Title: Characterization of inflammatory signaling networks
Description: Misregulation of immune signaling pathways leads to infectious, inflammatory, or autoimmune disorders. The IKK family of kinases are essential activators of the NFκB pathway and type I interferon signaling. While these kinases have been shown to play critical roles in diverse signaling pathways, few substrates of these kinases have been identified, and mechanisms of cross-talk between inflammatory signaling pathways are poorly understood. To this end, we have developed a positional scanning peptide library technology which can rapidly identify the optimal phosphorylation motifs for protein kinases. This method is superior to older methods because it is more sensitive, more generally applicable to diverse kinases, and more amenable to high-throughput analysis of kinases on a proteome-wide scale. It was hypothesized that determining the optimal phosphorylation motifs for the IKKs would facilitate the identification of novel IKK substrates, and, in turn, lead to a more complete understanding of the roles of these kinases in signal transduction pathways.

We utilized this newly-developed peptide library technology to identify the optimal phosphorylation motifs for IKKβ, IKKe, and TBK1. These data, in combination with bioinformatics and biochemical approaches, predicted a number of novel substrates of these kinases. Several predicted TBK1 and IKKe substrates have been validated in vitro , and a TBK1/IKKe phospho-substrate antibody has been developed which recognizes known and putative substrates in a kinase dependent manner. Ser381 of the K63 deubiquitinase A20 was predicted to be a likely site of IKKβ phosphorylation. While A20 is a known negative regulator of innate immune signaling pathways, the mechanisms regulating the activity of A20 are poorly understood. We show that IKKβ phosphorylates A20 in vitro and in vivo at serine 381, and we further show that this phosphorylation event increases the ability of A20 to inhibit the NFκB signaling pathway. Phosphorylation of A20 by IKKβ thus represents part of a novel feedback loop which regulates the duration of NFκB signaling following activation of innate immune signaling pathways. Taken together, these data provide evidence that the techniques described here provide an efficient and unbiased method for identifying novel kinase substrates.