The Cullin3 ubiquitin ligase functions as a Nedd8-bound heterodimer

Cullins are members of a family of scaffold proteins that assemble multisubunit ubiquitin ligase complexes to confer substrate specificity for the ubiquitination pathway. Cullin3 (Cul3) forms a catalytically inactive BTB-Cul3-Rbx1 (BCR) ubiquitin ligase, which becomes functional upon covalent attachment of the ubiquitin homologue neural-precursor-cell-expressed and developmentally down regulated 8 (Nedd8) near the C terminus of Cul3. Current models suggest that Nedd8 activates cullin complexes by providing a recognition site for a ubiquitin-conjugating enzyme. Based on the following evidence, we propose that Nedd8 activates the BCR ubiquitin ligase by mediating the dimerization of Cul3. First, Cul3 is found as a neddylated heterodimer bound to a BTB domain-containing protein in vivo. Second, the formation of a Cul3 heterodimer is mediated by a Nedd8 molecule, which covalently attaches itself to one Cul3 molecule and binds to the winged-helix B domain at the C terminus of the second Cul3 molecule. Third, complementation experiments revealed that coexpression of two distinct nonfunctional Cul3 mutants can rescue the ubiquitin ligase function of the BCR complex. Likewise, a substrate of the BCR complex binds heterodimeric Cul3, suggesting that the Cul3 complex is active as a dimer. These findings not only provide insight into the architecture of the active BCR complex but also suggest assembly as a regulatory mechanism for activation of all cullin-based ubiquitin ligases.     

Muf1, a novel Elongin BC-interacting leucine-rich repeat protein that can assemble with Cul5 and Rbx1 to reconstitute a ubiquitin ligase

The heterodimeric Elongin BC complex has been shown to interact in vitro and in mammalian cells with a conserved BC-box motif found in a growing number of proteins including RNA polymerase II elongation factor Elongin A, SOCS-box proteins, and the von Hippel-Lindau (VHL) tumor suppressor protein. Recently, the VHL-Elongin BC complex was found to interact with a module composed of Cullin family member Cul2 and RING-H2 finger protein Rbx1 to reconstitute a novel E3 ubiquitin ligase that activates ubiquitylation by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. In the context of the VHL ubiquitin ligase, Elongin BC functions as an adaptor that links the VHL protein to the Cul2/Rbx1 module, raising the possibility that the Elongin BC complex could function as an integral component of a larger family of E3 ubiquitin ligases by linking alternative BC-box proteins to Cullin/Rbx1 modules. In this report, we describe identification and purification from rat liver of a novel leucine-rich repeat-containing BC-box protein, MUF1, which we demonstrate is capable of assembling with a Cullin/Rbx1 module containing the Cullin family member Cul5 to reconstitute ubiquitin ligase activity. In addition, we show that the additional BC-box proteins Elongin A, SOCS1, and WSB1 are also capable of assembling with the Cul5/Rbx1 module to reconstitute potential ubiquitin ligases. Taken together, our findings identify MUF1 as a new member of the BC-box family of proteins, and they predict the existence of a larger family of Elongin BC-based E3 ubiquitin ligases.     

Ubiquitin ligase activity of Cul3-KLHL7 protein is attenuated by autosomal dominant retinitis pigmentosa causative mutation

Substrate-specific protein degradation mediated by the ubiquitin proteasome system (UPS) is crucial for the proper function of the cell. Proteins are specifically recognized and ubiquitinated by the ubiquitin ligases (E3s) and are then degraded by the proteasome. BTB proteins act as the substrate recognition subunit that recruits their cognate substrates to the Cullin 3-based multisubunit E3s. Recently, it was reported that missense mutations in KLHL7, a BTB-Kelch protein, are related to autosomal dominant retinitis pigmentosa (adRP). However, the involvement of KLHL7 in the UPS and the outcome of the adRP causative mutations were unknown. In this study, we show that KLHL7 forms a dimer, assembles with Cul3 through its BTB and BACK domains, and exerts E3 activity. Lys-48-linked but not Lys-63-linked polyubiquitin chain co-localized with KLHL7, which increased upon proteasome inhibition suggesting that KLHL7 mediates protein degradation via UPS. An adRP-causative missense mutation in the BACK domain of KLHL7 attenuated only the Cul3 interaction but not dimerization. Nevertheless, the incorporation of the mutant as a heterodimer in the Cul3-KLHL7 complex diminished the E3 ligase activity. Together, our results suggest that KLHL7 constitutes a Cul3-based E3 and that the disease-causing mutation inhibits ligase activity in a dominant negative manner, which may lead to the inappropriate accumulation of the substrates targeted for proteasomal degradation.     

Identification of Elongin C and Skp1 sequences that determine Cullin selection

The multiprotein von Hippel-Lindau (VHL) tumor suppressor and Skp1-Cul1-F-box protein (SCF) complexes belong to families of structurally related E3 ubiquitin ligases. In the VHL ubiquitin ligase, the VHL protein serves as the substrate recognition subunit, which is linked by the adaptor protein Elongin C to a heterodimeric Cul2/Rbx1 module that activates ubiquitylation of target proteins by the E2 ubiquitin-conjugating enzyme Ubc5. In SCF ubiquitin ligases, F-box proteins serve as substrate recognition subunits, which are linked by the Elongin C-like adaptor protein Skp1 to a Cul1/Rbx1 module that activates ubiquitylation of target proteins, in most cases by the E2 Cdc34. In this report, we investigate the functions of the Elongin C and Skp1 proteins in reconstitution of VHL and SCF ubiquitin ligases. We identify Elongin C and Skp1 structural elements responsible for selective interaction with their cognate Cullin/Rbx1 modules. In addition, using altered specificity Elongin C and F-box protein mutants, we investigate models for the mechanism underlying E2 selection by VHL and SCF ubiquitin ligases. Our findings provide evidence that E2 selection by VHL and SCF ubiquitin ligases is determined not solely by the Cullin/Rbx1 module, the target protein, or the integrity of the substrate recognition subunit but by yet to be elucidated features of these macromolecular complexes.     

Targeting of protein ubiquitination by BTB-Cullin 3-Roc1 ubiquitin ligases

The concentrations and functions of many cellular proteins are regulated by the ubiquitin pathway. Cullin family proteins bind with the RING-finger protein Roc1 to recruit the ubiquitin-conjugating enzyme (E2) to the ubiquitin ligase complex (E3). Cul1 and Cul7, but not other cullins, bind to an adaptor protein, Skp1. Cul1 associates with one of many F-box proteins through Skp1 to assemble various SCF-Roc1 E3 ligases that each selectively ubiquitinate one or more specific substrates. Here, we show that Cul3, but not other cullins, binds directly to multiple BTB domains through a conserved amino-terminal domain. In vitro, Cul3 promoted ubiquitination of Caenorhabditis elegans MEI-1, a katanin-like protein whose degradation requires the function of both Cul3 and BTB protein MEL-26. We suggest that in vivo there exists a potentially large number of BCR3 (BTB-Cul3-Roc1) E3 ubiquitin ligases.     

Neddylation-induced conformational control regulates cullin RING ligase activity in vivo

Cullin RING ligases (CRLs) constitute the largest family of ubiquitin ligases with diverse cellular functions. Conjugation of the ubiquitin-like molecule Nedd8 to a conserved lysine residue on the cullin scaffold is essential for the activity of CRLs. Using structural studies and in vitro assays, it has been demonstrated that neddylation stimulates CRL activity through conformational rearrangement of the cullin C-terminal winged-helix B domain and Rbx1 RING subdomain from a closed architecture to an open and dynamic structure, thus promoting ubiquitin transfer onto the substrate. Here, we tested whether the proposed mechanism operates in vivo in intact cells and applies to other CRL family members. To inhibit cellular neddylation, we used a cell line with tetracycline-inducible expression of a dominant-negative form of the Nedd8 E2 enzyme or treatment of cells with the Nedd8 E1 inhibitor MLN4924. Using these cellular systems, we show that different mutants of Cul2 and Cul3 and of Rbx1 that confer increased Rbx1 flexibility mimic neddylation and rescue CRL activity in intact cells. Our findings indicate that in vivo neddylation functions by inducing conformational changes in the C-terminal domain of Cul2 and Cul3 that free the RING domain of Rbx1 and bridge the gap for ubiquitin transfer onto the substrate.     

Characterization of cullin-based E3 ubiquitin ligases in intact mammalian cells--evidence for cullin dimerization

Cullin-based E3 ligases are a large family of ubiquitin ligases with diverse cellular functions. They are composed of one of six mammalian cullin homologues, the Ring finger containing protein Roc1/Rbx1 and cullin homologue-specific adapter and substrate recognition subunits. To be active, cullin-based ligases require the covalent modification of a conserved lysine residue in the cullin protein with the ubiquitin-like protein Nedd8. To characterize this family of E3 ligases in intact cells, we generated a cell line with tetracycline-inducible expression of a dominant-negative mutant of the Nedd8-conjugating enzyme Ubc12, a reported inhibitor of cullin neddylation. Using this cell line, we demonstrate that the substrate recognition subunit Skp2 and the adaptor protein Skp1 are subject to Ubc12-dependent autoubiquitination and degradation. In contrast, cullin protein stability is not regulated by neddylation in mammalian cells. We also provide evidence that Cul1 and Cul3, as well as their associated substrate recognition subunits Skp2 and Keap1, respectively, homooligomerize in intact cells, suggesting that cullin-based ligases are dimeric. Cul3, but not Cul1 homooligomerization is dependent on substrate recognition subunit dimer formation. As shown for other E3 ubiquitin ligases, dimerization may play a role in regulating the activity of cullin-based E3 ligases.     

Regulation of cullin RING E3 ubiquitin ligases by CAND1 in vivo

Cullin RING ligases are multi-subunit complexes consisting of a cullin protein which forms a scaffold onto which the RING protein Rbx1/2 and substrate receptor subunits assemble. CAND1, which binds to cullins that are not conjugated with Nedd8 and not associated with substrate receptors, has been shown to function as a positive regulator of Cullin ligases in vivo. Two models have been proposed to explain this requirement: (i) CAND1 sequesters cullin proteins and thus prevents autoubiquitination of substrate receptors, and (ii) CAND1 is required to promote the exchange of bound substrate receptors. Using mammalian cells, we show that CAND1 is predominantly cytoplasmically localized and that cullins are the major CAND1 interacting proteins. However, only small amounts of CAND1 bind to Cul1 in cells, despite low basal levels of Cul1 neddylation and approximately equal cytoplasmic endogenous protein concentrations of CAND1 and Cul1. Compared to F-box protein substrate receptors, binding of CAND1 to Cul1 in vivo is weak. Furthermore, preventing binding of F-box substrate receptors to Cul1 does not increase CAND1 binding. In conclusion, our study suggests that CAND1 does not function by sequestering cullins in vivo to prevent substrate receptor autoubiquitination and is likely to regulate cullin RING ligase activity via alternative mechanisms.     

A morphologic and semi-quantitative technique to analyze synthesis and release of specific proteins in cells

Cullins belong to a family of scaffold proteins that assemble multi-subunit ubiquitin ligase complexes to recruit protein substrates for ubiquitination via unique sets of substrate adaptor, such as Skp1 or Elongin B, and a substrate-binding protein with a conserved protein-protein interacting domain, such as l eucine-r ich r epeats (LRR), a WD40 domain, or a zinc-finger domain. In the case of the Cullin3 (Cul3), it forms a B TB-C ul3-R bx1 (BCR) ubiquitin ligase complex where it is believed that a BTB domain-containing protein performs dual functions where it serves as both the substrate adaptor and the substrate recognition protein. Tandem affinity purification and LC/MS-MS analysis of the BCR complex led to the identification of 10,225 peptides. After the SEQUEST algorithm and CDART program were used for protein identification and domain prediction, we discovered a group of C ul3-bound proteins that contain either the L RR or W D40 domain (CLWs). Further biochemical analysis revealed that the LRR domain-containing CLWs could bind both Cul3 and BTB domain-containing proteins. The dual binding role for the LRR domain-containing CLWs results in causing the BTB-domain protein to become a substrate instead of an adaptor. To further distinguish potential substrates from other components that are part of the BCR ubiquitin ligase complex, we altered the parameters in the SEQUEST algorithm to select for peptide fragments with a modified lysine residue. This method not only identifies the potential substrates of the BCR ubiquitin ligase complex, but it also pinpoints the lysine residue in which the post-translational modification occurs. Interestingly, none of the CLWs were identified by this method, supporting our hypothesis that CLWs were not potential substrates but rather additional components of the BCR ubiquitin ligase complex. Our study identified a new set of Cul3-binding proteins known as CLWs via tandem affinity purification and LC/MS-MS analysis. Subsequently, our biochemical analysis revealed that some CLWs modify binding of BTB domain-containing proteins to the complex, causing degradation of the BTB domain-containing protein. As these CLWs were excluded from our list of substrates, we propose that CLWs serve as unique Cul3 binding proteins that provide an alternative regulatory mechanism for the complex.     

PEST sequences mediate heat shock factor 2 turnover by interacting with the Cul3 subunit of the Cul3-RING ubiquitin ligase

Cullin-RING ubiquitin ligases promote the polyubiquitination and degradation of many important cellular proteins, which previous studies indicated can be targeted for degradation via interaction with BTB domain-containing subunits of this E3 ligase complex. PEST domains are known to promote the degradation of proteins that contain them. However, the molecular mechanism by which PEST sequences promote degradation of these proteins is not understood. Here we show that the PEST sequences of a short-lived protein called HSF2 interact with Cullin3, a subunit of a Cullin-RING E3 ubiquitin ligase, and that this interaction mediates the Cul3-dependent ubiquitination and degradation of HSF2. These results indicate how, at the molecular level, PEST sequences can promote the proteolysis of proteins that contain them. They also expand understanding of the mechanisms by which substrates can be recruited to Cullin-RING E3 ubiquitin ligases to include interactions between PEST sequences and Cul3.     

Characterization of the Cullin7 E3 ubiquitin ligase--heterodimerization of cullin substrate receptors as a novel mechanism to regulate cullin E3 ligase activity

Cul1 and Cul7 are cullin E3 ubiquitin ligase scaffold proteins. Cul1 is known to form a complex with the RING domain protein Rbx1 and one of approximately 70 different F-box proteins. F-box proteins function as substrate receptor subunits and recruit numerous substrates for poly-ubiquitination. Similarly to Cul1, Cul7 interacts with Rbx1, however, only one F-box protein, Fbxw8, has been shown to bind to Cul7. To date only few Cul7 E3 ubiquitin ligase substrates, including cyclin D1, IRS-1 and GRASP65, have been reported, and using Fbxw8 affinity purification, we were unable to identify additional substrate proteins. Here we provide evidence for a model in which Cul7-Rbx1 can promote the ubiquitination of Cul1 substrates by forming high order complexes with Cul1-Rbx1. Binding of Cul1-Rbx1 to Cul7-Rbx1 is mediated via heterodimerization of Fbxw8 with other F-box proteins which function to recruit substrates into the E3 ligase complex. The formation of this high order complex is likely to increase polyubiquitination efficiency.     

Arabidopsis has two redundant Cullin3 proteins that are essential for embryo development and that interact with RBX1 and BTB proteins to form multisubunit E3 ubiquitin ligase complexes in vivo

Cullin-based E3 ubiquitin ligases play important roles in the regulation of diverse developmental processes and environmental responses in eukaryotic organisms. Recently, it was shown in Schizosaccharomyces pombe, Caenorhabditis elegans, and mammals that Cullin3 (CUL3) directly associates with RBX1 and BTB domain proteins in vivo to form a new family of E3 ligases, with the BTB protein subunit functioning in substrate recognition. Here, we demonstrate that Arabidopsis thaliana has two redundant CUL3 (AtCUL3) genes that are essential for embryo development. Besides supporting anticipated specific AtCUL3 interactions with the RING protein AtRBX1 and representative Arabidopsis proteins containing a BTB domain in vitro, we show that AtCUL3 cofractionates and specifically associates with AtRBX1 and a representative BTB protein in vivo. Similar to the AtCUL1 subunit of the SKP1-CUL1-F-box protein-type E3 ligases, the AtCUL3 subunit of the BTB-containing E3 ligase complexes is subjected to modification and possible regulation by the ubiquitin-like protein Related to Ubiquitin in vivo. Together with the presence of large numbers of BTB proteins with diverse structural features and expression patterns, our data suggest that Arabidopsis has conserved AtCUL3-RBX1-BTB protein E3 ubiquitin ligases to target diverse protein substrates for degradation by the ubiquitin/proteasome pathway.     

Characterization of Cullin-box sequences that direct recruitment of Cul2-Rbx1 and Cul5-Rbx2 modules to Elongin BC-based ubiquitin ligases

The Elongin BC-box protein family includes the von Hippel-Lindau tumor suppressor and suppressor of cytokine signaling proteins, which are substrate recognition subunits of structurally related classes of E3 ubiquitin ligases composed of Elongin C-Elongin B-Cullin 2-Rbx1 (Cul2 ubiquitin ligases) or of Elongin C-Elongin B-Cullin 5-Rbx2 (Cul5 ubiquitin ligases). The Elongin BC complex acts as an adaptor that links a substrate recognition subunit to heterodimers of either Cullin 2 (Cul2) and RING finger protein Rbx1 or Cullin 5 (Cul5) and Rbx2. It has been shown ( Kamura, T., Maenaka, K., Kotoshiba, S., Matsumoto, M., Kohda, D., Conaway, R. C., Conaway, J. W., and Nakayama, K. I. (2004) Genes Dev. 18, 3055-3065 ) that interaction of BC-box proteins with their cognate Cul-Rbx module is determined by specific regions, called Cul2- or Cul5-boxes, located immediately downstream of their BC-boxes. Here, we investigate further the mechanisms governing assembly of BC-box proteins with their specific Cul-Rbx modules. Through purification and characterization of a larger collection of BC-box proteins that serve as substrate recognition subunits of Cul2 and Cul5 ubiquitin ligases and through structure-function studies, we define Cul2- and Cul5-boxes in greater detail. Although it previously appeared that there was little sequence similarity between Cul5- and Cul2-box motifs, analyses of newly identified BC-box proteins reveal that residues conserved in the Cul2-box represent a subset of those conserved in the Cul5-box. The sequence motif LPPhiP, which is conserved in most Cul5-boxes and has been suggested to specify assembly of Cul5 ligases, is compatible with Cul2 interaction. Finally, the spacing between BC- and Cullin-boxes is much more flexible than has been appreciated and can vary from as few as 3 and as many as approximately 80 amino acids. Taken together, our findings shed new light on the mechanisms by which BC-box proteins direct recruitment of Cullin-Rbx modules during reconstitution of ubiquitin ligases.     

BTB/POZ domain proteins are putative substrate adaptors for cullin 3 ubiquitin ligases

Cullins (CULs) are subunits of a prominent class of RING ubiquitin ligases. Whereas the subunits and substrates of CUL1-associated SCF complexes and CUL2 ubiquitin ligases are well established, they are largely unknown for other cullin family members. We show here that S. pombe CUL3 (Pcu3p) forms a complex with the RING protein Pip1p and all three BTB/POZ domain proteins encoded in the fission yeast genome. The integrity of the BTB/POZ domain, which shows similarity to the cullin binding proteins SKP1 and elongin C, is required for this interaction. Whereas Btb1p and Btb2p are stable proteins, Btb3p is ubiquitylated and degraded in a Pcu3p-dependent manner. Btb3p degradation requires its binding to a conserved N-terminal region of Pcu3p that precisely maps to the equivalent SKP1/F box adaptor binding domain of CUL1. We propose that the BTB/POZ domain defines a recognition motif for the assembly of substrate-specific RING/cullin 3/BTB ubiquitin ligase complexes.     

Cullin3 is a KLHL10-interacting protein preferentially expressed during late spermiogenesis

Kelch-like 10 (KLHL10) is a member of the BTB (Bric-a-brac, Tramtrack, and Broad-Complex)-kelch protein superfamily essential for spermiogenesis and male fertility. In a search for KLHL10-interacting proteins using a yeast two-hybrid assay, we identified Cullin3 (CUL3) as one of multiple KLHL10-interacting partners. Yeast cotransformation assays revealed that CUL3 bound the BTB/POZ domain of KLHL10. Northern blot and quantitative RT-PCR analyses demonstrated that Cul3 mRNA was preferentially expressed in the testis. In situ hybridization analysis localized Cul3 mRNA to spermatids in the adult testis. CUL3 protein was detected in elongating and elongated spermatids (steps 10-16) by immunofluorescent microscopy. The expression pattern of CUL3 resembles KLHL10. CUL3 was coimmunoprecipated with KLHL10, and KLHL10 was also detected in the CUL3 immunoprecipitants using testis lysates. These findings suggest that KLHL10, like other BTB/kelch proteins, interacts with CUL3 to form a CUL3-based ubiquitin E3 ligase that functions specifically in the testis to mediate protein ubiquitination during spermiogenesis.     

Hypertension-causing Mutations in Cullin3 Protein Impair RhoA Protein Ubiquitination and Augment the Association with Substrate Adaptors

Cullin-Ring ubiquitin ligases regulate protein turnover by promoting the ubiquitination of substrate proteins, targeting them for proteasomal degradation. It has been shown previously that mutations in Cullin3 (Cul3) causing deletion of 57 amino acids encoded by exon 9 (Cul3Δ9) cause hypertension. Moreover, RhoA activity contributes to vascular constriction and hypertension. We show that ubiquitination and degradation of RhoA is dependent on Cul3 in HEK293T cells in which Cul3 expression is ablated by either siRNA or by CRISPR-Cas9 genome editing. The latter was used to generate a Cul3-null cell line (HEK293T^Cul3KO). When expressed in these cells, Cul3Δ9 supported reduced ubiquitin ligase activity toward RhoA compared with equivalent levels of wild-type Cul3 (Cul3WT). Consistent with its reduced activity, binding of Cul3Δ9 to the E3 ubiquitin ligase Rbx1 and neddylation of Cul3Δ9 were impaired significantly compared with Cul3WT. Conversely, Cul3Δ9 bound to substrate adaptor proteins more efficiently than Cul3WT. Cul3Δ9 also forms unstable dimers with Cul3WT, disrupting dimers of Cul3WT complexes that are required for efficient ubiquitination of some substrates. Indeed, coexpression of Cul3WT and Cul3Δ9 in HEK293T^Cul3KO cells resulted in a decrease in the active form of Cul3WT. We conclude that Cul3Δ9-associated ubiquitin ligase activity toward RhoA is impaired and suggest that Cul3Δ9 mutations may act dominantly by sequestering substrate adaptors and disrupting Cul3WT complexes.     

Molecular recognition of Cullin3 by KCTDs: Insights from experimental and computational investigations

Recent investigations have highlighted a key role of the proteins of the KCTD (K-potassium channel tetramerization domain containing proteins) family in several fundamental biological processes. Despite the growing importance of KCTDs, our current understanding of their biophysical and structural properties is very limited. Biochemical characterizations of these proteins have shown that most of them act as substrate adaptor in E3 ligases during protein ubiquitination. Here we present a characterization of the KCTD5-Cullin3 interactions which are mediated by the KCTD5 BTB domain. Isothermal titration calorimetry experiments reveal that KCTD5 avidly binds the Cullin3 (Cul3). The complex presents a 5:5 stoichiometry and a dissociation constant of 59 nM. Molecular modeling and molecular dynamics simulations clearly indicate that the two proteins form a stable (KCTD5–Cul3)₅ pinwheel-shaped heterodecamer in which two distinct KCTD5 subunits cooperate in the binding of each cullin chain. Molecular dynamics simulations indicate that different types of interactions contribute to the stability of the assembly. Interestingly, residues involved in Cul3 recognitions are conserved in the KCTD5 orthologs and paralogs implicated in important biological processes. These residues are also rather well preserved in most of the other KCTD proteins. By using molecular modeling techniques, the entire ubiquitination system including the E3 ligase, the E2 conjugating enzyme and ubiquitin was generated. The analysis of the molecular architecture of this complex machinery provides insights into the ubiquitination processes which involve E3 ligases with a high structural complexity.