Chow K N, Dean D C
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Mol Cell Biol. 1996 Sep;16(9):4862-8. doi: 10.1128/MCB.16.9.4862.
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.
视网膜母细胞瘤蛋白（Rb）是一种肿瘤抑制因子，可调节细胞周期从G1期到S期的进程。此前，我们发现Rb是一种转录抑制因子，通过与细胞周期转录因子E2F家族相互作用而选择性地靶向启动子——当Rb通过E2F与启动子相连时，它不仅会阻断E2F的活性，还会结合周围的转录因子，阻止它们与基础转录复合物相互作用，从而对细胞周期基因的转录产生显性抑制作用。在此，我们研究了Rb的抑制基序。Rb口袋中的两个结构域A和B在物种间以及与Rb相关蛋白p107和p130中都是保守的，它们对于抑制活性都是必需的。分隔A和B的非保守间隔区则不是必需的。虽然单独的A或B都没有任何抑制活性，但令人惊讶的是，当这些结构域在不同的蛋白上共表达时，观察到了抑制活性。转染实验表明，一个结构域可以将另一个结构域招募到启动子上，形成一个既能与E2F相互作用又能对转录产生显性抑制作用的抑制基序。通过免疫共沉淀和体外结合实验，我们表明A和B直接相互作用，破坏这种相互作用的突变会抑制抑制活性。Rb口袋最初被定义为来自DNA肿瘤病毒（如腺病毒E1a）的癌蛋白的结合位点。我们提供的证据表明，E1a与由A和B相互作用形成的位点相互作用，并且这种与A和B的相互作用诱导或稳定了A - B的相互作用。
