GO:0031463 Cul3-RING ubiquitin ligase complex: Components, Assembly and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0031463 describes the Cul3-RING ubiquitin ligase complex (CRL3), a multi-subunit E3 ligase in which a CUL3 cullin scaffold and a RING-domain protein form the catalytic core, while a BTB-domain protein provides substrate specificity.
• CRL3 complexes are modular: different BTB-domain adapters recruit distinct substrates, allowing one catalytic core to regulate many cellular pathways.
• CRL3 activity is essential for striated muscle function in mice, linking the complex to tissue physiology.
• Viral proteins such as vaccinia A55 can subvert CUL3 to redirect substrate targeting, showing that CRL3 is a host-pathogen interface.
• Small molecules like UM171 can glue asymmetric CRL3-HDAC1/2 assemblies to degrade CoREST corepressors, demonstrating pharmacological control of CRL3.
• CRISPR-based knockout, point-mutation, knock-in and overexpression models are key tools for dissecting CRL3 subunit function and substrate specificity [1,4].
Description
The Cul3-RING ubiquitin ligase complex (CRL3) is a cellular_component defined in GO:0031463 as a ubiquitin ligase complex in which a cullin from the Cul3 subfamily and a RING domain protein form the catalytic core, while substrate specificity is conferred by a BTB-domain-containing protein. This architecture places CRL3 among the cullin-RING ligases (CRLs), a major class of E3 enzymes that catalyze the final step of ubiquitin transfer to target proteins. Because CRL3 complexes use interchangeable BTB adapters, they can recognize a wide range of substrates and thereby influence processes as diverse as protein turnover, signaling, and development. For researchers, GO:0031463 matters because it provides a precise annotation for experiments that map E3 ligase composition, substrate recruitment, and downstream phenotypes. Studies in mice have shown that Cullin-3-RING ubiquitin ligase activity is required for striated muscle function, establishing a physiological requirement for this complex in vivo. Structural and biochemical work has revealed how BTB adapters such as KLHL-12 bind peptide substrates, offering a template for understanding substrate selection. In parallel, viral proteins like vaccinia A55 can hijack CUL3, illustrating that CRL3 is a target of pathogen subversion. CRL3 also intersects with human disease biology and therapeutic discovery. The small molecule UM171 glues asymmetric CRL3-HDAC1/2 assemblies to degrade CoREST corepressors, showing that CRL3 can be chemically reprogrammed. In the kidney, the CUL3/KLHL3/WNK4 complex regulates NaCl cotransporter activity, and Camk2n1 deficiency reduces this activity, tying CRL3 to ion transport physiology. Together, these findings make GO:0031463 a focal point for studies of ubiquitin signaling, disease mechanisms, and targeted protein degradation [1,2,7].
Cul3-RING ubiquitin ligase complex At A Glance
| GO ID | GO:0031463 |
|---|---|
| GO term | Cul3-RING ubiquitin ligase complex |
| Ontology | cellular_component |
| Synonym | BC3B complex, BCR3 complex, CDL3 complex, CRL3 complex, cullin-RING ligase 3, SCF3 complex |
| Major function | E3 ubiquitin ligase activity; substrate recognition via BTB-domain proteins; ubiquitin transfer to target proteins |
| Catalytic core | CUL3 cullin and a RING-domain protein |
| Substrate specificity | Conferred by BTB-domain-containing proteins |
| Example adapters | KLHL-12, KBTBD6/KBTBD7, KLHL3 [5,8,7] |
| Physiological role | Required for striated muscle function in mice |
| Pharmacological relevance | Can be reprogrammed by molecular glues such as UM171 |
What Is GO:0031463?
In simple terms, GO:0031463 is the Gene Ontology annotation for a specific type of E3 ubiquitin ligase machine. The complex contains a CUL3 cullin protein and a RING-domain protein that together form the catalytic core, and it uses a BTB-domain-containing protein as the substrate-recognition module. This definition captures the essential composition of CRL3 complexes and distinguishes them from other cullin-RING ligases that use different cullin scaffolds and adapters.
Why Is Cul3-RING ubiquitin ligase complex Important in Cell Biology?
GO:0031463 is important because CRL3 complexes are central nodes in ubiquitin-dependent proteostasis and signaling, and their dysfunction or hijacking has been linked to muscle physiology, kidney ion transport, viral infection, and cancer-related corepressor degradation [1,2,3,4,7]. Because substrate specificity is encoded by BTB adapters, the complex offers a modular system for studying how a single catalytic core can regulate many substrates, and it is a promising target for chemical biology and therapeutic degradation strategies [1,2].
Cul3-RING ubiquitin ligase complex
• CRL3 complexes regulate protein turnover through ubiquitin-mediated degradation, influencing diverse cellular pathways.
• Cullin-3-RING ubiquitin ligase activity is required for striated muscle function in mice, linking CRL3 to tissue physiology.
• The CUL3/KLHL3/WNK4 complex controls NaCl cotransporter activity in the kidney, connecting CRL3 to ion transport and blood pressure biology.
• Viral proteins such as vaccinia A55 can subvert CUL3, making CRL3 a host-pathogen interface.
• Small molecules like UM171 can glue asymmetric CRL3-HDAC1/2 assemblies to degrade CoREST corepressors, showing therapeutic potential.
• BTB adapters such as KLHL-12 determine substrate selection, providing a basis for understanding specificity.
• CUL3-KBTBD6/KBTBD7 cooperates with GABARAP proteins to spatially restrict TIAM1-RAC1 signaling, linking CRL3 to cytoskeletal regulation.
• PEST sequences mediate heat shock factor 2 turnover by interacting with the Cul3 subunit, showing CRL3 regulation of stress-responsive transcription factors.
• CRL3 components are attractive targets for CRISPR-based functional genomics and drug discovery [1,2].
• Understanding CRL3 assembly and substrate recruitment can inform targeted protein degradation strategies [1,2].
What Happens During Cul3-RING ubiquitin ligase complex?
(未命名小节)
In simple terms: The complex acts like a tagging machine that attaches ubiquitin to specific proteins so they can be degraded or regulated.
CRL3 complexes catalyze the transfer of ubiquitin from an E2 enzyme to substrate proteins, a process that typically leads to proteasomal degradation or altered protein function. The catalytic core formed by CUL3 and a RING-domain protein brings the E2 and substrate into proximity, while the BTB-domain protein recruits the substrate. This modular arrangement allows different CRL3 complexes to target distinct substrates and thereby regulate many cellular processes.
Substrate Recruitment by BTB-Domain Proteins
In simple terms: Different adapter proteins decide which proteins get tagged.
Substrate specificity in CRL3 complexes is conferred by BTB-domain-containing proteins that bind both CUL3 and the substrate. Structural studies of KLHL-12, a substrate-specific adapter in a Cul3-RING E3 ligase complex, have elucidated how peptides bind to the adapter, providing insight into substrate recognition. Other adapters such as KBTBD6/KBTBD7 cooperate with GABARAP proteins to spatially restrict TIAM1-RAC1 signaling, demonstrating that adapter choice determines downstream signaling outcomes.
Structure and Composition of Cul3-RING ubiquitin ligase complex
In simple terms: The complex is built from a scaffold, a catalytic RING protein, and a substrate adapter.
The CRL3 complex consists of a CUL3 cullin scaffold, a RING-domain protein that forms the catalytic core, and a BTB-domain protein that provides substrate specificity. This composition is the defining feature of GO:0031463 and distinguishes CRL3 from other cullin-RING ligases. The assembly of these subunits is essential for E3 ligase activity, and disruption of any component can impair substrate ubiquitination [1,4].
Assembly and Regulation of CRL3 Complexes
In simple terms: The complex can be assembled and controlled in different ways depending on the cell context.
CRL3 assembly involves the association of CUL3 with a RING-domain protein and a BTB-domain adapter, and this assembly can be regulated by cellular signals and accessory proteins. For example, CUL3-KBTBD6/KBTBD7 cooperates with GABARAP proteins to spatially restrict TIAM1-RAC1 signaling, indicating that localization and accessory factors influence CRL3 function. In addition, viral proteins such as vaccinia A55 can subvert CUL3, showing that CRL3 activity can be redirected by pathogen-encoded factors.
Molecular Mechanism of Cul3-RING ubiquitin ligase complex
In simple terms: The complex uses a RING domain to transfer ubiquitin from an E2 enzyme to a target protein.
The RING-domain protein within the CRL3 complex facilitates the transfer of ubiquitin from a charged E2 enzyme to the substrate. The CUL3 scaffold positions the RING domain and the BTB-adapter-bound substrate for efficient ubiquitination. This mechanism is shared with other cullin-RING ligases, but the use of BTB-domain adapters gives CRL3 its unique substrate repertoire. Structural and biochemical studies of adapters like KLHL-12 have provided detailed views of how substrates are recognized and positioned for ubiquitination.
Regulation of CRL3 Activity
In simple terms: The complex can be turned on or off by cellular signals and by interacting proteins.
CRL3 activity can be regulated at the level of complex assembly, substrate availability, and post-translational modifications. For instance, PEST sequences mediate heat shock factor 2 turnover by interacting with the Cul3 subunit, linking CRL3 to stress-responsive transcription factor regulation. In the kidney, Camk2n1 deficiency reduces NaCl cotransporter activity through the CUL3/KLHL3/WNK4 complex, demonstrating physiological regulation of CRL3-dependent substrate targeting. Pharmacological regulation is also possible, as UM171 glues asymmetric CRL3-HDAC1/2 assembly to degrade CoREST corepressors.
Key Genes Involved in GO:0031463 Cul3-RING ubiquitin ligase complex
The following genes and proteins are core components or well-characterized adapters/substrates of the Cul3-RING ubiquitin ligase complex (GO:0031463).
| Gene | Major Role | Research Relevance |
|---|---|---|
| CUL3 | Scaffold cullin of the CRL3 catalytic core | Central to GO:0031463; required for striated muscle function in mice |
| RBX1 | RING-domain protein that forms the catalytic core with CUL3 | Essential for E3 ligase activity of CRL3 |
| KLHL12 | BTB-domain substrate adapter | Structural studies of peptide binding to KLHL-12 |
| KLHL3 | BTB-domain adapter in the CUL3/KLHL3/WNK4 complex | Regulates NaCl cotransporter activity in the kidney |
| KBTBD6 | BTB-domain adapter cooperating with GABARAP proteins | Spatially restricts TIAM1-RAC1 signaling |
| KBTBD7 | BTB-domain adapter cooperating with GABARAP proteins | Spatially restricts TIAM1-RAC1 signaling |
| WNK4 | Substrate of CUL3/KLHL3 complex | Regulates NaCl cotransporter activity |
| TIAM1 | Substrate of CUL3-KBTBD6/KBTBD7 | RAC1 signaling regulation |
| RAC1 | Small GTPase regulated by TIAM1 | Downstream of CRL3-dependent TIAM1 restriction |
| HSF2 | Heat shock transcription factor targeted by CUL3 | PEST sequences mediate HSF2 turnover via Cul3 |
| HDAC1 | Component recruited by UM171 to CRL3 | Degradation of CoREST corepressors |
| HDAC2 | Component recruited by UM171 to CRL3 | Degradation of CoREST corepressors |
| CoREST | Corepressor complex degraded via CRL3-HDAC1/2 | Target of UM171-mediated degradation |
| A55 | Vaccinia virus protein that subverts CUL3 | Viral hijacking of CRL3 |
| GABARAP | Accessory protein cooperating with KBTBD6/KBTBD7 | Spatial restriction of TIAM1-RAC1 signaling |
| CAMK2N1 | Regulator of CUL3/KLHL3/WNK4 complex | Deficiency reduces NaCl cotransporter activity |
| BTB-domain proteins | Generic substrate adapters for CRL3 | Confer substrate specificity to CRL3 complexes |
How Is Cul3-RING ubiquitin ligase complex Regulated?
CRL3 complexes are regulated at multiple levels, including assembly of the catalytic core with BTB adapters, substrate availability, and post-translational modifications. For example, PEST sequences mediate heat shock factor 2 turnover by interacting with the Cul3 subunit, linking CRL3 to stress-responsive transcription. In the kidney, Camk2n1 deficiency reduces NaCl cotransporter activity through the CUL3/KLHL3/WNK4 complex, showing physiological control of CRL3 substrate targeting. Pharmacological regulation is exemplified by UM171, which glues asymmetric CRL3-HDAC1/2 assembly to degrade CoREST corepressors.
Cul3-RING ubiquitin ligase complex and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| CUL3 | Striated muscle function | Cul3 knockout or point-mutation mouse models |
| KLHL3 | Kidney ion transport / NaCl cotransporter regulation | Klhl3 knockout or knock-in models |
| WNK4 | Kidney ion transport | Wnk4 knockout or point-mutation models |
| HDAC1/2 | Cancer-related corepressor degradation | CRISPR knock-in of degron tags or overexpression |
| A55 | Viral subversion of CRL3 | Viral protein overexpression in cell models |
CRL3 in Muscle Physiology and Disease
Cullin-3-RING ubiquitin ligase activity is required for striated muscle function in mice, indicating that loss of CRL3 function can impair muscle physiology. This finding links GO:0031463 to muscle-related disease mechanisms and suggests that CRL3 components may be relevant to myopathies or muscle wasting conditions.
CRL3 in Kidney Ion Transport and Hypertension
The CUL3/KLHL3/WNK4 complex regulates NaCl cotransporter activity in the kidney, and Camk2n1 deficiency reduces this activity. Because NaCl cotransporter activity influences salt handling and blood pressure, CRL3 dysfunction may contribute to renal ion transport disorders and hypertension-related phenotypes.
CRL3 as a Target of Viral Subversion
Vaccinia virus protein A55 subverts CUL3, demonstrating that pathogens can hijack CRL3 to redirect substrate targeting. This highlights CRL3 as a host-pathogen interface and suggests that viral proteins may be used as tools to study CRL3 function.
CRL3 and Cancer-Related Corepressor Degradation
UM171 glues asymmetric CRL3-HDAC1/2 assembly to degrade CoREST corepressors, showing that CRL3 can be pharmacologically directed to degrade cancer-relevant proteins. This positions CRL3 as a potential therapeutic target in cancers dependent on CoREST corepressor function.
From Cul3-RING ubiquitin ligase complex-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of CUL3 affect muscle function? | Cul3 knockout mouse or muscle-specific knockout |
| How does KLHL3 regulate WNK4 and NaCl cotransporter activity? | Klhl3 knockout or point-mutation kidney cell models |
| Can UM171 redirect CRL3 to degrade CoREST? | CRISPR knock-in of HDAC1/2 degron tags or overexpression |
| How does vaccinia A55 subvert CUL3? | Overexpression of A55 in human cell lines |
| What is the substrate specificity of KLHL-12? | Point mutations in KLHL-12 substrate-binding pocket |
| How does CUL3-KBTBD6/KBTBD7 restrict TIAM1-RAC1 signaling? | Knockout of KBTBD6/KBTBD7 or GABARAP |
How to Study the Cul3-RING ubiquitin ligase complex Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Co-immunoprecipitation | Protein-protein interactions | Identifying CRL3 subunits and substrates |
| Mass spectrometry | Protein abundance and ubiquitination sites | Ubiquitinome profiling of CRL3 targets |
| In vitro ubiquitination | E3 ligase activity | Testing CRL3 catalytic function |
| Structural biology (crystallography/cryo-EM) | Three-dimensional structure | Understanding BTB-adapter substrate binding |
| CRISPR knockout | Loss-of-function phenotypes | Testing CUL3, KLHL3, or KBTBD6/7 requirements [4,7,8] |
| Knock-in of degron tags | Induced degradation of target proteins | Studying UM171-mediated CRL3 degradation |
| Animal physiology | Tissue-level function | Muscle and kidney studies [4,7] |
| Viral protein overexpression | Pathogen-host interaction | Studying A55-mediated CUL3 subversion |
Proteomics and Ubiquitinome Analysis
Mass spectrometry-based proteomics can identify substrates and interactors of CRL3 complexes by comparing wild-type and mutant cells. Ubiquitinome profiling can reveal changes in ubiquitination sites upon CRL3 perturbation.
Structural Biology of CRL3 Components
Structural studies such as those on KLHL-12 provide atomic-level views of substrate recognition by BTB-domain adapters. These methods help explain how mutations in CRL3 components affect substrate binding.
Cell-Based Ubiquitination Assays
In vitro ubiquitination assays and cell-based degradation assays can measure CRL3 activity toward specific substrates. Such assays are useful for testing whether a candidate BTB adapter or substrate is functionally linked to CRL3.
Animal Models and Physiological Readouts
Mouse models have been used to show that Cullin-3-RING ubiquitin ligase activity is required for striated muscle function. Kidney physiology studies have used the CUL3/KLHL3/WNK4 axis to link CRL3 to NaCl cotransporter activity.
How CRISPR Can Be Used to Study GO:0031463 Cul3-RING ubiquitin ligase complex
Knockout
CRISPR knockout of CUL3, KLHL3, KBTBD6, KBTBD7, or other CRL3 components can reveal loss-of-function phenotypes in muscle, kidney, or signaling contexts [4,7,8]. Knockout models are useful for determining whether a candidate gene is required for CRL3-dependent processes.
Point Mutation
Point mutations in BTB-domain adapters such as KLHL-12 can disrupt substrate binding and help map interaction interfaces. Point mutations in CUL3 or RING-domain proteins can separate catalytic activity from scaffold functions.
Knock-in
Knock-in of degron tags or fluorescent tags into CRL3 substrates can enable real-time monitoring of degradation and localization. Knock-in models can also introduce disease-relevant mutations to study CRL3-related pathology.
Overexpression
Overexpression of viral proteins like vaccinia A55 can be used to study CRL3 subversion. Overexpression of BTB adapters or substrates can help identify dominant effects on CRL3 function.
How EDITGENE Supports Cul3-RING ubiquitin ligase complex Research
Researchers studying Cul3-RING ubiquitin ligase complex-related genes often need to determine whether a candidate gene is causally involved in CRL3 assembly, substrate recruitment, or downstream physiology. CRISPR-based models provide a direct way to test these hypotheses by deleting, mutating, tagging, or overexpressing the relevant genes in relevant cell types [1,4,7].
Contact EDITGENE today to design your custom CRISPR model for Cul3-RING ubiquitin ligase complex research.
Frequently Asked Questions About Cul3-RING ubiquitin ligase complex
What is GO:0031463?
GO:0031463 is the Gene Ontology cellular_component term for the Cul3-RING ubiquitin ligase complex, an E3 ligase in which CUL3 and a RING-domain protein form the catalytic core and a BTB-domain protein confers substrate specificity.
What genes are involved in the Cul3-RING ubiquitin ligase complex?
Core genes include CUL3 and RING-domain proteins, while BTB-domain adapters such as KLHL12, KLHL3, KBTBD6, and KBTBD7 provide substrate specificity [1,5,7,8].
What does the Cul3-RING ubiquitin ligase complex do?
It catalyzes ubiquitin transfer to substrate proteins, often leading to their degradation or functional regulation.
How is substrate specificity determined in CRL3 complexes?
Substrate specificity is conferred by BTB-domain-containing proteins that bind both CUL3 and the substrate.
Is CUL3 required for muscle function?
Yes, Cullin-3-RING ubiquitin ligase activity is required for striated muscle function in mice.
How does the CUL3/KLHL3/WNK4 complex affect the kidney?
It regulates NaCl cotransporter activity, and Camk2n1 deficiency reduces this activity through the complex.
Can CRL3 be targeted by drugs?
Yes, UM171 glues asymmetric CRL3-HDAC1/2 assembly to degrade CoREST corepressors, showing pharmacological control.
How do viruses interact with CRL3?
Vaccinia virus protein A55 subverts CUL3, redirecting the complex for viral benefit.
What research methods are used to study CRL3?
Common methods include co-immunoprecipitation, mass spectrometry, in vitro ubiquitination, structural biology, and CRISPR-based models [1,5].
What CRISPR models are available for CRL3 research?
Knockout, point-mutation, knock-in, and overexpression models can be generated for CUL3 and its adapters to test function and substrate specificity [1,4,7].
Conclusion
GO:0031463 defines the Cul3-RING ubiquitin ligase complex, a modular E3 ligase that uses CUL3 and a RING-domain protein for catalysis and BTB-domain proteins for substrate selection. Its roles in muscle function, kidney ion transport, viral subversion, and pharmacological degradation highlight its broad biological and therapeutic importance [2,3,4,7]. CRISPR-based models and functional genomics approaches will continue to clarify how CRL3 complexes are assembled, regulated, and targeted in health and disease [1,2].
References
- 1. Wang P et al.. 2020. CRL3s: The BTB-CUL3-RING E3 Ubiquitin Ligases.. Adv Exp Med Biol 1217:211-223 PMID: 31898230
- 2. Yeo MJR et al.. 2025. UM171 glues asymmetric CRL3-HDAC1/2 assembly to degrade CoREST corepressors.. Nature 639(8053):232-240 PMID: 39939761
- 3. Gao C et al.. 2019. Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55.. J Biol Chem 294(16):6416-6429 PMID: 30819806
- 4. Papizan JB et al.. 2018. Cullin-3-RING ubiquitin ligase activity is required for striated muscle function in mice.. J Biol Chem 293(23):8802-8811 PMID: 29653945
- 5. Zhao B et al.. 2020. Structural Elucidation of Peptide Binding to KLHL-12, a Substrate Specific Adapter Protein in a Cul3-Ring E3 Ligase Complex.. Biochemistry 59(8):964-969 PMID: 32032490
- 6. Xing H et al.. 2010. PEST sequences mediate heat shock factor 2 turnover by interacting with the Cul3 subunit of the Cul3-RING ubiquitin ligase.. Cell Stress Chaperones 15(3):301-8 PMID: 19768582
- 7. Zhang Y et al.. 2025. Camk2n1 deficiency reduces the NaCl cotransporter activity through the CUL3/KLHL3/WNK4 complex in the kidney.. Eur J Pharmacol 990:177270 PMID: 39798916
- 8. Genau HM et al.. 2015. CUL3-KBTBD6/KBTBD7 ubiquitin ligase cooperates with GABARAP proteins to spatially restrict TIAM1-RAC1 signaling.. Mol Cell 57(6):995-1010 PMID: 25684205
