the scfb-trcp–ubiquitin ligase complex associates specifically...

The SCFb-TRCP–ubiquitin ligase complexassociates specifically with phosphorylateddestruction motifs in IkBa and b-cateninand stimulates IkBa ubiquitination in vitroJeffrey T. Winston,1,5 Peter Strack,4,5 Peggy Beer-Romero,4 Claire Y. Chu,1 Stephen J. Elledge,1–3

and J. Wade Harper1,6

1Verna & Marrs McLean Department of Biochemistry, 2Department of Molecular and Human Genetics, 3Howard HughesMedical Institute, Baylor College of Medicine, Houston, Texas 77030 USA; 4Mitotix, Inc.,Cambridge, Massachusetts 02139 USA

Ubiquitin-mediated proteolysis has a central role in controlling the intracellular levels of several importantregulatory molecules such as cyclins, CKIs, p53, and IkBa. Many diverse proinflammatory signals lead to thespecific phosphorylation and subsequent ubiquitin-mediated destruction of the NF-kB inhibitor protein IkBa.Substrate specificity in ubiquitination reactions is, in large part, mediated by the specific association of theE3–ubiquitin ligases with their substrates. One class of E3 ligases is defined by the recently described SCFcomplexes, the archetype of which was first described in budding yeast and contains Skp1, Cdc53, and theF-box protein Cdc4. These complexes recognize their substrates through modular F-box proteins in aphosphorylation-dependent manner. Here we describe a biochemical dissection of a novel mammalian SCFcomplex, SCFb-TRCP, that specifically recognizes a 19-amino-acid destruction motif in IkBa (residues 21–41) ina phosphorylation-dependent manner. This SCF complex also recognizes a conserved destruction motif inb-catenin, a protein with levels also regulated by phosphorylation-dependent ubiquitination. EndogenousIkBa–ubiquitin ligase activity cofractionates with SCFb-TRCP. Furthermore, recombinant SCFb-TRCP assembledin mammalian cells contains phospho-IkBa-specific ubiquitin ligase activity. Our results suggest that anSCFb-TRCP complex functions in multiple transcriptional programs by activating the NF-kB pathway andinhibiting the b-catenin pathway.

[Key Words: Ubiquitin ligase; SCF complex; proteolysis; destruction motifs; NF-kB; b-catenin]

Received December 3, 1998; revised version accepted December 30, 1998.

The transcription factor NF-kB has a central role in cel-lular stress and inflammatory responses by controllingcytokine-inducible gene expression and lymphocytestimulation by antigens (Baeuerle and Baltimore 1996;Gilmore et al. 1996). In addition, NF-kB is required toblock cell death in response to tumor necrosis factor a(TNFa) and ionizing radiation, suggesting that it acts toregulate the transcription of survival genes (Beg and Bal-timore 1996; Liu et al. 1996; Van Antwerp et al. 1996;Wang et al. 1996). NF-kB is a ubiquitous heterodimericcomplex composed of a p65/RelA subunit and a p50 sub-unit. This complex is normally sequestered in an inac-tive form in the cytoplasm through interaction withmembers of a family of inhibitory proteins, the IkBs (Beget al. 1992; for review, see Baeuerle and Baltimore 1996).These proteins, when associated with NF-kB, obscure

the nuclear localization signal in NF-kB and also blockthe ability of NF-kB to bind DNA. In response to TNFaand other signals, IkBa is rapidly phosphorylated on twoserine residues near the amino terminus (Ser-32 and Ser-36 in IkBa) (Beg et al. 1993; Finco et al. 1994; Alkalay etal. 1995; Brown et al. 1995; Chen et al. 1995, 1996; Di-Donato et al. 1995; Lin et al. 1995). This modificationtriggers the rapid destruction of IkBa by ubiquitin-medi-ated proteolysis, thereby allowing NF-kB nuclear trans-location and target gene expression (Chen et al. 1995;Scherer et al. 1995; for review, see Hochstrasser 1996).Recent work has uncovered two IkBa kinases, IkKa andIkKb, that are responsible for signal-dependent phos-phorylation of IkBa (DiDonato et al. 1997; Mercurio etal. 1997; Regnier et al. 1997; Woronicz et al. 1997; Zandiet al. 1997, 1998). These proteins are part of a 700-kDprotein complex that is assembled through two struc-tural components IkKg/NEMO and IKAP (Cohen et al.1998; Rothwarf et al. 1998; Yamaoka et al. 1998) and areactivated by cytokines. In vitro, both IkKa and IkKb can

5Co-first authors.6Corresponding author.E-MAIL [email protected]; FAX (713) 796-9438.

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phosphorylate IkBa specifically on serines 32 and 36, butboth kinases are required for efficient IkBa phosphoryla-tion in vivo (Zandi et al. 1997).

Although the pathways leading to IkBa phosphoryla-tion have been described in detail, little is known aboutthe molecules responsible for ubiquitination. Ubiquitin-mediated proteolysis involves a cascade of ubiquitintransfer reactions in which the ubiquitin-activating en-zyme E1 uses ATP to form a high-energy thiolester bondwith ubiquitin, which is then transferred to members ofthe E2 ubiquitin-conjugating enzyme family (Hershko etal. 1983; Hochstrasser 1995). Ubiquitin is then trans-ferred from the E2 to lysine residues in the targetthrough an E3–ubiquitin ligase. E3s serve as adaptorsthat interact with both the target protein and the appro-priate E2, thereby providing specificity to the ubiquitintransfer reaction. In some cases, the E3 is also involvedin ubiquitin transfer (Scheffner et al. 1995; Rolfe et al.1995). Multiple rounds of ubiquitination of the initialconjugates lead to polyubiquitination, which targets theprotein for proteolysis by the 26S proteasome. Recentstudies have elaborated a modular ubiquitin ligase com-plex, the SCF–ubiquitin ligase, which mediates phos-phorylation-dependent ubiquitination of a large numberof proteins (for review, see Elledge and Harper 1998; Pat-ton et al. 1998b). The SCF is composed of Skp1, Cdc53/Cul1, and a specificity-conferring F-box protein (Bai et al.1996; Feldman et al. 1997; Skowyra et al. 1997; Patton etal. 1998a). F-box proteins contain two domains, an F-boxmotif that binds Skp1 and allows assembly into Skp1/Cdc53 complexes, and a second protein–protein interac-tion domain that interacts specifically with one or moretarget proteins (Bai et al. 1996). Cdc53/Cul1, in turn,interacts with both the E2 and the Skp1/F-box proteincomplex (Skowyra et al. 1997; Patton et al. 1998a). SCFcomplexes mediate phosphorylation-dependent destruc-tion of a wide array of regulatory proteins in yeast, in-cluding the Cdk inhibitors Sic1, Far1, and Rum1, G1 cy-clins, the transcription factor Gcn4, and the DNA repli-cation initiator proteins Cdc6 and Cdc18 (for review, seeElledge and Harper 1998; Patton et al. 1998b). In contrastwith yeast, targets of vertebrate SCF complexes remainlargely unknown. Previously, we identified four verte-brate proteins that contain the F-box motif, linking themto the ubiquitin pathway: mammalian cyclin F, Skp2,MD6, and Xenopus b-TRCP (b-transducin repeat-con-taining protein; Bai et al. 1996). b-TRCP was originallyidentified as a suppressor of a temperature-sensitive mu-tation in the budding yeast CDC15 gene (Spevak et al.1993), but its mechanism of suppression has not beendetermined. Recent genetic evidence has implicatedXenopus b-TRCP and its Drosphila homolog, slimb, incontrol of proteolysis in the Hedgehog and Wingless/Wnt signaling pathways (Jiang and Struhl 1998; Mari-kawa and Elinson 1998).

