Affinage

KLHL9

Kelch-like protein 9 · UniProt Q9P2J3

Length
617 aa
Mass
69.4 kDa
Annotated
2026-04-28
18 papers in source corpus 11 papers cited in narrative 10 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

KLHL9 is a substrate-recognition adaptor for CUL3-RBX1 E3 ubiquitin ligase complexes, directing ubiquitination of diverse substrates to control mitotic progression, nutrient signaling, insulin signaling, and mitophagy. KLHL9 binds substrates through its Kelch β-propeller domain and recruits CUL3 via its BTB domain; together with KLHL13, it ubiquitinates Aurora B to relocalize the chromosomal passenger complex from chromosomes to the central spindle during anaphase, a step required for proper chromosome alignment, midzone formation, and cytokinesis (PMID:17543862, PMID:18075312). Beyond mitosis, KLHL9 targets IRS1 for proteasomal degradation to modulate insulin signaling (PMID:31515271), ubiquitinates Rheb at the lysosome to promote amino acid-induced mTORC1 activation (PMID:39708321), mediates K63-linked ubiquitination of IMMT to initiate mitophagy (PMID:38834545), and drives proteasomal degradation of C/EBPβ/δ transcription factors whose loss underlies the mesenchymal glioblastoma phenotype (PMID:25303533). Multiplex CRISPR screening has defined the degron motifs recognized by the KLHL9/KLHL13 adaptor module, establishing a broader substrate repertoire for this E3 ligase complex (PMID:37735597).

Mechanistic history

Synthesis pass · year-by-year structured walk · 8 steps
  1. 2007 High

    Identification of KLHL9 as a CUL3 substrate adaptor that ubiquitinates Aurora B established the first molecular function for KLHL9 and explained how the chromosomal passenger complex is removed from chromosomes during the metaphase-to-anaphase transition.

    Evidence In vitro reconstituted ubiquitination with CUL3/KLHL9/KLHL13, direct binding assays, co-IP, and siRNA knockdown with mitotic phenotype readout in human cells

    PMID:17543862

    Open questions at the time
    • Whether KLHL9 and KLHL13 have non-redundant versus fully overlapping roles in Aurora B recognition
    • Structural basis of Aurora B degron recognition by the Kelch domain
    • In vivo validation in animal models
  2. 2007 Medium

    Linking CUL3-KLHL9/KLHL13 to spindle assembly checkpoint maintenance revealed that Aurora B ubiquitination by this complex prevents premature mitotic exit under spindle stress.

    Evidence siRNA knockdown of KLHL9/KLHL13 with Cyclin B degradation kinetics and SAC readout in nocodazole-treated cells

    PMID:18075312

    Open questions at the time
    • Single lab/method for SAC phenotype; independent confirmation needed
    • Whether SAC function is a direct consequence of Aurora B ubiquitination or involves additional substrates
  3. 2009 High

    Demonstration that KLHL21 (not KLHL9/KLHL13) recruits Aurora B to midzone microtubules established that distinct CUL3 adaptors non-redundantly regulate spatially separate pools of Aurora B during mitosis, defining KLHL9's role as specifically chromosomal rather than midzone-associated.

    Evidence Comparative siRNA knockdown, immunofluorescence localization, and in vitro ubiquitination assays in human cells

    PMID:19995937

    Open questions at the time
    • Whether KLHL9 has any midzone-related substrates beyond Aurora B
    • Temporal regulation of adaptor switching during mitotic progression
  4. 2014 Medium

    Discovery that KLHL9 loss activates C/EBPβ/δ transcription factors in glioblastoma expanded the substrate repertoire beyond mitotic targets and connected KLHL9 to tumor-suppressive degradation of mesenchymal master regulators.

    Evidence Lentiviral KLHL9 re-expression with proteasome inhibition, regulatory network inference, in vitro and in vivo tumor viability assays

    PMID:25303533

    Open questions at the time
    • Direct biochemical evidence that KLHL9/CUL3 ubiquitinates C/EBP proteins (e.g., reconstituted assay) is lacking
    • Whether KLHL13 is also required for C/EBP targeting
  5. 2019 Medium

    Identification of IRS1 as a KLHL9/KLHL13-CUL3 substrate linked the complex to insulin/metabolic signaling and showed that autophagy factor ATG16L1 normally antagonizes this degradation pathway.

    Evidence BioID proximity labeling, co-immunoprecipitation, siRNA knockdown with IRS1 protein level and insulin signaling readouts in ATG16L1-deficient cells

    PMID:31515271

    Open questions at the time
    • In vitro reconstitution of IRS1 ubiquitination by KLHL9/CUL3 not performed
    • Degron motif in IRS1 recognized by KLHL9 not mapped
    • Physiological relevance in whole-animal insulin signaling unknown
  6. 2023 High

    Systematic CRISPR screening defined the degron motifs recognized by KLHL9/KLHL13-CUL3 and expanded the validated substrate repertoire, establishing general rules for substrate selection by this adaptor pair.

    Evidence Multiplex CRISPR epistasis screening with site-saturation mutagenesis-based degron mapping in human cells

    PMID:37735597

    Open questions at the time
    • Structural co-crystal of KLHL9 Kelch domain with a degron peptide not yet available
    • Functional validation of all newly identified substrates in biological contexts
  7. 2024 High

    Discovery that KLHL9-CUL3-RBX1 ubiquitinates Rheb at the lysosome to activate mTORC1 in response to amino acids established a direct role for KLHL9 in nutrient sensing, independent of Akt signaling.

    Evidence In vitro ubiquitination reconstitution, KLHL9/CUL3/RBX1 knockout, lysosomal fractionation, and mTORC1 activity assays

    PMID:39708321

    Open questions at the time
    • Ubiquitin chain type on Rheb and whether it is degradative or signaling not fully resolved
    • Whether KLHL13 participates in Rheb ubiquitination at the lysosome
  8. 2024 High

    Demonstration that bacterial effector BipD hijacks KLHL9-KLHL13-CUL3 to K63-ubiquitinate the mitochondrial protein IMMT at K211 and trigger mitophagy revealed a new mode of pathogen exploitation and expanded KLHL9 substrates to mitochondrial targets.

    Evidence Co-IP, ubiquitome MS profiling, K211 site-directed mutagenesis, mitophagy and ROS assays in Burkholderia-infected cells

    PMID:38834545 PMID:39265641

    Open questions at the time
    • Whether KLHL9 ubiquitinates IMMT under physiological (infection-independent) conditions
    • Mechanism by which BipD redirects KLHL9 substrate specificity

Open questions

Synthesis pass · forward-looking unresolved questions
  • A high-resolution structural basis for KLHL9 substrate recognition, the full in vivo substrate landscape, and the regulatory logic governing KLHL9 versus KLHL13 specificity remain unresolved.
  • No crystal or cryo-EM structure of KLHL9 Kelch domain bound to any degron
  • Relative contributions of KLHL9 versus KLHL13 in heterodimeric versus homodimeric CUL3 complexes unclear
  • In vivo conditional knockout phenotype in mammals not reported

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140096 catalytic activity, acting on a protein 5 GO:0060090 molecular adaptor activity 4
Localization
GO:0005694 chromosome 2 GO:0005764 lysosome 1
Pathway
R-HSA-392499 Metabolism of proteins 5 R-HSA-1640170 Cell Cycle 3 R-HSA-162582 Signal Transduction 2 R-HSA-9612973 Autophagy 1
Partners
AURKBBIPDCUL3IMMTIRS1KLHL13RBX1RHEB
Complex memberships
CUL3-KLHL9-KLHL13 E3 ubiquitin ligaseCUL3-RBX1-KLHL9 E3 ubiquitin ligase

Evidence

Reading pass · 10 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2007 KLHL9 and KLHL13 serve as substrate-specific adaptors for a Cullin 3 (Cul3)-based E3 ubiquitin ligase complex. Aurora B directly binds to the substrate-recognition (Kelch) domain of KLHL9 and KLHL13 in vitro, co-immunoprecipitates with the Cul3 complex during mitosis, and is ubiquitylated in a Cul3-dependent manner in vivo and by reconstituted Cul3/KLHL9/KLHL13 ligase in vitro. This complex removes Aurora B (chromosomal passenger complex) from mitotic chromosomes, allowing its accumulation on the central spindle during anaphase, and is required for correct chromosome alignment, midzone/midbody formation, and completion of cytokinesis. In vitro binding assay, co-immunoprecipitation, in vitro ubiquitylation reconstitution, siRNA knockdown with mitotic phenotype readout Developmental cell High 17543862
2007 The Cul3/KLHL9/KLHL13 E3 ligase is required to maintain spindle assembly checkpoint (SAC) signaling; inactivation of this complex leads to premature degradation of Cyclin B and exit from the mitotic state in the presence of microtubule poisons, indicating a role in SAC maintenance through Aurora B ubiquitination. siRNA knockdown, cell cycle analysis, Cyclin B degradation assay Cell cycle (Georgetown, Tex.) Medium 18075312
2009 KLHL21 but not KLHL9/KLHL13 localizes to midzone microtubules in anaphase and recruits Aurora B and Cul3 to this region, establishing that different Cul3 adaptors non-redundantly regulate distinct pools of Aurora B at distinct subcellular localizations during mitosis. siRNA knockdown, immunofluorescence localization, in vitro ubiquitination assay The Journal of cell biology High 19995937
2017 The activity of the Cul3-KLHL9/KLHL13 complex is intact in SCCRO/DCUN1D1-deficient cells, demonstrating that SCCRO selectively neddylates cullins in a substrate-adaptor-dependent manner and that KLHL9/KLHL13-mediated Aurora B regulation does not require SCCRO-promoted neddylation. siRNA knockdown, functional rescue, immunofluorescence, ubiquitination assays The Journal of biological chemistry Medium 28620047
2019 KLHL9 and KLHL13, in complex with CUL3, interact with and promote proteasomal degradation of insulin receptor substrate-1 (IRS1). KLHL9/KLHL13 are novel IRS1 interactors identified by BioID proximity labeling and co-immunoprecipitation; siRNA knockdown of Klhl9, Klhl13, or Cul3 restored IRS1 protein levels and insulin signaling in ATG16L1-deficient cells. BioID proximity labeling, co-immunoprecipitation, siRNA knockdown, proteasome inhibition rescue The Journal of biological chemistry Medium 31515271
2023 Multiplex CRISPR screening identified substrates for the Cul3-KLHL9/KLHL13 E3 ligase complex and defined the degron motifs recognized, demonstrating that KLHL9/KLHL13 function together as a substrate adaptor module within the ubiquitin-proteasome system. Multiplex CRISPR screening, site-saturation mutagenesis, degron mapping Nature cell biology High 37735597
2023 Klhl9 and the autophagy pathway gene Cul3 are required for IFNγ-induced inhibition of murine norovirus replication in mouse cells, placing KLHL9 in the IFNγ-mediated intracellular immunity pathway. Genetic knockdown/knockout with viral replication readout, epistasis with autophagy genes mBio Low 37905813
2024 The CUL3-RBX1-KLHL9 E3 ubiquitin ligase complex translocates to the lysosome and ubiquitinates Rheb in response to amino acid stimulation. KLHL9 serves as an essential adaptor for CUL3-RBX1 to target Rheb on the lysosome. Deletion of CUL3, RBX1, or KLHL9 diminishes Rheb ubiquitination and reduces amino acid-induced mTORC1 activation without affecting lysosomal mTORC1 localization or Akt activity. Co-immunoprecipitation, in vitro ubiquitination assay, KLHL9/CUL3/RBX1 knockout, mTORC1 activity assay, lysosomal fractionation Cell reports High 39708321
2024 Burkholderia pseudomallei effector BipD hijacks the host KLHL9-KLHL13-CUL3 E3 ligase complex by binding to the BACK and Kelch domains of KLHL9 and KLHL13, resulting in K63-linked ubiquitination of the inner mitochondrial membrane protein IMMT at K211, initiating host mitophagy to reduce mitochondrial ROS and promote bacterial survival. Co-immunoprecipitation, ubiquitome profiling (MS), site-directed mutagenesis (K211 ubiquitination site), mitophagy/ROS assays Nature communications High 38834545 39265641
2014 KLHL9 deletions act as upstream activators of the mesenchymal glioblastoma master regulators C/EBPβ and C/EBPδ; rescue of KLHL9 expression induced proteasomal degradation of C/EBP proteins, abrogated the mesenchymal gene signature, and reduced tumor viability in vitro and in vivo. Lentiviral KLHL9 re-expression, proteasome inhibition, in vitro and in vivo tumor viability assays, regulatory network inference Cell Medium 25303533

Source papers

Stage 0 corpus · 18 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2013 Update on the Kelch-like (KLHL) gene family. Human genomics 209 23676014
2007 A Cul3-based E3 ligase removes Aurora B from mitotic chromosomes, regulating mitotic progression and completion of cytokinesis in human cells. Developmental cell 196 17543862
2014 Identification of causal genetic drivers of human disease through systems-level analysis of regulatory networks. Cell 139 25303533
2009 The Cul3-KLHL21 E3 ubiquitin ligase targets aurora B to midzone microtubules in anaphase and is required for cytokinesis. The Journal of cell biology 118 19995937
2023 Defining E3 ligase-substrate relationships through multiplex CRISPR screening. Nature cell biology 49 37735597
2018 Understanding the regulatory mechanisms of milk production using integrative transcriptomic and proteomic analyses: improving inefficient utilization of crop by-products as forage in dairy industry. BMC genomics 44 29843597
2019 Deficiency of the autophagy gene ATG16L1 induces insulin resistance through KLHL9/KLHL13/CUL3-mediated IRS1 degradation. The Journal of biological chemistry 34 31515271
2008 The contribution of large genomic deletions at the CDKN2A locus to the burden of familial melanoma. British journal of cancer 24 18612309
2007 A Cul3-based E3 ligase regulates mitosis and is required to maintain the spindle assembly checkpoint in human cells. Cell cycle (Georgetown, Tex.) 20 18075312
2017 Squamous cell carcinoma-related oncogene (SCCRO) neddylates Cul3 protein to selectively promote midbody localization and activity of Cul3KLHL21 protein complex during abscission. The Journal of biological chemistry 15 28620047
2014 Homozygous deletions at 3p22, 5p14, 6q15, and 9p21 result in aberrant expression of tumor suppressor genes in gastric cancer. Genes, chromosomes & cancer 15 25521327
2023 Autophagy gene-dependent intracellular immunity triggered by interferon-γ. mBio 14 37905813
2024 Burkholderia pseudomallei BipD modulates host mitophagy to evade killing. Nature communications 11 38834545
2019 Identification of Dysregulated Competitive Endogenous RNA Networks Driven by Copy Number Variations in Malignant Gliomas. Frontiers in genetics 9 31719831
2022 Significance of Chr9p22.1-p21.3 Deletion in Cancer Development: A Pan-cancer In Silico Analysis. Anticancer research 7 36288884
2024 The Cullin3-Rbx1-KLHL9 E3 ubiquitin ligase complex ubiquitinates Rheb and supports amino acid-induced mTORC1 activation. Cell reports 3 39708321
2024 Burkholderia pseudomallei BipD initiates mitophagy to evade killing by hijacking host KLHL9-KLHL13-CUL3 E3 ligase to ubiquitinate IMMT. Autophagy 1 39265641
2025 KLHL9-linked distal myopathy: a second family suggesting broad phenotypic variability. Neuromuscular disorders : NMD 0 40818927