ZFP91 activates the noncanonical NF-κB signaling pathway. A, MDA-MB231 cells were treated with kamebakaurin (KA) (10 μg/ml) for 5 h, and the extracted mRNAs were subject to Northern blot analysis using 28 S RNA as a control. B, 24 h after the transfection with titration of FLAG-ZFP91 and NF-κB reporter construct pNF-κB-Luc, the lysates of HEK293 cells were subject to the measurement of dual luciferase activity. Data represented as mean ± S.D. of three independent experiments. C, HEK293 cells transfected with titration of FLAG-ZFP91 were lysed, and the lysates were analyzed by immunoblot using the indicated antibodies for NIK, p52, p50, FLAG, and tubulin. D, HEK293 cells were co-transfected with FLAG-ZFP91 and Myc-NIK as indicated. The cell lysates were analyzed by immunoblot using the indicated antibodies for p52, p-IKKβ/α, IκB-α, Myc, FLAG, and tubulin.

ZFP91 stabilizes NIK and induces Lys⁶³-linked NIK ubiquitination. A, HEK293 cells were co-transfected with pNF-κB-Luc and Myc-NIK in combination with HA-Ub or FLAG-ZFP91 or together with HA-Ub and FLAG-ZFP91. Dual luciferase activity was measured 24 h after transfection. Data are represented as mean ± S.D. of three independent experiments. B, the lysates from HEK293 cells co-transfected with HA-Ub, FLAG-ZFP91, or both were subject to endogenous NIK and p52 immunoblots (top two panels). FLAG and tubulin immunoblots were also completed from the same cell lysates (bottom two panels). C, the lysates of HEK293 cells co-transfected with Myc-NIK, FLAG-ZFP91, and HA-Ub constructs as indicated were subject to immunoprecipitation (IP) using anti-Myc antibody, and the immune complexes were analyzed by immunoblots (IB; top two panels). FLAG, Myc, and tubulin immunoblot was completed from the same cell lysates (bottom panel). D, the lysates of HEK293 cells co-transfected with Myc-NIK and HA-UbK48R along with a control siRNA, ZFP91 siRNA, or FLAG-ZFP91 were incubated with anti-Myc antibody, and the immune complexes were analyzed by immunoblots (top two panels). The immunoblots using anti-ZFP91, anti-Myc, and anti-tubulin antibodies were completed from the same cell lysates (bottom two panels). E, the lysates of HEK293 cells co-transfected with Myc-NIK, FLAG-ZFP91, and HA-Ub constructs as indicated were subject to immunoprecipitation using anti-Myc antibody, a portion of the precipitated pellets were saved for immunoblots, and the remainder of the precipitated pellets were treated with 1% SDS, PBS to disrupt non-covalent interactions, and supernatants were diluted with lysis buffer, followed by a second immunoprecipitation using anti-Myc antibody. The immune complexes were analyzed by immunoblots (top two panels). FLAG immunoblot was completed from the same cell lysates.

ZFP91 is an E3 ligase for Lys⁶³-linked ubiquitination of NIK. A, in vitro ubiquitination assays were performed in reaction mixtures containing purified ZFP91, ubiquitin, E1 enzyme, and ATP in the presence of a panel of E2 ubiquitin-conjugating enzymes. Reactions were incubated at 37 °C for 1 h, and the reaction mixtures were detected by anti-HA immunoblot (upper panel). The amount of ZFP91 proteins used in the assay is shown in a FLAG immunoblot (lower panel). B, in vitro ubiquitination assays were performed in reaction mixtures containing purified ZFP91, ubiquitin, E1 enzyme, and ATP in the presence of an E2 ubiquitin-conjugating enzyme, UbcH13/Mms2. Reactions were incubated at 37 °C for 1 h, and the ubiquitinated ZFP91 was detected by an anti-HA and anti-FLAG immunoblot. C, in vitro ubiquitination assays were performed as described above using purified ZFP91 with Lys⁴⁸-only or Lys⁶³-only ubiquitin, and the ubiquitinated ZFP91 was detected by anti-Ub immunoblot (upper panel). The amount of ZFP91 proteins used in the assay is shown in the FLAG immunoblot (lower panel). D, in vitro ubiquitination assays were performed using ZFP91 and GST-NIK (aa 381–947) as described above and under “Experimental Procedures.” The reaction mixtures were immunoprecipitated with anti-GST antibody and analyzed by immunoblots (top two panels). Anti-Ub and -FLAG immunoblots were completed using the same samples (bottom two panels). IB, immunoblot; IP, immunoprecipitation.

