{"gene":"KSR2","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":2009,"finding":"KSR2 physically interacts with and modulates the activity of AMP-activated protein kinase (AMPK), regulating AMPK-dependent glucose uptake and fatty acid oxidation in mouse embryonic fibroblasts and glycolysis in a neuronal cell line.","method":"Co-immunoprecipitation, cellular metabolic assays (glucose uptake, fatty acid oxidation, glycolysis) in MEFs and neuronal cells, KSR2 knockout mice with metabolic phenotyping and hyperinsulinemic-euglycemic clamp studies","journal":"Cell metabolism","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP identifying AMPK interaction, multiple orthogonal cellular assays, in vivo KO phenotyping, replicated across labs in subsequent studies","pmids":["19883615"],"is_preprint":false},{"year":2009,"finding":"Calcineurin selectively interacts with KSR2 (but not KSR1) and dephosphorylates KSR2 on specific sites in response to Ca2+ signals, thereby regulating KSR2 localization and activity; KSR2 uniquely promotes Ca2+-mediated ERK cascade activation in pancreatic beta-cells and neuroblastoma cells.","method":"Proteomics/mass spectrometry comparison of KSR1 vs KSR2 binding partners, Co-IP, phosphorylation mapping, KSR2 depletion (RNAi) with Ca2+-stimulated ERK activation readout in INS1 and NG108 cells","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — proteomics-identified interaction confirmed by Co-IP, phosphosite mapping, functional loss-of-function in two cell lines, multiple orthogonal methods","pmids":["19560418"],"is_preprint":false},{"year":2009,"finding":"KSR2 functions as a scaffold for the RAF-MEK-ERK kinase module (similar to KSR1) but shows distinct RAF isoform specificity: KSR2 preferentially mediates A-RAF signaling whereas KSR1 mediates c-RAF signaling.","method":"Functional proteomics (affinity purification/mass spectrometry) of the KSR2 complex in HEK-293 cells with and without TNF-α stimulation, identifying ~100 associated proteins","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — proteomics interactome, single lab, RAF isoform specificity inferred from complex composition but not confirmed by mutagenesis or reconstitution","pmids":["19563921"],"is_preprint":false},{"year":2013,"finding":"Rare human KSR2 variants disrupt signaling through the Raf-MEK-ERK pathway and impair cellular fatty acid oxidation and glucose oxidation; these metabolic defects can be ameliorated by metformin treatment.","method":"Sequencing of 2,101 obese individuals and 1,536 controls; functional assays of KSR2 mutants in transfected cells measuring ERK signaling, fatty acid oxidation, and glucose oxidation; metformin rescue experiments","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple human variants functionally validated in cellular assays with ERK signaling and metabolic readouts, pharmacological rescue, large cohort","pmids":["24209692"],"is_preprint":false},{"year":2012,"finding":"KSR2 promotes ERK activation and cell transformation via its scaffold function, but its role in supporting anchorage-independent growth of tumor cells is dependent on AMPK signaling rather than MAP kinase signaling; constitutive AMPK activation complements KSR2 loss in metabolic signaling and anchorage-independent growth.","method":"KSR2 RNAi in MIN6 and NG108-15 tumor cell lines; rescue with constitutively active AMPK; MEK inhibition; expression of KSR2 mutant unable to interact with ERK; colony formation and proliferation assays","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined phenotype, epistasis via constitutive AMPK rescue and ERK-interaction-deficient mutant, single lab","pmids":["22801368"],"is_preprint":false},{"year":2014,"finding":"KSR2, through its associated calcineurin, is required for optimal store-operated calcium entry (SOCE); KSR2 deficiency impairs STIM1/ORAI1 puncta formation correlated with cytoskeleton disorganization, and calcineurin-dependent SOCE is KSR2-dependent.","method":"KSR2-knockout lymphocytes and fibroblasts, shKSR2 knockdown cells; Ca2+ flux measurements; STIM1/ORAI1 puncta imaging; cytoskeleton analysis; calcineurin inhibitor experiments","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined Ca2+ phenotype, imaging of STIM1/ORAI1 dynamics, pharmacological inhibition of calcineurin, single lab","pmids":["24672054"],"is_preprint":false},{"year":2002,"finding":"In C. elegans, ksr-2 (ortholog of mammalian KSR2) is required for Ras-mediated signaling during germline meiotic progression and acts redundantly with ksr-1; ksr-2; ksr-1 double mutants show severely reduced or absent diphosphorylated MPK-1 ERK, supporting KSR's role in promoting Raf/MEK/ERK cascade activation.","method":"C. elegans genetic analysis; double mutant construction; anti-dpERK immunofluorescence to quantify activated ERK levels","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in C. elegans ortholog, double mutant with direct ERK phosphorylation readout, established model organism system","pmids":["11882296"],"is_preprint":false},{"year":2016,"finding":"Brain-specific (Nestin-Cre driven) KSR2 deletion recapitulates obesity and glucose intolerance of global KSR2 knockout, demonstrating that KSR2 functions in the brain to regulate energy balance via feeding behavior and adaptive thermogenesis; however, brain KSR2 alone does not fully account for leptin sensitivity and AICAR response, indicating additional peripheral tissue roles.","method":"Conditional knockout using Nes-Cre × KSR2fl/fl; metabolic phenotyping including body composition, food intake, cold tolerance, glucose tolerance, insulin levels, AICAR and leptin response","journal":"Molecular metabolism","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — tissue-specific conditional KO with multiple metabolic phenotype readouts, single lab","pmids":["28180061"],"is_preprint":false},{"year":2016,"finding":"KSR2 disruption causes selective impairment of hepatic GH-stimulated JAK2/STAT5 signaling and reduced IGF-1/IGFBP3 expression in neonatal mice; this effect is cell non-autonomous, as isolated KSR2-null primary hepatocytes show normal GH-stimulated STAT5 phosphorylation, and IGF-1 restoration rescues body length defects.","method":"KSR2 knockout mouse GH injection studies; measurement of JAK2 and STAT5 phosphorylation in liver vs skeletal muscle; primary hepatocyte isolation and GH stimulation; adenoviral IGF-1 rescue experiment","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo vs in vitro dissociation establishing cell non-autonomy, rescue experiment, single lab","pmids":["27561547"],"is_preprint":false},{"year":2022,"finding":"14-3-3ζ physically complexes with KSR2, elevates KSR2 protein levels, and co-overexpression of both proteins hyperactivates MAPK signaling and confers sorafenib resistance in HCC cells.","method":"Co-immunoprecipitation; co-overexpression and knockdown experiments; MAPK signaling assays; flow cytometry for sorafenib resistance","journal":"Biomarker research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP identifying 14-3-3ζ/KSR2 interaction, functional co-overexpression/knockdown with signaling and drug resistance readouts, single lab","pmids":["35468812"],"is_preprint":false},{"year":2022,"finding":"KSR2 regulates bone formation by influencing adipocyte differentiation at the expense of osteoblasts in bone marrow; osteoblast-specific conditional deletion of KSR2 autonomously regulates bone formation, distinct from its hypothalamic role in feeding.","method":"Global KSR2 knockout mice (two genetic backgrounds); osteoblast-specific conditional KSR2 deletion; pair-feeding experiments; micro-CT and histology","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with specific cellular phenotype (adipocyte vs osteoblast fate), pair-feeding control to separate metabolic effects, single lab","pmids":["36342465"],"is_preprint":false},{"year":2025,"finding":"The self-renewal and clonogenicity-promoting function of KSR2 in SCLC-A cells is dependent on KSR2's interaction with ERK; a KSR2 mutant unable to interact with ERK fails to support colony formation and tumor initiation.","method":"KSR2 depletion in SCLC-A cell lines; colony-forming assay in vitro; tumor initiation assay in vivo; KSR2 ERK-interaction-deficient mutant rescue experiments","journal":"Molecular cancer research : MCR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined cellular phenotype, structure-function analysis using interaction-deficient mutant, in vitro and in vivo validation, single lab","pmids":["40063515"],"is_preprint":false},{"year":2026,"finding":"KSR2 competes with CRBN for binding to the K52 site of AMPKα1, inhibiting CRL4A E3 ubiquitin ligase complex-mediated K48-linked polyubiquitination and proteasomal degradation of AMPKα1, thereby stabilizing AMPKα1 and maintaining AMPK signaling in endothelial cells.","method":"Co-IP, CRISPR/Cas9 gene editing, endothelial-specific KSR2 overexpression (AAV9-ICAM2), global KSR2 KO and double KO (KSR2/ApoE) mouse models, atherosclerosis assays, ubiquitination assays","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic ubiquitination pathway defined by Co-IP and competition assays, multiple mouse models, single lab, 2026 publication not yet independently replicated","pmids":["41424849"],"is_preprint":false},{"year":2020,"finding":"SF3B1 promotes KSR2 expression by facilitating maturation of KSR2 pre-mRNA; loss of SF3B1 reduces KSR2 mRNA maturation and expression, and rescuing KSR2 expression partially restores cell growth upon SF3B1 knockdown in endometrial cancer cells.","method":"RNA-seq after SF3B1 knockdown; alternative splicing analysis; KSR2 rescue experiment in SF3B1-depleted cells measuring proliferation","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — RNA-seq with alternative splicing analysis, rescue experiment placing KSR2 downstream of SF3B1 in RNA processing, single lab","pmids":["33040078"],"is_preprint":false}],"current_model":"KSR2 is a molecular scaffold protein that promotes Raf/MEK/ERK signaling and uniquely interacts with AMPK (stabilizing AMPKα1 by competing with CRBN-CRL4A ubiquitin ligase-mediated degradation) and calcineurin (which dephosphorylates KSR2 to enable Ca2+-mediated ERK activation and store-operated calcium entry via STIM1/ORAI1), thereby integrating mitogenic and metabolic signaling to regulate energy expenditure, fatty acid and glucose oxidation, and cellular responses to calcium—with loss-of-function causing obesity, insulin resistance, and impaired substrate utilization in both mice and humans."},"narrative":{"mechanistic_narrative":"KSR2 is a molecular scaffold for the RAF-MEK-ERK kinase module that integrates mitogenic signaling with cellular energy and calcium homeostasis [PMID:19563921, PMID:11882296]. Like KSR1, it organizes the cascade to promote ERK activation, with a function evolutionarily conserved in the C. elegans ortholog ksr-2, which acts redundantly with ksr-1 to sustain MPK-1/ERK diphosphorylation during Ras-mediated germline signaling [PMID:11882296]. Distinct from KSR1, KSR2 physically associates with AMP-activated protein kinase and governs AMPK-dependent glucose uptake and fatty acid oxidation [PMID:19883615]; mechanistically, KSR2 competes with CRBN for the K52 site of AMPKα1, blocking CRL4A-mediated K48-linked polyubiquitination and proteasomal degradation to stabilize AMPKα1 [PMID:41424849]. It is also selectively bound and dephosphorylated by calcineurin in response to Ca2+, which directs KSR2 localization and uniquely enables Ca2+-mediated ERK activation; through this calcineurin association KSR2 is further required for store-operated calcium entry, supporting STIM1/ORAI1 puncta formation [PMID:19560418, PMID:24672054]. These activities make KSR2 a node coordinating substrate utilization and energy balance: rare human loss-of-function variants disrupt Raf-MEK-ERK signaling and impair cellular fatty acid and glucose oxidation in obese individuals, defects rescuable by metformin [PMID:24209692]. In vivo, KSR2 acts in the brain to regulate energy balance via feeding and adaptive thermogenesis, with additional peripheral roles [PMID:28180061], and autonomously controls bone formation by biasing marrow adipocyte versus osteoblast fate [PMID:36342465]. In cancer contexts, KSR2's scaffold and AMPK-coupled functions support anchorage-independent growth, ERK-dependent self-renewal in SCLC-A cells, and MAPK hyperactivation with drug resistance through partners including 14-3-3ζ [PMID:22801368, PMID:40063515, PMID:35468812].","teleology":[{"year":2002,"claim":"Established that the KSR2 ortholog functions in the Ras/Raf/MEK/ERK cascade in vivo, fixing KSR proteins as positive regulators of ERK activation rather than passive bystanders.","evidence":"C. elegans genetic epistasis with ksr-1;ksr-2 double mutants and anti-dpERK immunofluorescence","pmids":["11882296"],"confidence":"Medium","gaps":["Does not address mammalian KSR2-specific biochemistry","Redundancy with ksr-1 obscures KSR2-unique functions"]},{"year":2009,"claim":"Distinguished KSR2 from KSR1 by showing it physically engages AMPK and calcineurin, revealing KSR2 as an integrator of metabolic and calcium signaling beyond simple MAPK scaffolding.","evidence":"Reciprocal Co-IP, proteomics comparison of KSR1 vs KSR2 partners, phosphosite mapping, RNAi-based Ca2+/ERK readouts, and KSR2-KO mouse metabolic phenotyping","pmids":["19883615","19560418"],"confidence":"High","gaps":["Structural basis of calcineurin-dependent dephosphorylation sites not resolved","How AMPK binding mechanistically alters its activity not defined at this stage"]},{"year":2009,"claim":"Defined KSR2 as a RAF-MEK-ERK scaffold with RAF isoform preference distinct from KSR1, partially explaining functional divergence between the paralogs.","evidence":"Affinity purification/mass spectrometry of the KSR2 complex in HEK-293 cells +/- TNF-α","pmids":["19563921"],"confidence":"Medium","gaps":["A-RAF preference inferred from complex composition, not confirmed by mutagenesis or reconstitution","Single lab"]},{"year":2012,"claim":"Separated KSR2's growth-promoting role from its MAPK role, showing anchorage-independent growth depends on AMPK rather than MEK/ERK signaling.","evidence":"KSR2 RNAi in tumor lines with constitutively active AMPK rescue, MEK inhibition, and ERK-interaction-deficient mutant in colony assays","pmids":["22801368"],"confidence":"Medium","gaps":["Single lab","Generality across tumor types not established"]},{"year":2013,"claim":"Connected KSR2 mechanistic function to human disease, showing rare variants disrupt ERK signaling and substrate oxidation in obesity and are pharmacologically rescuable.","evidence":"Sequencing of 2,101 obese individuals/1,536 controls with functional ERK, fatty acid and glucose oxidation assays and metformin rescue","pmids":["24209692"],"confidence":"High","gaps":["Tissue-of-origin for human metabolic defect not resolved by cellular assays","Whether AMPK or ERK arm dominates the human phenotype unclear"]},{"year":2014,"claim":"Extended KSR2's calcineurin partnership to a defined cell-biological output—store-operated calcium entry—linking it to STIM1/ORAI1 and cytoskeletal organization.","evidence":"KSR2-KO and shKSR2 cells with Ca2+ flux, STIM1/ORAI1 puncta imaging, cytoskeleton analysis, and calcineurin inhibition","pmids":["24672054"],"confidence":"Medium","gaps":["Direct molecular link between KSR2 and STIM1/ORAI1 not established","Single lab"]},{"year":2016,"claim":"Localized KSR2's energy-balance function to the brain while revealing it also acts non-autonomously on hepatic GH/JAK2/STAT5-IGF-1 signaling, showing multi-tissue contributions.","evidence":"Nestin-Cre conditional KO metabolic phenotyping and KSR2-KO GH stimulation with hepatocyte and adenoviral IGF-1 rescue","pmids":["28180061","27561547"],"confidence":"Medium","gaps":["Identity of peripheral tissues accounting for leptin/AICAR phenotype unknown","Mediator of cell non-autonomous hepatic effect unidentified"]},{"year":2022,"claim":"Broadened KSR2's roles to bone formation and cancer drug resistance, and introduced 14-3-3ζ as a stabilizing physical partner that amplifies MAPK signaling.","evidence":"Osteoblast-specific conditional KO with micro-CT/pair-feeding; Co-IP and co-overexpression/knockdown of 14-3-3ζ with MAPK and sorafenib-resistance readouts","pmids":["36342465","35468812"],"confidence":"Medium","gaps":["Single lab for each finding","Mechanism by which 14-3-3ζ elevates KSR2 levels undefined"]},{"year":2025,"claim":"Demonstrated that KSR2's tumor-initiating function in SCLC-A is strictly ERK-interaction-dependent, validating the scaffold's MAPK arm as a driver in this context.","evidence":"KSR2 depletion with ERK-interaction-deficient mutant rescue in colony-forming and in vivo tumor initiation assays","pmids":["40063515"],"confidence":"Medium","gaps":["Single lab","Relationship to AMPK-dependent growth seen in other tumors not reconciled"]},{"year":2026,"claim":"Resolved the molecular mechanism of AMPK regulation, showing KSR2 stabilizes AMPKα1 by competing with CRBN at the K52 site to block CRL4A-mediated degradation.","evidence":"Co-IP and competition/ubiquitination assays with CRISPR editing and endothelial KSR2 overexpression/KO and ApoE double-KO atherosclerosis models","pmids":["41424849"],"confidence":"Medium","gaps":["2026 publication not yet independently replicated","Whether this competition operates outside endothelium not tested"]},{"year":null,"claim":"How KSR2's three biochemical activities—RAF-MEK-ERK scaffolding, AMPKα1 stabilization, and calcineurin-mediated calcium coupling—are coordinated within a single cell and which arm dominates in each tissue remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated structural model of KSR2 engaging RAF, AMPK, and calcineurin simultaneously","Tissue-specific weighting of each activity not mapped","Subcellular localization dynamics underlying signal switching uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,6,11]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,12]}],"localization":[],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,6,1]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,3]}],"complexes":["RAF-MEK-ERK module"],"partners":["AMPK","PPP3 (CALCINEURIN)","CRBN","STIM1","ORAI1","YWHAZ","ARAF"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6VAB6","full_name":"Kinase suppressor of Ras 2","aliases":[],"length_aa":950,"mass_kda":107.6,"function":"Location-regulated scaffold connecting MEK to RAF. Has very low protein kinase activity and can phosphorylate MAP2K1 at several Ser and Thr residues with very low efficiency (in vitro). Acts as MAP2K1/MEK1-dependent allosteric activator of BRAF; upon binding to MAP2K1/MEK1, dimerizes with BRAF and promotes BRAF-mediated phosphorylation of MAP2K1/MEK1 (PubMed:29433126). Interaction with BRAF enhances KSR2-mediated phosphorylation of MAP2K1 (in vitro). Blocks MAP3K8 kinase activity and MAP3K8-mediated signaling. Acts as a negative regulator of MAP3K3-mediated activation of ERK, JNK and NF-kappa-B pathways, inhibiting MAP3K3-mediated interleukin-8 production","subcellular_location":"Cytoplasm; Membrane","url":"https://www.uniprot.org/uniprotkb/Q6VAB6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/KSR2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/KSR2","total_profiled":1310},"omim":[{"mim_id":"610737","title":"KINASE SUPPRESSOR OF RAS 2; KSR2","url":"https://www.omim.org/entry/610737"},{"mim_id":"602539","title":"MITOGEN-ACTIVATED KINASE KINASE KINASE 3; MAP3K3","url":"https://www.omim.org/entry/602539"},{"mim_id":"191195","title":"MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE 8; MAP3K8","url":"https://www.omim.org/entry/191195"},{"mim_id":"176872","title":"MITOGEN-ACTIVATED PROTEIN KINASE KINASE 1; MAP2K1","url":"https://www.omim.org/entry/176872"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"brain","ntpm":5.4},{"tissue":"pituitary 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States","date":"2025-10-23","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.22.684065","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13817,"output_tokens":3635,"usd":0.047988,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11376,"output_tokens":3641,"usd":0.073953,"stage2_stop_reason":"end_turn"},"total_usd":0.121941,"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\": 2009,\n      \"finding\": \"KSR2 physically interacts with and modulates the activity of AMP-activated protein kinase (AMPK), regulating AMPK-dependent glucose uptake and fatty acid oxidation in mouse embryonic fibroblasts and glycolysis in a neuronal cell line.\",\n      \"method\": \"Co-immunoprecipitation, cellular metabolic assays (glucose uptake, fatty acid oxidation, glycolysis) in MEFs and neuronal cells, KSR2 knockout mice with metabolic phenotyping and hyperinsulinemic-euglycemic clamp studies\",\n      \"journal\": \"Cell metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP identifying AMPK interaction, multiple orthogonal cellular assays, in vivo KO phenotyping, replicated across labs in subsequent studies\",\n      \"pmids\": [\"19883615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Calcineurin selectively interacts with KSR2 (but not KSR1) and dephosphorylates KSR2 on specific sites in response to Ca2+ signals, thereby regulating KSR2 localization and activity; KSR2 uniquely promotes Ca2+-mediated ERK cascade activation in pancreatic beta-cells and neuroblastoma cells.\",\n      \"method\": \"Proteomics/mass spectrometry comparison of KSR1 vs KSR2 binding partners, Co-IP, phosphorylation mapping, KSR2 depletion (RNAi) with Ca2+-stimulated ERK activation readout in INS1 and NG108 cells\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — proteomics-identified interaction confirmed by Co-IP, phosphosite mapping, functional loss-of-function in two cell lines, multiple orthogonal methods\",\n      \"pmids\": [\"19560418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"KSR2 functions as a scaffold for the RAF-MEK-ERK kinase module (similar to KSR1) but shows distinct RAF isoform specificity: KSR2 preferentially mediates A-RAF signaling whereas KSR1 mediates c-RAF signaling.\",\n      \"method\": \"Functional proteomics (affinity purification/mass spectrometry) of the KSR2 complex in HEK-293 cells with and without TNF-α stimulation, identifying ~100 associated proteins\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — proteomics interactome, single lab, RAF isoform specificity inferred from complex composition but not confirmed by mutagenesis or reconstitution\",\n      \"pmids\": [\"19563921\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Rare human KSR2 variants disrupt signaling through the Raf-MEK-ERK pathway and impair cellular fatty acid oxidation and glucose oxidation; these metabolic defects can be ameliorated by metformin treatment.\",\n      \"method\": \"Sequencing of 2,101 obese individuals and 1,536 controls; functional assays of KSR2 mutants in transfected cells measuring ERK signaling, fatty acid oxidation, and glucose oxidation; metformin rescue experiments\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple human variants functionally validated in cellular assays with ERK signaling and metabolic readouts, pharmacological rescue, large cohort\",\n      \"pmids\": [\"24209692\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"KSR2 promotes ERK activation and cell transformation via its scaffold function, but its role in supporting anchorage-independent growth of tumor cells is dependent on AMPK signaling rather than MAP kinase signaling; constitutive AMPK activation complements KSR2 loss in metabolic signaling and anchorage-independent growth.\",\n      \"method\": \"KSR2 RNAi in MIN6 and NG108-15 tumor cell lines; rescue with constitutively active AMPK; MEK inhibition; expression of KSR2 mutant unable to interact with ERK; colony formation and proliferation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined phenotype, epistasis via constitutive AMPK rescue and ERK-interaction-deficient mutant, single lab\",\n      \"pmids\": [\"22801368\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"KSR2, through its associated calcineurin, is required for optimal store-operated calcium entry (SOCE); KSR2 deficiency impairs STIM1/ORAI1 puncta formation correlated with cytoskeleton disorganization, and calcineurin-dependent SOCE is KSR2-dependent.\",\n      \"method\": \"KSR2-knockout lymphocytes and fibroblasts, shKSR2 knockdown cells; Ca2+ flux measurements; STIM1/ORAI1 puncta imaging; cytoskeleton analysis; calcineurin inhibitor experiments\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined Ca2+ phenotype, imaging of STIM1/ORAI1 dynamics, pharmacological inhibition of calcineurin, single lab\",\n      \"pmids\": [\"24672054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"In C. elegans, ksr-2 (ortholog of mammalian KSR2) is required for Ras-mediated signaling during germline meiotic progression and acts redundantly with ksr-1; ksr-2; ksr-1 double mutants show severely reduced or absent diphosphorylated MPK-1 ERK, supporting KSR's role in promoting Raf/MEK/ERK cascade activation.\",\n      \"method\": \"C. elegans genetic analysis; double mutant construction; anti-dpERK immunofluorescence to quantify activated ERK levels\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in C. elegans ortholog, double mutant with direct ERK phosphorylation readout, established model organism system\",\n      \"pmids\": [\"11882296\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Brain-specific (Nestin-Cre driven) KSR2 deletion recapitulates obesity and glucose intolerance of global KSR2 knockout, demonstrating that KSR2 functions in the brain to regulate energy balance via feeding behavior and adaptive thermogenesis; however, brain KSR2 alone does not fully account for leptin sensitivity and AICAR response, indicating additional peripheral tissue roles.\",\n      \"method\": \"Conditional knockout using Nes-Cre × KSR2fl/fl; metabolic phenotyping including body composition, food intake, cold tolerance, glucose tolerance, insulin levels, AICAR and leptin response\",\n      \"journal\": \"Molecular metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — tissue-specific conditional KO with multiple metabolic phenotype readouts, single lab\",\n      \"pmids\": [\"28180061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"KSR2 disruption causes selective impairment of hepatic GH-stimulated JAK2/STAT5 signaling and reduced IGF-1/IGFBP3 expression in neonatal mice; this effect is cell non-autonomous, as isolated KSR2-null primary hepatocytes show normal GH-stimulated STAT5 phosphorylation, and IGF-1 restoration rescues body length defects.\",\n      \"method\": \"KSR2 knockout mouse GH injection studies; measurement of JAK2 and STAT5 phosphorylation in liver vs skeletal muscle; primary hepatocyte isolation and GH stimulation; adenoviral IGF-1 rescue experiment\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo vs in vitro dissociation establishing cell non-autonomy, rescue experiment, single lab\",\n      \"pmids\": [\"27561547\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"14-3-3ζ physically complexes with KSR2, elevates KSR2 protein levels, and co-overexpression of both proteins hyperactivates MAPK signaling and confers sorafenib resistance in HCC cells.\",\n      \"method\": \"Co-immunoprecipitation; co-overexpression and knockdown experiments; MAPK signaling assays; flow cytometry for sorafenib resistance\",\n      \"journal\": \"Biomarker research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP identifying 14-3-3ζ/KSR2 interaction, functional co-overexpression/knockdown with signaling and drug resistance readouts, single lab\",\n      \"pmids\": [\"35468812\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"KSR2 regulates bone formation by influencing adipocyte differentiation at the expense of osteoblasts in bone marrow; osteoblast-specific conditional deletion of KSR2 autonomously regulates bone formation, distinct from its hypothalamic role in feeding.\",\n      \"method\": \"Global KSR2 knockout mice (two genetic backgrounds); osteoblast-specific conditional KSR2 deletion; pair-feeding experiments; micro-CT and histology\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with specific cellular phenotype (adipocyte vs osteoblast fate), pair-feeding control to separate metabolic effects, single lab\",\n      \"pmids\": [\"36342465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The self-renewal and clonogenicity-promoting function of KSR2 in SCLC-A cells is dependent on KSR2's interaction with ERK; a KSR2 mutant unable to interact with ERK fails to support colony formation and tumor initiation.\",\n      \"method\": \"KSR2 depletion in SCLC-A cell lines; colony-forming assay in vitro; tumor initiation assay in vivo; KSR2 ERK-interaction-deficient mutant rescue experiments\",\n      \"journal\": \"Molecular cancer research : MCR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined cellular phenotype, structure-function analysis using interaction-deficient mutant, in vitro and in vivo validation, single lab\",\n      \"pmids\": [\"40063515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"KSR2 competes with CRBN for binding to the K52 site of AMPKα1, inhibiting CRL4A E3 ubiquitin ligase complex-mediated K48-linked polyubiquitination and proteasomal degradation of AMPKα1, thereby stabilizing AMPKα1 and maintaining AMPK signaling in endothelial cells.\",\n      \"method\": \"Co-IP, CRISPR/Cas9 gene editing, endothelial-specific KSR2 overexpression (AAV9-ICAM2), global KSR2 KO and double KO (KSR2/ApoE) mouse models, atherosclerosis assays, ubiquitination assays\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic ubiquitination pathway defined by Co-IP and competition assays, multiple mouse models, single lab, 2026 publication not yet independently replicated\",\n      \"pmids\": [\"41424849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SF3B1 promotes KSR2 expression by facilitating maturation of KSR2 pre-mRNA; loss of SF3B1 reduces KSR2 mRNA maturation and expression, and rescuing KSR2 expression partially restores cell growth upon SF3B1 knockdown in endometrial cancer cells.\",\n      \"method\": \"RNA-seq after SF3B1 knockdown; alternative splicing analysis; KSR2 rescue experiment in SF3B1-depleted cells measuring proliferation\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — RNA-seq with alternative splicing analysis, rescue experiment placing KSR2 downstream of SF3B1 in RNA processing, single lab\",\n      \"pmids\": [\"33040078\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"KSR2 is a molecular scaffold protein that promotes Raf/MEK/ERK signaling and uniquely interacts with AMPK (stabilizing AMPKα1 by competing with CRBN-CRL4A ubiquitin ligase-mediated degradation) and calcineurin (which dephosphorylates KSR2 to enable Ca2+-mediated ERK activation and store-operated calcium entry via STIM1/ORAI1), thereby integrating mitogenic and metabolic signaling to regulate energy expenditure, fatty acid and glucose oxidation, and cellular responses to calcium—with loss-of-function causing obesity, insulin resistance, and impaired substrate utilization in both mice and humans.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"KSR2 is a molecular scaffold for the RAF-MEK-ERK kinase module that integrates mitogenic signaling with cellular energy and calcium homeostasis [#2, #6]. Like KSR1, it organizes the cascade to promote ERK activation, with a function evolutionarily conserved in the C. elegans ortholog ksr-2, which acts redundantly with ksr-1 to sustain MPK-1/ERK diphosphorylation during Ras-mediated germline signaling [#6]. Distinct from KSR1, KSR2 physically associates with AMP-activated protein kinase and governs AMPK-dependent glucose uptake and fatty acid oxidation [#0]; mechanistically, KSR2 competes with CRBN for the K52 site of AMPKα1, blocking CRL4A-mediated K48-linked polyubiquitination and proteasomal degradation to stabilize AMPKα1 [#12]. It is also selectively bound and dephosphorylated by calcineurin in response to Ca2+, which directs KSR2 localization and uniquely enables Ca2+-mediated ERK activation; through this calcineurin association KSR2 is further required for store-operated calcium entry, supporting STIM1/ORAI1 puncta formation [#1, #5]. These activities make KSR2 a node coordinating substrate utilization and energy balance: rare human loss-of-function variants disrupt Raf-MEK-ERK signaling and impair cellular fatty acid and glucose oxidation in obese individuals, defects rescuable by metformin [#3]. In vivo, KSR2 acts in the brain to regulate energy balance via feeding and adaptive thermogenesis, with additional peripheral roles [#7], and autonomously controls bone formation by biasing marrow adipocyte versus osteoblast fate [#10]. In cancer contexts, KSR2's scaffold and AMPK-coupled functions support anchorage-independent growth, ERK-dependent self-renewal in SCLC-A cells, and MAPK hyperactivation with drug resistance through partners including 14-3-3ζ [#4, #11, #9].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established that the KSR2 ortholog functions in the Ras/Raf/MEK/ERK cascade in vivo, fixing KSR proteins as positive regulators of ERK activation rather than passive bystanders.\",\n      \"evidence\": \"C. elegans genetic epistasis with ksr-1;ksr-2 double mutants and anti-dpERK immunofluorescence\",\n      \"pmids\": [\"11882296\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not address mammalian KSR2-specific biochemistry\", \"Redundancy with ksr-1 obscures KSR2-unique functions\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Distinguished KSR2 from KSR1 by showing it physically engages AMPK and calcineurin, revealing KSR2 as an integrator of metabolic and calcium signaling beyond simple MAPK scaffolding.\",\n      \"evidence\": \"Reciprocal Co-IP, proteomics comparison of KSR1 vs KSR2 partners, phosphosite mapping, RNAi-based Ca2+/ERK readouts, and KSR2-KO mouse metabolic phenotyping\",\n      \"pmids\": [\"19883615\", \"19560418\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of calcineurin-dependent dephosphorylation sites not resolved\", \"How AMPK binding mechanistically alters its activity not defined at this stage\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined KSR2 as a RAF-MEK-ERK scaffold with RAF isoform preference distinct from KSR1, partially explaining functional divergence between the paralogs.\",\n      \"evidence\": \"Affinity purification/mass spectrometry of the KSR2 complex in HEK-293 cells +/- TNF-α\",\n      \"pmids\": [\"19563921\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"A-RAF preference inferred from complex composition, not confirmed by mutagenesis or reconstitution\", \"Single lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Separated KSR2's growth-promoting role from its MAPK role, showing anchorage-independent growth depends on AMPK rather than MEK/ERK signaling.\",\n      \"evidence\": \"KSR2 RNAi in tumor lines with constitutively active AMPK rescue, MEK inhibition, and ERK-interaction-deficient mutant in colony assays\",\n      \"pmids\": [\"22801368\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Generality across tumor types not established\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Connected KSR2 mechanistic function to human disease, showing rare variants disrupt ERK signaling and substrate oxidation in obesity and are pharmacologically rescuable.\",\n      \"evidence\": \"Sequencing of 2,101 obese individuals/1,536 controls with functional ERK, fatty acid and glucose oxidation assays and metformin rescue\",\n      \"pmids\": [\"24209692\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-of-origin for human metabolic defect not resolved by cellular assays\", \"Whether AMPK or ERK arm dominates the human phenotype unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extended KSR2's calcineurin partnership to a defined cell-biological output—store-operated calcium entry—linking it to STIM1/ORAI1 and cytoskeletal organization.\",\n      \"evidence\": \"KSR2-KO and shKSR2 cells with Ca2+ flux, STIM1/ORAI1 puncta imaging, cytoskeleton analysis, and calcineurin inhibition\",\n      \"pmids\": [\"24672054\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular link between KSR2 and STIM1/ORAI1 not established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Localized KSR2's energy-balance function to the brain while revealing it also acts non-autonomously on hepatic GH/JAK2/STAT5-IGF-1 signaling, showing multi-tissue contributions.\",\n      \"evidence\": \"Nestin-Cre conditional KO metabolic phenotyping and KSR2-KO GH stimulation with hepatocyte and adenoviral IGF-1 rescue\",\n      \"pmids\": [\"28180061\", \"27561547\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of peripheral tissues accounting for leptin/AICAR phenotype unknown\", \"Mediator of cell non-autonomous hepatic effect unidentified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Broadened KSR2's roles to bone formation and cancer drug resistance, and introduced 14-3-3ζ as a stabilizing physical partner that amplifies MAPK signaling.\",\n      \"evidence\": \"Osteoblast-specific conditional KO with micro-CT/pair-feeding; Co-IP and co-overexpression/knockdown of 14-3-3ζ with MAPK and sorafenib-resistance readouts\",\n      \"pmids\": [\"36342465\", \"35468812\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab for each finding\", \"Mechanism by which 14-3-3ζ elevates KSR2 levels undefined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated that KSR2's tumor-initiating function in SCLC-A is strictly ERK-interaction-dependent, validating the scaffold's MAPK arm as a driver in this context.\",\n      \"evidence\": \"KSR2 depletion with ERK-interaction-deficient mutant rescue in colony-forming and in vivo tumor initiation assays\",\n      \"pmids\": [\"40063515\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Relationship to AMPK-dependent growth seen in other tumors not reconciled\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Resolved the molecular mechanism of AMPK regulation, showing KSR2 stabilizes AMPKα1 by competing with CRBN at the K52 site to block CRL4A-mediated degradation.\",\n      \"evidence\": \"Co-IP and competition/ubiquitination assays with CRISPR editing and endothelial KSR2 overexpression/KO and ApoE double-KO atherosclerosis models\",\n      \"pmids\": [\"41424849\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"2026 publication not yet independently replicated\", \"Whether this competition operates outside endothelium not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How KSR2's three biochemical activities—RAF-MEK-ERK scaffolding, AMPKα1 stabilization, and calcineurin-mediated calcium coupling—are coordinated within a single cell and which arm dominates in each tissue remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated structural model of KSR2 engaging RAF, AMPK, and calcineurin simultaneously\", \"Tissue-specific weighting of each activity not mapped\", \"Subcellular localization dynamics underlying signal switching uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 6, 11]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 12]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 6, 1]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"complexes\": [\"RAF-MEK-ERK module\"],\n    \"partners\": [\"AMPK\", \"PPP3 (calcineurin)\", \"CRBN\", \"STIM1\", \"ORAI1\", \"YWHAZ\", \"ARAF\"]\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}