Regulation by phosphorylation of the relative affinities of the N-terminal transactivation domains of p53 for p300 domains and Mdm2.
The transcriptional activity of the tumour suppressor, p53, requires direct binding between its transactivation domain (TAD, 1-57) and the transcriptional coactivator, p300. We systematically assessed the role of TAD phosphorylation on binding of the p300 domains CH3, Taz1, Kix and IBiD. Thr18 phosphorylation increased the affinity up to sevenfold for CH3 and Taz1, with smaller increases from phosphorylation of Ser20, Ser15, Ser37, Ser33, Ser46 and Thr55. Binding of Kix and IBiD was less sensitive to phosphorylation. Strikingly, hepta-phosphorylation of all Ser and Thr residues increased binding 40- and 80-fold with CH3 and Taz1, respectively, but not with Kix or IBiD. Substitution of all phospho-sites with aspartates partially mimicked the effects of hepta-phosphorylation. Mdm2, the main negative regulator of p53, competes with p300 for binding to TAD. Binding of Mdm2 to TAD was reduced significantly only on phosphorylation of Thr18 (sevenfold) or by hepta-phosphorylation (24-fold). The relative affinities of Mdm2 and p300 for p53 TAD can thus be changed by up to three orders of magnitude by phosphorylation. Accordingly, phosphorylation of Thr18 and hepta-phosphorylation dramatically shifts the balance towards favouring the binding of p300 with p53, and is thus likely to be an important factor in its regulation.
Department of Chemistry, MRC Centre for Protein Engineering and Cambridge University, MRC Centre, Cambridge, UK.
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Teufel
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Bycroft
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Fersht
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D050881:E1A-Associated p300 Protein / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism*
D010446:Peptide Fragments; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D050881:E1A-Associated p300 Protein; C494360:EP300 protein, human; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2