Domains A and B in the Rb pocket interact to form a transcriptional repressor motif.
The retinoblastoma protein (Rb) is a tumor suppressor that regulates progression from the G1 phase to the S phase of the cell cycle. Previously, we found that Rb is a transcriptional repressor that is selectively targeted to promoters through an interaction with the E2F family of cell cycle transcription factors--when Rb is tethered to a promoter through E2F, it not only blocks E2F activity, it also binds surrounding transcription factors, preventing their interaction with the basal transcription complex, thus resulting in a dominant inhibitory effect on transcription of cell cycle genes. Here we examine the repressor motif of Rb. The two domains in the Rb pocket, A and B, which are conserved across species and in the Rb-related proteins p107 and p130, are both required for repressor activity. The nonconserved spacer separating A and B is not required. Although neither A nor B alone had any repressor activity, surprisingly, repressor activity was observed when the domains were coexpressed on separate proteins. Transfection assays suggest that one domain can recruit the other to the promoter to form a repressor motif that can both interact with E2F and have a dominant inhibitory effect on transcription. Using coimmunoprecipitation and in vitro binding assays, we show that A and B interact directly and that mutations which disrupt this interaction inhibit repressor activity. The Rb pocket was originally defined as the binding site for oncoproteins from DNA tumor viruses such as adenovirus E1a. We present evidence that E1a interacts with a site formed by the interaction of A and B and that this interaction with A and B induces or stabilizes the A-B interaction.
Chow
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Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Dean
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D017500:Adenovirus E1A Proteins / Q000378:metabolism; D001483:Base Sequence; D008969:Molecular Sequence Data; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry*; D016160:Retinoblastoma Protein / Q000737:chemistry*; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
D017500:Adenovirus E1A Proteins; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D014157:Transcription Factors