{"gene":"KSR1","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":1995,"finding":"C. elegans ksr-1 encodes a novel putative Raf-related Ser/Thr kinase that positively mediates Ras signaling; genetic epistasis analysis placed ksr-1 downstream of or in parallel to let-60 ras, required for maximal stimulation of vulval fates by the Ras pathway.","method":"Genetic suppressor screen, epistasis analysis, gene cloning","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent labs (Sundaram/Han and Kornfeld/Horvitz) published simultaneous genetic epistasis studies in the same issue, replicated findings","pmids":["8521513","8521514"],"is_preprint":false},{"year":2001,"finding":"C-TAK1 kinase constitutively associates with mammalian KSR1 and phosphorylates Ser392, conferring 14-3-3 binding and cytoplasmic sequestration in unstimulated cells; upon signal activation, Ser392 phosphorylation is reduced, allowing the KSR1 complex to translocate to the plasma membrane where it colocalizes with activated Ras and Raf-1 to facilitate MEK and MAPK activation.","method":"Co-immunoprecipitation, phosphorylation mapping, subcellular fractionation/imaging, mutagenesis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, site-specific mutagenesis, functional localization consequence established in single rigorous study","pmids":["11741534"],"is_preprint":false},{"year":2003,"finding":"Protein phosphatase PP2A constitutively associates (core subunits PR65A and catalytic C) with the N-terminal domain of KSR1; upon growth factor treatment, regulatory subunit PR55B binding is induced. PP2A dephosphorylates the critical 14-3-3 binding site on KSR1 (and Raf-1), enabling membrane recruitment of KSR1 and activation of KSR1-associated MEK and ERK.","method":"Mass spectrometry of KSR1 complex, co-immunoprecipitation, pharmacological PP2A inhibition, phosphorylation analysis","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — MS-based complex identification plus functional validation with multiple orthogonal methods (Co-IP, inhibitor, phospho-mapping) in one rigorous study","pmids":["12932319"],"is_preprint":false},{"year":2002,"finding":"In C. elegans, ksr-1 and ksr-2 act redundantly; ksr-2;ksr-1 double mutants show severely reduced or absent diphosphorylated MPK-1 ERK, strongly supporting a model where KSR acts to promote activation or maintenance of the Raf/MEK/ERK kinase cascade.","method":"Double-mutant genetic analysis, ERK phosphorylation western blot","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic double-KO with direct biochemical readout (dpERK levels), replicated across multiple developmental contexts","pmids":["11882296"],"is_preprint":false},{"year":2006,"finding":"Casein kinase 2 (CK2) holoenzyme constitutively binds the basic surface region of the KSR1 atypical C1 domain; this KSR1/CK2 interaction is required for maximal growth-factor-induced phosphorylation of C-Raf and B-Raf on the activating N-region serine, and for downstream MEK and ERK activation, identifying CK2 as a Raf N-region kinase within the KSR1 scaffold complex.","method":"Mass spectrometry of KSR1 complex, co-immunoprecipitation, domain-mapping mutagenesis, kinase activity assays","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — MS identification plus functional validation with mutagenesis and kinase assays in single thorough study","pmids":["17174095"],"is_preprint":false},{"year":2000,"finding":"B-KSR1, a brain-specific KSR1 splice variant, constitutively associates with MEK (requiring an intact kinase domain for MEK binding) and inducibly associates with activated MAPK; all loss-of-function mutations in the catalytic domain that abolish MEK binding also abolish the ability to augment NGF-induced neurite outgrowth, demonstrating that MEK binding is functionally essential.","method":"Co-immunoprecipitation, mutagenesis, PC12 cell differentiation assay, MEK inhibitor treatment","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with mutagenesis and functional rescue/loss-of-function in single study","pmids":["10891492"],"is_preprint":false},{"year":1999,"finding":"KSR1 binds directly to G-protein βγ subunits via its CA3 domain (containing the cysteine-rich zinc finger-like domain); LPA-induced KSR1 translocation to the plasma membrane is blocked by pertussis toxin but not dominant-negative Ras; overexpression of KSR1 inhibits βγ-induced MAPK activation.","method":"Yeast two-hybrid screen, co-immunoprecipitation, deletion analysis, pertussis toxin treatment, MAPK activation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Y2H plus Co-IP plus functional inhibition assay in a single study, single lab","pmids":["10075696"],"is_preprint":false},{"year":2004,"finding":"Phosphorylation of KSR1 at Ser392 and Thr274 regulates subcellular localization, protein stability, and duration of ERK activation. A KSR1 double mutant (T274V/S392A) promotes sustained ERK activation and accelerated cell-cycle progression in response to both PDGF and EGF, and is more stable than wild-type KSR1.","method":"Site-directed mutagenesis, cell proliferation assays, ERK phosphorylation kinetics, KSR1-/- MEF reconstitution","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KO/reconstitution with site-specific mutagenesis and multiple functional readouts in a single rigorous study","pmids":["15371409"],"is_preprint":false},{"year":2004,"finding":"KSR1 concentration-dependently regulates ERK activation intensity and duration: deletion of KSR1 eliminates prolonged ERK activation; low re-expression rescues signaling and transformation, while supraphysiologic expression inhibits these responses. Maximal association with all Raf/MEK/ERK cascade members occurs at the optimal KSR1 expression level.","method":"KSR1-/- MEF reconstitution with titrated expression, ERK activation assays, soft agar transformation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO/reconstitution with multiple expression levels and multiple orthogonal readouts","pmids":["15121859"],"is_preprint":false},{"year":2008,"finding":"14-3-3γ specifically binds KSR1 in an isoform-specific manner via its C-terminal stretch; this interaction protects KSR1 from EGF-induced dephosphorylation, impairs KSR1-mediated ERK2 activation and Ras signaling in Xenopus oocytes, and serves as the cytosolic anchor keeping KSR1 inactive. RNA interference of 14-3-3γ causes accumulation of KSR1 at the plasma membrane.","method":"In vitro binding assay, co-immunoprecipitation, Xenopus oocyte ERK activation assay, RNAi, confocal microscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro and in vivo binding, functional epistasis via RNAi, xenopus oocyte reconstitution, localization assay — multiple orthogonal methods","pmids":["18426801"],"is_preprint":false},{"year":2008,"finding":"IMP (E3 ubiquitin ligase) limits KSR1-dependent assembly of multivalent Raf·MEK complexes by disrupting KSR1 homo-oligomerization and B-Raf/c-Raf hetero-oligomerization, thereby impairing MEK recruitment to activated Raf and c-Raf kinase activation. Ras-GTP-induced IMP autoubiquitination relieves this constraint.","method":"Co-immunoprecipitation, ubiquitin ligase assay, RNAi, protein complex analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitin assay combined with RNAi functional readout, single lab","pmids":["18332145"],"is_preprint":false},{"year":2010,"finding":"Recombinant wild-type KSR1 (but not kinase-inactive D683A/D700A KSR1) undergoes serine autophosphorylation, phosphorylates MBP, and directly phosphorylates kinase-inactive MEK1 in vitro. KSR1-mediated TNF-induced cell survival requires both KSR1 kinase activity and MEK kinase activity, establishing KSR1 as a functional protein kinase with MEK1 as a substrate.","method":"Recombinant protein production in E. coli, in vitro kinase assay, site-directed mutagenesis (D683A/D700A), cell survival assay","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified recombinant proteins plus mutagenesis controls, functional validation in cells","pmids":["21144847"],"is_preprint":false},{"year":2004,"finding":"KSR1 kinase activity is an intrinsic property independent of associated MEK1: high-salt washing depleting 90% of KSR1-bound MEK1, or pre-treatment with MEK inhibitor PD98059 inactivating bound MEK1, did not alter KSR1 kinase activity in a two-stage in vitro reconstitution assay. A kinase-inactive KSR1 (D683A/D700A) that retains MEK1 binding lacks kinase activity.","method":"In vitro two-stage reconstitution kinase assay, high-salt MEK depletion, MEK inhibitor treatment, kinase-inactive mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple rigorous in vitro controls with reconstitution assay plus mutagenesis in a single study","pmids":["15084597"],"is_preprint":false},{"year":2011,"finding":"RAF inhibitors induce KSR1 binding to wild-type and oncogenic B-RAF (including V600E) but not C-RAF; this requires direct drug binding to B-RAF and conserved dimer interface residues in both proteins but is independent of RAS binding. KSR1 competes with C-RAF for inhibitor-induced B-RAF binding and thereby alters ERK cascade signaling.","method":"Co-immunoprecipitation in multiple cell lines, dimer interface mutagenesis, RAF inhibitor treatment panel","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with mutagenesis in multiple cell contexts, mechanistically well-defined","pmids":["21458265"],"is_preprint":false},{"year":2012,"finding":"VRK2A retains a fraction of KSR1 complexes on the endoplasmic reticulum surface by direct interaction between their C-terminal regions; MEK1 is incorporated into the basal VRK2A-KSR1 complex independently via the KSR1 CA5 region and VRK2A N-terminus. VRK2A overexpression increases KSR1 in the particulate fraction and prevents ERK1/2 incorporation after EGF stimulation, compartmentalizing MAPK signaling.","method":"Co-immunoprecipitation, subcellular fractionation, size-exclusion chromatography, VRK2A knockdown","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with domain mapping and functional fractionation, single lab","pmids":["22752157"],"is_preprint":false},{"year":2012,"finding":"The KSR1 CC-SAM domain (composed of a coiled coil and sterile α motif) targets KSR1 to specific signaling sites at the plasma membrane; membrane binding is mediated by helix α3 of the CC motif and mutations in α3 abolish plasma membrane targeting. The CC-SAM domain binds directly to micelles and bicelles in vitro.","method":"NMR spectroscopy, cell imaging, mutagenesis, in vitro lipid-binding assay","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure plus mutagenesis plus functional in vitro and cell validation in single rigorous study","pmids":["23250398"],"is_preprint":false},{"year":2011,"finding":"ERK1/2 directly phosphorylates KSR1 as a feedback mechanism; expression of feedback-deficient KSR1 promotes sustained ERK1/2 activation. In hippocampal neurons, feedback phosphorylation of KSR1 by ERK reduces KSR1 localization to dendritic spines (reversible by tetrodotoxin or PD184352), and restricts potentiation of excitatory postsynaptic currents.","method":"Western blot, immunocytochemistry, confocal imaging, electrophysiology, MEK inhibitor treatment","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct KSR1 phosphorylation by ERK with localization and functional electrophysiology readouts, single lab","pmids":["21471251"],"is_preprint":false},{"year":2013,"finding":"GEF-H1 acts as an adaptor protein linking PP2A B' subunits to KSR-1, thereby mediating dephosphorylation of KSR-1 Ser392 and activation of MAPK signaling in a positive feedback loop for oncogenic RAS. This function is independent of GEF-H1's RhoGEF activity.","method":"Co-immunoprecipitation, phosphorylation assay, siRNA knockdown, KRAS-transformed cell assays","journal":"Cancer cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with functional validation (phospho-mapping, rescue experiments), RhoGEF-independent mechanism defined","pmids":["24525234"],"is_preprint":false},{"year":2013,"finding":"AMPK phosphorylates BRAF at Ser729, which promotes BRAF association with 14-3-3 proteins and disrupts BRAF interaction with the KSR1 scaffolding protein, leading to attenuation of MEK-ERK signaling and impaired cell proliferation.","method":"In vitro kinase assay, co-immunoprecipitation, mutagenesis, cell proliferation assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay plus Co-IP with mutagenesis and functional cell readout in a single rigorous study","pmids":["24095280"],"is_preprint":false},{"year":2016,"finding":"Praja2 (PJA2) is the E3 ubiquitin ligase that ubiquitylates KSR1, leading to proteasomal degradation; growth factor/hormone stimulation induces KSR1 polyubiquitination coincident with decline in ERK1/2 signaling. Praja2-dependent KSR1 degradation modulates ERK activity in cancer cells and maintains pluripotency in embryonic stem cells.","method":"Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor treatment, knockdown/overexpression, in vivo tumor assay","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct E3 ligase-substrate relationship established by Co-IP, ubiquitination assay, and functional rescue, single lab","pmids":["27195677"],"is_preprint":false},{"year":2016,"finding":"ELK4 transcriptionally activates KDM5A, which removes H3K4me3 from the PJA2 promoter to suppress PJA2 expression; reduced PJA2 decreases ubiquitination and degradation of KSR1, thereby increasing KSR1 protein stability and promoting MAPK signaling and tumor progression.","method":"Dual luciferase reporter, ChIP assay, Co-IP, cycloheximide chase (protein stability), gain/loss-of-function assays","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP, luciferase, Co-IP, and stability assay in single study, but single lab","pmids":["34372882"],"is_preprint":false},{"year":2011,"finding":"KSR1 knockout in MEFs abrogates KSR1-mediated redistribution of MEK and ERK to caveolin-1-rich fractions; KSR1 physically interacts with caveolin-1, and this interaction is essential for optimal growth-factor-induced early-phase ERK activation, H-RasV12-induced senescence, and transformation.","method":"Co-immunoprecipitation, subcellular fractionation, KSR1-/- MEF reconstitution, transformation assay, mutagenesis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with fractionation and functional KO/reconstitution with mutagenesis in single rigorous study","pmids":["25002533"],"is_preprint":false},{"year":2009,"finding":"KSR1 is required for cell cycle reinitiation following DNA interstrand cross-link-induced G2/M arrest; a KSR1 mutant unable to bind ERK fails to rescue cell cycle re-entry, but constitutively active ERK alone is insufficient, demonstrating a specific requirement for the KSR1-ERK physical interaction in post-damage cell cycle recovery.","method":"KSR1-/- MEF reconstitution, cell cycle analysis, ERK binding mutant, gamma-H2AX foci assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO/reconstitution with ERK-binding mutant and DNA repair marker, single lab","pmids":["19147494"],"is_preprint":false},{"year":2007,"finding":"KSR1 undergoes caspase-dependent cleavage in apoptotic cells; cleavage destroys the scaffolding function of full-length KSR1 and generates a stable C-terminal fragment that inhibits ERK activation. A cleavage-resistant KSR1 maintains higher phosphoERK levels and reduces apoptotic signaling in response to TNFα/cycloheximide, whereas constitutive expression of the C-terminal fragment enhances apoptosis.","method":"Caspase cleavage assays, site-directed mutagenesis (cleavage-resistant mutant), ERK phosphorylation assay, cell death assay, in vivo mammary involution model","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — cleavage-resistant mutant combined with in vitro and in vivo validation, mechanistically well-defined","pmids":["17613518"],"is_preprint":false},{"year":2011,"finding":"KSR1 is required for efficient NK cell-mediated cytolysis and granule polarization; KSR1 is recruited to the immunological synapse during T-cell activation, and membrane recruitment of KSR1 is required for recruitment of active ERK to the synapse.","method":"KSR1-/- mouse cells, single-cell ERK activation analysis, confocal imaging, NK cytotoxicity assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with imaging and functional cytotoxicity readout, single lab","pmids":["19139278"],"is_preprint":false},{"year":2011,"finding":"KSR1 regulates PGC1α and ERRα expression to support oncogenic Ras-dependent anchorage-independent growth and metabolic capacity; this pathway is independent of ERK. Ectopic PGC1α rescues ERRα expression and anchorage-independent growth in KSR1-null RasV12 MEFs; PGC1α requires ERRα interaction for this rescue.","method":"KSR1-/- MEF reconstitution, ectopic expression rescue, ERRα inhibitor, metabolic assays, soft agar assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with rescue experiments and pharmacological validation, single lab","pmids":["21518958"],"is_preprint":false},{"year":2005,"finding":"KSR1 expression level titrates ERK and RSK activation to control adipogenic potential: KSR1 deletion prevents adipogenesis; low KSR1 rescues it by coordinating ERK and RSK activation with C/EBPβ synthesis and phosphorylation at the required moment; elevated KSR1 causes high sustained ERK that phosphorylates and inhibits PPARγ, blocking adipogenesis.","method":"KSR1-/- adipogenesis assay, KSR1 titration reconstitution, ERK/RSK activation kinetics, PPARγ phosphorylation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KO with multiple expression levels and multiple functional/biochemical readouts, single lab","pmids":["16107706"],"is_preprint":false},{"year":2021,"finding":"KSR1 and ERK promote EMT-like phenotype through preferential translation of EPSTI1 mRNA; EPSTI1 is required to induce the switch from E- to N-cadherin and coordinate migratory and invasive behavior. Ectopic EPSTI1 in KSR1-null CRC cells restores N-cadherin, ZEB1/Slug expression, migration, invasion, and anchorage-independent growth.","method":"KSR1 CRISPR knockout, ectopic expression rescue, migration/invasion assay, cadherin switching analysis","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with rescue experiments and multiple functional readouts, single lab","pmids":["33970103"],"is_preprint":false},{"year":2021,"finding":"Calmodulin binds directly to KSR1 in a Ca2+-dependent manner (both in vitro with purified proteins and from mammalian cell lysates); calmodulin antagonist CGS9343B impairs EGF-induced ERK activation in KSR1-overexpressing MEFs, blocks EGF-induced KSR1 membrane translocation, and impairs formation of KSR1-ERK and KSR1-pERK complexes.","method":"In vitro binding with purified proteins, co-immunoprecipitation, confocal microscopy, calmodulin antagonist treatment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro direct binding plus cellular Co-IP plus functional localization consequence in single rigorous study","pmids":["33766558"],"is_preprint":false},{"year":2024,"finding":"Ca2+/calmodulin binds to the CA3 domain of KSR1 in a collapsed mode engaging the α-helical KSR1-CA3 via hydrophobic interactions; mutation of KSR1-Phe355 to Asp reduces Ca2+/CaM binding by 76% and significantly impairs EGF-induced ERK activation, establishing that Ca2+/CaM binding to KSR1 promotes MAPK signaling.","method":"NMR spectroscopy, in silico molecular modeling, site-directed mutagenesis, in vitro binding assay, ERK activation assay","journal":"Protein science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR combined with mutagenesis and functional ERK activation readout in single study","pmids":["38591710"],"is_preprint":false},{"year":2016,"finding":"KSR1 promotes translation of Myc protein by a posttranscriptional mechanism in colon tumor cells; KSR1 and EPHB4 jointly support tumor survival through Myc and PGC1β expression, with KSR1 acting primarily at the translational level.","method":"KSR1 knockdown/overexpression, polysome profiling/translation analysis, western blotting, colony assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — translational regulation inferred from KD/OE with protein vs. mRNA comparison, single lab, single method type","pmids":["27273865"],"is_preprint":false},{"year":2017,"finding":"Oncogenic RAS and BRAF induce perinuclear relocalization of KSR1, CK2, and p-ERK1/2 to recycling endosomes and distinct endosomal populations; this spatial reorganization requires endocytosis, MEK-ERK and CK2 kinase activities, and the presence of KSR1. Perinuclear signaling complexes provide an essential platform for C/EBPβ phosphorylation and activation during RAS-induced senescence.","method":"Immunofluorescence/confocal imaging, KSR1 knockout MEFs, pharmacological inhibitors, Rab11 co-localization, C/EBPβ phosphorylation assay","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — imaging plus genetic KO plus biochemical readout, single lab with multiple orthogonal methods","pmids":["29259016"],"is_preprint":false},{"year":2011,"finding":"MARK2 binds and phosphorylates KSR1 on Ser392 in peripheral tissues; disruption of KSR1 in mark2-/- mice reverses the increased insulin sensitivity caused by MARK2 deletion, suggesting MARK2 negatively regulates insulin sensitivity through inhibitory phosphorylation of KSR1.","method":"Co-immunoprecipitation, in vitro kinase assay, double-knockout mouse model, glucose/insulin tolerance tests","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay plus double-KO epistasis in mouse, single lab","pmids":["22206009"],"is_preprint":false},{"year":2018,"finding":"Erbin interacts with KSR1 and displaces it from the RAF/MEK/ERK complex to prevent signal propagation; Erbin loss increases amplitude and duration of RAS/RAF signaling and promotes EMT.","method":"Co-immunoprecipitation, Erbin knockdown/overexpression, ERK pathway activation assays, 3D culture, mouse tumor model","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with displacement assay and functional validation in cells and in vivo, single lab","pmids":["29980571"],"is_preprint":false},{"year":2016,"finding":"KSR1 is a novel binding partner of Merlin (NF2 tumor suppressor); Merlin suppresses KSR1 function by inhibiting the binding between KSR1 and c-Raf, as demonstrated by immunoprecipitation. Proteomic analysis further identifies E3 ubiquitin ligase CRL4(DCAF1) as a KSR1-interacting protein.","method":"Co-immunoprecipitation, proteomics, functional knockdown assays in schwannoma cells","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP plus functional consequence of interaction with proteomic validation, single lab","pmids":["26549023"],"is_preprint":false},{"year":2013,"finding":"KSR1 overexpression decreases DBC1 phosphorylation, which reduces DBC1-SIRT1 interaction, enabling SIRT1 to deacetylate p53, thereby reducing p53 transcriptional activity. This positions KSR1 in a DBC1-SIRT1-p53 regulatory network in breast cancer cells.","method":"SILAC quantitative mass spectrometry, co-immunoprecipitation, luciferase reporter assay, western blotting","journal":"British journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — SILAC-MS with Co-IP and functional reporter assay validation, single lab","pmids":["24129246"],"is_preprint":false},{"year":2022,"finding":"Molecular dynamics simulations reveal that B-Raf's αG-helix Arg662 (vs. KSR1's Ala826 at equivalent position) is key: B-Raf Arg662 orients MEK1 Ser218 toward ATP for phosphorylation through interactions with the MEK1 activation loop, while KSR1 Ala826 results in fewer interactions with MEK1 A-loop, yielding a more flexible A-loop. The KSR1 P-rich loop of MEK1 plays a decisive role in MEK1 A-loop accessibility.","method":"Molecular dynamics simulation","journal":"Cellular and molecular life sciences : CMLS","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational simulation only, no experimental validation in this paper","pmids":["35508574"],"is_preprint":false},{"year":2022,"finding":"SHP2 physically interacts with KSR1 through its protein tyrosine phosphatase domain; during adaptive resistance to MEK inhibitors, activated SHP2 impairs this interaction with KSR1, releasing KSR1 to activate KSR1 and promote MAPK signaling. A KSR1 activating mutation (S269A) reduces the synergistic anti-proliferative effect of SHP2 and MEK inhibitor co-treatment.","method":"Co-immunoprecipitation (domain mapping), KSR1 knockdown, KSR1 S269A activating mutation, cell viability/proliferation assay, xenograft model","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-specific Co-IP with mutagenesis and in vivo validation, single lab","pmids":["36493900"],"is_preprint":false},{"year":2023,"finding":"KRASG12V promotes homo- and heterodimerization of BRAF via its RAS-binding domain; substantial KSR1 homo- and KSR1/BRAF heterodimerization occurs even without activated RAS and requires a salt bridge between the CC-SAM domain of KSR1 and the BRAF-specific region. The dimer interface is less critical for dimerization per se but essential for downstream signaling.","method":"Split Nanoluc luciferase complementation assay in living cells, loss-of-function dimerization interface mutants, RAF inhibitor treatment","journal":"Cell communication and signaling : CCS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — split luciferase PPI assay with mutagenesis in living cells, validated with multiple inhibitors, single lab","pmids":["37316874"],"is_preprint":false},{"year":2025,"finding":"KSR1 constitutively binds to YAP and MST1 and forms a complex with LATS1, functioning as a previously unrecognized scaffold of the Hippo pathway; KSR1 modulates YAP protein levels and transcriptional activity at least in part through the RhoA/actin axis.","method":"Co-immunoprecipitation, KSR1 knockdown/overexpression, YAP activity reporter assay, RhoA pathway analysis","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP binding and functional KD/OE, single lab, single paper","pmids":["41326667"],"is_preprint":false},{"year":2025,"finding":"Resistance to pan-RAF inhibitor exarafenib involves formation of RAS-mediated ARAF-KSR1 scaffolding complexes that maintain MAPK signaling despite drug treatment; this bypass is driven by RTK activation and RAS-GTP accumulation that specifically promotes ARAF-KSR1 complex assembly under drug pressure.","method":"Co-immunoprecipitation, drug resistance assays, ARAF/KSR1 knockdown, MEK inhibitor combination studies","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with functional resistance validation and combination therapy testing, single lab","pmids":["41654519"],"is_preprint":false},{"year":2026,"finding":"Soluble uric acid (UA) directly binds the KSR1 scaffold and hyperactivates MEK-ERK signaling, leading to chronic MAPK stimulation that upregulates inhibitory receptors (PD-1, Tim-3) on CD8+ T cells and promotes their exhaustion. Genetic disruption of the UA-KSR1-MAPK axis via Ksr1 knockdown restored T-cell effector activity.","method":"Direct binding assay (UA-KSR1), MEK-ERK phosphorylation analysis, Ksr1 knockdown, Tim-3 KO mouse model, in vivo tumor model","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding plus functional genetic validation in cells and in vivo, single lab","pmids":["42118604"],"is_preprint":false}],"current_model":"KSR1 functions primarily as a molecular scaffold that assembles Raf, MEK, and ERK into functional complexes at the plasma membrane to facilitate MAPK cascade activation; its membrane recruitment is regulated by phosphorylation—C-TAK1/MARK2-mediated phosphorylation of Ser392 confers 14-3-3 binding and cytoplasmic retention, while PP2A-mediated dephosphorylation (facilitated by GEF-H1) releases KSR1 to the membrane—and it also possesses intrinsic serine kinase activity capable of directly phosphorylating MEK1. KSR1 integrates multiple upstream inputs (G-protein βγ, CK2, caveolin-1, calmodulin/Ca2+, VRK2A-ER tethering, Erbin, Merlin) to modulate ERK signal amplitude, duration, and subcellular compartmentalization, and is itself subject to feedback phosphorylation by ERK and ubiquitin-mediated degradation via praja2. Beyond scaffolding the canonical Ras/MAPK cascade, KSR1 has now been identified as a scaffold for the Hippo pathway (binding YAP, MST1, LATS1) and regulates non-ERK outputs including PGC1α/ERRα-dependent metabolism, translational control of Myc and EPSTI1, and FLIP-mediated apoptosis resistance."},"narrative":{"mechanistic_narrative":"KSR1 is a molecular scaffold of the Ras/MAPK cascade that assembles Raf, MEK, and ERK into functional signaling complexes and tunes the amplitude, duration, and subcellular location of ERK output [PMID:15121859, PMID:25002533]. Originally defined genetically as a positive, Raf-related mediator of Ras signaling that acts downstream of or parallel to Ras and is required for maximal MPK-1/ERK diphosphorylation [PMID:8521513, PMID:8521514, PMID:11882296], KSR1 binds MEK constitutively (via an intact catalytic domain) and recruits activated ERK inducibly, an interaction functionally required for downstream cellular responses such as neurite outgrowth and post-DNA-damage cell-cycle re-entry [PMID:10891492, PMID:19147494]. Its activity is set largely by regulated membrane translocation: in resting cells C-TAK1/MARK2 phosphorylation of Ser392 creates a 14-3-3γ binding site that anchors KSR1 in the cytoplasm, while PP2A — recruited through the adaptor GEF-H1 — dephosphorylates this site to release KSR1 to the plasma membrane where it colocalizes with Ras and Raf [PMID:11741534, PMID:18426801, PMID:24525234, PMID:22206009]. Membrane targeting is mediated by the CC-SAM domain, which binds lipid directly, and KSR1 also engages caveolin-1 to redistribute MEK/ERK into caveolar fractions and Ca2+/calmodulin via its CA3 domain to promote translocation and ERK complex formation [PMID:23250398, PMID:25002533, PMID:33766558, PMID:38591710]. Within the scaffold KSR1 nucleates additional Raf-activating machinery, recruiting CK2 to phosphorylate the Raf N-region and undergoing salt-bridge-dependent homo- and BRAF-heterodimerization that is essential for signaling [PMID:17174095, PMID:37316874]. Beyond passive scaffolding KSR1 possesses intrinsic serine kinase activity independent of bound MEK and can directly phosphorylate MEK1 in vitro, an activity coupled to TNF-induced survival signaling [PMID:21144847, PMID:15084597]. KSR1 abundance is controlled by praja2-mediated ubiquitination and proteasomal degradation and by caspase cleavage during apoptosis, which destroys scaffold function and yields a dominant-negative C-terminal fragment [PMID:27195677, PMID:17613518]. Through these mechanisms KSR1 governs diverse outputs including adipogenesis, oncogenic Ras-driven transformation and senescence, and metabolic/translational programs (PGC1α/ERRα, Myc and EPSTI1 translation) that operate partly independent of ERK [PMID:15121859, PMID:21518958, PMID:16107706, PMID:33970103, PMID:29259016], and it has been identified as a scaffold of the Hippo pathway binding YAP, MST1, and LATS1 [PMID:41326667]. KSR1-RAF scaffolding complexes also drive adaptive resistance to RAF and MEK inhibitors [PMID:36493900, PMID:41654519].","teleology":[{"year":1995,"claim":"Established the existence and genetic placement of KSR as a positive Ras-pathway component, defining the central question of how a Raf-related kinase promotes Ras signaling.","evidence":"Genetic suppressor screen and epistasis analysis in C. elegans vulval development","pmids":["8521513","8521514"],"confidence":"High","gaps":["Did not establish biochemical mechanism or direct partners","Whether kinase activity is required left open"]},{"year":2002,"claim":"Showed KSR is required for the Raf/MEK/ERK cascade itself, linking the genetic phenotype to a direct biochemical readout of ERK activation.","evidence":"ksr-2;ksr-1 double-mutant analysis with dpERK western blot in C. elegans","pmids":["11882296"],"confidence":"High","gaps":["Redundancy complicates assigning specific molecular role","Does not distinguish scaffolding from catalytic contribution"]},{"year":2000,"claim":"Demonstrated that KSR1 physically binds MEK through its catalytic domain and that this binding is functionally essential, establishing the scaffolding model in mammalian cells.","evidence":"Co-IP, mutagenesis, and NGF-induced neurite outgrowth in PC12 cells","pmids":["10891492"],"confidence":"High","gaps":["ERK binding mode not resolved","Did not address membrane translocation triggers"]},{"year":2001,"claim":"Defined the phosphoswitch controlling KSR1 localization: C-TAK1 phosphorylation of Ser392 imposes 14-3-3-dependent cytoplasmic retention relieved upon stimulation, answering how membrane recruitment is gated.","evidence":"Co-IP, phospho-mapping, subcellular imaging, mutagenesis in mammalian cells","pmids":["11741534"],"confidence":"High","gaps":["Phosphatase reversing Ser392 not yet identified","In vivo physiological context of the switch unaddressed"]},{"year":2003,"claim":"Identified PP2A as the constitutive KSR1-associated phosphatase that dephosphorylates the 14-3-3 site to enable membrane recruitment, completing the recruitment cycle.","evidence":"Mass spectrometry of KSR1 complex, Co-IP, PP2A inhibition, phospho-analysis","pmids":["12932319"],"confidence":"High","gaps":["How growth factor induces PR55B subunit binding not defined","Adaptor linking PP2A to KSR1 not yet known"]},{"year":2004,"claim":"Quantified KSR1 as a concentration-dependent rheostat, showing optimal expression maximizes Raf/MEK/ERK association and that both deletion and overexpression impair signaling and transformation.","evidence":"KSR1-/- MEF reconstitution with titrated expression, ERK assays, soft agar; plus Ser392/Thr274 mutagenesis kinetics and stability","pmids":["15121859","15371409"],"confidence":"High","gaps":["Structural basis of optimal stoichiometry unresolved","Phosphodegron coupling to stability incompletely mapped"]},{"year":2004,"claim":"Resolved a long-standing controversy by demonstrating KSR1 possesses intrinsic kinase activity independent of bound MEK1.","evidence":"Two-stage in vitro reconstitution kinase assay with high-salt MEK depletion and kinase-inactive mutant","pmids":["15084597"],"confidence":"High","gaps":["Physiological substrate not identified in this study","Catalytic significance in vivo unclear"]},{"year":2006,"claim":"Identified CK2 within the KSR1 scaffold as a Raf N-region kinase, explaining how KSR1 promotes Raf activation rather than only colocalization.","evidence":"MS of KSR1 complex, Co-IP, domain mapping, kinase assays","pmids":["17174095"],"confidence":"High","gaps":["How CK2 docking is regulated by stimulation unknown","Structural arrangement of CK2-KSR1-Raf not defined"]},{"year":2008,"claim":"Defined 14-3-3γ isoform specificity and the E3 ligase IMP as opposing brakes on scaffold assembly, refining how KSR1 oligomerization and inactivity are enforced.","evidence":"In vitro/in vivo binding, RNAi, Xenopus oocyte assays (14-3-3γ); Co-IP and ubiquitin assays (IMP)","pmids":["18426801","18332145"],"confidence":"High","gaps":["IMP study Medium-confidence single lab","How Ras-GTP triggers IMP autoubiquitination mechanistically incomplete"]},{"year":2010,"claim":"Demonstrated KSR1 directly phosphorylates kinase-inactive MEK1 and that this catalytic function contributes to TNF-induced survival, establishing MEK1 as a KSR1 substrate.","evidence":"Recombinant kinase assay, D683A/D700A mutant, cell survival assay","pmids":["21144847"],"confidence":"High","gaps":["In vivo significance of KSR1 catalysis versus scaffolding unresolved","Stoichiometry relative to Raf-driven MEK phosphorylation unknown"]},{"year":2012,"claim":"Mapped the structural determinant of membrane targeting (CC-SAM helix α3) and a parallel ER-tethering mechanism via VRK2A, defining how KSR1 is compartmentalized.","evidence":"NMR, lipid-binding, mutagenesis, imaging (CC-SAM); Co-IP, fractionation, SEC (VRK2A)","pmids":["23250398","22752157"],"confidence":"High","gaps":["VRK2A study Medium-confidence single lab","Switch between ER retention and PM targeting not integrated"]},{"year":2013,"claim":"Identified GEF-H1 as the adaptor coupling PP2A to KSR1 in oncogenic RAS, and defined external inhibitory inputs (AMPK-BRAF, MARK2) regulating scaffold engagement and physiology.","evidence":"Co-IP, phospho-assays, siRNA in KRAS cells (GEF-H1); in vitro kinase and double-KO mouse (MARK2); kinase assay and Co-IP (AMPK-BRAF)","pmids":["24525234","22206009","24095280"],"confidence":"High","gaps":["MARK2 study Medium-confidence","Tissue-specific integration of opposing inputs incomplete"]},{"year":2011,"claim":"Established ERK-mediated feedback phosphorylation of KSR1 as a mechanism limiting signal duration and controlling synaptic localization and plasticity.","evidence":"Western blot, imaging, electrophysiology, MEK inhibitor in hippocampal neurons","pmids":["21471251"],"confidence":"Medium","gaps":["Single lab","Feedback phosphosites not exhaustively mapped"]},{"year":2011,"claim":"Linked KSR1 to membrane microdomain organization (caveolin-1) and to cellular processes beyond proliferation—immune synapse ERK recruitment and Ras-induced senescence.","evidence":"Co-IP, fractionation, KSR1-/- MEF reconstitution (caveolin-1); KSR1-/- cells, imaging, NK cytotoxicity (immune synapse)","pmids":["25002533","19139278"],"confidence":"Medium","gaps":["Immune-synapse study Medium-confidence single lab","How caveolin recruitment couples to senescence not mechanistically closed"]},{"year":2016,"claim":"Defined praja2 as the physiological E3 ligase degrading KSR1 to terminate ERK signaling, and an ELK4-KDM5A axis controlling praja2 levels and thus KSR1 stability.","evidence":"Co-IP, ubiquitination/proteasome assays, in vivo tumor (praja2); ChIP, luciferase, CHX chase (ELK4-KDM5A)","pmids":["27195677","34372882"],"confidence":"High","gaps":["ELK4-KDM5A arm Medium-confidence single lab","Signal triggering growth-factor-induced KSR1 ubiquitination not fully defined"]},{"year":2016,"claim":"Extended KSR1 to ERK-independent and translational outputs—PGC1α/ERRα metabolism, Myc translation, and later EPSTI1-driven EMT—broadening its role beyond canonical MAPK scaffolding.","evidence":"KSR1-/- reconstitution and rescue, metabolic and soft-agar assays (PGC1α); polysome/translation analysis (Myc); CRISPR KO and rescue, invasion assays (EPSTI1)","pmids":["21518958","27273865","33970103"],"confidence":"Medium","gaps":["Mechanism coupling scaffold to translational control unresolved","All single-lab studies"]},{"year":2017,"claim":"Showed oncogenic RAS/BRAF drives perinuclear/endosomal relocalization of KSR1-CK2-pERK complexes that serve as a platform for C/EBPβ activation during senescence, adding a spatial dimension to scaffold function.","evidence":"Confocal imaging, KSR1-/- MEFs, inhibitors, Rab11 colocalization, C/EBPβ phospho-assay","pmids":["29259016"],"confidence":"Medium","gaps":["Single lab","Trigger for endosomal redistribution not fully defined"]},{"year":2018,"claim":"Identified additional scaffold antagonists (Erbin, Merlin/NF2) that displace KSR1 from or restrict its Raf binding, integrating tumor-suppressor control of signal amplitude.","evidence":"Co-IP/displacement assays, knockdown/overexpression, 3D culture and tumor models (Erbin); Co-IP, proteomics in schwannoma (Merlin)","pmids":["29980571","26549023"],"confidence":"Medium","gaps":["Both Medium-confidence single-lab","Structural basis of displacement unresolved"]},{"year":2022,"claim":"Resolved the dimerization basis of scaffold function—salt-bridge-dependent KSR1 homo- and KSR1/BRAF heterodimers essential for signaling—and a structural rationale for why KSR1 is a weak MEK kinase relative to BRAF.","evidence":"Split Nanoluc PPI assay with interface mutants in living cells (dimerization); molecular dynamics simulation (catalytic comparison)","pmids":["37316874","35508574"],"confidence":"Medium","gaps":["MD study is computational with no experimental validation","Dimer stoichiometry in vivo not directly measured"]},{"year":2024,"claim":"Provided structural detail of Ca2+/calmodulin binding to the KSR1 CA3 domain and demonstrated functionally that this engagement promotes EGF-induced ERK activation.","evidence":"NMR, modeling, Phe355Asp mutagenesis, binding and ERK activation assays (building on 2021 calmodulin binding)","pmids":["38591710","33766558"],"confidence":"High","gaps":["How Ca2+ signals are physiologically integrated with growth-factor cues unclear","Downstream effect on Raf recruitment not detailed"]},{"year":2025,"claim":"Discovered KSR1 functions as a Hippo-pathway scaffold binding YAP, MST1, and LATS1, expanding its role to a second major signaling network.","evidence":"Co-IP, knockdown/overexpression, YAP reporter assay, RhoA pathway analysis","pmids":["41326667"],"confidence":"Medium","gaps":["Single lab single paper","Whether Hippo scaffolding is separable from MAPK scaffolding unknown"]},{"year":2025,"claim":"Defined KSR1 as a driver of therapeutic resistance and immune modulation: RAS-mediated ARAF-KSR1 and SHP2-released KSR1 complexes sustain MAPK under RAF/MEK inhibition, and soluble uric acid binds KSR1 to drive CD8+ T-cell exhaustion.","evidence":"Co-IP, drug-resistance and combination assays, knockdowns, xenografts (ARAF/SHP2); direct UA-KSR1 binding, knockdown, Tim-3 KO, tumor models (uric acid)","pmids":["41654519","36493900","42118604"],"confidence":"Medium","gaps":["All Medium-confidence single-lab studies","Direct small-molecule binding site on KSR1 for uric acid not structurally mapped"]},{"year":null,"claim":"How KSR1's intrinsic catalytic activity, its scaffolding of canonical MAPK, and its newer roles in the Hippo pathway and translational/metabolic control are integrated within a single protein remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified structural model of full-length KSR1 in complex with Raf/MEK/ERK","Relative in vivo weight of catalysis versus scaffolding unquantified","Mechanism coupling KSR1 to translational control of Myc/EPSTI1 undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[5,8,21]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[11,12]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[11,12]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[15]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,15,21]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,9]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[14]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[31]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3,8]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[37,40,41]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[22]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[23]}],"complexes":["KSR1-Raf-MEK-ERK scaffold complex","KSR1-CK2 complex","VRK2A-KSR1-MEK1 ER complex","KSR1-YAP-MST1-LATS1 Hippo complex"],"partners":["MEK1","BRAF","CK2","PP2A","14-3-3GAMMA","CALMODULIN","CAVEOLIN-1","YAP"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8IVT5","full_name":"Kinase suppressor of Ras 1","aliases":[],"length_aa":923,"mass_kda":102.2,"function":"Part of a multiprotein signaling complex which promotes phosphorylation of Raf family members and activation of downstream MAP kinases (By similarity). Independently of its kinase activity, acts as MAP2K1/MEK1 and MAP2K2/MEK2-dependent allosteric activator of BRAF; upon binding to MAP2K1/MEK1 or MAP2K2/MEK2, dimerizes with BRAF and promotes BRAF-mediated phosphorylation of MAP2K1/MEK1 and/or MAP2K2/MEK2 (PubMed:29433126). Promotes activation of MAPK1 and/or MAPK3, both in response to EGF and to cAMP (By similarity). Its kinase activity is unsure (By similarity). Some protein kinase activity has been detected in vitro, however the physiological relevance of this activity is unknown (By similarity)","subcellular_location":"Cytoplasm; Membrane; Cell membrane; Cell projection, ruffle membrane; Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/Q8IVT5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/KSR1","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"MAP2K1","stoichiometry":0.2},{"gene":"MAP2K2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/KSR1","total_profiled":1310},"omim":[{"mim_id":"619341","title":"PRAJA RING FINGER UBIQUITIN LIGASE 2; PJA2","url":"https://www.omim.org/entry/619341"},{"mim_id":"610737","title":"KINASE SUPPRESSOR OF RAS 2; KSR2","url":"https://www.omim.org/entry/610737"},{"mim_id":"602775","title":"SHOC2 LEUCINE-RICH REPEAT SCAFFOLD PROTEIN; SHOC2","url":"https://www.omim.org/entry/602775"},{"mim_id":"602678","title":"MAP/MICROTUBULE AFFINITY-REGULATING KINASE 3; MARK3","url":"https://www.omim.org/entry/602678"},{"mim_id":"601132","title":"KINASE SUPPRESSOR OF RAS 1; KSR1","url":"https://www.omim.org/entry/601132"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"pancreas","ntpm":104.2}],"url":"https://www.proteinatlas.org/search/KSR1"},"hgnc":{"alias_symbol":["RSU2"],"prev_symbol":["KSR"]},"alphafold":{"accession":"Q8IVT5","domains":[{"cath_id":"1.10.150.50","chopping":"34-172","consensus_level":"medium","plddt":83.6728,"start":34,"end":172},{"cath_id":"3.30.200.20","chopping":"599-689","consensus_level":"medium","plddt":82.9054,"start":599,"end":689},{"cath_id":"1.10.510.10","chopping":"693-883","consensus_level":"medium","plddt":83.6573,"start":693,"end":883}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IVT5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IVT5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IVT5-F1-predicted_aligned_error_v6.png","plddt_mean":60.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=KSR1","jax_strain_url":"https://www.jax.org/strain/search?query=KSR1"},"sequence":{"accession":"Q8IVT5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8IVT5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8IVT5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IVT5"}},"corpus_meta":[{"pmid":"8521513","id":"PMC_8521513","title":"The 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analysis, gene cloning\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent labs (Sundaram/Han and Kornfeld/Horvitz) published simultaneous genetic epistasis studies in the same issue, replicated findings\",\n      \"pmids\": [\"8521513\", \"8521514\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"C-TAK1 kinase constitutively associates with mammalian KSR1 and phosphorylates Ser392, conferring 14-3-3 binding and cytoplasmic sequestration in unstimulated cells; upon signal activation, Ser392 phosphorylation is reduced, allowing the KSR1 complex to translocate to the plasma membrane where it colocalizes with activated Ras and Raf-1 to facilitate MEK and MAPK activation.\",\n      \"method\": \"Co-immunoprecipitation, phosphorylation mapping, subcellular fractionation/imaging, mutagenesis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, site-specific mutagenesis, functional localization consequence established in single rigorous study\",\n      \"pmids\": [\"11741534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Protein phosphatase PP2A constitutively associates (core subunits PR65A and catalytic C) with the N-terminal domain of KSR1; upon growth factor treatment, regulatory subunit PR55B binding is induced. PP2A dephosphorylates the critical 14-3-3 binding site on KSR1 (and Raf-1), enabling membrane recruitment of KSR1 and activation of KSR1-associated MEK and ERK.\",\n      \"method\": \"Mass spectrometry of KSR1 complex, co-immunoprecipitation, pharmacological PP2A inhibition, phosphorylation analysis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — MS-based complex identification plus functional validation with multiple orthogonal methods (Co-IP, inhibitor, phospho-mapping) in one rigorous study\",\n      \"pmids\": [\"12932319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"In C. elegans, ksr-1 and ksr-2 act redundantly; ksr-2;ksr-1 double mutants show severely reduced or absent diphosphorylated MPK-1 ERK, strongly supporting a model where KSR acts to promote activation or maintenance of the Raf/MEK/ERK kinase cascade.\",\n      \"method\": \"Double-mutant genetic analysis, ERK phosphorylation western blot\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic double-KO with direct biochemical readout (dpERK levels), replicated across multiple developmental contexts\",\n      \"pmids\": [\"11882296\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Casein kinase 2 (CK2) holoenzyme constitutively binds the basic surface region of the KSR1 atypical C1 domain; this KSR1/CK2 interaction is required for maximal growth-factor-induced phosphorylation of C-Raf and B-Raf on the activating N-region serine, and for downstream MEK and ERK activation, identifying CK2 as a Raf N-region kinase within the KSR1 scaffold complex.\",\n      \"method\": \"Mass spectrometry of KSR1 complex, co-immunoprecipitation, domain-mapping mutagenesis, kinase activity assays\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — MS identification plus functional validation with mutagenesis and kinase assays in single thorough study\",\n      \"pmids\": [\"17174095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"B-KSR1, a brain-specific KSR1 splice variant, constitutively associates with MEK (requiring an intact kinase domain for MEK binding) and inducibly associates with activated MAPK; all loss-of-function mutations in the catalytic domain that abolish MEK binding also abolish the ability to augment NGF-induced neurite outgrowth, demonstrating that MEK binding is functionally essential.\",\n      \"method\": \"Co-immunoprecipitation, mutagenesis, PC12 cell differentiation assay, MEK inhibitor treatment\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with mutagenesis and functional rescue/loss-of-function in single study\",\n      \"pmids\": [\"10891492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"KSR1 binds directly to G-protein βγ subunits via its CA3 domain (containing the cysteine-rich zinc finger-like domain); LPA-induced KSR1 translocation to the plasma membrane is blocked by pertussis toxin but not dominant-negative Ras; overexpression of KSR1 inhibits βγ-induced MAPK activation.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, deletion analysis, pertussis toxin treatment, MAPK activation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Y2H plus Co-IP plus functional inhibition assay in a single study, single lab\",\n      \"pmids\": [\"10075696\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Phosphorylation of KSR1 at Ser392 and Thr274 regulates subcellular localization, protein stability, and duration of ERK activation. A KSR1 double mutant (T274V/S392A) promotes sustained ERK activation and accelerated cell-cycle progression in response to both PDGF and EGF, and is more stable than wild-type KSR1.\",\n      \"method\": \"Site-directed mutagenesis, cell proliferation assays, ERK phosphorylation kinetics, KSR1-/- MEF reconstitution\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO/reconstitution with site-specific mutagenesis and multiple functional readouts in a single rigorous study\",\n      \"pmids\": [\"15371409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"KSR1 concentration-dependently regulates ERK activation intensity and duration: deletion of KSR1 eliminates prolonged ERK activation; low re-expression rescues signaling and transformation, while supraphysiologic expression inhibits these responses. Maximal association with all Raf/MEK/ERK cascade members occurs at the optimal KSR1 expression level.\",\n      \"method\": \"KSR1-/- MEF reconstitution with titrated expression, ERK activation assays, soft agar transformation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO/reconstitution with multiple expression levels and multiple orthogonal readouts\",\n      \"pmids\": [\"15121859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"14-3-3γ specifically binds KSR1 in an isoform-specific manner via its C-terminal stretch; this interaction protects KSR1 from EGF-induced dephosphorylation, impairs KSR1-mediated ERK2 activation and Ras signaling in Xenopus oocytes, and serves as the cytosolic anchor keeping KSR1 inactive. RNA interference of 14-3-3γ causes accumulation of KSR1 at the plasma membrane.\",\n      \"method\": \"In vitro binding assay, co-immunoprecipitation, Xenopus oocyte ERK activation assay, RNAi, confocal microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro and in vivo binding, functional epistasis via RNAi, xenopus oocyte reconstitution, localization assay — multiple orthogonal methods\",\n      \"pmids\": [\"18426801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"IMP (E3 ubiquitin ligase) limits KSR1-dependent assembly of multivalent Raf·MEK complexes by disrupting KSR1 homo-oligomerization and B-Raf/c-Raf hetero-oligomerization, thereby impairing MEK recruitment to activated Raf and c-Raf kinase activation. Ras-GTP-induced IMP autoubiquitination relieves this constraint.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitin ligase assay, RNAi, protein complex analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitin assay combined with RNAi functional readout, single lab\",\n      \"pmids\": [\"18332145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Recombinant wild-type KSR1 (but not kinase-inactive D683A/D700A KSR1) undergoes serine autophosphorylation, phosphorylates MBP, and directly phosphorylates kinase-inactive MEK1 in vitro. KSR1-mediated TNF-induced cell survival requires both KSR1 kinase activity and MEK kinase activity, establishing KSR1 as a functional protein kinase with MEK1 as a substrate.\",\n      \"method\": \"Recombinant protein production in E. coli, in vitro kinase assay, site-directed mutagenesis (D683A/D700A), cell survival assay\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified recombinant proteins plus mutagenesis controls, functional validation in cells\",\n      \"pmids\": [\"21144847\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"KSR1 kinase activity is an intrinsic property independent of associated MEK1: high-salt washing depleting 90% of KSR1-bound MEK1, or pre-treatment with MEK inhibitor PD98059 inactivating bound MEK1, did not alter KSR1 kinase activity in a two-stage in vitro reconstitution assay. A kinase-inactive KSR1 (D683A/D700A) that retains MEK1 binding lacks kinase activity.\",\n      \"method\": \"In vitro two-stage reconstitution kinase assay, high-salt MEK depletion, MEK inhibitor treatment, kinase-inactive mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple rigorous in vitro controls with reconstitution assay plus mutagenesis in a single study\",\n      \"pmids\": [\"15084597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RAF inhibitors induce KSR1 binding to wild-type and oncogenic B-RAF (including V600E) but not C-RAF; this requires direct drug binding to B-RAF and conserved dimer interface residues in both proteins but is independent of RAS binding. KSR1 competes with C-RAF for inhibitor-induced B-RAF binding and thereby alters ERK cascade signaling.\",\n      \"method\": \"Co-immunoprecipitation in multiple cell lines, dimer interface mutagenesis, RAF inhibitor treatment panel\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with mutagenesis in multiple cell contexts, mechanistically well-defined\",\n      \"pmids\": [\"21458265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"VRK2A retains a fraction of KSR1 complexes on the endoplasmic reticulum surface by direct interaction between their C-terminal regions; MEK1 is incorporated into the basal VRK2A-KSR1 complex independently via the KSR1 CA5 region and VRK2A N-terminus. VRK2A overexpression increases KSR1 in the particulate fraction and prevents ERK1/2 incorporation after EGF stimulation, compartmentalizing MAPK signaling.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, size-exclusion chromatography, VRK2A knockdown\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with domain mapping and functional fractionation, single lab\",\n      \"pmids\": [\"22752157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The KSR1 CC-SAM domain (composed of a coiled coil and sterile α motif) targets KSR1 to specific signaling sites at the plasma membrane; membrane binding is mediated by helix α3 of the CC motif and mutations in α3 abolish plasma membrane targeting. The CC-SAM domain binds directly to micelles and bicelles in vitro.\",\n      \"method\": \"NMR spectroscopy, cell imaging, mutagenesis, in vitro lipid-binding assay\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure plus mutagenesis plus functional in vitro and cell validation in single rigorous study\",\n      \"pmids\": [\"23250398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ERK1/2 directly phosphorylates KSR1 as a feedback mechanism; expression of feedback-deficient KSR1 promotes sustained ERK1/2 activation. In hippocampal neurons, feedback phosphorylation of KSR1 by ERK reduces KSR1 localization to dendritic spines (reversible by tetrodotoxin or PD184352), and restricts potentiation of excitatory postsynaptic currents.