mβTrCP binds to phosphorylated IκBα. (A) In vitro binding. In vitro-translated ³⁵S-labeled IκBα (lanes 1,2,4,5) or S32A/S36A mutant (lanes 3,6) was phosphorylated by a MEKK1-activated IκB kinase complex and incubated with ³⁵S-labeled mβTrCP (lanes 1–3) or mβTrCPΔF (lanes 4–6) in RIPA buffer plus 0.1% SDS. Proteins associated with IκBα were coimmunoprecipitated with an IκBα-specific antibody and analyzed by SDS-PAGE. Lanes 7–10 show an aliquot of the in vitro-translated proteins, which is ∼40% of input in the immunoprecipitation experiments shown in lanes 1–6. (WT) Wild-type IκBα; (AA) S32A/S36A; (FL) full-length mβTrCP (wild type); (ΔF) F-box-deleted mutant of mβTrCP; (IP) immunoprecipitation; (IVT) in vitro translation. (B) mβTrCP forms a complex with phosphorylated IκBα, p65, and Skp1 in vivo. 293 cells were transfected with 3 μg of pcDNA₃–Myc–mβTrCP (lanes 2,3,6,7,10,11) or pcDNA3-Myc-mβTrCPΔF (lanes 4,5,8,9,12,13). After incubating the cells with 20 μm of MG132 for 30 min, the cells were treated with (lanes 3,5,7,9,11,13) or without (lanes 2,4,6,8,10,12) calyculin A (0.1 μm) for 10 min. Cell extracts were immunoprecipitated with antibodies against IκBα (lanes 2–5), p65 (lanes 6–9), or Myc (lanes 10–13), respectively, and the precipitated proteins analyzed by immunoblotting with antibodies against Myc, Skp1, IκBα, and p65, respectively. Lane 1 is 10 μg of 293 cell extract that also expresses mβTrCP. (C) mβTrCP binds directly to p-IκBα. 293 cells were transfected with pcDNA3 containing Myc–mβTrCP, together with Flag–IκBα (F-IκBα, lanes 1,2) or Flag–IκBαΔN mutant (FΔN, lanes 3,4). Following treatment of the transfected cells with MG132 and calyculin A, the cell extracts were immunoprecipitated with an anti-Flag antibody (M2, Kodak), and the precipitated proteins analyzed by immunoblotting with antibodies against Myc, Skp1, IκBα and p65, respectively.

Dominant-negative βTrCP inhibits the activation of NF-κB-dependent transcription. (A) Effect of mβTrCPΔF on induction of the NF-κB-dependent reporter gene by TNFα. 293 cells were transfected with pcDNA₃–mβTrCPΔF (0.01–3 μg as indicated), together with a NF-κB-dependent (●) or GAL4-dependent (□) luciferase reporter construct (p[κB]₃–tk–Luc, or Gal4–E1b–Luc, 10 ng each). The NF-κB reporter gene was stimulated with TNFα (20 ng/ml) for 6 hr prior to harvest, whereas the GAL4 reporter gene was activated by cotransfection with the strong activator GAL4–VP16 (200 ng). Reporter activity is expressed as fold induction of normalized luciferase activity in stimulated cells relative to that of unstimulated cells. TNFα stimulated the NF-κB reporter by 14-fold, whereas the GAL4–VP16 activation was >3000-fold. (B) Effect of mβTrCPΔF on the activation of NF-κB by IL-1β, NIK, MEKK1, and IKKβ. 293 cells were transfected with pcDNA₃–mβTrCPΔF and p[κB]₃–tk–Luc as described in A, together with 100 ng of NIK, MEKK1, and IKKβ expression constructs, respectively. Stimulation of cells with IL-1β (10 ng/ml) was carried out for 6 hr prior to harvest. Luciferase activity was determined and normalized as described in Materials and Methods.

βTrCP promotes ubiquitination of phosphorylated IκBα in vitro. (A) Reconstitution of IκBα ubiquitination. ³⁵S-labeled IκBα or mutants were incubated in an ATP-supplemented reconstitution system containing E1, Ubch5, Ub, p50/p65, IκB kinase and MEKK1Δ, as described in Materials and Methods. Ubiquitination was initiated by the addition of immunoprecipitated beads containing Myc–mβTrCP, which was expressed in 293 cells. Ubiquitinated products were analyzed by SDS-PAGE following immunoprecipitation with an anti-p65 antibody, which coprecipitated IκBα, as well as ubiquitinated IκBα (Chen et al. 1995). (Lane 1) No E3 was added; (lane 2) immunoprecipitated Myc–mβTrCPΔF was used in place of Myc–mβTrCP; (lane 3) normal rabbit IgG instead of Myc antibody was used to precipitate Myc–mβTrCP; (lane 4) no E2 was added; (lanes 5–8) Myc–mβTrCP (FL) beads were used to ubiquitinate wild-type and mutant IκBα as indicated; (lanes 9–12) 293 cell extracts (S100) were used to ubiquitinate wild-type and mutant IκBα as indicated. An aliquot of the immunoprecipitated beads used in the ubiquitination reaction was analyzed by western blotting using antibodies against Myc (middle panel) or Skp1 (bottom panel). (B) Quantitation of IκBα ubiquitination. Ubiquitinated IκBα shown in A was quantitated with the aid of PhosphorImager. Because only multiubiquitinated IκBα are degraded efficiently by the 26S proteasome, Ub_n–IκBα containing more than two Ub units (n > 2) was quantitated and expressed as the percentage of total radioactivity in each lane.

βTrCP is required for TNFα-induced degradation of IκBα. 293 cells were transfected with pCDNA3–Flag–IκBα (20 ng), together with increasing concentration of pcDNA₃–mβTrCPΔF (lanes 1,2, vector only; lanes 3,4, 0.01 μg; lanes 5,6, 0.1 μg; lanes 7,8, 1 μg). After treatment of cells with or without TNFα (20 ng/ml) for 30 min, cell extracts were analyzed by immunoblotting with antibodies against Flag, IκBα, or Myc, respectively.

Slimb is required for the Dorsal-dependent activation of twi and sna in Drosophila embryos. Wild-type (A,C) and slimb mutant (B,D) embryos were hybridized with twi (A,B) or sna (C,D) antisense RNA probes to visualize their expression. Embryos were oriented with anterior to the left and dorsal up. Wild-type embryos at the blastoderm stage express the Dorsal target genes twi and sna in the ventral region (A,C). In contrast, slimb mutant embryos at the same stage show diminished expression of both twi and sna in most of the ventral region, with residual expression detectable at the posterior pole (arrows in B and D).

A proposed SCF pathway for the signal-induced ubiquitination of IκBα. βTrCP/Slimb associates with Skp1 and another protein (possibly Cdc53-like) to form a SCF^βTrCP complex. Upon phosphorylation of IκB by an IκB kinase complex, βTrCP recruits IκBα to the SCF complex, allowing the associated E2, such as Ubch5, to ubiquitinate IκBα. Following ubiquitination, IκBα is rapidly degraded by the 26S proteasome, allowing NF-κB to translocate into the nucleus where it activates gene transcription. NF-κB is represented by a heterodimer of p50 and p65, whereas a multiubiquitin chain is represented by the branch structure on IκBα.