Regulation of IKK-epsilon and TRAF6 functions by posttranslational modifications

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Lipopolysaccaride (LPS) contained on the surface of gram-negative bacteria triggers complex intracellular signaling cascades activating transcription factors such as NF-ýB that serve to induce the expression of immune regulatory genes. The last years have witnessed the identification of many different components of the LPS-inducible intracellular signaling system, including the protein kinase IKKý and the ubiquitin E3 ligase TRAF6. As the control of their activity is not well known, this project aims to study their mutual regulation by posttranslational modifications. Preliminary results show that IKKý phosphorylates TRAF6 at 3 serines, while TRAF6 mediates inducible ubiquitination of IKKý at 9 lysines. To address the functional role of these posttranslational modifications, knock-out cells will be reconstituted to stably express these proteins in their wildtype and mutated forms that are changed in the modified amino acids. These cells will be analyzed by a large variety of experimental approaches including cell biological and biochemical experiments. The occurrence, kinetics and regulation of these modifications are traced by modification-specific antibodies. As recent evidence shows a role of IKKý in the nucleus, ChIP-Seq experiments will reveal a comprehensive map of genomic binding sites for this kinase. This basic research project will broaden our understanding of molecular processes that occur in proinflammatory signal transduction and are thus of biomedical interest.