Dissociation of DNA binding and in vitro transcriptional activities dependent on the C terminus of P53 proteins.
Wild type p53 protein requires posttranslational modification within a carboxy-terminal negative regulatory domain to activate DNA binding and transcription. Binding of monoclonal antibody PAb421 to the carboxy-terminal domain reproduces this activation. In the absence of PAb421, we found that wild type p53 bound actively to a template containing two copies of the p21WAF1p53 binding site. However, in an in vitro transcription assay with partially purified basal transcription factors, p53 only partially activated transcription from the same binding site and required PAb421 for full activation. Oncogenic missense mutant p53 proteins (N239 to S239, G245 to S245, R273 to H273) bound the WAF1 doublet significantly and were activated further by PAb421. However, these mutants were inactive in the transcription assay, even with PAb421. These results indicate that sequence-specific binding and transcriptional activities of p53 can be dissociated through C-terminal interactions and suggest that conformational changes induced by the mutations alter p53 interactions with basal transcription factors.
Kaku
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Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, New York 14263, USA.
Albor
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Kulesz-Martin
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53