CRL3s: The BTB-CUL3-RING E3 Ubiquitin Ligases

  • Pu Wang
  • Junbin Song
  • Dan YeEmail author
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1217)


The ubiquitin proteasome pathway is one of the major regulatory tools used by eukaryotic cells. The evolutionarily conserved cullin family proteins can assemble as many as >600 distinct E3 ubiquitin ligase complexes that regulate diverse cellular pathways. In most of Cullin-RING ubiquitin ligase (CRL) complexes, separate linker and adaptor proteins build the substrate recognition module. Differently, a single BTB-containing adaptor molecule utilizing two protein interaction sites can link the CUL3 scaffold to the substrate, forming as many as 188 CUL3-BTB E3 ligase complexes in mammals. Here, we review the most recent studies on CRL3 complexes, with a focus on the model for CUL3 assembly with its BTB-containing substrate receptors. Also, we summarize the current knowledge of CRL3 substrates and their relevant biological functions. Next, we discuss the mutual exclusivity of somatic mutations in KEAP1, NRF2, and CUL3 in human lung cancer. Finally, we highlight new strategies to expand CUL3 substrates and discuss outstanding questions remaining in the field.


CRL3 BTB NRF2-KEAP1-CUL3 Lung cancer 



Auxin-induced degradation


Ankyrin repeat and FYVE domain-containing 1


Anaphase-promoting complex


The antioxidant response element


Activating transcription factor 2


B-cell CLL/lymphoma 2


Bric-à-brac, tramtrack and broad


BTB-zinc finger


Beta-transducin repeat-containing E3 ubiquitin protein ligase


Centromere protein A


Cullin-RING ubiquitin ligase


Coronin 7




Damage-specific DNA binding protein 1


DDB1-binding WD40


Eukaryotic translation elongation factor 1 alpha 1


Epidermal growth factor receptor pathway substrate 15


Endosomal sorting complexes required for transport


Fragile histidine triad gene


Glutathione S-transferase A2


Homologous to the E6-AP Carboxyl Terminus


Intervening region


Kelch ECH associating protein 1


Kelch domain-containing protein


The Kelch-like


Lung squamous cell carcinomas


Meiosis inhibitor protein 1


Multivesicular bodies


Neural precursor cell expressed, developmentally downregulated 8


Nuclear factor, erythroid 2


Next-generation sequencing


NADPH:quinone oxidoreductase 1


Nuclear respiratory factor 1


Nuclear factor erythroid 2-related factor 2


Non-small cell lung cancer

p16 INK4a

Cyclin-dependent kinase inhibitor 2A


Polo-like kinases 1


Retinoblastoma 1


RING-In between-RING




Really Interesting New Gene


Reactive oxygen species




Secretory 31


S-Phase kinase associated protein 1


Suppressor of cytokine signaling


Speckle type BTB/POZ protein


Speckle type BTB/POZ protein like


Trans-Golgi network


Unc-51 like autophagy activating kinase 1


Whole-exome sequencing


WNK lysine deficient protein kinase 4



This work was supported by the National Key R&D Program of China (No.2016YFA0501800 to D.Y.) and the NSFC grant (No. 31871431, No. 81522033, No. 31821002 to D. Y.).


