Protein-protein Interactions Occur Between p53 Phosphoforms and ATM and 53BP1 at Sites of Exogenous DNA Damage

Radiat Res. 2011 May;175(5):588-98. doi: 10.1667/RR2084.1. Epub 2011 Mar 1.

Abstract

We have previously shown that the Ser15-phosphorylated p53 phosphoform, p53(Ser15), can localize at sites of ionizing radiation-induced DNA damage. In this study, we hypothesized that the non-specific DNA binding domain (NSDBD) of the p53 carboxy-terminus (C-terminus) mediates chromatin anchoring at sites of DNA damage to interact with two key mediators of the DNA damage response (DDR): ATM and 53BP1. Exogenous YFP-p53 fusion constructs expressing C-terminus deletion mutants of p53 were transfected into p53-null H1299 cells and tracked by microscopy and biochemistry to determine relative chromatin-binding pre- and postirradiation. We observed that exogenous YFP-p53(WT) and YFP-p53(Δ367-393) associated with ATM(Ser1981) and 53BP1 in the nuclear, chromatin-bound fractions after DNA damage. Of interest, YFP-p53(Δ1-299) fusion proteins, which lack transcriptional trans-activation and the Ser15-residue, bound to ATM(Ser1981) but not to 53BP1. In support of these data, we used subnuclear UV-microbeam and immunoprecipitation analyses of irradiated normal human fibroblasts (HDFs) that confirmed an interaction between endogenous p53 and ATM or 53BP1. Based on these observations, we propose a model whereby a pre-existing pool of p53 responds immediately to radiation-induced DNA damage using the C-terminus to spatially facilitate protein-protein interactions and the DDR at sites of DNA damage.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ataxia Telangiectasia Mutated Proteins
  • Cell Cycle Proteins / metabolism*
  • Cell Line
  • Chromatin / metabolism
  • DNA Damage*
  • DNA-Binding Proteins / metabolism*
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Phosphoproteins / chemistry
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Phosphorylation / radiation effects
  • Protein Binding / radiation effects
  • Protein Transport / radiation effects
  • Protein-Serine-Threonine Kinases / metabolism*
  • Sequence Deletion
  • Serine / metabolism
  • Signal Transduction / radiation effects
  • Tumor Suppressor Protein p53 / chemistry
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism*
  • Tumor Suppressor Proteins / metabolism*
  • Tumor Suppressor p53-Binding Protein 1

Substances

  • Cell Cycle Proteins
  • Chromatin
  • DNA-Binding Proteins
  • Intracellular Signaling Peptides and Proteins
  • Phosphoproteins
  • TP53BP1 protein, human
  • Tumor Suppressor Protein p53
  • Tumor Suppressor Proteins
  • Tumor Suppressor p53-Binding Protein 1
  • Serine
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Protein-Serine-Threonine Kinases