BACKGROUND: Amino acids 1-107 of the SV40 T antigen constitute a functionally important and complex region. Cellular proteins, Hsc70, Bub-1, Cul-7, and Rb, each of which is involved in cell growth control or genomic stability, bind within this portion of the T antigen. Mutational analysis has mapped the J domain/Hsc70, Bub-1, and the Rb binding motifs. Two regions of the T antigen have been implicated in Cul-7 binding. Mutation of F98A diminished Cul-7 binding, and deletion of amino acids 68-83 abolished it. The authors suggest, based on T-antigen structure, that F98 is inaccessible and that the F98A change altered the configuration of the upstream region, preventing Cul-7 binding. Our objective was to determine, by using monoclonal T-antigen antibodies, whether F98 is accessible and whether F98A substitution globally distorted the T-common region. METHODS: Cell-expressing T antigens, immunoprecipitation, and immunoblot were used to determine the accessibility of amino acids. CONCLUSION: Full-length T-antigen and N-terminal fragments containing F98A were immunoprecipitated by monoclonal antibody PAb902, which recognizes a conformation-dependent epitope within the first 82 amino acids. Therefore, this alteration does not globally distort the entire T-common region. Additionally, PAb416, which displaces Cul-7 from the T antigen and immunoprecipitates bound pRb peptides, depends on F98 for binding, implying that amino acid 98 is part of the epitope and accessible in the native T antigen.
BACKGROUND: Amino acids 1-107 of the SV40 T antigen constitute a functionally important and complex region. Cellular proteins, Hsc70, Bub-1, Cul-7, and Rb, each of which is involved in cell growth control or genomic stability, bind within this portion of the T antigen. Mutational analysis has mapped the J domain/Hsc70, Bub-1, and the Rb binding motifs. Two regions of the T antigen have been implicated in Cul-7 binding. Mutation of F98A diminished Cul-7 binding, and deletion of amino acids 68-83 abolished it. The authors suggest, based on T-antigen structure, that F98 is inaccessible and that the F98A change altered the configuration of the upstream region, preventing Cul-7 binding. Our objective was to determine, by using monoclonal T-antigen antibodies, whether F98 is accessible and whether F98A substitution globally distorted the T-common region. METHODS: Cell-expressing T antigens, immunoprecipitation, and immunoblot were used to determine the accessibility of amino acids. CONCLUSION: Full-length T-antigen and N-terminal fragments containing F98A were immunoprecipitated by monoclonal antibody PAb902, which recognizes a conformation-dependent epitope within the first 82 amino acids. Therefore, this alteration does not globally distort the entire T-common region. Additionally, PAb416, which displaces Cul-7 from the T antigen and immunoprecipitates bound pRb peptides, depends on F98 for binding, implying that amino acid 98 is part of the epitope and accessible in the native T antigen.