Direct Ubiquitination of β-Catenin by Siah-1 and Regulation by the Exchange Factor TBL1*

β-Catenin is a key component of the Wnt signaling pathway that functions as a transcriptional co-activator of Wnt target genes. Upon UV-induced DNA damage, β-catenin is recruited for polyubiquitination and subsequent proteasomal degradation by a unique, p53-induced SCF-like complex (SCF(TBL1)), comprised of Siah-1, Siah-1-interacting protein (SIP), Skp1, transducin β-like 1 (TBL1), and adenomatous polyposis coli (APC). Given the complexity of the various factors involved and the novelty of ubiquitination of the non-phosphorylated β-catenin substrate, we have investigated Siah-1-mediated ubiquitination of β-catenin in vitro and in cells. Overexpression and purification protocols were developed for each of the SCF(TBL1) proteins, enabling a systematic analysis of β-catenin ubiquitination using an in vitro ubiquitination assay. This study revealed that Siah-1 alone was able to polyubiquitinate β-catenin. In addition, TBL1 was shown to play a role in protecting β-catenin from Siah-1 ubiquitination in vitro and from Siah-1-targeted proteasomal degradation in cells. Siah-1 and TBL1 were found to bind to the same armadillo repeat domain of β-catenin, suggesting that polyubiquitination of β-catenin is regulated by competition between Siah-1 and TBL1 during Wnt signaling.

␤-Catenin is a key component of the Wnt signaling pathway that functions as a transcriptional co-activator of Wnt target genes. Upon UV-induced DNA damage, ␤-catenin is recruited for polyubiquitination and subsequent proteasomal degradation by a unique, p53-induced SCF-like complex (SCF(TBL1)), comprised of Siah-1, Siah-1-interacting protein (SIP), Skp1, transducin ␤-like 1 (TBL1), and adenomatous polyposis coli (APC). Given the complexity of the various factors involved and the novelty of ubiquitination of the non-phosphorylated ␤-catenin substrate, we have investigated Siah-1-mediated ubiquitination of ␤-catenin in vitro and in cells. Overexpression and purification protocols were developed for each of the SCF(TBL1) proteins, enabling a systematic analysis of ␤-catenin ubiquitination using an in vitro ubiquitination assay. This study revealed that Siah-1 alone was able to polyubiquitinate ␤-catenin. In addition, TBL1 was shown to play a role in protecting ␤-catenin from Siah-1 ubiquitination in vitro and from Siah-1-targeted proteasomal degradation in cells. Siah-1 and TBL1 were found to bind to the same armadillo repeat domain of ␤-catenin, suggesting that polyubiquitination of ␤-catenin is regulated by competition between Siah-1 and TBL1 during Wnt signaling.
␤-Catenin is a ubiquitous transcriptional activator in the canonical Wnt signaling pathway involved in cellular processes ranging from embryogenesis, cell proliferation, cell fate, and survival to adult stem cell differentiation and oncogenesis (1,2). Upon Wnt stimulation, ␤-catenin activates T cell factor (Tcf) 3 / lymphoid enhancer factors (Lef) initiating the expression of many genes including cyclin D1, c-Myc, Axin2, and vascular endothelial growth factor (VEGF) (1). Mutations in ␤-catenin and its regulatory factors, such as adenomatous polyposis coli (APC), are associated with increased levels of nuclear ␤-catenin and in turn, to breast, colorectal, ovarian, and other cancers (1,3,4).
Not surprisingly, the level and cellular localization of ␤-catenin are tightly regulated by a finely tuned balance of post-translational modifications and protein turnover (1,4). The most efficient means to lower the cellular levels of ␤-catenin is by polyubiquitination, leading to degradation in the 26 S proteasome. Defects in this protein degradation machinery or mutations in ␤-catenin that prevent the recognition or processing by this machinery often lead to the stabilization of ␤-catenin in its oncogenically active state (4).
Polyubiquitination involves the serial action of E1, E2, and E3 enzymes, of which the substrate-recruiting E3 ligating enzymes are the most diverse. ␤-Catenin is recognized and ubiquitinated by a growing number of E3 ligases. Of these, the most wellstudied is SCF ␤-TrCP , a multiprotein complex that itself is regulated through the canonical Wnt signaling pathway (5,6). In the absence of a Wnt ligand, ␤-catenin is phosphorylated by glycogen synthase kinase-3␤ (GSK-3␤), and it is the phosphorylated state of the protein that is recognized by SCF ␤-TrCP . Under conditions of genotoxic stress, activation of p53 occurs and an additional pathway for ␤-catenin degradation is initiated. p53 directly induces the expression of Siah-1 and in turn formation of a unique SCF-like complex (SCF(TBL1)) comprised of Siah-1, Siah-1-interacting protein (SIP), Skp1, transducin ␤-like 1 (TBL1), and APC (7,8). The physiological significance of Siah-1-targeted degradation of ␤-catenin is underscored by the discovery that this pathway is directly targeted by the viral oncoprotein latent membrane protein 1 (LMP1) (9). In addition, recent studies identified two drugs, hexachlorophene and isoreserpine, which attenuate the function of ␤-catenin through activation of Siah-1 and subsequent proteasomal degradation (10,11).
In addition to functioning as part of the SCF(TBL1) complex, Siah-1 alone has been shown to function as an E3 ligase. The Siah-1 RING E2-binding domain is linked to a substrate binding domain that directly recruits, and mediates polyubiquitination of many substrates including Deleted in Colorectal Cancer (DCC), nuclear co-repressor (NCoR), c-Myb, and synphilin-1 (12)(13)(14)(15). The ability of Siah-1 to serve as a simple E3 ligase as well as a component of an SCF-like complex raises the possibil-ity of redundancy in the polyubiquitination pathways that lead to degradation of ␤-catenin.
The involvement of TBL1 in the SCF(TBL1) E3 ligase complex is intriguing. Both TBL1 and its close isoform, TBL1-related protein (TBLR1) have been implicated as exchange factors between nuclear co-activator and co-repressor complexes in the regulation of nuclear receptors and transcription factors (16). Moreover, recent evidence shows that TBL1 acts as a coactivator of the Wnt signaling pathway by recruiting ␤-catenin to the promoter of Wnt target genes and stimulating their expression (17). Thus, TBL1 appears to play a role in both activation and repression of ␤-catenin activity.
Previous studies by Matsuzawa and Reed (8) extensively characterized the ubiquitination of non-phosphorylated ␤catenin through the action of SCF(TBL1) in cells. Here we report a combination of in vitro and cell-based assays of ␤-catenin ubiquitination by SCF(TBL1) and Siah-1 alone. Additionally, we mapped the physical interaction between Siah-1 and ␤-catenin and analyzed the effect of TBL1 on the Siah-1-mediated ubiquitination of ␤-catenin. These results highlight the role of TBL1 as a protector of ␤-catenin activity during Wnt signaling.
The proteins were overexpressed in the Escherichia coli BL21(DE3) strain. Cells were grown at 37°C until they reached A 600 of 0.6 -0.8 and were then induced with 0.5 mM isopropyl thiogalactoside for 3-4 h at 25°C for Siah-1 constructs, 30°C for ␤-catenin constructs and 37°C for SIP, Skp1, and UbcH5a. Purification of Siah-1, SIP, Skp1, and UbcH5a was performed by Ni-NTA (Qiagen) followed by a Source Q chromatography (18,20). Expression of 15 N-labeled Siah-SBD was carried in minimal medium supplemented with 15 NH 4 Cl as the sole nitrogen source following the same protocol. ␤-catenin constructs were affinity purified on glutathione-Sepharose 4B (Amersham Biosciences) followed by cleavage of the GST fusion tag, Source Q, and size exclusion chromatography, as previously described (19).
In Vitro Ubiquitination Assay of FL-␤-Catenin-All ubiquitination experiments were carried out at a final volume of 20 l including: E1(BostonBiochem) at 52 nM, His 6 -E2-UbcH5a at 0.6 M, ubiquitin (BostonBiochem) at 50 M, His 6 -MBP-Siah-1 at 0.18 M, SIP at 1.5 M, Skp1 at 1.5 M, and His 6 -TBL1 at 1.5 M. The assay was performed in ubiquitination buffer containing 100 mM NaCl, 1 mM dithiothreitol, 5 mM MgCl 2 , and 25 mM Tris-Cl at pH 7.5 for 0.25 M ␤-catenin-FL and (Nt) domain or at pH 8, for ␤-catenin (arm) and (armϩCt) constructs. The reactions were activated with 5 mM ATP and incubated at 30°C for different time periods, as indicated on the specific experiments. To stop the ubiquitination reaction, the samples were incubated for 15 min at 90°C after the addition of 5 l of SDSloading buffer. Reactions were resolved on a NuPAGE 4 -12% Bis-Tris gradient gel (Invitrogen) and detected by a SimplyBlue SafeStain (Invitrogen). Ubiquitination reactions analyzed by Western blotting using C-terminal ␤-catenin primary antibody (Cell Signaling) and visualized with goat anti-rabbit-horseradish peroxidase by SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).
NMR Samples and Chemical Shift Perturbation Assay-15 N-1 H TROSY HSQC spectra were acquired for 15 N-enriched Siah-SBD at 120 M in 25 mM Tris-Cl at pH 8, 100 mM NaCl, and 10 mM ␤ME in 90% H 2 O/10% D 2 O. One sample was free Siah-SBD, and the second also contained 90 M unlabeled ␤-catenin-FL. The NMR experiments were performed using a Bruker DRX 800 MHz spectrometer equipped with a Cryo-Probe. The 15 N-1 H TROSY HSQC spectra were acquired at 30°C with 128 scans. Data were processed using Topspin 2.0b (Bruker) and analyzed with Sparky (10).
Mapping Ubiquitination Sites on ␤-Catenin and Ubiquitin Chain Formation by Mass Spectrometry Analysis-Proteins were separated by SDS-PAGE and the gel was stained with SimplyBlue SafeStain (Invitrogen). Individual protein bands were excised, equilibrated in 50 mM NH 4 HCO 3 , reduced with dithiothreitol (3 mM in 100 mM NH 4 HCO 3 , 37°C for 15 min), and alkylated with iodoacetamide (6 mM in 50 mM NH 4 HCO 3 for 15 min). The gel slice was then dehydrated with acetonitrile

␤-Catenin Ubiquitination by Siah-1, Protection by TBL1
and rehydrated with 15 l of 12.5 mM NH 4 HCO 3 containing 0.01 g/l modified trypsin (Promega), and trypsin digestion was carried out for Ͼ2 h at 37°C. Peptides were extracted with 60% acetonitrile, 0.1% trifluoroacetic acid, and dried by vacuum centrifugation and resuspended in 10 l of 0.1% formic acid. LC-MS/MS analysis of the peptides was performed using a Thermo LTQ ion trap mass spectrometer equipped with a Thermo MicroAS autosampler and Thermo Surveyor HPLC pump, Nanospray source, and Xcalibur 2.0 SR2 instrument control. The peptides were resolved on a fused silica capillary column, 100 m ϫ 15 cm, packed with C18 resin (Jupiter C 18 , 5 m, 300 Å, Phenomonex, Torrance, CA) using a 95-min gradient of increasing acetonitrile with 0.1% formic acid. MS/MS scans were acquired using an isolation width of 2 m/z, and activation time of 30 ms, activation Q of 0.250, and 35% normalized collision energy using 1 microscan and maximum injection time of 100 for each scan. The MS/MS spectra of the peptides were acquired using data-dependent scanning (top-five) with dynamic exclusion (60 s exclusion, list size ϭ 50, repeat count ϭ 1). Individual MS/MS fragmentation spectra were then searched against the IPI_mouse data base (Feb 2008) allowing for complete carbamidomethylation of cysteine, and partial modification by oxidation of methionine. Partial modification of lysine by ϩ114 Da was also selected to detect ubiquitinated peptides (mass shift due to GG ubiquitin sequence that remains after trypsin digestion). All candidate spectra were manually inspected for verification of ubiquitination.
Cell Culture, siRNA Transfection, and Western Blot Analysis-Human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37°C in a 5% CO 2 /95% air atmosphere. For siRNA transfections, 1 ϫ 10 6 293T cells were seeded into 6-well plates for 12 h and then transfected with various amount of siRNA using Lipofectamine 2000 according to the manufacturer's pro-tocol (Invitrogen). Expression vector of Flag-Siah-1 or control empty vector were transfected using Fugene6 (Roche) 24 h after the transfection with siRNA. Cells were treated, unless otherwise indicated, with 20 mM LiCl. Nuclear and cytosolic extracts were prepared, and 5 g of extract were loaded on SDS-PAGE and followed by Western blot analysis using an enhanced chemiluminescence reagent (Thermo Scientific). siRNAs were synthesized by Dharmacon Research, and sequences are used as previously described (17). All antibodies used in the Western blot are commercially available: ␤-catenin (BD), TBL1 (Abcam), TBLR1 (Bethyl Laboratories), and ␣-tubulin (Santa Cruz Biotechnology).

RESULTS
In Vitro Ubiquitination of ␤-Catenin by the SCF(TBL1) Complex-To further investigate the molecular basis for ␤-catenin degradation by the SCF(TBL1) complex, we developed an in vitro ubiquitination assay using purified recombinant proteins. The E2-conjugating enzyme UbcH5a, Skp1, Siah-1, SIP, and the ␤-catenin substrate were overexpressed and purified from E. coli (18,19,22). A transient expression system was used for the expression of full-length TBL1 in mammalian 293-6E cells (21). The protocols for the in vitro ubiquitination reaction were developed based on previous reports (23), using proteins each purified to Ͼ95% homogeneity ( Fig. 1A). To ensure that the full length ␤-catenin substrate is properly folded, a circular dichroism (CD) experiment was performed (supplemental Fig. S1). The analysis of this spectrum correlates well with the ␣-helical secondary structure elements observed in the x-ray crystal structure of the protein (24).
The ubiquitination reaction was initiated by addition of ATP after mixing all components and incubated for 60 min at 30°C. Lane 5 in Fig. 1B shows the complete reaction with all components of the SCF(TBL1) present. In this reaction, all free ␤-catenin has been consumed, resulting in formation of a characteristic ubiquitin ladder. Control experiments show no ubiquitination of ␤-catenin when ubiquitin (lane 2), ␤-catenin substrate (lane 3), or ATP (lane 4) are excluded from the reactions (Fig. 1B). The absence of Siah-1 also results in no discernable ubiquitination of ␤-catenin, presumably because of the inability to co-localize the E2-conjugated ubiquitin and the substrate (Fig. 1B, lane 9). In contrast, ␤-catenin polyubiquitination is observed even in the absence of TBL1, Skp1 or SIP (Fig. 1B, lanes 6 -8). Together, these results show that the polyubiquitination of ␤-catenin by SCF(TBL1) can be reconstituted in vitro, and only Siah-1 is required for ␤-catenin polyubiquitination.
Siah-1 Directly Ubiquitinates ␤-Catenin-The results from the in vitro ubiquitination of ␤-catenin by the SCF(TBL1) com- plex were somewhat puzzling given the prevailing view of multiprotein SCF E3 complexes. Ideally, in the absence of any one component, the complex could not assemble and would lose its ubiquitinating activity. Our observation of ␤-catenin polyubiquitination in the absence of TBL1, Skp1, and SIP implied that non-phosphorylated ␤-catenin can be polyubiquitinated by an alternate Siah-1-mediated mechanism. Motivated by reports that Siah-1 serves as a simple E3 ligase and the observation that only Siah-1 is required for the ubiquitination of ␤-catenin (Fig.  1B, lane 9), a series of in vitro assays were performed to investigate if Siah-1 alone could serve as an E3 ligase for the ␤-catenin substrate, and if so, was the reaction modulated by any of the other SCF(TBL1) proteins.
A time course of the ubiquitination reaction with Siah-1 alone as an E3 ligase is shown in Fig. 2. Substantial monoubiquitination of ␤-catenin is observed within 10 min of activation of the reaction and both the extent of ubiquitination and the number of added ubiquitins increase over time (lanes 2-6). No ubiquitination was observed in reactions lacking ubiquitin, Siah-1 or ATP (lanes 7-9). Siah-1 robustly autoubiquitinates, but as shown in lane 11, the autoubiquitination reaction in the absence of ␤-catenin does not result in the distinctive ladder that is observed in the presence of the substrate (see lanes 6 versus 11). Immunoblotting with ␤-catenin antibody confirms that the species observed by Coomassie staining correspond to modified ␤-catenin substrate (Fig. 2, bottom panel). The specific pattern of two strong bands of ubiquitinated ␤-catenin seen in Fig. 1 can arise from multiple monoubiquitination events. To confirm that ␤-catenin is polyubiquitinated we performed the in vitro ubiquitination reaction using K(0)-ubiquitin (supplemental Fig. S2B). This reaction shows a substantial overall decrease in the ladder of K(0)-ubiquitinated ␤-catenin compared with the ladder observed with wt-ubiquitin, confirming that ␤-catenin is indeed polyubiquitinated.
The specificity of Siah-1 for ␤-catenin in the in vitro ubiquitination assay was confirmed using a substrate Skp1 that does not bind to Siah-1. Supplemental Fig. S2A shows that Skp1 is not ubiquitinated. To further demonstrate that Siah-1 ubiquitination requires physical interaction, the assay was performed using SIP, which is known to directly interact with Siah-1 (18). As anticipated, SIP is efficiently polyubiquitinated by Siah-1 (supplemental Fig. S2B). These results show that Siah-1 acting on its own as an E3 ligase specifically polyubiquitinates ␤-catenin in vitro, in a phosphorylation-independent manner.
Siah-1 Forms K11 Ubiquitin Chains on ␤-Catenin-To determine the specific ubiquitin chain assembled on ␤-catenin by Siah-1, the ubiquitinated substrate was characterized by tandem mass spectrometry. The products from the standard in vitro ubiquitination reactions were resolved on an SDS-PAGE gel and the second band, labeled ** (Fig. 2, lane 6) was excised, subjected to in-gel trypsin digest, followed by liquid chromatography-mass spectrometry (LC-MS/MS). The strategy involved identifying ubiquitin peptides from the trypsin proteolysis that contain lysine residues with the mass addition of 114.04 Da from the covalently attached ubiquitin signature peptide (-GG) that remains attached after trypsin cleavage. A single modified peptide sequence, 6 KTLTGK 11 -GG TITLEVEP-SDTIENVK 27 A, from ubiquitin was identified indicating a K11linked ubiquitin chain on ␤-catenin. A number of studies have already shown that formation of K11 ubiquitin chains can target the substrates for degradation in the cell (25,26). These data indicate that Siah-1/UbcH5a alone can assemble K11-linked ubiquitin chains on ␤-catenin that could lead to the proteasomal degradation of ␤-catenin in cells.
TBL1 Inhibits in Vitro Polyubiquitination of ␤-Catenin by Siah-1-Direct physical and functional interaction between TBL1 and ␤-catenin is well established (8,17). TBL1 and its isoform, TBLR1, have been shown to be required for the transcriptional activity of ␤-catenin in cells by mediating recruitment of ␤-catenin to the promoter of Tcf/Lef target genes during Wnt signaling (17). Moreover, in the SCF(TBL1) complex, TBL1 was shown to play the role of directly binding and recruiting non-phosphorylated ␤-catenin to the complex (8). However, our in vitro ubiquitination data indicate that the presence of TBL1 is not required for the recruitment and polyubiquitination of ␤-catenin by Siah-1.