We have used biochemical approaches to examinewhether IkBa ubiquitination might involve an SCF–ubiquitin ligase. Here we report that mammalianb-TRCP binds to the IkBa destruction motif in a phos-phorylation-dependent manner, thereby recruiting IkBa

into an SCF–ubiquitin ligase complex. Moreover,SCFb-TRCP components cofractionate with IkBa–ubi-quitin ligase activity from tissue culture cells andSCFb-TRCP can stimulate ubiquitination of phosphory-lated but not unphosphorylated IkBa in an in vitro re-constitution assay. We also demonstrate that the sameSCFb-TRCP complex recognizes a similar destruction mo-tif in b-catenin, a component of the TCF/Lef transcrip-tion factor complex that functions downstream of Wing-less/Wnt (for review, see Peifer 1997) and whose levelsare also controlled by phosphorylation-dependentubiquitin-mediated proteolysis (Aberle et al. 1997). Ourresults, together with the effects of loss-of-function mu-tations in the Drosophila b-TRCP homolog slimb (Jiangand Struhl 1998), suggest that a single SCFb-TRCP com-plex functions in diverse signaling pathways that im-pinge on transcription control mediated by cytokines(NF-kB), Wnt/Wingless (b-catenin), and Hedgehog [Cubi-tus interruptus (Ci)].

Results

Phosphorylation-dependent association of the IkBadestruction motif with Skp1

IkBa contains two serine residues at positions 32 and 36that are specifically phosphorylated by the IkK complexin response to TNFa stimulation. Phosphorylation ofboth of these residues is required for IkBa ubiquitinationin vivo. Previous studies have shown that a 21-amino-acid phosphopeptide containing this destruction motifcan block IkBa–ubiquitin ligase activity in crude cell ex-tracts and can block NF-kB activation in tissue culturecells (Yaron et al. 1997). In addition, this destructionmotif can confer signal-dependent destruction whenfused to heterologous proteins (Wulczyn et al. 1998).Given the role for SCF complexes in phosphorylation-dependent ubiquitination of various regulatory proteins,we sought to determine whether SCF complexes mightbe involved in IkBa ubiquitination. Synthetic 21-residuepeptides encompassing the IkBa destruction motif in ei-ther the doubly phosphorylated or unphosphorylatedforms (Fig. 1a) were immobilized on agarose beads andincubated with HeLa cell lysates. Proteins stably associ-ated with these beads were then examined for the pres-ence of Skp1 by immunoblotting (Fig. 1b). Skp1 wasreadily detected in proteins bound to the phospho-IkBapeptide but not the unphosphorylated peptide. We esti-mate that ∼1% of the total Skp1 in these lysates stablyassociated with the phospho-IkBa peptide under theseconditions.

Recognition of phosphorylated destruction motifsin IkBa and b-catenin by the WD-40repeat-containing F-box protein b-TRCP

The ability of a phospho-IkBa peptide to associate withSkp1 suggested the existence of an F-box protein capableof recognizing the IkBa destruction motif. Our previousstudies identified three vertebrate F-box proteins (Skp2,

SCFb-TRCP, an IkBa–ubiquitin ligase

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MD6, and Xenopus b-TCRP) based on homology to theF-box sequence in human cyclin F and the budding yeastprotein Cdc4 (Bai et al. 1996). Recently, we have identi-fied cDNAs encoding 20 distinct mouse and/or humanF-box proteins, including the WD-40 repeat-containingprotein b-TRCP, a leucine-rich repeat containing F-boxprotein F1, and a number of F-box proteins lackingknown protein–protein interaction motifs outside the F-box (Fig. 1c; J. Winston, S.J. Elledge, and J.W. Harper, inprep.). Using in vitro translation products, we askedwhether members of a collection of these F-box proteinscould associate with IkBa peptides. Only one, b-TRCP,was found to associate with the phospho-IkBa destruc-tion motif, and this interaction was dependent on phos-phorylation (Fig. 1c). Mouse and human b-TRCP are95% identical and both interact equally well with IkBain this assay (data not shown for human b-TRCP). Ouranalysis included two other WD-40-containing F-boxproteins, human MD6 and Met30, the closest homolog

of b-TRCP in budding yeast (31% identity). Importantly,neither of these proteins associated with phospho-IkBa(Fig. 1c), suggesting that the interaction of b-TRCP withphospho-IkBa is highly specific.

Previous studies in Drosophila have demonstratedthat mutations in the b-TRCP homolog slimb led to ac-cumulation of Armadillo, the Drosophila homolog ofb-catenin (Jiang and Struhl 1998). b-Catenin is known tobe ubiquitinated in a glycogen synthase kinase 3b(GSK3b)-dependent manner and contains a motif withina cluster of candidate GSK3b phosphorylation sites thatis closely related to the IkBa destruction motif (Fig. 1a;Ikeda et al. 1998). Although the GSK3b phosphorylationsites in b-catenin are not known, we hypothesized basedon the sequence similarity between IkBa and b-cateninthat Ser-33 and Ser-37 might represent relevant phos-phorylation sites. A b-catenin-derived peptide contain-ing phosphoserine residues at these two positions asso-ciated with b-TRCP but not other F-box proteins tested,

Figure 1. The F-box protein b-TRCP associ-ates with phosphorylated destruction motifs inIkBa and b-catenin. (a) Sequences of the IkBa

(p21) and b-catenin peptides used in this study.The positions of phosphorylation in the pep-tides are shown as is the consensus sequencefor the destruction motif. (f) Hydrophobicamino acid. (b) Phosphorylation-specific asso-ciation of the IkBa destruction motif withSkp1 in vitro. HeLa cell proteins (600 µg) wereincubated with phosphorylated or unphos-phorylated IkBa peptides (p21) immobilized onbeads. Bound proteins were immunoblottedwith anti-Skp1 antibodies. Approximately 1%of the Skp1 contained in these lysates re-mained bound to the phosphorylated IkBa

beads. (c) b-TRCP specifically associated withphosphorylated IkBa and b-catenin destructionmotifs. A panel of [35S]methionine-labeled invitro-translated F-box proteins was used inbinding reactions with IkBa (lanes 1–3) orb-catenin (lanes 4–6) peptide beads. Bound pro-teins were analyzed by SDS-PAGE and autora-diography. (Right) The domain structures ofeach F-box protein. (d) The pattern of expres-sion of b-TRCP at day 11.5 during mouse de-velopment was determined by in situ hybrid-ization. The dark-field signal from the b-TRCPriboprobe is shown in red. (hb) Hindbrain; (fb)forebrain; (h) heart; (l) lung; (li) liver. (e) Chro-mosomal localization of b-TRCP. A bacmidcontaining human b-TRCP DNA was hybrid-ized to metaphase chromosomes (blue) and de-tected using fluorescein. The position of hy-bridization (yellow) is 10q24 (indicated by ar-rows). (f) b-TRCP is localized in the cytoplasm.HeLa cells were transiently transfected withpCMV–HA–b-TRCP and subcellular localiza-tion determined after 48 hr by indirect immu-nofluorescence. Anti-HA localization, red; nu-clei stained with DAPI, blue.

