The overall objective of this proposal is to understand the molecular and biochemical basis underlying the regulation of the p53 tumor suppressor protein in mammalian systems. Our approach is to focus on a specific cellular process that modifies the properties of the p53 protein and thus its functions in response to DNA damage caused by UV radiation or cisplatin treatment. Hence, utilizing p53 as a probe, we have identified and purified a stress-responsive kinase complex that specifically targets serine 392 which is evolutionarily conserved and plays a critical role in regulating p53 activity. This newly identified kinase complex contains chromatin-related transcriptional elongation factor FACTp140/SSRP1 and the alpha, alpha' and beta subunits of casein kinase II (CKII). Molecular delineation of its role in regulating p53 function will shed light on the mechanisms of DNA damage-induced p53 signaling.
Three specific aims are to dissect the mechanisms of p53 regulation by this kinase in response to DNA damage in experimental and biological contexts. 1) To determine how the substrate selectivity of the FACTp140/SSRP1-associated p53 kinase complex is achieved. Using the C-terminal domain of p53 as a substrate, the serine 392 kinase will be purified and characterized from UV- or cisplatin-treated HeLa cell nuclear extracts using conventional chromatography and affinity columns. Kinetic analysis of this kinase will be performed to examine the biochemical mechanism of its substrate specificity. Molecular, biochemical and cell biological methods will be developed to determine protein-protein interactions among the components of the complex and their association with p53 in vitro and in vivo. Interacting domains will be mapped, and the effect of deletion mutants of these kinase components on the substrate selectivity will be assessed. 2) To determine the mechanism by which the FACTp140-SSRP1-associated p53 kinase is activated in response to DNA damage. The activation of this kinase in cells after DNA damage or transcriptional attenuation will be analyzed. Co- localization of the p53 S392 kinase components after DNA damage and the effect of cisplatin-damaged DNA on the assembly and activity of this kinase complex in vitro will be analyzed. The effects of dominant negative mutants of FACTp140 or of SSRP1 on the p53 S392 phosphorylation will be assessed after DNA damage. 3) To determine whether the FACTp140-SSRP1-associated p53 kinase regulates p53 stability and activity after DNA damage. The effects of this kinase on MDM2-mediated p53 ubiquitination in vitro and in vivo, p53 stability and transcriptional activity- will be evaluated in various cells after DNA damage. These studies will systematically elucidate the novel multi- subunit protein kinase complex and will also contribute to a better understanding of a different p53 regulation mechanism in response to DNA damage. This investigation has important implications for anti-cancer drug development.
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