Inhibition of E2F-1 transactivation by direct binding of the retinoblastoma protein.
Loss of a functional retinoblastoma tumor suppressor gene product, pRB, is a key step in the development of many human tumors. pRB is a negative regulator of cell proliferation and appears to participate in control of entry into the S phase of the cell cycle. The recent demonstration that pRB binds to transcription factor E2F has provided a model for the mechanism of pRB-mediated growth regulation. Since adenovirus E1A proteins dissociate the pRB-E2F complexes and stimulate E2F-dependent transcription, it has been suggested that pRB inhibits E2F transactivation. Although some evidence for this hypothesis has been provided, it has not been possible to determine the mechanism of pRB-mediated inhibition of E2F transactivation. In this study, we constructed mutants of E2F-1 that do not bind to pRB yet retain the ability to transactivate the adenovirus E2 promoter through E2F DNA-binding sites. We demonstrated that transactivation mediated by the wild-type E2F-1 protein was inhibited by overexpression of wild-type pRB but not by a naturally occurring mutant of pRB. Transactivation mediated by mutants of E2F-1 which do not bind to pRB was not affected by overexpression of wild-type pRB. Furthermore, when the E2F-1 transactivation domain was fused to the GAL4 DNA-binding domain, pRB inhibited GAL4-E2F-1 transactivation through GAL4 sites. Expression of pRB did not inhibit transactivation mediated by GAL4-E2F-1 mutant constructs which were devoid of pRB binding. In conclusion, these data demonstrate that pRB inhibits E2F-dependent transactivation by direct protein-protein interaction.
Helin
K
K
Massachusetts General Hospital Cancer Center, Charlestown 02129.
Harlow
E
E
Fattaey
A
A
D001665:Binding Sites; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D005656:Fungal Proteins / Q000378:metabolism; D006801:Humans; D016296:Mutagenesis; D016160:Retinoblastoma Protein / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D029701:Saccharomyces cerevisiae Proteins; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D029701:Saccharomyces cerevisiae Proteins; D050680:Transcription Factor DP1; D014157:Transcription Factors; D004247:DNA