NEMO binds ubiquitinated IRAK-1 but not ubiquitinated TRAF6. (A) 293/IL-1R/TLR4 cells transfected with HA-tagged K63-only ubiquitin were stimulated or not with IL-1 for the indicated amounts of time, harvested, and lysed with detergent. The lysates were heated at 95°C in the presence of 1% SDS for 5 min and diluted to a final concentration of 0.1% SDS, and IRAK-1 and TRAF6 were immunoprecipitated (IP). The amounts of K63-linked polyubiquitinated IRAK-1 and TRAF6 were determined by immunoblotting (IB) with anti-HA. The amounts of immunoprecipitated IRAK-1 and TRAF6 were determined by immunoblotting with anti-IRAK-1 and anti-TRAF6. (B) 293/IL-1R/TLR4 cells were stimulated with or without IL-1 for 5 min, harvested, and lysed. Lysates were either heated as for panel A and immunoprecipitated with anti- IRAK-1 or anti-TRAF6 or they were incubated with GST- or GST-NEMO-coated beads. The eluted material and lysates were immunoblotted with anti-Ub (left panels) or anti-IRAK-1 and anti-TRAF6 (right panels). (C) NEMO was immunoprecipitated from cell lysates made from 293/IL-1R/TLR4 cells stimulated with or without IL-1 for 5 min and immunoblotted for IRAK-1, TRAF6, and NEMO. IRAK-1 and TRAF6 in the lysates were detected by immunoblotting with anti-IRAK-1 and anti-TRAF6. (D) NEMO was immunoprecipitated from lysates made from 293/IL-1R/TLR4 cells that had been stimulated with LPS for the indicated times and immunoblotted for IRAK-1 and NEMO.

K63-linked polyubiquitin binding of NEMO is required for IRAK-1 binding and IL-1-induced IκB degradation. (A) Cell lysates from 293/IL-1R/TLR4 cells that had been stimulated with or without IL-1 (10 ng/ml) for 5 min were incubated with beads coated with GST, GST-NEMO, or GST-NEMO^L329P and immunoblotted (IB) with anti-IRAK-1. (B) NEMO was immunoprecipitated (IP) from cell lysates from NEMO-deficient MEFs stably reconstituted with HA-tagged NEMO (HA-NEMO) or HA-tagged NEMO^L329P (HA- NEMO^L329P) that had been stimulated with or without IL-1 (5 ng/ml) for 5 min and immunoblotted with anti-IRAK-1 or anti-HA. (C) NEMO-deficient MEFs (-/-) or NEMO-deficient MEFs reconstituted with HA-NEMO or HA-NEMO^L329P were stimulated with IL-1 (1 ng/ml) for the indicated periods of time. The cells were harvested and lysed, and the amounts of IκB and NEMO were determined by immunoblotting. (D) HA-NEMO or HA-NEMO^L329P cells were stimulated with 0.2 or 0.5 ng/ml IL-1 for 10 min, harvested, and lysed, and the amount of IκB was determined by immunoblotting. The lanes were reordered for clarity. (E) IL-1- and LPS-induced NF-κB luciferase activity as measured by relative light units (RLU) in NEMO-deficient MEFs transiently transfected with vector (-), NEMO, or NEMO^L329P.

TRAF6 E3 activity is required for K63-linked polyubiquitination of IRAK-1. (A) GST-TRAF6-coated beads were incubated with in vitro-translated and ³⁵S-labeled IRAK-1, washed extensively, and incubated in the presence or absence of ATP, and/or Ubc13/Uev1A, and ubiquitin. IRAK-1 ubiquitination was detected using a phosphorimager. No IRAK-1 binding was detected with beads coated with GST alone. (C) Cell lysates from wild-type (WT) or TRAF6-deficient (T6-/-) MEFs that were stimulated with or without IL-1 for 5 min were incubated with GST-NEMO-coated beads and immunoblotted (IB) with anti-IRAK-1. (C) NEMO was immunoprecipitated (IP) from lysates of WT or TRAF6-deficient MEFs that had been stimulated with or without IL-1 for 5 min and immunoblotted with anti-IRAK-1 and anti-NEMO. (D) Cell lysates from T6^−/− MEFs or T6^−/− MEFs reconstituted with wild-type TRAF6 (T6-WT) or TRAF6^C70A (T6-C70A) were immunoblotted for TRAF6. (E) Lysates made from unstimulated or IL-1-stimulated T6^−/−, T6-WT, or T6-C70A MEFs were incubated with GST-NEMO-coated beads and immunoblotted for IRAK-1. (F) IL-1-induced NF-κB DNA binding in T6^−/−, T6-WT, and T6-C70A MEFs was assessed in an electrophoretic mobility shift assay. (G) IL-1-induced p38 dual phosphorylation (p-p38) was measured in lysates of T6^−/−, T6-WT, and T6-C70A MEFs by immunoblotting.

IRAK-1 lysines 134 and 180 are required for TRAF6-mediated and IL-1-induced ubiquitination. (A) In vitro ubiquitination of in vitro-translated and ³⁵S-labeled IRAK-1 (WT), IRAK-1^K134R (K134R), IRAK-1^K180R (K180R), or IRAK-1^K134/180R (K134/180R) by GST-TRAF6 in the presence of ATP, Ubc13/Uev1A, and K63-only ubiquitin (K63O). Lanes 1 to 4 are the in vitro-translated products offered to the GST-TRAF6-coated beads. The ubiquitination reactions are shown in lanes 5 to 12. (B) IRAK-1 was either immunoprecipitated (IP) with anti-IRAK-1 or pulled down with GST-NEMO-coated beads from lysates of unstimulated and IL-1-stimulated IRAK-1-deficient MEFs retrovirally transduced with vector (-), WT, K134R, K180R, or K134/180R. IRAK-1 immunoprecipitates were immunoblotted (IB) with anti-Ub, and GST-NEMO-bound material was blotted with anti-IRAK-1. Expression of IRAK-1 and β-actin in the lysates is shown in the bottom two rows.

IRAK-1 lysines 134 and 180 are required for IL-1-induced NF-κB but not p38 activation. (A) NF-κB activation in 293/IL-1R/TLR4 cells transiently transfected with increasing amounts of IRAK-1 (WT), IRAK-1^K134R (K134R), IRAK-1^K180R (K180R), or IRAK-1^K134/180R (K134/180R). Values were normalized to β-galactosidase activity and are expressed as the induction level compared to luciferase in cells transfected with empty vector (-). Expression of WT, K134R, K180R, and K134/180R was confirmed by immunoblotting (IB) with anti-Flag. A large nonspecific band just below IRAK-1 is denoted by ns. (B) IL-1- and LPS-induced NF-κB luciferase activity was measured in IRAK-1-deficient MEFs transiently transfected with an NF-κB-driven luciferase reporter in combination with vector (-), WT, K134R, K180R, or K134/180R. Each transfection was performed in triplicate, and error bars represent the standard errors of the means. This experiment is representative of five independent experiments. (C) IL-1-induced p38 activation was measured in IRAK-1-deficient MEFs transiently transfected with vector, IRAK-1, or IRAK-1^K134/180R. The amount of phosphorylated (p-p38) and total p38 was determined by immunoblotting.