Phosphorylation of Thr18 and Ser20 of p53 in Ad-p53-induced apoptosis

Neuro Oncol. 2008 Jun;10(3):275-91. doi: 10.1215/15228517-2008-015. Epub 2008 Apr 28.

Abstract

The p53 protein plays a critical role in inducing cell cycle arrest or apoptosis. Because p53 is inactivated in human gliomas, restoring p53 function is a major focus of glioma therapy. The most clinically tested strategy for replacing p53 has been adenoviral-mediated p53 gene therapy (Ad-p53). In addition to their therapeutic implications, investigations into Ad-p53 provide model systems for understanding p53's ability to induce cell cycle arrest versus apoptosis, particularly because wild-type p53 cells are resistant to Ad-p53-induced apoptosis. Here we use Ad-p53 constructs to test the hypothesis that simultaneous phosphorylation of p53 at threonine 18 (Thr18) and serine 20 (Ser20) is causally associated with p53-mediated apoptosis. Studies using phosphorylation-specific antibodies demonstrated that p53-induced apoptosis correlates with phosphorylation of p53 at Thr18 and Ser20 but not with carboxy-terminal phosphorylation (Ser392). To prove a causal relationship between apoptosis and Thr18 and Ser20 phosphorylation of p53, the effects of an adenoviral p53 construct that was not phosphorylated (Ad-p53) was compared with a Thr18/Ser20 phosphomimetic construct (Ad-p53-18D20D) in wild-type p53 gliomas. Whereas treatment with Ad-p53 resulted only in cell cycle arrest, treatment with Ad-p53-18D20D induced dramatic apoptosis. Microarray and Western blot analyses showed that only Ad-p53-18D20D was capable of inducing expression of apoptosis-inducing proteins. Chromatin immunoprecipitation assays indicated that the protein product of Ad-p53-18D20D, but not Ad-p53, was capable of binding to apoptosis-related genes. We thus conclude that phosphorylation of Thr18 and Ser20 is sufficient for inducing p53-mediated apoptosis in glioma cells. These results have implications for p53 gene therapy and inform other strategies that aim to restore p53 function.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviridae
  • Apoptosis / physiology*
  • Blotting, Western
  • Cell Cycle / physiology
  • Cell Line, Tumor
  • Flow Cytometry
  • Fluorescent Antibody Technique
  • Gene Expression
  • Gene Expression Regulation
  • Genetic Therapy / methods*
  • Genetic Vectors
  • Humans
  • Immunoprecipitation
  • Oligonucleotide Array Sequence Analysis
  • Phosphorylation
  • Polymerase Chain Reaction
  • Serine / metabolism*
  • Threonine / metabolism*
  • Tumor Suppressor Protein p53 / metabolism*

Substances

  • Tumor Suppressor Protein p53
  • Threonine
  • Serine