Control of NOD2 and Rip2-dependent innate immune activation by GEF-H1

Abstract

Background Genetic variants of nucleotide-binding oligomerization domain 2 (NOD2) lead to aberrant microbial recognition and can cause chronic inflammatory diseases in patients with Crohn’s disease. Methods We utilized gene specific siRNA mediated knockdown and expression of GEF-H1 in wildtype, Rip2- and Nod2-deficient macrophages, HCT-116 and HEK 293 cells to determine the role of GEF-H1 in NOD2 and Rip2 mediated NF-kB dependent induction of proinflammatory cytokine expression. Confocal microscopy was used to determine subcellular distribution of GEF-H1, Rip2 and NOD2. Results We identified guanine nucleotide exchange factor H1 (GEF-H1) as an unexpected component of innate immune regulation during microbial pattern recognition by NOD2. Surprisingly, GEF-H1 mediated tyrosine phosphorylation of Rip2, which occurred during signaling by NOD2, but not in the presence of the 3020insC variant of NOD2 associated with Crohn’s disease. GEF-H1 functioned downstream of NOD2 as part of Rip2-containing signaling complexes and was responsible for phosphorylation of Rip2 by Src tyrosine kinase. Human Rip2 variants lacking the tyrosine target of GEF-H1-mediated phosphorylation were unable to mediate NF-κB activation in Rip2-deficient macrophages and failed to transduce NOD2 signaling. GEF-H1 is required downstream of NOD2 as part of Rip2-containing signaling complexes and was responsible for tyrosine phosphorylation of Rip2. Conclusion GEF-H1 connects tyrosine kinase function to NOD-like receptor signaling and is fundamental to the regulation of microbial recognition by ubiquitous innate immune mechanisms mediated by Rip2 kinase.

NOD-Like Receptors: Guardians of Intestinal Mucosal Barriers

“…Interestingly, Keestra et al discovered a similar relationship between NOD1 and RAC1 induced by the Salmonella virulence factor SopE (50). In line with RAC1, guanine nucleotide exchange factor H1 (GEF-H1) also activates Rho GTPases, and colocalizes with NOD1 and NOD2 to serve as a modulator of RIP2-dependent NF-B activation (30,118). These findings are remarkable because this 1) assigns a new role to Rho GTPases within immune defense and 2) indicates that NOD1 and NOD2 not only detect PGN but also are involved in sensing cytoskeleton reorganization induced by invasive bacteria.…”

NOD1 and NOD2: Beyond Peptidoglycan Sensing

“…In addition, GEF-H1 interaction with NOD1 is required for the detection of iE-DAP fragments [30]. Further, GEF-H1 also promotes MDP-induced NOD2 activation [40]. Rac1 and Rac2 mutated in the C-terminus to prevent membrane insertion of the small GTPases were unable to induce an inflammatory response, suggesting that localization of NOD1 and NOD2 to the cell membrane is essential for formation of a functional protein complex.…”

Detection of enteric pathogens by the nodosome

“…Furthermore, NOD2 colocalizes with GEF-H1; a GEF for RHOA [45]. Evidence for an interaction of NOD1 with the activated forms of RAC1 and CDC42 comes from the observation that these proteins can be coimmunoprecipitated [40].…”