Person:

Chun, Eunyoung

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.

Cereblon (CRBN) is a substrate receptor protein for the CRL4A E3 ubiquitin ligase complex. In this study, we report on a new regulatory role of CRBN in TLR4 signaling. CRBN overexpression leads to suppression of NF-κB activation and production of pro-inflammatory cytokines including IL-6 and IL-1β in response to TLR4 stimulation. Biochemical studies revealed interactions between CRBN and TAK1, and TRAF6 proteins. The interaction between CRBN and TAK1 did not affect the association of the TAB1 and TAB2 proteins, which have pivotal roles in the activation of TAK1, whereas the CRBN-TRAF6 interaction critically affected ubiquitination of TRAF6 and TAB2. Binding mapping results revealed that CRBN interacts with the Zinc finger domain of TRAF6, which contains the ubiquitination site of TRAF6, leading to attenuation of ubiquitination of TRAF6 and TAB2. Functional studies revealed that CRBN-knockdown THP-1 cells show enhanced NF-κB activation and p65- or p50-DNA binding activities, leading to up-regulation of NF-κB-dependent gene expression and increased pro-inflammatory cytokine levels in response to TLR4 stimulation. Furthermore, Crbn−/− mice exhibit decreased survival in response to LPS challenge, accompanied with marked enhancement of pro-inflammatory cytokines, such as TNF-α and IL-6. Taken together, our data demonstrate that CRBN negatively regulates TLR4 signaling via attenuation of TRAF6 and TAB2 ubiquitination.