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whereas the unphosphorylated peptide failed to associatewith b-TRCP (Fig. 1c).

NF-kB is a ubiquitous transcription factor. As assessedby in situ hybridization, b-TRCP is also expressedthroughout the developing mouse embryo (day 11.5 post-coitum), with the highest levels in the brain, lung, andliver (Fig. 1d). b-TRCP is largely, if not exclusively, cy-toplasmic, as assessed in HeLa cells transiently express-ing an HA-tagged b-TRCP protein (Fig. 1f). The gene forhuman b-TRCP lies on chromosome 10q24, as deter-mined by in situ hybridization of metaphase chromo-somes with b-TRCP genomic DNA (Fig. 1e). Cytogeneticdata indicate that this region of the genome is altered ina limited number of cancer types (see Discussion).

Association of SCFb-TRCP with IkBa and b-catenindestruction motifs

Having identified b-TRCP as a candidate F-box proteinfor IkBa and b-catenin, we next sought to demonstratethat b-TRCP forms an SCF complex in mammalian cellsand that this complex recognizes IkBa and b-catenin de-struction motifs. Although there are six mammalianCullin homologs, the interaction of Skp1 with this fam-ily appears to be restricted to Cul1 (Michel and Xiong1998). 293T cells were transfected with various combi-nations of plasmids expressing epitope-tagged b-TRCP,

Cul1, or Skp1 and anti-Myc immune complexes fromcell lysates analyzed by immunoblotting. b-TRCPMyc as-sociated with both transfection-derived Skp1HA (Fig. 2a,lanes 4,5) and Cul1HA (lane 5). In contrast, Cul1HA andSkp1HA were not precipitated from control lysates lack-ing b-TRCPMyc (lane 6). In the absence of transfection ofSkp1 and Cul1, b-TRCPMyc associated with endogenousSkp1 (lane 3) and Cul1 (data not shown). Analogous re-sults were obtained when Cul1HA was immunoprecipi-tated with anti-HA antibodies from cells expressingCul1HA, b-TRCPMyc, and Skp1Myc (Fig. 2c). Thus,b-TRCP can form an SCF complex in vivo analogous tothat found previously for other F-box proteins (Skowyraet al. 1997; Lisztwan et al. 1998; Lyapina et al. 1998;Michel and Xiong 1998).

Next, we asked whether the SCFb-TRCP complex couldassociate with the IkBa destruction motif peptide. Asshown in Figure 3a, the SCFb-TRCP complex readily as-sociated with phosphorylated IkBa peptide beads (lanes6,8,10) but was not retained on unphosphorylated IkBabeads (lanes 5,7,9). Although Cul1 associates at low lev-els with agarose beads containing IkBa peptides in theabsence of b-TRCPMyc expression (lanes 11,12) and withagarose beads alone (data not shown), the associationwith the phospho-IkBa peptide was greatly enhanced byexpression of b-TRCPMyc (lane 10). Consistent with theresults in Figure 1b, endogenous Skp1 was observed in

Figure 2. b-TRCP associates with Skp1 and Cul1 in tissue culturecells. 293T cells were transfected with the indicated plasmids andlysates (0.5 µg of protein/250 µl) used for immunoprecipitation asdescribed in Materials and Methods. Immune complexes or crude ly-sates from each transfection were analyzed for the presence of Skp1,Cul1, and b-TRCP by immunoblotting. (a) Anti-b-TRCPMyc immunecomplexes. Blots were probed first for b-TRCP, stripped, and probedfor Skp1 and Cul1. The bands indicated by the asterisk indicate theposition of b-TRCP whose antibody was not efficiently stripped fromthe blot. (b) Crude cell lysates (50 µg) corresponding to extracts usedin a. (c) Anti-Cul1HA immune complexes (lanes 1–6) and correspond-ing cell lysates (50 µg) (lanes 7–13). The positions of both epitope-tagged and endogenous Skp1 are shown.






association with phospho-IkBa peptide beads in a phos-phorylation-dependent manner in the absence of trans-fection of b-TRCP (lanes 1–4), but when the levels ofb-TRCP were increased by transfection, the quantity ofendogenous Skp1 associated with b-TRCP increased sub-stantially (Fig. 3a, lanes 4,6). Analogous results were ob-tained in a more limited series of binding reactions em-ploying b-catenin-derived peptides (Fig. 3b).

Although it was clear that destruction motif peptidescan bind the SCFb-TRCP complex, it was necessary todemonstrate that this complex also recognized the en-dogenous ubiquitination substrate, the IkK-phosphory-lated IkBa/NF-kB complex. To generate this substrate,IkBa/p50/p65 complexes were produced in insect cellsand purified to near homogeneity (Fig. 4a). When incu-bated with ATP and purified IkK-b, the IkBa protein un-derwent a mobility shift reminiscent of that observedupon phosphorylation in vivo, and this phosphorylatedIkBa protein was recognized by phosphospecific anti-bodies directed at Ser-32 of IkBa (Fig. 4b, lane 2). In ad-dition, microsequencing of IkK-treated IkBa confirmedthat both Ser-32 and Ser-36 were phosphorylated (datanot shown). To examine whether SCFb-TRCP could rec-ognize this complex, binding reactions were performedusing immobilized SCFb-TRCP complexes isolated from293T cells transiently expressing Myc-tagged b-TRCP ormock transfected cells as a control and either phosphory-lated or unphosphorylated IkBa/NF-kB complexes. Theb-TRCPMyc immune complexes contain endogenousSkp1 (Fig. 2, lane 3) and Cul1 (data not shown) as deter-mined by immunoblotting. Both IkBa and p50 werefound to associate with the SCFb-TRCP complex but not

control immune complex in a phosphorylation-depen-dent manner (Fig. 3c). Similar results were obtained withGST–b-TRCP complexes purified from insect cells (datanot shown).

Biochemical association of endogenous IkBa–ubiquitinligase activity with b-TRCP

Crude cell lysates from the human monocyte cell lineTHP.1 contain potent IkBa–ubiquitin ligase activity (Fig.4c). In the context of an IkBa/NF-kB complex, efficientIkBa ubiquitination by these lysates is dependent onphosphorylation by IkK (Fig. 4c). As reported earlier(Yaron et al. 1997), this IkBa–ubiquitin ligase activity isstrongly inhibited by phosphorylated IkBa destructionmotif peptides (Fig. 4d, lanes 4,5) but not by nonphos-phorylatable destruction box peptides (lanes 2,3). Thus,this assay reiterates the requirements for IkBa ubiquiti-nation observed in vivo. Greater than 95% of the IkBa–ubiquitin ligase activity in these extracts can be precipi-tated with 30%–50% ammonium sulfate (Fig. 4e, lane 1)and can be further purified by chromatography on a phe-nyl–Sepharose column (Fig. 4e; see Materials and Meth-ods). Peak fractions of IkBa–ubiquitin ligase activity (Fig.4e, lanes 8,9) elute at 0.5 M ammonium sulfate.

