Essential role for the d-Asb11 cul5 Box domain for proper notch signaling and neural cell fate decisions in vivo

ECS (Elongin BC-Cul2/Cul5-SOCS-box protein) ubiquitin ligases recruit substrates to E2 ubiquitin-conjugating enzymes through a SOCS-box protein substrate receptor, an Elongin BC adaptor and a cullin (Cul2 or Cul5) scaffold which interacts with the RING protein. In vitro studies have shown that the conserved amino acid sequence of the cullin box in SOCS-box proteins is required for complex formation and function. However, the in vivo importance of cullin boxes has not been addressed. To explore the biological functions of the cullin box domain of ankyrin repeat and SOCS-box containing protein 11 (d-Asb11), a key mediator of canonical Delta-Notch signaling, we isolated a zebrafish mutant lacking the Cul5 box (Asb11(Cul)). We found that homozygous zebrafish mutants for this allele were defective in Notch signaling as indicated by the impaired expression of Notch target genes. Importantly, asb11(Cul) fish were not capable to degrade the Notch ligand DeltaA during embryogenesis, a process essential for the initiation of Notch signaling during neurogenesis. Accordingly, proper cell fate specification within the neurogenic regions of the zebrafish embryo was impaired. In addition, Asb11(Cul) mRNA was defective in the ability to transactivate a her4::gfp reporter DNA when injected in embryos. Thus, our study reporting the generation and the characterization of a metazoan organism mutant in the conserved cullin binding domain of the SOCS-box demonstrates a hitherto unrecognized importance of the SOCS-box domain for the function of this class of cullin-RING ubiquitin ligases and establishes that the d-Asb11 cullin box is required for both canonical Notch signaling and proper neurogenesis.     

ASB proteins interact with Cullin5 and Rbx2 to form E3 ubiquitin ligase complexes

The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins from ASB1 to ASB18 and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. ASB2 was recently shown to interact with a certain Cul-Rbx module to form an E3 ubiquitin (Ub) ligase complex, but the functional composition of the ASB-containing E3 Ub ligase complexes remains to be characterized. Here, we show that ASB proteins interact with Cul5-Rbx2 but neither Cul2 nor Rbx1 in cells. Mutational analysis revealed that the highly conserved amino acid sequences of the BC box and Cul5 box in the SOCS box of ASB proteins were essential for the interaction with Cul5-Rbx2. Although ASB proteins show slight divergences from the consensus sequences of the BC box and Cul5 box, all five tested ASB proteins bound to Cul5-Rbx2. Furthermore, all three tested ASB complexes containing Cul5-Rbx2 were found to have E3 Ub ligase activity. These findings suggest that the ASB family proteins interact with Cul5-Rbx2 to form E3 Ub ligases and play significant roles via a ubiquitination-mediated pathway.     

The SCF ubiquitin ligase: an extended look

The SCF E3 ubiquitin ligases select specific proteins for ubiquitination (and typically destruction) by coupling variable adaptor (F box) proteins that bind protein substrates to a conserved catalytic engine containing a cullin, Cul1, and the Rbx1/Roc1 RING finger protein. A new crystal structure of the SCF(Skp2) ubiquitin ligase shows the molecular organization of this complex and raises important questions as to how substrate ubiquitination is accomplished.     

The SOCS box domain of SOCS3: structure and interaction with the elonginBC-cullin5 ubiquitin ligase

Suppressor of cytokine signalling 3 (SOCS3) is responsible for regulating the cellular response to a variety of cytokines, including interleukin 6 and leukaemia inhibitory factor. Identification of the SOCS box domain led to the hypothesis that SOCS3 can associate with functional E3 ubiquitin ligases and thereby induce the degradation of bound signalling proteins. This model relies upon an interaction between the SOCS box, elonginBC and a cullin protein that forms the E3 ligase scaffold. We have investigated this interaction in vitro using purified components and show that SOCS3 binds to elonginBC and cullin5 with high affinity. The SOCS3-elonginBC interaction was further characterised by determining the solution structure of the SOCS box-elonginBC ternary complex and by deletion and alanine scanning mutagenesis of the SOCS box. These studies revealed that conformational flexibility is a key feature of the SOCS-elonginBC interaction. In particular, the SOCS box is disordered in isolation and only becomes structured upon elonginBC association. The interaction depends upon the first 12 residues of the SOCS box domain and particularly on a deeply buried, conserved leucine. The SOCS box, when bound to elonginBC, binds tightly to cullin5 with 100 nM affinity. Domains upstream of the SOCS box are not required for elonginBC or cullin5 association, indicating that the SOCS box acts as an independent binding domain capable of recruiting elonginBC and cullin5 to promote E3 ligase formation.     

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.     

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.     

The SOCS-Box of HIV-1 Vif Interacts with ElonginBC by Induced-Folding to Recruit Its Cul5-Containing Ubiquitin Ligase Complex

The HIV-1 viral infectivity factor (Vif) protein recruits an E3 ubiquitin ligase complex, comprising the cellular proteins elongin B and C (EloBC), cullin 5 (Cul5) and RING-box 2 (Rbx2), to the anti-viral proteins APOBEC3G (A3G) and APOBEC3F (A3F) and induces their polyubiquitination and proteasomal degradation. In this study, we used purified proteins and direct in vitro binding assays, isothermal titration calorimetry and NMR spectroscopy to describe the molecular mechanism for assembly of the Vif-EloBC ternary complex. We demonstrate that Vif binds to EloBC in two locations, and that both interactions induce structural changes in the SOCS box of Vif as well as EloBC. In particular, in addition to the previously established binding of Vif''s BC box to EloC, we report a novel interaction between the conserved Pro-Pro-Leu-Pro motif of Vif and the C-terminal domain of EloB. Using cell-based assays, we further show that this interaction is necessary for the formation of a functional ligase complex, thus establishing a role of this motif. We conclude that HIV-1 Vif engages EloBC via an induced-folding mechanism that does not require additional co-factors, and speculate that these features distinguish Vif from other EloBC specificity factors such as cellular SOCS proteins, and may enhance the prospects of obtaining therapeutic inhibitors of Vif function.     

Mechanism of cullin3 e3 ubiquitin ligase dimerization

Cullin E3 ligases are the largest family of ubiquitin ligases with diverse cellular functions. One of seven cullin proteins serves as a scaffold protein for the assembly of the multisubunit ubiquitin ligase complex. Cullin binds the RING domain protein Rbx1/Rbx2 via its C-terminus and a cullin-specific substrate adaptor protein via its N-terminus. In the Cul3 ubiquitin ligase complex, Cul3 substrate receptors contain a BTB/POZ domain. Several studies have established that Cul3-based E3 ubiquitin ligases exist in a dimeric state which is required for binding of a number of substrates and has been suggested to promote ubiquitin transfer. In two different models, Cul3 has been proposed to dimerize either via BTB/POZ domain dependent substrate receptor homodimerization or via direct interaction between two Cul3 proteins that is mediated by Nedd8 modification of one of the dimerization partners. In this study, we show that the majority of the Cul3 proteins in cells exist as dimers or multimers and that Cul3 self-association is mediated via the Cul3 N-terminus while the Cul3 C-terminus is not required. Furthermore, we show that Cul3 self-association is independent of its modification with Nedd8. Our results provide evidence for BTB substrate receptor dependent Cul3 dimerization which is likely to play an important role in promoting substrate ubiquitination.     

A role for Saccharomyces cerevisiae Cul8 ubiquitin ligase in proper anaphase progression

We have undertaken a study of the yeast cullin family members Cul3 and Cul8, as little is known about their biochemical and physiological functions. We demonstrate that these cullins are associated in vivo with ubiquitin ligase activity. We show that Cul3 and Cul8 are functionally distinct from Cdc53 and do not interact with ySkp1, suggesting that they target substrates by Skp1- and possibly F-box protein-independent mechanisms. Whereas null mutants of CUL3 appear normal, yeast cells lacking CUL8 have a slower growth rate and are delayed in their progress through anaphase. The anaphase delay phenotype can be complemented by ectopic expression of Cul8 but not by any other yeast or human cullins, nor by a cul8 mutant deficient in binding to RING finger protein Roc1. Deletion of the RAD9 gene suppressed the anaphase delay phenotype of cul8delta, suggesting that loss of Cul8 function may compromise genomic integrity. These results indicate that in addition to the anaphase promoting complex, mitotic progression may involve another E3 ubiquitin ligase mediated by Cul8 protein.     

Targeting SUMO E1 to ubiquitin ligases: a viral strategy to counteract sumoylation

SUMO-1 (small ubiquitin-related modifier-1) is a ubiquitin-like family member that is conjugated to its substrates through three discrete enzymatic steps, activation (involving the E1 enzyme (SAE1/SAE2)), conjugation (involving the E2 enzyme), and substrate modification (through the cooperation of the E2 and E3 protein ligases). The adenoviral protein Gam1 inactivates E1, both in vitro and in vivo, followed by SAE1/SAE2 degradation. We have shown here that Gam1 possesses a C-terminal SOCS domain that allows its interaction with two cellular cullin RING (really interesting new gene) ubiquitin ligases. We demonstrate that Gam1 is necessary for the recruitment of SAE1/SAE2 into Cul2/5-EloB/C-Roc1 ubiquitin ligase complexes and for subsequent SAE1 ubiquitylation and degradation. The degradation of SAE2 is not tightly related to Gam1 but is a consequent effect of SAE1 disappearance. These results reveal the mechanism by which a viral protein inactivates and subsequently degrades an essential cellular enzyme, arresting a key regulatory pathway.     

Structural insight into the human immunodeficiency virus Vif SOCS box and its role in human E3 ubiquitin ligase assembly

Human immunodeficiency virus (HIV) virion infectivity factor (Vif) causes the proteasome-mediated destruction of human antiviral protein APOBEC3G by tethering it to a cellular E3 ubiquitin ligase composed of ElonginB, ElonginC, Cullin5, and Rbx2. It has been proposed that HIV Vif hijacks the E3 ligase through two regions within its C-terminal domain: a BC box region that interacts with ElonginC and a novel zinc finger motif that interacts with Cullin5. We have determined the crystal structure of the HIV Vif BC box in complex with human ElonginB and ElonginC. This complex presents direct structural evidence of the recruitment of a human ubiquitin ligase by a viral BC box protein that mimics the conserved interactions of cellular ubiquitin ligases. We further mutated conserved hydrophobic residues in a region downstream of the Vif BC box. These mutations demonstrate that this region, the Vif Cullin box, composes a third E3-ligase recruiting site critical for interaction between Vif and Cullin5. Furthermore, our homology modeling reveals that the Vif Cullin box and zinc finger motif may be positioned adjacent to the N terminus of Cullin5 for interaction with loop regions in the first cullin repeat of Cullin5.     

MUF1/Leucine-Rich Repeat Containing 41 (LRRC41), a Substrate of RhoBTB-Dependent Cullin 3 Ubiquitin Ligase Complexes, Is a Predominantly Nuclear Dimeric Protein

RhoBTB (BTB stands for broad-complex, tramtrack, bric à brac) proteins are tumor suppressors involved in the formation of cullin 3 (Cul3)-dependent ubiquitin ligase complexes. However, no substrates of RhoBTB-Cul3 ubiquitin ligase complexes have been identified. We identified MUF1 (LRRC41, leucine-rich repeat containing 41) as a potential interaction partner of RhoBTB3 in a two-hybrid screening on a mouse brain cDNA library. MUF1 is a largely uncharacterized protein containing a leucine-rich repeat and, interestingly, a BC-box that serves as a linker in multicomponent, cullin 5 (Cul5)-based ubiquitin ligases. We confirmed the interaction of MUF1 with all three mammalian RhoBTB proteins using immunoprecipitation. We characterized MUF1 in terms of expression profile and subcellular localization, the latter also with respect to RhoBTB proteins. We found out that MUF1 is a ubiquitously expressed nuclear protein that, upon coexpression with RhoBTB, partially retains in the cytoplasm, where both proteins colocalize. We also show that MUF1 is able to dimerize similarly to other leucine-rich repeat-containing proteins. To explore the significance of MUF1-RhoBTB interaction within Cul-ligase complexes and the mechanism of MUF1 degradation, we performed a protein stability assay and found that MUF1 is degraded in the proteasome in a Cul5-independent manner by RhoBTB3-Cul3 ubiquitin ligase complex. Finally, we explored a possible heterodimerization of Cul3 and Cul5 and indeed discovered that these two cullins are capable of forming heterodimers. Thus, we have identified MUF1 as the first substrate for RhoBTB-Cul3 ubiquitin ligase complexes. Identification of substrates of these complexes will result in better understanding of the tumor suppressor function of RhoBTB.     

