The NEDD8 Pathway Is Essential for SCFβ-TrCP-mediated Ubiquitination and Processing of the NF-κB Precursor p105*

The p50 subunit of NF-κB is generated by limited processing of the precursor p105. IκB kinase-mediated phosphorylation of the C-terminal domain of p105 recruits the SCFβ-TrCP ubiquitin ligase, resulting in rapid ubiquitination and subsequent processing/degradation of p105. NEDD8 is known to activate SCF ligases following modification of their cullin component. Here we show that NEDDylation is required for conjugation and processing of p105 by SCFβ-TrCP following phosphorylation of the molecule. In a crude extract, a dominant negative E2 enzyme, UBC12, inhibits both conjugation and processing of p105, and inhibition is alleviated by an excess of WT- UBC12. In a reconstituted cell-free system, ubiquitination of p105 was stimulated only in the presence of all three components of the NEDD8 pathway, E1, E2, and NEDD8. A Cul-1 mutant that cannot be NEDDylated could not stimulate ubiquitination and processing of p105. Similar findings were observed also in cells. It should be noted that NEDDylation is required only for the stimulated but not for basal processing of p105. Although the mechanisms that underlie processing of p105 are largely obscure, it is clear that NEDDylation and the coordinated activity of SCFβ-TrCP on both p105 and IκBα serve as an important regulatory mechanism controlling NF-κB activity.

NF-B 1 is a member of the Rel family of transcription factors that are known to regulate basic processes, such as the immune and proinflammatory responses, development and differentiation, malignant transformation, and apoptosis (for a recent review, see Ref. 1). NF-B is a dimeric complex composed in many cases of p50 or p52 and p65 subunits. p50 and p52 are synthesized as inactive precursors, p105 and p100, respectively, which undergo ubiquitin-mediated limited processing that removes the C-terminal ankyrin repeat-containing domain of the molecule to yield the p50 or p52 N-terminal active subunit (2)(3)(4)(5). Intracellular localization plays an important role in NF-B regulation, with the inactive proteins retained in the cytosol. Regulation is achieved via two major pathways: 1) control of p105/p100 processing and 2) interaction of the p50/ p65 heterodimer with inhibitory molecules, IBs. Processing of p105 occurs under both basal and activated conditions. Several studies suggested that all the motifs that are required for basal processing or processing that may occur co-translationally are contained within the N-terminal ϳ550 amino residues (6 -9), whereas signal-induced processing requires phosphorylation of serine residues 923 and 927 (10 -12). In quiescent cells, the active heterodimeric complex is retained in the cytosol bound to a member of the IB family of inhibitory proteins. Following stimulation, specific IB kinases (IKKs) are activated, leading to phosphorylation of the inhibitor on serine residues 32 and 36, its rapid ubiquitination by the SCF ␤-TrCP ubiquitin ligase, and its subsequent degradation by the 26 S proteasome. Following degradation of IB, the NF-B active p50/p52⅐p65 heterodimeric complex translocates into the nucleus and activates target genes (for a recent review, see Ref. 13).
The ubiquitin proteolytic pathway plays key roles in regulating the levels of many proteins involved in diverse cellular processes. Proteins targeted for degradation are first tagged by a polyubiquitin chain in a three-step cascade reaction involving ubiquitin activation (catalyzed by the ubiquitin activation enzyme, E1), ubiquitin transfer (catalyzed by a ubiquitin carrier protein, E2), and ubiquitin ligation (catalyzed by a ubiquitinprotein ligase, E3). Tagged proteins are then degraded by the 26 S proteasome complex (for recent reviews on the ubiquitin pathway, see for example Refs. 14 and 15). There are four major classes of ubiquitin ligases classified according to a common motif shared by one of the enzyme components. HECT (homologous to E6-AP carboxyl terminus) domain proteins are represented by E6-AP (E6-associated protein) and contain within the HECT domain a conserved cysteine residue to which the activated ubiquitin is transferred from E2. The other three groups, the U-box-containing ligases (16), the PHD domaincontaining ligases (17), and the RING finger-containing E3s function differently and serve as scaffolds that position the substrate and the E2 optimally for direct ubiquitin transfer. The SCF ␤-TrCP involved in p105 processing (see below) belongs to the RING finger-containing ligases. It is a complex that contains three commonly shared subunits, Skp1, Cullin-1, and the RING finger protein Rbx1-Roc1-Hrt1, and an F-box protein which is a variable substrate-binding subunit involved in recognition of phosphorylated target proteins (18). The F-box protein ␤-TrCP recognizes the common motif DpSGDpS, where pS is phosphoserine and is a hydrophobic residue, that is found in IBs, ␤-catenin, and HIV-Vpu, for example (13).
