Fig. 2. IκKβ-mediated processing of p105 requires the C-terminal domain of the precursor molecule. (A) Transfection of cells with constitutively active IκKβ significantly stimulates processing of p105. Cos-7 cells were transiently transfected with a cDNA coding for wild-type p105 with or without a cDNA coding for IκKβ. Processing was monitored in a pulse–chase labeling and immunoprecipitation experiment as described in Materials and methods. (B) Enhanced processing of p105 requires active IκKβ. Cos-7 cells were transiently transfected with a cDNA coding for wild-type p105 without or with cDNAs coding for either constitutively active (IκKβ-SS>EE) or inactive (IκKβ-SS>AA) IκKβs (see Materials and methods). Processing was monitored as described in (A). (C) The acidic domain of p105 (residues 446–454) is not required for IκKβ-mediated processing of the molecule. Cos-7 cells were transiently transfected with cDNAs coding for either wild-type p105 or p105-Δ446–454, with or without a cDNA coding for constitutively active IκKβ. Processing was monitored as described in (A). (D) The C-terminal phosphorylation domain of p105 (residues 918–934) is required for IκKβ-mediated processing of the precursor molecule. Cos-7 cells were transiently transfected with cDNAs coding for either wild-type p105 or p105-Δ918–934, with or without a cDNA coding for IκKβ. Processing was monitored as described in (A).

Fig. 4. ΔF-box β-TrCP1 inhibits processing of wild-type p105 but not p105-Δ918–934. (A) Effect of ΔF-box β-TrCP1 on processing of wild-type p105 and p105-Δ918–934 in the presence of IκKβ. Cos-7 cells were transiently transfected with cDNAs coding for either wild-type p105 or p105-Δ918–934. cDNAs coding for ΔF-box β-TrCP1 and IκKβ were co-transfected as indicated. Processing was monitored in a pulse–chase labeling and immunoprecipitation experiment as described in Materials and methods. (B) Effect of ΔF-box β-TrCP1 on processing of wild-type p105 and p105-Δ918–934 in the absence of IκKβ. Cos-7 cells were transiently transfected with cDNAs coding for either wild-type p105 or p105-Δ918–934. A cDNA coding for ΔF-box β-TrCP was co-transfectd as indicated. Processing was monitored as described in (A). Quantitative analysis of processing ([p50/p50 + p105] × 100) is presented (bottom of panels) following subtraction of background radioactivity (time 0). (C) The C-terminal domain of p105 is phosphorylated in vivo under non-stimulated conditions. (C1) Phosphorylation of wild-type and Δ918–934 p105s in vivo. Cos-7 cells were transiently transfected with cDNAs coding for either wild-type p105 or p105-Δ918–934. Cells were labeled with H₃[³²P]O₄, and the labeled p105 was precipitated and visualized as described in Materials and methods. (C2) Western blot analysis of expressed wild-type p105 and p105-Δ918–934. Cos-7 cells were transfected with cDNAs coding for either wild-type p105 or p105-Δ918–934 as described in (A). Proteins were analyzed using anti-p50 antibody and western blot analysis.

Fig. 6. The IκBα phosphopeptide specifically inhibits conjugation and processing of p105 in vitro. (A) The IκBα phosphopeptide inhibits conjugation of p105. Conjugation of p105 in a cell-free system was monitored as described in Materials and methods in the absence or presence of ATP, the IκBα-derived phosphopeptide (pP) or the S32,36A control peptide (S/A) as indicated. (B) The IκBα phosphopeptide inhibits processing of p105. Processing of p105 in a cell-free system was monitored as described in Materials and methods and in (A). (C) Effect of the IκBα-derived phosphopeptide on processing of wild-type p105, p105-Δ918–934 and p105-Δ446–454. Processing of wild-type p105, p105-Δ918–934 and p105-Δ446–454 was monitored in a cell-free system as described in Materials and methods in the absence or presence of ATP and the IκBα-derived phosphopeptide (pP), as indicated.

Fig. 8. β-TrCP, but not Skp2, is the ubiquitin ligase involved in conjugation and processing of phosphorylated p105 in vitro. (A1) h-β-TrCP reconstitutes p105 conjugation. ³⁵S-labeled p105 translated in wheat germ extract was subjected to in vitro kinase assay in the presence of IκKβ, and conjugation was carried out as described in Materials and methods. Recombinant and purified SCF^h-β-TrCP (lanes 1 and 2), wheat germ extract (75 µg of protein; lanes 3 and 4) or crude HeLa extract (75 µg of protein; lanes 5 and 6) were used as the source of E3. (A2) IκKβ-dependent phosphorylation of p105 is required for its β-TrCP-mediated conjugation. ³⁵S-labeled p105 (as in A1) was subjected to in vitro kinase assay in the presence (lanes 1–5) or absence (lane 6) of IκKβ as described in Materials and methods, and conjugation was monitored as described. Purified recombinant tetrameric SCF^β-TrCP was used at 0.75 (1×; lane 3) and 1.5 (2×; lanes 4 and 6) µg. (B) SCF^β-TrCP reconstitutes processing of p105. ³⁵S-labeled p105 (as in A1) was subjected to in vitro kinase assay as described in Materials and methods. Processing was carried out in a complete (lanes 1 and 2) or Skp1-depleted (lanes 3 and 4) HeLa cell extract in the absence (lane 3) or presence (lane 4) of purified SCF^β-TrCP as described in Materials and methods. (C) Skp2 is not required for conjugation of phosphorylated p105. (C1) Conjugation of p27^Kip1 requires Skp2. In vitro trans lated ³⁵S-labeled p27^Kip1 was incubated in the presence of Skp2-immunodepleted synchronized HeLa extract as described in Materials and methods. Recombinant and purified Skp2 (0.5 µg) were added when indicated. (C2) Conjugation of p105 is Skp2 independent. ³⁵S-labeled p105 (as in A1) was subjected to in vitro kinase assay in the presence of IκKβ as described in Materials and methods. Conjugation was monitored in the presence of complete or Skp2-immunodepleted HeLa cell extract as indicated and described in Materials and methods. All notes are as described in the legend to Figure 1.

