The transcriptional coactivators CREB binding protein (CBP) and p300 regulate numerous signal transduction pathways in eukaryotic cells by activation or repression of gene expression. They are essential for such basic cellular functions as growth, differentiation, apoptosis, DNA repair, and embryonic development. They also function as tumor suppressors and regulate key genes that control cellular proliferation, tumorigenesis, and cancer progression. CBP and p300 mediate crosstalk between many signaling pathways that regulate the response to cellular stress, thereby playing a key role in cell fate decisions ? determining whether cells should survive and proliferate or undergo apoptosis. CBP and p300 are central hubs in the signaling circuitry of the cell; the amount of CBP/p300 in the cell is limiting and regulatory proteins must compete for binding. CBP and p300 are absolutely required for activation of the tumor suppressor p53 and regulation of p53-mediated stress response pathways. They also perform an indispensable function in the response to hypoxia, by activating transcription of oxygen stress genes controlled by the hypoxia inducible transcription factor HIF-1? and by acting as a central hub in a negative feedback circuit in which the protein CITED2 downregulates HIF-1? transactivation by competition for the TAZ1 domain of CBP/p300. The overarching goals of the present proposal are to elucidate the structural and molecular basis by which CBP and p300 perform their central regulatory roles to control the hypoxic response and to protect against oncogenic transformation. Preliminary data show that the CBP/p300 TAZ1 domain and the activation domains of HIF-1? and CITED2 function cooperatively to create a unidirectional hypersensitive molecular switch that efficiently displaces the HIF- 1? activation domain from CBP/p300 to downregulate transcription of HIF-1? responsive genes. The intermolecular interactions that drive this switch will be investigated using affinity measurements and stopped flow kinetics, and the structural and dynamic features of the system will be characterized by NMR to provide insights into the detailed molecular mechanism by which this hypersensitive allosteric switch functions. Switch-like behavior has also been observed in competition between the p65 subunit of NF?B and HIF-1?, and between p53 and p65 for binding to TAZ1. Biophysical measurements will be performed to determine the mechanism of p65, p53, and HIF-1? competition. Given the central role of CBP/p300 as concentration-limited hubs in critical cellular signaling networks, it is likely that allostery may be a general mechanism by which disordered signaling proteins compete for their targets.
The proposed research will address the mechanisms by which CREB binding protein (CBP) and p300 function as tumor suppressors and regulate key genes that control cell proliferation, tumor formation, and tumor progression. This research will provide new molecular level understanding of the roles of CBP and p300 in activation of the tumor suppressor p53, and in the regulation of the cellular response to oxygen deficiency (hypoxia), a common phenomenon in growing tumor cells. It will provide new knowledge of the key molecular interactions by which CBP and p300 guard against or promote cancer, interactions which are of direct interest as potential targets for design of novel therapeutic agents.
