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. ? ? ?
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