Processing/degradation of p105 is mediated by two distinct structural motifs. The first resides adjacent to the glycine-rich region and contains two lysine residues that serve as ubiquitin anchors and a downstream acidic domain that may serve as an E3-binding site. This motif is probably involved in basal/constitutive and/or co-translational processing/degradation of p105, providing resting cells with the low amount of p50 required for their activity under non-stimulated conditions (8). The second motif is involved in signal-induced processing/degradation of p105 and is dependent upon IKK-mediated phosphorylation, SCF ␤-TrCP -mediated ubiquitination, and limited processing/degradation of the molecule (10 -12). In addition, the C-terminal domain of p105 contains seven ankyrin repeats between the glycine-rich region and the IKK/TrCP recognition domain. These repeats bind free p50 subunits that inhibit processing of the precursor (19 -21). Following stimulation, inhibition is alleviated via phosphorylation and ubiquitination of the C-terminal domain that lead to rapid processing/degradation of p105. The released free p50 subunits are then translocated into the nucleus (22).
NEDD8 is a mammalian member of ubiquitin-like (UbL) proteins, which modify proteins in a manner similar to ubiquitination, except that in most cases it is a single moiety of the modifying protein that is attached to the substrate (reviewed recently in Ref. 23). NEDDylation requires the coordinated action of APP-BPI/Uba3 (a heterodimeric E1-like enzyme) and UBC12 (an E2-like enzyme) (24,25). A possible role for an E3-like enzyme has been demonstrated recently for SUMO conjugation in yeast (26), although it is not clear whether a ligase is required in all reactions involving UbL protein modification. NEDD8 has been shown to modify members of the cullin/cdc53 family (23), which are the only known substrates of the pathway. Some of the cullins are members of ubiquitin ligase complexes; Cul-1 serves as a core subunit in the SCF ubiquitin ligase family, whereas Cul-2 and Cul-5 assemble with Elongin B and C to form similar ligase complexes (27,28). The NEDD8 pathway is essential for cell viability in fission yeast (29), but under basal conditions it is dispensable in the budding yeast (30,31). In mammalian cells, the NEDD8 pathway is essential for cell cycle progression and morphogenesis (32). NEDD8 is conjugated to Cul-1 at lysine residue 720 (Ref. 33; see also below), and this modification stimulates the ubiquitin ligase activity of SCF ␤-TrCP toward IB␣ (see, for example, Ref. 34) and of SCF Skp2 toward p27 kip1 (35,36). Mechanistically, it appears that NEDD8 up-regulates the SCF complex activity through a conformational change of Cul-1 that may promote efficient formation of an E2-E3 complex (37,38). Taken together, it seems that Cul-1 modification by NEDD8 is a universal regulated mechanism that has a profound effect on the ubiquitin ligase activity of SCF complexes and probably of other related cullin-containing complexes, thus affecting the levels of key cellular proteins.
Although it is recognized that NEDD8 modification stimulates the activity of SCF ubiquitin ligases, a role for the NEDD8 pathway in p105 ubiquitination that affects NF-B activation has not been established. Here we show that NEDD8 modification is required for efficient SCF ␤-TrCP -mediated ubiquitination and processing of p105 following phosphorylation of the molecule by IKK␤. Thus, NEDD8 modification serves as an additional regulatory mechanism controlling NF-B transcriptional activity, linking together the ubiquitin and NEDD8 pathways with signal-induced NF-B activation.

