This work was supported by the Mid-career Researcher Program through an NRF Grant (NRF-2014R1A2A1A11053221). Author contributions YM and SMW performed and experiments. initially reported as a candidate gene for a mild form of autosomal recessive non-syndromic mental retardation.1, 2 Subsequently, different cellular roles of the CRBN protein have been characterized and identified. CRBN interacts with the cytoplasmic region of large-conductance calcium-activated potassium channels, regulating its surface expression.3 In the retina, CRBN interacts with voltage-gated chloride channel-2 (ClC-2), thereby influencing assembly or cellular targeting of ClC-2.4 CRBN interacts with the by CRL4CRBN E3 ubiquitin ligase,14 indicating that CRBN-binding immune modulatory drugs (IMiDs) differentially regulate CRL4CRBN E3 ubiquitin ligase activity. So far, many different functions of E3 ubiquitin ligases have been reported.15, 16, 17 Several E3 ubiquitin ligases have a crucial role in regulating immune receptor and cellular signaling, and in modulating immune homeostasis and activation.18, 19, 20 Among them, tumor necrosis factor (TNF) receptor associated factor Vanillylacetone 6 (TRAF6), has a pivotal role in innate signaling, including signaling of toll-like receptors (TLRs).21, 22 In TLRs-mediated signaling, TRAF6 associates with the dimeric ubiquitin-conjugating enzyme Ubc13/Uev1A and functions as both an adaptor and an E3 ubiquitin ligase-conjugating K63-linked ubiquitin chain attaching to itself and other proteins.23, 24 TRAF6 ubiquitination involves the activation of ubiquitin-dependent kinase TAK1, along with binding to TAK1 by several different proteins, such as TAK1-binding protein (TAB)1, TAB2, TAB3, and TAB4.25, 26, 27 TAB2 is ubiquitinated by TRAF6, which facilitates assembly of a Toll/interleukin-1 (IL-1) signaling Vanillylacetone complex containing TRAF6, TAK1, and Iresults, mice exhibited increased mortality, accompanied with marked enhancements of the pro-inflammatory cytokines after challenge with lipopolysaccharide (LPS) closed bar in mock), whereas a significant decrease could be seen in CRBN-transfected cells treated with LPS (Figure 1a, closed bar in mock closed bar in HA-CRBN). In addition, CRBN overexpression significantly inhibited the p65-DNA binding activity induced by LPS stimulation, as compared with mock-transfected cells (Figure 1b, closed bar in mock closed bar in HA-CRBN). To verify the results observed in 293/TLR4 cells, we transfected THP-1 human monocytic cells with an NF-closed bars in HA-CRBN). TLR4 stimulation induces NF-production were greatly enhanced (Figures 1d and e, open bar closed bar in mock), whereas marked decreases could be detected in HA-CRBN-transfected THP-1 cells (Figures 1d and e, closed bar in mock closed bars in HA-CRBN), suggesting that CRBN overexpression negatively regulates NF-(e) were analyzed by Mouse monoclonal to p53 enzyme-linked immunosorbent assay (ELISA). All error bars represent S.D. of the mean from triplicate samples. *production were also Vanillylacetone significantly attenuated in both Ctrl and CRBNKD THP-1 cells overexpressed by Flag-CRBN, as compared with mock-transfected cells (Figure 2g, IL-6 and IL-1were analyzed by ELISA. All error bars represent S.D. of the mean from triplicate samples. *were analyzed by ELISA. All error bars represent S.D. of the mean from triplicate samples. *lane 8). To verify the result, Flag-TRAF6 and HA-Ub were co-transfected into HEK293T cells with different concentrations of Myc-CRBN. According to expressions of Myc-CRBN, the ubiquitination of TRAF6 gradually decreased (Figure 4e, lane 2 lanes 3C5), indicating that CRBN attenuates the ubiquitination of TRAF6. Open in a separate window Figure 4 CRBN interacts with TRAF6 and inhibits the ubiquitination of TRAF6. (a) HEK293T cells were transfected with mock, HA-CRBN, or Flag-TRAF6, as indicated. At 38?h after transfection, transfected cells Vanillylacetone were extracted, immunoprecipitated with anti-Flag antibody, and then immunoblotting was performed with anti-Flag or anti-HA antibodies. (b) A schematic presentation of TRAF6 wt and its truncated mutants used in this study: TRAF6 110-522, TRAF6 260-522, and TRAF6 349-522. (c) HEK293T cells were transfected with HA-CRBN, Flag-TRAF6 wt, Flag-TRAF6 110-522, Flag-TRAF6 260-522, or Flag-TRAF6 349-522, as indicated. At 38?h after transfection, transfected cells were extracted, immunoprecipitated with an anti-Flag antibody, and then immunoblotting was performed with anti-Flag or anti-HA antibodies. (d) HEK293T cells were transfected with mock, Myc-CRBN, Flag-TRAF6, or HA-Ub, as indicated. At 38?h after transfection, transfected cells were extracted, immunoprecipitated with an anti-Flag antibody, and then immunoblotting was performed with anti-Flag, anti-Myc, or anti-HA antibodies. (e) HEK293T cells were transfected with mock, HA-Ub, and Flag-TRAF6 in the absence or presence of different concentrations of Myc-CRBN. At 38?h after transfection, transfected cells were extracted, immunoprecipitated with an Vanillylacetone anti-Flag antibody, and then immunoblotting was performed with anti-Flag, anti-Myc, or anti-HA antibodies. (f) THP-1 cells were infected with a lentivirus containing shRNA.