  1. Bade D, Pauleau AL, Wendler A, Erhardt S (2014) The E3 ligase CUL3/RDX controls centromere maintenance by ubiquitylating and stabilizing CENP-A in a CAL1-dependent manner. Dev Cell 28:508–519PubMedCrossRefGoogle Scholar
  2. Bai C, Sen P, Hofmann K, Ma L, Goebl M, Harper JW, Elledge SJ (1996) SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell 86:263–274PubMedPubMedCentralCrossRefGoogle Scholar
  3. Bardwell VJ, Treisman R (1994) The POZ domain: a conserved protein-protein interaction motif. Genes Dev 8:1664–1677PubMedCrossRefGoogle Scholar
  4. Beck J, Maerki S, Posch M, Metzger T, Persaud A, Scheel H, Hofmann K, Rotin D, Pedrioli P, Swedlow JR et al (2013) Ubiquitylation-dependent localization of PLK1 in mitosis. Nat Cell Biol 15:430–439PubMedCrossRefGoogle Scholar
  5. Bonifacino JS, Glick BS (2004) The mechanisms of vesicle budding and fusion. Cell 116:153–166PubMedCrossRefGoogle Scholar
  6. Buetow L, Huang DT (2016) Structural insights into the catalysis and regulation of E3 ubiquitin ligases. Nat Rev Mol Cell Biol 17:626–642PubMedPubMedCentralCrossRefGoogle Scholar
  7. Cancer Genome Atlas Research N (2012) Comprehensive genomic characterization of squamous cell lung cancers. Nature 489:519–525CrossRefGoogle Scholar
  8. Cancer Genome Atlas Research N (2014) Comprehensive molecular profiling of lung adenocarcinoma. Nature 511:543–550CrossRefGoogle Scholar
  9. Chaharbakhshi E, Jemc JC (2016) Broad-complex, tramtrack, and bric-a-brac (BTB) proteins: critical regulators of development. Genesis 54:505–518PubMedPubMedCentralCrossRefGoogle Scholar
  10. Christ L, Raiborg C, Wenzel EM, Campsteijn C, Stenmark H (2017) Cellular functions and molecular mechanisms of the ESCRT membrane-scission machinery. Trends Biochem Sci 42:42–56PubMedCrossRefGoogle Scholar
  11. Clark-Maguire S, Mains PE (1994a) Localization of the mei-1 gene product of Caenorhabditis elegans, a meiotic-specific spindle component. J Cell Biol 126:199–209PubMedCrossRefGoogle Scholar
  12. Clark-Maguire S, Mains PE (1994b) mei-1, a gene required for meiotic spindle formation in Caenorhabditis elegans, is a member of a family of ATPases. Genetics 136:533–546PubMedPubMedCentralGoogle Scholar
  13. Combes G, Alharbi I, Braga LG, Elowe S (2017) Playing polo during mitosis: PLK1 takes the lead. Oncogene 36:4819–4827PubMedCrossRefGoogle Scholar
  14. Cullinan SB, Gordan JD, Jin J, Harper JW, Diehl JA (2004) The Keap1-BTB protein is an adaptor that bridges NRF2 to a Cul3-based E3 ligase: oxidative stress sensing by a Cul3-Keap1 ligase. Mol Cell Biol 24:8477–8486PubMedPubMedCentralCrossRefGoogle Scholar
  15. Cummings CM, Bentley CA, Perdue SA, Baas PW, Singer JD (2009) The Cul3/Klhdc5 E3 ligase regulates p60/katanin and is required for normal mitosis in mammalian cells. J Biol Chem 284:11663–11675PubMedPubMedCentralCrossRefGoogle Scholar
  16. Dow MR, Mains PE (1998) Genetic and molecular characterization of the caenorhabditis elegans gene, mel-26, a postmeiotic negative regulator of mei-1, a meiotic-specific spindle component. Genetics 150:119–128PubMedPubMedCentralGoogle Scholar
  17. Frank R, Scheffler M, Merkelbach-Bruse S, Ihle MA, Kron A, Rauer M, Ueckeroth F, Konig K, Michels S, Fischer R et al (2018) Clinical and pathological characteristics of KEAP1- and NFE2L2-mutated non-small cell lung carcinoma (NSCLC). Clin Cancer Res 24:3087–3096PubMedCrossRefPubMedCentralGoogle Scholar
