DNA damage induced phosphorylation and regulation of NF-kappaB

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To respond to threats from the environment or infectious agents, organisms 'activate' specialized groups of proteins that regulate the response of different genes, thus allowing cells of the body to adapt and survive, or fight the infection. Among these types of proteins, the Nuclear Factor kappa B (NF-kB) family is of particular importance as a regulator of the immune, inflammatory and stress responses. Because of the central role it plays, NF-kB can respond to a large number of different stimuli that include bacteria, viruses, inflammatory proteins and cell stresses such as DNA damage. Although the effect of DNA damage is not studied to the same extent as the response due to infection and inflammation, it is of great importance and can occur through exposure to environmental genotoxins. Many common cancer therapies that rely on DNA damage as their mechanism of killing tumour cells also active this protein family. Moreover, the production of reactive oxygen species (ROS) during inflammation or as a result of ageing can also lead to DNA damage and trigger 'activation' of NF-kB. The consequences of NF-kB activation in response to these stimuli can vary enormously, depending on the type of stimulation, the cell type in which it is occurring and the presence of other proteins activated at the same time. These differences manifest themselves as activation or repression of different genes by NF-kB, which can vary according to the context. Thus the effect of activating NF-kB on the cell and the organism will also vary depending on the type of stimulus. These differences include effects on cell proliferation, survival, production of inflammatory proteins and generation of ROS. This is also true of DNA damage, where different types of DNA damage and DNA damaging agent can result in very different effects on NF-kB function; this in turn differs from NF-kB activated in response to inflammatory stimuli. To a large extent, these differences can be explained by modifications to the protein structure through addition of small functional chemical groups, such as phosphate, a process termed post-translational modification (PTM). Such PTM is a form of code that rapidly regulates protein function in cells. Preliminary data from the Eyers group, looking at the effect of the cytokine TNF on the NF-kB protein RelA, has indicated that the level and complexity of PTMs on this protein is much greater than previous thought. In this proposal we will extend this analysis and use state of the art techniques to characterise the multiple PTMs that occur in response to different types of DNA damage. This analysis will include not only the identification of the sites of modification but also how they change during time and whether they occur simultaneously on the same molecules or are present separately on different molecules (generating a mix of differently modified proteins). In parallel with the analysis in the Eyers group, the Perkins group will determine the function and importance of these PTMs. This includes the identification of the enzymes, called kinases that regulate the addition of phosphate groups to different positions in the NF-kB protein. Furthermore, based the analysis in the Eyers lab, amino acids in the NF-kB protein RelA will be mutated such that it can no longer be modified in this manner, thus allowing the importance of these modifications on the regulation of the DNA-damage-induced response of NF-kB to be assessed. These experiments will give new insights into the regulation of NF-kB by DNA damage during inflammation, ageing and cancer chemotherapy. They will also provide a template for future analysis to investigate other aspects of NF-kB activity (different stimuli, context and activation pathways) while also serving more generally as a relevant example of a stimulus responsive network.