\",\n      \"method\": \"Western blot, immunocytochemistry, confocal imaging, electrophysiology, MEK inhibitor treatment\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct KSR1 phosphorylation by ERK with localization and functional electrophysiology readouts, single lab\",\n      \"pmids\": [\"21471251\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"GEF-H1 acts as an adaptor protein linking PP2A B' subunits to KSR-1, thereby mediating dephosphorylation of KSR-1 Ser392 and activation of MAPK signaling in a positive feedback loop for oncogenic RAS. This function is independent of GEF-H1's RhoGEF activity.\",\n      \"method\": \"Co-immunoprecipitation, phosphorylation assay, siRNA knockdown, KRAS-transformed cell assays\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with functional validation (phospho-mapping, rescue experiments), RhoGEF-independent mechanism defined\",\n      \"pmids\": [\"24525234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"AMPK phosphorylates BRAF at Ser729, which promotes BRAF association with 14-3-3 proteins and disrupts BRAF interaction with the KSR1 scaffolding protein, leading to attenuation of MEK-ERK signaling and impaired cell proliferation.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, mutagenesis, cell proliferation assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay plus Co-IP with mutagenesis and functional cell readout in a single rigorous study\",\n      \"pmids\": [\"24095280\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Praja2 (PJA2) is the E3 ubiquitin ligase that ubiquitylates KSR1, leading to proteasomal degradation; growth factor/hormone stimulation induces KSR1 polyubiquitination coincident with decline in ERK1/2 signaling. Praja2-dependent KSR1 degradation modulates ERK activity in cancer cells and maintains pluripotency in embryonic stem cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor treatment, knockdown/overexpression, in vivo tumor assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct E3 ligase-substrate relationship established by Co-IP, ubiquitination assay, and functional rescue, single lab\",\n      \"pmids\": [\"27195677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ELK4 transcriptionally activates KDM5A, which removes H3K4me3 from the PJA2 promoter to suppress PJA2 expression; reduced PJA2 decreases ubiquitination and degradation of KSR1, thereby increasing KSR1 protein stability and promoting MAPK signaling and tumor progression.\",\n      \"method\": \"Dual luciferase reporter, ChIP assay, Co-IP, cycloheximide chase (protein stability), gain/loss-of-function assays\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP, luciferase, Co-IP, and stability assay in single study, but single lab\",\n      \"pmids\": [\"34372882\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"KSR1 knockout in MEFs abrogates KSR1-mediated redistribution of MEK and ERK to caveolin-1-rich fractions; KSR1 physically interacts with caveolin-1, and this interaction is essential for optimal growth-factor-induced early-phase ERK activation, H-RasV12-induced senescence, and transformation.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, KSR1-/- MEF reconstitution, transformation assay, mutagenesis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with fractionation and functional KO/reconstitution with mutagenesis in single rigorous study\",\n      \"pmids\": [\"25002533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"KSR1 is required for cell cycle reinitiation following DNA interstrand cross-link-induced G2/M arrest; a KSR1 mutant unable to bind ERK fails to rescue cell cycle re-entry, but constitutively active ERK alone is insufficient, demonstrating a specific requirement for the KSR1-ERK physical interaction in post-damage cell cycle recovery.\",\n      \"method\": \"KSR1-/- MEF reconstitution, cell cycle analysis, ERK binding mutant, gamma-H2AX foci assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO/reconstitution with ERK-binding mutant and DNA repair marker, single lab\",\n      \"pmids\": [\"19147494\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"KSR1 undergoes caspase-dependent cleavage in apoptotic cells; cleavage destroys the scaffolding function of full-length KSR1 and generates a stable C-terminal fragment that inhibits ERK activation. A cleavage-resistant KSR1 maintains higher phosphoERK levels and reduces apoptotic signaling in response to TNFα/cycloheximide, whereas constitutive expression of the C-terminal fragment enhances apoptosis.\",\n      \"method\": \"Caspase cleavage assays, site-directed mutagenesis (cleavage-resistant mutant), ERK phosphorylation assay, cell death assay, in vivo mammary involution model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cleavage-resistant mutant combined with in vitro and in vivo validation, mechanistically well-defined\",\n      \"pmids\": [\"17613518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"KSR1 is required for efficient NK cell-mediated cytolysis and granule polarization; KSR1 is recruited to the immunological synapse during T-cell activation, and membrane recruitment of KSR1 is required for recruitment of active ERK to the synapse.\",\n      \"method\": \"KSR1-/- mouse cells, single-cell ERK activation analysis, confocal imaging, NK cytotoxicity assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with imaging and functional cytotoxicity readout, single lab\",\n      \"pmids\": [\"19139278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"KSR1 regulates PGC1α and ERRα expression to support oncogenic Ras-dependent anchorage-independent growth and metabolic capacity; this pathway is independent of ERK. Ectopic PGC1α rescues ERRα expression and anchorage-independent growth in KSR1-null RasV12 MEFs; PGC1α requires ERRα interaction for this rescue.\",\n      \"method\": \"KSR1-/- MEF reconstitution, ectopic expression rescue, ERRα inhibitor, metabolic assays, soft agar assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with rescue experiments and pharmacological validation, single lab\",\n      \"pmids\": [\"21518958\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"KSR1 expression level titrates ERK and RSK activation to control adipogenic potential: KSR1 deletion prevents adipogenesis; low KSR1 rescues it by coordinating ERK and RSK activation with C/EBPβ synthesis and phosphorylation at the required moment; elevated KSR1 causes high sustained ERK that phosphorylates and inhibits PPARγ, blocking adipogenesis.\",\n      \"method\": \"KSR1-/- adipogenesis assay, KSR1 titration reconstitution, ERK/RSK activation kinetics, PPARγ phosphorylation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with multiple expression levels and multiple functional/biochemical readouts, single lab\",\n      \"pmids\": [\"16107706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"KSR1 and ERK promote EMT-like phenotype through preferential translation of EPSTI1 mRNA; EPSTI1 is required to induce the switch from E- to N-cadherin and coordinate migratory and invasive behavior. Ectopic EPSTI1 in KSR1-null CRC cells restores N-cadherin, ZEB1/Slug expression, migration, invasion, and anchorage-independent growth.\",\n      \"method\": \"KSR1 CRISPR knockout, ectopic expression rescue, migration/invasion assay, cadherin switching analysis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with rescue experiments and multiple functional readouts, single lab\",\n      \"pmids\": [\"33970103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Calmodulin binds directly to KSR1 in a Ca2+-dependent manner (both in vitro with purified proteins and from mammalian cell lysates); calmodulin antagonist CGS9343B impairs EGF-induced ERK activation in KSR1-overexpressing MEFs, blocks EGF-induced KSR1 membrane translocation, and impairs formation of KSR1-ERK and KSR1-pERK complexes.\",\n      \"method\": \"In vitro binding with purified proteins, co-immunoprecipitation, confocal microscopy, calmodulin antagonist treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro direct binding plus cellular Co-IP plus functional localization consequence in single rigorous study\",\n      \"pmids\": [\"33766558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Ca2+/calmodulin binds to the CA3 domain of KSR1 in a collapsed mode engaging the α-helical KSR1-CA3 via hydrophobic interactions; mutation of KSR1-Phe355 to Asp reduces Ca2+/CaM binding by 76% and significantly impairs EGF-induced ERK activation, establishing that Ca2+/CaM binding to KSR1 promotes MAPK signaling.\",\n      \"method\": \"NMR spectroscopy, in silico molecular modeling, site-directed mutagenesis, in vitro binding assay, ERK activation assay\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR combined with mutagenesis and functional ERK activation readout in single study\",\n      \"pmids\": [\"38591710\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"KSR1 promotes translation of Myc protein by a posttranscriptional mechanism in colon tumor cells; KSR1 and EPHB4 jointly support tumor survival through Myc and PGC1β expression, with KSR1 acting primarily at the translational level.\",\n      \"method\": \"KSR1 knockdown/overexpression, polysome profiling/translation analysis, western blotting, colony assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — translational regulation inferred from KD/OE with protein vs. mRNA comparison, single lab, single method type\",\n      \"pmids\": [\"27273865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Oncogenic RAS and BRAF induce perinuclear relocalization of KSR1, CK2, and p-ERK1/2 to recycling endosomes and distinct endosomal populations; this spatial reorganization requires endocytosis, MEK-ERK and CK2 kinase activities, and the presence of KSR1. Perinuclear signaling complexes provide an essential platform for C/EBPβ phosphorylation and activation during RAS-induced senescence.\",\n      \"method\": \"Immunofluorescence/confocal imaging, KSR1 knockout MEFs, pharmacological inhibitors, Rab11 co-localization, C/EBPβ phosphorylation assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — imaging plus genetic KO plus biochemical readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"29259016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MARK2 binds and phosphorylates KSR1 on Ser392 in peripheral tissues; disruption of KSR1 in mark2-/- mice reverses the increased insulin sensitivity caused by MARK2 deletion, suggesting MARK2 negatively regulates insulin sensitivity through inhibitory phosphorylation of KSR1.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, double-knockout mouse model, glucose/insulin tolerance tests\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay plus double-KO epistasis in mouse, single lab\",\n      \"pmids\": [\"22206009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Erbin interacts with KSR1 and displaces it from the RAF/MEK/ERK complex to prevent signal propagation; Erbin loss increases amplitude and duration of RAS/RAF signaling and promotes EMT.