The MDM2 ubiquitination signal in the DNA-binding domain of p53 forms a docking site for calcium calmodulin kinase superfamily members

Ashley L Craig, Jennifer A Chrystal, Jennifer A Fraser, Nathalie Sphyris, Yao Lin, Ben J Harrison, Mary T Scott, Irena Dornreiter, Ted R Hupp

Research output: Contribution to journalArticlepeer-review

Abstract

Genetic and biochemical studies have shown that Ser(20) phosphorylation in the transactivation domain of p53 mediates p300-catalyzed DNA-dependent p53 acetylation and B-cell tumor suppression. However, the protein kinases that mediate this modification are not well defined. A cell-free Ser(20) phosphorylation site assay was used to identify a broad range of calcium calmodulin kinase superfamily members, including CHK2, CHK1, DAPK-1, DAPK-3, DRAK-1, and AMPK, as Ser(20) kinases. Phosphorylation of a p53 transactivation domain fragment at Ser(20) by these enzymes in vitro can be mediated in trans by a docking site peptide derived from the BOX-V domain of p53, which also harbors the ubiquitin signal for MDM2. Evaluation of these calcium calmodulin kinase superfamily members as candidate Ser(20) kinases in vivo has shown that only CHK1 or DAPK-1 can stimulate p53 transactivation and induce Ser(20) phosphorylation of p53. Using CHK1 as a prototypical in vivo Ser(20) kinase, we demonstrate that (i) CHK1 protein depletion using small interfering RNA can attenuate p53 phosphorylation at Ser(20), (ii) an enhanced green fluorescent protein (EGFP)-BOX-V fusion peptide can attenuate Ser(20) phosphorylation of p53 in vivo, (iii) the EGFP-BOX-V fusion peptide can selectively bind to CHK1 in vivo, and (iv) the Deltap53 spliced variant lacking the BOX-V motif is refractory to Ser(20) phosphorylation by CHK1. These data indicate that the BOX-V motif of p53 has evolved the capacity to bind to enzymes that mediate either p53 phosphorylation or ubiquitination, thus controlling the specific activity of p53 as a transcription factor.
Original languageEnglish
Pages (from-to)3542-55
Number of pages14
JournalMolecular and Cellular Biology
Volume27
Issue number9
DOIs
Publication statusPublished - May 2007

Keywords

  • Amino Acid Motifs
  • Apoptosis Regulatory Proteins
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Cell Line, Tumor
  • Checkpoint Kinase 2
  • DNA
  • Death-Associated Protein Kinases
  • Enzyme Activation
  • Gene Deletion
  • Humans
  • Molecular Sequence Data
  • Peptide Fragments
  • Protein Binding
  • Protein Kinases
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-mdm2
  • Sequence Homology, Amino Acid
  • Transcriptional Activation
  • Tumor Suppressor Protein p53
  • Ubiquitin

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