Having partially purified components of the IkBa–ubiquitin ligase, we examined whether b-TRCP andother SCF components were contained in active frac-tions from the phenyl–Sepharose column. Skp1 has anextended elution profile, but both Skp1 and Cul1 arecontained in the active fractions 7 and 8 (Fig. 4e). Skp1and Cul1 can interact with multiple F-box proteins, and

Figure 3. Association of SCFb-TRCP with IkBa and b-catenin destruction motifs and with theIkBa/NF-kB complex. (a,b) Cell lysates (0.3 µg of protein/150 µl) from Fig. 2 were used in IkBa

(a) and b-catenin (b) peptide bead binding reactions as described in Materials and Methods.Bound proteins were analyzed by immunoblotting with the indicated antibodies. (c) Phos-phorylation-dependent association of b-TRCPMyc with the IkBa/p50/p65 complex in vitro.b-TRCPMyc immune complexes (lanes 2,5) corresponding to those in Fig. 2a (lane 3) or controlcomplexes (lanes 3,6) corresponding to those in Fig. 2a (lane 1) were used in binding reactionswith either IkBa/p50/p65 or IkK-b phosphorylated IkBa/p50/p65 complexes (see Materials andMethods). Bound proteins were separated by SDS-PAGE and immunoblotted using anti-p50 oranti-IkBa antibodies. The asterisk (lanes 1,4) indicates the positions of 15% of the input IkBa

complexes used in the binding reaction.

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the identity of the F-box protein in complexes with Cul1and Skp1 is likely to affect elution properties on phenyl–Sepharose. In contrast with Skp1, b-TRCP levels peak infraction 7, as determined using affinity-purified carboxy-terminal antibodies, coincident with maximal activity(Fig. 4e, lane 8). b-TRCP was also detected in active frac-tion 8 (Fig. 4e, lane 9). This fraction contained lowerlevels of IkBa–ubiquitin ligase activity, as assessed bythe extent of conjugation, consistent with the lower lev-els of b-TRCP. As shown below, under some gel condi-tions the b-TRCP protein is resolved into a closelyspaced doublet of proteins at 58–60 kD. Interestingly,although fraction 6 contains detectable levels of Skp1and b-TRCP, it lacks detectable Cul1 and IkBa–ubiquitinligase activity (Fig. 4e, lane 7). Likewise, fraction 9 con-taining Skp1 and Cul1 but no b-TRCP is also inactive(Fig. 4e, lane 10).

Consistent with a role for Skp1 in the IkBa–ubiquitinligase, antibodies against Skp1, but not control GST an-tibodies, deplete IkBa–ubiquitin ligase activity from the

active phenyl–Sepharose fractions (Fig. 5a). As expected,Skp1 and Cul1 are largely depleted from these activeextracts (lane 2). Importantly, the majority of b-TRCP isalso removed by Skp1 antibodies (Fig. 5a, lane 2).b-TRCP migrated as a closely spaced doublet at 58 and60 kD. The faster migrating form, corresponding to∼80% of the total, was essentially depleted by anti-Skp1antibodies (lane 2), when compared with control GST-depleted extracts. The source of the heterogeneity inb-TRCP is not known at present, but it is possible thatthe more slowly migrating form is not associated withSkp1 or is dislodged from Skp1 by the anti-Skp1 antibod-ies. We also note that Cul1 migrated as a doublet (Fig.5a). The slower migrating form is likely to correspond toa form of the protein conjugated to NEDD8, a homologof Rub1 that is known to be covalently linked to Cdc53in budding yeast (for review, see Hochstrasser 1998).

We also found that phospho-IkBa destruction motifpeptides (but not the nonphosphorylatable counterparts)were able to deplete ubiquitin ligase activity from both

Figure 4. IkBa–ubiquitin ligase activity from human cells cofractionates with b-TRCP. (a) IkBa/p50/p65 complexes were purified tonear homogeneity from insect cells as described in Materials and Methods. Proteins were separated by SDS-PAGE and stained withCoomassie blue. (b) Phosphorylation of the IkBa/p50/p65 complex by IkK-b. The IkBa complex (lanes 1,2) or a nonphosphorylatableIkBa mutant (S32/36A) complex (lane 3) was incubated in the presence of ATP and IkK-b as indicated in Materials and Methods.Products were analyzed by immunoblotting with anti-IkBa antibodies to detect a mobility shift accompanying phosphorylation thatis absent in the nonphosphorylatable mutant (top) or with antibodies that specifically detect the Ser-32-phosphorylated form of IkBa

(bottom). (c) Ubiquitination of IkBa complexes by crude cell lysates was performed as described in Materials and Methods. Phos-phorylation leads to a ∼10- to 20-fold increase in ubiquitin conjugates relative to the unphosphorylated complex, whereas no activityis observed with the nonphosphorylatable IkBa complexes. (d) Inhibition of IkBa ubiquitination by phosphorylated IkBa destructionmotif peptides but not by nonphosphorylatable destruction motif peptides (p19). Ubiquitination reactions were performed with crudecell extracts and IkK-b phosphorylated IkBa complexes in the presence or absence of phosphorylated or nonphosphorylatable destruc-tion motif peptides. Specific inhibition of ubiquitination was observed with the phosphorylated peptide. (e) Cofractionation of b-TRCPwith endogenous IkBa–ubiquitin ligase activity. Crude extracts from THP.1 cells were precipitated with ammonium sulfate. Solubi-lized proteins containing ubiquitin ligase activity were fractionated using a phenyl–Sepharose column and activity in each fractiondetermined as described in Materials and Methods. Aliquots of column fractions were assayed for b-TRCP, Cul1, and Skp1 byimmunoblotting. Fractions containing b-TRCP, Cul1, and Skp1 (fractions 7,8) contain IkBa–ubiquitin ligase activity.






active fractions from the phenyl–Sepharose column (Fig.5b) and crude cell extracts (Fig. 5c). Skp1, Cul1, andb-TRCP were all associated with the phosphorylated de-struction motif beads but not with the nonphosphorylat-able destruction motif (Fig. 5, b, lanes 4 and 5, and c,lanes 3 and 4). Although the levels of Skp1 and Cul1 insupernatants were essentially unaffected (Fig. 5, b, lanes1–3, and c, lanes 1 and 2), the level of b-TRCP in thesupernatant from both crude and purified fractions wassubstantially reduced (∼80% for the crude lysate and>90% for the phenyl–Sepharose fraction) (Fig. 5b,c, lanes1,2). These data are consistent with b-TRCP-associatedSkp1 being a small fraction of the total Skp1/Cul1 com-plexes present in the cell (see Fig. 1b). Currently avail-able antibodies against b-TRCP were unable to immu-nodeplete b-TRCP from crude lysates or purified frac-tions, prohibiting a direct analysis of the effects ofremoval of b-TRCP on IkBa–ubiquitin ligase activity.Nevertheless, the finding that depletion of SCFb-TRCP

from either crude lysates or purified fraction correlateswith loss of ubiquitin ligase activity strongly implicatesthis SCF complex as being involved in IkBa ubiquitination.

