Adaptor Protein Self-Assembly Drives the Control of a Cullin-RING Ubiquitin Ligase

Structure Article

Wesley J. Errington,1 M. Qasim Khan,1 Stephanie A. Bueler,2 John L. Rubinstein,1,2,3 Avijit Chakrabartty,1,3,4 and Gilbert G. Prive´ 1,3,4,* 1Department of Biochemistry, University of Toronto, 1 Kings College Circle, Toronto, Ontario M5S 1A8, Canada 2The Hospital for Sick Children Research Institute, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada 3Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada 4Ontario Cancer Institute, Campbell Family Institute for Cancer Research, 101 College Street, Toronto, Ontario M5G 1L7, Canada *Correspondence: [email protected] DOI 10.1016/j.str.2012.04.009

SUMMARY the much more numerous and more varied E3s. Interestingly, a number of reports have demonstrated that E3s, in particular The E3 ligases recruit substrate proteins for targeted those of the CRL family, can exert active roles in regulating ubiquitylation. Recent insights into the mechanisms substrate ubiquitylation (Deshaies et al., 2010; Duda et al., of ubiquitylation demonstrate that E3 ligases can 2011; Gallagher et al., 2006) demonstrating that E3 ligase possess active regulatory properties beyond those complexes can function beyond simple assembly scaffolds on of a simple assembly scaffold. Here, we describe which ubiquitylation occurs. the dimeric structure of the E3 ligase adaptor protein The CRLs are the largest family of E3s and are constructed from modular protein subunits (Petroski and Deshaies, 2005). SPOP (speckle-type POZ protein) in complex with ˚ The defining feature of a CRL is the central cullin scaffold protein the N-terminal domain of Cul3 at 2.4 A resolution. that interacts with a substrate adaptor protein via its N-terminal We find that SPOP forms large oligomers that can domain, and an E2-recruiting RING protein via its C-terminal form heteromeric species with the closely related domain. The human genome encodes seven cullin proteins paralog SPOPL. In combination, SPOP and SPOPL (Cul1, 2, 3, 4a, 4b, 5, and 7), two associated variant RING (SPOP-like) form a molecular rheostat that can fine- proteins (Rbx1 and Rbx2) and a large number of substrate adap- tune E3 ubiquitin ligase activity by affecting the olig- tors. Many of the characterized and predicted CRL substrate omeric state of the E3 complex. We propose that adaptors belong to the BTB (bric a` brac, tramtrack, broad- adaptor protein self-assembly provides a graded complex) superfamily of proteins (Furukawa et al., 2003; Geyer level of regulation of the SPOP/Cul3 E3 ligase toward et al., 2003; Pintard et al., 2003; Xu et al., 2003). BTB proteins its multiple protein substrates. interact with cullins via their BTB domain and recruit substrates for ubiquitylation through either an associated protein domain on the same polypeptide chain, or, in the case of the BTB proteins Skp1 and Elongin C, through recruitment of an addi- INTRODUCTION tional F box or SOCS box adaptor protein (Stogios et al., 2005). Speckle-type POZ protein (SPOP) is a member of the MATH- Ubiquitylation, the covalent attachment of ubiquitin onto a tar- BTB protein family and functions as a CRL adaptor protein in geted substrate protein, is a versatile form of post-translational complex with Cul3. SPOP has roles in maintaining normal modification that was originally characterized as a molecular cellular growth and development as indicated by the high inci- tag directing eukaryotic proteins for proteolytic turnover by the dence of both SPOP gene overexpression and SPOP gene 26S proteasome (Hershko and Ciechanover, 1998). Ubiquityla- copy loss in a number of human cancers (Berger et al., 2011; tion has since become appreciated for its non-proteolytic regu- Kan et al., 2010). SPOP has been shown to directly target ten lation of various cellular events, such as DNA repair (Hoege et al., proteins for ubiquitylation via its MATH domain, including the 2002) and subcellular localization (Li et al., 2003). The process of polycomb protein Bmi1 (Herna´ ndez-Mun˜ oz et al., 2005), the ubiquitin conjugation occurs through a conserved three-enzyme apoptosis factor Daxx (Kwon et al., 2006), the pancreatic and cascade that is, in general, not dependent on the protein duodenal homeobox protein Pdx1 (Liu et al., 2004), the inositol substrate or the type of ubiquitin tag that it receives (Pickart, kinase PIPKIIb (Bunce et al., 2008), and the Hedgehog signaling 2001). A ubiquitin moiety is first transferred from an E1 activating transcription factors Gli2 and Gli3 (Wang et al., 2010). Addition- enzyme onto an E2 conjugating enzyme. Subsequently, an E3 ally, a recent report has indicated a tumor-suppressor role for ubiquitin ligase, acting as a substrate specificity factor and cata- SPOP in part through mediating the polyubiquitylation and lyst, facilitates ubiquitin transfer through proximal recruitment of subsequent proteolysis of the oncogenic steroid receptor both the target substrate and the E2 conjugating enzyme. Within SRC-3 (Li et al., 2011). the ubiquitylation cascade, E1 and E2 enzymes have been the To gain a greater understanding of the role of SPOP in the subject of detailed structural and mechanistic studies (Huang regulation of its various target proteins, we have undertaken et al., 2005, 2007). Fewer details are known, however, regarding structural and functional studies of the assembly and activity of

Figure 1. Structure of the SPOP BTB Domain in Complex with the N-Terminal Domain of Cul3 (A) Domain architecture of human SPOP. (B) Crystal structure of the 2:2 heterotetrameric SPOP/Cul3 complex shown in two orthogonal views. The two chains of the SPOP BTB dimer are yellow and red and the two chains of Cul3NTD are blue. (C) Cartoon depiction of the heterotetrameric crystal structure in (B) in the context of a full-length protein assembly. (D) Model of the full-length functional SPOP/Cul3 ubiquitin ligase based around a dimeric BTB domain. The structure in (B) was used as the scaffold on which to model overlapping homologous structures from the PDB structures 3HQI (Zhuang et al., 2009), 1LDK (Zheng et al., 2002), 1FBV (Zheng et al., 2000), and 3A33 (Sakata et al., 2010). The SPOP homodimer is yellow and red, Cul3 is blue, Rbx1 is purple, and the thioester-linked UbcH5b-ubiquitin complex is green and gray. See also Figures S1 and S2. the SPOP/Cul3/Rbx1 CRL (CRL3SPOP). We present the crystal In agreement with an earlier report (Zhuang et al., 2009), structure of the Cul3 N-terminal domain (Cul3NTD) in complex SPOPBTB forms a homodimer with a tightly packed hydrophobic with the dimeric SPOP BTB domain (SPOPBTB) and find that interface between helices a1, a2, and a3 of the two monomers a larger construct of SPOP drives high molecular weight and a strand-exchanged amino terminal region, similar to what complexes of Cul3. In contrast, the closely related paralog has been observed for other BTB domain structures (Stogios SPOPL (SPOP-like) appears to be limited to dimeric complexes et al., 2005). In contrast to the dimeric BTB structures of BCL6 and has a reduced level of E3 activity. In combination, SPOP (also known as ZBTB27) (Ahmad et al., 2003), PLZF (also known partners with SPOPL to create a molecular rheostat that is as ZBTB16) (Ahmad et al., 1998), gigaxonin (also known as capable of fine-tuning E3 valency and activity. We propose KLHL16) (Zhuang et al., 2009), and KLHL11 (Protein Data Bank that this mechanism produces a nuanced control with which [PDB] ID 3I3N), which have an interchain antiparallel b sheet CRL3SPOP/SPOPL can precisely effect regulation of its numerous between b1 and b50, the swapped region in SPOP makes contact substrate proteins. only with a60 and does not adopt a b strand conformation (Fig- ure S1 available online). Structural variability in this N-terminal RESULTS region has also been observed in the BTB domains of FAZF and Miz-1 (Stogios et al., 2010). Crystal Structure of a Dimeric SPOPBTB/Cul3NTD The Cul3NTD comprises a series of five-helix cullin repeats Complex producing an extended arc-shaped structure (Figure 1), similar SPOP is a 374 residue protein that possesses a centrally located to what has been seen in Cul1 (Zheng et al., 2002), Cul4a (Angers BTB domain that interacts with Cul3NTD. To examine the details et al., 2006) and Cul5 (PDB ID 2WZK) (Figure S2). Each Cul3NTD of the BTB/Cul3 assembly, we determined the crystal structure chain associates with a surface that is distal to the BTB/BTB of SPOPBTB in complex with Cul3NTD to a resolution of 2.4 A˚ dimer interface in the heterotetrameric SPOPBTB/Cul3NTD (Figure 1; Table 1). complex (Figure 1B; Figure S1). There are no Cul3/Cul3

NTD Table 1. SPOPBTB/Cul3NTD Data Collection and Reﬁnement Cul3 interface. Additional contributions to the SPOP/Cul3 Statistics complex are made through residues in the BTB a5 and a6 helical Data Collection hairpin and residues in the BACK domain, as discussed in the following section. Space group C2 The modular CRLs employ a single cullin protein to engage Cell dimensions multiple substrate proteins though a combinatorial array of a b c ˚ , , (A) 213.16, 76.94, 85.83 adaptor proteins. Thus, Cul1 uses Skp1 to engage multiple F- a, b, g ( ) 90, 108.34, 90 box substrate-binding proteins, while Cul2 interacts with Elon- Resolution (A˚ ) 19.6-2.40 (2.49–2.40) ginC to form complexes with a large number of SOCS box

Rsym 0.052 (0.495) proteins (Pintard et al., 2004). In contrast, a diverse set of BTB I / sI 20.4 (2.2) domain adaptor proteins link directly to both Cul3 and to Completeness (%) 95.6 (74.0) substrates with a single polypeptide chain (Furukawa et al., 2003; Geyer et al., 2003; Pintard et al., 2003; Xu et al., 2003). Redundancy 2.9 This shifts the main combinatorial site from the Skp1/F-box Reﬁnement and Elongin C/SOCS box interfaces in CRL1s and CRL2s to ˚ Resolution (A) 19.6–2.40 the Cul3/BTB interface in CRL3s. It is not clear how multiple No. reﬂections 49,397 BTB domain proteins bind to the same surface of Cul3 given

Rwork / Rfree 0.211 / 0.263 the high divergence of BTB domain sequences. We identified No. atoms a Cul3-binding signature by constructing a multiple sequence Protein 6755 (four chains) alignment of the BTB domains of SPOP and four additional Water 46 bona fide Cul3 adaptor proteins (Keap1, KLHL1, KLHL20 and RhoBTB3) (Figure 2B). Conserved features in the a3-b4 loop B-factors (A˚ 2) emerged as the most significant element in the Cul3 binding Protein 86.5 BTB domains. The a3-b4 loops of Cul3 adaptors are 10 residues Water 69.0 long and possess a core f-x-E motif, where f represents Rmsd a hydrophobic residue (most often an aliphatic Met or Leu Bond lengths (A˚ ) 0.013 residue), x represents a non-conserved residue, and E repre- Bond angles () 1.45 sents a glutamate residue. The majority of known Cul3 adaptor Values in parentheses are for the highest resolution shell. proteins belong to BTB-BACK-Kelch proteins of the KLHL and KBTBD families, and an expanded multiple sequence alignment shows strong conservation of both loop length and interactions, as expected. The active complex formed by full- the f-x-E motif in the BTB domains of 37 of the 47 human length Cul3 includes the RING protein Rbx1 and an E2 (Fig- KLHL/KBTBD proteins (Figure S3). In contrast, the a3-b4 loop ure 1C). To examine the architecture of the active assembly, is highly divergent in length and sequence in all of the 42 human we used the SPOPBTB/Cul3NTD crystal structure as a template ZBTB (BTB-zinc finger) proteins (Figure S3), including PLZF and the similarity between the first cullin repeats of Cul3 and (Figure 2B). Cul1, which have a Ca rmsd of 1.4 A˚ (Figure S2B), to construct The f-x-E motif in SPOP corresponds to residues M233, E234, a model of a CRL based around a dimeric BTB domain (Fig- and E235. Within the SPOP BTB domain, M233 makes the single ure 1D). This model generates a suprafacial architecture in which largest contribution to the SPOPBTB/Cul3NTD interaction, burying both substrates and E2 ubiquitin-conjugating enzymes are posi- 112 A˚ 2 of accessible surface area. Consistent with this observa- tioned on the same face of the CRL. This architecture supports tion, we measured a Kd of 1.0 ± 0.1 mM between wild-type previous models for a dimeric CRL3 (Stogios et al., 2005; Zhuang SPOPBTB and Cul3NTD by isothermal titration calorimetry (ITC), et al., 2009), and a similar suprafacial organization has been but a SPOPBTB M233E mutant disrupted Cul3NTD binding (Fig- proposed in the dimerization of a CRL1Cdc4 complex driven by ure 2C). A surface representation of the interface shows the the unrelated D-domain of the F-box protein Cdc4 (Tang et al., M233 side chain buried in a hydrophobic cavity between the 2007). This suprafacial architecture differs, however, from the H1-H2 loop and helix H5 of Cul3 (Figure 3G). Crystal structures antiparallel, ‘‘crossed-dimer’’ architecture generated through of the uncomplexed Cul3 binders SPOP, gigaxonin and Fbx4 mediated dimerization of CRL1Fbx4 (Li and Hao, 2010). KLHL11 have weak electron density in the BTB a3-b4 loop region, indicating that a disordered-to-ordered transition of this SPOPBTB/Cul3NTD Interaction Surface loop occurs upon Cul3 binding. Each SPOPBTB/Cul3NTD interface buries 825 A2 of surface area Next, we considered the basis for cullin selection by BTB involving non-contiguous regions from both proteins. The Cul3 proteins. The only other reported BTB/cullin structure is a interaction surface within SPOPBTB involves the a3, b4, and a5 Skp1BTB/Cul1 complex (Zheng et al., 2002). The overall path of secondary structural elements, while the SPOP interaction the BTB and cullin backbones are similar in the two complexes, surface of Cul3NTD involves helices H2 and H5 (Figures 2 and and in general, equivalent BTB and cullin side chains are found 3). The largest SPOPBTB contribution to the Cul3NTD interface at the interface (Figure 3). The main differences are in the resides within the a3-b4 loop of the SPOP BTB domain (Figures SPOP and Skp1 a3-b4 loops (Figure 3) and in the orientation of 2B and 3A). Together, the ten residues of the a3-b4 loop bury 341 an equivalent side chain in the two cullins, Y42 in Cul1 and F54 A2 of surface area and make up 42% of the total SPOPBTB/ in Cul3 (Figures 3G and 3H). As a result of the different rotamers

Figure 2. The a3-b4 Loop Makes a Signiﬁ- cant Contribution to the Interface of the BTB/Cul3 Complex (A) Close-up view of the SPOP/Cul3 interface. Cul3NTD is blue; SPOP BTB domain is red. The a3- b4 loop is indicated by the dashed rectangle. (B) Multiple sequence alignment from various BTB domains: SPOP, four Cul3 binding partners (Keap1, KLHL1, KLHL20, and RhoBTB3), and the BTB zinc-ﬁnger protein PLZF. Highly conserved and similar residues are highlighted in red. The upper bar graph shows the contribution of each SPOP BTB residue to the interface with Cul3 as a percentage of the total buried surface area. The position of the a3-b4 loop is indicated by the dashed rectangle. (C) ITC data of the interaction between Cul3NTD and wild-type or M233E mutant SPOPBTB. See also Figure S3.

gigaxonin (PDB 3HVE) show that the BACK domain consists of 3–4 repeats of a paired helix motif. In these proteins, the 3-box corresponds to the first paired at this position, the Cul1 surface is relatively flat at the Skp1 helix repeat within the larger, structured BACK domain. In addi- interface, while the Cul3 surface forms a deep pocket that tion, the last two helices of the BTB domains of SPOP and the accommodates the SPOP M233 side chain. Notably, we observe known KLHL/KBTBD structures form an additional paired helix the absence of the f-x-E motif in both Skp1 and Elongin C that is contiguous with the paired helix repeats from the BACK (Figure 3A). domain (Zhuang et al., 2009). Structure predictions of the C-terminal region of SPOP (resi- The 3-Box Exists as a Motif within the Larger BACK dues 302–374) using HHpred (So¨ ding et al., 2005) and I-TASSER Domain (Roy et al., 2010) identified the BACK domains from the proteins The BTB domain is a requisite element for recruitment of KBTBD4, KLHL11, and gigaxonin as the top-scoring templates substrate adaptors to cullin scaffolds, with the notable excep- from PDB. The I-TASSER model generated from the restrained tions of Cul4a and Cul4b that interact with a b-propeller domain refinement of the threading-aligned regions extends to the C of the protein DDB1 (Angers et al., 2006). However, an additional terminus of the protein (Figure 4A). In this model, the region motif has been identified C-terminal to the BTB domain that following the BTB domain consists of two repeats of a helical extends the cullin-binding surface and enhances the affinity of hairpin motif. The first of these repeats corresponds to the the complex. A paired helix motif termed the 3-box has been 3-box motif and agrees with the SPOPBTB-3box structure (Zhuang described in a number of BTB proteins that, in a role analogous et al., 2009), while the second hairpin motif spans residues to that of the F-box and SOCS box, extends the interaction 330–359. Residues 360–374 contain a consensus nuclear local- surface of the BTB protein with Cul3 (Zhuang et al., 2009). In ization signal (NLS), which modeled as a short a-helical segment. agreement with that study, we found that extending the Crystal structures of NLS regions of CLIC4 show a-helical constructs of SPOP and the BBK proteins KLHL1 and KLHL20 structures that adopt extended structures in complex with to include additional C-terminal residues beyond the BTB importin-a (Mynott et al., 2011). Overall, the results of the domain significantly enhanced the robustness and affinity of secondary structure prediction and modeling indicate that the their interactions with Cul3NTD (Figures S4A and S5C; Zhuang SPOP302-359 region is structured and contiguous with the BTB et al., 2009). domain and consists of an atypical BACK domain. The KLHL/KBTBD proteins constitute the largest recognized Because we were only successful obtaining crystals of the family of Cul3 adaptors in humans (Figure S3; Stogios et al., SPOP BTB domain in complex with Cul3NTD, we could not 2005). These proteins share a common structural organization directly assess the structural contribution of the minimal 3-box consisting of an N-terminal BTB domain, immediately followed or the larger BACK domain to the Cul3 interaction. To gain further by an all a-helical BACK domain of 130 residues (Stogios and insight into the expanded interface and the structure of a Prive´ , 2004) and a C-terminal Kelch repeat domain. In SPOP, BTB-BACK/Cul3 complex, we expanded our SPOPBTB/Cul3NTD the substrate-binding module is a MATH domain that is structure to include the modeled C-terminal region of SPOP. N-terminal to the BTB domain, while in the KLHL/KBTBD This indicated that the consecutive BTB a5/a6 and BACK/ proteins, the Kelch repeat substrate-binding domain follows 3-box helical hairpins make contacts with the H2-H3 loop of the BTB-BACK region. Structures of the BACK domain from Cul3 (Figure S4B). In total, we predict that the inclusion of the the proteins KBTBD4 (PDB 2EQX), KLHL11 (PDB 3I3N) and BACK domain extends the SPOP/Cul3 interface by 215 A˚ 2.

The 3-box helical hairpin contributes to 165 A˚ 2 of the expanded affected, we generated a SPOP construct containing the interface, with the additional 50 A˚ 2 from the second helical MATH, BTB, and 3-box motif (SPOPMATH-BTB-3-box). The trunca- hairpin. In total, the SPOPBTB-BACK/Cul3 interface buries tion of the C-terminal half of the BACK domain does not affect 1040 A˚ 2 of surface area. Cul3 binding, because Zhuang et al. (2009) report a Kd of 17 nM for the interaction between a Cul3 N-terminal fragment The BACK Domain Promotes High-Order SPOP and an SPOP BTB-3-box construct (residues 172-329), and we Self-Assembly and Enhances E3 Activity measured a comparable Kd for the interaction between Cul3NTD Self-assembly is a feature common of many E3s, and a number and SPOPBTB-BACK (Figure S5C). The E3 ubiquitin ligase activity of domains have been identified that are capable of mediating mediated by SPOPMATH-BTB-BACK was significantly greater than the oligomerization of the complexes. SPOPBTB exists as a that of SPOPMATH-BTB-3-box (Figure 4F), indicating that the large homodimeric species in the SPOPBTB/Cul3NTD crystal structure SPOP oligomers were more active than the dimers. Analytical and in solution as measured by analytical size exclusion chroma- SEC experiments confirmed that the SPOP oligomers were tography and sedimentation equilibrium experiments, in agree- competent to associate with Cul3 (Figure S4C). ment with previous reports (Zhuang et al., 2009). We observed, however, that longer constructs of SPOP that included both An 18 Amino-Acid BACK Element Limits the Self- the BTB and BACK domains (SPOPBTB-BACK) generated oligo- Assembly and Activity of a SPOP Paralog mers with apparent molecular weights significantly larger than The only other MATH-BTB protein in humans is SPOPL, an un- that of the dimer. The SPOPBTB-3box construct reported earlier characterized gene product that shares an overall 85% did not form associations larger then dimers (Zhuang et al., sequence identity with SPOP. SPOPL is widely expressed in 2009). Analytical size exclusion chromatography (SEC) of human tissues and is highly conserved among vertebrate SPOPBTB-BACK resulted in a broad, tailing elution profile, indi- species. The high degree of similarity with SPOP indicates that cating a heterogeneous population of high molecular weight SPOPL also functions as a CRL3 adaptor protein. The two oligomers (Figure 4B). In addition, sedimentation equilibrium proteins are expected to have identical substrate specificities, data indicated a population-weighted average of 450 kDa for given that the SPOP and SPOPL MATH domains share 95% SPOPBTB-BACK and 25 kDa for SPOPBTB at concentrations of sequence identity with full conservation of substrate-binding 25 mM when fit to single-species models (Figure 4C). The plot residues (Figure 5A). We confirmed that SPOP and SPOPL of ln(concentration) versus the square of the radius was strongly have similar affinities for both Cul3NTD and the SPOP substrate curved, providing additional evidence for the existence of a macroH2A by ITC (Figure S5). heterogeneous self-assembly. Consistent with this result, nega- The most striking difference between SPOP and SPOPL is an tive stain electron microscopy revealed that SPOPBTB-BACK 18 amino-acid element in SPOPL that is encoded by a unique exists as a heterogeneous population of assemblies ranging exon within the BACK domain coding sequence (Figure 5A). from 5 to 20 nm in diameter (Figure 4D). We generated a model of the SPOPL BACK domain using The SPOPBTB-BACK SEC elution profiles could be shifted to I-TASSER, and the most consistent model positioned the higher and lower molecular assemblies in a concentration depen- SPOPL element between the first and second a-helical hairpins dent manner. This behavior was not observed for SPOPBTB, of the BACK domain (Figures S6A and S6B). Within the context of which maintained a dimeric state across all examined concentra- the Cul3 complex, the SPOPL element is positioned distal to tions (Figure 4E). The absence of monomeric species could be the Cul3 binding surface, consistent with the similar Cul3 accounted for by the very low dimer dissociation constant that binding affinities of SPOP and SPOPL (Figures S6C and S6D). has been observed for other BTB domains (Stogios et al., SPOPLBTB-BACK was dimeric in solution at concentrations up to 2010). Taken together, these data indicate that the BACK domain 2.0 mM by SEC, and is devoid of the higher-order assemblies can promote formation of large (>500 kDa), multimeric (>25mers), observed with SPOP. Deletion of the element within dynamic, and dissociable SPOP assemblies in solution. SPOPLBTB-BACK resulted in the formation of oligomers, similar To gain insight into how the C-terminal region produces large to those observed with SPOP (Figure 5B), indicating that this SPOP assemblies, we next examined the self-association element disrupts the oligomerization surface found in the properties of the SPOP BACK domain. We introduced four SPOP BACK domain. Finally, SPOPL was significantly less dimer-disrupting point mutations into the SPOP BTB domain robust than SPOP in its E3 ligase activity, and deletion of the (Zhuang et al., 2009) in order to isolate the BACK domain from BACK element increased SPOPL-mediated ubiquitylation to the effects of BTB-mediated dimerization (SPOPBTB-BACK-Ddimer). a level comparable with SPOP (Figure 5C). Overall, these results SPOPBTB-BACK-Ddimer had an elution profile consistent with show that the conserved 18 amino-acid element within the a tetrameric species by SEC, and sedimentation equilibrium SPOPL BACK domain disrupts higher-order assembly and forms data with SPOPBTB-BACK-Ddimer were best described by a mono- a less active CRL3 complex. mer-pentamer equilibrium model (Figure S4D–S4F). Under identical conditions, a SPOPLBTB-BACK-Ddimer construct was SPOPL Is Capable of Attenuating SPOP Self-Assembly monomeric by SEC. Together, these data indicate that the and E3 Ubiquitin Ligase Activity SPOP BACK domain can form homo-oligomers independent of The high degree of SPOP/SPOPL sequence identity led us next BTB homo-dimerization. to address whether these two proteins can interact with one We next examined the effect of the BACK domain oligomeriza- another. SPOPBTB-BACK and SPOPLBTB-BACK heterocomplexes tion on the E3 ubiquitin ligase activity of SPOP-Cul3 toward were observed by SEC experiments as indicated by a peak shift a model substrate. To ensure that Cul3 binding would not be in the elution profile. However, no interaction was observed

Figure 3. Comparison of the BTB/Cullin Interfaces of the SPOP/Cul3 and Skp1/Cul1 Crystal Structures and an EloC/Cul5 Model (A) Structure-based sequence alignment of the cullin-binding regions of the SPOP, EloC, and Skp1 BTB domains. An engineered deletion in the Skp1 a3-b4 loop and disorder within the EloC a3-b4 loop are indicated by lowercase sequence. The residue-based surface areas of SPOP and Skp1 buried by Cul3 and Cul1, respectively, are shown as bar graphs. The a3-b4 loops of SPOP and Skp1 are red and green, respectively. The square brackets denote the omission of a noninteracting, intervening region of sequence. (B) Structure-based sequence alignment of the BTB-binding regions of Cul3, Cul5, and Cul1. The residue-based surface areas of Cul3 and Cul1 buried by SPOP and Skp1, respectively, are shown as bar graphs.

between SPOPBTB-BACK and SPOPLBTB-BACK-Ddimer, indicating interface and abolish binding (Xu et al., 2003). However, four of that SPOP and SPOPL interact via BTB-BTB heterodimerization these (243, 245, 283 and 285) correspond to buried residues (Figure 6A). Next, we examined the effect of SPOPL on SPOP and are expected to disrupt the BTB fold. In addition to self-assembly. By varying the SPOP:SPOPL molar ratio, we engineered changes, disease mutations have been found in generated a continuum of species ranging in size from the Cul3-binding BTB proteins; an L95F mutation in KLHL9 high-order SPOP oligomers to the low-molecular weight SPOPL (corresponding to SPOP position I243) is associated with an in- dimer (Figure 6B). We also observed that SPOPL was capable of herited form of distal myopathy (Cirak et al., 2010), and KLHL3 driving SPOP into a narrow distribution of lower molecular weight mutations A77E, M78V, and E85A (corresponding to SPOP species. Furthermore, ubiquitylation assays performed on the A227 and M228 in a3, and E235 in the f-x-E motif) cause hyper- heteromeric samples demonstrated that shifting the size of the tension and electrolyte abnormalities (Boyden et al., 2012). An oligomers by increasing the proportion of SPOPL correlated additional KLHL3 mutation, C164F (SPOP A314), corresponds with a decrease in the E3 ubiquitin ligase activity (Figure 6C). to a buried residue in the 3-box and may affect Cul3 binding Together these results indicate that SPOPL is capable of inhi- by perturbing the BACK domain structure. In terms of Cul3, biting SPOP self-assembly and E3 ubiquitin ligase activity in a binding to BTB protein is lost with helix H2 mutations S53A/ ratiometric and dose-dependent manner. F54A (Pintard et al., 2003), Y58G (Geyer et al., 2003), or L52A/ E55A (Xu et al., 2003), or H5 helix mutation Y125A/R128A DISCUSSION (Xu et al., 2003). All of these mutations involve residues that make major contributions to the SPOP/Cul3 interaction surface Within the BTB-BACK-Kelch and MATH-BTB families, the BTB (Figures 2 and 3). a3/b4 loop, the a5/a6 hairpin helices and the 3-box motif with The RING superfamily consists of many hundreds of E3s that the BACK domain determine specificity for Cul3. Thus, the pres- differ greatly in size, structure, subunit stoichiometry, and mode ence of the f-x-E motif and the BACK signature within a BTB of regulation (reviewed in Deshaies and Joazeiro, 2009). protein should be taken as strong indication of CRL3 adaptor Common within the RING superfamily, however, is a remarkable protein function. Other modes of BTB recognition in SCF3 propensity to self-assemble into oligomers. To accomplish this complexes appear to be possible, however, since several feat, E3s have co-opted various oligomeric domains into their members of the more distantly related KCTD family of BTB multidomain architectures. RING, UBA, TRAF, and BTB domain proteins also bind Cul3 (Bayo´ n et al., 2008; Canettieri domains, for example, can yield a variety of oligomeric states, et al., 2010; De Smaele et al., 2011) yet lack these determinants ranging from discrete dimeric species to high-order assemblies (Dementieva et al., 2009). In addition, alternative architectures of (Kentsis et al., 2002; McMahon et al., 2006; Park et al., 1999; Pe- BTB/Cul3 assembly may also occur. For example, Btbd6a has schard et al., 2007). Disruption of self-assembly through domain been shown as a common binding partner to both Cul3 and deletion or site-directed mutagenesis significantly reduces the the ZBTB protein PLZF (Sobieszczuk et al., 2010). E3 ligase activity and the length of the resulting polyubiquitin Interestingly, ‘‘loop 3’’ in Skp1 and Elongin C has been shown chains on the target protein (Poyurovsky et al., 2007; Tang to be an important determinant guiding Cul1 versus Cul2 selec- et al., 2007). Detailed insights into discrete oligomeric species tivity (Yan et al., 2004). This loop is at an equivalent position to have been provided through crystal structures of such the a3/b4 motif that we describe here in SPOPBTB, but each complexes as the UBA domain dimerization of c-Cbl (Peschard has its own unique sequence signature. Specifically, swapping et al., 2007) and the D-domain dimerization of SCFCdc4 (Tang this loop between Skp1 and ElonginC was capable of promoting et al., 2007), and a proposed tetrameric KCTD/Cul3 complex non-native Skp1/Cul2 and EloC/Cul1 complexes. This loop is (Correale et al., 2011). often poorly structured in uncomplexed forms of BTB domains, Fewer details, however, are known of the mechanisms that indicating that signature elements in this region become more govern high-order E3 assemblies such as those formed by ordered upon forming interaction interfaces with their respective Mdm2, BRCA1 and numerous other RING proteins. In this cullins. work, we demonstrate that SPOP possesses two types of Mutations in interface regions have been shown to disrupt oligomerization domains, the BTB domain and the BACK BTB/Cul3 assemblies. Mutations in the Cul3 adaptor protein domain, and that these function in tandem to generate large Mel26 at residues M243A/I245A/D247A and D281A/Y283A/ dynamic assemblies that rapidly associate and dissociate in L285A (corresponding to SPOP residues V241/I243/D245 and a concentration dependent manner. We propose that a network D278/Y280/L282, respectively) are in the vicinity of the Cul3 of homomeric interactions creates a malleable catalytic surface

(C) Detailed view of the SPOPBTB/Cul3 interface. The interacting side chains of SPOP and Cul3 are red and blue, respectively. Secondary structural elements are labeled in white. (D) Detailed view of a homology model of the EloC/Cul5 interface based on the PDB structures 2C9W (Bullock et al., 2006) and 2WZK. The interacting side chains of EloC and Cul5 are gray and blue, respectively. Secondary structural elements are labeled in white. (E) Detailed view of the Skp1/Cul1 interface (PDB 1LDK). The interacting side chains of Skp1 and Cul1 are green and blue, respectively. Secondary structural elements are labeled in white. (F) Structural superposition of the SPOP and Skp1 (PDB 1LDK) BTB domains. The a3-b4 loops of the SPOP and Skp1 BTB domains are red and green, respectively. (G) The Cul3 surface representation is blue. Residues within the a3-b4 loops of the SPOP BTB domain are red. (H) The Cul1 surface representation is blue. Residues within the a3-b4 loops of the Skp1 BTB domain are green.

Figure 4. Dynamic SPOP Self-Assembly Is Mediated by Tandem Oligomeric BTB and BACK Domains (A) Predicted 3D structure of the SPOP BACK domain. The BTB domain is red, the first helical hairpin of the BACK domain corresponding to the 3-box is green. The second BACK helical hairpin is gray. The NLS is blue. (B) The BACK domain creates a large increase in molecular size and heterogeneity of SPOP. SEC profiles of SPOPBTB and SPOPBTB-BACK at protein concentrations of 40 mM are shown, along with a corresponding Coomassie-stained SDS-PAGE (inset). (C) Sedimentation equilibrium data of 25 mM samples of SPOPBTB-BACK and SPOPBTB collected at 8,000 rpm and 20,000 rpm, respectively, and fit to a single species model. (D) Negative stain electron micrographs of SPOPBTB-BACK. The four panels are views from four different regions of the grid. The scale bar represents 30 nm. BTB-BACK (E) SEC profiles of SPOP at protein concentrations of 10 (dark blue), 20, 40, 60, 120 and 200 mM (orange). The void volume (vo) and molecular mass markers in kDa are indicated with arrows. Inset: SEC profiles of SPOPBTB at protein concentrations of 10, 20, 30, 50 and 100 mM. 1-391 MATH-BTB-BACK (F) Western blots detecting the ubiquitylation of ThxHis6-HA-Puc that is mediated by SPOP including (SPOP ) and excluding (SPOPMATH-BTB-3box) of the full-length BACK domain. See also Figure S4. with considerable dynamic range that is subject to regulation by model for CRL3Keap1, BTB-mediated dimerization of Keap1 both the thermodynamic properties of the system and by cellular leads to the rigid, bivalent tethering of the substrate Nrf2, factors. positioning it for optimal E2-mediated ubiquitin conjugation Specific models have been proposed to explain how E3 self- (McMahon et al., 2006). Additionally, in a report of CRL1Fbx4 assembly might lead to activation of the complex. In a proposed (Li and Hao, 2010), evidence is provided in which dimerization

Figure 5. SPOPL Harbors a Unique Element that Impedes BACK-Mediated Assembly and E3 Ligase Activity (A) Amino acid sequence alignment of SPOP and SPOPL. Identical residues in the MATH domain are colored in purple; the BTB domain in red; and the BACK domain in green. The positions of the 3-box and SPOPL insert are indicated. (B) Overlay of size exclusion chromatography proﬁles for SPOPBTB-BACK (colored in blue), SPOPLBTB-BACK (colored in red) and SPOPLBTB-BACK/Dinsert (colored in black). Molecular mass markers (listed in kDa) are indicated with arrows at the top of the x axis. Inset: SDS-PAGE analysis of the same three proteins. 1-391 MATH-BTB-BACK MATH-BTB-BACK (C) Western blots detecting the ubiquitylation of ThxHis6-HA-Puc that is mediated by SPOP , SPOPL , and SPOPLMATH-BTB-BACK/Dinsert. See also Figure S5.

Figure 6. SPOP/SPOPL: A Molecular Rheostat Capable of Fine-Tuning E3 Ubiquitin Ligase Activity (A) Overlay of analytical SEC profiles of SPOPBTB-BACK alone (solid line) or pre-incubated with an equal molar ratio of SPOPLBTB-BACK (dash-dot line) or SPOPLBTB-BACK Ddimer (dotted line). The double-headed arrow indicates a peak shift between traces indicating a positive interaction with dimer-competent SPOP and SPOPL. Molecular weight markers are indicated by arrows at the top of the x axis. Asterisks denote the elution position of the uncomplexed SPOPLBTB-BACK and SPOPLBTB-BACK Ddimer. (B) A continuum of oligomeric populations are accessible through varied SPOP/SPOPL heteromerization. Overlay of size exclusion chromatography profiles of SPOP:SPOPL (i–vii) pre-incubated at the indicated relative molar ratios. Molecular mass markers in kDa are indicated with arrows. 1-391 (C) Western blots detecting the ubiquitylation of ThxHis6-HA-Puc mediated by each of the heteromeric SPOP/SPOPL assemblies (i–vii) from (B). SDS-PAGE analysis of the SPOP/SPOPL input of each ubiquitylation reaction is shown in the lower panel. The asterisks denote a contaminating degradation product. Molecular mass markers in kDa are indicated. See also Figure S6. of the F-box protein Fbx4 induces conformational changes that Moreover, kinetic analyses of CRL-mediated polyubiquitylation promote CRL assembly and E3 ligase activity. have revealed that due to a high rate of substrate-E3 dissocia- While it is possible and perhaps likely that each of the varied tion, only 10% of initial substrate-E3 encounters result in and numerous instances of E3 oligomerization is accompanied covalent attachment of a ubiquitin moiety to the substrate by a type-specific mechanism for activation, such as those (Pierce et al., 2009). Thus, reducing the off-rate of the substrate proposed above, we hypothesize that a general mechanism from the E3 through high-avidity, multivalent interactions would may additionally underlie the activating properties of E3 self- enhance the rate-limiting step of polyubiquitylation. assembly. We speculate that E3 self-assembly enhances the Additional mechanisms of self-assembly-mediated activity apparent catalytic efficiency toward a locally bound substrate. enhancement specific to CRLs are possible. Not only would Two parallel mechanisms may be involved. self-assembly serve to recruit high effective concentrations of First, as has been suggested in a study of SCFFbw7, self- the core CRL subunits, but also associated regulatory machinery assembly serves to enhance substrate-E3 avidity through the would similarly be affected. For example, the enzyme-catalyzed presentation of multiple substrate-binding domains (Hao et al., conjugation of the ubiquitin-like protein Nedd8 (a process 2007). Reciprocally, substrate-E3 avidity would be further termed neddylation) to an acceptor lysine near the cullin C enhanced by multiple E3 binding motifs within a single substrate, terminus enhances CRL E3 ligase activity (Deshaies et al., a property especially common among the numerous SPOP 2010; Duda et al., 2011). Thus, higher oligomeric forms of substrates (Zhang et al., 2009; Zhuang et al., 2009). Indeed, a CRL may be capable of supporting faster rates of neddylation. cooperative multivalent interactions are important for SPOP- Though Nedd8 modification was not included in this report, it mediated repression of Gli (Zhang et al., 2009) and for threshold has been previously observed that UbcH5b mediated ubiquityla- effects in the regulation of phosphodegron-containing tion of the Nedd8 acceptor site can mimic the effects of neddy- substrates (Nash et al., 2001; Tang et al., 2012). lation (Duda et al., 2008). Second, we propose that E3 self-assembly enhances the Self-assembly is an attractive property to dynamically regu- catalytic rate of ubiquitylation by increasing the effective late E3 activity. Two recent reports have identified roles for concentration of the E2 conjugating enzyme. These parallel phosphorylation in controlling the oligomerization and activity properties would serve to retain dynamically associated of SCFFbx4 (Barbash et al., 2008) and Mdm2 (Cheng et al., substrates in close proximity to hyper-catalytic concentrations 2009). Our examination of a closely related and functionally un- of E2 ubiquitin-conjugating enzymes, thus increasing both the characterized paralog of SPOP indicates an additional means rate and processivity of polyubiquitin chain formation. In support of controlling E3 self-assembly and activity. We demonstrate of this model, it was demonstrated that reinforcing the associa- that SPOPL is capable of associating with SPOP via BTB- tion of the substrate protein Sic1 could mitigate the lost activity BTB heterodimer formation where it functions to restrict self- incurred by disrupting the SCFcdc4 dimer (Tang et al., 2007). assembly through its possession of an inhibitory structural

Figure 7. Proposed Model for Oligomer-Mediated Activity Enhancement and Rheostatic Control of SPOP (A) Oligomerization enhances the apparent catalytic efficiency of an E3 ubiquitin ligase through two general mechanisms. (1) Enhanced substrate avidity through presentation of multiple binding domains. (2) Significant increase in the effective concentration of the E2 ubiquitin-conjugating enzyme. (B) Ratiometric incorporation of SPOPL is capable of restricting SPOP self-assembly and modulating the resulting E3 ubiquitin ligase activity. element within its BACK domain. Thus, the SPOP/SPOPL EXPERIMENTAL PROCEDURES system is able to fine-tune substrate ubiquitylation via the ratiometric incorporation of the SPOPL negative regulator. Such Protein Expression and Purification The BTB proteins were expressed from a modified pET32a vector (Novagen) a mechanism would allow an E3 to effect subtle perturbations encoding an N-terminal thioredoxin carrier protein, a 6xHis tag and a thrombin in the proteolytic half-life of cellular proteins (Figure 7). More- cleavage site. The plasmid constructs were transformed into Escherichia over, because ubiquitylation is capable of mediating non- coli BL21 cells and protein expression was induced with 1 mM IPTG followed proteolytic fates, it is possible that SPOPL incorporation may by overnight shaking and incubation at 15C. Proteins were purified by Ni-NTA also serve to shift the reaction toward monoubiquitylation. affinity and SEC. Where indicated, thioredoxin-His6 tags were removed by This would allow substrates to escape proteasomal degrada- thrombin cleavage followed by an additional round of Ni-NTA affinity and SEC. Residue ranges in the various SPOP and SPOPL constructs were tion and possibly be directed toward non-proteolytic fates SPOPBTB, 177-301; SPOPBTB-3-box, 177-329; SPOPBTB-BACK, 177-359, such as subcellular relocalization. Mdm2, for example, through SPOPMATH-BTB-3-box, 28-329; SPOPMATH-BTB-BACK, 28-359; SPOPLBTB-BACK, a concentration dependent mechanism, is capable of directing 177-377; and SPOPLMATH-BTB-BACK, 28-377. Cul3NTD (residues 20-381) incor- p53 to either the proteasome or for nuclear export (Li et al., porating the stabilizing mutations I342R/L346D were generated based on the 2003). Interestingly, SPOP displays similar multi-functionality. ‘‘Split-n-Coexpress’’ method (Li et al., 2005). Cul3NTD was subcloned into While SPOP-mediated ubiquitylation of SRC-3 (Li et al., 2011) either the modified pET32a or pGEX-4T1 vectors and was expressed and and Daxx (Kwon et al., 2006) target these proteins for proteol- purified as described above. ysis, SPOP-mediated monoubiquitylation of mH2A is necessary Crystallization and Structure Determination for its recruitment to the inactive X chromosome (Herna´ ndez- Data were collected on a crystal of SPOP residues 177-319 in complex with Mun˜ oz et al., 2005). In conclusion, we propose a general model selenomethionine-substituted Cul3NTD. Full details are described in the in which self-assembly increases the effective catalytic effi- Supplemental Experimental Procedures. ciency of the E3, and a mechanism by which the activity can Ubiquitylation Assays be attenuated through ratiometric incorporation of a self- Ubiquitylation assays were assembled in ligase buffer (50 mM Tris-Cl pH 7.5, assembly inhibitor. 150 mM NaCl, 10 mM MgCl2, 5 mM ATP, and 0.5 mM TCEP) in 25 ml volumes

containing 200 nM Uba1, 2 mM UbcH5b, 75 mM ubiquitin, 5 mM HA-Puc1-391, Boyden, L.M., Choi, M., Choate, K.A., Nelson-Williams, C.J., Farhi, A., Toka, and 2.5 mM of the CRL3SPOP/SPOPL. SPOPMATH-BTB-BACK,SPOPMATH-BTB-3box, H.R., Tikhonova, I.R., Bjornson, R., Mane, S.M., Colussi, G., et al. (2012). BTB-BACK 1-391 SPOPL , and HA-Puc were expressed as thioredoxin-His6 fusion Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte proteins in BL21 as described above. Reactions were stopped at the indicated abnormalities. Nature 482, 98–102. times by the addition of SDS loading dye. His6-Uba1 and GST-Cul3/Rbx1 were Bullock, A.N., Debreczeni, J.E., Edwards, A.M., Sundstro¨ m, M., and Knapp, S. expressed in the baculovirus-Sf9 cell system. Ubiquitin and UbcH5b were (2006). Crystal structure of the SOCS2-elongin C-elongin B complex deﬁnes purchased form BostonBiochem (Cambridge, MA). Titration experiments a prototypical SOCS box ubiquitin ligase. Proc. Natl. Acad. Sci. USA 103, were performed by pre-incubating the indicated ratios of SPOP and SPOPL 7637–7642. for 90 minutes at 37C followed by fractionation by analytical SEC. Peak Bunce, M.W., Boronenkov, I.V., and Anderson, R.A. (2008). Coordinated fractions were pooled, standardized for total protein concentration by activation of the nuclear ubiquitin ligase Cul3-SPOP by the generation of absorbance at 280 nm, and used in the standard ubiquitylation assay. The phosphatidylinositol 5-phosphate. J. Biol. Chem. 283, 8678–8686. ubiquitylation reactions were resolved by SDS-PAGE and visualized by western blot against the HA epitope of Puc1-391. Canettieri, G., Di Marcotullio, L., Greco, A., Coni, S., Antonucci, L., Infante, P., Pietrosanti, L., De Smaele, E., Ferretti, E., Miele, E., et al. (2010). Histone ACCESSION NUMBERS deacetylase and Cullin3-REN(KCTD11) ubiquitin ligase interplay regulates Hedgehog signalling through Gli acetylation. Nat. Cell Biol. 12, 132–142. Coordinates and structure factors for the reported crystal structure have been Cheng, Q., Chen, L., Li, Z., Lane, W.S., and Chen, J. (2009). ATM activates p53 deposited with the Protein Data Bank under accession code 4EOZ. by regulating MDM2 oligomerization and E3 processivity. EMBO J. 28, 3857– 3867. SUPPLEMENTAL INFORMATION Cirak, S., von Deimling, F., Sachdev, S., Errington, W.J., Herrmann, R., Bo¨ nnemann, C., Brockmann, K., Hinderlich, S., Lindner, T.H., Steinbrecher, Supplemental Information includes six ﬁgures and Supplemental Experi- A., et al. (2010). Kelch-like homologue 9 mutation is associated with an early mental Procedures and can be found with this article online at doi:10.1016/ onset autosomal dominant distal myopathy. Brain 133, 2123–2135. j.str.2012.04.009. Correale, S., Pirone, L., Di Marcotullio, L., De Smaele, E., Greco, A., Mazza` , D., Moretti, M., Alterio, V., Vitagliano, L., Di Gaetano, S., et al. (2011). Molecular ACKNOWLEDGMENTS organization of the cullin E3 ligase adaptor KCTD11. Biochimie 93, 715–724.

