Aberrant NOD2 signaling causes granulomatous inflammatory disease. Patients with loss-of-function NOD2 alleles are prone to the development of Crohn's disease, an inflammatory disorder of the gastrointestinal tract. In contrast, patients with gain-of-function NOD2 mutations develop Early Onset Sarcoidosis (EOS), an inflammatory disorder characterized by noncaseating granulomas that cause lung, liver and eye damage. The fact that both loss-of-function polymorphisms and gain-of-function mutations both cause inflammatory diseases is likely due to the fact that NOD2 functions as a rheostat to help maintain normal immunologic homeostasis. This rheostat function begins upon bacterial invasion of the cell whereupon NOD2 binds to a breakdown product of bacterial peptidoglycan. This activates NOD2 such that it can modulate the innate immune system to help tailor the adaptive immune response to eradicate the offending pathogen. Either too much or too little NOD2 activation can be deleterious, and this imbalance is central to the development of inflammatory disease. NOD2 and its obligate kinase RIP2 are part of a positive regulatory circuit in which intracellular bacterial recognition causes the NOD2:RIP2 complex to be activated. In addition to stimulating autophagy, bacteriocidal activity, MHC Class II presentation and MAPK activation, the NOD2:RIP2 complex activates NF-?B. Both NOD2 and RIP2 are NF-?B regulated genes, and as such, their activation causes a positive feedback loop in which activation of NOD2:RIP2 stimulates further activation and further inflammation. Additionally, NOD2 and RIP2 expression are stimulated by a variety of mediators of inflammation, including TNF and IFN. Given this, in the prior granting period, we hypothesized that inhibiting this positive regulatory circuit might be efficacious in treating inflammatory disease. We were successful in identifying nanomolar inhibitors of RIP2's kinase activity. Despite this, a troubling fact remains: We still don't know what the kinase activity of RIP2 is doing in the cell. Some studies have shown that the kinase activity is dispensable for NOD2 activity while others have shown that it's essential. Our work has helped clarify this as we showed that RIP2 was misclassified as a serine-threonine kinase. It is actually a dual specificity kinase, meaning that t phosphorylates serines, threonines and tyrosines. Our own work has shown that inhibition of RIP2 attenuates the acute NOD2 inflammatory response. While my lab has found that RIP2's kinase activity helps regulate NF-?B, we don't know its role in regulating other NOD2-driven responses like autophagy or MAPK signaling. The uncertainty regarding RIP2's kinase activity takes on added importance given the interest of pharmaceutical companies in inhibiting RIP2 in inflammatory diseases like sarcoidosis, asthma, IBD and inflammatory arthritis. Understanding the kinase activity is essential if the goal is to inhibit RIP2 in inflammatory disease and to then determine efficacy and response in those diseases. This knowledge is also essential to predict outcomes of RIP2 inhibition in inflammatory disease. This grant application aims to answer these key questions.