Stimulation of IkBa ubiquitination by an SCFb-TRCP

complex in vitro

The results described thus far are consistent with a rolefor SCFb-TRCP in IkBa ubiquitination. If b-TRCP func-

tions as a specificity factor for ubiquitination of IkBathrough an SCF-dependent pathway, it should be pos-sible to confer IkBa ubiquitination activity by introduc-ing b-TRCP into a system that lacks such an activity.Although budding yeast contains a number of E2 en-zymes that could potentially support IkBa ubiquitina-tion, its closest homolog to b-TRCP, Met30, does notassociate with the IkBa destruction motif (Fig. 1c). Wetherefore anticipated that yeast extracts shown previ-ously to support SCF-dependent ubiquitination of Cln2and Sic1 (Deshaies et al. 1995; Skowyra et al. 1997;Verma et al. 1997) would be inactive toward either phos-phorylated or unphosphorylated IkBa and this was thecase (Fig. 6c, lanes 4,5). However, when these same re-action mixtures were supplemented with Flag-taggedSCFb-TRCP complexes isolated from 293T cells (Fig. 6a),IkBa ubiquitination was observed (Fig. 6c, lane 7). Theactivity was dependent upon phosphorylation of IkBa(lane 6) and was absent in reaction mixtures containinganti-Flag immunoprecipitates from mock-transfectedcells (lanes 8,9). Moreover, SCFb-TRCP was unable tostimulate ubiquitination when mixed with E1, ATP, andubiquitin in the absence of yeast extract (lane 10), indi-cating that a specific E2 activity is not efficiently immu-noprecipitated with the SCFb-TRCP complex. As ex-pected, the active SCFb-TRCP complexes associated withphosphorylated IkBa while control immune complex didnot (Fig. 6b).

Figure 5. Depletion of IkBa–ubiquitin ligase activity by anti-Skp1 antibodies and destruction motif peptides correlates with removalof b-TRCP. (a) Ubiquitin ligase activity from phenyl–Sepharose fractions 7 and 8 was depleted with antibodies against Skp1 or GSTand the supernatant assayed for ubiquitination activity toward phosphorylated IkBa (bottom). The levels of Skp1, Cul1, and b-TRCPin the supernatants from the depleted fractions were determined by immunoblotting (top). (b,c) Endogenous b-TRCP associates withphosphorylated IkBa destruction motif peptides during depletion of IkBa–ubiquitin ligase activity. Crude cell extracts (b) or activefractions from a phenyl–Sepharose column (c) were incubated with beads containing either phosphorylated or nonphosphorylatableIkBa peptides and the supernatants assayed for ubiquitin ligase activity (bottom panels). The levels of Skp1, Cul1, and b-TRCP in thesupernatant and associated with destruction motif peptides were determined by immunoblotting (top). b-TRCP is associated with thephosphorylated destruction motif peptides and is substantially depleted from active ubiquitin ligase fractions.

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In an alternative approach, we tested whether recom-binant GST–b-TRCP, purified to near homogeneity frominsect cells (data not shown), could support ubiquitina-tion of IkBa in the mammalian ubiquitination systemdescribed in Figure 4. As noted above, fraction 9 from thephenyl–Sepharose column contains Cul1 and Skp1 butlacks detectable b-TRCP and IkBa ubiquitination activ-ity (Fig. 6d, lane 1). However, when this fraction wassupplemented with GST–b-TRCP, a potent phosphory-lation-dependent IkBa–ubiquitin ligase activity was gen-erated (Fig. 6d, lane 2). This activity was not observedwhen this fraction was supplemented with purified GSTprotein (Fig. 6d, lane 3). Also, addition of a phosphory-lated IkBa destruction motif peptide (but not the unphos-phorylated peptide) completely blocked IkBa ubiquitina-tion (Fig. 6d, lanes 5,6). Finally, the activity was not ob-served when the GST–b-TRCP protein was incubated inthe reaction conditions lacking the phenyl–Sepharosefraction, suggesting a requirement for Cul1 and Skp1(Fig. 6d, lane 7). Taken together, these two assay systemsprovide compelling evidence that SCFb-TRCP functionsas an IkB–ubiquitin ligase.

Discussion

Activation of NF-kB involves an extensive signal trans-

duction pathway that culminates in the destruction ofthe NF-kB inhibitor IkBa. Although the protein kinasepathways that control the timing of NF-kB activationhave been defined, the molecules responsible for the ac-tual ubiquitination events have not been elucidated. Inthis work, we provide biochemical evidence that theWD-40-containing F-box protein, b-TRCP, functions as aspecificity factor in an SCF complex to promote signal-dependent ubiquitination of IkBa (Fig. 7). A role forb-TRCP in controlling IkBa ubiquitination is supportedby the following findings. (1) Destruction of IkBa isknown to require IkK-dependent phosphorylation of resi-dues (Ser-32 and Ser-36) located in a destruction motif.b-TRCP and its SCF complex associate with this IkBadestruction motif and with the IkBa/NF-kB complex in amanner that is dependent upon phosphorylation of theIkBa destruction motif. A variety of other F-box proteins,including two other WD-40 containing F-box proteins(Met30 and MD6), failed to associate with either phos-phorylated or unphosphorylated IkBa destruction motifs,pointing to the specificity of the interaction withb-TRCP. We believe that the interaction betweenb-TRCP and the IkBa destruction motif is direct, as pep-tide beads containing this motif precipitate GST–b-TRCP from insect cell lysates in the absence of otherabundant proteins (J. Winston, S. Elledge, and J. Harper,

Figure 6. Stimulation of IkBa–ubiquitinligase activity by SCFb-TRCP in vitro. (a)Flag-tagged SCFb-TRCP was prepared aftertransient transfection in 293T cells by im-munoprecipitation along with a Flag im-mune complex from mock-transfectedcells. Immune complexes were analyzedfor the presence of Cul1HA, Skp1Myc, andb-TRCPFlag by immunoblotting (lanes 3,4).Crude lysates used for immunoprecipita-tion are shown as controls (lanes 1,2). (b)b-TRCPFlag immune complexes associatewith phosphorylated IkBa in vitro. Im-mune complexes (10 µl beads) from (a)were incubated with 15 nM phosphory-lated IkBa/NF-kB complexes in a total vol-ume of 100 µl. Washed beads were sub-jected to SDS-PAGE and IkBa determinedby immunobloting. The asterisk indicatesa sample containing 15% of the input IkBa

complex. (c) Stimulation of IkBa–ubiqui-tin ligase activity by SCFb-TRCP in vitro.Yeast extracts (supplemented with E1,ubiquitin, and an ATP-regenerating sys-tem) were incubated with unphosphory-lated or phosphorylated IkBa/NF-kB com-plexes (25 nM) in the presence of 10 µl ofcontrol immune complexes (lanes 8,9) orb-TRCPFlag immune complexes (lanes

6,7). After 90 min, reaction mixtures were separated by SDS-PAGE and IkBa detected by immunoblotting with anti-IkBa antibodies.As controls, untreated IkBa complexes (lanes 1,2), supplemented yeast lysates (lane 3), and an SCFb-TRCP immune complex reactionmixture containing all components except the yeast extract (lane 10) were also included. (d) Reconstitution of IkBa ubiquitinationactivity in mammalian extracts by addition of purified GST–b-TRCP. Reaction mixtures, prepared as described in Materials andMethods, contained E1, ubiquitin, ATP, HQ unbound as a source of E2 activity, and other components as indicated (lanes 1–6). Controlreactions (lanes 7,8) lacked phenyl–Sepharose fraction 9. After 90 min, products were analyzed by SDS-PAGE and immunoblottingwith anti-IkBa antibodies.