\",\n      \"method\": \"Co-immunoprecipitation, Erbin knockdown/overexpression, ERK pathway activation assays, 3D culture, mouse tumor model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with displacement assay and functional validation in cells and in vivo, single lab\",\n      \"pmids\": [\"29980571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"KSR1 is a novel binding partner of Merlin (NF2 tumor suppressor); Merlin suppresses KSR1 function by inhibiting the binding between KSR1 and c-Raf, as demonstrated by immunoprecipitation. Proteomic analysis further identifies E3 ubiquitin ligase CRL4(DCAF1) as a KSR1-interacting protein.\",\n      \"method\": \"Co-immunoprecipitation, proteomics, functional knockdown assays in schwannoma cells\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP plus functional consequence of interaction with proteomic validation, single lab\",\n      \"pmids\": [\"26549023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"KSR1 overexpression decreases DBC1 phosphorylation, which reduces DBC1-SIRT1 interaction, enabling SIRT1 to deacetylate p53, thereby reducing p53 transcriptional activity. This positions KSR1 in a DBC1-SIRT1-p53 regulatory network in breast cancer cells.\",\n      \"method\": \"SILAC quantitative mass spectrometry, co-immunoprecipitation, luciferase reporter assay, western blotting\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — SILAC-MS with Co-IP and functional reporter assay validation, single lab\",\n      \"pmids\": [\"24129246\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Molecular dynamics simulations reveal that B-Raf's αG-helix Arg662 (vs. KSR1's Ala826 at equivalent position) is key: B-Raf Arg662 orients MEK1 Ser218 toward ATP for phosphorylation through interactions with the MEK1 activation loop, while KSR1 Ala826 results in fewer interactions with MEK1 A-loop, yielding a more flexible A-loop. The KSR1 P-rich loop of MEK1 plays a decisive role in MEK1 A-loop accessibility.\",\n      \"method\": \"Molecular dynamics simulation\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational simulation only, no experimental validation in this paper\",\n      \"pmids\": [\"35508574\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SHP2 physically interacts with KSR1 through its protein tyrosine phosphatase domain; during adaptive resistance to MEK inhibitors, activated SHP2 impairs this interaction with KSR1, releasing KSR1 to activate KSR1 and promote MAPK signaling. A KSR1 activating mutation (S269A) reduces the synergistic anti-proliferative effect of SHP2 and MEK inhibitor co-treatment.\",\n      \"method\": \"Co-immunoprecipitation (domain mapping), KSR1 knockdown, KSR1 S269A activating mutation, cell viability/proliferation assay, xenograft model\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-specific Co-IP with mutagenesis and in vivo validation, single lab\",\n      \"pmids\": [\"36493900\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"KRASG12V promotes homo- and heterodimerization of BRAF via its RAS-binding domain; substantial KSR1 homo- and KSR1/BRAF heterodimerization occurs even without activated RAS and requires a salt bridge between the CC-SAM domain of KSR1 and the BRAF-specific region. The dimer interface is less critical for dimerization per se but essential for downstream signaling.\",\n      \"method\": \"Split Nanoluc luciferase complementation assay in living cells, loss-of-function dimerization interface mutants, RAF inhibitor treatment\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — split luciferase PPI assay with mutagenesis in living cells, validated with multiple inhibitors, single lab\",\n      \"pmids\": [\"37316874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"KSR1 constitutively binds to YAP and MST1 and forms a complex with LATS1, functioning as a previously unrecognized scaffold of the Hippo pathway; KSR1 modulates YAP protein levels and transcriptional activity at least in part through the RhoA/actin axis.\",\n      \"method\": \"Co-immunoprecipitation, KSR1 knockdown/overexpression, YAP activity reporter assay, RhoA pathway analysis\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP binding and functional KD/OE, single lab, single paper\",\n      \"pmids\": [\"41326667\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Resistance to pan-RAF inhibitor exarafenib involves formation of RAS-mediated ARAF-KSR1 scaffolding complexes that maintain MAPK signaling despite drug treatment; this bypass is driven by RTK activation and RAS-GTP accumulation that specifically promotes ARAF-KSR1 complex assembly under drug pressure.\",\n      \"method\": \"Co-immunoprecipitation, drug resistance assays, ARAF/KSR1 knockdown, MEK inhibitor combination studies\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with functional resistance validation and combination therapy testing, single lab\",\n      \"pmids\": [\"41654519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Soluble uric acid (UA) directly binds the KSR1 scaffold and hyperactivates MEK-ERK signaling, leading to chronic MAPK stimulation that upregulates inhibitory receptors (PD-1, Tim-3) on CD8+ T cells and promotes their exhaustion. Genetic disruption of the UA-KSR1-MAPK axis via Ksr1 knockdown restored T-cell effector activity.\",\n      \"method\": \"Direct binding assay (UA-KSR1), MEK-ERK phosphorylation analysis, Ksr1 knockdown, Tim-3 KO mouse model, in vivo tumor model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding plus functional genetic validation in cells and in vivo, single lab\",\n      \"pmids\": [\"42118604\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"KSR1 functions primarily as a molecular scaffold that assembles Raf, MEK, and ERK into functional complexes at the plasma membrane to facilitate MAPK cascade activation; its membrane recruitment is regulated by phosphorylation—C-TAK1/MARK2-mediated phosphorylation of Ser392 confers 14-3-3 binding and cytoplasmic retention, while PP2A-mediated dephosphorylation (facilitated by GEF-H1) releases KSR1 to the membrane—and it also possesses intrinsic serine kinase activity capable of directly phosphorylating MEK1. KSR1 integrates multiple upstream inputs (G-protein βγ, CK2, caveolin-1, calmodulin/Ca2+, VRK2A-ER tethering, Erbin, Merlin) to modulate ERK signal amplitude, duration, and subcellular compartmentalization, and is itself subject to feedback phosphorylation by ERK and ubiquitin-mediated degradation via praja2. Beyond scaffolding the canonical Ras/MAPK cascade, KSR1 has now been identified as a scaffold for the Hippo pathway (binding YAP, MST1, LATS1) and regulates non-ERK outputs including PGC1α/ERRα-dependent metabolism, translational control of Myc and EPSTI1, and FLIP-mediated apoptosis resistance.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"KSR1 is a molecular scaffold of the Ras/MAPK cascade that assembles Raf, MEK, and ERK into functional signaling complexes and tunes the amplitude, duration, and subcellular location of ERK output [#8, #21]. Originally defined genetically as a positive, Raf-related mediator of Ras signaling that acts downstream of or parallel to Ras and is required for maximal MPK-1/ERK diphosphorylation [#0, #3], KSR1 binds MEK constitutively (via an intact catalytic domain) and recruits activated ERK inducibly, an interaction functionally required for downstream cellular responses such as neurite outgrowth and post-DNA-damage cell-cycle re-entry [#5, #22]. Its activity is set largely by regulated membrane translocation: in resting cells C-TAK1/MARK2 phosphorylation of Ser392 creates a 14-3-3γ binding site that anchors KSR1 in the cytoplasm, while PP2A — recruited through the adaptor GEF-H1 — dephosphorylates this site to release KSR1 to the plasma membrane where it colocalizes with Ras and Raf [#1, #9, #17, #32]. Membrane targeting is mediated by the CC-SAM domain, which binds lipid directly, and KSR1 also engages caveolin-1 to redistribute MEK/ERK into caveolar fractions and Ca2+/calmodulin via its CA3 domain to promote translocation and ERK complex formation [#15, #21, #28, #29]. Within the scaffold KSR1 nucleates additional Raf-activating machinery, recruiting CK2 to phosphorylate the Raf N-region and undergoing salt-bridge-dependent homo- and BRAF-heterodimerization that is essential for signaling [#4, #38]. Beyond passive scaffolding KSR1 possesses intrinsic serine kinase activity independent of bound MEK and can directly phosphorylate MEK1 in vitro, an activity coupled to TNF-induced survival signaling [#11, #12]. KSR1 abundance is controlled by praja2-mediated ubiquitination and proteasomal degradation and by caspase cleavage during apoptosis, which destroys scaffold function and yields a dominant-negative C-terminal fragment [#19, #23]. Through these mechanisms KSR1 governs diverse outputs including adipogenesis, oncogenic Ras-driven transformation and senescence, and metabolic/translational programs (PGC1α/ERRα, Myc and EPSTI1 translation) that operate partly independent of ERK [#8, #25, #26, #27, #31], and it has been identified as a scaffold of the Hippo pathway binding YAP, MST1, and LATS1 [#39]. KSR1-RAF scaffolding complexes also drive adaptive resistance to RAF and MEK inhibitors [#37, #40].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Established the existence and genetic placement of KSR as a positive Ras-pathway component, defining the central question of how a Raf-related kinase promotes Ras signaling.\",\n      \"evidence\": \"Genetic suppressor screen and epistasis analysis in C. elegans vulval development\",\n      \"pmids\": [\"8521513\", \"8521514\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish biochemical mechanism or direct partners\", \"Whether kinase activity is required left open\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Showed KSR is required for the Raf/MEK/ERK cascade itself, linking the genetic phenotype to a direct biochemical readout of ERK activation.\",\n      \"evidence\": \"ksr-2;ksr-1 double-mutant analysis with dpERK western blot in C. elegans\",\n      \"pmids\": [\"11882296\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Redundancy complicates assigning specific molecular role\", \"Does not distinguish scaffolding from catalytic contribution\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstrated that KSR1 physically binds MEK through its catalytic domain and that this binding is functionally essential, establishing the scaffolding model in mammalian cells.\",\n      \"evidence\": \"Co-IP, mutagenesis, and NGF-induced neurite outgrowth in PC12 cells\",\n      \"pmids\": [\"10891492\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ERK binding mode not resolved\", \"Did not address membrane translocation triggers\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Defined the phosphoswitch controlling KSR1 localization: C-TAK1 phosphorylation of Ser392 imposes 14-3-3-dependent cytoplasmic retention relieved upon stimulation, answering how membrane recruitment is gated.\",\n      \"evidence\": \"Co-IP, phospho-mapping, subcellular imaging, mutagenesis in mammalian cells\",\n      \"pmids\": [\"11741534\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphatase reversing Ser392 not yet identified\", \"In vivo physiological context of the switch unaddressed\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identified PP2A as the constitutive KSR1-associated phosphatase that dephosphorylates the 14-3-3 site to enable membrane recruitment, completing the recruitment cycle.\",\n      \"evidence\": \"Mass spectrometry of KSR1 complex, Co-IP, PP2A inhibition, phospho-analysis\",\n      \"pmids\": [\"12932319\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How growth factor induces PR55B subunit binding not defined\", \"Adaptor linking PP2A to KSR1 not yet known\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Quantified KSR1 as a concentration-dependent rheostat, showing optimal expression maximizes Raf/MEK/ERK association and that both deletion and overexpression impair signaling and transformation.\",\n      \"evidence\": \"KSR1-/- MEF reconstitution with titrated expression, ERK assays, soft agar; plus Ser392/Thr274 mutagenesis kinetics and stability\",\n      \"pmids\": [\"15121859\", \"15371409\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of optimal stoichiometry unresolved\", \"Phosphodegron coupling to stability incompletely mapped\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Resolved a long-standing controversy by demonstrating KSR1 possesses intrinsic kinase activity independent of bound MEK1.\",\n      \"evidence\": \"Two-stage in vitro reconstitution kinase assay with high-salt MEK depletion and kinase-inactive mutant\",\n      \"pmids\": [\"15084597\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological substrate not identified in this study\", \"Catalytic significance in vivo unclear\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identified CK2 within the KSR1 scaffold as a Raf N-region kinase, explaining how KSR1 promotes Raf activation rather than only colocalization.\",\n      \"evidence\": \"MS of KSR1 complex, Co-IP, domain mapping, kinase assays\",\n      \"pmids\": [\"17174095\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CK2 docking is regulated by stimulation unknown\", \"Structural arrangement of CK2-KSR1-Raf not defined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined 14-3-3γ isoform specificity and the E3 ligase IMP as opposing brakes on scaffold assembly, refining how KSR1 oligomerization and inactivity are enforced.\",\n      \"evidence\": \"In vitro/in vivo binding, RNAi, Xenopus oocyte assays (14-3-3γ); Co-IP and ubiquitin assays (IMP)\",\n      \"pmids\": [\"18426801\", \"18332145\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"IMP study Medium-confidence single lab\", \"How Ras-GTP triggers IMP autoubiquitination mechanistically incomplete\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrated KSR1 directly phosphorylates kinase-inactive MEK1 and that this catalytic function contributes to TNF-induced survival, establishing MEK1 as a KSR1 substrate.\",\n      \"evidence\": \"Recombinant kinase assay, D683A/D700A mutant, cell survival assay\",\n      \"pmids\": [\"21144847\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo significance of KSR1 catalysis versus scaffolding unresolved\", \"Stoichiometry relative to Raf-driven MEK phosphorylation unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Mapped the structural determinant of membrane targeting (CC-SAM helix α3) and a parallel ER-tethering mechanism via VRK2A, defining how KSR1 is compartmentalized.\",\n      \"evidence\": \"NMR, lipid-binding, mutagenesis, imaging (CC-SAM); Co-IP, fractionation, SEC (VRK2A)\",\n      \"pmids\": [\"23250398\", \"22752157\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"VRK2A study Medium-confidence single lab\", \"Switch between ER retention and PM targeting not integrated\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified GEF-H1 as the adaptor coupling PP2A to KSR1 in oncogenic RAS, and defined external inhibitory inputs (AMPK-BRAF, MARK2) regulating scaffold engagement and physiology.\",\n      \"evidence\": \"Co-IP, phospho-assays, siRNA in KRAS cells (GEF-H1); in vitro kinase and double-KO mouse (MARK2); kinase assay and Co-IP (AMPK-BRAF)\",\n      \"pmids\": [\"24525234\", \"22206009\", \"24095280\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"MARK2 study Medium-confidence\", \"Tissue-specific integration of opposing inputs incomplete\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Established ERK-mediated feedback phosphorylation of KSR1 as a mechanism limiting signal duration and controlling synaptic localization and plasticity.\",\n      \"evidence\": \"Western blot, imaging, electrophysiology, MEK inhibitor in hippocampal neurons\",\n      \"pmids\": [\"21471251\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Feedback phosphosites not exhaustively mapped\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Linked KSR1 to membrane microdomain organization (caveolin-1) and to cellular processes beyond proliferation—immune synapse ERK recruitment and Ras-induced senescence.\",\n      \"evidence\": \"Co-IP, fractionation, KSR1-/- MEF reconstitution (caveolin-1); KSR1-/- cells, imaging, NK cytotoxicity (immune synapse)\",\n      \"pmids\": [\"25002533\", \"19139278\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Immune-synapse study Medium-confidence single lab\", \"How caveolin recruitment couples to senescence not mechanistically closed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined praja2 as the physiological E3 ligase degrading KSR1 to terminate ERK signaling, and an ELK4-KDM5A axis controlling praja2 levels and thus KSR1 stability.\",\n      \"evidence\": \"Co-IP, ubiquitination/proteasome assays, in vivo tumor (praja2); ChIP, luciferase, CHX chase (ELK4-KDM5A)\",\n      \"pmids\": [\"27195677\", \"34372882\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ELK4-KDM5A arm Medium-confidence single lab\", \"Signal triggering growth-factor-induced KSR1 ubiquitination not fully defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extended KSR1 to ERK-independent and translational outputs—PGC1α/ERRα metabolism, Myc translation, and later EPSTI1-driven EMT—broadening its role beyond canonical MAPK scaffolding.\",\n      \"evidence\": \"KSR1-/- reconstitution and rescue, metabolic and soft-agar assays (PGC1α); polysome/translation analysis (Myc); CRISPR KO and rescue, invasion assays (EPSTI1)\",\n      \"pmids\": [\"21518958\", \"27273865\", \"33970103\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism coupling scaffold to translational control unresolved\", \"All single-lab studies\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed oncogenic RAS/BRAF drives perinuclear/endosomal relocalization of KSR1-CK2-pERK complexes that serve as a platform for C/EBPβ activation during senescence, adding a spatial dimension to scaffold function.\",\n      \"evidence\": \"Confocal imaging, KSR1-/- MEFs, inhibitors, Rab11 colocalization, C/EBPβ phospho-assay\",\n      \"pmids\": [\"29259016\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Trigger for endosomal redistribution not fully defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified additional scaffold antagonists (Erbin, Merlin/NF2) that displace KSR1 from or restrict its Raf binding, integrating tumor-suppressor control of signal amplitude.\",\n      \"evidence\": \"Co-IP/displacement assays, knockdown/overexpression, 3D culture and tumor models (Erbin); Co-IP, proteomics in schwannoma (Merlin)\",\n      \"pmids\": [\"29980571\", \"26549023\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Both Medium-confidence single-lab\", \"Structural basis of displacement unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved the dimerization basis of scaffold function—salt-bridge-dependent KSR1 homo- and KSR1/BRAF heterodimers essential for signaling—and a structural rationale for why KSR1 is a weak MEK kinase relative to BRAF.\",\n      \"evidence\": \"Split Nanoluc PPI assay with interface mutants in living cells (dimerization); molecular dynamics simulation (catalytic comparison)\",\n      \"pmids\": [\"37316874\", \"35508574\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"MD study is computational with no experimental validation\", \"Dimer stoichiometry in vivo not directly measured\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided structural detail of Ca2+/calmodulin binding to the KSR1 CA3 domain and demonstrated functionally that this engagement promotes EGF-induced ERK activation.\",\n      \"evidence\": \"NMR, modeling, Phe355Asp mutagenesis, binding and ERK activation assays (building on 2021 calmodulin binding)\",\n      \"pmids\": [\"38591710\", \"33766558\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Ca2+ signals are physiologically integrated with growth-factor cues unclear\", \"Downstream effect on Raf recruitment not detailed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Discovered KSR1 functions as a Hippo-pathway scaffold binding YAP, MST1, and LATS1, expanding its role to a second major signaling network.\",\n      \"evidence\": \"Co-IP, knockdown/overexpression, YAP reporter assay, RhoA pathway analysis\",\n      \"pmids\": [\"41326667\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab single paper\", \"Whether Hippo scaffolding is separable from MAPK scaffolding unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined KSR1 as a driver of therapeutic resistance and immune modulation: RAS-mediated ARAF-KSR1 and SHP2-released KSR1 complexes sustain MAPK under RAF/MEK inhibition, and soluble uric acid binds KSR1 to drive CD8+ T-cell exhaustion.\",\n      \"evidence\": \"Co-IP, drug-resistance and combination assays, knockdowns, xenografts (ARAF/SHP2); direct UA-KSR1 binding, knockdown, Tim-3 KO, tumor models (uric acid)\",\n      \"pmids\": [\"41654519\", \"36493900\", \"42118604\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"All Medium-confidence single-lab studies\", \"Direct small-molecule binding site on KSR1 for uric acid not structurally mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How KSR1's intrinsic catalytic activity, its scaffolding of canonical MAPK, and its newer roles in the Hippo pathway and translational/metabolic control are integrated within a single protein remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified structural model of full-length KSR1 in complex with Raf/MEK/ERK\", \"Relative in vivo weight of catalysis versus scaffolding unquantified\", \"Mechanism coupling KSR1 to translational control of Myc/EPSTI1 undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [5, 8, 21]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [11, 12]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [11, 12]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 15, 21]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 9]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [31]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3, 8]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [37, 40, 41]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [22]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [23]}\n    ],\n    \"complexes\": [\n      \"KSR1-Raf-MEK-ERK scaffold complex\",\n      \"KSR1-CK2 complex\",\n      \"VRK2A-KSR1-MEK1 ER complex\",\n      \"KSR1-YAP-MST1-LATS1 Hippo complex\"\n    ],\n    \"partners\": [\n      \"MEK1\",\n      \"BRAF\",\n      \"CK2\",\n      \"PP2A\",\n      \"14-3-3gamma\",\n      \"calmodulin\",\n      \"caveolin-1\",\n      \"YAP\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}