  18. Furukawa M, Xiong Y (2005) BTB protein Keap1 targets antioxidant transcription factor NRF2 for ubiquitination by the Cullin 3-Roc1 ligase. Mol Cell Biol 25:162–171PubMedPubMedCentralCrossRefGoogle Scholar
  19. Furukawa M, He YJ, Borchers C, Xiong Y (2003) Targeting of protein ubiquitination by BTB-Cullin 3-Roc1 ubiquitin ligases. Nat Cell Biol 5:1001–1007PubMedCrossRefPubMedCentralGoogle Scholar
  20. Geyer R, Wee S, Anderson S, Yates J, Wolf DA (2003) BTB/POZ domain proteins are putative substrate adaptors for cullin 3 ubiquitin ligases. Mol Cell 12:783–790CrossRefGoogle Scholar
  21. Glotzer M, Murray AW, Kirschner MW (1991) Cyclin is degraded by the ubiquitin pathway. Nature 349:132–138PubMedCrossRefGoogle Scholar
  22. Green RA, Paluch E, Oegema K (2012) Cytokinesis in animal cells. Annu Rev Cell Dev Biol 28:29–58PubMedCrossRefGoogle Scholar
  23. Gschweitl M, Ulbricht A, Barnes CA, Enchev RI, Stoffel-Studer I, Meyer-Schaller N, Huotari J, Yamauchi Y, Greber UF, Helenius A et al (2016) A SPOPL/Cullin-3 ubiquitin ligase complex regulates endocytic trafficking by targeting EPS15 at endosomes. Elife 5:e13841PubMedPubMedCentralCrossRefGoogle Scholar
  24. Haas AL, Warms JV, Hershko A, Rose IA (1982) Ubiquitin-activating enzyme. Mechanism and role in protein-ubiquitin conjugation. J Biol Chem 257:2543–2548PubMedGoogle Scholar
  25. Hast BE, Cloer EW, Goldfarb D, Li H, Siesser PF, Yan F, Walter V, Zheng N, Hayes DN, Major MB (2014) Cancer-derived mutations in KEAP1 impair NRF2 degradation but not ubiquitination. Cancer Res 74:808–817PubMedCrossRefGoogle Scholar
  26. He YJ, McCall CM, Hu J, Zeng Y, Xiong Y (2006) DDB1 functions as a linker to recruit receptor WD40 proteins to CUL4-ROC1 ubiquitin ligases. Genes Dev 20:2949–2954PubMedPubMedCentralCrossRefGoogle Scholar
  27. Hershko A, Heller H, Elias S, Ciechanover A (1983) Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J Biol Chem 258:8206–8214PubMedGoogle Scholar
  28. Huang G, Kaufman AJ, Xu K, Manova K, Singh B (2017) Squamous cell carcinoma-related oncogene (SCCRO) neddylates Cul3 protein to selectively promote midbody localization and activity of Cul3(KLHL21) protein complex during abscission. J Biol Chem 292:15254–15265PubMedPubMedCentralCrossRefGoogle Scholar
  29. Huotari J, Meyer-Schaller N, Hubner M, Stauffer S, Katheder N, Horvath P, Mancini R, Helenius A, Peter M (2012) Cullin-3 regulates late endosome maturation. Proc Natl Acad Sci U S A 109:823–828PubMedPubMedCentralCrossRefGoogle Scholar
  30. Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD, Yamamoto M (1999) Keap1 represses nuclear activation of antioxidant responsive elements by NRF2 through binding to the amino-terminal Neh2 domain. Genes Dev 13:76–86PubMedPubMedCentralCrossRefGoogle Scholar
  31. Jin L, Pahuja KB, Wickliffe KE, Gorur A, Baumgartel C, Schekman R, Rape M (2012) Ubiquitin-dependent regulation of COPII coat size and function. Nature 482:495–500PubMedPubMedCentralCrossRefGoogle Scholar
  32. Kamura T, Maenaka K, Kotoshiba S, Matsumoto M, Kohda D, Conaway RC, Conaway JW, Nakayama KI (2004) VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases. Genes Dev 18:3055–3065PubMedPubMedCentralCrossRefGoogle Scholar
