Stoichiometric phosphorylation of human p53 at Ser315 stimulates p53-dependent transcription

J P Blaydes, M G Luciani, S Pospisilova, H M Ball, B Vojtesek, T R Hupp

Research output: Contribution to journalArticlepeer-review

Abstract

p53 protein activity as a transcription factor can be activated in vivo by antibodies that target its C-terminal negative regulatory domain suggesting that cellular enzymes that target this domain may play a role in stimulating p53-dependent gene expression. A phospho-specific monoclonal antibody to the C-terminal Ser(315) phospho-epitope was used to determine whether phosphorylation of endogenous p53 at Ser(315) can be detected in vivo, whether steady-state Ser(315) phosphorylation increases or decreases in an irradiated cell, and whether this phosphorylation event activates or inhibits p53 in vivo. A native phospho-specific IgG binding assay was developed for quantitating the extent of p53 phosphorylation at Ser(315) where one, two, three, or four phosphates/tetramer could be defined after in vitro phosphorylation by cyclin-dependent protein kinases. Using this assay, near-stoichiometric Ser(315) phosphorylation of endogenous p53 protein was detected in vivo after UV irradiation of MCF7 and A375 cells, coinciding with elevated p53-dependent transcription. Transfection of the p53 gene with an alanine mutation at the Ser(315) site into Saos-2 cells gave rise to a form of p53 protein with a substantially reduced specific activity as a transcription factor. The treatment of cells with the cyclin-dependent protein kinase inhibitor Roscovitine promoted a reduction in the specific activity of endogenous p53 or ectopically expressed p53. These results indicate that the majority of p53 protein has been phosphorylated at Ser(315) after irradiation damage and identify a cyclin-dependent kinase pathway that plays a role in stimulating p53 function.
Original languageEnglish
Pages (from-to)4699-708
Number of pages10
JournalJournal of Biological Chemistry
Volume276
Issue number7
DOIs
Publication statusPublished - 16 Feb 2001

Keywords

  • Antibodies, Monoclonal
  • DNA
  • Enzyme Inhibitors
  • Humans
  • Phosphorylation
  • Phosphoserine
  • Point Mutation
  • Purines
  • Transcription Factors
  • Transcriptional Activation
  • Tumor Cells, Cultured
  • Tumor Suppressor Protein p53
  • Ultraviolet Rays

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