EXPERIMENTAL PROCEDURES
Materials-Materials for SDS-PAGE and Bradford reagent for protein concentration determination were from Bio-Rad. L-[ 35 S]Methionine (Ͼ1000 Ci/mmol; ϳ50 mCi/ml) for in vitro translation, prestained molecular weight markers, and immobilized Protein A were obtained from Amersham Biosciences. Tissue culture sera and media were from Biological Industries, Bet Haemek, Israel, or Sigma. Rabbit anti-NF-B1 p50 antibody that recognizes both p105 and p50 was from Santa Cruz, and peroxidase-conjugated goat anti-rabbit antibody was from Jackson ImmunoResearch Laboratories. Antibody to the His-tag and Ni-NTA resin were from Qiagen. Anti-hemagglutinin, anti-FLAG, and anti-T7 were from Roche Molecular Biochemicals, Sigma, and Novagen, respectively. Ubiquitin, dithiothreitol, ATP, ATP␥S, phosphocreatine, creatine phosphokinase, 2-deoxyglucose, isopropyl-␤-D-thiogalactopyranoside, and Tris buffer were from Sigma. Hexokinase and FuGENE TM 6 transfection reagent were from Roche Molecular Biochemicals. Protease inhibitor mixture was from Calbiochem. Reagents for enhanced chemiluminescence (ECL) were from Pierce. A wheat germ extract-based coupled transcription-translation kit was from Promega. Restriction and modifying enzymes were from New England Biolabs. Oligonucleotides were synthesized by Biotechnology General, Rehovot, Israel. All other reagents were of the highest analytical grade.
Cell Lines-COS-7 cells were grown at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics (penicillin/streptomycin). Hi-5 insect cells were grown at 27°C in Grace medium supplemented with 10% fetal calf serum, lactalbumin hydrolysate, yeastolate, and antibiotics (penicillin/streptomycin and gentamycin). Transfections were carried out using either the Fu-GENE TM reagent or the calcium phosphate method (39).
Plasmids and Construction of Mutants-Wild-type human p105 cDNAs used for in vitro translation (pT7␤105) and for transient transfection (pCI-neo) were described previously (4,8). cDNA coding for the constitutively active (S176E,S180E) IKK␤ was as described (11,40). WT-UBC12 cDNA was generated by reverse transcriptase-PCR of RNA prepared from a UOK-111 human kidney carcinoma cell line using the Trizol kit (Invitrogen) according to the manufacturer's instructions. It was subcloned in-frame with the His 6 tag into pcDNA3.1 for cell expression or into pT7-7 for bacterial expression. Dominant negative (DN) UBC12(C111S) mutants for mammalian cell and bacterial expression were generated by site-directed mutagenesis using the QuikChange TM kit (Stratagene). Sequencing was determined using the ABI 310 autosequencer. cDNAs coding for Cul-1(K696R) for both cell expression and expression of protein in insect cells were as described (37). Note that two Cul-1 proteins were described, one of them has an extra 24 amino acids near the N-terminal region; therefore the previously reported modified K720R (33)(34)(35) corresponds to K696R of the shorter Cul-1 (37). cDNA coding for His-NEDD8 for bacterial expression in pT7-7 was a kind gift from Dr. Keiji Tanaka.
Transient Transfections and Processing of p105 in Cells-COS-7 cells were transiently transfected with 1.5 g of WT p105 cDNA along with ϳ1.5 g of each of the cDNAs coding for the constitutively active IKK␤ and WT-UBC12, DN-UBC12(C111S), or Cul-1(K696R) as indicated. An empty vector was added when necessary to maintain an equal amount of DNA in all transfections. Transfections were carried out by using either FuGENE TM reagent or the calcium phosphate method. 48 h after transfection, cells were harvested and lysed. Equal amounts of protein were resolved via SDS-PAGE (10%) and blotted onto nitrocellulose paper. Processing of p105 was monitored by Western blot analysis using anti-p50 antibody and ECL.
In Vitro Phosphorylation of p105-p105 protein was translated in vitro using a wheat germ extract-based transcription-translation coupled kit in the presence of L-[ 35 S]methionine according to the manufacturer's instructions. Labeled p105 was phosphorylated in a cell-free system in a 25-l reaction mixture that contained 5 mM MgCl 2 , 0.5 mM ATP, and 0.4 g of baculovirus-expressed IKK␤. The reaction mixture was incubated for 20 min at 30°C.
