Structural basis for CBP/p300 transcriptional regulation

描述（由申请人提供）：CREB结合蛋白（CBP）和p300是模块化转录共激活因子，整合真核细胞中的多种信号转导途径，对生长、分化、凋亡和DNA修复至关重要。CBP/p300在胚胎发育中起关键作用，是许多致癌事件的靶点。它们还具有肿瘤抑制的功能，是许多细胞和肿瘤病毒癌蛋白转录活动所必需的。CBP和p300在调节肿瘤抑制因子p53的稳定性和转录活性中发挥重要作用。这些蛋白之间的相互作用是高度动态的，并在基因毒性应激反应中通过p53反活化结构域在多个位点的磷酸化来调节。腺病毒癌蛋白E1A与细胞因子竞争，隔离CBP/p300，破坏正常的细胞周期控制，诱导细胞永生。本研究将利用广泛的蛋白质构建文库来研究p53和E1A与CBP的TAZ1、TAZ2、KIX和核辅激活子结合域的相互作用。将确定p53的二部n端反激活结构域与CBP结构域形成的复合物的结构，以获得结构域之间相互作用的动态相互作用以及CBP/p300激活p53调控转录的分子基础的新见解。p53反活化结构域的单位点和多位点磷酸化对这些结构域相互作用的影响将被研究。将确定E1A（非致癌性AdV5和强致癌性AdV12血清型）与CBP/p300结构域形成的复合物的结构，以便深入了解E1A与其他转录因子竞争以隔离CBP/p300并破坏细胞转录机制的分子基础。最后，两个三元复合物将被表征，在HDM2、p53和CBP的TAZ1结构域之间，以及在视网膜母细胞瘤蛋白Rb、E1A的口袋结构域和TAZ2结构域之间。该研究强调弱的、竞争的相互作用以及多个结合位点和多个伙伴之间的协同相互作用，将为CBP/p300的分子识别机制，以及它们介导p53和病毒癌蛋白在肿瘤抑制、生长和进展中的关键相互作用的分子基础提供新的和基础的见解。

DESCRIPTION (provided by applicant): CREB binding protein (CBP) and p300 are modular transcriptional coactivators that integrate numerous signal transduction pathways in eukaryotic cells, and are essential for growth, differentiation, apoptosis, and DNA repair. CBP/p300 play critical roles in embryonic development and are the targets of many oncogenic events. They also function as tumor suppressors and are required for the transcriptional activities of many cellular and tumor virus oncoproteins. CBP and p300 play an essential role in mediating the stability and transcriptional activity of the tumor suppressor p53. The interactions between these proteins are highly dynamic and are modulated by phosphorylation of the p53 transactivation domain at multiple sites in response to genotoxic stress. The adenoviral oncoprotein E1A competes with cellular factors to sequester CBP/p300, disrupting normal cell cycle control to induce cell immortalization. This research will utilize an extensive library of protein constructs to investigate the interactions of p53 and E1A with the TAZ1, TAZ2, KIX, and nuclear coactivator binding domains of CBP. The structures of the complexes formed by the bipartite N-terminal transactivation domain of p53 with the CBP domains will be determined, to obtain new insights into the dynamic interplay of interactions between the domains and into the molecular basis for CBP/p300 activation of p53-regulated transcription. The effects of phosphorylation of the p53 transactivation domain, at single and multiple sites, on interactions with these domains will be investigated. Structures will be determined for the complexes formed by E1A (both non-oncogenic AdV5 and strongly oncogenic AdV12 serotypes), with domains of CBP/p300, in order to obtain insights into the molecular basis by which E1A competes with other transcription factors to sequester CBP/p300 and subvert the cellular transcription machinery. Finally, two ternary complexes will be characterized, between HDM2, p53, and the TAZ1 domain of CBP, and between the pocket domain of the retinoblastoma protein Rb, E1A and the TAZ2 domain. With its emphasis on weak, competing interactions and the synergistic interplay between multiple binding sites and multiple partners, this research will provide new and fundamental insights into the mechanisms of molecular recognition by CBP/p300, and the molecular basis by which they mediate critical interactions with p53 and viral oncoproteins in tumor suppression, growth and progression.