The Crohn’s disease susceptibility protein, NOD2, coordinates signaling responses upon intracellular exposure to bacteria. Because dysregulation of this coordination can lead to inflammatory disease, maintaining appropriate activation of the NOD2 signaling pathway is paramount in immunologic homeostasis.
Although NOD2 is known to activate NF-κB, little is known about the molecular mechanisms by which NOD2 coordinates functionally separate signaling pathways such as NF-κB, JNK, and p38 to regulate cytokine responses, or the mechanisms by which NOD2 signaling is terminated. Given that one of the characteristics of Crohn’s disease is an altered cytokine response to normal bacterial flora, the coupling and termination of signaling pathways is likely to be important for Crohn’s-disease pathophysiology.
Coordination of NOD2 induced NF-κB and MAPK signaling is achieved in part at the level of the MAP3K’s. We identify the MAP3K, MEKK4, as a binding partner of RIP2. This MEKK4:RIP2 complex dissociates upon exposure to the NOD2 agonist, MDP, allowing NOD2 to bind to RIP2 and activate NF-κB. MEKK4 thus sequesters RIP2 to inhibit the NOD2:RIP2 complex from activating NF-κB signaling pathways. MEKK4 also helps dictate signal specificity downstream of NOD2 activation as knockdown of MEKK4 causes increased NF-κB activity, absent p38 activity, and hypo-responsiveness to TLR2 and TLR4 agonists.
TRAF proteins are involved in several inflammatory signaling networks. We identify the atypical TRAF family member, TRAF4, as a key negative regulator of NOD2 signaling. TRAF4 inhibits NOD2-induced NF-κB activation and directly binds to NOD2 to inhibit NOD2-induced bacterial killing. We find that two consecutive glutamate residues in NOD2 are required for interaction with TRAF4 and inhibition of NOD2 signaling.
Finally, we find that IKKα phosphorylates TRAF4 at S426 to enhance its function as a negative regulator of NOD2 signaling. Phosphorylation of this site affects TRAF4’s ability to bind NOD2, and allows it to inhibit NOD2 signaling. Structurally, Serine-426 resides within an exaggerated β-bulge in TRAF4 that is not present in the other TRAF proteins, and phosphorylation of this site provides a structural basis for the atypical function of TRAF4 and its atypical role in NOD2 signaling.