title: 14-3-3 activation of DNA binding of p53 by enhancing its association into tetramers.
abstract: Activation of the tumour suppressor p53 on DNA damage involves post-translational modification by phosphorylation and acetylation. Phosphorylation of certain residues is critical for p53 stabilization and plays an important role in DNA-binding activity. The 14-3-3 family of proteins activates the DNA-binding affinity of p53 upon stress by binding to a site in its intrinsically disordered C-terminal domain containing a phosphorylated serine at 378. We have screened various p53 C-terminal phosphorylated peptides for binding to two different isoforms of 14-3-3, epsilon and gamma. We found that phosphorylation at either S366 or T387 caused even tighter binding to 14-3-3. We made by semi-synthesis a tetrameric construct comprised of the tetramerization plus C-terminal domains of p53 that was phosphorylated on S366, S378 and T387. It bound 10 times tighter than did the monomeric counterpart to dimeric 14-3-3. We showed indirectly from binding curves and directly from fluorescence-detection analytical ultracentrifugation that 14-3-3 enhanced the binding of sequence-specific DNA to p53 by causing p53 dimers to form tetramers at lower concentrations. If the in vitro data extrapolate to in vivo, then it is an attractive hypothesis that p53 activity may be subject to control by accessory proteins lowering its tetramer-dimer dissociation constant from its normal value of 120-150 nM.
authors.affiliation: MRC Laboratory of Molecular Biology and MRC Centre for Protein Engineering, Hills Road, Cambridge, CB2 0QH, UK.
authors.forename: Sridharan
authors.initials: S
authors.lastname: Rajagopalan
authors.forename: Agnes M
authors.initials: AM
authors.lastname: Jaulent
authors.forename: Mark
authors.initials: M
authors.lastname: Wells
authors.forename: Dmitry B
authors.initials: DB
authors.lastname: Veprintsev
authors.forename: Alan R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D005454:Fluorescence Polarization; D010455:Peptides / Q000378:metabolism; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D010455:Peptides; D010748:Phosphopeptides; D016159:Tumor Suppressor Protein p53; D004247:DNA