{"gene":"RAF1","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":1992,"finding":"Raf-1 directly phosphorylates and activates MAP kinase-kinase (MAPKK/MEK) at serine/threonine residues in vitro, establishing MAPKK as the first identified physiological substrate of c-Raf-1 and placing Raf-1 as the immediate upstream activator of MAPKK in vivo.","method":"In vitro kinase assay with purified c-Raf-1 and partially purified MAPKK; phosphatase 2A inactivation/reactivation assay; v-raf-transformed cell analysis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified proteins, replicated across transformed cell and in vitro systems, foundational result confirmed by multiple subsequent studies","pmids":["1322500"],"is_preprint":false},{"year":1993,"finding":"Activated Ras-GTP (but not effector domain mutant Ras-Ile36Ala) specifically binds Raf-1 and is required for formation of complexes containing MAPKK activity, demonstrating that Ras-GTP recruits Raf-1 and MAPKK into a signaling complex in a GTP- and effector-domain-dependent manner.","method":"Affinity pulldown using immobilized Ras variants (wild-type, G12V, GMP-PNP-loaded, I36A effector mutant) with cell lysates; direct MAPKK activity assays on Ras-bound complexes","journal":"Science","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — biochemical reconstitution with multiple Ras mutants, replicated and confirmed by numerous subsequent studies","pmids":["8503013"],"is_preprint":false},{"year":1993,"finding":"PKC-alpha directly phosphorylates and activates Raf-1 both in vitro and in vivo, including at Ser499; mutations at Ser499 or Ser259 block PKC-alpha-mediated (but not Ras+Lck-mediated) Raf-1 activation, demonstrating a direct PKC-alpha→Raf-1 activation mechanism distinct from Ras-dependent activation.","method":"In vitro phosphorylation assay with purified PKC-alpha and Raf-1; site-directed mutagenesis of Ser499 and Ser259; in vivo activation assays in NIH3T3 cells; transformation cooperation assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with purified proteins plus mutagenesis plus in vivo validation, multiple orthogonal methods","pmids":["8321321"],"is_preprint":false},{"year":1994,"finding":"14-3-3 zeta and 14-3-3 beta proteins bind the amino-terminal regulatory region of Raf-1 (identified by yeast two-hybrid), and expression of 14-3-3 proteins in Xenopus oocytes enhances Raf-1 activity and promotes Raf-1-dependent oocyte maturation; dominant-negative Raf-1 blocks these effects.","method":"Yeast two-hybrid screen; Xenopus oocyte functional assay; dominant-negative Raf-1 epistasis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid identification plus functional validation in Xenopus oocytes, replicated by multiple subsequent studies","pmids":["7935795"],"is_preprint":false},{"year":1994,"finding":"PKA inhibits Raf-1 by direct phosphorylation of the Raf-1 kinase domain, independently of weakening Raf-1/Ras interaction; PKA phosphorylation can downregulate Raf-1 kinase activity even after prior activation by PKC-alpha or amino-terminal truncation, and the isolated kinase domain lacking the Ras-binding domain is still susceptible.","method":"In vitro phosphorylation assays with purified PKA and Raf-1 proteins; kinase domain fragment analysis; sequential activation/inhibition assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified proteins, multiple mechanistic controls (Ras-binding domain deletion, sequential activation experiments), single lab","pmids":["7935389"],"is_preprint":false},{"year":1994,"finding":"Raf-1 forms a specific signaling complex with Ras and MEK-1 but not MEK-2; MEK-1 binding to Ras requires RAF-1 as a bridge, and a proline-rich region of MEK-1 containing a phosphorylation site is essential for complex formation.","method":"Immobilized Ras pulldown from NIH 3T3 cell lysates; MEK-1 and MEK-2 immunodetection; exogenous RAF-1 addition to lysates; MEK-1 mutant analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — biochemical pulldown with multiple controls, specificity for MEK-1 over MEK-2 demonstrated, single lab with orthogonal methods","pmids":["7969158"],"is_preprint":false},{"year":1994,"finding":"Raf-1 associates with Fyn and Src SH2 domains in a serine-phosphorylation-dependent (not tyrosine-phosphorylation-dependent) manner; co-expression of Raf-1 with full-length Fyn/Src results in co-immunoprecipitation, tyrosine phosphorylation of Raf-1, and stimulation of Raf-1 kinase activity.","method":"Co-immunoprecipitation; SH2 domain binding assay; baculovirus/Sf9 co-expression; site-directed mutagenesis of Src SH2 Arg175; kinase activity assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and baculovirus co-expression with multiple domain mutants, single lab","pmids":["7517401"],"is_preprint":false},{"year":1994,"finding":"Enzymatic characterization of c-Raf-1 shows Km for ATP of 11.6 µM and for MAPKK of 0.8 µM; c-Raf-1 has highly restricted substrate specificity, with MAPKK as the preferred substrate; active c-Raf-1 elutes as a multimeric complex (>150 kDa) on gel filtration.","method":"In vitro kinase assay with purified baculovirus-expressed His-tagged c-Raf-1; Km determination; substrate panel screening; gel-filtration chromatography","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — rigorous in vitro enzyme characterization with purified proteins, multiple substrate specificity controls, single lab","pmids":["8108400"],"is_preprint":false},{"year":1995,"finding":"Raf-1 activation requires its Ras-binding domain (residues 53-132), active kinase function, tyrosine phosphorylation at Y340/Y341, constitutive serine phosphorylation at S621, and an