Ubiquitination of ECSIT is crucial for the activation of p65/p50 NF-κBs in Toll-like receptor 4 signaling

Mol Biol Cell. 2015 Jan 1;26(1):151-60. doi: 10.1091/mbc.E14-08-1277. Epub 2014 Oct 29.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Cell Line
  • Cell Nucleus / metabolism
  • Gene Expression
  • Gene Knockdown Techniques
  • Humans
  • NF-kappa B p50 Subunit / genetics
  • NF-kappa B p50 Subunit / metabolism*
  • Signal Transduction*
  • Toll-Like Receptor 4 / genetics
  • Toll-Like Receptor 4 / metabolism*
  • Transcription Factor RelA / genetics
  • Transcription Factor RelA / metabolism*
  • Ubiquitination


  • Adaptor Proteins, Signal Transducing
  • Ecsit protein, human
  • NF-kappa B p50 Subunit
  • NFKB1 protein, human
  • RELA protein, human
  • TLR4 protein, human
  • Toll-Like Receptor 4
  • Transcription Factor RelA