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

Record type: Article

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 Ser315 phospho-epitope was used to determine whether phosphorylation of endogenous p53 at Ser315 can be detected in vivo, whether steady-state Ser315 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 Ser315 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 Ser315 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 Ser315 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 Ser315 after irradiation damage and identify a cyclin-dependent kinase pathway that plays a role in stimulating p53 function.

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Citation

Blaydes, Jeremy P., Luciani, M. Gloria, Pospisilova, Sarka, Ball, Helen M., Vojtesek, Borek and Hupp, Ted R. (2001) Stoichiometric phosphorylation of human p53 at Ser315 stimulates p53-dependent transcription The Journal of Biological Chemistry, 276, (7), pp. 4699-4708. (doi:10.1074/jbc.M003485200).

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Published date: 2001

Identifiers

Local EPrints ID: 26221
URI: http://eprints.soton.ac.uk/id/eprint/26221
ISSN: 0021-9258
PURE UUID: 481aba18-cc9c-46bc-bb8c-e57721db1d06

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Date deposited: 21 Apr 2006
Last modified: 17 Jul 2017 16:08

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Contributors

Author: M. Gloria Luciani
Author: Sarka Pospisilova
Author: Helen M. Ball
Author: Borek Vojtesek
Author: Ted R. Hupp

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