Fig. 1. The acidic domain (residues 443–454) and the C-terminal IκK phosphorylation site of p105 are both required for processing of p105 in vitro. (A) Sequence of amino acid residues 443–454 and 918–934 of p105. The asterisks denote aspartate residues 448, 451 and 452, which were replaced by alanine in the experiments described in (B) and (C). The plus signs denote Asp443, Glu445 and Ser450, which were replaced by alanine in the same experiments. Acidic and serine residues in the 443–454 sequence and serine and threonine residues in the 918–934 sequence are in bold. (B) ATP-dependent conjugation of wild-type-p105, p105-D(*)A and p105-D,E,S(*,+)A. Conjugation of the p105 acidic domain mutants was carried out in a crude HeLa cell extract as described in Materials and methods. (C) ATP-dependent processing of wild-type p105 and p105-D,E,S(*,+)A. Processing of wild-type p105 and p105-D,E,S(*,+)A was carried out in a crude HeLa cell extract as described in Materials and methods. (D) ATP-dependent processing of wild-type p105, p105-Δ446–454, p105-Δ918–934 and p105-Δ446–454/Δ918–934. Processing of the different p105 deletion mutants was carried out as described in (C). Conj. denotes conjugates. p50 and p105 denote the site of migration of the two molecules, respectively.

Fig. 3. IκKβ-dependent phosphorylation of p105 requires residues 918–934. The effect of IκKβ was monitored using wild-type p105 (lanes 1–5) or p105-Δ918–934 (lanes 6–10). Translated [³⁵S]methionine-labeled (lanes 1–3 and 6–8) or unlabeled (lanes 4 and 5, and 9 and 10; an amount equivalent to the labeled protein) p105s were incubated in the presence of unlabeled (lanes 1–3 and 6–8) or [γ-³²P]ATP (lanes 4 and 5, and 9 and 10) as described in Materials and methods. IκKβ was added as indicated. Following incubation for 25 min, calf intestine alkaline phosphatase (10 U) was added where indicated, and the incubation continued for an additional 5 min. p105 was precipitated using anti-p50 antibody and immobilized protein A. Samples were resolved via SDS–PAGE and proteins visualized via phosphoimaging. p-p105 denotes phosphorylated p105.

Fig. 5. ΔF-box β-TrCP1 and ΔF-box β-TrCP2, but not ΔF-box Skp2, inhibit processing of p105. (A) Effect of ΔF-box β-TrCP1 and 2 on the processing of p105. Cos-7 cells were transiently transfected with a cDNA coding for wild-type p105. A cDNA coding for ΔF-box β-TrCP1 (A1) or ΔF-box β-TrCP2 (A2) was co-transfected where indicated. Processing of p105 was monitored in a pulse–chase labeling and immunoprecipitation experiment as described in Materials and methods. (B) Effect of ΔF-box Skp2 on processing of wild-type p105. Cos-7 cells were transiently transfected with a cDNA coding for wild-type p105. cDNAs coding for ΔF-box Skp2 or ΔF-box β-TrCP1 were co-transfected where indicated. Processing of p105 was monitored as described in (A).

Fig. 7. Wild-type p105, but not p105-Δ918–934, associates physically with ΔF-box β-TrCP following phosphorylation. (A) TNF-α stimulates physical association between p105 and β-TrCP in cells. Cos-7 cells were transiently transfected with cDNAs coding for His₆-tagged ΔF-box β-TrCP and either wild-type p105 or p105-Δ918–934 as indicated. After 40 h, TNF-α was added as described in Materials and methods. Cells were disrupted, His-TrCP was immobilized, and proteins were visualized via western blot analysis using anti-p50 antibody as described in Materials and methods. The site of migration of wild-type p105 is marked by separation of in vitro translated labeled p105 (lane 5). (B) Association between p105 and β-TrCP in vitro requires prior IκK-mediated phosphorylation. In vitro translated and labeled wild-type p105 (lanes 1–3) or p105-Δ918–934 (lanes 4 and 5) were incubated in the absence (lane 1) or presence (lanes 2–5) of IκK and ATP as described in Materials and methods. His-tagged β-TrCP was added when indicated. Following incubation, the complex was immobilized and proteins resolved and visualized as described in Materials and methods.

Fig. 9. Two ubiquitin ligases are involved in p105 processing, one of them, SCF^β-TrCP, requires the presence of residues 918–934 and, in particular, serine residues 922, 924 and 933. (A) SCF^β-TrCP conjugates wild-type and Δ446–454, but not Δ918–934 and Δ446–454-S922,924,933A p105s. In vitro translated and kinase-phosphorylated wild-type (lanes 1–3), Δ446–454 (lanes 4–6), Δ918–934 (lanes 7–9) and Δ446–454-S922,924,933A (lanes 10–12) p105s were subjected to in vitro conjugation in a reconstituted cell-free system as described in Materials and methods. When indicated, Ubc5Hc and SCF^h-β-TrCP were added as described. (B) Conjugation of wild-type and Δ918–934 p105s in crude HeLa extract. In vitro translated and kinase-phosphorylated wild-type (lanes 1 and 2) and Δ918–934 (lanes 3 and 4) p105s were subjected to in vitro conjugation as described in Materials and methods. The cell-free system contained crude HeLa cell extract as a source of the conjugating enzymes. Proteins were resolved and visualized as described in Materials and methods.