Effect of the NEDD8 Pathway on in Vitro Ubiquitination and Processing of p105-In vitro ubiquitination and processing of p105 were monitored in crude HeLa extract as described (4,8,11). Ubiquitin-p105 conjugates were generated also in a reconstituted cell-free system as described (11). Briefly, phosphorylated [ 35 S]methionine-labeled p105 was incubated in a reaction mixture that contained in a final volume of 20 l the following components: 250 ng of purified E1 (41), 0.5 g of recombinant UBCH5C, ϳ1.5 g of recombinant SCF ␤-TrCP , 40 mM Tris-HCl (pH 7.6), 5 mM MgCl 2 , 2 mM dithiothreitol, 5 g of ubiquitin, 5 mM ATP␥S, and 0.5 g of Ubal (a specific inhibitor of certain isopeptidases, see Ref. 42). To remove all endogenous components of the NEDDylation machinery, labeled p105 was immunoprecipitated from the reaction mixture in which it was synthesized by using anti-p50. The immunoprecipitate, isolated on Protein A beads, was used as a source for the substrate (ϳ10,000 cpm). When indicated, NEDD8 (6 g), APP-BP1/ Uba3 (ϳ0.5 g), and Ubc12 (0.8 g) were added. All reaction mixtures were preincubated for 5 min at 37°C prior to the addition of the labeled substrate. Mixtures were incubated for 1 h at 37°C. Incubation was accompanied by gentle swirling. Following incubation, reaction mixtures were resolved via SDS-PAGE (7.5 or 10% polyacrylamide as indicated). Gels were dried, and proteins were visualized using phosphorimaging (Fuji, Tokyo, Japan).
Protein Concentration-Protein concentration was determined according to Bradford (43) using bovine serum albumin as a standard.

The NEDD8 Conjugation Pathway Is Required for p105
Ubiquitination-Ubiquitination of p105 appears to require the activity of the SCF ␤-TrCP E3 ligase complex that is preceded by IKK␤-mediated phosphorylation of specific serine residues at the C-terminal domain of the molecule (10 -12). It has been recently shown that NEDD8 modification of Cul-1 activates the ubiquitin ligase activity of SCF complexes (33)(34)(35)(36)(37)(38). Because a role for SCF ␤-TrCP in p105 processing has been established only recently, and because processing of p105 is only weakly dependent on signaling and occurs also under basal conditions (8), we wanted to clarify the role of NEDDylation in SCF ␤-TrCPmediated p105 ubiquitination and processing. Ubiquitin conjugation assays were carried out in a cell-free system containing phosphorylated p105, E1, UBCH5C, and SCF ␤-TrCP . Labeled p105 was immunoprecipitated from the mixture in which it was synthesized to isolate it from endogenous components of the NEDDylation machinery. As can be seen in Fig. 1 (lane 2), the three ubiquitin conjugating enzymes, E1, UBCH5C, and SCF ␤-TrCP , are not sufficient to promote conjugation of phosphorylated p105. It is the addition of the three components of the NEDDylation machinery, APP-BP1/Uba3, UBC12, and NEDD8, that stimulated conjugation significantly and resulted in conversion of almost all the labeled substrate into high molec- In vitro translated and labeled p105 was phosphorylated in a cell-free system by IKK␤ and immunoprecipitated with anti-p50 as described under "Experimental Procedures." All samples contained purified E1 and recombinant UBCH5C. WT SCF ␤-TrCP complex as well as the three components of the NEDD8 pathway were added as indicated. Samples were incubated and resolved on 7.5% SDS-PAGE, and proteins were visualized using phosphorimaging as described under "Experimental Procedures." Points of migration of p105 as well as p105-ubiquitin adducts (Conj.) are marked.

FIG. 2. SCF ␤-TrCP that contains Cul-1(K696R) fails to ubiquinate p105.
In vitro translated and labeled p105 was phosphorylated as described in the legend to Fig. 1 and under "Experimental Procedures." Since wheat germ extract does not contain a measurable SCF ␤-TrCP activity, we used the crude translation mixture rather then immunoprecipitated p105 as a source for the radiolabeled substrate. The NEDD8 pathway components were all provided by the crude wheat germ extract. All samples contained purified E1 and recombinant UBCH5C. WT and mutant Cul-1(K696R) SCF ␤-TrCP complexes were added as indicated. Samples were incubated and resolved on 7.5% SDS-PAGE, and proteins were visualized using phosphorimaging as described under "Experimental Procedures." Points of migration of p105 and of p105-ubiquitin adducts (Conj.) are marked.
