDM2 can be switched between targeting p53 or MDMX by stress signals, thus having both negative and positive effects on p53 function.”
    Quote in the paperSubstantial claim
52 of 52

  • The paper relies on this without support.

    Caffeine's inhibition of MDMX phosphorylation and degradation after radiation is due to inhibition of ATM/ATR rather than other caffeine effects.

    “MCF7 cells were treated with caffeine to inhibit ATM/ATR.”
    “This treatment partially inhibited the mobility shift and degradation of MDMX after radiation (Figure 3B).”

  • The paper relies on this without support.

    MDMX siRNA and stable knockdown specifically reduce MDMX levels without off-target effects.

    “U2OS cells were transfected with MDMX siRNA for 48 h, followed by treatment with 10 Gy gamma irradiation.”
    “Similar results were also observed using MCF7 cells with stable knockdown of MDMX (data not shown).”

  • The paper relies on this without support.

    The Chk2-A347 mutant is kinase-dead and can act as a dominant-negative inhibitor of Chk2.

    “kinase-dead Chk2-A347 mutant, activated myr-Akt1 and CK1a had no effect (Figure 9A).”
    “In wild-type HCT116 cells, Chk2-A347 reduced S367 phosphorylation, possibly through dominant-negative effect (Figure 9A).”
3 of 3

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  • Phosphorylation of Mdm2 and Mdmx at serine residues close to the RING domain by ATM or ATM-dependent kinases leads to accelerated auto-degradation of both Mdm2 and Mdmx, with consequent p53 accumulation and activation.

    “Additionally, phosphorylation of Mdm2 and Mdmx at serine residues close to the RING domain by ATM or ATM-dependent kinases leads to accelerated auto-degradation of both Mdm2 [54] and Mdmx [41,55–58], with consequent p53 accumulation and activation.”

    From The p53 orchestra: Mdm2 and Mdmx set the tone (2010) · cites this paper as [55]

  • Chk1 and Chk2 failed to phosphorylate S386 and S429 in vitro, but phosphorylated MDMX S367.

    “Furthermore, Chk1 and Chk2 failed to phosphorylate S386 and S429 in vitro, but phosphorylated the positive control site MDMX S367 (Chen et al, 2005).”

    From ATM activates p53 by regulating MDM2 oligomerization and E3 processivity (2009) · cites this paper as Chen et al, 2005

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 8A names a different phosphosite antibody

    Minor

    The Figure 8A caption says the kinase products were probed with PS467, whereas the described assay and antibody are for S367. The caption therefore identifies a different phosphosite from the one reported in the results.

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Authors
Chen, L., Gilkes, D. M., Pan, Y., Lane, W. S., & Chen, J.
Affiliations
  • Moffitt Cancer Center (US)
  • Harvard University (US)
Corresponding author
Chen, Jiandong
ORCID
Venue
The EMBO Journal
Publisher
Springer Science and Business Media LLC
Published
2005-09-15
Volume
24
Issue
19
Pages
3411-3422
ISSN
  • 0261-4189 (ISSN-L)
  • 1460-2075
DOI
10.1038/sj.emboj.7600812
PMC
PMC1276172
OpenAlex
W2116326380
Semantic Scholar
74f10c564d416135fbbc705c0184d57a01fc3e2a
Cited by
260
References
43
Citations by year
2026: 5 · 2025: 4 · 2024: 8 · 2023: 2 · 2022: 8 · 2021: 10 · 2020: 10 · 2019: 8 · 2018: 10 · 2017: 9 · 2016: 17 · 2015: 17 · 2014: 15 · 2013: 20 · 2012: 20
FWCI
6.41
Type
journal article
Language
en
Open access
Licence
Locations
  • The EMBO Journal (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • American Cancer Society
  • National Institutes of Health
Topics
  • Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
Keywords
p53 activation, MDMX, MDM2, ATM kinase, Chk2, DNA damage response, protein phosphorylation, ubiquitination, protein degradation, ionizing radiation, E3 ubiquitin ligase, tumor suppressor
MeSH terms
Cells, Cultured, Chromatography, High Pressure Liquid, DNA Damage, DNA-Binding Proteins, Electrophoresis, Polyacrylamide Gel, Gene Expression Regulation, Humans, Nuclear Proteins, Phosphorylation, Proto-Oncogene Proteins, Mass Spectrometry, Blotting, Western, Tumor Suppressor Protein p53, Protein Serine-Threonine Kinases, Cell Cycle Proteins, Tumor Suppressor Proteins, RNA Interference, RNA, Small Interfering, Immunoprecipitation, Proto-Oncogene Proteins c-mdm2, Ataxia Telangiectasia Mutated Proteins, Checkpoint Kinase 2

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-08.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-08.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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