Inhibition of RIP2's tyrosine kinase activity limits NOD2-driven cytokine responses

Abstract

Upon intracellular bacterial exposure, the Crohn's disease and sarcoidosis susceptibility protein NOD2 (nucleotide oligomerization domain protein 2) binds to the protein kinase RIP2 (receptor-interacting protein 2) to coordinate NF-kB (nuclear factor k B)-mediated cytokine responses. While RIP2 clearly has kinase activity, the function of its kinase domain has been enigmatic. Although originally classified as a serine-threonine kinase based on homology scans, we find that RIP2 also has tyrosine kinase activity. RIP2 undergoes autophosphorylation on Tyr 474 (Y474). This phosphorylation event is necessary for effective NOD2 signaling and does not occur in the presence of the most common Crohn's disease-associated NOD2 allele. Given this tyrosine kinase activity, a small-molecule inhibitor screen designed to identify pharmacologic agents that inhibit RIP2's tyrosine kinase activity was performed. At nanomolar concentrations, the EGFR (epidermal growth factor receptor) tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) were found to inhibit both RIP2 tyrosine phosphorylation and MDP (muramyl dipeptide)-induced cytokine release in a variety of NOD2 hyperactivation states. This effect is specific for RIP2 and does not depend on EGFR. The finding that RIP2 has tyrosine kinase activity and the finding that gefitinib and erlotinib, two agents already used clinically for cancer chemotherapy, can inhibit this activity suggest that RIP2's tyrosine kinase activity could be targeted specifically in the treatment of inflammatory diseases.[Keywords: NOD2; RIP2; ITCH; innate immunity; tyrosine kinase] Supplemental material is available at http://www.genesdev.org.

A RIPK2 inhibitor delays NOD signalling events yet prevents inflammatory cytokine production

“…WEHI-345 inhibits RIPK2 activity in vitro with an IC 50 of 134 nM, while IC 50 against other RIP Kinases is greater than 10 mM. Treatment with WEHI-345 significantly inhibited IL-6 secretion from Raw 267.4 cells at 50 nM, showing a 10-fold stronger inhibition than erlotinib and gefitinib in an identical assay 22 . In cells, WEHI-345 blocked NOD-dependent cytokine production but spared necroptosis ARTICLE signalling mediated by RIPK1 and RIPK3.…”

“…Upon activation, RIPK2 autophosphorylates on Ser176 and on Tyr474 (refs 22,25). While Ser176 phosphorylation is essential for RIPK2 activity, phosphorylation of Tyr474 enhances, but is not essential for signalling 22 .…”

“…RIPK2 was originally identified as a serine-threonine kinase based on sequence homology [19][20][21] , but has recently been reclassified as a dual-specificity kinase 22 . Earlier reports suggested that the kinase activity of RIPK2 was dispensable for NF-kB activation and cytokine production 23 .…”

“…Among them are the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors Gefitinib and Eroltinib 22 and the p38, mitogen-activated protein kinase (MAPK) and Src inhibitor SB-203580 (refs 26,27). Pharmacological inhibition of RIPK2 kinase activity via these relatively nonspecific inhibitors caused a reduction in RIPK2 stability and a reduction in cytokine production upon NOD stimulation.…”

NF-κB in immunobiology

“…The CARDcontaining kinase RIP2 is required for NF-κB activation. The function of RIP2 tyrosine kinase activity is unclear, although it was recently shown that kinase inhibitors could prevent MDP-induced cytokine responses [79]. Although RIP2 binds to NEMO, it is not thought that RIP2 tyrosine kinase activity mediates IKK phosphorylation directly.…”