unpubl.). (2) b-TRCP forms a complex with two proteins,Skp1 and Cul1, that have been linked previously to phos-phorylation-dependent ubiquitination. b-TRCP is local-ized in the cytoplasm where IkBa ubiquitination isthought to occur. (3) b-TRCP copurifies with IkBa–ubiq-uitin ligase activity from tissue culture cells, and theseactive fractions also contain Cul1 and Skp1. (4) Deple-tion of b-TRCP with either anti-Skp1 antibodies or phos-phorylated destruction motif peptides correlates withloss of IkBa–ubiquitin ligase activity. (5) SCFb-TRCP com-plexes stimulated phosphorylation-dependent IkBa–ubiquitin ligase activity when supplemented with E1,ubiquitin, ATP, and a yeast extract. These yeast extractslack IkBa–ubiquitin ligase despite the presence of mul-tiple SCF complexes (Bai et al. 1996; Patton et al.1998a,b), providing further evidence of a role for b-TRCPas a specificity factor for IkBa, but provide E2 activitiesand possibly other components that support IkBa ubiq-uitination by the b-TRCP complex. (6) Addition ofb-TRCP to fractions containing Cul1 and Skp1 but lack-ing IkBa–ubiquitination activity leads to robust ubiqui-tination activity that is phosphorylation dependent and

inhibited by a phosphorylated IkBa destruction motifpeptide. At present, we have been unable to reconstituteIkBa–ubiquitin ligase activity using SCFb-TRCP com-plexes isolated from transfected cells and column frac-tions depleted of b-TRCP by either anti-Skp1 antibodiesor phospho-IkBa peptides. This may reflect removal of anessential factor by depletion that is not present in suffi-cient levels in the transiently expressed SCF complex tosupport IkBa ubiquitination but are provided in trans byyeast extracts or undepleted mammalian extracts. Takentogether, these data provide strong evidence that SCFb-

TRCP functions in IkBa ubiquitination. After submissionof this paper, Yaron et al. (1998) reported that b-TRCP isa component of the IkBa–ubiquitin ligase and demon-strated that mutants lacking the F-box stabilize IkBa andblock NF-kB activation in vivo. However, no data link-ing b-TRCP to an SCF-dependent process was presented,and it was suggested that b-TRCP might function inde-pendently of Cul1 and Skp1. Our data provide compel-ling and complementary biochemical evidence thatb-TRCP functions in the context of an SCF pathway, aresult that has important mechanistic implications and

Figure 7. Schematic representation of theproposed pathways controlling ubiquitin-mediated proteolysis of IkBa and b-catenin. b-TRCP, an F-box protein, is acomponent of an SCF–ubiquitin ligase. Inresponse to appropriate signals (i.e.,TNFa), the IkK complex is activated andphosphorylates IkBa in complexes withNF-kB on Ser-32 and Ser-36. This complexis then recognized by b-TRCP in an SCFcomplex, facilitating ubiquitination byan E1- and E2-dependent mechanism. b-Catenin, in complexes with APC, axin,and GSK3b, is phosphorylated on Ser-33and Ser-37. This phosphorylated b-catenincan then associate with SCFb-TRCP, result-ing in ubiquitination. It is not clear atpresent whether b-catenin alone or theAPC/b-catenin complex is the relevanttarget. Yellow ovals indicate phosphoryla-tion.

Winston et al.





further implicates the SCF pathway in phosphorylation-dependent ubiquitination reactions. Currently, the iden-tity of the E2(s) involved in IkBa ubiquitination in vivo isunknown, as is the nature of the heterogeneity observedwith b-TRCP. We note, however, that other F-box pro-teins including Skp2 are modified by phosphorylation(Lisztwan et al. 1998), and such modifications could po-tentially play regulatory roles. The methods we have em-ployed offer two general approaches for determiningwhether a particular ubiquitination process involves anSCF complex: (1) Depletion of active fractions with Skp1antibodies, and (2) the use of substrates as affinity re-agents to examine association with cloned F-box pro-teins. The expanding number of F-box protein sequencesavailable will greatly facilitate the identification of SCF-dependent processes through these types of approaches.

The sequence conservation of the IkBa destructionmotif with a region of b-catenin implicated in its turn-over, coupled with a genetic requirement for the b-TRCPhomolog slimb in turnover of the b-catenin homolog Ar-madillo (Jiang and Struhl 1998), led us to addresswhether b-TRCP might interact directly with b-catenin.Phosphorylation of serine residues 33 and 37 was suffi-cient to allow for a peptide spanning this region to asso-ciate with b-TRCP and its SCF complex but not otherF-box proteins. b-Catenin is a component of the Wing-less/Wnt signaling pathway and functions with Tcf/Leftranscription factors to regulate patterning and other de-velopmental decisions (Peifer 1997). Recent work has re-vealed that expression of a b-TRCP protein lacking theF-box leads to accumulation of b-catenin and ectopic ac-tivation of the Wnt pathway in Xenopus (Marikawa andElinson 1998) and b-catenin stabilization in mammaliancells (Latres et al. 1999). This, together with our datalinking b-TRCP to direct recognition of the phosphory-lated b-catenin destruction motif, strongly implicatesSCFb-TRCP as the b-catenin–ubiquitin ligase (Fig. 7). Thelevels of b-catenin are regulated by the APC (adenoma-tous polyposis coli) tumor suppressor protein, axin, andthe protein kinase GSK3b (Korinek et al. 1997; Morin etal. 1997; Rubinfeld et al. 1997). Formation of an APC/axin/GSK3b/b-catenin complex is thought to be re-quired to allow appropriate phosphorylation of b-cateninby GSK3b (Hart et al. 1998; Ikeda et al. 1998) and in theabsence of Wnt signaling, b-catenin levels remain lowdue to constitutive phosphorylation and ubiquitin-medi-ated proteolysis. Wnt signaling inactivates GSK3b, lead-ing to increased levels of b-catenin and activation oftranscription (Peifer 1997). Mutations in either the APCgene or in b-catenin allow for b-catenin accumulation(Morin et al. 1997; Rubinfeld et al. 1997). Such muta-tions are found in a large fraction of colon cancers (Morinet al. 1997) and have also been observed in melanoma(Rubinfeld et al. 1997), prostate cancer (Voeller et al.1998), and experimentally induced cancers (Dashwood etal. 1998). Interestingly, stabilizing mutations in b-cateninare localized to its destruction motif and include muta-tions in both the phosphoacceptor sites S33 and S37 andother residues in the consensus b-TRCP recognition mo-tif including D32, G34, and I35. Mutations in these resi-

dues would be expected to weaken or abolish associationof b-catenin with b-TRCP, leading to accumulation ofb-catenin.

The role for b-TRCP in b-catenin turnover suggeststhat it might function as a tumor suppressor. We local-ized the human b-TRCP gene to 10q24. This region dis-plays genetic abnormalities in a limited number of pros-tatic, melanocytic, and neural cancers (Parmiter et al.1988; Lundgren et al. 1992; Rasheed et al. 1992). How-ever, a preliminary analysis of four colon cancers whichare wild-type for b-catenin and APC failed to reveal mu-tations in the b-TRCP protein (Sparks et al. 1998). Giventhe role for b-TRCP in IkBa ubiquitination, it is conceiv-able that mutations that inactivate b-TRCP are incom-patible with transformation because of loss of survivalpathways dependent on NF-kB activation (Beg and Balti-more 1996; Van Antwerp et al. 1996; Wang et al. 1996).