intact zinc finger (C165/C168); S259A mutation reduces but does not abolish activation efficiency; the zinc finger is not required for Ras binding itself.","method":"In vitro activation assay using purified plasma membranes from transformed cells; panel of Raf-1 point and deletion mutants expressed in baculovirus; kinase assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution in vitro with purified membranes and extensive mutational analysis, single lab","pmids":["7623807"],"is_preprint":false},{"year":1995,"finding":"Protein phosphatases (both serine/threonine and tyrosine phosphatases) inactivate purified Raf-1; 14-3-3 zeta or HSP90 block phosphatase-mediated inactivation; GTP-loading of plasma membranes from transformed cells inactivates Raf-1 via phosphatases present in the membrane, suggesting membrane-localized phosphatases regulate Raf-1.","method":"In vitro phosphatase treatment of purified Raf-1 (from Sf9 cells co-expressing Ras and Src-Y527F); GTP-loading of plasma membranes; phosphatase inhibitor controls","journal":"Science","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified protein and membranes, single lab","pmids":["7604263"],"is_preprint":false},{"year":1996,"finding":"Bcl-2 targets Raf-1 kinase to mitochondria; mitochondria-targeted active Raf-1 protects cells from apoptosis and phosphorylates BAD, whereas plasma membrane-targeted Raf-1 phosphorylates ERK-1/2 but does not protect from apoptosis; kinase-inactive Raf-1 abrogates Bcl-2-mediated apoptosis suppression.","method":"GFP-Raf-1 fusion protein localization; mitochondrial and plasma membrane targeting constructs; BAD phosphorylation assay; cell death assays; kinase-dead Raf-1 mutant","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct subcellular targeting experiments with functional readouts, multiple targeting constructs and kinase-dead controls, foundational result replicated in subsequent studies","pmids":["8929532"],"is_preprint":false},{"year":1996,"finding":"BAG-1 specifically binds to and activates Raf-1 kinase; bacterially produced BAG-1 increases Raf-1 kinase activity in vitro; BAG-1 and Raf-1 co-immunoprecipitate from mammalian and insect cells.","method":"Co-immunoprecipitation from mammalian cells and baculovirus-infected insect cells; in vitro kinase activation assay with bacterially produced BAG-1; yeast two-hybrid","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, in vitro activation with bacterially produced protein, and yeast two-hybrid, single lab","pmids":["8692945"],"is_preprint":false},{"year":1996,"finding":"Coumermycin-induced dimerization of a modified Raf-1 (fused to gyrase B) is sufficient to activate Raf-1 and stimulate the MAP kinase cascade in the absence of membrane components, indicating that Raf oligomerization per se promotes activation.","method":"Chemical dimerization (coumermycin/gyrase B fusion); MAP kinase cascade activation assay in cells; absence-of-membrane-component controls","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — chemical-genetic dimerization system with clean controls, replicated by companion paper (PMID 8774885)","pmids":["8774884"],"is_preprint":false},{"year":1996,"finding":"FK506-induced oligomerization of FKBP12-Raf-1 activates Raf kinase activity in a Ras-GTP-dependent manner, demonstrating that oligomerization promotes Raf activation through a Ras-dependent mechanism.","method":"Chemical dimerization (FKBP12-FK1012A system); Raf kinase activity assay; dominant-negative Ras epistasis to show Ras dependence","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — chemical-genetic oligomerization system with Ras epistasis controls, companion to PMID 8774884","pmids":["8774885"],"is_preprint":false},{"year":1996,"finding":"14-3-3 zeta binds bivalently to both the amino- and carboxy-termini of c-Raf-1; activated Ras displaces 14-3-3 zeta specifically from the N-terminal site; S259A mutation in the N-terminal domain prevents 14-3-3 binding at that site; only unphosphorylated 14-3-3 zeta binds the N-terminus of Raf-1.","method":"In vivo and in vitro binding assays; mutant Raf-1 fragments; co-expression of activated Ras","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo binding with multiple mutants, single lab","pmids":["8637718"],"is_preprint":false},{"year":1997,"finding":"The 14-3-3 zeta amphipathic groove (Lys49 being critical) mediates binding to Raf-1; the K49E mutation dramatically disrupts 14-3-3 zeta/Raf-1 interaction; this same site is used to bind exoenzyme S, indicating a common structural binding determinant.","method":"Crystal structure of 14-3-3 zeta; charge-reversal mutagenesis (K49E, R56E, R60E); in vitro binding assays; circular dichroism; partial proteolysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure-guided mutagenesis with binding assays and structural validation, single lab","pmids":["9153224"],"is_preprint":false},{"year":1998,"finding":"Autoinhibition mediated by the N-terminal regulatory region of Raf-1 (involving the cysteine-rich domain) suppresses kinase activity; disruption of this autoinhibition by cysteine-rich domain mutation or by Y340D phosphomimetic mutation increases Raf-1 activity, demonstrating an intramolecular repression mechanism.","method":"Site-directed mutagenesis of cysteine-rich domain and Y340D; kinase activity assays; regulatory domain co-expression inhibition experiments","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with kinase activity readout, single lab, moderate follow-up","pmids":["9689060"],"is_preprint":false},{"year":1998,"finding":"PAK3 phosphorylates Raf-1 on Ser338 both in vitro and in vivo, and this phosphorylation positively regulates Raf-1 activity; PAK3 is regulated by Rho-family GTPases Rac and Cdc42, linking these pathways to Raf-1 activation.","method":"In vitro kinase assay (PAK3 phosphorylating Raf-1); in vivo phosphorylation assays; phospho-specific antibodies","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay plus in vivo phosphorylation, replicated by subsequent PAK1 and other PAK studies","pmids":["9823899"],"is_preprint":false},{"year":1999,"finding":"RKIP (Raf kinase inhibitor protein) binds Raf-1, MEK, and ERK in vitro, co-immunoprecipitates with Raf-1 and MEK from cell lysates, and competitively disrupts the Raf-1/MEK interaction without being a substrate; RKIP overexpression inhibits MEK/ERK activation and AP-1-dependent transcription; RKIP downregulation activates MEK/ERK signaling.","method":"Yeast two-hybrid screen; in vitro binding assays; co-immunoprecipitation; confocal microscopy colocalization; antisense RNA and antibody microinjection; reporter gene assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (co-IP, in vitro binding, loss-of-function, gain-of-function), replicated by subsequent studies","pmids":["10490027"],"is_preprint":false},{"year":1999,"finding":"Activated Raf-1 is phosphorylated on both S338 (by PAK pathway, Ras-dependent) and Y341 (by Src); phosphorylation at both sites is required for full Raf-1 activation; Ras-GTP binding is required for both phosphorylation events to occur, likely at the plasma membrane; B-Raf differs in having constitutive S445 phosphorylation not regulated by Ras.","method":"Phospho-specific antisera; co-expression of oncogenic Ras and activated Src; mutagenesis of S338, S339, Y340, Y341; kinase activity assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — phospho-specific antibodies combined with systematic mutagenesis and co-expression experiments, multiple orthogonal methods","pmids":["10205168"],"is_preprint":false},{"year":1999,"finding":"Phosphatidylserine (inner plasma membrane phospholipid) displaces 14-3-3 from Raf-1 and increases Raf-1 kinase activity; 14-3-3 removal from activated Raf-1 by phosphopeptides eradicates kinase activity of soluble Raf-1, indicating 14-3-3 maintains Raf-1 activity once activated.","method":"In vitro incubation of Raf-1 with phosphatidylserine; phosphopeptide competition assays; kinase activity measurements","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution experiments with multiple conditions, single lab","pmids":["10445849"],"is_preprint":false},{"year":2000,"finding":"MEKK1 binds endogenous ERK2, MEK1, and Raf-1, suggesting it can assemble all three proteins of the ERK2 MAP kinase module into a complex.","method":"Co-immunoprecipitation of endogenous proteins","journal":"The Journal of biological chemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP experiment, no functional validation of the complex, single lab","pmids":["10969079"],"is_preprint":false},{"year":2000,"finding":"Raf-1 associates with vimentin via GST-Raf-1 pulldown; vimentin is not a direct Raf-1 substrate but is phosphorylated by Raf-1-associated kinases including casein kinase 2; Raf-1 activation status correlates with vimentin phosphorylation; selective Raf-1 activation induces vimentin network rearrangement independently of MEK/ERK.","method":"GST-Raf-1 pulldown; co-immunoprecipitation; in vitro kinase assays; MEK inhibitor controls; conditional estrogen-regulated Raf-1 mutant system","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pulldown, co-IP, kinase assays, and MEK-independent functional readout, single lab","pmids":["11023985"],"is_preprint":false},{"year":2001,"finding":"Ser259 dephosphorylation by PP1 and PP2A is a critical early step in Ras-dependent Raf-1 activation; serine phosphatase inhibition blocks S259 dephosphorylation and prevents Raf-1 activation; S259A Raf-1 mutant is relatively resistant to phosphatase inhibitors and is constitutively membrane-associated.","method":"In vitro Raf-1 activation assay with serine phosphatase inhibitors; S259A Raf-1 mutant; sucrose gradient fractionation of plasma membrane microdomains","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro activation system with pharmacological and genetic tools, single lab","pmids":["11494123"],"is_preprint":false},{"year":2001,"finding":"Mitogens stimulate Raf-1 S259 dephosphorylation concomitant with Raf-1 membrane accumulation and activation; blocking S259 dephosphorylation inhibits membrane recruitment and activation; S259A mutant is constitutively membrane-localized; membrane-tethered Raf-1-CAAX is activated independently of S259 dephosphorylation, placing S259 dephosphorylation upstream of membrane recruitment.","method":"Phospho-S259 antibody; pharmacological phosphatase inhibition; S259A and Raf-1-CAAX constructs; cell fractionation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic and pharmacological tools with clear epistasis, independently confirmed by PMID 11494123","pmids":["11756411"],"is_preprint":false},{"year":2001,"finding":"Active PAK1 directly associates with Raf-1 under physiological conditions; active PAK (T423E or N-terminal truncation) binds Raf-1 more strongly than wild-type; kinase-dead PAK barely binds Raf-1; extent of PAK-Raf-1 binding correlates with Raf-1 S338 phosphorylation and MAPK activation; the Raf-1 binding site maps to the C-terminus of the PAK catalytic domain.","method":"Co-immunoprecipitation under physiological conditions; PAK mutant analysis; in vitro phosphorylation of Raf-1 S338; MAPK activation assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with multiple mutants and correlated kinase activity, single lab","pmids":["11733498"],"is_preprint":false},{"year":2001,"finding":"Raf-1 MEK kinase activity (assessed via Y340F/Y341F knock-in mutation abolishing Raf-1 kinase activity toward MEK) is not essential for normal mouse development or ERK activation; however, Raf-1 knockout causes embryonic lethality with vascular defects and increased apoptosis, and ERK activation is normal in both knockout and kinase-dead knock-in cells, revealing a kinase-independent essential function.","method":"Gene targeting (knockout and Y340F/Y341F knock-in mice); in vitro MEK kinase assay; embryonic phenotype analysis; ERK activation assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — rigorous gene targeting with two independent alleles (null and kinase-dead knock-in), clean genetic separation of kinase-dependent vs. kinase-independent functions","pmids":["11296227"],"is_preprint":false},{"year":2002,"finding":"PKA phosphorylates Raf-1 on S43, S259, and S621 in vitro and in vivo; S259 phosphorylation is the main mechanism of PKA-mediated Raf-1 inhibition (S259A mutant largely resistant to PKA inhibition); PKA also reduces S338 phosphorylation of Raf-1 in a S259-dependent manner.","method":"In vitro PKA phosphorylation mapping; in vivo cAMP stimulation; S259A, S43A mutants; ERK activation assays; cAMP kinetics correlated with ERK deactivation","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — phosphorylation site mapping in vitro and in vivo, multiple site mutants with clear functional phenotypes, single lab","pmids":["11971957"],"is_preprint":false},{"year":2002,"finding":"Ser259 dephosphorylation is an essential step in Raf-1 activation; phospho-Ser259 Raf-1 is refractory to mitogenic stimulation; S259A mutation elevates kinase activity by enhancing Ras binding and constitutive membrane recruitment, which facilitates S338 phosphorylation; S259A also improves functional coupling to MEK.","method":"Phospho-S259 antibody; S259A Raf-1 mutant; Ras binding assays; membrane recruitment assays; MEK activation assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal assays with mechanistic mutants, independently confirmed by companion studies","pmids":["11782426"],"is_preprint":false},{"year":2003,"finding":"PKC-dependent phosphorylation of RKIP on Ser153 causes RKIP to dissociate from Raf-1 and instead associate with GRK-2, thereby simultaneously relieving Raf-1 inhibition and blocking GPCR internalization; this switch mechanism was demonstrated in cardiomyocytes.","method":"Co-immunoprecipitation; RKIP S153 phosphorylation analysis; GRK-2 binding assays; cardiomyocyte functional assays; GPCR signaling readouts","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — mechanistic switch demonstrated with co-IP, mutagenesis, and physiological readout in cardiomyocytes, single lab but multiple orthogonal methods","pmids":["14654844"],"is_preprint":false},{"year":2004,"finding":"Raf-1 associates directly with Rok-alpha (a Rho-effector kinase); Raf-1-deficient keratinocytes and fibroblasts show cortical actin bundles, disordered vimentin cytoskeleton, and impaired migration due to hyperactivity and incorrect plasma membrane localization of Rok-alpha; reintroduction of either wild-type or kinase-dead Raf-1 rescues cell shape and migration defects, demonstrating a kinase-independent spatial regulatory role.","method":"Conditional Raf-1 gene ablation; cell migration assays; actin/vimentin cytoskeleton imaging; Rok-alpha localization and activity assays; rescue with kinase-dead Raf-1","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout with kinase-dead rescue, multiple cellular phenotype readouts, orthogonal imaging and biochemical analyses","pmids":["15753127"],"is_preprint":false},{"year":2004,"finding":"Cardiac-specific Raf-1 knockout causes left ventricular systolic dysfunction and cardiomyocyte apoptosis without affecting MEK/ERK activation; instead, ASK1, JNK, and p38 kinase activities are elevated; ablation of ASK1 rescues the cardiac phenotype, placing Raf-1 upstream of ASK1 suppression in a MEK/ERK-independent survival pathway.","method":"Cre-loxP cardiac-specific knockout; echocardiography; kinase activity assays (MEK, ERK, ASK1, JNK, p38); ASK1 double-knockout rescue","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — tissue-specific knockout with genetic rescue and clear epistasis establishing Raf-1→ASK1 suppression independent of MEK/ERK","pmids":["15467832"],"is_preprint":false},{"year":2004,"finding":"Raf-1 associates directly with K8 (keratin 8) independently of Raf-1 kinase activity or Ras-Raf interaction; K18 is a physiological Raf-1 substrate; Raf-1 activation during oxidative/toxin stress disrupts keratin-Raf association in a phosphorylation-dependent manner; 14-3-3 residues essential for Raf-1 binding also regulate keratin association.","method":"Co-immunoprecipitation; kinase-dead and Ras-binding-defective Raf-1 mutants; in vivo and in vitro phosphorylation assays; 14-3-3 binding-site mutants","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with multiple Raf-1 mutants and substrate phosphorylation assay, single lab","pmids":["15314064"],"is_preprint":false},{"year":2005,"finding":"ERK-mediated feedback phosphorylation at six proline-directed sites (five are ERK targets) in Raf-1 following mitogen stimulation inhibits the Ras/Raf-1 interaction and desensitizes Raf-1 to further stimuli; dephosphorylation by PP2A and prolyl isomerization by Pin1 return Raf-1 to a signaling-competent state.","method":"Mass spectrometry-based phosphorylation site identification; MEK inhibitor treatments; in vitro phosphorylation by ERK; Ras-Raf binding assays; PP2A and Pin1 co-immunoprecipitation and functional assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — mass spectrometry site identification combined with in vitro kinase assays, binding assays, and phosphatase/isomerase functional validation, multiple orthogonal methods","pmids":["15664191"],"is_preprint":false},{"year":2005,"finding":"Wild-type B-Raf forms a complex with C-Raf in a Ras-dependent manner, whereas kinase-impaired B-Raf mutants bind C-Raf independently of Ras; B-Raf activates C-Raf through a mechanism involving 14-3-3-mediated hetero-oligomerization and C-Raf transphosphorylation; C-Raf activation segment phosphorylation and 14-3-3 binding to C-Raf are required.","method":"Co-immunoprecipitation; Ras-dependence assays; kinase activity assays; 14-3-3 binding analysis; activation segment phosphorylation assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple B-Raf mutants, co-IP, and mechanistic dissection with multiple orthogonal experiments, single lab but comprehensive","pmids":["16364920"],"is_preprint":false},{"year":2005,"finding":"RKIP inhibits Raf-1 by preventing PAK and Src family kinase phosphorylation of Raf-1 kinase domain (acting after membrane recruitment); phosphomimetic mutations at PAK and Src phosphorylation sites on Raf-1 prevent RKIP association; RKIP has no effect on B-Raf activation despite binding B-Raf.","method":"RKIP overexpression and depletion; Raf-1 phosphomimetic mutants; PAK and Src kinase co-IP; MEK/ERK and DNA synthesis assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple phosphomimetic mutants and RKIP depletion, single lab","pmids":["15886202"],"is_preprint":false},{"year":2005,"finding":"Raf-1 is required for wound healing in vivo and migration of keratinocytes/fibroblasts in vitro; Raf-1 physically associates with Rok-alpha; Raf-1 loss causes Rok-alpha hyperactivity and mislocalization; these phenotypes are rescued by kinase-dead Raf-1, establishing a kinase-independent function as a spatial regulator of Rho-Rok-alpha signaling.","method":"Conditional gene ablation; wound healing assay; in vitro cell migration; actin/vimentin cytoskeleton analysis; Rok-alpha localization; kinase-dead rescue","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout with kinase-dead rescue and multiple cellular readouts (same paper as PMID 15753127)","pmids":["15753127"],"is_preprint":false},{"year":2005,"finding":"Raf-1 controls the proapoptotic kinase MST2 by preventing its dimerization and recruiting a phosphatase that removes activating phosphorylations; both functions require Raf-1 binding to MST2 and are independent of Raf-1 kinase activity and the ERK pathway; MST2 siRNA reverts apoptosis hypersensitivity of Raf-1−/− fibroblasts.","method":"Raf-1 knockout cells; MST2 siRNA rescue; kinase-dead Raf-1 reconstitution; MST2 dimerization and phosphorylation assays; apoptosis assays","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic rescue with kinase-dead Raf-1 and MST2 siRNA, single lab; companion to the full paper establishing the Raf-1/MST2 interaction","pmids":["15701972"],"is_preprint":false},{"year":2005,"finding":"CNK1 mediates Src-dependent tyrosine phosphorylation and activation of Raf-1 by forming a trimeric complex with preactivated Raf-1 and activated Src; CNK1 regulates Raf-1 activation in a concentration-dependent manner typical of a scaffold protein; CNK1 knockdown by siRNA interferes with Src-dependent ERK activation.","method":"Co-immunoprecipitation; CNK1 siRNA knockdown; ERK activation assays; scaffold dose-response analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and siRNA with functional readout, single lab","pmids":["15845549"],"is_preprint":false},{"year":2005,"finding":"Raf-1 contains an N-terminal autoinhibitory domain; interaction of this domain with the catalytic domain is blocked by active H-Ras binding; Raf-1 and B-Raf use distinct autoregulatory mechanisms—Raf-1 requires regulated S338 phosphorylation while B-Raf has constitutive S445 phosphorylation.","method":"Co-immunoprecipitation of regulatory and catalytic domains; kinase activity assays; mutagenesis of S338/S445; active H-Ras co-expression","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain co-IP and mutagenesis with kinase activity readouts, single lab","pmids":["15710605"],"is_preprint":false},{"year":2005,"finding":"HCV NS5A binds to the C-terminal domain of NS5A and associates with Raf-1, colocalizing with Raf-1 in the HCV replication complex; NS5A-Raf-1 interaction increases Raf-1 phosphorylation at S338; Raf-1 inhibition by BAY43-9006 or siRNA knockdown attenuates HCV replication.","method":"Co-immunoprecipitation; confocal colocalization; phospho-S338 assay; small molecule and siRNA inhibition of Raf-1; viral replication assay","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, colocalization, and functional knockdown, single lab","pmids":["16405965"],"is_preprint":false},{"year":2008,"finding":"PAK5 directly associates with Raf-1 (but not A-Raf or B-Raf), phosphorylates Raf-1 at S338, activates Raf-1 kinase activity, and targets a subpopulation of Raf-1 to mitochondria.","method":"Co-immunoprecipitation; in vitro S338 phosphorylation assay; subcellular fractionation to mitochondria; kinase activity assay","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, in vitro kinase assay, and subcellular fractionation, single lab","pmids":["18465753"],"is_preprint":false},{"year":2009,"finding":"C-Raf paradoxically inhibits B-Raf(V600E) kinase activity by forming B-Raf(V600E)-C-Raf complexes; this inhibitory effect is specific to C-Raf among Raf family members; impaired C-Raf binding to B-Raf(V600E) elevates oncogenic potential; oncogenic Ras and sorafenib stabilize B-Raf(V600E)-C-Raf complexes, impairing MAPK activation.","method":"Co-immunoprecipitation; B-Raf/C-Raf interaction mutants; ERK phosphorylation assays; proliferation assays; C-Raf ectopic expression and depletion","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple co-IP experiments with binding mutants, functional proliferation and signaling readouts, single lab but comprehensive","pmids":["19917255"],"is_preprint":false},{"year":2009,"finding":"Raf-1 functions as an endogenous inhibitor of the Rho-dependent kinase Rok-alpha in the context of a Ras-induced Raf-1:Rok-alpha complex; Raf-1-induced Rok-alpha inhibition allows STAT3 phosphorylation and Myc expression, promoting dedifferentiation in Ras-induced skin tumors; this is kinase-independent.","method":"Conditional Raf-1 knockout in Ras-induced skin tumors; Rok-alpha activity and co-IP assays; STAT3 phosphorylation and Myc expression analysis; kinase-dead Raf-1 rescue","journal":"Cancer cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional tumor knockout with molecular mechanistic readouts and kinase-dead validation, single lab but comprehensive in vivo/in vitro approach","pmids":["19647225"],"is_preprint":false},{"year":2013,"finding":"PDE8A associates with Raf-1 with picomolar affinity; the PDE8A binding site on Raf-1 maps to amino acids 454-465 of PDE8A; PDE8A protects Raf-1 from PKA-mediated inhibitory phosphorylation at S259, thereby enhancing Raf-1-stimulated ERK signaling; disruption of this interaction reduces ERK activation and the cellular response to EGF.","method":"Co-immunoprecipitation; affinity measurement; peptide array mapping; cell-permeable disrupting peptide; catalytically inactive PDE8A dominant negative; PDE8A−/− mice; Drosophila PDE8 deletion","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (co-IP, peptide array, disrupting peptide, genetic knockout in mice and flies), single lab but comprehensive","pmids":["23509299"],"is_preprint":false},{"year":2014,"finding":"Raf-1 regulates both the MST2-LATS and MEK-ERK pathways through competing protein interactions; Akt phosphorylation of MST2 and LATS1 feedback phosphorylation of Raf-1 Ser259 create signaling switches; Raf-1 Ser259 mutation simultaneously drives both apoptosis (MST2) and proliferation (MEK), but concomitant MST2 downregulation switches the outcome to proliferation and transformation.","method":"Mathematical modelling combined with experimental validation; Raf-1 S259 mutant; MST2 knockdown; Akt phosphorylation assays; LATS1 kinase assays; apoptosis and proliferation readouts","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — integrated mathematical and experimental approach with multiple genetic tools and functional readouts, single lab","pmids":["24929361"],"is_preprint":false},{"year":2022,"finding":"USP7 deubiquitinates Raf-1 by binding to the PVDS motif in the CR2 region of Raf-1; USP7 decreases K6, K11, K27, K33, and K48-linked polyubiquitination of Raf-1 and reduces threonine phosphorylation of Raf-1, thereby inhibiting ERK1/2 pathway activation, G2/M transition, and cell proliferation.","method":"Co-immunoprecipitation; ubiquitination assays; USP7 DUB activity assays; phosphorylation assays; cell cycle analysis; proliferation assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, ubiquitination mapping, and functional knockdown with multiple readouts, single lab","pmids":["35948545"],"is_preprint":false},{"year":2009,"finding":"The C-terminal 14-3-3 binding site of Raf-1 (S621) acts as an activation switch; mutations preventing 14-3-3 binding at S621 render Raf-1 inactive by specifically disrupting its capacity to bind ATP; 14-3-3 proteins function as critical cofactors that maintain Raf-1 in an ATP-binding-competent conformation.","method":"S621 mutagenesis; ATP-binding assays; 14-3-3 phosphopeptide competition; MEK binding assays; in vitro kinase activity","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic mutagenesis with ATP-binding and MEK-binding dissection, single lab","pmids":["19595761"],"is_preprint":false}],"current_model":"RAF1 (C-Raf) is a serine/threonine kinase that functions as the canonical link between RAS-GTP (which recruits and promotes RAF1 oligomerization at the plasma membrane) and the MEK-ERK cascade, but also exerts essential kinase-independent functions by physically suppressing the pro-apoptotic kinases MST2 and Rok-alpha; its activity is controlled by a multi-layered phosphorylation code (activating: S338 by PAK1/3, Y341 by Src; inhibitory: S259 by PKA and LATS1, feedback sites by ERK), regulated by 14-3-3 proteins that maintain its ATP-binding-competent conformation, modulated by interacting proteins including RKIP (competitive inhibitor of MEK binding), BAG-1 and PDE8A (activators), and targeted to mitochondria by Bcl-2 where it phosphorylates BAD to suppress apoptosis."