Characterization of the in vitro interaction between SV40 T antigen and p53: mapping the p53 binding site.
An efficient in vitro system for generating soluble complexes between simian virus 40 T antigen and the cellular protein p53 was developed. A p53 cDNA was inserted 3' to the SP6 promoter in pGEM-1 (Promega-Biotec) and transcribed by SP6 polymerase. In vitro translation of the cRNA generated p53 which was immunoprecipitable with all five monoclonal antibodies tested (PAb122, PAb421, PAb242, PAb246, and PAb248). The p53 sedimented at about 8-10 S in sucrose gradients, possibly corresponding to a tetramer. T-antigen-p53 complexes were produced by the addition of immunoaffinity-purified T antigen to p53-containing translation lysates. Equivalent amounts of p53 were immunoprecipitated with the anti-T-antigen antibodies PAb416, PAb419, and PAb101, suggesting that in vitro made p53 complexed mostly to a population of T-antigen molecules that had matured at least 15 min in the cell. The complexes sedimented at 18-20 S in sucrose gradients. In order to map the p53 binding site on T antigen, p53 was complexed in vitro to labeled proteolytic fragments of T antigen. A 46K fragment, spanning residues 131-517, was immunoprecipitated with the anti-p53 monoclonal PAb122 and therefore is likely to contain the p53 binding site. This region contains T-antigen sequences necessary for the efficient transformation of nonpermissive cells and for the induction of cellular rRNA synthesis. It also contains the binding sites for DNA polymerase alpha and ATP. We suggest a possible role for T-p53 complexes in T-antigen-associated functions.
Schmieg
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School of Life and Health Sciences, University of Delaware, Newark 19716.
Simmons
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D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D001665:Binding Sites; D004247:DNA / Q000235:genetics; D009363:Neoplasm Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013539:Simian virus 40 / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53
D000952:Antigens, Polyomavirus Transforming; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA