Hypophosphorylation of Mdm2 augments p53 stability

Christine Blattner, Trevor Hay, David W. Meek, David P. Lane

    Research output: Contribution to journalArticle

    122 Citations (Scopus)

    Abstract

    The Mdm2 protein mediates ubiquitylation and degradation of p53 and is a key regulator of this tumor suppressor. More recently, it has been shown that Mdm2 is highly phosphorylated within its central acidic domain. In order to address the issue of how these modifications might regulate Mdm2 function, putative phosphorylation sites within this domain were substituted, individually or in pairs, with alanine residues. Mutants with serine-to-alanine substitutions between residues 244 and 260 abolished or at least reduced the capacity of Mdm2 to promote p53 degradation. In each case, loss of degradation function was independent of the ability to bind to p53 or p14ARF. Moreover, each of the Mdm2 mutants completely retained the capacity to act as a ubiquitin ligase in vivo. Thus, ubiquitylation and degradation can be uncoupled. Two-dimensional phosphopeptide mapping coupled with the use of phospho-specific antibodies revealed that Mdm2 is phosphorylated physiologically at several sites within this region, consistent with the idea that phosphorylation is important for Mdm2 activity. Strikingly, treatment of cells with ionizing radiation resulted in a significant decrease in the phosphorylation of residues that are important for p53 turnover. This hypophosphorylation preceded p53 accumulation. These findings indicate that Mdm2 contributes an additional function toward the degradation of p53 that is distinct from its ubiquitin ligase activity and is regulated by phosphorylation. Our model suggests that hypophosphorylation of Mdm2 in response to ionizing irradiation inactivates this novel function, thereby contributing to p53 stabilization.
    Original languageEnglish
    Pages (from-to)6170-6182
    Number of pages13
    JournalMolecular and Cellular Biology
    Volume22
    Issue number17
    DOIs
    Publication statusPublished - Sep 2002

    Fingerprint

    Phosphorylation
    Ubiquitination
    Ligases
    Ubiquitin
    Alanine
    Proto-Oncogene Proteins c-mdm2
    Tumor Suppressor Protein p14ARF
    Phospho-Specific Antibodies
    Phosphopeptides
    Ionizing Radiation
    Serine
    Neoplasms

    Keywords

    • Nuclear proteins
    • Protein processing
    • Proto-oncogene proteins physiology
    • Tumour suppressor protein p53 chemistry
    • Mdm2 protein
    • p53
    • Phosphorylation
    • Hypophosphorylation

    Cite this

    Blattner, Christine ; Hay, Trevor ; Meek, David W. ; Lane, David P. / Hypophosphorylation of Mdm2 augments p53 stability. In: Molecular and Cellular Biology. 2002 ; Vol. 22, No. 17. pp. 6170-6182.
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    abstract = "The Mdm2 protein mediates ubiquitylation and degradation of p53 and is a key regulator of this tumor suppressor. More recently, it has been shown that Mdm2 is highly phosphorylated within its central acidic domain. In order to address the issue of how these modifications might regulate Mdm2 function, putative phosphorylation sites within this domain were substituted, individually or in pairs, with alanine residues. Mutants with serine-to-alanine substitutions between residues 244 and 260 abolished or at least reduced the capacity of Mdm2 to promote p53 degradation. In each case, loss of degradation function was independent of the ability to bind to p53 or p14ARF. Moreover, each of the Mdm2 mutants completely retained the capacity to act as a ubiquitin ligase in vivo. Thus, ubiquitylation and degradation can be uncoupled. Two-dimensional phosphopeptide mapping coupled with the use of phospho-specific antibodies revealed that Mdm2 is phosphorylated physiologically at several sites within this region, consistent with the idea that phosphorylation is important for Mdm2 activity. Strikingly, treatment of cells with ionizing radiation resulted in a significant decrease in the phosphorylation of residues that are important for p53 turnover. This hypophosphorylation preceded p53 accumulation. These findings indicate that Mdm2 contributes an additional function toward the degradation of p53 that is distinct from its ubiquitin ligase activity and is regulated by phosphorylation. Our model suggests that hypophosphorylation of Mdm2 in response to ionizing irradiation inactivates this novel function, thereby contributing to p53 stabilization.",
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    Hypophosphorylation of Mdm2 augments p53 stability. / Blattner, Christine; Hay, Trevor; Meek, David W.; Lane, David P.

    In: Molecular and Cellular Biology, Vol. 22, No. 17, 09.2002, p. 6170-6182.

    Research output: Contribution to journalArticle

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    T1 - Hypophosphorylation of Mdm2 augments p53 stability

    AU - Blattner, Christine

    AU - Hay, Trevor

    AU - Meek, David W.

    AU - Lane, David P.

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    AB - The Mdm2 protein mediates ubiquitylation and degradation of p53 and is a key regulator of this tumor suppressor. More recently, it has been shown that Mdm2 is highly phosphorylated within its central acidic domain. In order to address the issue of how these modifications might regulate Mdm2 function, putative phosphorylation sites within this domain were substituted, individually or in pairs, with alanine residues. Mutants with serine-to-alanine substitutions between residues 244 and 260 abolished or at least reduced the capacity of Mdm2 to promote p53 degradation. In each case, loss of degradation function was independent of the ability to bind to p53 or p14ARF. Moreover, each of the Mdm2 mutants completely retained the capacity to act as a ubiquitin ligase in vivo. Thus, ubiquitylation and degradation can be uncoupled. Two-dimensional phosphopeptide mapping coupled with the use of phospho-specific antibodies revealed that Mdm2 is phosphorylated physiologically at several sites within this region, consistent with the idea that phosphorylation is important for Mdm2 activity. Strikingly, treatment of cells with ionizing radiation resulted in a significant decrease in the phosphorylation of residues that are important for p53 turnover. This hypophosphorylation preceded p53 accumulation. These findings indicate that Mdm2 contributes an additional function toward the degradation of p53 that is distinct from its ubiquitin ligase activity and is regulated by phosphorylation. Our model suggests that hypophosphorylation of Mdm2 in response to ionizing irradiation inactivates this novel function, thereby contributing to p53 stabilization.

    KW - Nuclear proteins

    KW - Protein processing

    KW - Proto-oncogene proteins physiology

    KW - Tumour suppressor protein p53 chemistry

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    KW - Hypophosphorylation

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    DO - 10.1128/MCB.22.17.6170-6182.2002

    M3 - Article

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    JO - Molecular and Cellular Biology

    JF - Molecular and Cellular Biology

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