ZFP91 interacts with NIK. A, the lysates of HEK293 cells co-transfected with FLAG-ZFP91 and Myc-NIK were incubated with IgG and anti-FLAG antibody, and the immune complexes were analyzed by immunoblots using appropriate antibodies. B, the lysates of HEK293 cells treated with proteasomal inhibitor MG132 (10 μm) for the final 4 h were incubated with IgG and anti-ZFP91 antibody, and the immune complexes were subject to immunoblots using the indicated antibodies. C, FLAG-ZFP91 and GST-NIK purified from Sf21 cells were incubated in 0.1% BSA binding buffer. The associated complexes were incubated with IgG and anti-NIK antibody and then analyzed by immunoblots using appropriate antibodies. D and E, lysates of HEK293 cells co-transfected with the indicated FLAG-ZFP91 and Myc-NIK constructs were incubated with anti-FLAG antibody, and the immune complexes were analyzed by immunoblots (top two panels). FLAG and Myc immunoblots were completed from the same cell lysates (bottom panels). NS, nonspecific bands. IP, immunoprecipitation; IB, immunoblot.

ZnF domain 2 of ZFP91 is critical for Lys⁶³-linked ubiquitination of NIK. A, the lysates of HEK293 cells co-transfected with Myc-NIK and HA-Ub along with FLAG-ZFP91FL (aa 1–570), FLAG-ZFP91N (aa 1–242), or FLAG-ZFP91C (aa 242–570) were incubated with anti-Myc antibody, and the immune complexes were analyzed by immunoblots (top two panels). A FLAG, Myc, and tubulin immunoblot was completed from the same cell lysates (bottom panel). NS, nonspecific bands. B, the lysates of HEK293 cells co-transfected with Myc-NIK and HA-Ub along with various FLAG-ZFP91 constructs as indicated were incubated with anti-Myc antibody, and the immune complexes were analyzed by immunoblots (top two panels). A FLAG, Myc, and tubulin immunoblot was completed from the same cell lysates (bottom panel). C, the lysates of HEK293 cells co-transfected with Myc-NIK and HA-Ub along with FLAG-ZFP91 or point mutants of conserved cysteines in ZnF1 MT (C313A/C318A), ZnF2 MT (C344A/C349A), ZnF3 MT (C374A/C377A), ZnF4 MT (C402A/C405A), or ZnF5 MT (C432A/C435A) were incubated with anti-Myc antibody, and the immune complexes were analyzed by immunoblots (top two panels). A FLAG, Myc, and tubulin immunoblot was completed from the same cell lysates (bottom). D, the lysates of HEK293 cells co-transfected with Myc-NIK and HA-Ub48K (treated with proteasomal inhibitor MG132 for the final 4 h) or HA-Ub63K alone or together with FLAG-tagged TRAF2, cIAP1, and ZFP91, as indicated, were incubated with anti-Myc antibody, and the immune complexes were analyzed by immunoblots (top two panels). A FLAG, Myc, and tubulin immunoblot was completed from the same cell lysates (bottom). IP, immunoprecipitation; IB, immunoblot.

ZFP91 mediates the Lys⁶³-linked ubiquitination and phosphorylation of NIK in CD40 signaling. A, the lysates of HEK293 cells co-transfected with FLAG-ZFP91 and HA-Ub together with Myc-NIK wild type, Myc-NIK K429A/K430A (KK429/430AA), or Myc-NIK T559A were incubated with anti-Myc antibody. Immunoblot analyses for immunoprecipitated proteins were completed by using anti-HA and Myc antibodies (top two panels) from the immune complexes and anti-phospho-NIK, p52, FLAG, Myc, and tubulin antibodies (bottom four panels) from the same cell lysates. B, lysates from HEK293 cells co-transfected with NF-κB luciferase reporter construct pNF-κB-Luc, Myc-NIK, and FLAG-ZFP91 along with HA-Ub, HA-Ub Lys⁴⁸-only (HA-UbK48^only), or HA-Ub Lys⁶³-only (HA-UbK63^only), as indicated, were analyzed by luciferase activity and immunoblot. C, MDA-MB231 cells transfected with a control or ZFP91 siRNA stimulated with CD154 for the indicated time. Cell lysates were subject to immunoblots using anti-p52, NIK, ZFP91, and tubulin antibodies. D, MDA-MB231 cells co-transfected with Myc-NIK and a control or ZFP91 siRNA stimulated with CD154 for 6 h. Cell lysates were immunoprecipitated with anti-Myc antibody, and the immune complexes were analyzed with an antibody specific for Lys⁶³-linked ubiquitin. The immunoblots (IB) using anti-ZFP91, anti-Myc, and anti-tubulin antibodies were completed from the same cell lysates. E, MDA-MB231 cells transfected with a control or ZFP91 siRNA stimulated with CD154 for the indicated time. RNAs were isolated from cells, reverse-transcribed, and analyzed by real-time PCR for IL-6, IL-8, MCP1, TNF-α, BAFF, and CCL19. Data are represented as mean ± S.D. (error bars) of three independent experiments. IB, immunoblot; IP, immunoprecipitation.