To obtain further insights into these observations, we compared Siah-1 in vitro ubiquitination assays performed under identical conditions for ␤-catenin alone and preincubated with SCF(TBL1) (Fig. 3). To specifically monitor ubiquitination of ␤-catenin, the reactions were detected by Western blot with ␤-catenin antibody, as reported previously (27). We note that

␤-Catenin Ubiquitination by Siah-1, Protection by TBL1
although species with long polyubiquitin chains are indeed generated in the reaction (Fig. 2), the amount of highly polyubiquitinated ␤-catenin molecules is below the detection limit and only the first few bands are observed using this approach.
Interestingly, Siah-1 alone appeared to ubiquitinate ␤-catenin at a faster rate than the SCF(TBL1) complex. Fig. 3 presents a time-course of ␤-catenin ubiquitination by Siah-1 in the absence (lanes 2-6) and presence (lanes 7-11) of SCF(TBL1). The data show that free ␤-catenin is depleted by 60 min in the absence of SCF(TBL1), whereas a significant amount of nonubiquitinated ␤-catenin still remains when SCF(TBL1) is present in the reaction (Fig. 3, lanes 6 versus lane 11). Furthermore, monoubiquitination of ␤-catenin by Siah-1 alone is observed at the 5-min point, and two additional bands of ubiquitinated ␤-catenin also appear at 10 and 15 min (lanes 2-4). In contrast, only one band of ubiquitinated ␤-catenin is observed at 15 min after preincubation with SCF(TBL1) (lane 9). This clearly shows that the formation of ubiquitin chains on ␤-catenin is delayed in the presence of all SCF(TBL1) proteins. Because the differences between the time courses are modest, the experiments were repeated several times, including using proteins from different purification stocks. A high reproducibility was observed between experiments, which serves to validate the observations.
To further investigate if a single component from the SCF(TBL1) is able to attenuate Siah-1-dependent ubiquitination of ␤-catenin, the effect of preincubating SIP, Skp1, or TBL1 with ␤-catenin was tested. Lanes 12-16 in Fig. 3 show that the addition of TBL1 alone has an effect similar to the SCF(TBL1) complex (lanes 7-11). Together, these results show that TBL1 inhibits the in vitro polyubiquitination of ␤-catenin by Siah-1.
TBL1 Inhibits Siah-1-mediated ␤-Catenin Degradation in Cells-It has previously been shown that expression of Siah-1 correlates with reduced levels of ␤-catenin in cells (7-9, 28). Overexpression of Siah-1 in the absence of a Wnt ligand reduced levels of ␤-catenin and lowered the induction of Tcf/ Lef target genes (7,8). It has also been shown that Wnt activation stimulates interaction of TBL1 and TBLR1 with ␤-catenin (17). In the presence of a Wnt ligand, expression of Siah-1 also decreased the amount of expressed Tcf/Lef target genes, therefore promoting degradation of ␤-catenin, but the effect was not as substantial as in the absence of a Wnt ligand (7).
To determine the effect of TBL1/TBLR1 on ␤-catenin degradation during Wnt signaling we examined the levels of ␤-catenin in cells. It is important to note that the endogenous level of Siah-1 in cells is extremely low and does not rise to appreciable levels until p53 is activated. Thus, endogenous Siah-1 cannot ordinarily be observed on Western blots. In essence, because Siah-1 is not prevalent in quiescent cells, the "normal" condition when Siah-1 is actively involved in regulating ␤-catenin corresponds only when there is stress to the cells so that Siah-1 is up-regulated through activation of p53. Consequently, the standard and now well-accepted approach to study the effects of Siah-1 is via overexpression (7,8,(12)(13)(14)(15).
To study the roles of non-canonical pathways for ␤-catenin degradation, the basal phosphorylation-dependent polyubiquitination of ␤-catenin by the SCF ␤-TrCP complex must be down regulated. The standard protocol for this is to use LiCl to inhibit GSK-3␤ kinase, which suppresses the phosphorylation of ␤-catenin and therefore its ability to be recognized and degraded by the SCF ␤-TrCP complex (3,17).
In our experiments, small interfering RNA (siRNA) was used to knock down the expression of TBL1 and TBLR1 in HEK293T cells, and LiCl is added after transfection of Siah-1 to monitor the effect on degradation of ␤-catenin. The amount of nuclear and cytoplasmic ␤-catenin was detected over a period of time by Western blot analysis. More than 80% reduction of TBL1 and TBLR1 expression was achieved in 293T cells by the siRNA (Fig. 4A, bottom panels). Expression of Siah-1 leads to a significant decrease in the level of nuclear ␤-catenin over the time course of Wnt induction when TBL1 and TBLR1 are knocked down. In contrast, the presence of TBL1 and TBLR1 is seen to protect ␤-catenin from Siah-1-mediated degradation in the nucleus (Fig. 4A). The same effect was observed with cytoplasmic ␤-catenin, where a significant decrease in the level of ␤-catenin is seen when TBL1 and TBLR1 are knocked down (Fig. 4B).
To ensure that the level of transfected Siah-1 is comparable to previously published data and to the amount of protein expressed upon activation of p53 during DNA damage, we measured the level of Siah-1 after transfection and upon addition of adriamycin (7,8,29). The data confirm that our transfection protocol does not result in gross overexpression of Siah-1, and in fact the level of Siah-1 is similar to the level observed upon activation of p53 (supplemental Fig. S5). Taken together, our results indicate that upon Wnt signaling, TBL1 and TBLR1 serve to protect ␤-catenin from Siah-1-induced degradation.
Siah-1 Ubiquitinates ␤-Catenin at Lysines Outside the TBL1 Binding Site-The core of ␤-catenin is an armadillo (arm) repeat domain, which has an elongated ␣-helical structure that facilitates the interaction of the majority of ␤-catenin substrates (19,24). The interaction site of TBL1 with ␤-catenin was previously mapped to the N-terminal region of this (arm)

␤-Catenin Ubiquitination by Siah-1, Protection by TBL1
domain, specifically residues 134 -467 (17). To test if TBL1 directly blocks access to the lysine residues targeted for ubiquitination by Siah-1, mass spectrometry was used to identify the ubiquitination sites on ␤-catenin (30,31). The standard in vitro ubiquitination reaction was carried out, and the reaction products were run on an SDS-PAGE gel (Fig. 5A). Bands 1 and 2 were excised, digested by trypsin and analyzed by LC-MS/MS. Lys 666 or Lys 671 at the C-terminal (Ct) domain of ␤-catenin (Ct: residues 665-781) were identified as the predominant sites of ubiquitination in both bands 1 and 2 (Fig. 5B). Notably, in all of the MS/MS experiments performed, no peptides with both Lys residues ubiquitinated were ever observed. Together, these data show the lysine residues on ␤-catenin targeted for polyubiquitination by Siah-1 are well outside the TBL1 binding site (Fig. 5C).
An NMR chemical shift perturbation experiment was performed to confirm the direct physical interaction between Siah-1 and ␤-catenin.

␤-Catenin Ubiquitination by Siah-1, Protection by TBL1
100 kDa, which ordinarily would lead to slow tumbling times in solution and near complete loss of signal intensity. In such solutions, useful information on binding can be obtained when examining the early part of the titration curve for systems that exhibit binding in the M range (32,33). Upon addition of a substoichiometric amount of ␤-catenin to 15 N-Siah-SBD, spectral changes were observed including loss of intensity and perturbation of chemical shifts for a subset of peaks in the spectrum ( Fig. 6C and for full spectrum: supplemental Fig. S6). These observations confirm a relatively weak, but specific interaction between Siah-1 and ␤-catenin.

DISCUSSION
Efficient polyubiquitination and degradation of ␤-catenin during genotoxic stress is critical to preventing constitutive cell proliferation and preserving genomic stability. Upon UV-induced DNA damage, ␤-catenin is targeted for polyubiquitination and subsequent proteasomal degradation by a p53-induced mechanism that does not require phosphorylation of the substrate (7,8). The critical protein in this pathway is Siah-1, which mediates an efficient depletion of ␤-catenin, thereby down-regulating transcription of Wnt target genes. Siah-1-mediated degradation of ␤-catenin was initially demonstrated through the formation of an SCF-like complex (SCF(TBL1)), comprised of Siah-1, SIP, Skp1, and TBL1 (8). We established an in vitro ubiquitination assay with reconstituted SCF(TBL1) to investigate the mechanism of action of this complex, but interestingly we found that Siah-1 alone functions as an E3 ligase that is able to directly bind and polyubiquitinate ␤-catenin in vitro (Figs. 2 and 6C).
In addition to demonstrating that polyubiquitination of ␤-catenin can occur through a direct interaction with Siah-1, we established that Siah-1/UbcH5 assembles K11 ubiquitin chains at a novel ␤-catenin ubiquitination site. We have also found that during Wnt signaling TBL1, a transcriptional co-activator of ␤-catenin, plays a role in protecting ␤-catenin from Siah-1-mediated polyubiquitination and proteasomal degradation. TBL1 and Siah-1 were found to bind ␤-catenin at the (arm) domain. The absence of polyubiquitination of TBL1 by Siah-1 in the in vitro ubiquitination assay (supplemental Fig. S2A) rules out the possibility of TBL1 competing with ␤-catenin as a substrate for Siah-1. Whereas inhibition of ␤-catenin ubiquitination via an allosteric binding effect cannot be ruled out, our results support a model in which TBL1 protection of ␤-catenin from polyubiquitination by Siah-1 is caused by direct competition for the Siah-1 binding site.