Dissection of the HIV Vif Interaction with Human E3 Ubiquitin Ligase

The human immunodeficiency virus type 1 (HIV-1) protein Vif recruits the host E3 ubiquitin ligase, composed of cullin 5 (Cul5), Rbx2, Elongin B, and Elongin C (EloBC), to polyubiquitinate the antiviral protein APOBEC3G. Multiple regions in the C-terminal half of Vif interact with the E3 ligase. We have purified individual regions of Vif and investigated their thermodynamic contributions to the ligase assembly in vitro using isothermal titration calorimetry and fluorescence anisotropy. Our results quantify the high-affinity interactions between the Vif BC box and EloBC and between the Vif zinc finger and Cul5, as well as the modest interaction between the Vif cullin box and Cul5. Our purified Vif constructs also provide direct biochemical evidence that the Vif cullin box, containing the PPLP region, leads to the dimerization of Vif-EloBC complexes but not Cul5-Vif-EloBC complexes.HIV Vif antagonizes the human antiviral protein APOBEC3G by hijacking the human Elongin B/C (EloBC)-cullin-SOCS box (ECS)-type E3 ubiquitin ligase, resulting in the polyubiquitination of APOBEC3G and subsequently its proteasomal degradation. Canonical ECS-type ubiquitin ligases consist of a cullin scaffold protein to which adaptor and substrate receptor proteins bind at the N terminus. HIV Vif serves as a substrate receptor protein—its N terminus recruits APOBEC3G, while multiple C-terminal regions assemble with the E3 ligase (9, 13, 24). The E3 ligase interacting regions include a zinc finger (residues 100 to 140), a BC box (residues 141 to 154), and a cullin box (residues 155 to 176) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.(A) A sequence schematic of Vif showing the regions that interact with A3G, A3F, EloBC, and Cul5. (B) An illustration of the assembly of the Vif-E3 ubiquitin ligase. (C) A homology model of Vif-Cul5-EloBC, where the Vif BC box-EloBC is actual structural data (PDB ID 3DCG).Vif binds the cullin adaptor proteins EloB and EloC through the BC-box region (24). The BC box is a loop-helix motif with the consensus sequence (T/S)LxxxCxxx(V/L/I) (7), and it also exists in cellular proteins that interact with EloBC. While Vif does not fit this consensus perfectly, it still binds EloBC with high affinity, and this interaction is lost upon mutation or deletion of consensus BC-box residues (10, 24, 25). This interaction has been described previously for the cellular proteins VHL (15), SOCS2 (3), SOCS3 (1), SOCS4 (4), and recently HIV Vif (14).Both the Vif zinc finger and cullin box interact with the E3 ligase scaffold protein cullin 5 (Cul5) (11, 12, 20, 21). It has been established that the zinc finger is required for Vif to bind Cul5. Mutation of critical histidine or cysteine residues in this region or the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) abolishes the Vif-Cul5 interaction (8, 11, 25). The sequence of the Vif cullin box is not as conserved as those of cellular SOCS-box proteins, which have a defined structure and determine the specificities of their respective cullins (6). The role of the Vif cullin box is not clear, but it has been suggested to promote dimerization of Vif, involving the conserved PPLP region (22, 23), and has recently been implicated in APOBEC3G binding (5, 17). While its importance in Cul5 binding has been demonstrated in coimmunoprecipitation experiments (14), experimental data also exist showing that the Vif zinc finger alone still immunoprecipitates Cul5 (11, 21).To dissect the assembly of the Vif-E3 ubiquitin ligase, we quantified the binding interactions between various C-terminal Vif constructs, EloBC, and Cul5 by isothermal titration calorimetry (ITC) and fluorescence polarization (FP). We additionally probed the effects of the cullin box on Vif dimerization.     

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.     

Ubiquitin crosstalk connecting cellular processes

Cullin 4 (Cul4), a member of the evolutionally conserved cullin protein family, serves as a scaffold to assemble multisubunit ubiquitin E3 ligase complexes. Cul4 interacts with the Ring finger-containing protein ROC1 through its C-terminal cullin domain and with substrate recruiting subunit(s) through its N-terminus. Previous studies have demonstrated that Cul4 E3 ligase ubiquitylates key regulators in cell cycle control and mediates their degradation through the proteasomal pathway, thus contributing to genome stability. Recent studies from several groups have revealed that Cul4 E3 ligase can target histones for ubiquitylation, and importantly, ubiquitylation of histones may facilitate the cellular response to DNA damage. Therefore, histone ubiquitylation by Cul4 E3 ligase constitutes a novel mechanism through which Cul4 regulates chromatin function and maintains genomic integrity. We outline these studies and suggest that histone ubiquitylation might play important roles in Cul4-regualted chromatin function including the cellular response to DNA damage and heterochromatin gene silencing.     

VprBP binds full-length RAG1 and is required for B-cell development and V(D)J recombination fidelity

The N-terminus of full-length RAG1, though dispensable for RAG1/2 cleavage activity, is required for efficient V(D)J recombination. This region supports RING E3 ubiquitin ligase activity in vitro, but whether full-length RAG1 functions as a single subunit or a multi-subunit E3 ligase in vivo is unclear. We show the multi-subunit cullin RING E3 ligase complex VprBP/DDB1/Cul4A/Roc1 associates with full-length RAG1 through VprBP. This complex is assembled into RAG protein-DNA complexes, and supports in-vitro ubiquitylation activity that is insensitive to RAG1 RING domain mutations. Conditional B lineage-specific VprBP disruption arrests B-cell development at the pro-B-to-pre-B cell transition, but this block is bypassed by expressing rearranged immunoglobulin transgenes. Mice with a conditional VprBP disruption show modest reduction of D-J(H) rearrangement, whereas V(H)-DJ(H) and V(κ)-J(κ) rearrangements are severely impaired. D-J(H) coding joints from VprBP-insufficent mice show longer junctional nucleotide insertions and a higher mutation frequency in D and J segments than normal. These data suggest full-length RAG1 recruits a cullin RING E3 ligase complex to ubiquitylate an unknown protein(s) to limit error-prone repair during V(D)J recombination.     

ELA1 and CUL3 are required along with ELC1 for RNA polymerase II polyubiquitylation and degradation in DNA-damaged yeast cells

Treatment of yeast and human cells with DNA-damaging agents elicits lysine 48-linked polyubiquitylation of Rpb1, the largest subunit of RNA polymerase II (Pol II), which targets Pol II for proteasomal degradation. However, the ubiquitin ligase (E3) responsible for Pol II polyubiquitylation has not been identified in humans or the yeast Saccharomyces cerevisiae. Here we show that elongin A (Ela1) and cullin 3 (Cul3) are required for Pol II polyubiquitylation and degradation in yeast cells, and on the basis of these and other observations, we propose that an E3 comprised of elongin C (Elc1), Ela1, Cul3, and the RING finger protein Roc1 (Rbx1) mediates this process in yeast cells. This study provides, in addition to the identification of the E3 required for Pol II polyubiquitylation and degradation in yeast cells, the first evidence for a specific function in yeast for a member of the elongin C/BC-box protein/cullin family of ligases. Also, these observations raise the distinct possibility that the elongin C-containing ubiquitin ligase, the von Hippel-Lindau tumor suppressor complex, promotes Pol II polyubiquitylation and degradation in human cells.