Phosphorylation of human p53 on Thr-55.
The pleiotropic function of p53 is believed to be greatly influenced by phosphorylation, and several sites on p53 are known to be targets for distinct protein kinases. In this study, we observed that affinity-purified p53 from overexpressing cells was phosphorylated by a co-purified protein kinase in vitro. To identify phosphorylation site(s), the resulting phosphorylated p53 protein was subjected to primary and secondary proteolytic cleavage, and phosphopeptides were fractionated by a two-dimensional peptide gel system. Phosphoamino acid analysis and manual Edman degradation of the isolated phosphopeptides enabled us to unequivocally identify Thr-55 as the major phosphorylation site on p53. Furthermore, comparative phosphopeptide mapping data suggest that DNA-PK is not the kinase responsible for this phosphorylation. Significantly, using a phospho-specific antibody for Thr-55, we have shown that Thr-55 is indeed phosphorylated in vivo. These data define Thr-55 as a novel phosphorylation site and for the first time show threonine phosphorylation of human p53.
Department of Biochemistry, University of California, Riverside, California 92521, USA.
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Gatti
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Li
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Traugh
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Liu
D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D006801:Humans; D009154:Mutation; D009687:Nuclear Proteins; D010449:Peptide Mapping; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016159:Tumor Suppressor Protein p53; D010769:Phosphothreonine; D013912:Threonine; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases