Figure 2
XIAP interacts with BCL10 and promotes BCL10 K63 polyubiquitination and BCL10-directed NF-κB activation. (A) Interaction between endogenous XIAP and BCL10 in macrophages. BMDMs were activated with curdlan, and total cell lysates were prepared at the indicated time points. Total cell lysates (1 mg) were immunoprecipitated with anti-BCL10, and the presence of XIAP, MALT1, and BCL10 was determined. (B) BCL10 interacts with the BIR1 domain of XIAP. FLAG-tagged full-length (FL), deleted RING finger (ΔR), N-terminal (N), C-terminal (C), BIR1, BIR2, or BIR3 of XIAP were cotransfected with BCL10-Myc into 293T. Total cell lysates were immunoprecipitated by anti-Myc, and the presence of XIAP and its mutants in immune complexes was detected by anti-FLAG. (C) FL-XIAP, but neither XIAPΔRF nor XIAPΔBIR1, promotes BCL10 polyubiquitination. HA-ubiquitin, BCL10, XIAP, XIAPΔRF, or XIAPΔBIR1 (ΔB) was transfected into 293T cells as indicated. Total cell lysates were prepared and immunoprecipitated by anti-BCL10, and Ub-containing protein was detected by anti-HA. (D) BCL10 K63 ubiquitination was induced by XIAP in vitro. In vitro ubiquitination assays were conducted in reaction mixtures containing human ubiquitin-K63, human E1, E2 (Ubc13), BCL10-FLAG, XIAP, or XIAPΔRF, as indicated. BCL10-FLAG was loaded on protein G magnetic beads through anti-FLAG. Reactions proceeded at 30°C for 1 hour. BCL10-FLAG-protein G was pulled down, separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blotted with ubiquitin antibody (upper panel). The input is shown in the lower panel. (E) Lys31 and Lys63 on BCL10 were required for XIAP-mediated K63 ubiquitination on BCL10. WT-BCL10 or BCL10-KKRR (K31R, K63R) was cotransfected with XIAP and K63 Ub into 293T cells. Total cell lysates were immunoprecipitated by anti-BCL10. Ubiquitinated BCL10 of the immune complexes was detected by anti-HA antibody. (F) XIAP enhances BCL10-activated κB reporter. κB-Luc was transfected with EGFP, XIAP, XIAPΔRF, XIAPΔBIR1, and BCL10, as indicated, into 293T cells, and luciferase activity was determined 24 hours later. EGFP was used to determine transfection efficiency. ***P < .001 for paired Student t test. (A-F) Each experiment was repeated 3 times with similar results.

XIAP interacts with BCL10 and promotes BCL10 K63 polyubiquitination and BCL10-directed NF-κB activation. (A) Interaction between endogenous XIAP and BCL10 in macrophages. BMDMs were activated with curdlan, and total cell lysates were prepared at the indicated time points. Total cell lysates (1 mg) were immunoprecipitated with anti-BCL10, and the presence of XIAP, MALT1, and BCL10 was determined. (B) BCL10 interacts with the BIR1 domain of XIAP. FLAG-tagged full-length (FL), deleted RING finger (ΔR), N-terminal (N), C-terminal (C), BIR1, BIR2, or BIR3 of XIAP were cotransfected with BCL10-Myc into 293T. Total cell lysates were immunoprecipitated by anti-Myc, and the presence of XIAP and its mutants in immune complexes was detected by anti-FLAG. (C) FL-XIAP, but neither XIAPΔRF nor XIAPΔBIR1, promotes BCL10 polyubiquitination. HA-ubiquitin, BCL10, XIAP, XIAPΔRF, or XIAPΔBIR1 (ΔB) was transfected into 293T cells as indicated. Total cell lysates were prepared and immunoprecipitated by anti-BCL10, and Ub-containing protein was detected by anti-HA. (D) BCL10 K63 ubiquitination was induced by XIAP in vitro. In vitro ubiquitination assays were conducted in reaction mixtures containing human ubiquitin-K63, human E1, E2 (Ubc13), BCL10-FLAG, XIAP, or XIAPΔRF, as indicated. BCL10-FLAG was loaded on protein G magnetic beads through anti-FLAG. Reactions proceeded at 30°C for 1 hour. BCL10-FLAG-protein G was pulled down, separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blotted with ubiquitin antibody (upper panel). The input is shown in the lower panel. (E) Lys31 and Lys63 on BCL10 were required for XIAP-mediated K63 ubiquitination on BCL10. WT-BCL10 or BCL10-KKRR (K31R, K63R) was cotransfected with XIAP and K63 Ub into 293T cells. Total cell lysates were immunoprecipitated by anti-BCL10. Ubiquitinated BCL10 of the immune complexes was detected by anti-HA antibody. (F) XIAP enhances BCL10-activated κB reporter. κB-Luc was transfected with EGFP, XIAP, XIAPΔRF, XIAPΔBIR1, and BCL10, as indicated, into 293T cells, and luciferase activity was determined 24 hours later. EGFP was used to determine transfection efficiency. ***P < .001 for paired Student t test. (A-F) Each experiment was repeated 3 times with similar results.

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