NEDD8 pathway on p105 ubiquitination and processing in vitro and in vivo. Cul-1 is conjugated to NEDD8 at lysine 696 (or lysine 720 depending on the Cul-1 protein species used; see above and Refs. 33-35 and 37). We generated a point mutant Cul-1 in which this lysine is substituted with arginine and therefore cannot be conjugated to NEDD8. Ubiquitin conjugation of phosphorylated p105 was monitored in a reconstituted cell-free system that contains either the WT or the mutant Cul-1(K696R) SCF ␤-TrCP complex. Only the WT enzyme was able to catalyze conjugation of p105, whereas the enzyme that contains the mutant Cul-1 was inactive ( Fig. 2; compare lane 2 to lane 3). In this experiment, the NEDD8 pathway components were provided by the wheat germ cell extract in which p105 was translated. It is clear that the conjugates are generated from p105, as its concentration diminishes in the presence of the WT enzyme but not in that of the mutant one.
To link p105 phosphorylation and Cul-1 NEDDylation, we demonstrated that only a phosphorylated substrate that is incubated in the presence of the WT SCF complex and the three components of the NEDD8 pathway can be conjugated (Fig. 3,  lane 3). Non-phosphorylated p105 (lane 7) or phosphorylated p105 that is incubated in the presence of mutant Cul-1-containing SCF complex (lane 4) cannot be conjugated.
Mutant UBC12 Exerts a Dominant Negative Effect on p105 Ubiquitination-The requirement for NEDD8 modification in p105 ubiquitination was further studied by using a point mutant UBC12 in which the active site cysteine 111 was substituted with serine. The thiol group of this cysteine residue serves as a transient acceptor during the transfer of NEDD8 from the E1 (APP-BP1/Uba3) to the target substrate. A serine residue in this position forms a stable ester with glycine 76 of NEDD8 that is not cleaved to transfer activated NEDD8 to the target protein. This results in sequestration of NEDD8 and a dominant negative inhibition of NEDD8 conjugation (44). Addition of the mutant UBC12 to ubiquitination assays carried out in crude HeLa cell extract blocked p105 conjugate formation in a dose-dependent manner (Fig. 4A, compare lanes 4 and  5 to lane 2; Fig. 4B, compare lanes 3-5 to lane 2). Inhibition was not complete, as ubiquitination proceeds via the basal site of p105 even in the presence of a high concentration of DN-UBC12 (Fig. 4, A and B, lanes 5). It should be noted that the cell extract contains a sufficient amount of UBC12 (as well as of all other components of the NEDD8 pathway) to support maximal conjugation, and addition of exogenous WT-UBC12 does not stimulate conjugation further (and even inhibits it slightly; Fig.  4A, compare lane 3 to lane 2). Importantly, inhibition of conjugation by DN-UBC12 could be alleviated by the addition of WT-UBC12 (Fig. 4B, compare lanes 6 -8 to lane 5). As expected, the dominant negative UBC12 had no effect on conjugation of FIG. 3. Ubiquitination of p105 by SCF ␤-TrCP requires phosphorylation followed by the activity of the NEDD8 pathway. In vitro translated and labeled p105 was phosphorylated, immunoprecipitated, and subjected to ubiquitination (lanes 1-4) as described in the legend to Fig. 1 and under "Experimental Procedures." Parallel samples were incubated with non-phosphorylated p105 (lanes 5-8). All samples contained purified E1 and recombinant UBCH5C. WT and Cul-1(K696R) SCF ␤-TrCP complexes, as well as all three components of the NEDD8 pathway (APP-BP1/Uba3, UBC12, and NEDD8) were added as indicated. Samples were incubated and resolved on 7.5% SDS-PAGE, and proteins were visualized via phosphorimaging as described under "Experimental Procedures." Points of migration of p105 and p105-ubiquitin adducts (Conj.) are marked. FIG. 4. A catalytic site mutant UBC12(C111S) exerts a dominant negative effect on p105 ubiquitination. In vitro translated and labeled p105 was phosphorylated as described in the legend to Fig. 1, and the crude translation mixture was used as a source for the substrate as described in the legend to Fig. 2. Ubiquitination of labeled p105 was carried out in crude HeLa cell extract as described under "Experimental Procedures," and the different enzymes were added as indicated. Panel A, a mutant UBC12 (C111S) inhibits in vitro ubiquitination of p105. WT-(0.8 