We thank Frank Sicheri, Steve Orlicky, Yang-Chieh Chou, and Cheryl Arrow- De Smaele, E., Di Marcotullio, L., Moretti, M., Pelloni, M., Occhione, M.A., smith for baculovirus expression reagents. Lu Chen and Doug Kuntz estab- Infante, P., Cucchi, D., Greco, A., Pietrosanti, L., Todorovic, J., et al. (2011). lished the baculovirus expression methods. We thank the Advanced Photon Identification and characterization of KCASH2 and KCASH3, 2 novel Cullin3 Source at the Argonne National Laboratory for access to the SBC-CAT 19 ID adaptors suppressing histone deacetylase and Hedgehog activity in medullo- 13 synchrotron facilities. Argonne is operated by UChicago Argonne, LLC, for blastoma. Neoplasia , 374–385. the U.S. Department of Energy, Office of Biological and Environmental Dementieva, I.S., Tereshko, V., McCrossan, Z.A., Solomaha, E., Araki, D., Xu, Research under contract DE-AC02-06CH11357. This research was funded C., Grigorieff, N., and Goldstein, S.A. (2009). Pentameric assembly of potas- in part by the Ontario Ministry of Health and Long Term Care. The views sium channel tetramerization domain-containing protein 5. J. Mol. Biol. 387, expressed do not necessarily reflect those of the OMOHLTC. W.J.E. was 175–191. supported by a fellowship from the Canadian Cancer Society. G.G.P. was Deshaies, R.J., and Joazeiro, C.A. (2009). RING domain E3 ubiquitin ligases. funded by grants from the CCSRI and the Samuel Waxman Cancer Research Annu. Rev. Biochem. 78, 399–434. Foundation. Deshaies, R.J., Emberley, E.D., and Saha, A. (2010). Control of cullin-ring ubiquitin ligase activity by nedd8. Subcell. Biochem. 54, 41–56. Received: February 10, 2012 Revised: April 16, 2012 Duda, D.M., Borg, L.A., Scott, D.C., Hunt, H.W., Hammel, M., and Schulman, Accepted: April 19, 2012 B.A. (2008). Structural insights into NEDD8 activation of cullin-RING ligases: Published online: May 24, 2012 conformational control of conjugation. Cell 134, 995–1006. Duda, D.M., Scott, D.C., Calabrese, M.F., Zimmerman, E.S., Zheng, N., and REFERENCES Schulman, B.A. (2011). Structural regulation of cullin-RING ubiquitin ligase complexes. Curr. Opin. Struct. Biol. 21, 257–264. Ahmad, K.F., Engel, C.K., and Prive´ , G.G. (1998). Crystal structure of the BTB Furukawa, M., He, Y.J., Borchers, C., and Xiong, Y. (2003). Targeting of protein domain from PLZF. Proc. Natl. Acad. Sci. USA 95, 12123–12128. ubiquitination by BTB-Cullin 3-Roc1 ubiquitin ligases. Nat. Cell Biol. 5, 1001– Ahmad, K.F., Melnick, A., Lax, S., Bouchard, D., Liu, J., Kiang, C.L., Mayer, S., 1007. Takahashi, S., Licht, J.D., and Prive´ , G.G. (2003). Mechanism of SMRT core- pressor recruitment by the BCL6 BTB domain. Mol. Cell 12, 1551–1564. Gallagher, E., Gao, M., Liu, Y.C., and Karin, M. (2006). Activation of the E3 ubiquitin ligase Itch through a phosphorylation-induced conformational Angers, S., Li, T., Yi, X., MacCoss, M.J., Moon, R.T., and Zheng, N. (2006). change. Proc. Natl. Acad. Sci. USA 103, 1717–1722. Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery. Nature 443, 590–593. Geyer, R., Wee, S., Anderson, S., Yates, J., and Wolf, D.A. (2003). BTB/POZ domain proteins are putative substrate adaptors for cullin 3 ubiquitin ligases. Barbash, O., Zamfirova, P., Lin, D.I., Chen, X., Yang, K., Nakagawa, H., Lu, F., Mol. Cell 12, 783–790. Rustgi, A.K., and Diehl, J.A. (2008). Mutations in Fbx4 inhibit dimerization of the SCF(Fbx4) ligase and contribute to cyclin D1 overexpression in human cancer. Hao, B., Oehlmann, S., Sowa, M.E., Harper, J.W., and Pavletich, N.P. (2007). Cancer Cell 14, 68–78. Structure of a Fbw7-Skp1-cyclin E complex: multisite-phosphorylated substrate recognition by SCF ubiquitin ligases. Mol. Cell 26, 131–143. Bayo´ n, Y., Trinidad, A.G., de la Puerta, M.L., Del Carmen Rodrı´guez, M., Bogetz, J., Rojas, A., De Pereda, J.M., Rahmouni, S., Williams, S., Herna´ ndez-Mun˜ oz, I., Lund, A.H., van der Stoop, P., Boutsma, E., Muijrers, I., Matsuzawa, S., et al. (2008). KCTD5, a putative substrate adaptor for cullin3 Verhoeven, E., Nusinow, D.A., Panning, B., Marahrens, Y., and van Lohuizen, ubiquitin ligases. FEBS J. 275, 3900–3910. M. (2005). Stable X chromosome inactivation involves the PRC1 Polycomb Berger, M.F., Lawrence, M.S., Demichelis, F., Drier, Y., Cibulskis, K., complex and requires histone MACROH2A1 and the CULLIN3/SPOP ubiquitin 102 Sivachenko, A.Y., Sboner, A., Esgueva, R., Pflueger, D., Sougnez, C., et al. E3 ligase. Proc. Natl. Acad. Sci. USA , 7635–7640. (2011). The genomic complexity of primary human prostate cancer. Nature Hershko, A., and Ciechanover, A. (1998). The ubiquitin system. Annu. Rev. 470, 214–220. Biochem. 67, 425–479.

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