Recent evidence shows that evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) interacts with tumor necrosis factor receptor-associated factor 6 (TRAF6), is ubiquitinated, and contributes to bactericidal activity during Toll-like receptor (TLR) signaling. Here we report a new regulatory role for ECSIT in TLR4 signaling. On TLR4 stimulation, endogenous ECSIT formed a molecular complex with p65/p50 NF-κB proteins. Our biochemical studies showed that ECSIT specifically interacted with p65/p50 NF-κB proteins, which colocalized in the nucleus. Of interest, these effects were critically dependent on ubiquitination of the ECSIT lysine (K) 372 residue. K372A mutant ECSIT did not interact with p65/p50 NF-κB proteins and markedly attenuated nuclear colocalization. In addition, ECSIT-knockdown THP-1 cells could not activate NF-κB DNA-binding activities of p65 and p50, production of proinflammatory cytokines, or NF-κB-dependent gene expression in response to TLR4 stimulation. However, these activities were markedly restored by expressing the wild-type ECSIT protein but not the K372A mutant ECSIT protein. These data strongly suggest that the ubiquitination of ECSIT might have a role in the regulation of NF-κB activity in TLR4 signaling.
© 2015 Wi, Park, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).