title: Two distinct motifs within the p53 transactivation domain bind to the Taz2 domain of p300 and are differentially affected by phosphorylation.
abstract: The tumor suppressor p53 functions as a transcriptional activator for many genes, including several key genes involved in cell cycle arrest and apoptosis. Following DNA damage-induced stress, p53 undergoes extensive posttranslational modification, resulting in increased stability and activity. Two critical cofactors for p53-mediated transactivation are the histone acetyltransferase paralogues CREB-binding protein (CBP) and p300. The N-terminal transactivation domain of p53 interacts with several domains of CBP/p300, including the Taz2 domain. Here, we report the effects of specific p53 phosphorylations on its interaction with the Taz2 domain of p300. Using a competitive fluorescence anisotropy assay, we determined that monophosphorylation of p53 at Ser(15) or Thr(18) increased the affinity of p53(1-39) for Taz2, and diphosphorylations at Ser(15) and Ser(37) or Thr(18) and Ser(20) further increased the affinity. In addition, we identified a second binding site for Taz2 within p53 residues 35-59. This second site bound Taz2 with a similar affinity as the first site, but the binding was unaffected by phosphorylation. Thus, p53 posttranslational modification modulates only one of the two binding sites for p300 Taz2. Further investigation of Taz2 binding to p53(1-39) or p53(35-59) by isothermal titration calorimetry indicated that upon complex formation, the change in heat capacity at constant pressure, DeltaC(p), was negative for both sites, suggesting the importance of hydrophobic interactions. However, the more negative value of DeltaC(p) for Taz2 binding to the first (-330 cal/(mol.K)) compared to the second site (-234 cal/(mol.K)) suggests that the importance of nonpolar and polar interactions differs between the two sites.
authors.affiliation: Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Lisa M Miller
authors.initials: LM
authors.lastname: Jenkins
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamaguchi
authors.forename: Ryo
authors.initials: R
authors.lastname: Hayashi
authors.forename: Scott
authors.initials: S
authors.lastname: Cherry
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Tropea
authors.forename: Maria
authors.initials: M
authors.lastname: Miller
authors.forename: Alexander
authors.initials: A
authors.lastname: Wlodawer
authors.forename: Ettore
authors.initials: E
authors.lastname: Appella
authors.forename: Sharlyn J
authors.initials: SJ
authors.lastname: Mazur
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002151:Calorimetry; D002942:Circular Dichroism; D050881:E1A-Associated p300 Protein / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013816:Thermodynamics; D015533:Transcriptional Activation / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D050881:E1A-Associated p300 Protein; C494360:EP300 protein, human; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2