g; lane 3) and DN-UBC12 (1.2 g and 4.8 g; lanes 4 and 5, respectively; marked by a wedge) were added as indicated. Panel B, inhibition of p105 ubiquitination by DN-UBC12 is alleviated by an excess of the WT enzyme. The DN-UBC12 was added at 1.2, 3, and 4.8 g (lanes 3-5), whereas the WT-UBC12 was added at 0.8, 1.6, and 2.4 g (lanes 6 -8) to reaction mixtures that already contain 4.8 g of DN-UBC12. Results presented in panels A and B were quantified where 100% represents conjugate formation in the crude extract in the absence of WT-or DN-UBC12. Numbers represent % inhibition relative to this value. Panel C, conjugation of p105 that lacks the ␤-TrCP-binding site is not affected by DN-UBC12. WT-and DN-UBC12 were added at 0.8 and 4.8 g, respectively, as indicated. The p105 protein used in this experiment lacks the ␤-TrCP-binding site (p105⌬918 -934). Samples were incubated and resolved on 7.5% SDS-PAGE, and proteins were visualized via phosphorimaging as described under "Experimental Procedures." Points of migration of p105 as well as p105-ubiquitin adducts (Conj.) are marked. p105 that lacks the C-terminal IKK␤ phosphorylation and ␤-⌻rCP-binding site (Fig. 4C, compare lane 3 to lane 2). This protein is conjugated most probably by a ligase that is distinct from the SCF complex and that recognizes the substrate in a constitutive manner following binding to its basal site (8). Subsequent to inhibition of conjugation, addition of the mutant UBC12 inhibited significantly, although not completely, processing of p105 in crude HeLa extract (Fig. 5A, compare lane 4  to lanes 3 and 2). Similar to inhibition of conjugation, inhibition of processing could also be alleviated by the addition of excess WT-UBC12 (Fig. 5B, compare lane 4 to lane 3). Taken together, these results suggest that NEDD8 modification of Cul-1 is required for efficient ubiquitination of p105 by the SCF ␤-TrCP complex, and blockage of the pathway either by a dominant negative UBC12 or mutant Cul-1 could abolish this activity significantly.
In Vivo Processing of p105 Requires the NEDD8 Pathway-Having established a role for NEDD8 modification in the cellfree assays in vitro, it was important to assess the requirement for NEDDylation of Cul-1 on p105 processing in vivo. We transfected COS-7 cells with cDNAs coding for p105, IKK␤, and either dominant negative UBC12 or mutant Cul-1(K696R). Because p105 processing proceeds via independent mechanisms under basal and stimulated conditions, and because NEDDylation is expected to have an effect only on the stimulated mode, we monitored the effect of the dominant negative components of the NEDD8 pathway on IKK␤-mediated processing and compared it to the effect in the absence of the kinase. In cells transfected with mutant UBC12, IKK␤-mediated p105 processing was completely inhibited (Fig. 6A, compare lane 4 to lane 2). There was no effect whatsoever on basal processing (compare lane 3 to lane 1). In cells transfected with mutant Cul-1(K696R), processing of p105 was also inhibited, although to a lesser extent then in the DN-UBC12-containing cells (Fig. 6B, compare lanes 4 and 3 to lane 2). Here too the effect was completely dependent on co-transfection with IKK (not shown). FIG. 6. The NEDD8 pathway is required for processing of p105 in vivo. cDNAs coding for p105, IKK␤, DN-UBC12, and DN-Cul-1(K696R) were transfected into COS-7 cells as indicated and as described under "Experimental Procedures." Expression of p105 and p50 was monitored using Western blot analysis with anti-p50 antibody and ECL as described under "Experimental Procedures." Panel A, IKK␤mediated processing/degradation of p105 but not basal processing is inhibited by DN-UBC12. cDNAs coding for p105, IKK␤, and DN-UBC12 were transfected into COS-7 cells as indicated, and processing of p105 to p50 was monitored after 48 h. Data were quantified and presented (percent of processing; p50/p50ϩp105). Panel B, DN-UBC12 and DN-Cul-1(K696R) inhibit IKK␤-mediated processing of p105. cDNAs coding for p105, IKK␤, DN-UBC12, and DN-Cul-1(K696R) were transfected into COS-7 cells as indicated, and processing of p105 to p50 was monitored after 48 h. Equal amounts of protein were resolved via SDS-PAGE (10%) and blotted onto nitrocellulose paper. Data were quantified as described under panel A.

