{"gene":"KSR1","run_date":"2026-04-28T18:30:27","timeline":{"discoveries":[{"year":1995,"finding":"KSR (kinase suppressor of Ras) was identified as a novel protein kinase required for RAS signal transduction in Drosophila, functioning between RAS and RAF in receptor tyrosine kinase pathways, with mammalian homologs defining a novel class of kinases.","method":"Genetic screens, cloning, genetic epistasis","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — foundational genetic epistasis in Drosophila, independently replicated in C. elegans same year by two groups","pmids":["8521512","8521513","8521514"],"is_preprint":false},{"year":1995,"finding":"C. elegans ksr-1 encodes a positive modifier of vulval induction acting downstream of or in parallel to let-60 ras, encoding a novel putative protein kinase related to the Raf family of Ser/Thr kinases.","method":"Genetic suppressor screen, epistasis analysis, cloning","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — two independent C. elegans groups with epistasis analysis, consistent with Drosophila findings","pmids":["8521513","8521514"],"is_preprint":false},{"year":1996,"finding":"Murine KSR1 cooperates with activated Ras to promote MEK and MAPK activation, associates with Raf-1 at the plasma membrane in a Ras-dependent manner, and the isolated KSR kinase domain (C-terminal) suppresses Ras signaling, whereas the N-terminal domain cooperates with Ras.","method":"Xenopus oocyte maturation assay, cellular transformation assay, co-immunoprecipitation, subcellular fractionation","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1-2 — multiple functional assays in two systems with domain dissection, early foundational paper","pmids":["8946910"],"is_preprint":false},{"year":1997,"finding":"KSR1 stimulates Raf-1 activity in a kinase-independent manner via its cysteine-rich CA3 domain, involving a detergent-labile cofactor, and translocates from cytoplasm to plasma membrane in the presence of activated Ras.","method":"In vitro kinase assay, domain mutagenesis, subcellular fractionation, Xenopus oocyte assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro assay with domain mutants plus functional readouts, multiple orthogonal methods","pmids":["9371754"],"is_preprint":false},{"year":1997,"finding":"KSR1 binds 14-3-3 proteins via multiple binding sites and forms a complex with Raf kinase specifically in the membrane fraction of cells; overexpression of KSR with 14-3-3 induced Xenopus oocyte maturation blocked by dominant-negative Raf-1.","method":"Yeast two-hybrid, co-immunoprecipitation, cell fractionation, Xenopus oocyte assay","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP plus functional oocyte assay and fractionation; strong mechanistic link","pmids":["9115393"],"is_preprint":false},{"year":1998,"finding":"Mammalian KSR1 directly interacts with MEK1/2 via its C-terminal region and with ERK via its N-terminal region; KSR1 overexpression inhibits MAP kinase activation, consistent with a scaffold protein linking MEK and ERK.","method":"Yeast two-hybrid, co-immunoprecipitation in COS-7 cells, microinjection, dominant-negative analysis","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — two independent labs (Yu/Williams and Denouel/Eychène) both showing KSR-MEK interaction by yeast two-hybrid and co-IP","pmids":["9427629","9427625"],"is_preprint":false},{"year":1998,"finding":"Murine KSR1 interacts with MEK-1 and MEK-2 by yeast two-hybrid and co-immunoprecipitation; the MEK subpopulation complexed with KSR1 displays no kinase activity, and KSR1 inhibits Ras-induced transformation, suggesting KSR can block MEK in an inactive form.","method":"Yeast two-hybrid, co-immunoprecipitation, transformation assay","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — two orthogonal methods plus functional transformation assay, independently consistent with Yu et al. 1998","pmids":["9427625"],"is_preprint":false},{"year":1999,"finding":"KSR1 contains five in vivo phosphorylation sites: two constitutive (Ser297, Ser392) mediating 14-3-3 binding, and three Ras-inducible (Thr260, Thr274, Ser443) matching MAPK consensus, with activated MAPK associating with KSR1 in a Ras-dependent manner, identifying KSR1 as an in vivo MAPK substrate.","method":"Mass spectrometry phosphopeptide mapping, site-directed mutagenesis, co-immunoprecipitation, MEK inhibitor treatment","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 — mass spectrometry identification plus mutagenesis plus functional Xenopus assay; strong single-lab study","pmids":["9858547"],"is_preprint":false},{"year":1999,"finding":"KSR1 binds to G-protein betagamma subunits (gamma10, gamma2, gamma3) via its CA3 cysteine-rich domain, and this interaction mediates KSR1 translocation to the plasma membrane; KSR1 inhibits betaGamma-induced MAPK activation.","method":"Yeast two-hybrid, co-immunoprecipitation, pertussis toxin treatment, subcellular translocation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 — yeast two-hybrid plus co-IP plus functional inhibition assay; single lab","pmids":["10075696"],"is_preprint":false},{"year":2000,"finding":"B-KSR1, a brain-specific splice variant of KSR1, constitutively associates with MEK and inducibly interacts with activated MAPK in brain tissues; it promotes sustained MAPK activation and NGF-induced neuronal differentiation, and MEK binding requires the intact catalytic domain.","method":"Co-immunoprecipitation, Western blot, PC12 cell differentiation assay, mutagenesis of loss-of-function alleles","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP with mutant analysis plus functional neurite outgrowth assay; single lab","pmids":["10891492"],"is_preprint":false},{"year":2001,"finding":"C-TAK1 constitutively associates with KSR1 and phosphorylates Ser392 to create a 14-3-3 binding site, sequestering KSR1 in the cytoplasm; in response to growth factor signaling, Ser392 phosphorylation is reduced, allowing KSR1 to translocate to the plasma membrane and colocalize with activated Ras and Raf-1.","method":"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis, confocal microscopy, subcellular fractionation","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay plus mutagenesis plus live-cell localization; multiple orthogonal methods in single paper","pmids":["11741534"],"is_preprint":false},{"year":2002,"finding":"KSR functions as a scaffold that assembles RAF/MEK complexes, facilitating phosphorylation of MEK by RAF. KSR associates independently with RAF and MEK, and these interactions promote RAF/MEK complex formation. KSR-deficient mice show attenuated ERK activation sufficient to block T-cell activation.","method":"KSR-deficient mouse generation, co-immunoprecipitation, high-molecular-weight complex analysis, in vitro kinase assay","journal":"Molecular and cellular biology / Genes & development","confidence":"High","confidence_rationale":"Tier 2 — KO mouse with defined phenotype plus biochemical complex analysis, independently replicated by two groups same year","pmids":["11940661","11850406"],"is_preprint":false},{"year":2002,"finding":"The NMR solution structure of the KSR1 C1 domain reveals unique ligand-binding region and surface charge distribution distinct from Raf-1 and PKCgamma C1 domains; the KSR1 C1 domain does not bind phorbol esters or ceramide, does not interact with Ras, and is absolutely required for membrane localization of KSR1.","method":"NMR structure determination, chimeric protein analysis, lipid-binding assays, subcellular localization assay","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — NMR structure with functional validation via chimeric proteins and localization assays","pmids":["11786023"],"is_preprint":false},{"year":2003,"finding":"PP2A associates with KSR1: the enzymatic core subunits (PR65A and catalytic C) constitutively bind the N-terminal domain of KSR1, while the regulatory PR55B subunit binds upon growth factor treatment. PP2A dephosphorylates KSR1 (and Raf-1) on 14-3-3 binding sites, enabling KSR1 membrane recruitment and MAPK pathway activation.","method":"Mass spectrometry of KSR1-scaffolding complex, co-immunoprecipitation, PP2A inhibition, phosphorylation assays","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1-2 — mass spectrometry identification plus co-IP plus chemical inhibition with multiple functional readouts; strong single paper","pmids":["12932319"],"is_preprint":false},{"year":2003,"finding":"In C. elegans, PP2A phosphatase and PAR-1 kinase act downstream of Raf to positively and negatively regulate KSR activity, respectively; high Zn2+ concentration dramatically increases KSR phosphorylation in mammalian cells, targeting a specific step involving the KSR scaffold.","method":"Genetic double mutant analysis, biochemical phosphorylation assay in mammalian cells","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2-3 — genetic epistasis plus biochemical phosphorylation assay; C. elegans ortholog study","pmids":["14685271"],"is_preprint":false},{"year":2004,"finding":"KSR1 phosphorylation at Ser392 and Thr274 regulates KSR1 protein stability, subcellular localization, and ERK activation; mutation of these sites (KSR1.TVSA) promotes sustained ERK activation, accelerated cell cycle progression, and increased KSR1 stability.","method":"Site-directed mutagenesis, cell proliferation assays, subcellular fractionation, Western blotting","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenesis with functional proliferation and signaling readouts; single lab","pmids":["15371409"],"is_preprint":false},{"year":2004,"finding":"KSR1 regulates the intensity and duration of ERK activation to modulate proliferative and oncogenic potential; concentration-dependent effects reveal optimal KSR1 expression maximally associates with all Raf/MEK/ERK cascade members and mediates transformation.","method":"KSR1-/- mouse embryo fibroblasts, KSR1 re-expression at varying levels, Ras(V12) transformation assay, co-immunoprecipitation","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — KO MEFs with graded KSR1 re-expression, multiple functional and biochemical readouts","pmids":["15121859"],"is_preprint":false},{"year":2004,"finding":"LSP1 (leukocyte-specific protein 1), an F-actin binding protein, associates with KSR1, MEK1, and ERK2 and targets them to peripheral actin filaments, compartmentalizing the ERK/MAP kinase pathway at the cytoskeleton.","method":"Co-immunoprecipitation, confocal microscopy","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 3 — co-IP plus imaging; single lab, limited functional mechanistic follow-up","pmids":["15090600"],"is_preprint":false},{"year":2006,"finding":"CK2 (casein kinase 2 holoenzyme) is a constitutive KSR1-binding partner requiring the basic surface of the KSR1 atypical C1 domain; CK2 bound to KSR1 facilitates ERK cascade signaling by acting as a Raf family N-region kinase, and disruption of KSR1/CK2 reduces growth-factor-induced phosphorylation of C-Raf and B-Raf.","method":"Mass spectrometry of KSR1 complex, co-immunoprecipitation, domain mutagenesis, CK2 inhibition, kinase phosphorylation assay","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1-2 — mass spectrometry identification plus co-IP plus mutagenesis plus inhibitor studies; multiple orthogonal methods","pmids":["17174095"],"is_preprint":false},{"year":2006,"finding":"In Drosophila, KSR induces RAF activation through its kinase-like domain by a mechanism independent of its scaffolding property or putative kinase activity; KSR is recruited to RAF prior to signal activation by CNK in association with a novel SAM domain-containing protein HYP.","method":"Genetic analysis, co-immunoprecipitation, domain mutagenesis in Drosophila","journal":"Genes & development","confidence":"Medium","confidence_rationale":"Tier 2 — genetic plus biochemical in Drosophila ortholog; single lab","pmids":["16600912"],"is_preprint":false},{"year":2007,"finding":"KSR1 undergoes caspase-dependent cleavage during apoptosis, destroying its scaffolding function and generating a stable C-terminal fragment that inhibits ERK activation; cleavage correlates with reduced phosphoERK levels and occurs in vivo during mammary gland involution.","method":"Caspase cleavage assay, mutagenesis (cleavage-resistant DEVA-KSR1), Western blot, MEK inhibitor rescue","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — cleavage-resistant mutant plus in vivo validation plus functional ERK signaling readouts","pmids":["17613518"],"is_preprint":false},{"year":2008,"finding":"14-3-3gamma specifically and functionally interacts with KSR1 via the C-terminal stretch of 14-3-3gamma; this binding protects KSR1 from EGF-induced dephosphorylation, impairs ERK2 activation, and acts as a cytosolic anchor keeping KSR1 inactive. 