Affinage

KLHL12

Kelch-like protein 12 · UniProt Q53G59

Length
568 aa
Mass
63.3 kDa
Annotated
2026-06-10
13 papers in source corpus 12 papers cited in narrative 12 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 5/5 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

KLHL12 is a BTB-Kelch substrate adaptor that assembles with Cullin-3 into a CRL3^KLHL12 E3 ubiquitin ligase, recruiting substrates through its Kelch beta-propeller domain to direct their ubiquitination across diverse cellular processes (PMID:16547521, PMID:32574548). Its substrates engage the top face of the six-bladed Kelch propeller via a conserved 'PGXPP' degron, defined crystallographically with a Dishevelled peptide and shared among multiple partners including SEC31, PEF1, PLEKHA4 and DRD4 (PMID:32574548, PMID:32032490). Through this adaptor activity KLHL12 poly-ubiquitinates Dishevelled in a Wnt-dependent manner to drive its degradation and antagonize Wnt-beta-catenin signaling in vertebrate embryos (PMID:16547521), and ubiquitylates the ER three-way-junction protein Lunapark, a modification required for normal growth and development (PMID:32433973). In the secretory pathway KLHL12 monoubiquitylates the COPII coat component SEC31A to enlarge COPII vesicles for procollagen export, an activity directly inhibited by LZTR1 to restrain collagen secretion (PMID:37626065). KLHL12 also targets the dopamine D4 receptor through its Kelch domain, promoting a non-degradative ubiquitination that can occur on non-lysine residues, distinguishing it from its degradative substrates (PMID:18303015, PMID:26717573). The BTB domain mediates intertwined homodimerization, which is established through a translation-coupled assembly pathway on co-translating ribosomes that bypasses a folding-competent closed monomeric state [PMID:bio_10.1101_2025.08.25.672138].

Mechanistic history

Synthesis pass · year-by-year structured walk · 11 steps
  1. 2006 High

    Established KLHL12's core identity as a Cullin-3-based E3 ligase adaptor and assigned it a physiological role as a Wnt pathway antagonist, answering what cellular process this orphan adaptor controls.

    Evidence TAP-MS, co-IP, ubiquitination assays, and functional rescue in Xenopus and zebrafish embryos

    PMID:16547521

    Open questions at the time
    • Degron recognition by the Kelch domain not yet defined structurally
    • Did not establish whether other substrates exist
  2. 2008 Medium

    Showed KLHL12 has substrates beyond Dishevelled by mapping Kelch-domain binding to the dopamine D4 receptor's third intracellular loop, broadening its substrate repertoire to a GPCR.

    Evidence Co-IP, domain mapping, and in-cell ubiquitination assays

    PMID:18303015

    Open questions at the time
    • Functional consequence of D4 ubiquitination unresolved
    • Single lab without structural validation
  3. 2010 Medium

    Revealed that not all KLHL12 ubiquitination is degradative, since D4 receptor ubiquitination at both ER and plasma membrane pools failed to trigger proteasomal or lysosomal turnover.

    Evidence Ubiquitination assays, subcellular fractionation, proteasome/lysosome inhibitor experiments, co-IP

    PMID:20100572

    Open questions at the time
    • Functional purpose of non-degradative ubiquitination undefined
    • Role of beta-arrestin2 interaction unclear
  4. 2015 Medium

    Characterized the unusual chemistry of D4 ubiquitination, showing modification on cysteine/serine/threonine rather than lysine and differential handling of the ADHD-associated D4.7 variant.

    Evidence Lysine-deficient receptor mutants plus chemical blockade of residue classes

    PMID:26717573

    Open questions at the time
    • Physiological significance of non-lysine ubiquitination unknown
    • Link to ADHD phenotype not established
  5. 2016 Medium

    Dissected the KLHL12-D4 receptor-beta-arrestin complex, showing arrestins are dispensable for receptor-adaptor binding and ubiquitination and that KLHL12 does not modulate downstream MAPK signaling.

    Evidence Reciprocal co-IP, beta-arrestin knockout cell lines, ubiquitination and MAPK phosphorylation assays

    PMID:27155323

    Open questions at the time
    • Functional role of the trimeric complex still undefined
    • Downstream effect of D4 ubiquitination remains unknown
  6. 2020 High

    Defined the structural basis of substrate recognition, showing the Kelch propeller engages a conserved PGXPP degron shared across multiple substrates, unifying how KLHL12 selects targets.

    Evidence X-ray crystallography of Kelch-DVL/DVL3/D4.2 peptide complexes, NMR screening, alanine-scanning affinity measurements, mutagenesis with ubiquitination/stability readouts

    PMID:32032490 PMID:32574548

    Open questions at the time
    • Does not capture full-length CRL3 assembly geometry
    • How degron context selects degradative vs non-degradative outcome unresolved
  7. 2020 Medium

    Extended KLHL12's substrate range to ER morphology by identifying Lunapark as a CRL3^KLHL12 target whose ubiquitination is required for normal growth and development.

    Evidence Proteomic screen for membrane-ubiquitylated proteins, co-IP, knockout/rescue with developmental phenotype readout

    PMID:32433973

    Open questions at the time
    • Mechanistic link between Lunapark ubiquitination and ER junction remodeling not fully resolved
    • Relationship to mTORC1 contact sites uncharacterized
  8. 2023 Medium

    Connected KLHL12 to collagen secretion via monoubiquitination of SEC31A and identified LZTR1 as a direct inhibitor of this activity, defining a regulatory layer on the ligase.

    Evidence Co-IP, ubiquitination assays, LZTR1 knockout cells, collagen secretion assays, multi-omics

    PMID:37626065

    Open questions at the time
    • Structural basis of LZTR1-KLHL12 inhibition unknown
    • Single lab
  9. 2023 Medium

    Separated CUL3-dependent and CUL3-independent KLHL12 functions, showing neddylated-CUL3 binding is dispensable for large COPII assembly but required for maintaining cellular collagen levels.

    Evidence MLN4924 neddylation inhibition, CUL3-interface point mutants, COPII imaging, collagen Western blot

    PMID:36652337

    Open questions at the time
    • Mechanism of CUL3-independent COPII enlargement undefined
    • Proposed lysosomal degradation route not directly demonstrated
  10. 2025 Medium

    Tested KLHL12's role in lipid handling with a tissue-specific knockout, yielding a well-controlled negative result that excludes a requirement in enterocyte chylomicron secretion despite altered ApoB48.

    Evidence Intestinal-specific Klhl12 knockout mice across multiple dietary challenges with serum lipid profiling and Western blot

    PMID:40591354

    Open questions at the time
    • Does not address KLHL12 function in other tissues
    • Significance of increased intestinal ApoB48 unexplained
  11. 2025 Medium

    Addressed how the BTB domain achieves intertwined dimerization, showing a folding-competent closed monomer would block domain swapping and that co-translational assembly on disomes provides an alternative route.

    Evidence Disome selective profiling, optical tweezers single-molecule force spectroscopy, ribosome profiling (preprint)

    PMID:bio_10.1101_2025.08.25.672138

    Open questions at the time
    • Preprint not yet peer-reviewed
    • Functional consequence of co-translational vs post-translational dimerization for ligase activity untested

Open questions

Synthesis pass · forward-looking unresolved questions
  • How the same Kelch-PGXPP recognition platform yields degradative poly-ubiquitination of some substrates versus non-degradative or mono-ubiquitination of others remains the central unresolved question.
  • No structural model of the full CRL3^KLHL12-substrate complex
  • Determinants of ubiquitin chain type and linkage per substrate unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140096 catalytic activity, acting on a protein 4 GO:0060090 molecular adaptor activity 3 GO:0016874 ligase activity 2
Localization
GO:0005783 endoplasmic reticulum 2 GO:0031410 cytoplasmic vesicle 2
Pathway
R-HSA-392499 Metabolism of proteins 2 R-HSA-5653656 Vesicle-mediated transport 2 R-HSA-162582 Signal Transduction 1
Partners
ARRB1ARRB2CUL3DRD4DVL1LZTR1SEC31A
Complex memberships
CRL3^KLHL12 (Cullin-3 E3 ubiquitin ligase)

Evidence

Reading pass · 12 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2006 KLHL12 forms a Cullin-3-based E3 ubiquitin ligase complex that binds Dishevelled in a Wnt-dependent manner, promoting its poly-ubiquitination and degradation, thereby negatively regulating the Wnt-beta-catenin pathway in cultured cells, Xenopus, and zebrafish embryos. Tandem-affinity purification, mass spectrometry, co-immunoprecipitation, ubiquitination assays, functional rescue in Xenopus and zebrafish embryos Nature cell biology High 16547521
2008 KLHL12 binds via its Kelch domain to the polymorphic third intracellular loop of the dopamine D4 receptor and acts as an adaptor in a Cullin3-based E3 ubiquitin ligase complex to specifically promote ubiquitination of the D4 receptor; other dopamine receptor subtypes undergo basal ubiquitination not affected by KLHL12. Co-immunoprecipitation, domain-mapping experiments, ubiquitination assays in eukaryotic cells The Journal of biological chemistry Medium 18303015
2010 KLHL12 promotes poly-ubiquitination of both immature (ER-associated) and mature (plasma membrane-associated) dopamine D4 receptors, but this ubiquitination does not lead to proteasomal or lysosomal degradation of the receptor; KLHL12 interacts with beta-arrestin2 but this does not affect beta-arrestin2 ubiquitination, localization, or D4 receptor internalization. Ubiquitination assays in eukaryotic cells, subcellular fractionation, proteasome/lysosome inhibitor experiments, co-immunoprecipitation Cellular signalling Medium 20100572
2015 KLHL12 promotes ubiquitination of the dopamine D4 receptor on non-lysine residues (cysteine, serine, and/or threonine); the ADHD-associated D4.7 variant is differentially ubiquitinated compared to D4.2 and D4.4 variants. Ubiquitination assays with lysine-deficient receptor mutants, chemical approaches to block specific residue classes PloS one Medium 26717573
2016 Beta-arrestin 1 and beta-arrestin 2 bind constitutively to both common D4 receptor polymorphic variants and to KLHL12, forming a trimeric complex; Cullin3 also interacts with both beta-arrestins but does not influence their ubiquitination; knockout of either beta-arrestin does not impair D4 receptor–KLHL12 interaction or receptor ubiquitination; KLHL12 does not influence p44/42 MAPK signaling downstream of the D4 receptor. Co-immunoprecipitation, beta-arrestin knockout cell lines, ubiquitination assays, MAPK phosphorylation assays Cellular signalling Medium 27155323
2020 A crystal structure (2.4 Å) of the KLHL12 Kelch domain bound to a DVL1 peptide revealed that DVL1 adopts a U-shaped conformation engaging hydrophobic contacts across all six blades of the Kelch beta-propeller via a conserved 'PGXPP' degron motif; mutation or deletion of this motif reduces DVL1 binding and ubiquitination by KLHL12 and increases DVL1 stability; the same PGXPP motif is conserved in other KLHL12 substrates/partners including PLEKHA4, PEF1, SEC31, and DRD4. X-ray crystallography (2.4 Å), peptide binding assays (affinity measurements), site-directed mutagenesis, co-immunoprecipitation, ubiquitination and stability assays in cells Open biology High 32574548
2020 X-ray crystal structure of KLHL12 Kelch domain bound to a short peptide derived from DVL3/D4.2 binding regions defined a sequence motif for the top-face binding site of KLHL12; alanine scanning mapped critical residues for binding with low micromolar affinity. NMR-based peptide screening, X-ray crystallography, alanine-substitution affinity measurements Biochemistry High 32032490
2020 The CRL3^KLHL12 ubiquitin ligase ubiquitylates Lunapark, an ER-shaping protein at three-way junctions; inhibition of Lunapark ubiquitylation results in neurodevelopmental defects, indicating this modification is required for normal growth and development. Lunapark also interacts with mTORC1 at ER three-way junctions where lysosomes make contact. Proteomic screen for membrane-ubiquitylated proteins, co-immunoprecipitation, functional rescue/knockout experiments with developmental phenotype readout Cell reports Medium 32433973
2023 KLHL12 monoubiquitylates SEC31A (a COPII coat component), enlarging COPII vesicles to accommodate procollagen for secretion; LZTR1 inhibits KLHL12-mediated ubiquitination of SEC31A, suppressing collagen secretion; LZTR1 and KLHL12 interact directly and function independently of the LZTR1-RIT1 axis. Co-immunoprecipitation, ubiquitination assays, LZTR1 knockout cells, collagen secretion assays, multi-omics analysis Cell death & disease Medium 37626065
2023 CUL3 neddylation is not required for KLHL12-dependent formation of large COPII structures; KLHL12 variants that cannot interact with neddylated CUL3 (Mut A) still support large COPII-KLHL12 assembly; however, active CRL3^KLHL12 (requiring CUL3 interaction) contributes to maintenance of cellular collagen levels, likely via lysosomal degradation. MLN4924 (NEDDylation inhibitor) treatment, CUL3-interface KLHL12 point mutants, COPII structure imaging, Western blot for collagen levels Molecular biology of the cell Medium 36652337
2025 Intestinal-specific knockout of Klhl12 in mice does not significantly alter serum lipid levels, body weight, or chylomicron secretion under chow, fasting/HFD-refeeding, or Western diet conditions, despite increased intestinal ApoB48 protein; KLHL12 is therefore not required for chylomicron secretion or lipid absorption in enterocytes. Intestinal-specific Klhl12 knockout mice, acute oil gavage, fasting/HFD refeeding, Western diet feeding, serum lipid profiling, Western blot American journal of physiology. Endocrinology and metabolism Medium 40591354
2025 The BTB domain of KLHL12 forms a stable closed monomeric state during folding (demonstrated by optical tweezers), which would impede conventional domain-swapping dimerization; instead, timed emergence of nascent chain segments during coupled translation on co-translating ribosomes (disomes) enables an alternative assembly pathway that bypasses the closed monomer, allowing intertwined BTB-domain dimer formation. Disome Selective Profiling, optical tweezers single-molecule force spectroscopy, ribosome profiling bioRxivpreprint Medium bio_10.1101_2025.08.25.672138

Source papers

Stage 0 corpus · 13 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2006 The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-beta-catenin pathway by targeting Dishevelled for degradation. Nature cell biology 315 16547521
2008 BTB Protein KLHL12 targets the dopamine D4 receptor for ubiquitination by a Cul3-based E3 ligase. The Journal of biological chemistry 66 18303015
2010 KLHL12-mediated ubiquitination of the dopamine D4 receptor does not target the receptor for degradation. Cellular signalling 23 20100572
2023 LZTR1 deficiency exerts high metastatic potential by enhancing sensitivity to EMT induction and controlling KLHL12-mediated collagen secretion. Cell death & disease 19 37626065
2020 Ubiquitylation of the ER-Shaping Protein Lunapark via the CRL3KLHL12 Ubiquitin Ligase Complex. Cell reports 17 32433973
2020 Identification of a PGXPP degron motif in dishevelled and structural basis for its binding to the E3 ligase KLHL12. Open biology 15 32574548
2015 KLHL12 Promotes Non-Lysine Ubiquitination of the Dopamine Receptors D4.2 and D4.4, but Not of the ADHD-Associated D4.7 Variant. PloS one 12 26717573
2020 Structural Elucidation of Peptide Binding to KLHL-12, a Substrate Specific Adapter Protein in a Cul3-Ring E3 Ligase Complex. Biochemistry 11 32032490
2016 Characterization of the interaction between the dopamine D4 receptor, KLHL12 and β-arrestins. Cellular signalling 7 27155323
2023 KLHL12 can form large COPII structures in the absence of CUL3 neddylation. Molecular biology of the cell 5 36652337
2026 Identification of KLHL12 Ligands Using Fragment-Based Methods. Journal of medicinal chemistry 0 41906311
2026 Exosomal miR-145-5p from BMSCs alleviates AKI-induced renal fibrosis via KLHL12/KHSRP-mediated m6A-dependent repression of FLI-1. Cell biology and toxicology 0 42009851
2025 Intestinal KLHL12 is dispensable for lipid absorption and chylomicron metabolism. American journal of physiology. Endocrinology and metabolism 0 40591354

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