DISCUSSION
We have recently identified SCF ␤-TrCP as the ubiquitin ligase complex mediating phosphorylation-dependent p105 processing (11). In the current study we show that NEDD8 modification of the Cul-1 component of the SCF complex is required for efficient ubiquitination and processing of p105 by the SCF ␤-TrCP following phosphorylation of the precursor by IKK␤. Several lines of evidence support our conclusion. We show that in a cell-free system, the addition of all three NEDD8 pathway components is required to reconstitute p105 ubiquitination (Figs. 1 and 3). Not surprisingly, the NEDD8 pathway could not support conjugation of p105 that was not phosphorylated (Fig. 3) or lacks the IKK␤ and ␤-TrCP recognition site (Fig. 4C). These findings strongly support the notion that NEDDylation is required specifically for p105 processing which is dependent on IKK-mediated phosphorylation followed by SCF ␤-TrCP -catalyzed ubiquitination. Consistent with these results, a mutant Cul-1(K696R)-containing the SCF ␤-TrCP complex that cannot be modified by NEDD8 is completely inactive and cannot catalyze ubiquitination (Figs. 2 and 3). Furthermore, a catalytic site mutant UBC12(C111S) inhibited, in a cell-free assay, p105 ubiquitination ( Fig. 4) and subsequent processing (Fig. 5). Finally, our findings have been corroborated in vivo: inhibition of the NEDD8 pathway, either by using a DN-UBC12 or a mutant Cul-1, blocked completely phosphorylation-mediated p105 processing (Fig. 6). Here too, inhibition was limited to the IKK␤-stimulated processing (and the phosphorylated precursor) and not to the basal processing.
We have previously reported that p105 is targeted for ubiquitination and processing by two distinct, basal/constitutive (8) and signal-induced (11), recognition motifs that are utilized under different physiological conditions. The two motifs are probably recognized by distinct E2 and E3 enzymes (11). According to our proposed model (8,11,22), following stimulation and consumption of cell stores of NF-B, newly synthesized p105 molecules may undergo co-or post-translational processing (7), using the basal/constitutive recognition motif. The p50 subunits generated during this process dock to the emerging ankyrin repeat domain at the C-terminal half of p105 and inhibit processing of these precursors (22). The completely synthesized p105 with the docked p50 subunits serves as an inactive storage by sequestering these subunits in the cytosol and inhibiting their translocation into the nucleus. Following stimulation, the SCF ␤-TrCP ubiquitin ligase is recruited to the IKKmediated phosphorylation motif at the C-terminal domain, leading to rapid polyubiquitination and subsequent processing/ degradation of p105 with release of the docked p50 molecules and an additional p50 subunit released from processed p105 (11,12,22). Our results further support this model by underscoring the role of SCF ␤-TrCP as the ligase involved in phosphorylated p105 processing.
NEDDYlation clearly adds an additional layer of control to an already tightly regulated, signal-induced pathway. It is a complex cycle involving control of the extent of NEDD8 modification (34,37), regulated deNEDDylation by the COP9 signalosome (45,46) and possibly other C-terminal hydrolases (47), and down-regulation of NEDD8 by its specific recruitment to the proteasome (48). An interesting question in this context is why p105 processing requires such an elaborate mechanism. Processing of p105 is mostly constitutive and is stimulated ϳ2.0-fold following stimulation. This is due, most probably, to the presence of two sites of recognition in p105, one operating under non-stimulated conditions and the other following signaling (see above). Even this site was found to be partially phosphorylated in resting cells. In contrast, processing of p100 to yield p52 is mostly signal-induced (49, 50), although, we know little on this process. Because the two molecules are homologous, it is possible that the evolution of the cumbersome regulatory mechanism that involves signaling, IKK activation, IKK-mediated phosphorylation, NEDDylation of the Cul-1 component of the SCF complex, and ubiquitination of p105 at the second, signal-induced site, was not directed toward p105 but rather at p100. p52, which is derived from p100, is required for lymphoid tissue development for example (see Ref. 49) and cannot be replaced by p50. p105 that provides the cell with the NF-B transcriptional activator required for many of its basic needs, some independent on one another, had to evolve additional motifs acting under basal conditions that allows the molecule to be relieved from its tight signaling control and processed mostly in a constitutive manner.