A Novel Motif in the Crohn's Disease Susceptibility Protein, NOD2, Allows TRAF4 to Down-regulate Innate Immune Responses

Abstract

The Crohn's disease and early onset sarcoidosis susceptibility protein, NOD2, coordinates innate immune signaling pathways. Because dysregulation of this coordination can lead to inflammatory disease, maintaining appropriate activation of the NOD2 signaling pathway is paramount in immunologic homeostasis. In this work, we identify the atypical tumor necrosis factor-associated factor (TRAF) family member, TRAF4, as a key negative regulator of NOD2 signaling. TRAF4 inhibits NOD2-induced NF-B 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 because mutation of these residues abrogated both TRAF4 binding and inhibition of NOD2. This work identifies a novel negative regulator of NOD2 signaling. Additionally, it defines a TRAF4 binding motif within NOD2 involved in termination of innate immune signaling responses.Upon intracellular exposure to bacterial products, the Crohn's disease susceptibility protein, NOD2 (nucleotidebinding oligomerization domain 2), initiates innate immune inflammatory signaling pathways to help tailor the adaptive immune system such that it can eradicate offending pathogens (1-3). Both the induction and the termination of NOD2 signaling are important to immune homeostasis and must be tightly controlled. Genetic variants of NOD2 result in dysregulated signaling and are associated with inflammatory disorders, including Crohn's disease, early onset sarcoidosis (EOS), 2 and Blau syndrome (4 -6). Although the signaling cascade linking NOD2 stimulation to NF-B activation has been extensively studied, the mechanisms by which NOD2, itself, is regulated remain elusive.Upon intracellular exposure to a breakdown product of bacterial peptidoglycan, muramyl dipeptide (MDP), NOD2 binds to the scaffolding protein kinase, RIP2 (receptor-interacting protein 2), via caspase recruitment domain interactions (1, 7). Following activation of the NOD2-RIP2 complex, the (CARD) IKK scaffolding protein IKK␥ (NEMO) becomes polyubiquitinated via Lys 63 linkages on Lys 285 of NEMO. These ubiquitin chains on NEMO are thought to help nucleate TAK1 (TNF receptor-associated kinase 1) such that it can phosphorylate the activation loop of IKK␤ and ultimately activate the NF-B transcription factors (8 -11).Previous work from this laboratory shows that one point of coordination for NOD2-induced innate immune signaling occurs at the level of the mitogen-activated protein kinase kinase kinases (MAP3Ks). MAP3Ks are the upper tier of a sequential cascade of MAPK-activating kinases that ultimately leads to MAPK activation. Specifically, the MAP3K, MEKK4, was identified as a regulator of NOD2. Both basally and in response to MDP, MEKK4 helps the cell to maintain low levels of NF-B activity (12). In addition to phosphorylating MAP2Ks, the MAP3K proteins act as scaffolding proteins for signaling complexes (13). This is evidenced by the fact that one atypical tumor necrosis recepto...

IκB Kinase α Phosphorylation of TRAF4 Downregulates Innate Immune Signaling

“…The L1007insC is a loss-offunction mutation in that it has decreased capacity for IKK signalosome activation and subsequent NF-B activation (27,36,49). We have previously shown that TRAF4 maintains binding to L1007insC NOD2 (34). However, cotransfection of HEK293T cells with Omni-TRAF4 and HA-L1007insC failed to induce TRAF4 phosphorylation (Fig.…”

“…Similar to the atypical nature of IKK␣, TRAF4 is an atypical member of the TRAF superfamily of proteins (25). Unlike the other six TRAF family members, TRAF4 plays a negative regulatory role in NF-B activation (34,47). Since S426 within the predicted IKK␣ phosphorylation motif of TRAF4 is also conserved across species (Fig.…”

“…Phospho-IKK motif antibody was generated as previously described (19). NTAP-TRAF4, Omni-TRAF4, HA-NOD2, GST-IKK␤, and GST-IKKε were used as previously described (18,19,34,37). GST-IKK␣ was generated by PCR cloning into the BamHI and NotI sites of pEBG.…”

“…Recombinant full-length human IKK␣ containing an N-terminal GST tag was purchased from Millipore. A 5= Flag-fused retroviral TRAF4 was generated as previously described for generation of stable macrophages and HT29 cells (34). Flag beads (M2) and streptavidin agarose were purchased from Sigma.…”

“…Unlike other TRAF family members that work to positively regulate inflammatory signaling downstream of agonists like TNF, lipopolysaccharide (LPS), or IL-1, TRAF4 negatively regulates innate immune signaling. TRAF4 binds directly to the Nod-like receptor (NLR) family member, NOD2, to inhibit NF-B activation (34). Additionally, TRAF4 also binds to TRAF6 and TRIF to dampen Toll-like receptor (TLR)-mediated NF-B and interferon (IFN) activation (47).…”