A novel Aurora-A-mediated phosphorylation of p53 inhibits its interaction with MDM2.

Crosstalk between Aurora-A kinase and p53 has been proposed. While the genetic amplification of Aurora-A has been observed in many human cancers, how p53 is regulated by Aurora-A remains ambiguous. In this study, Aurora-A-mediated phosphorylation of p53 was analyzed by mass spectrometry in order to identify a new phosphorylation site. ...
Subsequently, the functional consequences of such phosphorylation were examined.In vitro phosphorylation of p53 by Aurora-A was performed and the phosphorylated protein was then digested with trypsin and enriched for phosphopeptides by immobilized metal affinity chromatography. Subsequently, a combination of β-elimination and Michael addition was applied to the phosphopeptides in order to facilitate the identification of phosphorylation sites by MS. The functional consequences of the novel phosphorylation of p53 on the protein-protein interactions, protein stability and transactivation activity were then examined using co-immunoprecipitation, Western blotting and reporter assays.Ser-106 of p53 was identified as a novel site phosphorylated by Aurora-A. A serine-to-alanine mutation at this site was found to attenuate Aurora-A-mediated phosphorylation in vitro. In addition, phosphate-sensitive Phos-tag SDS-PAGE was used to confirm that the Ser-106 of p53 is in vivo phosphorylated by Aurora-A. Finally, co-immunoprecipitation studies suggested that Ser-106 phosphorylation of p53 decreases its interaction with MDM2 and prolongs the half-life of p53.The inhibition of the interaction between p53 and MDM2 by a novel Aurora-A-mediated p53 phosphorylation was identified in this study and this provides important information for further investigations into the interaction between p53 and Aurora-A in terms of cancer biology.
Mesh Terms:
Aurora Kinases, Cell Line, Tumor, HEK293 Cells, Humans, Neoplasms, Peptide Mapping, Phosphorylation, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins c-mdm2, Serine, Tumor Suppressor Protein p53
Biochim. Biophys. Acta
Date: Feb. 01, 2013
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