Function of TBL1 as Transcriptional Co-activator and Corepressor of ␤-Catenin-TBL1 appears to serve two roles in regulating the activity of ␤-catenin. Besides the initially identified role of TBL1 in recruiting ␤-catenin to the SCF(TBL1) complex, it has also been shown to function as a transcriptional co-activator of ␤-catenin in recruiting it to the promoter site of Wnt target genes (left side, Fig. 7) (17). Our results indicated that TBL1 can inhibit the polyubiquitination of ␤-catenin by Siah-1 in vitro (Fig. 3) and stabilize ␤-catenin in cells by protecting it from Siah-1-mediated ubiquitination and proteasomal degradation (Fig. 4).
We note that the in vitro ubiquitination of ␤-catenin by Siah-1 is not an extremely efficient reaction. Liu and co-workers (7,35) showed that APC is required to observe Siah-1-mediated degradation of ␤-catenin in cells and that APC directly interacts with Siah-1 and with ␤-catenin. Our data indicate that Siah-1 binding to ␤-catenin is very dynamic and weak (Fig. 6C). It is likely that APC serves as a molecular bridge for Siah-1 and ␤-catenin, thus stabilizing the complex to increase the efficiency of ubiquitin chain formation in cells. The situation in cells would also be different because TBL1 recruits ␤-catenin to the Tcf/Lef transcription factors, which in effect protects ␤-catenin from access by Siah-1. This interpretation is sup-  APRIL 30, 2010 • VOLUME 285 • NUMBER 18 ported by the inhibitory effect of TBL1 in the cell-based experiments (Fig. 4). Furthermore, our data indicate that TBL1 (TBLR1) forms a complex with ␤-catenin in the cytoplasm and nucleus, because ␤-catenin can be protected from Siah-1-induced degradation in both cellular compartments (Fig. 4). Together, our results suggest a model in which down-regulation of Wnt target genes during DNA damage involves binding of ␤-catenin by Siah-1 and polyubiquitination of free non-phosphorylated ␤-catenin (Fig. 7). Once a complex between ␤-catenin and TBL1 (TBR1) is formed upon Wnt stimulation, it binds the Wnt target gene promoter and transcription factors such as Tcf, therefore limiting the ability of Siah-1 to access and polyubiquitinate ␤-catenin. We propose that the activation of Wnt target genes during genotoxic stress and the regulation of cellular processes such as cell proliferation and apoptosis depend on the balance of Siah-1-mediated degradation of ␤-catenin versus TBL1 (TBLR1)-facilitated protection and activation of ␤-catenin-targeted genes.

␤-Catenin Ubiquitination by Siah-1, Protection by TBL1
Role of SCF(TBL1) Components SIP and Skp1 in ␤-Catenin Polyubiquitination and Degradation-Our results leave open the question as to whether Siah-1 is sufficient for the recruitment and polyubiquitination of ␤-catenin in cells or additional components from the SCF(TBL1) complex are necessary. Siah-1 has a substrate binding domain for recruiting substrates and a RING domain for binding the E2-ubiquitin complex. It has been shown to act alone in polyubiquitinating many substrates such as DCC, NCoR, and synphilin-1 (12,13,15). Our data also indicate that Siah-1 can function as a simple E3 ligase for polyubiquitination of ␤-catenin. If Siah-1 functions on its own, what is the role of SIP? One possibility is that SIP, like DCC and NCoR, is simply another target of Siah-1 for polyubiquitination and degradation. In vitro ubiquitination reactions show an efficient polyubiquitination of SIP in the presence or absence of Skp1, TBL1, and ␤-catenin (supplemental Fig. S2B). It has been reported that overexpression of SIP mutants that cannot interact with Siah-1 prevent the efficient degradation of ␤-catenin in HEK293T cells, and the level of SIP is essential for ␤-catenin degradation in gastric and renal cancer cells (35)(36)(37). However, overexpression of only Siah-1 is sufficient for ␤-catenin degradation in cells, and the in vitro ubiquitination of ␤-catenin is not affected by the addition of SIP ( Fig. 4 and supplemental Fig. S3B) (7,9). It is possible that the interaction between SIP and Siah-1 is important for the proper localization of Siah-1 or for protection of Siah-1 from autoubiquitination and degradation.
The role of Skp1 as an adaptor is to provide an anchor in SCF ligases between Cullins, in our case SIP, and the F Box-protein TBL1. Interaction between SIP and Skp1 has been demonstrated by affinity chromatography and NMR chemical shift perturbation experiments (18). However, evidence for an effective interaction between the F Box domain of TBL1 and Skp1 is lacking. A wealth of biochemical and structural data have established that the binding between Skp1 and F Box domains from Skp2 or ␤-TrCP is very strong with a half-life of the Skp1-Skp2 complex longer than 9 h, and the surface area of the core interface of more than 2000 Å 2 (38,39). TBL1 has a putative F Box domain that appears to have a very low affinity for Skp1. Previous studies using the Drosophila homologue of TBL1, Ebi have been unsuccessful to detect an interaction between Ebi and Skp1 (40). We have also been unable to detect this interaction by pull down experiments with purified proteins, co-expression of Skp1 and TBL1 followed by pull-downs and negative gel shift assays. Using the very sensitive NMR chemical shift perturbation assay, we found an extremely weak (K D ϳmM) interaction between Skp1 and TBL1-(1-170) (data not shown). Thus, it is unclear how Skp1 is able to play the role of the adaptor in the SCF(TBL1) complex. Although additional experiments are required to systematically evaluate if the SCF(TBL1) does indeed form in vivo as postulated, the role of Siah-1 in degradation of ␤-catenin and the modulation of this activity by TBL1 are clear.
