Characterization of an E1A-CBP Interaction Defines a Novel Transcriptional Adapter Motif (TRAM) in CBP/p300

The adenovirus E1A protein subverts cellular processes to induce mitotic activity in quiescent cells. Important targets of E1A include members of the transcriptional adapter family containing CBP/p300. Competition for CBP/p300 binding by various cellular transcription factors has been suggested as a means of integrating different signalling pathways and may also represent a potential mechanism by which E1A manipulates cell fate. Here we describe the characterization of the interaction between E1A and the C/H3 region of CBP. We define a novel conserved 12-residue transcriptional adapter motif (TRAM) within CBP/p300 that represents the binding site for both E1A and numerous cellular transcription factors. We also identify a sequence (FPESLIL) within adenovirus E1A that is required to bind the CBP TRAM. Furthermore, an E1A peptide containing the FPESLIL sequence is capable of preventing the interaction between CBP and TRAM-binding transcription factors, such as p53, E2F, and TFIIB, thus providing a molecular model for E1A action. As an in vivo demonstration of this model, we used a small region of CBP containing a functional TRAM that can bind to the p53 protein. The CBP TRAM binds p53 sequences targeted by the cellular regulator MDM2, and we demonstrate that an MDM2-p53 interaction can be disrupted by the CBP TRAM, leading to stabilization of cellular p53 levels and the activation of p53-dependent transcription. Transcriptional activation of p53 by the CBP TRAM is abolished by wild-type E1A but not by a CBP-binding-deficient E1A mutant.

腺病毒E1A蛋白可劫持细胞正常进程，以诱导静息细胞产生有丝分裂活性。E1A的重要靶标包括含有CBP/p300的转录适配器家族成员。此前研究提出，多种细胞转录因子对CBP/p300结合位点的竞争，是整合不同信号通路的一种方式，这也可能是E1A调控细胞命运的潜在机制之一。本研究对E1A与CBP的C/H3区域之间的相互作用进行了表征。我们在CBP/p300中鉴定出一个全新的保守12残基转录适配器基序（transcriptional adapter motif, TRAM），该基序是E1A与多种细胞转录因子的结合位点。我们还在腺病毒E1A中鉴定出一段结合CBP TRAM所必需的序列（FPESLIL）。此外，一段包含FPESLIL序列的E1A肽段能够阻断CBP与TRAM结合型转录因子（如p53、E2F及TFIIB）之间的相互作用，这为E1A的作用机制提供了分子模型。为在体内验证该模型，我们使用了一段包含功能性TRAM的CBP小片段，该片段能够结合p53蛋白。CBP TRAM可结合细胞调控因子MDM2所靶向的p53序列，我们证实CBP TRAM能够破坏MDM2-p53的相互作用，进而使细胞内p53蛋白水平稳定，并激活p53依赖性转录。野生型E1A可完全阻断CBP TRAM对p53的转录激活作用，而无法结合CBP的E1A突变体则无此效果。