},"narrative":{"mechanistic_narrative":"RAF1 (c-Raf-1) is a serine/threonine kinase that serves as the proximal activator of the MEK-ERK cascade, directly phosphorylating and activating MEK (MAPKK) as its principal physiological substrate with highly restricted specificity [PMID:1322500, PMID:8108400]. Its activation is initiated when GTP-loaded Ras binds the RAF1 Ras-binding domain in an effector-domain-dependent manner, recruiting RAF1 and bridging it to MEK-1 to assemble a Ras-RAF1-MEK signaling complex at the plasma membrane [PMID:8503013, PMID:7969158, PMID:7623807]. Activation is gated by a multi-layered phosphorylation code: it requires dephosphorylation of the inhibitory site S259 by PP1/PP2A as an early licensing step that permits membrane recruitment, followed by activating phosphorylation at S338 (by PAK kinases, downstream of Rac/Cdc42) and at Y341 (by Src), with oligomerization itself sufficient to drive activation [PMID:11494123, PMID:11756411, PMID:9823899, PMID:10205168, PMID:8774884, PMID:8774885]. RAF1 is held in an autoinhibited conformation by its N-terminal regulatory/cysteine-rich domain, relieved upon Ras binding, and 14-3-3 proteins bind bivalently to its N- and C-termini to maintain the kinase in an ATP-binding-competent state [PMID:9689060, PMID:15710605, PMID:8637718, PMID:19595761]. The pathway is negatively regulated by PKA, which inhibits RAF1 chiefly through S259 phosphorylation, by RKIP, which competitively blocks the RAF1-MEK interaction and prevents PAK/Src phosphorylation, and by ERK-mediated feedback phosphorylation that uncouples RAF1 from Ras [PMID:11971957, PMID:10490027, PMID:15886202, PMID:15664191]. Beyond catalysis, gene-targeting in mice established an essential kinase-independent function: RAF1 promotes cell survival by physically binding and suppressing the pro-apoptotic kinases MST2 and ASK1, and by spatially restraining the Rho-effector kinase Rok-alpha to control cytoskeletal organization and migration [PMID:11296227, PMID:15701972, PMID:15467832, PMID:15753127, PMID:19647225]. RAF1 also localizes to mitochondria via Bcl-2, where it phosphorylates BAD to block apoptosis, and integrates pro-survival and pro-proliferative outputs through competing MST2-LATS and MEK-ERK interactions [PMID:8929532, PMID:24929361].","teleology":[{"year":1992,"claim":"Established RAF1's catalytic output by identifying its first physiological substrate, placing it as the direct upstream activator of the MAP kinase cascade.","evidence":"In vitro kinase assay with purified c-Raf-1 and MAPKK; v-raf-transformed cell analysis","pmids":["1322500"],"confidence":"High","gaps":["Did not define how RAF1 itself is activated","Substrate specificity beyond MAPKK not yet quantified"]},{"year":1993,"claim":"Connected RAF1 to upstream Ras, showing GTP-loaded Ras directly binds RAF1 to nucleate a signaling complex, and that PKC-alpha provides a Ras-independent activation input.","evidence":"Ras-variant affinity pulldowns with MAPKK activity assays; in vitro PKC-alpha phosphorylation and S499/S259 mutagenesis","pmids":["8503013","8321321"],"confidence":"High","gaps":["Mechanism by which Ras binding activates RAF1 catalysis not resolved","Membrane recruitment step not yet defined"]},{"year":1994,"claim":"Defined the regulatory protein and structural requirements for RAF1 activity — 14-3-3 binding, complex assembly with MEK-1, Src/Fyn association, and the architecture required for activation.","evidence":"Yeast two-hybrid and Xenopus oocyte assays; Ras-MEK1 bridging pulldowns; co-IP with Src/Fyn SH2 domains; enzymatic Km characterization","pmids":["7935795","7969158","7517401","8108400","7935389"],"confidence":"High","gaps":["Specific activating phosphosites not yet mapped","How 14-3-3 binding affects catalysis mechanistically unresolved"]},{"year":1995,"claim":"Dissected the mutational requirements for RAF1 activation and showed phosphatases and chaperone/14-3-3 dynamically regulate the active state.","evidence":"Purified-membrane reconstitution with RAF1 mutant panel; in vitro phosphatase treatment with 14-3-3/HSP90 protection","pmids":["7623807","7604263"],"confidence":"High","gaps":["Order of phosphorylation events not established","Identity of membrane phosphatases not defined"]},{"year":1996,"claim":"Demonstrated that oligomerization per se drives RAF1 activation and revealed a mitochondrial, MEK-independent pro-survival branch via BAD phosphorylation.","evidence":"Coumermycin- and FKBP12/FK506-induced dimerization systems; Bcl-2-mediated mitochondrial targeting with BAD phosphorylation and apoptosis assays; BAG-1 co-IP and in vitro activation","pmids":["8774884","8774885","8929532","8692945","8637718"],"confidence":"High","gaps":["How dimerization mechanistically activates the kinase domain not resolved","Relationship between membrane and mitochondrial pools unclear"]},{"year":1997,"claim":"Provided the structural basis for 14-3-3/RAF1 recognition by mapping the binding determinant to the 14-3-3 amphipathic groove.","evidence":"Crystal structure of 14-3-3 zeta with charge-reversal mutagenesis (K49E) and binding assays","pmids":["9153224"],"confidence":"High","gaps":["RAF1-side structural determinants not crystallized here","Functional consequence on RAF1 conformation addressed only later"]},{"year":1998,"claim":"Established intramolecular autoinhibition and identified PAK3-mediated S338 phosphorylation as a positive activating input linking Rho-family GTPases to RAF1.","evidence":"Cysteine-rich domain and Y340D mutagenesis with kinase assays; in vitro and in vivo PAK3 phosphorylation of S338","pmids":["9689060","9823899"],"confidence":"High","gaps":["How autoinhibition is relieved by Ras not fully mechanistically resolved","Which PAK isoform acts physiologically not yet settled"]},{"year":1999,"claim":"Integrated the activating phosphorylation code (S338/Y341, Ras-dependent), identified RKIP as a competitive RAF1-MEK inhibitor, and showed 14-3-3 maintains the active conformation.","evidence":"Phospho-specific antisera with Ras/Src co-expression and mutagenesis; RKIP yeast two-hybrid, co-IP, and gain/loss of function; phosphatidylserine and phosphopeptide competition assays","pmids":["10205168","10490027","10445849"],"confidence":"High","gaps":["Spatial coordination of S338 and Y341 phosphorylation not fully resolved","RKIP regulation by upstream signals not yet defined"]},{"year":2001,"claim":"Placed S259 