  33. Katsuoka F, Motohashi H, Ishii T, Aburatani H, Engel JD, Yamamoto M (2005) Genetic evidence that small maf proteins are essential for the activation of antioxidant response element-dependent genes. Mol Cell Biol 25:8044–8051PubMedPubMedCentralCrossRefGoogle Scholar
  34. King RW, Peters JM, Tugendreich S, Rolfe M, Hieter P, Kirschner MW (1995) A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B. Cell 81:279–288PubMedCrossRefGoogle Scholar
  35. Kobayashi A, Kang MI, Okawa H, Ohtsuji M, Zenke Y, Chiba T, Igarashi K, Yamamoto M (2004) Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of NRF2. Mol Cell Biol 24:7130–7139PubMedPubMedCentralCrossRefGoogle Scholar
  36. Koiwai K, Maezawa S, Hayano T, Iitsuka M, Koiwai O (2008) BPOZ-2 directly binds to eEF1A1 to promote eEF1A1 ubiquitylation and degradation and prevent translation. Genes Cells 13:593–607PubMedCrossRefGoogle Scholar
  37. Kurz T, Pintard L, Willis JH, Hamill DR, Gonczy P, Peter M, Bowerman B (2002) Cytoskeletal regulation by the Nedd8 ubiquitin-like protein modification pathway. Science 295:1294–1298PubMedCrossRefGoogle Scholar
  38. Liu CC, Lin YC, Chen YH, Chen CM, Pang LY, Chen HA, Wu PR, Lin MY, Jiang ST, Tsai TF et al (2016) Cul3-KLHL20 ubiquitin ligase governs the turnover of ULK1 and VPS34 complexes to control autophagy termination. Mol Cell 61:84–97PubMedCrossRefGoogle Scholar
  39. Lo SC, Hannink M (2006) CAND1-mediated substrate adaptor recycling is required for efficient repression of NRF2 by Keap1. Mol Cell Biol 26:1235–1244PubMedPubMedCentralCrossRefGoogle Scholar
  40. Lo SC, Li X, Henzl MT, Beamer LJ, Hannink M (2006) Structure of the Keap1:NRF2 interface provides mechanistic insight into NRF2 signaling. EMBO J 25:3605–3617PubMedPubMedCentralCrossRefGoogle Scholar
  41. Lyapina SA, Correll CC, Kipreos ET, Deshaies RJ (1998) Human CUL1 forms an evolutionarily conserved ubiquitin ligase complex (SCF) with SKP1 and an F-box protein. Proc Natl Acad Sci U S A 95:7451–7456PubMedPubMedCentralCrossRefGoogle Scholar
  42. Ma J, Chang K, Peng J, Shi Q, Gan H, Gao K, Feng K, Xu F, Zhang H, Dai B et al (2018) SPOP promotes ATF2 ubiquitination and degradation to suppress prostate cancer progression. J Exp Clin Cancer Res 37:145PubMedPubMedCentralCrossRefGoogle Scholar
  43. Maeda I, Ohta T, Koizumi H, Fukuda M (2001) In vitro ubiquitination of cyclin D1 by ROC1-CUL1 and ROC1-CUL3. FEBS Lett 494:181–185PubMedCrossRefPubMedCentralGoogle Scholar
  44. Maekawa M, Tanigawa K, Sakaue T, Hiyoshi H, Kubota E, Joh T, Watanabe Y, Taguchi T, Higashiyama S (2017) Cullin-3 and its adaptor protein ANKFY1 determine the surface level of integrin beta1 in endothelial cells. Biol Open 6:1707–1719PubMedPubMedCentralCrossRefGoogle Scholar
  45. McIntosh JR (2016) Mitosis. Cold Spring Harb Perspect Biol 8Google Scholar
  46. McMahon M, Lamont DJ, Beattie KA, Hayes JD (2010) Keap1 perceives stress via three sensors for the endogenous signaling molecules nitric oxide, zinc, and alkenals. Proc Natl Acad Sci U S A 107:18838–18843PubMedPubMedCentralCrossRefGoogle Scholar
  47. Merlet J, Burger J, Gomes JE, Pintard L (2009) Regulation of cullin-RING E3 ubiquitin-ligases by neddylation and dimerization. Cell Mol Life Sci 66:1924–1938PubMedPubMedCentralCrossRefGoogle Scholar
  48. Metzger MB, Pruneda JN, Klevit RE, Weissman AM (2014) RING-type E3 ligases: master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination. Biochim Biophys Acta 1843:47–60PubMedCrossRefPubMedCentralGoogle Scholar
  49. Metzger T, Kleiss C, Sumara I (2013) CUL3 and protein kinases: insights from PLK1/KLHL22 interaction. Cell Cycle 12:2291–2296PubMedPubMedCentralCrossRefGoogle Scholar
  50. Michel JJ, Xiong Y (1998) Human CUL-1, but not other cullin family members, selectively interacts with SKP1 to form a complex with SKP2 and cyclin A. Cell Growth Differ 9:435–449PubMedGoogle Scholar
  51. Moghe S, Jiang F, Miura Y, Cerny RL, Tsai MY, Furukawa M (2012) The CUL3-KLHL18 ligase regulates mitotic entry and ubiquitylates Aurora-A. Biol Open 1:82–91PubMedCrossRefGoogle Scholar
  52. Motohashi H, Yamamoto M (2004) NRF2-Keap1 defines a physiologically important stress response mechanism. Trends Mol Med 10:549–557PubMedCrossRefGoogle Scholar
  53. Negrini S, Gorgoulis VG, Halazonetis TD (2010) Genomic instability--an evolving hallmark of cancer. Nat Rev Mol Cell Biol 11:220–228PubMedCrossRefGoogle Scholar
  54. Nguyen T, Sherratt PJ, Huang HC, Yang CS, Pickett CB (2003) Increased protein stability as a mechanism that enhances NRF2-mediated transcriptional activation of the antioxidant response element. Degradation of NRF2 by the 26 S proteasome. J Biol Chem 278:4536–4541PubMedCrossRefGoogle Scholar
  55. Nikonova AS, Astsaturov I, Serebriiskii IG, Dunbrack RL Jr, Golemis EA (2013) Aurora A kinase (AURKA) in normal and pathological cell division. Cell Mol Life Sci 70:661–687PubMedCrossRefGoogle Scholar
  56. Ohta T, Michel JJ, Schottelius AJ, Xiong Y (1999) ROC1, a homolog of APC11, represents a family of cullin partners with an associated ubiquitin ligase activity. Mol Cell 3:535–541PubMedCrossRefGoogle Scholar
  57. Petroski MD, Deshaies RJ (2005) Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol 6:9–20PubMedPubMedCentralCrossRefGoogle Scholar
  58. Pickart CM, Eddins MJ (2004) Ubiquitin: structures, functions, mechanisms. Biochim Biophys Acta 1695:55–72PubMedCrossRefPubMedCentralGoogle Scholar
  59. Pickart CM, Rose IA (1985) Functional heterogeneity of ubiquitin carrier proteins. J Biol Chem 260:1573–1581PubMedGoogle Scholar
  60. Pintard L, Kurz T, Glaser S, Willis JH, Peter M, Bowerman B (2003a) Neddylation and deneddylation of CUL-3 is required to target MEI-1/Katanin for degradation at the meiosis-to-mitosis transition in C. elegans. Curr Biol 13:911–921CrossRefGoogle Scholar
  61. Pintard L, Willis JH, Willems A, Johnson JL, Srayko M, Kurz T, Glaser S, Mains PE, Tyers M, Bowerman B et al (2003b) The BTB protein MEL-26 is a substrate-specific adaptor of the CUL-3 ubiquitin-ligase. Nature 425:311–316CrossRefGoogle Scholar
  62. Rojo de la Vega M, Chapman E, Zhang DD (2018) NRF2 and the hallmarks of Cancer. Cancer Cell 34:21–43PubMedCrossRefGoogle Scholar
  63. Rushmore TH, Morton MR, Pickett CB (1991) The antioxidant responsive element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional activity. J Biol Chem 266:11632–11639PubMedGoogle Scholar
  64. Shibata S, Zhang J, Puthumana J, Stone KL, Lifton RP (2013) Kelch-like 3 and Cullin 3 regulate electrolyte homeostasis via ubiquitination and degradation of WNK4. Proc Natl Acad Sci USA 110:7838–7843CrossRefGoogle Scholar
