The N-terminal interferon-binding domain (IBiD) homology domain of p300 binds to peptides with homology to the p53 transactivation domain.
Two high affinity Ser-20-phospho-LXXLL p53-binding domains of p300 map to the C-terminal interferon-binding domain (IBiD) and N-terminal IBiD homology domain (IHD) regions. Purified fractions of a recombinant IHD miniprotein are active in a set of in vitro assays highlighting its affinity to the N-terminal LXXLL domain of p53 including (i) dose-dependent binding to Ser-20-phosphorylated p53 tetramers; (ii) DNA-stimulated binding to p53 tetramers; and (iii) inhibition of MDM2-mediated p53 ubiquitination. The active component of the IHD miniprotein was localized to a 75-amino-acid fragment corresponding to amino acids 401-475 on human p300. This minimal IHD miniprotein can function in vivo as a p53-binding polypeptide in assays including: (i) complex formation with VP16-LXXLL peptide motifs in the two-hybrid assay; (ii) action as a dominant negative inhibitor of p53 from p21 luciferase templates; and (iii) attenuation of endogenous p21 protein levels. Further, we show here that the IRF-1-dependent stabilization and reactivation of p53DeltaPRO protein (LXXLL+/PXXP-) can be neutralized by the minimal IHD miniprotein, suggesting that IHD can bind to the p53 LXXLL domain in vivo. Phage-peptide display to the IHD miniprotein gave rise to an LSQXTFSXLXXLL consensus binding site that displays significant homology to the LXXLL transactivation domain of p53. These data validate the IHD scaffold as an independent LXXLL peptide-binding domain within the p300 protein, complementing the known peptide-binding domains including IBiD, C/H1, and C/H3.
Finlan
Lee
L
University of Edinburgh, Division of Oncology, CRUK Cell Signaling Unit, South Crewe Road, Edinburgh EH4 2XU, Scotland, United Kingdom.
Hupp
Ted R
TR
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D017353:Gene Deletion; D017930:Genes, Reporter; D006801:Humans; D007150:Immunohistochemistry; D007313:Insecta; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D015534:Trans-Activators / Q000737:chemistry*; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry*; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000737:chemistry
C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D009687:Nuclear Proteins; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D015534:Trans-Activators; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D012694:Serine; D008156:Luciferases