The SCF(TBL1) complex closely resembles the Drosophila Sina/Phyl/Ebi complex. In Drosophila, the transcriptional repressor Ttk88 is recruited and polyubiquitinated by the Sina (Siah-1 homologue)/Phyllopod (Phyl) complex for proteasomal degradation (41). Phyl functions as an adaptor of Sina in recruiting different substrates for polyubiquitination during Drosophila neurogenesis (42). Both Phyl and mammalian SIP contain a Siah-binding motif or a degron sequence that has been crystallized in complex with Siah-SBD, demonstrating an almost identical interaction (43). Ttk88 can directly bind to Sina and Phyl, but it also requires Ebi (a homologue of TBL1) for FIGURE 7. Model of the mechanism of TBL1-mediated activation and Siah-1-induced polyubiquitination of ␤-catenin. Left, during Wnt activation, the degradation of ␤-catenin is inhibited leading to its accumulation in the cytoplasm and translocation to the nucleus. TBL1/TBLR1 forms a complex with ␤-catenin and mutually co-localize to the promoter site of Wnt target genes, stimulating their transcription. Right, upon DNA damage during Wnt signaling, Siah-1 targets free ␤-catenin in the cytoplasm and nucleus for polyubiquitination and proteasomal degradation, resulting in down-regulation of Wnt target gene transcription.

␤-Catenin Ubiquitination by Siah-1, Protection by TBL1
an efficient polyubiquitination and degradation of Ttk88. In contrast to TBL1, which has been shown to directly bind ␤-catenin, Ebi has been shown to have a strong affinity for Sina and Phyl, but weak and indirect interaction with the substrate Ttk88 (40). The similarity between mammalian and Drosophila Siah/Sina-mediated ubiquitin complexes suggests the formation of a Siah-1-mediated E3 ligase that does not resemble the conventional SCF complex. Siah-1 and Sina can directly bind and recruit the substrate for polyubiquitination, but they also require the assistance of the adaptor protein SIP/Phyl. Skp1 has been demonstrated to be dispensable to the function of the complex by us and others (40). Most interestingly Ebi does not interact directly with the Ttk88 substrate, but with Sina and Phyl, whereas TBL1 appears to play a dual role in facilitating both activation and degradation of protein substrates.
Tight Regulation of ␤-Catenin by Multiple E3 Ligases-The existence of multiple ubiquitination pathways leading to degradation implies the physiological importance of tightly regulating ␤-catenin. Besides the major E3 ligases SCF ␤-TrCP and the p53-induced Siah-1, polyubiquitination of ␤-catenin is also initiated by a recently identified single subunit E3 ligase, Jade-1 (44). Interestingly, membrane-associated ␤-catenin is regulated by a different set of E3 ligases, such as Hakai and the muscle-specific Ozz E3 ubiquitin ligase (45,46). Thus the mechanism of recognition, recruitment, and polyubiquitination of ␤-catenin by different E3 ligases as well as the signaling event initiating the degradation of the substrate appears to vary substantially. The activation of the SCF ␤-TrCP depends on the presence or absence of a Wnt ligand, whereas Siah-1-mediated ␤-catenin degradation is induced by activation of p53 during genotoxic stress. In the SCF ␤-TrCP complex, ␤-TrCP binds the phosphorylated N-terminal domain of ␤-catenin leading to the attachment of ubiquitin chains on Lys 19 and Lys 49 (47,48). Here, we show that Siah-1 binds the non-phosphorylated ␤-catenin, leading to the attachment of ubiquitin chains at the very C-terminal region of the (arm) domain on Lys 666 and Lys 671 . Notably, this coincides with the binding surface of most transcription co-activator complexes such as p300/CBP and TRRAP/TIP60 histone acetyltransferases (HATs) and the PAF1 complex (49). Thus polyubiquitination of ␤-catenin by Siah-1 could prevent the transcription of many Wnt target genes. Mutations in ␤-catenin can lead to inefficient degradation and therefore greater accumulation in the nucleus. The corresponding overactivation of Tcf/Lef genes by ␤-catenin is believed to be responsible for the initiation and progression of many types of cancer (3). Understanding the uniqueness and precise mechanism of action of each E3 ubiquitin ligase targeting ␤-catenin for degradation and the molecular basis for defective activity of mutants is an important goal for understanding the accumulation of ␤-catenin in the cell and its relationship to oncogenesis.