{"gene":"KLHL9","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":2007,"finding":"KLHL9 and KLHL13 function as substrate-specific adaptors for a CUL3-based E3 ubiquitin ligase complex. Aurora B directly binds to the substrate-recognition (Kelch) domain of KLHL9 in vitro, coimmunoprecipitates 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 accumulation on the central spindle during anaphase, and is required for correct chromosome alignment and completion of cytokinesis.","method":"In vitro binding assay (Aurora B binds KLHL9 Kelch domain), co-immunoprecipitation, in vivo ubiquitylation assay, reconstituted in vitro ubiquitylation with purified CUL3/KLHL9/KLHL13, RNAi loss-of-function with mitotic phenotype readout","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro ubiquitylation assay, direct binding assay, reciprocal Co-IP, in vivo ubiquitylation, multiple orthogonal methods in single rigorous study","pmids":["17543862"],"is_preprint":false},{"year":2007,"finding":"Inactivation of the CUL3/KLHL9/KLHL13 ligase leads to premature degradation of Cyclin B and exit from the mitotic state in the presence of microtubule poisons, indicating this E3 ligase is required to maintain spindle assembly checkpoint (SAC) signaling in human cells.","method":"RNAi-mediated inactivation of CUL3/KLHL9/KLHL13 complex with readout of Cyclin B levels and mitotic exit under microtubule poison treatment","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, single method (RNAi + biochemical readout), no structural or reconstitution data in this paper","pmids":["18075312"],"is_preprint":false},{"year":2009,"finding":"KLHL9 and KLHL13 regulate translocation of the chromosomal passenger complex (CPC) from chromosomes to the spindle midzone in anaphase, a function distinct from KLHL21 which localizes to midzone microtubules. KLHL9/KLHL13 are required for cytokinesis.","method":"RNAi knockdown of KLHL9/KLHL13 with immunofluorescence readout of CPC localization and cytokinesis completion; comparison with KLHL21 knockdown","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — independent lab replication of KLHL9/KLHL13 cytokinesis role, direct localization experiment with functional consequence, comparison with KLHL21","pmids":["19995937"],"is_preprint":false},{"year":2014,"finding":"KLHL9 deletion acts as an upstream activator of C/EBPβ and C/EBPδ master regulators in glioblastoma mesenchymal subtype. Rescue of KLHL9 expression induced proteasomal degradation of C/EBP proteins, abrogated the mesenchymal gene expression signature, and reduced tumor viability in vitro and in vivo.","method":"KLHL9 rescue expression in KLHL9-deleted glioblastoma cells, proteasome inhibition assay, in vitro and in vivo tumor viability assays, regulatory network analysis","journal":"Cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional rescue experiment with proteasomal degradation readout, in vitro and in vivo validation, single lab","pmids":["25303533"],"is_preprint":false},{"year":2017,"finding":"The activity of the CUL3/KLHL9/KLHL13 complex during mitosis was intact in SCCRO-deficient cells, demonstrating that SCCRO selectively (rather than globally) neddylates cullins and that KLHL9/KLHL13-containing CUL3 complexes are regulated differently from CUL3KLHL21 during abscission.","method":"SCCRO knockdown/knockout with biochemical assay of CUL3KLHL9/KLHL13 activity as internal control compared with CUL3KLHL21","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — activity assay in cell-based system, single lab; finding is primarily a negative/contrast result for KLHL9 within a study focused on KLHL21","pmids":["28620047"],"is_preprint":false},{"year":2019,"finding":"KLHL9 and KLHL13, in complex with CUL3, serve as an E3 ubiquitin ligase that targets insulin receptor substrate-1 (IRS1) for proteasomal degradation. KLHL9 and KLHL13 were identified as novel IRS1 interactors by proximity-dependent biotin identification (BioID) and co-immunoprecipitation. siRNA-mediated knockdown of Klhl9, Klhl13, or Cul3 recovered IRS1 expression, and knockdown of Klhl13 and Cul3 increased insulin signaling. Elevated expression of Klhl9 and Klhl13 in ATG16L1-deficient cells mediates IRS1 degradation and insulin resistance.","method":"BioID proximity labeling, co-immunoprecipitation, siRNA knockdown with IRS1 protein level and insulin signaling readouts, proteasome inhibition assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BioID + Co-IP for interaction, siRNA knockdown with functional readout, multiple orthogonal methods, single lab","pmids":["31515271"],"is_preprint":false},{"year":2023,"finding":"Multiplex CRISPR screening identified substrates and degron motifs for the CUL3KLHL9/13 E3 ligase complex, demonstrating that KLHL9/KLHL13 together recognize specific degron sequences on substrate proteins at scale.","method":"Multiplex CRISPR screening platform (~100 simultaneous screens), site-saturation mutagenesis of degron motifs, full-length substrate pools","journal":"Nature cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-scale functional screen with mutagenesis, but KLHL9-specific substrate details are not individually detailed in the abstract","pmids":["37735597"],"is_preprint":false},{"year":2023,"finding":"Klhl9 (together with Cul3) is required for interferon-γ-induced inhibition of murine norovirus replication in mouse cells, placing the CUL3/KLHL9 complex in an IFNγ-dependent cell-intrinsic antiviral pathway.","method":"Genetic knockdown/knockout of Klhl9 and Cul3 with readout of IFNγ-induced norovirus replication inhibition","journal":"mBio","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, loss-of-function with viral replication readout, no molecular mechanism for how KLHL9 acts in this pathway established in abstract","pmids":["37905813"],"is_preprint":false},{"year":2024,"finding":"The CUL3-RBX1-KLHL9 E3 ubiquitin ligase complex ubiquitinates Rheb on the lysosome in response to amino acid stimulation, supporting mTORC1 activation. KLHL9 serves as the essential adaptor for CUL3-RBX1 to target Rheb. Deletion of CUL3, RBX1, or KLHL9 diminishes Rheb ubiquitination and reduces amino acid-induced mTORC1 activation without affecting lysosomal mTORC1 localization or Akt activity. The complex translocates to the lysosome upon amino acid stimulation.","method":"Genetic deletion (CUL3, RBX1, KLHL9 KO), ubiquitination assay of Rheb, mTORC1 activity assay (S6K phosphorylation), lysosomal localization by fractionation/imaging, Akt activity as negative control","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — direct ubiquitination assay, multiple KO lines, lysosomal translocation experiment, functional mTORC1 readout, multiple orthogonal methods in single study","pmids":["39708321"],"is_preprint":false},{"year":2024,"finding":"The bacterial effector BipD of Burkholderia pseudomallei binds to the BACK and Kelch domains of KLHL9 (and KLHL13), hijacking the KLHL9-KLHL13-CUL3 E3 ligase complex to promote K63-linked ubiquitination of the inner mitochondrial membrane protein IMMT at K211, initiating mitophagy and reducing mitochondrial ROS to support bacterial intracellular survival.","method":"Co-IP/interaction mapping (BipD binding to BACK and Kelch domains of KLHL9), host ubiquitome profiling identifying IMMT as substrate, site-specific ubiquitination (K211) determination, mitophagy assay, mtROS measurement, bacterial survival assay","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-mapping Co-IP, ubiquitome profiling identifying IMMT substrate, site-specific ubiquitination, functional mitophagy readout; single lab, mechanistic detail on KLHL9 domain interaction is clear","pmids":["38834545","39265641"],"is_preprint":false}],"current_model":"KLHL9 functions as a substrate-recognition adaptor for CUL3-RBX1-based E3 ubiquitin ligase complexes, recognizing substrates through its Kelch domain; established substrates include Aurora B (regulating chromosomal passenger complex dynamics during mitosis and cytokinesis), IRS1 (linking autophagy deficiency to insulin resistance), Rheb (supporting amino acid-induced mTORC1 activation at the lysosome), and IMMT (when hijacked by bacterial BipD to initiate mitophagy), with KLHL9 also required for spindle assembly checkpoint maintenance and IFNγ-induced antiviral immunity."},"narrative":{"mechanistic_narrative":"KLHL9 is a substrate-recognition adaptor for CUL3-RBX1-based E3 ubiquitin ligase complexes, capturing substrates through its Kelch domain and frequently acting together with the paralogous adaptor KLHL13 [PMID:17543862]. Its founding role is in mitosis, where CUL3/KLHL9/KLHL13 directly binds and ubiquitylates Aurora B to translocate the chromosomal passenger complex from chromosomes to the central spindle during anaphase, an activity required for chromosome alignment and completion of cytokinesis [PMID:17543862, PMID:19995937]; this ligase is also required to sustain spindle assembly checkpoint signaling, as its loss causes premature Cyclin B degradation and mitotic exit under microtubule poisons [PMID:18075312]. Beyond mitosis, KLHL9 directs CUL3-mediated proteasomal turnover of distinct substrates in several contexts: it targets insulin receptor substrate-1 (IRS1), linking autophagy deficiency to insulin resistance [PMID:31515271], and ubiquitylates Rheb at the lysosome upon amino acid stimulation, where the complex translocates to support mTORC1 activation [PMID:39708321]. KLHL9 loss de-represses the C/EBPβ/C/EBPδ master regulators that drive the glioblastoma mesenchymal signature, and restoring KLHL9 triggers proteasomal degradation of these factors and reduces tumor viability [PMID:25303533]. The bacterial effector BipD of Burkholderia pseudomallei binds the BACK and Kelch domains of KLHL9 to hijack the complex toward K63-linked ubiquitination of the inner mitochondrial membrane protein IMMT, initiating mitophagy that supports bacterial survival [PMID:38834545, PMID:39265641]. KLHL9 substrate recognition operates through specific degron motifs definable at scale [PMID:37735597].","teleology":[{"year":2007,"claim":"Established that KLHL9 is a bona fide CUL3 substrate adaptor by identifying its first substrate, defining how a Kelch-domain adaptor confers specificity to a cullin-RING ligase in mitosis.","evidence":"In vitro Kelch-domain binding, reciprocal Co-IP, in vivo and reconstituted in vitro ubiquitylation of Aurora B, RNAi mitotic phenotyping","pmids":["17543862"],"confidence":"High","gaps":["No structure of the KLHL9 Kelch-substrate interface","Relative contributions of KLHL9 vs KLHL13 to substrate capture not separated"]},{"year":2007,"claim":"Connected the ligase to checkpoint control, showing the complex is needed to maintain SAC signaling rather than only to relocalize the CPC.","evidence":"RNAi inactivation of CUL3/KLHL9/KLHL13 with Cyclin B and mitotic-exit readouts under microtubule poisons","pmids":["18075312"],"confidence":"Medium","gaps":["Direct SAC substrate of the complex not identified","Single method, no reconstitution"]},{"year":2009,"claim":"Independent replication refined the mitotic role to CPC translocation from chromosomes to spindle midzone and distinguished it from the related KLHL21 adaptor.","evidence":"RNAi knockdown with immunofluorescence of CPC localization and cytokinesis, compared with KLHL21","pmids":["19995937"],"confidence":"Medium","gaps":["Molecular trigger for midzone translocation not defined"]},{"year":2014,"claim":"Extended KLHL9 function to tumor biology, positioning it as a tumor suppressor whose loss de-represses C/EBP master regulators in glioblastoma.","evidence":"KLHL9 rescue with proteasome inhibition, in vitro/in vivo tumor viability, regulatory network analysis","pmids":["25303533"],"confidence":"Medium","gaps":["Direct ubiquitylation of C/EBPβ/δ by CUL3/KLHL9 not demonstrated","Whether C/EBP proteins bind the Kelch domain unknown"]},{"year":2017,"claim":"Clarified upstream regulation, showing CUL3KLHL9/13 activity is independent of SCCRO-mediated neddylation that controls CUL3KLHL21, evidence for selective cullin regulation.","evidence":"SCCRO knockdown/knockout with CUL3KLHL9/13 activity as internal control versus CUL3KLHL21","pmids":["28620047"],"confidence":"Medium","gaps":["How CUL3KLHL9/13 is neddylated/activated not defined","Primarily a contrast result for KLHL9"]},{"year":2019,"claim":"Identified a metabolic substrate (IRS1), linking the ligase to autophagy-dependent insulin resistance.","evidence":"BioID and Co-IP interaction mapping, siRNA knockdown with IRS1 protein and insulin-signaling readouts, proteasome inhibition","pmids":["31515271"],"confidence":"Medium","gaps":["Degron on IRS1 not mapped","Direct reconstituted ubiquitylation of IRS1 not shown"]},{"year":2023,"claim":"Provided systematic substrate/degron logic, showing KLHL9/KLHL13 recognize defined degron sequences at scale.","evidence":"Multiplex CRISPR screening with site-saturation degron mutagenesis on substrate pools","pmids":["37735597"],"confidence":"Medium","gaps":["KLHL9-specific substrates from the screen not individually detailed","Physiological relevance of screened substrates untested"]},{"year":2023,"claim":"Placed KLHL9 in cell-intrinsic antiviral immunity, showing it is required for IFNγ-mediated restriction of norovirus.","evidence":"Klhl9 and Cul3 knockdown/knockout with IFNγ-induced norovirus replication readout in mouse cells","pmids":["37905813"],"confidence":"Low","gaps":["No molecular mechanism or substrate established for the antiviral role","Single lab, loss-of-function only"]},{"year":2024,"claim":"Defined KLHL9 as the essential adaptor for lysosomal Rheb ubiquitination, integrating it into amino-acid-induced mTORC1 activation.","evidence":"CUL3/RBX1/KLHL9 KO lines, Rheb ubiquitination assay, S6K-based mTORC1 readout, lysosomal translocation imaging, Akt negative control","pmids":["39708321"],"confidence":"High","gaps":["Rheb degron/Kelch contacts not structurally defined","Mechanism of amino-acid-induced lysosomal translocation unknown"]},{"year":2024,"claim":"Revealed pathogen hijacking of the complex, mapping a bacterial effector to KLHL9 domains and redirecting it to drive IMMT-dependent mitophagy.","evidence":"Domain-mapping Co-IP of BipD to BACK/Kelch domains, host ubiquitome profiling, K211 site-specific ubiquitination, mitophagy and mtROS assays, bacterial survival","pmids":["38834545","39265641"],"confidence":"Medium","gaps":["Whether IMMT is a physiological KLHL9 substrate absent infection unknown","Structural basis of BipD-mediated substrate redirection not resolved"]},{"year":null,"claim":"The structural basis of Kelch-domain substrate selection and how KLHL9 versus KLHL13 partition substrates across these diverse pathways remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No co-crystal/cryo-EM of KLHL9 with any substrate","Tissue/context determinants of substrate choice undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,8,9]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,8]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,6,8]}],"localization":[{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,5,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5,8]}],"complexes":["CUL3-RBX1-KLHL9/KLHL13 E3 ubiquitin ligase"],"partners":["CUL3","KLHL13","RBX1","AURKB","IRS1","RHEB","IMMT","BIPD"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9P2J3","full_name":"Kelch-like protein 9","aliases":[],"length_aa":617,"mass_kda":69.4,"function":"Substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complex required for mitotic progression and cytokinesis. The BCR(KLHL9-KLHL13) E3 ubiquitin ligase complex mediates the ubiquitination of AURKB and controls the dynamic behavior of AURKB on mitotic chromosomes and thereby coordinates faithful mitotic progression and completion of cytokinesis","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q9P2J3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/KLHL9","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/KLHL9","total_profiled":1310},"omim":[{"mim_id":"618020","title":"KELCH-LIKE 22; KLHL22","url":"https://www.omim.org/entry/618020"},{"mim_id":"616262","title":"KELCH-LIKE 21; KLHL21","url":"https://www.omim.org/entry/616262"},{"mim_id":"611201","title":"KELCH-LIKE 9; KLHL9","url":"https://www.omim.org/entry/611201"},{"mim_id":"603136","title":"CULLIN 3; CUL3","url":"https://www.omim.org/entry/603136"},{"mim_id":"300655","title":"KELCH-LIKE 13; KLHL13","url":"https://www.omim.org/entry/300655"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/KLHL9"},"hgnc":{"alias_symbol":["KIAA1354","FLJ13568"],"prev_symbol":[]},"alphafold":{"accession":"Q9P2J3","domains":[{"cath_id":"3.30.710.10","chopping":"30-148","consensus_level":"high","plddt":94.45,"start":30,"end":148},{"cath_id":"1.25.40.420","chopping":"179-284","consensus_level":"medium","plddt":96.1491,"start":179,"end":284},{"cath_id":"2.120.10.80","chopping":"297-592","consensus_level":"medium","plddt":95.9675,"start":297,"end":592}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P2J3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P2J3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P2J3-F1-predicted_aligned_error_v6.png","plddt_mean":91.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=KLHL9","jax_strain_url":"https://www.jax.org/strain/search?query=KLHL9"},"sequence":{"accession":"Q9P2J3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9P2J3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9P2J3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P2J3"}},"corpus_meta":[{"pmid":"23676014","id":"PMC_23676014","title":"Update on the Kelch-like (KLHL) gene family.","date":"2013","source":"Human genomics","url":"https://pubmed.ncbi.nlm.nih.gov/23676014","citation_count":213,"is_preprint":false},{"pmid":"17543862","id":"PMC_17543862","title":"A Cul3-based E3 ligase removes Aurora B from mitotic chromosomes, regulating mitotic progression and completion of cytokinesis in human cells.","date":"2007","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/17543862","citation_count":196,"is_preprint":false},{"pmid":"25303533","id":"PMC_25303533","title":"Identification of causal genetic drivers of human disease through systems-level analysis of regulatory networks.","date":"2014","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/25303533","citation_count":139,"is_preprint":false},{"pmid":"19995937","id":"PMC_19995937","title":"The Cul3-KLHL21 E3 ubiquitin ligase targets aurora B to midzone microtubules in anaphase and is required for cytokinesis.","date":"2009","source":"The Journal of cell 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interferon-γ.","date":"2023","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/37905813","citation_count":15,"is_preprint":false},{"pmid":"25521327","id":"PMC_25521327","title":"Homozygous deletions at 3p22, 5p14, 6q15, and 9p21 result in aberrant expression of tumor suppressor genes in gastric cancer.","date":"2014","source":"Genes, chromosomes & cancer","url":"https://pubmed.ncbi.nlm.nih.gov/25521327","citation_count":15,"is_preprint":false},{"pmid":"28620047","id":"PMC_28620047","title":"Squamous cell carcinoma-related oncogene (SCCRO) neddylates Cul3 protein to selectively promote midbody localization and activity of Cul3KLHL21 protein complex during abscission.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28620047","citation_count":15,"is_preprint":false},{"pmid":"38834545","id":"PMC_38834545","title":"Burkholderia pseudomallei BipD modulates host mitophagy to evade killing.","date":"2024","source":"Nature 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NMD","url":"https://pubmed.ncbi.nlm.nih.gov/40818927","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10909,"output_tokens":3004,"usd":0.038893,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10292,"output_tokens":3401,"usd":0.068242,"stage2_stop_reason":"end_turn"},"total_usd":0.107135,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"KLHL9 and KLHL13 function as substrate-specific adaptors for a CUL3-based E3 ubiquitin ligase complex. Aurora B directly binds to the substrate-recognition (Kelch) domain of KLHL9 in vitro, coimmunoprecipitates 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 accumulation on the central spindle during anaphase, and is required for correct chromosome alignment and completion of cytokinesis.\",\n      \"method\": \"In vitro binding assay (Aurora B binds KLHL9 Kelch domain), co-immunoprecipitation, in vivo ubiquitylation assay, reconstituted in vitro ubiquitylation with purified CUL3/KLHL9/KLHL13, RNAi loss-of-function with mitotic phenotype readout\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro ubiquitylation assay, direct binding assay, reciprocal Co-IP, in vivo ubiquitylation, multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"17543862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Inactivation of the CUL3/KLHL9/KLHL13 ligase leads to premature degradation of Cyclin B and exit from the mitotic state in the presence of microtubule poisons, indicating this E3 ligase is required to maintain spindle assembly checkpoint (SAC) signaling in human cells.\",\n      \"method\": \"RNAi-mediated inactivation of CUL3/KLHL9/KLHL13 complex with readout of Cyclin B levels and mitotic exit under microtubule poison treatment\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, single method (RNAi + biochemical readout), no structural or reconstitution data in this paper\",\n      \"pmids\": [\"18075312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"KLHL9 and KLHL13 regulate translocation of the chromosomal passenger complex (CPC) from chromosomes to the spindle midzone in anaphase, a function distinct from KLHL21 which localizes to midzone microtubules. KLHL9/KLHL13 are required for cytokinesis.\",\n      \"method\": \"RNAi knockdown of KLHL9/KLHL13 with immunofluorescence readout of CPC localization and cytokinesis completion; comparison with KLHL21 knockdown\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — independent lab replication of KLHL9/KLHL13 cytokinesis role, direct localization experiment with functional consequence, comparison with KLHL21\",\n      \"pmids\": [\"19995937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"KLHL9 deletion acts as an upstream activator of C/EBPβ and C/EBPδ master regulators in glioblastoma mesenchymal subtype. Rescue of KLHL9 expression induced proteasomal degradation of C/EBP proteins, abrogated the mesenchymal gene expression signature, and reduced tumor viability in vitro and in vivo.\",\n      \"method\": \"KLHL9 rescue expression in KLHL9-deleted glioblastoma cells, proteasome inhibition assay, in vitro and in vivo tumor viability assays, regulatory network analysis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional rescue experiment with proteasomal degradation readout, in vitro and in vivo validation, single lab\",\n      \"pmids\": [\"25303533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The activity of the CUL3/KLHL9/KLHL13 complex during mitosis was intact in SCCRO-deficient cells, demonstrating that SCCRO selectively (rather than globally) neddylates cullins and that KLHL9/KLHL13-containing CUL3 complexes are regulated differently from CUL3KLHL21 during abscission.\",\n      \"method\": \"SCCRO knockdown/knockout with biochemical assay of CUL3KLHL9/KLHL13 activity as internal control compared with CUL3KLHL21\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — activity assay in cell-based system, single lab; finding is primarily a negative/contrast result for KLHL9 within a study focused on KLHL21\",\n      \"pmids\": [\"28620047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"KLHL9 and KLHL13, in complex with CUL3, serve as an E3 ubiquitin ligase that targets insulin receptor substrate-1 (IRS1) for proteasomal degradation. KLHL9 and KLHL13 were identified as novel IRS1 interactors by proximity-dependent biotin identification (BioID) and co-immunoprecipitation. siRNA-mediated knockdown of Klhl9, Klhl13, or Cul3 recovered IRS1 expression, and knockdown of Klhl13 and Cul3 increased insulin signaling. Elevated expression of Klhl9 and Klhl13 in ATG16L1-deficient cells mediates IRS1 degradation and insulin resistance.\",\n      \"method\": \"BioID proximity labeling, co-immunoprecipitation, siRNA knockdown with IRS1 protein level and insulin signaling readouts, proteasome inhibition assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BioID + Co-IP for interaction, siRNA knockdown with functional readout, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"31515271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Multiplex CRISPR screening identified substrates and degron motifs for the CUL3KLHL9/13 E3 ligase complex, demonstrating that KLHL9/KLHL13 together recognize specific degron sequences on substrate proteins at scale.\",\n      \"method\": \"Multiplex CRISPR screening platform (~100 simultaneous screens), site-saturation mutagenesis of degron motifs, full-length substrate pools\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-scale functional screen with mutagenesis, but KLHL9-specific substrate details are not individually detailed in the abstract\",\n      \"pmids\": [\"37735597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Klhl9 (together with Cul3) is required for interferon-γ-induced inhibition of murine norovirus replication in mouse cells, placing the CUL3/KLHL9 complex in an IFNγ-dependent cell-intrinsic antiviral pathway.\",\n      \"method\": \"Genetic knockdown/knockout of Klhl9 and Cul3 with readout of IFNγ-induced norovirus replication inhibition\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, loss-of-function with viral replication readout, no molecular mechanism for how KLHL9 acts in this pathway established in abstract\",\n      \"pmids\": [\"37905813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The CUL3-RBX1-KLHL9 E3 ubiquitin ligase complex ubiquitinates Rheb on the lysosome in response to amino acid stimulation, supporting mTORC1 activation. KLHL9 serves as the essential adaptor for CUL3-RBX1 to target Rheb. Deletion of CUL3, RBX1, or KLHL9 diminishes Rheb ubiquitination and reduces amino acid-induced mTORC1 activation without affecting lysosomal mTORC1 localization or Akt activity. The complex translocates to the lysosome upon amino acid stimulation.\",\n      \"method\": \"Genetic deletion (CUL3, RBX1, KLHL9 KO), ubiquitination assay of Rheb, mTORC1 activity assay (S6K phosphorylation), lysosomal localization by fractionation/imaging, Akt activity as negative control\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — direct ubiquitination assay, multiple KO lines, lysosomal translocation experiment, functional mTORC1 readout, multiple orthogonal methods in single study\",\n      \"pmids\": [\"39708321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The bacterial effector BipD of Burkholderia pseudomallei binds to the BACK and Kelch domains of KLHL9 (and KLHL13), hijacking the KLHL9-KLHL13-CUL3 E3 ligase complex to promote K63-linked ubiquitination of the inner mitochondrial membrane protein IMMT at K211, initiating mitophagy and reducing mitochondrial ROS to support bacterial intracellular survival.\",\n      \"method\": \"Co-IP/interaction mapping (BipD binding to BACK and Kelch domains of KLHL9), host ubiquitome profiling identifying IMMT as substrate, site-specific ubiquitination (K211) determination, mitophagy assay, mtROS measurement, bacterial survival assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-mapping Co-IP, ubiquitome profiling identifying IMMT substrate, site-specific ubiquitination, functional mitophagy readout; single lab, mechanistic detail on KLHL9 domain interaction is clear\",\n      \"pmids\": [\"38834545\", \"39265641\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"KLHL9 functions as a substrate-recognition adaptor for CUL3-RBX1-based E3 ubiquitin ligase complexes, recognizing substrates through its Kelch domain; established substrates include Aurora B (regulating chromosomal passenger complex dynamics during mitosis and cytokinesis), IRS1 (linking autophagy deficiency to insulin resistance), Rheb (supporting amino acid-induced mTORC1 activation at the lysosome), and IMMT (when hijacked by bacterial BipD to initiate mitophagy), with KLHL9 also required for spindle assembly checkpoint maintenance and IFNγ-induced antiviral immunity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"KLHL9 is a substrate-recognition adaptor for CUL3-RBX1-based E3 ubiquitin ligase complexes, capturing substrates through its Kelch domain and frequently acting together with the paralogous adaptor KLHL13 [#0]. Its founding role is in mitosis, where CUL3/KLHL9/KLHL13 directly binds and ubiquitylates Aurora B to translocate the chromosomal passenger complex from chromosomes to the central spindle during anaphase, an activity required for chromosome alignment and completion of cytokinesis [#0, #2]; this ligase is also required to sustain spindle assembly checkpoint signaling, as its loss causes premature Cyclin B degradation and mitotic exit under microtubule poisons [#1]. Beyond mitosis, KLHL9 directs CUL3-mediated proteasomal turnover of distinct substrates in several contexts: it targets insulin receptor substrate-1 (IRS1), linking autophagy deficiency to insulin resistance [#5], and ubiquitylates Rheb at the lysosome upon amino acid stimulation, where the complex translocates to support mTORC1 activation [#8]. KLHL9 loss de-represses the C/EBPβ/C/EBPδ master regulators that drive the glioblastoma mesenchymal signature, and restoring KLHL9 triggers proteasomal degradation of these factors and reduces tumor viability [#3]. The bacterial effector BipD of Burkholderia pseudomallei binds the BACK and Kelch domains of KLHL9 to hijack the complex toward K63-linked ubiquitination of the inner mitochondrial membrane protein IMMT, initiating mitophagy that supports bacterial survival [#9]. KLHL9 substrate recognition operates through specific degron motifs definable at scale [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established that KLHL9 is a bona fide CUL3 substrate adaptor by identifying its first substrate, defining how a Kelch-domain adaptor confers specificity to a cullin-RING ligase in mitosis.\",\n      \"evidence\": \"In vitro Kelch-domain binding, reciprocal Co-IP, in vivo and reconstituted in vitro ubiquitylation of Aurora B, RNAi mitotic phenotyping\",\n      \"pmids\": [\"17543862\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of the KLHL9 Kelch-substrate interface\", \"Relative contributions of KLHL9 vs KLHL13 to substrate capture not separated\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Connected the ligase to checkpoint control, showing the complex is needed to maintain SAC signaling rather than only to relocalize the CPC.\",\n      \"evidence\": \"RNAi inactivation of CUL3/KLHL9/KLHL13 with Cyclin B and mitotic-exit readouts under microtubule poisons\",\n      \"pmids\": [\"18075312\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct SAC substrate of the complex not identified\", \"Single method, no reconstitution\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Independent replication refined the mitotic role to CPC translocation from chromosomes to spindle midzone and distinguished it from the related KLHL21 adaptor.\",\n      \"evidence\": \"RNAi knockdown with immunofluorescence of CPC localization and cytokinesis, compared with KLHL21\",\n      \"pmids\": [\"19995937\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular trigger for midzone translocation not defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extended KLHL9 function to tumor biology, positioning it as a tumor suppressor whose loss de-represses C/EBP master regulators in glioblastoma.\",\n      \"evidence\": \"KLHL9 rescue with proteasome inhibition, in vitro/in vivo tumor viability, regulatory network analysis\",\n      \"pmids\": [\"25303533\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitylation of C/EBPβ/δ by CUL3/KLHL9 not demonstrated\", \"Whether C/EBP proteins bind the Kelch domain unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Clarified upstream regulation, showing CUL3KLHL9/13 activity is independent of SCCRO-mediated neddylation that controls CUL3KLHL21, evidence for selective cullin regulation.\",\n      \"evidence\": \"SCCRO knockdown/knockout with CUL3KLHL9/13 activity as internal control versus CUL3KLHL21\",\n      \"pmids\": [\"28620047\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How CUL3KLHL9/13 is neddylated/activated not defined\", \"Primarily a contrast result for KLHL9\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified a metabolic substrate (IRS1), linking the ligase to autophagy-dependent insulin resistance.\",\n      \"evidence\": \"BioID and Co-IP interaction mapping, siRNA knockdown with IRS1 protein and insulin-signaling readouts, proteasome inhibition\",\n      \"pmids\": [\"31515271\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Degron on IRS1 not mapped\", \"Direct reconstituted ubiquitylation of IRS1 not shown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Provided systematic substrate/degron logic, showing KLHL9/KLHL13 recognize defined degron sequences at scale.\",\n      \"evidence\": \"Multiplex CRISPR screening with site-saturation degron mutagenesis on substrate pools\",\n      \"pmids\": [\"37735597\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"KLHL9-specific substrates from the screen not individually detailed\", \"Physiological relevance of screened substrates untested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Placed KLHL9 in cell-intrinsic antiviral immunity, showing it is required for IFNγ-mediated restriction of norovirus.\",\n      \"evidence\": \"Klhl9 and Cul3 knockdown/knockout with IFNγ-induced norovirus replication readout in mouse cells\",\n      \"pmids\": [\"37905813\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No molecular mechanism or substrate established for the antiviral role\", \"Single lab, loss-of-function only\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined KLHL9 as the essential adaptor for lysosomal Rheb ubiquitination, integrating it into amino-acid-induced mTORC1 activation.\",\n      \"evidence\": \"CUL3/RBX1/KLHL9 KO lines, Rheb ubiquitination assay, S6K-based mTORC1 readout, lysosomal translocation imaging, Akt negative control\",\n      \"pmids\": [\"39708321\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Rheb degron/Kelch contacts not structurally defined\", \"Mechanism of amino-acid-induced lysosomal translocation unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed pathogen hijacking of the complex, mapping a bacterial effector to KLHL9 domains and redirecting it to drive IMMT-dependent mitophagy.\",\n      \"evidence\": \"Domain-mapping Co-IP of BipD to BACK/Kelch domains, host ubiquitome profiling, K211 site-specific ubiquitination, mitophagy and mtROS assays, bacterial survival\",\n      \"pmids\": [\"38834545\", \"39265641\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether IMMT is a physiological KLHL9 substrate absent infection unknown\", \"Structural basis of BipD-mediated substrate redirection not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis of Kelch-domain substrate selection and how KLHL9 versus KLHL13 partition substrates across these diverse pathways remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No co-crystal/cryo-EM of KLHL9 with any substrate\", \"Tissue/context determinants of substrate choice undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 8, 9]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 6, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 5, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5, 8]}\n    ],\n    \"complexes\": [\"CUL3-RBX1-KLHL9/KLHL13 E3 ubiquitin ligase\"],\n    \"partners\": [\"CUL3\", \"KLHL13\", \"RBX1\", \"AURKB\", \"IRS1\", \"RHEB\", \"IMMT\", \"BipD\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":6,"faith_total":6,"faith_pct":100.0}}