The transcription factor NF-kappaB is regulated primarily through its association with the inhibitory protein IkappaB. The NF-kappaB/IkappaB complex is normally sequestered in the cytoplasm until cells are stimulated with an agonist such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and lipopolysaccharides (LPS). Upon stimulation of cells, IkappaB is rapidly phosphorylated by an IkappaB kinase complex (IKK) and then degraded by the ubiquitin-proteasome pathway. The degradation of 1kappaB allows NF-kappaB to enter the nucleus to turn on downstream genes, many of which play pivotal roles in the life and death of cells. A key step in the NF-kappaB signaling pathway is the phosphorylation of IkappaB by the IKK complex. This kinase complex integrates signals from multiple pathways including those emanating from IL-1 and LPS. Genetics studies have demonstrated that TRAF6, a RING finger domain protein, and components of the IKK complex are essential for NF-kappaB activation in response to IL-1 and LPS. However, it is not known how TRAF6 activates IKK. Recent studies by the principal investigator's laboratory have shown that the activation of IKK complex by TRAF6 requires a dimeric ubiquitin conjugating enzyme complex, Ubc13/Uev1A, and the formation of a unique polyubiquitin chain linked through lysine-63 (K63) of ubiquitin. The goal of this proposal is to understand the novel mechanisms of ubiquitin-dependent activation of the IKK complex. Specifically, experiments are proposed to 1) study the structure and function of Ubcl3/Uev1A and TRAF6 as related to IKK activation; 2) identify and characterize additional factors required for IKK activation by TKAF6, including the ubiquitination target; 3) identify and characterize the K63-linked polyubiquitin chain binding protein; 4) investigate the mechanisms of ubiquitin-dependent activation of IKK by TRAF6. Taken together, these studies should fill significant gaps in the NF-kappaB signaling pathway, and provide the important pieces that together are required to solve the puzzle of IKB kinase activation by ubiquitin. Given the importance of NF-kappaB in human diseases, information gained from the proposed studies will be of direct relevance to biomedicine, including discovery of novel therapeutic targets.
Showing the most recent 10 out of 39 publications