Our results indicate that an SCFb-TRCP complex func-tions in two critical transcriptional control pathways, inone case by inactivating an inhibitor of transcription andin the other case by inhibiting an activator of transcrip-tion (Fig. 7). Genetic data in Drosophila suggest that theb-TRCP homolog slimb may also regulate the Hedgehogpathway (Jiang and Struhl 1998). In slimb mutants, theCi transcription factor accumulates inappropriately, al-though the question of whether this regulation is directremains to be determined. Moreover, other recent stud-ies have revealed that b-TRCP is coopted by the HIVprotein Vpu to facilitate destruction of the CD4 protein(Margottin et al. 1998). Interestingly, Vpu contains ab-TRCP recognition motif very similar to that found inIkBa and b-catenin and phosphorylation is required for itto recruit CD4 to b-TRCP and to bind b-TRCP in thetwo-hybrid system. Further studies are required to deter-mine whether any of the many proteins containing theDSGfXS motif are also substrates for SCFb-TRCP. In ad-dition, we note that the anti-inflammatory effects of as-pirin are mediated through inhibition of IkK activity(Grilli et al. 1996; Yin et al. 1998), thereby blocking NF-kB activation. Molecules that selectively block recogni-tion of IkBa by b-TRCP may also constitute an alterna-tive therapeutic target for anti-inflammatory agents.

Materials and methods

Plasmid and baculovirus construction

cDNAs encoding mouse and human homologs of Xenopusb-TRCP, human Skp2, and human MD6 were obtained as ex-pressed sequence tags and the sequences determined by auto-mated DNA sequencing. The sequence of human b-TRCP wasreported previously. The sequence of mouse b-TRCP was de-posited in GenBank (accession no. AF110396). The sources ofother novel F-box proteins will be reported elsewhere (J. Win-ston, D. Koepp, S.J. Elledge, and J.W. Harper, in prep.). Openreading frames were amplified by PCR using Expand high-fidel-ity polymerase (Boehringer Mannheim) and cloned into theunivector derivative pUNI10 as NdeI–SalI fragments. Similarly,human Skp1 was cloned into pUNI10 as NdeI–BamHI fragment.In vitro Cre-recombinase-mediated plasmid fusion (Lui et al.1998) of univector plasmids with various pHOST recipient plas-






mids was then used to create plasmids for expression of proteinsas amino-terminal Myc3, Flag, or Ha3 fusion proteins undercontrol of the CMV/T7 promoter, and a GST fusion protein forexpression in insect cells. Coding sequences for His6-taggedIkBa, IkK-b, p50, and p65 were amplified by PCR and baculovi-ruses generated using the Bac-to-Bac system (Invitrogen). Plas-mids for expression of Cul1HA (Lisztwan et al. 1998) and cyclinF (Bai et al. 1996) were from previous studies. GST fusion pro-teins were purified from insect cells and eluted from glutathi-one–Sepharose beads as described previously (Skowyra et al.1997).

Antibodies

Polyclonal antibodies against human Skp1 and Cul1 were gen-erated in rabbits after injection of GST–Skp1 or GST–Cul1(536–815) produced in bacteria. Antisera were depleted of anti-GSTreactivity using immobilized bacterial GST protein and thenaffinity purified using immobilized GST–Skp1 or GST–Cul1(536–815). Antibodies against the carboxyl terminusof b-TRCP were generated in rabbits using the peptideNETRSPSRTYTYISR. Antibodies were affinity purified usingthis peptide coupled to Affigel-10 (Bio-Rad). Monoclonal anti-bodies recognizing Myc, Ha, or Flag epitopes were obtainedfrom BabCO or Sigma. Antibodies against the phosphorylatedIkBa destruction motif were from New England Biolabs. Allother antibodies were from Santa Cruz Biotechnology.

In vitro binding

Twenty-one (p21, residues 21–42) and 19 (p19, residues 23–41)amino acid peptides containing the IkBa destruction motif weresynthesized in both the unphosphorylated and doubly phos-phorylated forms where serines 32 and 36 are phosphorylated asdescribed previously (Yaron et al. 1997). Analogous unphos-phorylated and doubly phosphorylated peptides overlapping acandidate destruction motif in b-catenin were also synthesized.p21 and b-catenin peptides were coupled to Affigel (Bio-Rad) at2 mg/ml of resin and p19 peptides were coupled to cyanogenbromide activated Sepharose (Pharmacia) at 10 mg/ml resin.Coupling efficiencies were determined to be >90% by analyticalreverse-phase HPLC of reaction supernatants. To examine as-sociation of Skp1 containing complexes with destruction mo-tifs, binding reactions were performed in a total volume of 120µl of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.35% NP-40, 1mM DTT, 0.75 mM EDTA, and 5 µg/ml antipain, leupeptin, andaprotinin using 15 µl of peptide beads and the indicated quantityof cell lysate or column fraction. After incubation at 4°C for ∼1hr, beads were washed three times with 1 ml of binding buffer[20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 1 mM

DTT, 1 mM EDTA, and 5 µg/ml antipain, leupeptin, and apro-tinin] prior to SDS-PAGE (13.5%) and immunoblotting usingthe indicated antibodies. Antibody detection was accomplishedusing ECL (Amersham). To examine association of F-box pro-teins with peptide beads, [35S]-methionine-labeled in vitrotranslation products of various F-box proteins were employed.Comparable quantities of translation products (typically 5–10µl) were diluted to 120 µl with binding buffer prior to incubationwith 15 µl of peptide beads (4°C, 1 hr). Beads were washed threetimes with binding buffer prior to electrophoresis and autoradi-ography.

In vivo interactions

To examine assembly of SCFb-TRCP complexes in mammalian

cells, 293T cells were transfected with various combinations ofplasmids (1 µg each/10-cm dish) expressing Myc, Flag, or Ha-tagged Skp1, b-TRCP (mouse), and Cul1 under control of theCMV promoter using lipofectamine (GIBCO/BRL). The totalDNA level was kept constant at 10 µg using empty vector DNAand each transfection contained 100 ng of pCMV–GFP DNA tofacilitate determination of transfection efficiency (typically>90%). Forty hours post-transfection, cells were lysed in bind-ing buffer supplemented with 10 mM b-glycerol phosphate, 5mM NaF, and 1 mM p-nitrophenylphosphate (30 min on ice).After centrifugation (14,000 rpm, 20 min), lysates (1 mg/0.5 ml)were subjected to immunoprecipitation using 10 µl of anti-Mycor anti-HA immobilized on agarose beads, or used in peptidebead binding reactions as described above. Washed immunecomplexes were separated by SDS-PAGE and immunoblottedusing the indicated antibodies. In some experiments, immunecomplexes from transfected cells were used for ubiquitinationreactions (see below) or for binding to IkBa/NF-kB complexes.For binding experiments, immobilized SCF complexes from one10-cm dish of transfected 293T cells (10 µl of beads) were incu-bated with 15 nM IkBa/NF-kB complexes in a total volume of100 µl of binding buffer for 60 min. Beads were washed threetimes with 600 µl of binding buffer prior to immunoblotting forp50 and IkBa.