  65. Singer JD, Gurian-West M, Clurman B, Roberts JM (1999) Cullin-3 targets cyclin E for ubiquitination and controls S phase in mammalian cells. Genes Dev 13:2375–2387PubMedPubMedCentralCrossRefGoogle Scholar
  66. Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, Hoque MO, Herman JG, Baylin SB, Sidransky D, Gabrielson E et al (2006) Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer. PLoS Med 3:e420PubMedPubMedCentralCrossRefGoogle Scholar
  67. Stogios PJ, Downs GS, Jauhal JJ, Nandra SK, Prive GG (2005) Sequence and structural analysis of BTB domain proteins. Genome Biol 6:R82PubMedPubMedCentralCrossRefGoogle Scholar
  68. Sumara I, Quadroni M, Frei C, Olma MH, Sumara G, Ricci R, Peter M (2007) A Cul3-based E3 ligase removes Aurora B from mitotic chromosomes, regulating mitotic progression and completion of cytokinesis in human cells. Dev Cell 12:887–900CrossRefGoogle Scholar
  69. Wakabayashi N, Itoh K, Wakabayashi J, Motohashi H, Noda S, Takahashi S, Imakado S, Kotsuji T, Otsuka F, Roop DR et al (2003) Keap1-null mutation leads to postnatal lethality due to constitutive NRF2 activation. Nat Genet 35:238–245PubMedCrossRefGoogle Scholar
  70. Xu L, Wei Y, Reboul J, Vaglio P, Shin TH, Vidal M, Elledge SJ, Harper JW (2003) BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3. Nature 425:316–321CrossRefGoogle Scholar
  71. Yesbolatova A, Tominari Y, Kanemaki MT (2019) Ligand-induced genetic degradation as a tool for target validation. Drug Discov Today Technol 31:91–98PubMedCrossRefGoogle Scholar
  72. Yuan WC, Lee YR, Lin SY, Chang LY, Tan YP, Hung CC, Kuo JC, Liu CH, Lin MY, Xu M et al (2014) K33-linked Polyubiquitination of Coronin 7 by Cul3-KLHL20 ubiquitin E3 ligase regulates protein trafficking. Mol Cell 54:586–600PubMedCrossRefGoogle Scholar
  73. Zhang DD, Hannink M (2003) Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of NRF2 and for stabilization of NRF2 by chemopreventive agents and oxidative stress. Mol Cell Biol 23:8137–8151PubMedPubMedCentralCrossRefGoogle Scholar
  74. Zhang DD, Lo SC, Cross JV, Templeton DJ, Hannink M (2004) Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex. Mol Cell Biol 24:10941–10953PubMedPubMedCentralCrossRefGoogle Scholar
  75. Zheng N, Shabek N (2017) Ubiquitin ligases: structure, function, and regulation. Annu Rev Biochem 86:129–157PubMedPubMedCentralCrossRefGoogle Scholar
  76. Zhu M, Fahl WE (2001) Functional characterization of transcription regulators that interact with the electrophile response element. Biochem Biophys Res Commun 289:212–219PubMedCrossRefGoogle Scholar
  77. Zhuang M, Calabrese MF, Liu J, Waddell MB, Nourse A, Hammel M, Miller DJ, Walden H, Duda DM, Seyedin SN et al (2009) Structures of SPOP-substrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases. Mol Cell 36:39–50PubMedPubMedCentralCrossRefGoogle Scholar
  78. Zollman S, Godt D, Prive GG, Couderc JL, Laski FA (1994) The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc Natl Acad Sci U S A 91:10717–10721PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2020

Authors and Affiliations

  1. 1.Molecular and Cell Biology LabInstitutes of Biomedical Sciences, Shanghai Medical College, Fudan UniversityShanghaiChina

Personalised recommendations