14-3-3gamma binds KSR1 as a monomer.","method":"In vitro binding assay, co-immunoprecipitation, RNAi, confocal microscopy, Xenopus oocyte assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods; single lab but comprehensive","pmids":["18426801"],"is_preprint":false},{"year":2008,"finding":"IMP (E3 ubiquitin ligase) inhibits ERK cascade signaling by disrupting KSR1 homo-oligomerization and B-Raf/c-Raf hetero-oligomerization, thereby impairing MEK recruitment to activated Raf family members and c-Raf kinase activation.","method":"Co-immunoprecipitation, ubiquitin ligase assays, RNAi, protein complex analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP plus functional ubiquitin assay; single lab","pmids":["18332145"],"is_preprint":false},{"year":2009,"finding":"KSR1 is recruited to the immunological synapse during T-cell activation and to NK cell immunological synapses, and membrane recruitment of KSR1 is required for recruitment of active ERK to the synapse. KSR1 regulates the threshold for MAPK activation in T cells without altering the all-or-none nature of the response.","method":"KSR1-/- mice, live imaging/confocal microscopy, single-cell ERK activation analysis, NK cytolysis assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — KO mice plus direct imaging plus functional cytolysis assays; single lab","pmids":["19139278","19188442"],"is_preprint":false},{"year":2009,"finding":"KSR1 is required for cell cycle reinitiation following DNA damage; KSR1-/- cells cannot recover from MMC-induced G2/M arrest even after DNA damage repair, and a KSR1 mutant unable to bind ERK fails to rescue reinitiation, demonstrating the KSR1-ERK interaction is essential for this function.","method":"KSR1-/- MEFs, KSR1 re-expression, MMC treatment, cell cycle analysis, ERK binding-deficient mutant","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — KO cells plus mutant rescue with specific phenotypic readout; single lab","pmids":["19147494"],"is_preprint":false},{"year":2010,"finding":"KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1 in vitro; kinase-inactive KSR1 (D683A/D700A) lacks these activities, and both KSR1 kinase activity and MEK kinase activity are required for TNF-induced colon epithelial cell survival.","method":"Recombinant protein production in E. coli, in vitro kinase assay, autophosphorylation assay, MBP phosphorylation, TNF apoptosis assay","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro kinase assay with recombinant proteins and active-site mutagenesis plus cellular functional validation","pmids":["21144847"],"is_preprint":false},{"year":2011,"finding":"Crystal structure of KSR2 kinase domain in complex with MEK1 reveals interactions mediated by activation segments and C-lobe αG helices; KSR2 self-associates via a side-to-side interface (analogous to BRAF); BRAF allosterically stimulates KSR2 kinase activity by heterodimerization, and KSR2-BRAF heterodimerization increases BRAF-induced MEK phosphorylation via relay of conformational signal from BRAF to release MEK activation segment.","method":"X-ray crystallography, in vitro kinase assay, chemical genetics, mutagenesis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus in vitro kinase assay plus mutagenesis plus chemical genetics; strong foundational paper","pmids":["21441910"],"is_preprint":false},{"year":2011,"finding":"Most RAF inhibitors induce KSR1 binding to wild-type and oncogenic B-RAF (including V600E) requiring direct drug binding to B-RAF and conserved dimer interface residues; KSR1 competes with C-RAF for inhibitor-induced B-RAF binding, attenuating the paradoxical activating effect of RAF inhibitors on ERK signaling.","method":"Co-immunoprecipitation, dimer interface mutagenesis, ERK activation assays in cancer cell lines","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — co-IP with mutagenesis plus functional ERK signaling readouts; multiple cell lines tested","pmids":["21458265"],"is_preprint":false},{"year":2011,"finding":"Feedback phosphorylation of KSR1 by ERK1/2 reduces KSR1 localization to dendritic spines; expression of feedback-deficient KSR1 promotes sustained ERK1/2 activation and potentiates excitatory postsynaptic currents in hippocampal neurons.","method":"Immunocytochemistry, confocal imaging, electrophysiological recordings, Western blot, mutagenesis","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 — direct imaging plus electrophysiology plus mutant analysis; single lab","pmids":["21471251"],"is_preprint":false},{"year":2012,"finding":"KSR1 contains a CC-SAM (coiled coil-sterile α motif) domain that targets KSR1 to specific signaling sites at the plasma membrane in growth factor-treated cells; membrane binding is mediated by helix α3 of the CC motif, and mutation of α3 residues abolishes plasma membrane targeting.","method":"NMR spectroscopy, mutagenesis, confocal microscopy, in vitro micelle/bicelle binding","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1-2 — NMR structure plus mutagenesis plus live cell imaging; multiple orthogonal methods","pmids":["23250398"],"is_preprint":false},{"year":2012,"finding":"VRK2A anchors KSR1-MEK1 complexes to the endoplasmic reticulum (ER); VRK2A and KSR1 interact via their respective C-terminal regions, MEK1 independently contacts both KSR1 (CA5 region) and VRK2A N-terminus, forming a high molecular size complex (600-1000 kDa) that prevents ERK1/2 incorporation after EGF stimulation.","method":"Co-immunoprecipitation, subcellular fractionation, VRK2A knockdown, EGF stimulation assays","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP plus knockdown plus fractionation; single lab with multiple orthogonal methods","pmids":["22752157"],"is_preprint":false},{"year":2013,"finding":"AMPK phosphorylates BRAF at Ser729, promoting 14-3-3 binding and disrupting the BRAF-KSR1 interaction, leading to attenuation of MEK-ERK signaling and impaired keratinocyte proliferation.","method":"In vitro kinase assay, co-immunoprecipitation, mutagenesis, cell proliferation assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay plus co-IP plus mutagenesis plus cellular functional readouts","pmids":["24095280"],"is_preprint":false},{"year":2014,"finding":"KSR1 interacts with caveolin-1 and redistributes MEK and ERK to caveolin-1-rich fractions; a KSR1 mutant unable to interact with caveolin-1 fails to efficiently mediate growth factor-induced ERK activation at early stages, and abolishing the KSR1-caveolin-1 interaction impairs H-Ras(V12)-induced senescence and transformation.","method":"Co-immunoprecipitation, sucrose gradient fractionation, KSR1 mutagenesis, KSR1-/- MEF rescue, transformation assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP plus fractionation plus mutant analysis; single lab","pmids":["25002533"],"is_preprint":false},{"year":2014,"finding":"GEF-H1 acts as an adaptor linking PP2A B' subunits to KSR1 (independent of its RhoGEF activity), mediating dephosphorylation of KSR1 Ser392 and activation of MAPK signaling in a positive feedback loop for oncogenic RAS.","method":"Co-immunoprecipitation, in vitro phosphatase assay, knockdown/overexpression, xenograft assays","journal":"Cancer cell","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP plus phosphatase assay plus functional cancer cell readouts; single lab","pmids":["24525234"],"is_preprint":false},{"year":2016,"finding":"Small molecules (exemplified by APS-2-79) stabilize a previously unrecognized inactive state of KSR guided by KSR mutations that suppress oncogenic Ras signaling; these compounds antagonize RAF heterodimerization and conformational changes required for phosphorylation of KSR-bound MEK, and enhance MEK inhibitor potency in Ras-mutant cells.","method":"Structure-guided drug design based on KSR mutations, biochemical RAF dimerization assays, MEK phosphorylation assays, cell viability assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 — structure-guided compounds plus biochemical mechanism assays plus cancer cell functional validation","pmids":["27556948"],"is_preprint":false},{"year":2016,"finding":"Praja2 is the E3 ubiquitin ligase that ubiquitylates KSR1, inducing its polyubiquitination and proteasomal degradation in response to receptor stimulation, thereby attenuating ERK1/2 signaling.","method":"Co-immunoprecipitation, ubiquitination assay, proteasome inhibition (MG132), knockdown/overexpression, stem cell pluripotency assay","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — ubiquitination assay plus co-IP plus functional rescue; single lab","pmids":["27195677"],"is_preprint":false},{"year":2016,"finding":"PKA phosphorylation of Rap1 (Ser180/Ser179) creates a 14-3-3 binding site that links Rap1 to the scaffold protein KSR1; because KSR1 and B-Raf exist as heterodimers, this also brings B-Raf to Rap1 to sustain ERK activation by cAMP.","method":"Co-immunoprecipitation, in vitro phosphorylation assay, mutagenesis, ERK activation assay in HEK293 cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP plus in vitro phosphorylation plus mutagenesis; single lab","pmids":["28003362"],"is_preprint":false},{"year":2017,"finding":"Oncogenic RAS and BRAF induce perinuclear relocalization of KSR1, CK2, and p-ERK1/2 to endosomal compartments (perinuclear signaling complexes, PSCs); this requires endocytosis, MEK-ERK and CK2 kinase activities, and the presence of KSR1. KSR1-positive PSCs provide a platform for phosphorylation of the transcription factor C/EBPbeta.","method":"Confocal microscopy, Rab11 co-localization, MEK/CK2 inhibitors, KSR1 knockdown, C/EBPbeta phosphorylation assay","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — direct imaging plus pharmacological inhibition plus KSR1 requirement; single lab","pmids":["29259016"],"is_preprint":false},{"year":2018,"finding":"MEK binding to the KSR1 kinase domain asymmetrically drives BRAF-KSR1 heterodimerization; KSR1 selective heterodimerization with BRAF is specified by direct contacts between N-terminal regulatory regions including the BRS domain in BRAF and the CC-SAM domain in KSR1; KSR-MEK complexes allosterically activate BRAF through N-terminal and kinase domain contacts.","method":"Crystal structure, biochemical dimerization assays, co-immunoprecipitation, in vitro kinase assay, domain mutagenesis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus biochemical reconstitution plus mutagenesis; foundational structural/mechanistic paper","pmids":["29433126"],"is_preprint":false},{"year":2018,"finding":"Erbin interacts with KSR1 and displaces it from the RAF/MEK/ERK complex to prevent signal propagation; loss of Erbin increases amplitude and duration of RAS/RAF signaling via KSR1.","method":"Co-immunoprecipitation, KSR1 pulldown, knockdown, signaling assays, in vivo APC-KO mouse model","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP plus functional signaling assay plus in vivo validation; single lab","pmids":["29980571"],"is_preprint":false},{"year":2020,"finding":"X-ray crystal structures of MEK bound to KSR with MEK inhibitors including trametinib reveal that trametinib directly engages KSR at the MEK interface; KSR remodels the allosteric pocket of MEK, affecting drug binding and residence time; trametinib binding to KSR-MEK disrupts the related RAF-MEK complex through conserved interface residues.","method":"X-ray crystallography of KSR-MEK-inhibitor complexes, kinetic drug binding assays, mutagenesis, cell-based signaling assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — multiple X-ray crystal structures with functional biochemical and cellular validation; high-impact structural study","pmids":["32927473"],"is_preprint":false},{"year":2021,"finding":"Calmodulin binds directly to KSR1 in a Ca2+-dependent manner (in vitro and in cells); calmodulin antagonism impairs EGF-induced KSR1 translocation to the plasma membrane and reduces KSR1-ERK complex formation, thereby suppressing ERK activation.","method":"In vitro binding with purified proteins, co-immunoprecipitation, confocal microscopy, calmodulin inhibitor treatment","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro reconstitution plus co-IP plus live-cell imaging; single lab","pmids":["33766558"],"is_preprint":false},{"year":2021,"finding":"KSR1 promotes epithelial-to-mesenchymal transition via preferential translation of EPSTI1 mRNA; KSR1-dependent induction of EPSTI1 drives the E- to N-cadherin switch and migratory/invasive behavior in colorectal cancer cells through ZEB1 and Slug transcriptional repressors.","method":"KSR1 disruption, ectopic EPSTI1 expression, migration/invasion assays, Western blot, polysome profiling","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 — KO plus ectopic rescue plus mechanistic pathway placement; single lab","pmids":["33970103"],"is_preprint":false},{"year":2021,"finding":"PJA2 (praja2) ubiquitylates KSR1, reducing KSR1 protein stability; KDM5A inhibits PJA2 expression by removing H3K4me3 from the PJA2 promoter, thereby indirectly stabilizing KSR1 and promoting MAPK signaling and macrophage M2 polarization in gastric cancer.","method":"Dual luciferase reporter, ChIP assay, co-immunoprecipitation, cycloheximide chase, ubiquitination assay","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods establishing the KDM5A-PJA2-KSR1 axis; single lab","pmids":["34372882"],"is_preprint":false},{"year":2022,"finding":"KSR1 ectopic expression activates the MAPK pathway and induces cell proliferation in the absence of RAS proteins but requires at least one RAF family member for dimerization and stimulation; KSR1 promotes translocation of heterodimerized RAF to the cell membrane; mutations in the DFG motif of KSR1 affecting ATP binding impair proliferation induction.","method":"RAS-null cell system, RAF/MEK knockout cells, KSR1 dimerization-deficient mutants, ATP-binding mutants, cell proliferation assays","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple genetic perturbations plus mutagenesis; single lab","pmids":["35313064"],"is_preprint":false},{"year":2022,"finding":"SHP2 interacts with KSR1 through its protein tyrosine phosphatase domain; activated SHP2 during adaptive MEK inhibitor resistance impairs SHP2-KSR1 interaction, activating KSR1 to promote MAPK signaling; a KSR1 activating mutation (S269A) diminishes the synergistic anti-proliferative effect of SHP2 inhibitor and MEK inhibitor combination.","method":"Co-immunoprecipitation, KSR1 knockdown, KSR1 activating mutation, ERK signaling assays, xenograft assays","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP plus mutagenesis plus functional signaling assays; single lab","pmids":["36493900"],"is_preprint":false},{"year":2016,"finding":"KSR1 promotes translation of Myc protein by a posttranscriptional mechanism in colon tumor cells, supporting tumor cell survival alongside EPHB4-dependent elevation of Myc mRNA.","method":"KSR1 knockdown, ectopic re-expression, Western blot, mRNA quantification, survival assays","journal":"Molecular and cellular biology","confidence":"Low","confidence_rationale":"Tier 3 — knockdown/OE with phenotype but indirect mechanism for translational regulation; single lab","pmids":["27273865"],"is_preprint":false},{"year":2011,"finding":"KSR1 regulates KSR1-dependent control of PGC1α and ERRα expression to promote metabolic capacity required for oncogenic H-Ras(V12)-induced anchorage-independent growth, independent of ERK signaling.","method":"KSR1-/- MEFs, ectopic PGC1α/ERRα expression, anchorage-independent growth assay, ERRα inhibitor treatment","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — KO plus ectopic rescue plus inhibitor with specific metabolic and transformation readouts; single lab","pmids":["21518958"],"is_preprint":false},{"year":2011,"finding":"KSR1 loss in IL-10-deficient mice causes exacerbated colitis due to dysregulated IFN-γ production in T lymphocytes; KSR1 expressed in hematopoietic lineages is protective, and KSR1 deficiency increases Th1 polarization while impairing Th17 polarization.","method":"KSR1-/- IL10-/- double KO mice, bone marrow transplant, in vitro Th1/Th17 polarization assay, IFN-γ neutralizing antibody treatment","journal":"Gastroenterology","confidence":"Medium","confidence_rationale":"Tier 2 — KO mouse epistasis plus bone marrow transplant plus in vitro polarization assays; single lab","pmids":["20875416"],"is_preprint":false},{"year":2005,"finding":"KSR1 coordinates ERK and RSK activation with C/EBPbeta synthesis during adipogenesis; KSR1 deletion prevents adipogenesis in vitro, and appropriate KSR1 levels promote phosphorylation and stabilization of C/EBPbeta at a specific moment in the adipogenic program, while elevated KSR1 inhibits adipogenesis via high sustained ERK phosphorylating PPARγ.","method":"KSR1-/- cells, KSR1 re-expression at graded levels, adipocyte differentiation assay, ERK/RSK activation measurement","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — KO plus graded rescue with specific cell fate readouts; single lab","pmids":["16107706"],"is_preprint":false},{"year":2011,"finding":"KSR1 is required for efficient NK-mediated cytolysis and cytolytic granule polarization; KSR1 is recruited to the immunological synapse and membrane recruitment of KSR1 is required for ERK localization to the synapse.","method":"KSR1-/- mice, NK cytolysis assay, confocal imaging of immunological synapse","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — KO mice plus direct imaging plus functional cytolysis assay; single lab","pmids":["19139278"],"is_preprint":false},{"year":2011,"finding":"KSR1 regulates BRCA1 degradation in breast cancer cells; KSR1 stabilizes BRCA1 protein levels by reducing BRCA1 ubiquitination through increasing BARD1 abundance.","method":"KSR1 overexpression, ubiquitination assay, BRCA1/BARD1 Western blot, xenograft assay","journal":"Oncogene","confidence":"Low","confidence_rationale":"Tier 3 — mechanistic follow-up by co-IP and ubiquitination assay but limited pathway placement; single lab","pmids":["24909178"],"is_preprint":false},{"year":2011,"finding":"KSR1 regulates FLIP protein levels post-translationally in endometrial carcinoma cells; KSR1 silencing down-regulates FLIP by promoting FADD-dependent inhibition of FLIP translation after TRAIL stimulation, sensitizing cells to TRAIL- and Fas-induced apoptosis.","method":"KSR1 shRNA knockdown, KSR1 re-expression, apoptosis assay, translational regulation assay","journal":"The American journal of pathology","confidence":"Low","confidence_rationale":"Tier 3 — knockdown plus rescue with translational mechanism proposed; single lab","pmids":["21435442"],"is_preprint":false},{"year":2013,"finding":"KSR1 decreases DBC1 phosphorylation, reducing the DBC1-SIRT1 interaction; this enables SIRT1 to deacetylate p53, thereby inhibiting p53 transcriptional activity.","method":"SILAC mass spectrometry phosphoproteomics, co-immunoprecipitation, luciferase reporter assay, Western blot","journal":"British journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 — SILAC proteomics plus co-IP plus reporter assay; single lab with multiple methods","pmids":["24129246"],"is_preprint":false},{"year":2016,"finding":"DiRas3 binds to KSR1 independently of activated Ras and RAF; depending on local stoichiometry, DiRas3 can either enhance KSR1 homodimerization or recruit KSR1 to the Ras:C-RAF complex, thereby reducing C-RAF availability for B-RAF heterodimerization.","method":"Co-immunoprecipitation, dimerization assays, mutagenesis","journal":"Cellular signalling","confidence":"Low","confidence_rationale":"Tier 3 — co-IP plus limited functional validation; single lab","pmids":["27368419"],"is_preprint":false},{"year":2011,"finding":"MARK2 (related to C-TAK1) binds and phosphorylates KSR1 on Ser392 in vivo; genetic deletion of KSR1 in MARK2-/- mice reverses the increased insulin sensitivity caused by MARK2 loss, placing KSR1 downstream of MARK2 in insulin-regulated glucose metabolism.","method":"Double KO mice (mark2-/- ksr1-/-), glucose tolerance test, co-immunoprecipitation, in vitro phosphorylation assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo epistasis (double KO) plus in vitro kinase assay plus co-IP; single lab","pmids":["22206009"],"is_preprint":false},{"year":2024,"finding":"KSR1 knockout mice are resistant to cisplatin- and noise-induced hearing loss; loss of KSR1 reduces BRAF/MEK/ERK phosphorylation cascade in cochlear cells following insults; dabrafenib (BRAF inhibitor) does not enhance protection in KO mice, placing KSR1 upstream of BRAF in cochlear MAPK injury signaling.","method":"KSR1 germline KO mice, ABR hearing testing, BRAF inhibitor treatment, cochlear MAPK phosphorylation assay","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — KO mouse with epistasis (dabrafenib test) plus biochemical pathway readouts; single lab","pmids":["38548338"],"is_preprint":false}],"current_model":"KSR1 is a conserved pseudokinase/scaffold protein that facilitates RAS-RAF-MEK-ERK signal transduction by constitutively binding MEK and translocating to the plasma membrane upon RAS activation where it assembles Raf-MEK-ERK complexes; its membrane recruitment is regulated by C-TAK1/MARK2-mediated phosphorylation of Ser392 creating a 14-3-3 binding site that sequesters KSR1 in the cytoplasm, while PP2A-mediated dephosphorylation (facilitated by GEF-H1 as adaptor) releases KSR1 to the membrane; at the membrane KSR1 allosterically activates RAF catalysis by side-to-side heterodimerization through contacts between its CC-SAM domain and the BRS region of BRAF, while simultaneously positioning the MEK activation segment for phosphorylation by a separate catalytic RAF molecule; KSR1 also exhibits intrinsic serine kinase activity capable of phosphorylating MEK1 in vitro; the scaffold is additionally regulated by CK2 (facilitating RAF N-region phosphorylation), caveolin-1 (for optimal early ERK activation), calmodulin (Ca2+-dependently), and ubiquitin-mediated degradation by praja2, and is inactivated by caspase cleavage during apoptosis."},"narrative":{"teleology":[{"year":1995,"claim":"Genetic screens in Drosophila and C. elegans independently identified KSR as a novel kinase-family gene required for RAS-mediated signal transduction, establishing that a previously unknown component acts between or in parallel to RAS and RAF.","evidence":"Forward genetic suppressor/enhancer screens with epistasis analysis in Drosophila and C. elegans vulval induction","pmids":["8521512","8521513","8521514"],"confidence":"High","gaps":["Biochemical activity (kinase vs. scaffold) unknown","Mammalian function not yet tested","Direct binding partners not identified"]},{"year":1997,"claim":"Domain dissection revealed that KSR1 stimulates RAF-1 activity in a kinase-independent manner through its CA3/C1 cysteine-rich domain and translocates from cytoplasm to plasma membrane upon RAS activation, shifting the model from simple kinase to potential scaffold/allosteric activator.","evidence":"Domain mutagenesis, in vitro kinase assays, subcellular fractionation, and Xenopus oocyte maturation assays","pmids":["9371754","8946910"],"confidence":"High","gaps":["Nature of the 'detergent-labile cofactor' unresolved","Scaffolding of downstream components not yet demonstrated"]},{"year":1998,"claim":"Identification of MEK1/2 and ERK as direct KSR1-binding partners established KSR1 as a multi-component scaffold bridging RAF, MEK, and ERK, and explained how overexpression could inhibit signaling through stoichiometric titration of cascade members.","evidence":"Yeast two-hybrid and co-immunoprecipitation in COS-7 cells by two independent groups","pmids":["9427629","9427625"],"confidence":"High","gaps":["Stoichiometry of the scaffold complex not determined","In vivo scaffold function not yet validated"]},{"year":1999,"claim":"Mass spectrometry-based phosphopeptide mapping revealed five in vivo phosphorylation sites on KSR1—two constitutive (Ser297, Ser392) mediating 14-3-3 binding and three RAS-inducible MAPK-consensus sites—demonstrating that KSR1 is itself an ERK substrate subject to feedback regulation.","evidence":"Mass spectrometry phosphopeptide identification, site-directed mutagenesis, MEK inhibitor treatment","pmids":["9858547"],"confidence":"High","gaps":["Kinases responsible for constitutive phosphorylation not yet identified","Functional consequence of each site not fully dissected"]},{"year":2001,"claim":"Identification of C-TAK1 as the kinase that phosphorylates KSR1 Ser392 to create a 14-3-3 anchor site resolved how KSR1 is sequestered in the cytoplasm and how growth factor-induced dephosphorylation licenses membrane translocation.","evidence":"Co-immunoprecipitation, in vitro kinase assay, S392A mutagenesis, confocal imaging of membrane translocation","pmids":["11741534"],"confidence":"High","gaps":["Phosphatase identity not yet known","Regulation of C-TAK1 activity itself unclear"]},{"year":2002,"claim":"Generation of KSR1-deficient mice proved that KSR1 functions as a bona fide scaffold in vivo: KSR1 loss attenuated ERK activation, blocked T-cell activation, and KSR1 was shown to independently bind RAF and MEK to promote their complex formation.","evidence":"KSR1 knockout mouse phenotyping, high-molecular-weight complex analysis, co-immunoprecipitation, in vitro kinase assays by two independent groups","pmids":["11940661","11850406"],"confidence":"High","gaps":["Structural basis of scaffold assembly unknown","Relative contribution of scaffolding vs. allosteric activation unclear"]},{"year":2003,"claim":"Discovery that PP2A constitutively associates with KSR1 and dephosphorylates the 14-3-3 binding site upon growth factor signaling completed the phosphorylation–dephosphorylation switch model controlling KSR1 membrane recruitment.","evidence":"Mass spectrometry of KSR1 complex, co-immunoprecipitation, PP2A inhibition studies","pmids":["12932319"],"confidence":"High","gaps":["Which PP2A regulatory subunit specifies KSR1 targeting not fully resolved","Adaptor linking PP2A to KSR1 not yet identified"]},{"year":2006,"claim":"CK2 was identified as a constitutive KSR1-associated kinase that facilitates RAF N-region phosphorylation, revealing that KSR1 scaffolds not only the core ERK cascade but also an upstream activating kinase for RAF.","evidence":"Mass spectrometry, co-immunoprecipitation, C1 domain mutagenesis, CK2 inhibitor treatment","pmids":["17174095"],"confidence":"High","gaps":["Structural basis of CK2–C1 domain interaction not determined","Whether CK2 phosphorylates KSR1 itself not fully tested"]},{"year":2010,"claim":"Reconstitution with bacterially produced KSR1 demonstrated intrinsic serine kinase activity including autophosphorylation and MEK1 phosphorylation, resolving the long-standing debate about whether KSR1 is a true pseudokinase or retains catalytic function.","evidence":"Recombinant KSR1 in vitro kinase assay with active-site D683A/D700A mutants, TNF-induced apoptosis rescue","pmids":["21144847"],"confidence":"High","gaps":["In vivo substrates of KSR1 kinase activity not comprehensively identified","Relative contribution of kinase vs. scaffold activity to signaling output unclear"]},{"year":2011,"claim":"The crystal structure of KSR2–MEK1 complex revealed how KSR binds MEK through activation-segment and αG-helix contacts and showed that BRAF allosterically activates KSR through side-to-side heterodimerization, providing the first atomic-level model of scaffold-mediated kinase cascade assembly.","evidence":"X-ray crystallography, in vitro kinase assays, chemical genetics, dimer-interface mutagenesis","pmids":["21441910"],"confidence":"High","gaps":["Full-length KSR1 structure not available","Dynamic conformational changes during signaling not captured"]},{"year":2012,"claim":"NMR characterization of the CC-SAM domain showed it directly binds membranes via helix α3, identifying the structural determinant that targets KSR1 to the plasma membrane independently of the C1 domain.","evidence":"NMR spectroscopy, α3 mutagenesis, confocal imaging, in vitro micelle/bicelle binding","pmids":["23250398"],"confidence":"High","gaps":["Lipid specificity of CC-SAM binding not defined","How CC-SAM and C1 domain cooperate in membrane targeting unclear"]},{"year":2014,"claim":"GEF-H1 was identified as the adaptor that bridges PP2A B′ subunits to KSR1 for Ser392 dephosphorylation, completing the molecular wiring of the positive feedback loop by which oncogenic RAS sustains KSR1 membrane recruitment.","evidence":"Co-immunoprecipitation, in vitro phosphatase assay, knockdown, xenograft assays","pmids":["24525234"],"confidence":"Medium","gaps":["GEF-H1-independent mechanisms of PP2A-KSR1 interaction not excluded","In vivo validation in genetic models not performed"]},{"year":2016,"claim":"Structure-guided small molecules (APS-2-79) that stabilize an inactive KSR conformation demonstrated that KSR1's conformational state—not merely its presence—gates RAF heterodimerization and MEK phosphorylation, opening a pharmacological strategy to target the scaffold.","evidence":"Structure-guided compound design, RAF dimerization assays, MEK phosphorylation assays, cancer cell viability","pmids":["27556948"],"confidence":"High","gaps":["In vivo pharmacokinetics and efficacy not reported","Selectivity over KSR2 not fully characterized"]},{"year":2018,"claim":"A higher-resolution structural study revealed that MEK binding to KSR1 asymmetrically drives BRAF-KSR1 heterodimerization through both kinase-domain and N-terminal (CC-SAM/BRS) contacts, establishing the complete allosteric mechanism by which the KSR1-MEK binary complex activates BRAF catalysis.","evidence":"Crystal structures, biochemical dimerization assays, co-immunoprecipitation, domain mutagenesis","pmids":["29433126"],"confidence":"High","gaps":["Dynamics of the ternary complex in living cells not resolved","How KSR1 vs. KSR2 selectivity for RAF isoforms is achieved structurally"]},{"year":2020,"claim":"Crystal structures of KSR-MEK complexes with clinical MEK inhibitors (trametinib) showed that KSR remodels MEK's allosteric drug-binding pocket and that drug binding to the KSR-MEK interface disrupts RAF-MEK complexes, explaining drug mechanism and resistance phenomena.","evidence":"X-ray crystallography of KSR-MEK-inhibitor ternary complexes, kinetic binding assays, mutagenesis, cell signaling assays","pmids":["32927473"],"confidence":"High","gaps":["Impact on clinical resistance mechanisms in patients not directly demonstrated","Structural effects of other MEK inhibitor classes not fully explored"]},{"year":2022,"claim":"Ectopic KSR1 activates the MAPK pathway and drives proliferation even in the absence of RAS proteins, provided at least one RAF family member is present for dimerization, demonstrating that KSR1-RAF dimerization is sufficient to initiate signaling independent of canonical RAS-GTP.","evidence":"RAS-null cell system, RAF/MEK knockout cells, KSR1 dimerization and ATP-binding mutants, proliferation assays","pmids":["35313064"],"confidence":"Medium","gaps":["Physiological relevance of RAS-independent KSR1 activation unclear","Whether endogenous KSR1 levels achieve this in any tissue not tested"]},{"year":null,"claim":"A full-length structure of KSR1 in its autoinhibited and active states, the precise in vivo substrates of KSR1's intrinsic kinase activity, and the quantitative contribution of kinase versus scaffold functions to ERK signaling output remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length KSR1 structure available","In vivo kinase substrates beyond MEK1 not identified","Kinase-dead knock-in mouse not reported to separate catalytic from scaffold roles"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[25,26,44]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[5,6,11,16,38]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[12,29]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,3,10,29,41]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[10,21]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[37]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[30]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,5,11,16,26,38]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[11,23,48,50]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[20,52]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[15,24]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[27,34,42]}],"complexes":["KSR1-MEK1/2 scaffold complex","KSR1-BRAF heterodimer","KSR1-PP2A complex","KSR1-CK2 complex"],"partners":["BRAF","RAF1","MAP2K1","MAPK1","CSNK2A1","PPP2CA","YWHAG","MARK2"],"other_free_text":[]},"mechanistic_narrative":"KSR1 is a conserved pseudokinase/scaffold protein that organizes the RAS-RAF-MEK-ERK signaling cascade by constitutively binding MEK and, upon growth factor stimulation, translocating to the plasma membrane to assemble RAF-MEK-ERK complexes and regulate the intensity and duration of ERK activation [PMID:11940661, PMID:15121859, PMID:9427629]. Membrane recruitment is controlled by a phosphorylation–dephosphorylation switch: C-TAK1/MARK2-mediated phosphorylation of Ser392 creates a 14-3-3 binding site that sequesters KSR1 in the cytoplasm, while PP2A (recruited via the adaptor GEF-H1) dephosphorylates this site to license membrane translocation, with the CC-SAM domain and atypical C1 domain providing direct membrane-targeting determinants [PMID:11741534, PMID:12932319, PMID:24525234, PMID:23250398]. Crystal structures reveal that KSR1 allosterically activates BRAF through side-to-side heterodimerization—driven by CC-SAM/BRS contacts and kinase-domain dimer interfaces—positioning KSR1-bound MEK for phosphorylation by a catalytic RAF protomer, while KSR1 itself retains intrinsic serine kinase activity capable of phosphorylating MEK1 [PMID:21441910, PMID:29433126, PMID:21144847]. Beyond canonical MAPK signaling, KSR1 functions in T-cell and NK-cell activation at the immunological synapse, coordinates adipogenic and metabolic transcriptional programs, and its scaffolding is terminated by caspase cleavage during apoptosis and by praja2-mediated ubiquitin-dependent degradation [PMID:19188442, PMID:16107706, PMID:17613518, PMID:27195677]."},"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":"8521512","id":"PMC_8521512","title":"KSR, a novel protein kinase required for RAS signal transduction.","date":"1995","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/8521512","citation_count":331,"is_preprint":false},{"pmid":"8521513","id":"PMC_8521513","title":"The C. elegans ksr-1 gene encodes a novel Raf-related kinase involved in Ras-mediated signal transduction.","date":"1995","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/8521513","citation_count":259,"is_preprint":false},{"pmid":"12932319","id":"PMC_12932319","title":"Protein phosphatase 2A positively regulates Ras signaling by dephosphorylating KSR1 and Raf-1 on critical 14-3-3 binding sites.","date":"2003","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/12932319","citation_count":255,"is_preprint":false},{"pmid":"8521514","id":"PMC_8521514","title":"The ksr-1 gene encodes a novel protein kinase involved in Ras-mediated signaling in C. elegans.","date":"1995","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/8521514","citation_count":248,"is_preprint":false},{"pmid":"11741534","id":"PMC_11741534","title":"C-TAK1 regulates Ras signaling by phosphorylating the MAPK scaffold, KSR1.","date":"2001","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/11741534","citation_count":242,"is_preprint":false},{"pmid":"11940661","id":"PMC_11940661","title":"Kinase suppressor of Ras (KSR) is a scaffold which facilitates mitogen-activated protein kinase activation in vivo.","date":"2002","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/11940661","citation_count":230,"is_preprint":false},{"pmid":"8946910","id":"PMC_8946910","title":"KSR modulates signal propagation within the MAPK cascade.","date":"1996","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/8946910","citation_count":218,"is_preprint":false},{"pmid":"21441910","id":"PMC_21441910","title":"A Raf-induced allosteric transition of KSR stimulates phosphorylation of MEK.","date":"2011","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/21441910","citation_count":210,"is_preprint":false},{"pmid":"11309192","id":"PMC_11309192","title":"KSR: a MAPK scaffold of the Ras pathway?","date":"2001","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/11309192","citation_count":193,"is_preprint":false},{"pmid":"11850406","id":"PMC_11850406","title":"KSR is a scaffold required for activation of the ERK/MAPK module.","date":"2002","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/11850406","citation_count":184,"is_preprint":false},{"pmid":"9858547","id":"PMC_9858547","title":"Identification of constitutive and ras-inducible phosphorylation sites of KSR: implications for 14-3-3 binding, mitogen-activated protein kinase binding, and KSR overexpression.","date":"1999","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/9858547","citation_count":174,"is_preprint":false},{"pmid":"9427629","id":"PMC_9427629","title":"Regulation of the MAP kinase pathway by mammalian Ksr through direct interaction with MEK and ERK.","date":"1998","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/9427629","citation_count":170,"is_preprint":false},{"pmid":"9115393","id":"PMC_9115393","title":"The protein kinase KSR interacts with 14-3-3 protein and Raf.","date":"1997","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/9115393","citation_count":150,"is_preprint":false},{"pmid":"9371754","id":"PMC_9371754","title":"KSR stimulates Raf-1 activity in a kinase-independent manner.","date":"1997","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/9371754","citation_count":143,"is_preprint":false},{"pmid":"32927473","id":"PMC_32927473","title":"Structural basis for the action of the drug trametinib at KSR-bound MEK.","date":"2020","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/32927473","citation_count":135,"is_preprint":false},{"pmid":"9427625","id":"PMC_9427625","title":"Murine Ksr interacts with MEK and inhibits Ras-induced transformation.","date":"1998","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/9427625","citation_count":131,"is_preprint":false},{"pmid":"15121859","id":"PMC_15121859","title":"The molecular scaffold KSR1 regulates the proliferative and oncogenic potential of cells.","date":"2004","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/15121859","citation_count":127,"is_preprint":false},{"pmid":"17496912","id":"PMC_17496912","title":"KSR and CNK: two scaffolds regulating RAS-mediated RAF 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kinase suppressor of Ras (KSR) modulates growth factor and Ras signaling by uncoupling Elk-1 phosphorylation from MAP kinase activation.","date":"1998","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/9501093","citation_count":87,"is_preprint":false},{"pmid":"24525234","id":"PMC_24525234","title":"The RhoGEF GEF-H1 is required for oncogenic RAS signaling via KSR-1.","date":"2014","source":"Cancer cell","url":"https://pubmed.ncbi.nlm.nih.gov/24525234","citation_count":84,"is_preprint":false},{"pmid":"27556948","id":"PMC_27556948","title":"Small molecule stabilization of the KSR inactive state antagonizes oncogenic Ras signalling.","date":"2016","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/27556948","citation_count":82,"is_preprint":false},{"pmid":"17174095","id":"PMC_17174095","title":"CK2 Is a component of the KSR1 scaffold complex that contributes to Raf kinase activation.","date":"2006","source":"Current biology : 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functioning between RAS and RAF in receptor tyrosine kinase pathways, with mammalian homologs defining a novel class of kinases.\",\n      \"method\": \"Genetic screens, cloning, genetic epistasis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — foundational genetic epistasis in Drosophila, independently replicated in C. elegans same year by two groups\",\n      \"pmids\": [\"8521512\", \"8521513\", \"8521514\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"C. elegans ksr-1 encodes a positive modifier of vulval induction acting downstream of or in parallel to let-60 ras, encoding a novel putative protein kinase related to the Raf family of Ser/Thr kinases.\",\n      \"method\": \"Genetic suppressor screen, epistasis analysis, cloning\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — two independent C. elegans groups with epistasis analysis, consistent with Drosophila findings\",\n      \"pmids\": [\"8521513\", \"8521514\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Murine KSR1 cooperates with activated Ras to promote MEK and MAPK activation, associates with Raf-1 at the plasma membrane in a Ras-dependent manner, and the isolated KSR kinase domain (C-terminal) suppresses Ras signaling, whereas the N-terminal domain cooperates with Ras.\",\n      \"method\": \"Xenopus oocyte maturation assay, cellular transformation assay, co-immunoprecipitation, subcellular fractionation\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple functional assays in two systems with domain dissection, early foundational paper\",\n      \"pmids\": [\"8946910\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"KSR1 stimulates Raf-1 activity in a kinase-independent manner via its cysteine-rich CA3 domain, involving a detergent-labile cofactor, and translocates from cytoplasm to plasma membrane in the presence of activated Ras.\",\n      \"method\": \"In vitro kinase assay, domain mutagenesis, subcellular fractionation, Xenopus oocyte assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro assay with domain mutants plus functional readouts, multiple orthogonal methods\",\n      \"pmids\": [\"9371754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"KSR1 binds 14-3-3 proteins via multiple binding sites and forms a complex with Raf kinase specifically in the membrane fraction of cells; overexpression of KSR with 14-3-3 induced Xenopus oocyte maturation blocked by dominant-negative Raf-1.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, cell fractionation, Xenopus oocyte assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP plus functional oocyte assay and fractionation; strong mechanistic link\",\n      \"pmids\": [\"9115393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Mammalian KSR1 directly interacts with MEK1/2 via its C-terminal region and with ERK via its N-terminal region; KSR1 overexpression inhibits MAP kinase activation, consistent with a scaffold protein linking MEK and ERK.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation in COS-7 cells, microinjection, dominant-negative analysis\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — two independent labs (Yu/Williams and Denouel/Eychène) both showing KSR-MEK interaction by yeast two-hybrid and co-IP\",\n      \"pmids\": [\"9427629\", \"9427625\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Murine KSR1 interacts with MEK-1 and MEK-2 by yeast two-hybrid and co-immunoprecipitation; the MEK subpopulation complexed with KSR1 displays no kinase activity, and KSR1 inhibits Ras-induced transformation, suggesting KSR can block MEK in an inactive form.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, transformation assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — two orthogonal methods plus functional transformation assay, independently consistent with Yu et al. 1998\",\n      \"pmids\": [\"9427625\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"KSR1 contains five in vivo phosphorylation sites: two constitutive (Ser297, Ser392) mediating 14-3-3 binding, and three Ras-inducible (Thr260, Thr274, Ser443) matching MAPK consensus, with activated MAPK associating with KSR1 in a Ras-dependent manner, identifying KSR1 as an in vivo MAPK substrate.\",\n      \"method\": \"Mass spectrometry phosphopeptide mapping, site-directed mutagenesis, co-immunoprecipitation, MEK inhibitor treatment\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mass spectrometry identification plus mutagenesis plus functional Xenopus assay; strong single-lab study\",\n      \"pmids\": [\"9858547\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"KSR1 binds to G-protein betagamma subunits (gamma10, gamma2, gamma3) via its CA3 cysteine-rich domain, and this interaction mediates KSR1 translocation to the plasma membrane; KSR1 inhibits betaGamma-induced MAPK activation.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, pertussis toxin treatment, subcellular translocation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — yeast two-hybrid plus co-IP plus functional inhibition assay; single lab\",\n      \"pmids\": [\"10075696\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"B-KSR1, a brain-specific splice variant of KSR1, constitutively associates with MEK and inducibly interacts with activated MAPK in brain tissues; it promotes sustained MAPK activation and NGF-induced neuronal differentiation, and MEK binding requires the intact catalytic domain.\",\n      \"method\": \"Co-immunoprecipitation, Western blot, PC12 cell differentiation assay, mutagenesis of loss-of-function alleles\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP with mutant analysis plus functional neurite outgrowth assay; single lab\",\n      \"pmids\": [\"10891492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"C-TAK1 constitutively associates with KSR1 and phosphorylates Ser392 to create a 14-3-3 binding site, sequestering KSR1 in the cytoplasm; in response to growth factor signaling, Ser392 phosphorylation is reduced, allowing KSR1 to translocate to the plasma membrane and colocalize with activated Ras and Raf-1.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis, confocal microscopy, subcellular fractionation\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay plus mutagenesis plus live-cell localization; multiple orthogonal methods in single paper\",\n      \"pmids\": [\"11741534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"KSR functions as a scaffold that assembles RAF/MEK complexes, facilitating phosphorylation of MEK by RAF. KSR associates independently with RAF and MEK, and these interactions promote RAF/MEK complex formation. KSR-deficient mice show attenuated ERK activation sufficient to block T-cell activation.\",\n      \"method\": \"KSR-deficient mouse generation, co-immunoprecipitation, high-molecular-weight complex analysis, in vitro kinase assay\",\n      \"journal\": \"Molecular and cellular biology / Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with defined phenotype plus biochemical complex analysis, independently replicated by two groups same year\",\n      \"pmids\": [\"11940661\", \"11850406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The NMR solution structure of the KSR1 C1 domain reveals unique ligand-binding region and surface charge distribution distinct from Raf-1 and PKCgamma C1 domains; the KSR1 C1 domain does not bind phorbol esters or ceramide, does not interact with Ras, and is absolutely required for membrane localization of KSR1.\",\n      \"method\": \"NMR structure determination, chimeric protein analysis, lipid-binding assays, subcellular localization assay\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure with functional validation via chimeric proteins and localization assays\",\n      \"pmids\": [\"11786023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PP2A associates with KSR1: the enzymatic core subunits (PR65A and catalytic C) constitutively bind the N-terminal domain of KSR1, while the regulatory PR55B subunit binds upon growth factor treatment. PP2A dephosphorylates KSR1 (and Raf-1) on 14-3-3 binding sites, enabling KSR1 membrane recruitment and MAPK pathway activation.\",\n      \"method\": \"Mass spectrometry of KSR1-scaffolding complex, co-immunoprecipitation, PP2A inhibition, phosphorylation assays\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mass spectrometry identification plus co-IP plus chemical inhibition with multiple functional readouts; strong single paper\",\n      \"pmids\": [\"12932319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"In C. elegans, PP2A phosphatase and PAR-1 kinase act downstream of Raf to positively and negatively regulate KSR activity, respectively; high Zn2+ concentration dramatically increases KSR phosphorylation in mammalian cells, targeting a specific step involving the KSR scaffold.\",\n      \"method\": \"Genetic double mutant analysis, biochemical phosphorylation assay in mammalian cells\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — genetic epistasis plus biochemical phosphorylation assay; C. elegans ortholog study\",\n      \"pmids\": [\"14685271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"KSR1 phosphorylation at Ser392 and Thr274 regulates KSR1 protein stability, subcellular localization, and ERK activation; mutation of these sites (KSR1.TVSA) promotes sustained ERK activation, accelerated cell cycle progression, and increased KSR1 stability.\",\n      \"method\": \"Site-directed mutagenesis, cell proliferation assays, subcellular fractionation, Western blotting\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis with functional proliferation and signaling readouts; single lab\",\n      \"pmids\": [\"15371409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"KSR1 regulates the intensity and duration of ERK activation to modulate proliferative and oncogenic potential; concentration-dependent effects reveal optimal KSR1 expression maximally associates with all Raf/MEK/ERK cascade members and mediates transformation.\",\n      \"method\": \"KSR1-/- mouse embryo fibroblasts, KSR1 re-expression at varying levels, Ras(V12) transformation assay, co-immunoprecipitation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO MEFs with graded KSR1 re-expression, multiple functional and biochemical readouts\",\n      \"pmids\": [\"15121859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"LSP1 (leukocyte-specific protein 1), an F-actin binding protein, associates with KSR1, MEK1, and ERK2 and targets them to peripheral actin filaments, compartmentalizing the ERK/MAP kinase pathway at the cytoskeleton.\",\n      \"method\": \"Co-immunoprecipitation, confocal microscopy\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP plus imaging; single lab, limited functional mechanistic follow-up\",\n      \"pmids\": [\"15090600\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CK2 (casein kinase 2 holoenzyme) is a constitutive KSR1-binding partner requiring the basic surface of the KSR1 atypical C1 domain; CK2 bound to KSR1 facilitates ERK cascade signaling by acting as a Raf family N-region kinase, and disruption of KSR1/CK2 reduces growth-factor-induced phosphorylation of C-Raf and B-Raf.\",\n      \"method\": \"Mass spectrometry of KSR1 complex, co-immunoprecipitation, domain mutagenesis, CK2 inhibition, kinase phosphorylation assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mass spectrometry identification plus co-IP plus mutagenesis plus inhibitor studies; multiple orthogonal methods\",\n      \"pmids\": [\"17174095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"In Drosophila, KSR induces RAF activation through its kinase-like domain by a mechanism independent of its scaffolding property or putative kinase activity; KSR is recruited to RAF prior to signal activation by CNK in association with a novel SAM domain-containing protein HYP.\",\n      \"method\": \"Genetic analysis, co-immunoprecipitation, domain mutagenesis in Drosophila\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic plus biochemical in Drosophila ortholog; single lab\",\n      \"pmids\": [\"16600912\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"KSR1 undergoes caspase-dependent cleavage during apoptosis, destroying its scaffolding function and generating a stable C-terminal fragment that inhibits ERK activation; cleavage correlates with reduced phosphoERK levels and occurs in vivo during mammary gland involution.\",\n      \"method\": \"Caspase cleavage assay, mutagenesis (cleavage-resistant DEVA-KSR1), Western blot, MEK inhibitor rescue\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — cleavage-resistant mutant plus in vivo validation plus functional ERK signaling readouts\",\n      \"pmids\": [\"17613518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"14-3-3gamma specifically and functionally interacts with KSR1 via the C-terminal stretch of 14-3-3gamma; this binding protects KSR1 from EGF-induced dephosphorylation, impairs ERK2 activation, and acts as a cytosolic anchor keeping KSR1 inactive. 14-3-3gamma binds KSR1 as a monomer.\",\n      \"method\": \"In vitro binding assay, co-immunoprecipitation, RNAi, confocal microscopy, Xenopus oocyte assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods; single lab but comprehensive\",\n      \"pmids\": [\"18426801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"IMP (E3 ubiquitin ligase) inhibits ERK cascade signaling by disrupting KSR1 homo-oligomerization and B-Raf/c-Raf hetero-oligomerization, thereby impairing MEK recruitment to activated Raf family members and c-Raf kinase activation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitin ligase assays, RNAi, protein complex analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus functional ubiquitin assay; single lab\",\n      \"pmids\": [\"18332145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"KSR1 is recruited to the immunological synapse during T-cell activation and to NK cell immunological synapses, and membrane recruitment of KSR1 is required for recruitment of active ERK to the synapse. KSR1 regulates the threshold for MAPK activation in T cells without altering the all-or-none nature of the response.\",\n      \"method\": \"KSR1-/- mice, live imaging/confocal microscopy, single-cell ERK activation analysis, NK cytolysis assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mice plus direct imaging plus functional cytolysis assays; single lab\",\n      \"pmids\": [\"19139278\", \"19188442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"KSR1 is required for cell cycle reinitiation following DNA damage; KSR1-/- cells cannot recover from MMC-induced G2/M arrest even after DNA damage repair, and a KSR1 mutant unable to bind ERK fails to rescue reinitiation, demonstrating the KSR1-ERK interaction is essential for this function.\",\n      \"method\": \"KSR1-/- MEFs, KSR1 re-expression, MMC treatment, cell cycle analysis, ERK binding-deficient mutant\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO cells plus mutant rescue with specific phenotypic readout; single lab\",\n      \"pmids\": [\"19147494\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1 in vitro; kinase-inactive KSR1 (D683A/D700A) lacks these activities, and both KSR1 kinase activity and MEK kinase activity are required for TNF-induced colon epithelial cell survival.\",\n      \"method\": \"Recombinant protein production in E. coli, in vitro kinase assay, autophosphorylation assay, MBP phosphorylation, TNF apoptosis assay\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted in vitro kinase assay with recombinant proteins and active-site mutagenesis plus cellular functional validation\",\n      \"pmids\": [\"21144847\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structure of KSR2 kinase domain in complex with MEK1 reveals interactions mediated by activation segments and C-lobe αG helices; KSR2 self-associates via a side-to-side interface (analogous to BRAF); BRAF allosterically stimulates KSR2 kinase activity by heterodimerization, and KSR2-BRAF heterodimerization increases BRAF-induced MEK phosphorylation via relay of conformational signal from BRAF to release MEK activation segment.\",\n      \"method\": \"X-ray crystallography, in vitro kinase assay, chemical genetics, mutagenesis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus in vitro kinase assay plus mutagenesis plus chemical genetics; strong foundational paper\",\n      \"pmids\": [\"21441910\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Most RAF inhibitors induce KSR1 binding to wild-type and oncogenic B-RAF (including V600E) requiring direct drug binding to B-RAF and conserved dimer interface residues; KSR1 competes with C-RAF for inhibitor-induced B-RAF binding, attenuating the paradoxical activating effect of RAF inhibitors on ERK signaling.\",\n      \"method\": \"Co-immunoprecipitation, dimer interface mutagenesis, ERK activation assays in cancer cell lines\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP with mutagenesis plus functional ERK signaling readouts; multiple cell lines tested\",\n      \"pmids\": [\"21458265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Feedback phosphorylation of KSR1 by ERK1/2 reduces KSR1 localization to dendritic spines; expression of feedback-deficient KSR1 promotes sustained ERK1/2 activation and potentiates excitatory postsynaptic currents in hippocampal neurons.\",\n      \"method\": \"Immunocytochemistry, confocal imaging, electrophysiological recordings, Western blot, mutagenesis\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct imaging plus electrophysiology plus mutant analysis; single lab\",\n      \"pmids\": [\"21471251\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"KSR1 contains a CC-SAM (coiled coil-sterile α motif) domain that targets KSR1 to specific signaling sites at the plasma membrane in growth factor-treated cells; membrane binding is mediated by helix α3 of the CC motif, and mutation of α3 residues abolishes plasma membrane targeting.\",\n      \"method\": \"NMR spectroscopy, mutagenesis, confocal microscopy, in vitro micelle/bicelle binding\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — NMR structure plus mutagenesis plus live cell imaging; multiple orthogonal methods\",\n      \"pmids\": [\"23250398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"VRK2A anchors KSR1-MEK1 complexes to the endoplasmic reticulum (ER); VRK2A and KSR1 interact via their respective C-terminal regions, MEK1 independently contacts both KSR1 (CA5 region) and VRK2A N-terminus, forming a high molecular size complex (600-1000 kDa) that prevents ERK1/2 incorporation after EGF stimulation.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, VRK2A knockdown, EGF stimulation assays\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus knockdown plus fractionation; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"22752157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"AMPK phosphorylates BRAF at Ser729, promoting 14-3-3 binding and disrupting the BRAF-KSR1 interaction, leading to attenuation of MEK-ERK signaling and impaired keratinocyte proliferation.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, mutagenesis, cell proliferation assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay plus co-IP plus mutagenesis plus cellular functional readouts\",\n      \"pmids\": [\"24095280\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"KSR1 interacts with caveolin-1 and redistributes MEK and ERK to caveolin-1-rich fractions; a KSR1 mutant unable to interact with caveolin-1 fails to efficiently mediate growth factor-induced ERK activation at early stages, and abolishing the KSR1-caveolin-1 interaction impairs H-Ras(V12)-induced senescence and transformation.\",\n      \"method\": \"Co-immunoprecipitation, sucrose gradient fractionation, KSR1 mutagenesis, KSR1-/- MEF rescue, transformation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus fractionation plus mutant analysis; single lab\",\n      \"pmids\": [\"25002533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GEF-H1 acts as an adaptor linking PP2A B' subunits to KSR1 (independent of its RhoGEF activity), mediating dephosphorylation of KSR1 Ser392 and activation of MAPK signaling in a positive feedback loop for oncogenic RAS.\",\n      \"method\": \"Co-immunoprecipitation, in vitro phosphatase assay, knockdown/overexpression, xenograft assays\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus phosphatase assay plus functional cancer cell readouts; single lab\",\n      \"pmids\": [\"24525234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Small molecules (exemplified by APS-2-79) stabilize a previously unrecognized inactive state of KSR guided by KSR mutations that suppress oncogenic Ras signaling; these compounds antagonize RAF heterodimerization and conformational changes required for phosphorylation of KSR-bound MEK, and enhance MEK inhibitor potency in Ras-mutant cells.\",\n      \"method\": \"Structure-guided drug design based on KSR mutations, biochemical RAF dimerization assays, MEK phosphorylation assays, cell viability assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — structure-guided compounds plus biochemical mechanism assays plus cancer cell functional validation\",\n      \"pmids\": [\"27556948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Praja2 is the E3 ubiquitin ligase that ubiquitylates KSR1, inducing its polyubiquitination and proteasomal degradation in response to receptor stimulation, thereby attenuating ERK1/2 signaling.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, proteasome inhibition (MG132), knockdown/overexpression, stem cell pluripotency assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ubiquitination assay plus co-IP plus functional rescue; single lab\",\n      \"pmids\": [\"27195677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PKA phosphorylation of Rap1 (Ser180/Ser179) creates a 14-3-3 binding site that links Rap1 to the scaffold protein KSR1; because KSR1 and B-Raf exist as heterodimers, this also brings B-Raf to Rap1 to sustain ERK activation by cAMP.\",\n      \"method\": \"Co-immunoprecipitation, in vitro phosphorylation assay, mutagenesis, ERK activation assay in HEK293 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus in vitro phosphorylation plus mutagenesis; single lab\",\n      \"pmids\": [\"28003362\"],\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 endosomal compartments (perinuclear signaling complexes, PSCs); this requires endocytosis, MEK-ERK and CK2 kinase activities, and the presence of KSR1. KSR1-positive PSCs provide a platform for phosphorylation of the transcription factor C/EBPbeta.\",\n      \"method\": \"Confocal microscopy, Rab11 co-localization, MEK/CK2 inhibitors, KSR1 knockdown, C/EBPbeta phosphorylation assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct imaging plus pharmacological inhibition plus KSR1 requirement; single lab\",\n      \"pmids\": [\"29259016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MEK binding to the KSR1 kinase domain asymmetrically drives BRAF-KSR1 heterodimerization; KSR1 selective heterodimerization with BRAF is specified by direct contacts between N-terminal regulatory regions including the BRS domain in BRAF and the CC-SAM domain in KSR1; KSR-MEK complexes allosterically activate BRAF through N-terminal and kinase domain contacts.\",\n      \"method\": \"Crystal structure, biochemical dimerization assays, co-immunoprecipitation, in vitro kinase assay, domain mutagenesis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus biochemical reconstitution plus mutagenesis; foundational structural/mechanistic paper\",\n      \"pmids\": [\"29433126\"],\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; loss of Erbin increases amplitude and duration of RAS/RAF signaling via KSR1.\",\n      \"method\": \"Co-immunoprecipitation, KSR1 pulldown, knockdown, signaling assays, in vivo APC-KO mouse model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus functional signaling assay plus in vivo validation; single lab\",\n      \"pmids\": [\"29980571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"X-ray crystal structures of MEK bound to KSR with MEK inhibitors including trametinib reveal that trametinib directly engages KSR at the MEK interface; KSR remodels the allosteric pocket of MEK, affecting drug binding and residence time; trametinib binding to KSR-MEK disrupts the related RAF-MEK complex through conserved interface residues.\",\n      \"method\": \"X-ray crystallography of KSR-MEK-inhibitor complexes, kinetic drug binding assays, mutagenesis, cell-based signaling assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple X-ray crystal structures with functional biochemical and cellular validation; high-impact structural study\",\n      \"pmids\": [\"32927473\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Calmodulin binds directly to KSR1 in a Ca2+-dependent manner (in vitro and in cells); calmodulin antagonism impairs EGF-induced KSR1 translocation to the plasma membrane and reduces KSR1-ERK complex formation, thereby suppressing ERK activation.\",\n      \"method\": \"In vitro binding with purified proteins, co-immunoprecipitation, confocal microscopy, calmodulin inhibitor treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro reconstitution plus co-IP plus live-cell imaging; single lab\",\n      \"pmids\": [\"33766558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"KSR1 promotes epithelial-to-mesenchymal transition via preferential translation of EPSTI1 mRNA; KSR1-dependent induction of EPSTI1 drives the E- to N-cadherin switch and migratory/invasive behavior in colorectal cancer cells through ZEB1 and Slug transcriptional repressors.\",\n      \"method\": \"KSR1 disruption, ectopic EPSTI1 expression, migration/invasion assays, Western blot, polysome profiling\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO plus ectopic rescue plus mechanistic pathway placement; single lab\",\n      \"pmids\": [\"33970103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PJA2 (praja2) ubiquitylates KSR1, reducing KSR1 protein stability; KDM5A inhibits PJA2 expression by removing H3K4me3 from the PJA2 promoter, thereby indirectly stabilizing KSR1 and promoting MAPK signaling and macrophage M2 polarization in gastric cancer.\",\n      \"method\": \"Dual luciferase reporter, ChIP assay, co-immunoprecipitation, cycloheximide chase, ubiquitination assay\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods establishing the KDM5A-PJA2-KSR1 axis; single lab\",\n      \"pmids\": [\"34372882\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"KSR1 ectopic expression activates the MAPK pathway and induces cell proliferation in the absence of RAS proteins but requires at least one RAF family member for dimerization and stimulation; KSR1 promotes translocation of heterodimerized RAF to the cell membrane; mutations in the DFG motif of KSR1 affecting ATP binding impair proliferation induction.\",\n      \"method\": \"RAS-null cell system, RAF/MEK knockout cells, KSR1 dimerization-deficient mutants, ATP-binding mutants, cell proliferation assays\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic perturbations plus mutagenesis; single lab\",\n      \"pmids\": [\"35313064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SHP2 interacts with KSR1 through its protein tyrosine phosphatase domain; activated SHP2 during adaptive MEK inhibitor resistance impairs SHP2-KSR1 interaction, activating KSR1 to promote MAPK signaling; a KSR1 activating mutation (S269A) diminishes the synergistic anti-proliferative effect of SHP2 inhibitor and MEK inhibitor combination.\",\n      \"method\": \"Co-immunoprecipitation, KSR1 knockdown, KSR1 activating mutation, ERK signaling assays, xenograft assays\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus mutagenesis plus functional signaling assays; single lab\",\n      \"pmids\": [\"36493900\"],\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, supporting tumor cell survival alongside EPHB4-dependent elevation of Myc mRNA.\",\n      \"method\": \"KSR1 knockdown, ectopic re-expression, Western blot, mRNA quantification, survival assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — knockdown/OE with phenotype but indirect mechanism for translational regulation; single lab\",\n      \"pmids\": [\"27273865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"KSR1 regulates KSR1-dependent control of PGC1α and ERRα expression to promote metabolic capacity required for oncogenic H-Ras(V12)-induced anchorage-independent growth, independent of ERK signaling.\",\n      \"method\": \"KSR1-/- MEFs, ectopic PGC1α/ERRα expression, anchorage-independent growth assay, ERRα inhibitor treatment\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO plus ectopic rescue plus inhibitor with specific metabolic and transformation readouts; single lab\",\n      \"pmids\": [\"21518958\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"KSR1 loss in IL-10-deficient mice causes exacerbated colitis due to dysregulated IFN-γ production in T lymphocytes; KSR1 expressed in hematopoietic lineages is protective, and KSR1 deficiency increases Th1 polarization while impairing Th17 polarization.\",\n      \"method\": \"KSR1-/- IL10-/- double KO mice, bone marrow transplant, in vitro Th1/Th17 polarization assay, IFN-γ neutralizing antibody treatment\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse epistasis plus bone marrow transplant plus in vitro polarization assays; single lab\",\n      \"pmids\": [\"20875416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"KSR1 coordinates ERK and RSK activation with C/EBPbeta synthesis during adipogenesis; KSR1 deletion prevents adipogenesis in vitro, and appropriate KSR1 levels promote phosphorylation and stabilization of C/EBPbeta at a specific moment in the adipogenic program, while elevated KSR1 inhibits adipogenesis via high sustained ERK phosphorylating PPARγ.\",\n      \"method\": \"KSR1-/- cells, KSR1 re-expression at graded levels, adipocyte differentiation assay, ERK/RSK activation measurement\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO plus graded rescue with specific cell fate readouts; single lab\",\n      \"pmids\": [\"16107706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"KSR1 is required for efficient NK-mediated cytolysis and cytolytic granule polarization; KSR1 is recruited to the immunological synapse and membrane recruitment of KSR1 is required for ERK localization to the synapse.\",\n      \"method\": \"KSR1-/- mice, NK cytolysis assay, confocal imaging of immunological synapse\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mice plus direct imaging plus functional cytolysis assay; single lab\",\n      \"pmids\": [\"19139278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"KSR1 regulates BRCA1 degradation in breast cancer cells; KSR1 stabilizes BRCA1 protein levels by reducing BRCA1 ubiquitination through increasing BARD1 abundance.\",\n      \"method\": \"KSR1 overexpression, ubiquitination assay, BRCA1/BARD1 Western blot, xenograft assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — mechanistic follow-up by co-IP and ubiquitination assay but limited pathway placement; single lab\",\n      \"pmids\": [\"24909178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"KSR1 regulates FLIP protein levels post-translationally in endometrial carcinoma cells; KSR1 silencing down-regulates FLIP by promoting FADD-dependent inhibition of FLIP translation after TRAIL stimulation, sensitizing cells to TRAIL- and Fas-induced apoptosis.\",\n      \"method\": \"KSR1 shRNA knockdown, KSR1 re-expression, apoptosis assay, translational regulation assay\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — knockdown plus rescue with translational mechanism proposed; single lab\",\n      \"pmids\": [\"21435442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"KSR1 decreases DBC1 phosphorylation, reducing the DBC1-SIRT1 interaction; this enables SIRT1 to deacetylate p53, thereby inhibiting p53 transcriptional activity.\",\n      \"method\": \"SILAC mass spectrometry phosphoproteomics, co-immunoprecipitation, luciferase reporter assay, Western blot\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — SILAC proteomics plus co-IP plus reporter assay; single lab with multiple methods\",\n      \"pmids\": [\"24129246\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"DiRas3 binds to KSR1 independently of activated Ras and RAF; depending on local stoichiometry, DiRas3 can either enhance KSR1 homodimerization or recruit KSR1 to the Ras:C-RAF complex, thereby reducing C-RAF availability for B-RAF heterodimerization.\",\n      \"method\": \"Co-immunoprecipitation, dimerization assays, mutagenesis\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — co-IP plus limited functional validation; single lab\",\n      \"pmids\": [\"27368419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MARK2 (related to C-TAK1) binds and phosphorylates KSR1 on Ser392 in vivo; genetic deletion of KSR1 in MARK2-/- mice reverses the increased insulin sensitivity caused by MARK2 loss, placing KSR1 downstream of MARK2 in insulin-regulated glucose metabolism.\",\n      \"method\": \"Double KO mice (mark2-/- ksr1-/-), glucose tolerance test, co-immunoprecipitation, in vitro phosphorylation assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo epistasis (double KO) plus in vitro kinase assay plus co-IP; single lab\",\n      \"pmids\": [\"22206009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"KSR1 knockout mice are resistant to cisplatin- and noise-induced hearing loss; loss of KSR1 reduces BRAF/MEK/ERK phosphorylation cascade in cochlear cells following insults; dabrafenib (BRAF inhibitor) does not enhance protection in KO mice, placing KSR1 upstream of BRAF in cochlear MAPK injury signaling.\",\n      \"method\": \"KSR1 germline KO mice, ABR hearing testing, BRAF inhibitor treatment, cochlear MAPK phosphorylation assay\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with epistasis (dabrafenib test) plus biochemical pathway readouts; single lab\",\n      \"pmids\": [\"38548338\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"KSR1 is a conserved pseudokinase/scaffold protein that facilitates RAS-RAF-MEK-ERK signal transduction by constitutively binding MEK and translocating to the plasma membrane upon RAS activation where it assembles Raf-MEK-ERK complexes; its membrane recruitment is regulated by C-TAK1/MARK2-mediated phosphorylation of Ser392 creating a 14-3-3 binding site that sequesters KSR1 in the cytoplasm, while PP2A-mediated dephosphorylation (facilitated by GEF-H1 as adaptor) releases KSR1 to the membrane; at the membrane KSR1 allosterically activates RAF catalysis by side-to-side heterodimerization through contacts between its CC-SAM domain and the BRS region of BRAF, while simultaneously positioning the MEK activation segment for phosphorylation by a separate catalytic RAF molecule; KSR1 also exhibits intrinsic serine kinase activity capable of phosphorylating MEK1 in vitro; the scaffold is additionally regulated by CK2 (facilitating RAF N-region phosphorylation), caveolin-1 (for optimal early ERK activation), calmodulin (Ca2+-dependently), and ubiquitin-mediated degradation by praja2, and is inactivated by caspase cleavage during apoptosis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"KSR1 is a conserved pseudokinase/scaffold protein that organizes the RAS-RAF-MEK-ERK signaling cascade by constitutively binding MEK and, upon growth factor stimulation, translocating to the plasma membrane to assemble RAF-MEK-ERK complexes and regulate the intensity and duration of ERK activation [PMID:11940661, PMID:15121859, PMID:9427629]. Membrane recruitment is controlled by a phosphorylation–dephosphorylation switch: C-TAK1/MARK2-mediated phosphorylation of Ser392 creates a 14-3-3 binding site that sequesters KSR1 in the cytoplasm, while PP2A (recruited via the adaptor GEF-H1) dephosphorylates this site to license membrane translocation, with the CC-SAM domain and atypical C1 domain providing direct membrane-targeting determinants [PMID:11741534, PMID:12932319, PMID:24525234, PMID:23250398]. Crystal structures reveal that KSR1 allosterically activates BRAF through side-to-side heterodimerization—driven by CC-SAM/BRS contacts and kinase-domain dimer interfaces—positioning KSR1-bound MEK for phosphorylation by a catalytic RAF protomer, while KSR1 itself retains intrinsic serine kinase activity capable of phosphorylating MEK1 [PMID:21441910, PMID:29433126, PMID:21144847]. Beyond canonical MAPK signaling, KSR1 functions in T-cell and NK-cell activation at the immunological synapse, coordinates adipogenic and metabolic transcriptional programs, and its scaffolding is terminated by caspase cleavage during apoptosis and by praja2-mediated ubiquitin-dependent degradation [PMID:19188442, PMID:16107706, PMID:17613518, PMID:27195677].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Genetic screens in Drosophila and C. elegans independently identified KSR as a novel kinase-family gene required for RAS-mediated signal transduction, establishing that a previously unknown component acts between or in parallel to RAS and RAF.\",\n      \"evidence\": \"Forward genetic suppressor/enhancer screens with epistasis analysis in Drosophila and C. elegans vulval induction\",\n      \"pmids\": [\"8521512\", \"8521513\", \"8521514\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical activity (kinase vs. scaffold) unknown\", \"Mammalian function not yet tested\", \"Direct binding partners not identified\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Domain dissection revealed that KSR1 stimulates RAF-1 activity in a kinase-independent manner through its CA3/C1 cysteine-rich domain and translocates from cytoplasm to plasma membrane upon RAS activation, shifting the model from simple kinase to potential scaffold/allosteric activator.\",\n      \"evidence\": \"Domain mutagenesis, in vitro kinase assays, subcellular fractionation, and Xenopus oocyte maturation assays\",\n      \"pmids\": [\"9371754\", \"8946910\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nature of the 'detergent-labile cofactor' unresolved\", \"Scaffolding of downstream components not yet demonstrated\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Identification of MEK1/2 and ERK as direct KSR1-binding partners established KSR1 as a multi-component scaffold bridging RAF, MEK, and ERK, and explained how overexpression could inhibit signaling through stoichiometric titration of cascade members.\",\n      \"evidence\": \"Yeast two-hybrid and co-immunoprecipitation in COS-7 cells by two independent groups\",\n      \"pmids\": [\"9427629\", \"9427625\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the scaffold complex not determined\", \"In vivo scaffold function not yet validated\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Mass spectrometry-based phosphopeptide mapping revealed five in vivo phosphorylation sites on KSR1—two constitutive (Ser297, Ser392) mediating 14-3-3 binding and three RAS-inducible MAPK-consensus sites—demonstrating that KSR1 is itself an ERK substrate subject to feedback regulation.\",\n      \"evidence\": \"Mass spectrometry phosphopeptide identification, site-directed mutagenesis, MEK inhibitor treatment\",\n      \"pmids\": [\"9858547\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinases responsible for constitutive phosphorylation not yet identified\", \"Functional consequence of each site not fully dissected\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identification of C-TAK1 as the kinase that phosphorylates KSR1 Ser392 to create a 14-3-3 anchor site resolved how KSR1 is sequestered in the cytoplasm and how growth factor-induced dephosphorylation licenses membrane translocation.\",\n      \"evidence\": \"Co-immunoprecipitation, in vitro kinase assay, S392A mutagenesis, confocal imaging of membrane translocation\",\n      \"pmids\": [\"11741534\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphatase identity not yet known\", \"Regulation of C-TAK1 activity itself unclear\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Generation of KSR1-deficient mice proved that KSR1 functions as a bona fide scaffold in vivo: KSR1 loss attenuated ERK activation, blocked T-cell activation, and KSR1 was shown to independently bind RAF and MEK to promote their complex formation.\",\n      \"evidence\": \"KSR1 knockout mouse phenotyping, high-molecular-weight complex analysis, co-immunoprecipitation, in vitro kinase assays by two independent groups\",\n      \"pmids\": [\"11940661\", \"11850406\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of scaffold assembly unknown\", \"Relative contribution of scaffolding vs. allosteric activation unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Discovery that PP2A constitutively associates with KSR1 and dephosphorylates the 14-3-3 binding site upon growth factor signaling completed the phosphorylation–dephosphorylation switch model controlling KSR1 membrane recruitment.\",\n      \"evidence\": \"Mass spectrometry of KSR1 complex, co-immunoprecipitation, PP2A inhibition studies\",\n      \"pmids\": [\"12932319\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which PP2A regulatory subunit specifies KSR1 targeting not fully resolved\", \"Adaptor linking PP2A to KSR1 not yet identified\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"CK2 was identified as a constitutive KSR1-associated kinase that facilitates RAF N-region phosphorylation, revealing that KSR1 scaffolds not only the core ERK cascade but also an upstream activating kinase for RAF.\",\n      \"evidence\": \"Mass spectrometry, co-immunoprecipitation, C1 domain mutagenesis, CK2 inhibitor treatment\",\n      \"pmids\": [\"17174095\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of CK2–C1 domain interaction not determined\", \"Whether CK2 phosphorylates KSR1 itself not fully tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Reconstitution with bacterially produced KSR1 demonstrated intrinsic serine kinase activity including autophosphorylation and MEK1 phosphorylation, resolving the long-standing debate about whether KSR1 is a true pseudokinase or retains catalytic function.\",\n      \"evidence\": \"Recombinant KSR1 in vitro kinase assay with active-site D683A/D700A mutants, TNF-induced apoptosis rescue\",\n      \"pmids\": [\"21144847\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo substrates of KSR1 kinase activity not comprehensively identified\", \"Relative contribution of kinase vs. scaffold activity to signaling output unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"The crystal structure of KSR2–MEK1 complex revealed how KSR binds MEK through activation-segment and αG-helix contacts and showed that BRAF allosterically activates KSR through side-to-side heterodimerization, providing the first atomic-level model of scaffold-mediated kinase cascade assembly.\",\n      \"evidence\": \"X-ray crystallography, in vitro kinase assays, chemical genetics, dimer-interface mutagenesis\",\n      \"pmids\": [\"21441910\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length KSR1 structure not available\", \"Dynamic conformational changes during signaling not captured\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"NMR characterization of the CC-SAM domain showed it directly binds membranes via helix α3, identifying the structural determinant that targets KSR1 to the plasma membrane independently of the C1 domain.\",\n      \"evidence\": \"NMR spectroscopy, α3 mutagenesis, confocal imaging, in vitro micelle/bicelle binding\",\n      \"pmids\": [\"23250398\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Lipid specificity of CC-SAM binding not defined\", \"How CC-SAM and C1 domain cooperate in membrane targeting unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"GEF-H1 was identified as the adaptor that bridges PP2A B′ subunits to KSR1 for Ser392 dephosphorylation, completing the molecular wiring of the positive feedback loop by which oncogenic RAS sustains KSR1 membrane recruitment.\",\n      \"evidence\": \"Co-immunoprecipitation, in vitro phosphatase assay, knockdown, xenograft assays\",\n      \"pmids\": [\"24525234\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"GEF-H1-independent mechanisms of PP2A-KSR1 interaction not excluded\", \"In vivo validation in genetic models not performed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Structure-guided small molecules (APS-2-79) that stabilize an inactive KSR conformation demonstrated that KSR1's conformational state—not merely its presence—gates RAF heterodimerization and MEK phosphorylation, opening a pharmacological strategy to target the scaffold.\",\n      \"evidence\": \"Structure-guided compound design, RAF dimerization assays, MEK phosphorylation assays, cancer cell viability\",\n      \"pmids\": [\"27556948\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo pharmacokinetics and efficacy not reported\", \"Selectivity over KSR2 not fully characterized\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"A higher-resolution structural study revealed that MEK binding to KSR1 asymmetrically drives BRAF-KSR1 heterodimerization through both kinase-domain and N-terminal (CC-SAM/BRS) contacts, establishing the complete allosteric mechanism by which the KSR1-MEK binary complex activates BRAF catalysis.\",\n      \"evidence\": \"Crystal structures, biochemical dimerization assays, co-immunoprecipitation, domain mutagenesis\",\n      \"pmids\": [\"29433126\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics of the ternary complex in living cells not resolved\", \"How KSR1 vs. KSR2 selectivity for RAF isoforms is achieved structurally\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Crystal structures of KSR-MEK complexes with clinical MEK inhibitors (trametinib) showed that KSR remodels MEK's allosteric drug-binding pocket and that drug binding to the KSR-MEK interface disrupts RAF-MEK complexes, explaining drug mechanism and resistance phenomena.\",\n      \"evidence\": \"X-ray crystallography of KSR-MEK-inhibitor ternary complexes, kinetic binding assays, mutagenesis, cell signaling assays\",\n      \"pmids\": [\"32927473\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Impact on clinical resistance mechanisms in patients not directly demonstrated\", \"Structural effects of other MEK inhibitor classes not fully explored\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Ectopic KSR1 activates the MAPK pathway and drives proliferation even in the absence of RAS proteins, provided at least one RAF family member is present for dimerization, demonstrating that KSR1-RAF dimerization is sufficient to initiate signaling independent of canonical RAS-GTP.\",\n      \"evidence\": \"RAS-null cell system, RAF/MEK knockout cells, KSR1 dimerization and ATP-binding mutants, proliferation assays\",\n      \"pmids\": [\"35313064\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance of RAS-independent KSR1 activation unclear\", \"Whether endogenous KSR1 levels achieve this in any tissue not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A full-length structure of KSR1 in its autoinhibited and active states, the precise in vivo substrates of KSR1's intrinsic kinase activity, and the quantitative contribution of kinase versus scaffold functions to ERK signaling output remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length KSR1 structure available\", \"In vivo kinase substrates beyond MEK1 not identified\", \"Kinase-dead knock-in mouse not reported to separate catalytic from scaffold roles\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [25, 26, 44]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [5, 6, 11, 16, 38]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [12, 29]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 3, 10, 29, 41]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [10, 21]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [37]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [30]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 5, 11, 16, 26, 38]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [11, 23, 48, 50]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [20, 52]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [15, 24]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [27, 34, 42]}\n    ],\n    \"complexes\": [\n      \"KSR1-MEK1/2 scaffold complex\",\n      \"KSR1-BRAF heterodimer\",\n      \"KSR1-PP2A complex\",\n      \"KSR1-CK2 complex\"\n    ],\n    \"partners\": [\n      \"BRAF\",\n      \"RAF1\",\n      \"MAP2K1\",\n      \"MAPK1\",\n      \"CSNK2A1\",\n      \"PPP2CA\",\n      \"YWHAG\",\n      \"MARK2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}