Identification of mutations in p53 that affect its binding to SV40 large T antigen by using the yeast two-hybrid system.
The tumor suppressor p53 protein binds to the products of several viral oncogenes, including SV40 large T antigen. We reconstructed the p53-T antigen interaction in the yeast two-hybrid system, a genetic assay that uses the reconstitution of the activity of a transcriptional activator to detect protein-protein interactions. Using mutants of T antigen known to be defective in binding to p53, we demonstrate that the two-hybrid system is more sensitive than immunoprecipitation in the detection of weak interactions. We mutagenized the murine p53 gene and screened in the yeast assay for decreased reporter gene expression indicative of the failure of p53 to bind T antigen. This screen identified 34 p53 mutants, almost all of which contain at least one mutation in the conserved domains frequently found mutated in human cancers. These results support the idea that the function of the wild-type p53 protein requires residues involved in binding to T antigen, and indicate that this approach may be generally applicable in the analysis of protein-protein interactions.
Li
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B
Graduate Program In Molecular and Cellular Biology, State University of New York, Stony Brook 11794.
Fields
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D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D016158:Genes, p53; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D016159:Tumor Suppressor Protein p53