title: SV40 T-Antigen Amino Acid Changes that Disrupt Cul-7 or Bub-1 Binding Do Not Globally Distort the T-Common Region.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biology, Elizabethtown College, Elizabethtown, Pa., USA.
authors.forename: Jane F
authors.initials: JF
authors.lastname: Cavender
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Tevethia
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D002472:Cell Transformation, Viral; D044842:Cullin Proteins / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming; C506299:CUL7 protein, human; D044842:Cullin Proteins; D000939:Epitopes; C584277:BUB1 protein, human; D017346:Protein Serine-Threonine Kinases