Novel phosphorylation sites of human tumour suppressor protein p53 at Ser20 and Thr18 that disrupt the binding of mdm2 (mouse double minute 2) protein are modified in human cancers

A L Craig, L Burch, B Vojtesek, J Mikutowska, A Thompson, T R Hupp

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

The ability to separate the isoforms of human tumour suppressor protein p53 expressed in insect cells using heparin-Sepharose correlates with differences in the isoelectric point of p53, demonstrating that p53 can be heterogeneously modified and providing support for the use of insect cells as a model system for identifying novel signalling pathways that target p53. One p53 isoform that was reduced in its binding to the monoclonal antibody DO-1 could be stimulated in its binding to DO-1 by prior incubation with protein phosphatases, suggesting the presence of a previously unidentified N-terminal phosphorylation site capable of masking the DO-1 epitope. A synthetic peptide from the N-terminal domain of p53 containing phosphate at Ser(20) inhibited DO-1 binding, thus identifying the phosphorylation site responsible for DO-1 epitope masking. Monoclonal antibodies overlapping the DO-1 epitope were developed that are specific for phospho-Thr(18) (adjacent to the DO-1 epitope) and phospho-Ser(20) (within the DO-1 epitope) to determine whether direct evidence could be obtained for novel phosphorylation sites in human p53. A monoclonal antibody highly specific for phospho-Ser(20) detected significant phosphorylation of human p53 expressed in insect cells, whereas the relative proportion of p53 modified at Thr(18) was substantially lower. The relevance of these two novel phosphorylation sites to p53 regulation in human cells was made evident by the extensive phosphorylation of human p53 at Thr(18) and Ser(20) in a panel of human breast cancers with a wild-type p53 status. Phospho-Ser(20) or phospho-Thr(18) containing p53 peptides are as effective as the phospho-Ser(15) peptide at reducing mdm2 (mouse double minute 2) protein binding, indicating that the functional effects of these phosphorylation events might be to regulate the binding of heterologous proteins to p53. These results provide evidence in vivo for two novel phosphorylation sites within p53 at Ser(20) and Thr(18) that can affect p53 protein-protein interactions and indicate that some human cancers might have amplified one or more Ser(20) and Thr(18) kinase signalling cascades to modulate p53 activity.
Original languageEnglish
Pages (from-to)133-41
Number of pages9
JournalBiochemical Journal
Volume342 ( Pt 1)
Publication statusPublished - 15 Aug 1999

Keywords / Materials (for Non-textual outputs)

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal
  • Antibody Specificity
  • Breast Neoplasms
  • Cell Line
  • Epitopes
  • Humans
  • Isoelectric Point
  • Nuclear Proteins
  • Peptide Fragments
  • Phosphoric Monoester Hydrolases
  • Phosphorylation
  • Phosphoserine
  • Phosphothreonine
  • Protein Isoforms
  • Protein Processing, Post-Translational
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-mdm2
  • Recombinant Proteins
  • Signal Transduction
  • Spodoptera
  • Tumor Suppressor Protein p53


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