In vitro ubiquitination

To generate IkBa/NF-kB complexes as a substrate for ubiquiti-nation, insect cells were coinfected with viruses expressingHis6–IkBa, p50, and p65 and complexes purified on nickel–NTAresin. Proteins were eluted with imidazole and the heterotri-meric complex size-selected by gel filtration. To generate phos-phorylated IkBa, the IkBa/p50/p65 complex (3 µM) was incu-bated with IkK-b (500 nM) purified from insect cells and 300 µM

ATP, 10 mM MgCl2, 1 mM DTT, 50 mM Tris (pH 7.5) for 2 hr at25°C. IkBa–ubiquitin ligase activity in THP.1 cell extracts andcolumn fractions was assayed as described previously (Chen etal. 1995) using 25 nM phosphorylated or unphosphorylatedIkBa/NF-kB complexes, HQ unbound (33 µg), and 200 nM E1.Proteins were separated by 4%–20% Novex SDS-PAGE, trans-ferred to nitrocellulose, and IkBa revealed with anti-IkBa anti-bodies. THP.1 cell extracts were prepared by Dounce homog-enization (10 strokes) in eight volumes (wt/vol) of buffer con-taining 50 mM Tris, 1 mM DTT, 1 mM EDTA, 1 mM EGTA, 1×Boehringer Mannheim protease inhibitor cocktail (EDTA-free)and centrifuged at 25,000g for 25 min. The filtered supernatantwas fractionated on a POROS HQ column equilibrated in dialy-sis buffer lacking glycerol to obtain unbound fraction (HQ un-bound) or precipitated with ammonium sulfate. The 30%–50%ammonium sulfate cut equivalent to 1 gram of cell paste wassolubilized in 3 ml of dialysis buffer (25 mM Tris at pH 7.5, 10%glycerol, 1 mM DTT, 1 mM Benzamidine, 0.4 mM PMSF). Thesolubilized pellet (2 ml) was adjusted to 1.2 M ammonium sul-fate, loaded to phenyl–Sepharose column (Pharmacia 5/5)equilibrated in dialysis buffer containing 1.2 M ammonium sul-fate but without glycerol, and bound proteins eluted with a 20column volume gradient 1.2–0 M ammonium sulfate, collectingtwo-column volume fractions followed by dialysis. To depleteIkBa–ubiquitin ligase activity from crude extracts (5 mg/ml) orphenyl–Sepharose fractions (0.7 mg/ml), p19 or pp19 beads (5 µl)were incubated with 30 µl of sample for 2 hr. Unbound materialwas removed for ubiquitination assays or immunoblotting andproteins associated with washed beads were subjected to SDS-PAGE and immunoblotting. Skp1 was immunodepleted by pre-coupling 30 µl of protein G beads (Pierce) with 1 ml of anti-Skp1

Winston et al.





or anti-GST mouse monoclonal IgG1 antibodies (TransductionLaboratories) at 45 µg/ml for 2 hr. Washed beads were incubatedwith phenyl–Sepharose pools for 3 hr as indicated above.

To assay IkBa ubiquitination activity in yeast extracts, 50 µgof crude yeast extract (Deshaies et al. 1995) was supplementedwith 100 ng of human E1, 200 µg of ubiquitin, 2 mM ATP (to-gether with an ATP regenerating system), 10 µM LLNL, and 25nM of IkBa/NF-kB complexes as substrate in a total volume of10 µl (50 mM Tris-HCl at pH 7.5, 5 mM MgCl2, 0.6 mM DTT).Reaction mixtures were incubated with SCF immune com-plexes or control immune complexes from transfected 293Tcells (10 µl of beads per assay) for 90 min at 30°C and productsanalyzed by SDS-PAGE and immunoblotting with antibodiesagainst IkBa. We estimate that the reaction mixture contained50–100 ng of SCF complexes. To assay IkBa ubiquitination ac-tivity in reconstituted mammalian extracts, GST–b-TRCP orGST proteins purified from insect cells (200 ng) were added to100 µg of phenyl–Sepharose fraction 9 followed by HQ unbound(33 µg), E1 (200 ng), ubiquitin (5 µM), and ATP (2 mM) in a finalvolume of 15 µl (90 min). In some cases, IkBa peptides wereadded at 50 µg/ml.

Chromosomal localization and in situ hybridization

Genomic clones (Bacmids 171I9 and 350H4) that hybridize tothe human b-TRCP cDNA were obtained from Research Ge-netics. The chromosomal localization of the human b-TRCPgene was determined by fluorescence in situ hybridization bythe Baylor College of Medicine FISH facility. RNA expressionanalysis by in situ hybridization was performed as described(Parker et al. 1995) using antisense mouse b-TRCP sequences asa probe and sense sequences as a negative control (data notshown).

Acknowledgments

We are extremely grateful to Mark Rolfe at Mitotix for his ef-forts in coordinating this work. We thank W. Krek for a Cul1expression plasmid; C. Wong for in situ hybridization; D. Koeppfor F-box protein expression constructs; S. Glass for molecularbiology; I. Chiu and A. Frederick for p50 cloning; M. Pelletier forIkK reaction optimization; and M. Caligiuri, A. Theodoras, L.Brizuela, G. Draetta, R. Copeland, K. Auger, K. Wee, and D.Skowyra for insightful discussions. This work is supported bygrants from the National Institutes of Health [National Insti-tute on Aging (NIA)], the Welch Foundation, and the BaylorSpecialized Program of Research Excellence in Prostate Cancerto J.W.H and S.J.E.; and by the Dupont Pharmaceutical Com-pany to Mitotix, Inc. J.W. is supported by a postdoctoral traininggrant from NIA. S.J.E. is an Investigator with the HowardHughes Medical Institute.

The publication costs of this article were defrayed in part bypayment of page charges. This article must therefore be herebymarked ‘advertisement’ in accordance with 18 USC section1734 solely to indicate this fact.

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Erratum

Genes & Development 13: 270–283 (1999)

The SCFb-TRCP–ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IkBa andb-catenin and stimulates IkBa ubiquitination in vitroJefferey T. Winston, Peter Strack, Peggy Beer-Romero, Claire Y. Chu, Stephen J. Elledge, and J. Wade Harper

Figure 3a of the above article was misprinted. The correct figure appears below with its legend in its entirety.

Figure 3. Association of SCFb-TRCP with IkBa and b-catenin destruction motifs and with the IkBa/NF-kB complex. (a,b) Cell lysates (0.3 µg of protein/150 µl) from Fig. 2 were used in IkBa (a) andb-catenin (b) peptide bead binding reactions as described in Materials and Methods. Bound proteinswere analyzed by immunoblotting with the indicated antibodies. (c) Phosphorylation-dependentassociation of b-TRCPMyc with the IkBa/p50/p65 complex in vitro. b-TRCPMyc immune complexes(lanes 2,5) corresponding to those in Fig. 2a (lane 3) or control complexes (lanes 3,6) correspondingto those in Fig. 2a (lane 1) were used in binding reactions with either IkBa/p50/p65 or IkK-bphosphorylated IkBa/p50/p65 complexes (see Materials and Methods). Bound proteins were sepa-rated by SDS-PAGE and immunoblotted using anti-p50 or anti-IkBa antibodies. The asterisk (lanes1,4) indicates the positions of 15% of the input IkBa complexes used in the binding reaction.

1050 GENES & DEVELOPMENT 13:000–000 © 1999 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/99 $5.00; www.genesdev.org

13:1999, Genes Dev. Jeffrey T. Winston, Peter Strack, Peggy Beer-Romero, et al.

ubiquitination in vitroαBκstimulates I-catenin andβ and αBκphosphorylated destruction motifs in I

ubiquitin ligase complex associates specifically with−-TRCPβThe SCF

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