A competitive inhibitor assay for SV40 T/pRB complex formation employing extracts of SV40-transformed human lung cells.
Simian virus 40 (SV40) large T antigen (T), a potent dominant oncogene product, forms a specific complex with the human retinoblastoma protein (pRb), a cellular growth suppressor. We have used a recombinant pRb fusion protein (GST-Rb) in combination with extracts from a line of SV40-transformed human lung cells (WI-26 VA4) to develop a simple, non-radioactive assay to rapidly screen for competitive inhibitors of T/pRb binding. We illustrate the use of the assay by demonstrating that several short peptides containing the signature sequence, Leu-X-Cys-X-Glu, can inhibit T/pRb complex formation. In contrast, peptides containing the related motif, Leu-X-Glu-X-Glu, including two peptides derived from the transcription factor E2F, are inactive in this assay. These results show that Glu cannot substitute for Cys in the Leu-X-Cys-X-Glu motif. This assay will facilitate the identification of agents that are inhibitors of T/pRb complex formation and that might exert effects on cellular growth regulation.
Breese
K
K
Department of Microbiology, Immunology & Molecular Genetics, Albany Medical College, New York 12208.
Friedrich
T
T
Figge
J
J
D000595:Amino Acid Sequence; D000952:Antigens, Polyomavirus Transforming / Q000032:analysis / Q000378:metabolism*; D001667:Binding, Competitive; D001681:Biological Assay; D015153:Blotting, Western; D002457:Cell Extracts / Q000494:pharmacology*; D002461:Cell Line, Transformed; D002846:Chromatography, Affinity; D006801:Humans; D008168:Lung / Q000166:cytology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000032:analysis / Q000378:metabolism*
D000952:Antigens, Polyomavirus Transforming; D002457:Cell Extracts; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein