Affinage

KLHL20

Kelch-like protein 20 · UniProt Q9Y2M5

Length
609 aa
Mass
68.0 kDa
Annotated
2026-04-28
23 papers in source corpus 14 papers cited in narrative 14 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

KLHL20 is the substrate-recognition subunit of a Cullin 3–ROC1 RING E3 ubiquitin ligase that controls diverse cellular processes—including autophagy termination, interferon-induced apoptosis, hypoxia/HIF-1α signaling, post-Golgi trafficking, and neurite outgrowth—by targeting specific substrates for ubiquitination. Its BTB domain engages Cul3 while its six-bladed Kelch β-propeller recruits substrates (DAPK1, PML, ULK1, VPS34/Beclin-1, PDZ-RhoGEF, coronin 7, SERINC5, ZBTB7A), with substrate recognition often gated by prior phosphorylation (CDK1/2 for PML, p38 for PDZ-RhoGEF, ULK1 autophosphorylation) or proline isomerization by Pin1 (PMID:20389280, PMID:21840486, PMID:21670212, PMID:26687681). KLHL20 catalyzes both K48-linked degradative and K33-linked non-degradative polyubiquitin chains in a substrate-dependent manner: K33-linked chains on coronin 7 direct its TGN targeting and post-Golgi carrier biogenesis, whereas K48-linked chains drive proteasomal turnover of DAPK1, PML, ULK1, and other substrates (PMID:24768539, PMID:35474067). Its activity is spatially regulated by TGN localization and by interferon-induced sequestration into PML nuclear bodies, which separates it from cytoplasmic substrates such as DAPK1, and is negatively regulated by the pseudo-substrate KLHL39, which competes for its Kelch domain (PMID:20389280, PMID:25619834).

Mechanistic history

Synthesis pass · year-by-year structured walk · 12 steps
  1. 2003 Medium

    Establishing that KLHL20 is a BTB/Kelch-repeat protein that binds F-actin and transiently localizes to cell–cell contacts revealed an initial cytoskeletal scaffolding function downstream of Rac1.

    Evidence Overexpression, dominant-negative constructs, and Rac1-CA co-expression in MDCK cells

    PMID:14668487

    Open questions at the time
    • Single cell type (MDCK); relevance to non-epithelial contexts unknown
    • Mechanism linking KLHL20 to Rac1-induced actin assembly not resolved at the molecular level
  2. 2007 Medium

    Demonstrating that KLHL20 interacts with the RhoGEF ECT2 upon VEGF stimulation and is required for RhoA activation linked its function to Rho-family GTPase signaling in angiogenesis.

    Evidence Co-IP of KLHL20–ECT2, RhoA-GTP pull-down, siRNA knockdown, and sprouting angiogenesis assay in endothelial cells

    PMID:17395875

    Open questions at the time
    • Whether KLHL20 ubiquitinates ECT2 or acts as a scaffold was not determined
    • No independent replication in a second endothelial model
  3. 2010 High

    Identification of KLHL20 as a Cul3 substrate adaptor that polyubiquitinates DAPK1 for proteasomal degradation established its E3 ligase identity, and its IFN-induced sequestration into PML nuclear bodies explained how IFN stabilizes DAPK to trigger apoptosis and autophagy.

    Evidence Co-IP, in vitro ubiquitination, proteasome-inhibitor rescue, domain mapping (BTB→Cul3; Kelch→DAPK), immunofluorescence of PML-body sequestration

    PMID:20389280

    Open questions at the time
    • Ubiquitin chain linkage type on DAPK1 not specified
    • Mechanism of KLHL20 recruitment into PML bodies not defined
  4. 2011 High

    Showing that Cul3-KLHL20 degrades PML in a CDK1/2-phosphorylation and Pin1-isomerization dependent manner under hypoxia, with HIF-1α transcriptionally upregulating KLHL20, revealed a feedforward loop amplifying HIF-1α signaling in cancer.

    Evidence Ubiquitination assay, CDK/Pin1 inhibitors, HIF-1α ChIP, siRNA, xenograft tumor models

    PMID:21840486

    Open questions at the time
    • Whether the CDK1/2-Pin1 priming mechanism applies to other KLHL20 substrates is untested
    • Structural basis of phospho-PML recognition by KLHL20 Kelch domain unknown
  5. 2011 High

    Demonstrating Cul3-KLHL20-mediated degradation of PDZ-RhoGEF, primed by p38 phosphorylation, to restrict RhoA and promote neurotrophin-driven neurite outgrowth established a second phosphorylation-gated substrate and a neuronal function for the ligase.

    Evidence In vitro ubiquitination, p38 inhibitor, RhoA-GTP assay, siRNA in primary hippocampal/cortical neurons, neurite outgrowth measurement

    PMID:21670212

    Open questions at the time
    • In vivo neuronal phenotype of Klhl20 knockout not reported
    • Specific phosphorylation sites on PDZ-RhoGEF mediating KLHL20 recognition not mapped
  6. 2014 High

    Discovery that KLHL20 localizes to the TGN and catalyzes non-degradative K33-linked polyubiquitination of coronin 7, recognized by Eps15 to direct TGN targeting and post-Golgi carrier formation, expanded the ligase's repertoire beyond degradative ubiquitination.

    Evidence In vitro ubiquitination with K33R mutants, Crn7-Eps15 interaction mapping, vesicle trafficking assay, forced-TGN-targeting rescue

    PMID:24768539

    Open questions at the time
    • Whether other KLHL20 substrates receive K33 chains at the TGN is unexplored
    • Structural basis of K33 linkage specificity not determined
  7. 2015 High

    Identification of KLHL39 as a competitive inhibitor that binds the KLHL20 Kelch domain, displacing substrates (PML, DAPK) and disrupting Cul3 association, established a dedicated endogenous negative regulator of the Cul3-KLHL20 ligase.

    Evidence Reciprocal Co-IP, domain mapping, ubiquitination/stability assays, siRNA, in vivo metastasis model

    PMID:25619834

    Open questions at the time
    • Regulation of KLHL39 expression or activity is not characterized
    • Whether KLHL39 affects all KLHL20 substrates equally is unknown
  8. 2015 High

    Showing that Cul3-KLHL20 ubiquitinates ULK1 (gated by ULK1 autophosphorylation) and coordinately degrades ATG13, VPS34, Beclin-1, and ATG14 during prolonged starvation established KLHL20 as the central feedback terminator of autophagy.

    Evidence In vitro ubiquitination, autophosphorylation mutants, siRNA/knockout, pulse-chase, starvation assay, diabetic mouse muscle-atrophy model

    PMID:26687681

    Open questions at the time
    • Whether ULK1 autophagy-complex components are direct or indirect KLHL20 substrates not fully delineated
    • Upstream signal that activates KLHL20 during prolonged starvation not identified
  9. 2019 High

    A 1.1-Å crystal structure of the KLHL20 Kelch domain bound to a DAPK1 degron peptide provided the first atomic-resolution view of substrate recognition, showing the 'LPDLV' motif inserts into the β-propeller central pocket contacting all six blades.

    Evidence X-ray crystallography at 1.1 Å, peptide binding assays, mutagenesis

    PMID:31279627

    Open questions at the time
    • Whether the same pocket accommodates PML, ULK1, coronin 7, and other substrates is not structurally resolved
    • No co-structure with full-length substrate or with the Cul3 complex
  10. 2022 High

    Demonstrating that Cul3-KLHL20 ubiquitinates SERINC5 at K130 with both K33 and K48 chains—K33 controlling plasma-membrane delivery and K48 controlling surface downregulation—showed dual-linkage regulation of a single substrate with functional consequences for HIV-1 restriction.

    Evidence Linkage-specific ubiquitin mutants, K130 mutagenesis, flow cytometry, HIV-1 infection assay

    PMID:35474067

    Open questions at the time
    • Determinants selecting K33 versus K48 chain assembly on the same substrate are unknown
    • Whether KLHL20 directly affects HIV-1 pathogenesis in vivo not tested
  11. 2022 Medium

    Validation of KLHL20 as a recruitable E3 ligase for PROTAC-mediated degradation of BET-family proteins demonstrated its pharmacological tractability beyond its endogenous substrate scope.

    Evidence Macrocyclic KLHL20 ligand–JQ1 PROTAC (BTR2004), Western blot for BRD2/3/4 degradation in cells

    PMID:36328355

    Open questions at the time
    • Structural basis of macrocycle–KLHL20 interaction not determined
    • Selectivity and in vivo utility of KLHL20-based PROTACs not characterized
  12. 2023 Medium

    Linking p53-induced KLHL20 expression to ubiquitin-dependent degradation of the proto-oncogene ZBTB7A, opposed by HSP90-mediated stabilization, placed KLHL20 downstream of p53 tumor-suppressor signaling.

    Evidence Co-IP, ubiquitination assay, p53 overexpression, HSP90 inhibitor (17-AAG), ZBTB7A stability measurement

    PMID:37011832

    Open questions at the time
    • KLHL20-binding degron on ZBTB7A not mapped
    • Awaits independent replication; single-lab study

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key unresolved questions include how the same Cul3-KLHL20 complex selects between K33 and K48 chain assembly, the structural basis by which diverse substrates engage the central Kelch pocket, the upstream signals controlling KLHL20's spatial distribution between TGN and nucleus, and the physiological consequences of Klhl20 loss in whole-organism models.
  • No Klhl20-null mouse phenotype reported
  • Chain-linkage selectivity determinants unresolved
  • Full-length Cul3-KLHL20-substrate ternary structure unavailable

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140096 catalytic activity, acting on a protein 7 GO:0008092 cytoskeletal protein binding 1
Localization
GO:0005794 Golgi apparatus 2 GO:0005829 cytosol 2 GO:0005634 nucleus 1
Pathway
R-HSA-392499 Metabolism of proteins 6 R-HSA-162582 Signal Transduction 2 R-HSA-5653656 Vesicle-mediated transport 2 R-HSA-5357801 Programmed Cell Death 1 R-HSA-9612973 Autophagy 1
Partners
CRN7CUL3DAPK1ECT2KLHL39PMLSERINC5ULK1
Complex memberships
Cul3-KLHL20-ROC1 E3 ubiquitin ligase

Evidence

Reading pass · 14 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2003 KLHL20 (KLEIP) contains BTB/POZ and six kelch-repeat domains, binds F-actin directly, and localizes transiently to cell-cell contact sites in MDCK cells during contact induction but not at mature junctions; constitutively active Rac1 enhances its recruitment and F-actin assembly at adhesion sites, and the N-terminal half of KLHL20 (lacking the actin-binding site) acts dominantly negative to inhibit Rac1-induced actin assembly. Overexpression, dominant-negative constructs, E-cadherin bead assay, cytochalasin B treatment, constitutively active Rac1 co-expression, immunofluorescence localization in MDCK cells Molecular biology of the cell Medium 14668487
2007 KLHL20 (KLEIP) acts downstream of VEGF to activate RhoA GTPase in endothelial cells; KLHL20 physically associates with the RhoGEF ECT2 upon VEGF stimulation, and KLHL20 depletion blunts VEGF-induced RhoA activation and sprouting angiogenesis without affecting ERK1/2 activation. siRNA knockdown, Co-IP (KLHL20-ECT2 interaction), RhoA-GTP pull-down assay, sprouting angiogenesis in-gel assay, ERK1/2 activation assay Circulation research Medium 17395875
2010 KLHL20 forms a Cul3-KLHL20-ROC1 E3 ubiquitin ligase complex that binds DAPK via its Kelch-repeat domain and Cul3 via its BTB domain, polyubiquitinates DAPK, and drives its proteasomal degradation; IFN-α/γ treatment sequesters KLHL20 into PML nuclear bodies, separating it from DAPK and thereby stabilizing DAPK to mediate IFN-induced cell death and autophagy. Co-IP, ubiquitination assay, proteasome inhibitor rescue, siRNA knockdown, immunofluorescence (KLHL20 nuclear body sequestration), domain-mapping experiments The EMBO journal High 20389280
2011 The Cul3-KLHL20 E3 ligase mediates hypoxia-induced proteasomal degradation of PML; CDK1/2 phosphorylation and Pin1 isomerization cooperate to prime PML for KLHL20 recognition, and HIF-1 transcriptionally upregulates KLHL20 as part of a positive feedback loop to maximize HIF-1α induction, metabolic reprogramming, EMT, and angiogenesis. Co-IP, ubiquitination assay, proteasome inhibitor rescue, CDK1/2 inhibitor treatment, Pin1 requirement, HIF-1α ChIP/reporter assay, siRNA knockdown, xenograft tumor models Cancer cell High 21840486
2011 The Cul3-KLHL20 E3 ligase targets PDZ-RhoGEF for ubiquitin-dependent proteasomal degradation; p38 MAPK phosphorylates PDZ-RhoGEF to enable its recruitment to KLHL20, and this degradation restricts RhoA activity to facilitate growth cone spreading and neurite outgrowth in hippocampal and cortical neurons in response to neurotrophins (BDNF, NT-3). Co-IP, ubiquitination assay, proteasome inhibitor rescue, p38 inhibitor treatment, RhoA activity assay, siRNA knockdown in primary neurons, neurite outgrowth assay The Journal of cell biology High 21670212
2014 KLHL20 localizes to the trans-Golgi network (TGN) and the Cul3-KLHL20 E3 ligase catalyzes non-degradative K33-linked polyubiquitination on coronin 7 (Crn7); this K33-ubiquitin chain is recognized by Eps15, enabling Crn7 targeting to the TGN, TGN-pool F-actin assembly, and post-Golgi transport carrier biogenesis. Immunofluorescence localization (TGN), Co-IP, in vitro ubiquitination assay, ubiquitin chain linkage mutants (K33R), Crn7-Eps15 interaction assay, vesicle trafficking assay, rescue experiments with forced TGN targeting of Crn7 Molecular cell High 24768539
2015 KLHL20 is a substrate adaptor of Cul3 that ubiquitinates ULK1 upon autophagy induction; ULK1 autophosphorylation facilitates its recruitment to KLHL20, leading to ULK1 ubiquitination and proteasomal degradation; KLHL20 also governs degradation of ATG13, VPS34, Beclin-1, and ATG14 during prolonged starvation, constituting a feedback mechanism to terminate autophagy. Co-IP, in vitro ubiquitination assay, ULK1 autophosphorylation mutants, siRNA/knockout, pulse-chase half-life measurement, starvation-induced autophagy assay, diabetic mouse model (muscle atrophy phenotype) Molecular cell High 26687681
2015 KLHL39, another BTB-Kelch protein that does not bind Cul3, binds to the substrate-binding Kelch domain of KLHL20, disrupting substrate (PML, DAPK) binding to KLHL20 and KLHL20 binding to Cul3, thereby acting as a negative regulator of the Cul3-KLHL20 E3 ligase complex. Co-IP, domain-mapping, ubiquitination assay, stability assay, siRNA knockdown, invasion/migration assays, in vivo metastasis model Oncogene High 25619834
2019 A 1.1-Å crystal structure of the KLHL20 Kelch domain bound to a DAPK1 peptide reveals that the 'LPDLV'-containing motif in the DAPK1 death domain inserts as a loose helical turn into the central pocket of the β-propeller, contacting all six blades via salt-bridge and hydrophobic interactions including key tryptophan and cysteine residues. X-ray crystallography (1.1-Å resolution), peptide binding assays, mutagenesis of binding motif Structure High 31279627
2022 The Cul3-KLHL20 E3 ligase, localized to the TGN, polyubiquitinates SERINC5 at lysine 130 via K33/K48-linked ubiquitin chains; K33-linked ubiquitination determines SERINC5 plasma membrane expression (post-Golgi trafficking), while K48-linked ubiquitination contributes to SERINC5 downregulation from the cell surface, modulating HIV-1 restriction. Co-IP, ubiquitin linkage-specific assays (K33R and K48R mutants), site-directed mutagenesis of K130, flow cytometry for surface expression, HIV-1 infection assay Nature communications High 35474067
2022 A synthetic macrocyclic ligand engaging KLHL20 was developed and used to construct a PROTAC (BTR2004) linking KLHL20 to JQ1, validating that Cul3-KLHL20 can be recruited to polyubiquitinate and degrade BET family proteins (BRD2, BRD3, BRD4) in cells. PROTAC/degrader chemistry, ubiquitin-proteasome-dependent degradation assay, Western blot, cellular target engagement Genes & development Medium 36328355
2023 p53 upregulates KLHL20 expression, and Cul3-KLHL20 mediates ubiquitin-proteasomal degradation of the proto-oncogene ZBTB7A; HSP90 stabilizes ZBTB7A and opposes this KLHL20-dependent degradation. Co-IP (ZBTB7A-HSP90, ZBTB7A-KLHL20), ubiquitination assay, HSP90 inhibitor (17-AAG) treatment, p53 overexpression, Western blot for ZBTB7A stability Biochimica et biophysica acta. Gene regulatory mechanisms Medium 37011832
2026 ZFTA is identified as a novel KLHL20 substrate; ZFTA peptides bind the KLHL20 Kelch domain with ~35 µM affinity via 'fuzzy binding' (feature-specific rather than sequence-specific recognition of disordered regions), with specificity for KLHL20 over KLHL12. Biosensor (SPR/BLI) binding analysis, AlphaFold2-based structural modeling, peptide truncation and alanine scanning Chembiochem Low 41762441
2026 In high glucose conditions, KLHL20 expression is transcriptionally suppressed, reducing DAPK1 ubiquitination and proteasomal degradation; Co-IP confirmed KLHL20-DAPK1 physical interaction, and KLHL20 overexpression restores DAPK1 ubiquitination and protects renal tubular cells from mitochondrial apoptosis through DAPK1 as the downstream effector. Co-IP, ubiquitination assay, KLHL20 overexpression, DAPK1 knockdown, protein half-life measurement, HK-2 cells and db/db mouse model Biochemical and biophysical research communications Medium 41637987

Source papers

Stage 0 corpus · 23 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2015 Cul3-KLHL20 Ubiquitin Ligase Governs the Turnover of ULK1 and VPS34 Complexes to Control Autophagy Termination. Molecular cell 186 26687681
2011 A Cullin3-KLHL20 Ubiquitin ligase-dependent pathway targets PML to potentiate HIF-1 signaling and prostate cancer progression. Cancer cell 151 21840486
2014 K33-Linked Polyubiquitination of Coronin 7 by Cul3-KLHL20 Ubiquitin E3 Ligase Regulates Protein Trafficking. Molecular cell 134 24768539
2010 The Cullin 3 substrate adaptor KLHL20 mediates DAPK ubiquitination to control interferon responses. The EMBO journal 104 20389280
2011 PDZ-RhoGEF ubiquitination by Cullin3-KLHL20 controls neurotrophin-induced neurite outgrowth. The Journal of cell biology 44 21670212
2015 KLHL39 suppresses colon cancer metastasis by blocking KLHL20-mediated PML and DAPK ubiquitination. Oncogene 33 25619834
2019 Structural Basis for Recruitment of DAPK1 to the KLHL20 E3 Ligase. Structure (London, England : 1993) 32 31279627
2003 Novel kelch-like protein, KLEIP, is involved in actin assembly at cell-cell contact sites of Madin-Darby canine kidney cells. Molecular biology of the cell 32 14668487
2007 The BTB-Kelch protein KLEIP controls endothelial migration and sprouting angiogenesis. Circulation research 29 17395875
2014 Angiopoietin-1 is regulated by miR-204 and contributes to corneal neovascularization in KLEIP-deficient mice. Investigative ophthalmology & visual science 27 24917145
2016 Cul3-KLHL20 ubiquitin ligase: physiological functions, stress responses, and disease implications. Cell division 18 27042198
2022 Cul3-KLHL20 E3 ubiquitin ligase plays a key role in the arms race between HIV-1 Nef and host SERINC5 restriction. Nature communications 17 35474067
2022 A synthetic KLHL20 ligand to validate CUL3KLHL20 as a potent E3 ligase for targeted protein degradation. Genes & development 15 36328355
2012 KLEIP deficiency in mice causes progressive corneal neovascular dystrophy. Investigative ophthalmology & visual science 14 22511632
2016 KLHL20 links the ubiquitin-proteasome system to autophagy termination. Autophagy 13 26985984
2014 Kelch-like ECT2-interacting protein KLEIP regulates late-stage pulmonary maturation via Hif-2α in mice. Disease models & mechanisms 10 24785085
2022 De novo missense variants in the E3 ubiquitin ligase adaptor KLHL20 cause a developmental disorder with intellectual disability, epilepsy, and autism spectrum disorder. Genetics in medicine : official journal of the American College of Medical Genetics 6 36214804
2024 KLHL20 and its role in cell homeostasis: A new perspective and therapeutic potential. Life sciences 3 39233199
2023 Post-translational regulation of proto-oncogene ZBTB7A expression by p53 status in cancer cells: HSP90-dependent stabilization vs. p53-KLHL20-ubiquitin proteasomal degradation. Biochimica et biophysica acta. Gene regulatory mechanisms 3 37011832
2026 KLHL20 alleviates high glucose-induced mitochondrial apoptosis in renal tubular cells by targeting DAPK1 for ubiquitination and degradation. Biochemical and biophysical research communications 0 41637987
2026 Identification of ZFTA as a Novel KLHL20 Substrate and Mechanistic Insights Into Fuzzy Binding of Disordered Peptides via Biosensor Analysis and Computational Modelling. Chembiochem : a European journal of chemical biology 0 41762441
2025 Temporal and Spatial Characterization of CUL3KLHL20-Driven Targeted Degradation of BET Family BRD Proteins by the Macrocycle-Based Degrader BTR2004. ACS chemical biology 0 40891966
2024 Temporal and Spatial Characterization of CUL3KLHL20-driven Targeted Degradation of BET family, BRD Proteins by the Macrocycle-based Degrader BTR2004. bioRxiv : the preprint server for biology 0 39677683