title: Ubiquitination of ECSIT is crucial for the activation of p65/p50 NF-κBs in Toll-like receptor 4 signaling.
abstract: 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.
authors.affiliation: Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea.
authors.forename: Sae
authors.initials: S
authors.lastname: Mi Wi
authors.affiliation: Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea.
authors.forename: Jeongho
authors.initials: J
authors.lastname: Park
authors.affiliation: Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY 10065.
authors.forename: Jae-Hyuck
authors.initials: JH
authors.lastname: Shim
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, and Department of Medicine, Harvard Medical School, Boston, MA 02115 echun@hsph.harvard.edu thylee@skku.edu.
authors.forename: Eunyoung
authors.initials: E
authors.lastname: Chun
authors.affiliation: Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea echun@hsph.harvard.edu thylee@skku.edu.
authors.forename: Ki-Young
authors.initials: KY
authors.lastname: Lee
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006801:Humans; D052002:NF-kappa B p50 Subunit / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C518657:Ecsit protein, human; D052002:NF-kappa B p50 Subunit; C489548:NFKB1 protein, human; C498143:RELA protein, human; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051996:Transcription Factor RelA