dephosphorylation upstream of membrane recruitment in the activation sequence and confirmed direct PAK1-RAF1 association driving S338 phosphorylation.","evidence":"Phospho-S259 antibody with phosphatase inhibitors, S259A and RAF1-CAAX constructs, cell fractionation; PAK1 co-IP with mutant panel","pmids":["11756411","11494123","11733498"],"confidence":"High","gaps":["Phosphatase recruitment mechanism to S259 not fully defined","Some co-IP findings from single labs"]},{"year":2001,"claim":"Genetically separated RAF1's catalytic and non-catalytic roles, revealing an essential kinase-independent survival function despite dispensability of MEK kinase activity for ERK activation and development.","evidence":"Knockout and Y340F/Y341F kinase-dead knock-in mice; in vitro MEK kinase and ERK activation assays; embryonic phenotyping","pmids":["11296227"],"confidence":"High","gaps":["Molecular identity of the kinase-independent effector not defined in this study","Mechanism of vascular/apoptosis defect unresolved"]},{"year":2002,"claim":"Defined PKA-mediated inhibition acting primarily through S259 and refined S259 dephosphorylation as the rate-limiting activation step that enhances Ras binding and membrane recruitment.","evidence":"In vitro/in vivo PKA phosphorylation site mapping with S259A/S43A mutants; phospho-S259 antibody with Ras binding and MEK activation assays","pmids":["11971957","11782426"],"confidence":"High","gaps":["Cross-talk between PKA and the dephosphorylation machinery not fully mapped"]},{"year":2004,"claim":"Identified the kinase-independent effectors of RAF1's survival/cytoskeletal roles — spatial suppression of Rok-alpha and suppression of cardiac ASK1 — and a keratin association.","evidence":"Conditional RAF1 ablation with kinase-dead rescue, Rok-alpha localization/activity, cytoskeleton imaging; cardiac-specific knockout with ASK1 double-knockout rescue; K8/K18 co-IP and phosphorylation","pmids":["15753127","15467832","15314064"],"confidence":"High","gaps":["Structural basis of RAF1-Rok-alpha and RAF1-ASK1 binding not resolved","Keratin findings from single lab"]},{"year":2005,"claim":"Established ERK feedback phosphorylation as a desensitization mechanism, characterized RAF1-MST2 suppression, and defined RAF1-B-Raf hetero-oligomerization and additional scaffolds/regulators.","evidence":"Mass-spec phosphosite mapping with Pin1/PP2A functional assays; RAF1 knockout/MST2 siRNA rescue; B-Raf/C-Raf co-IP and 14-3-3 analysis; CNK1 scaffold and RKIP phosphomimetic studies","pmids":["15664191","15701972","16364920","15845549","15886202","15710605"],"confidence":"High","gaps":["Several interaction findings rest on single-lab co-IP","Stoichiometry of hetero-oligomers in cells not resolved"]},{"year":2009,"claim":"Revealed RAF1 as a paradoxical inhibitor of oncogenic B-Raf(V600E), a kinase-independent suppressor of Rok-alpha promoting tumor dedifferentiation, and clarified the 14-3-3/S621 ATP-binding switch.","evidence":"B-Raf/C-Raf co-IP with binding mutants and proliferation assays; conditional RAF1 knockout in Ras-induced skin tumors with STAT3/Myc readouts; S621 mutagenesis with ATP-binding assays","pmids":["19917255","19647225","19595761"],"confidence":"High","gaps":["Structural basis of paradoxical B-Raf inhibition not fully defined","S621 ATP-binding mechanism from single lab"]},{"year":2014,"claim":"Synthesized RAF1 as a node balancing competing MST2-LATS apoptotic and MEK-ERK proliferative outputs through S259-centered switches.","evidence":"Mathematical modelling with RAF1 S259 mutant, MST2 knockdown, Akt/LATS1 phosphorylation, and apoptosis/proliferation readouts","pmids":["24929361"],"confidence":"High","gaps":["Quantitative thresholds for switch behavior context-dependent","In vivo relevance of competing-interaction model not tested here"]},{"year":2022,"claim":"Identified post-translational control of RAF1 stability and ERK output via USP7-mediated deubiquitination.","evidence":"Co-IP, ubiquitin-linkage mapping, DUB assays, and cell cycle/proliferation analysis","pmids":["35948545"],"confidence":"Medium","gaps":["Single-lab study","Physiological/in vivo role of USP7-RAF1 axis not established"]},{"year":null,"claim":"How the multiple regulatory inputs (phosphorylation code, 14-3-3, oligomerization, autoinhibition) are integrated into a single activation trajectory at atomic resolution, and how the catalytic versus scaffolding pools are partitioned in vivo, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No full-length active RAF1 structure in the corpus","Spatial/temporal partitioning of kinase-dependent vs kinase-independent functions not quantified","Mechanism coupling S259 dephosphorylation to conformational activation not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,7,10,17,19]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,10,17]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[47]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[37,30,31,43]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,5,24,28]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[10,41]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[20]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,19,33]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[10,31,37,45]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[46]}],"complexes":["Ras-RAF1-MEK1 complex","B-Raf/C-Raf heterodimer","RAF1:Rok-alpha complex","RAF1:MST2 complex"],"partners":["RAS","MAP2K1","BRAF","YWHAZ","RKIP","MST2","ROCK1","PDE8A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P04049","full_name":"RAF proto-oncogene serine/threonine-protein kinase","aliases":["Proto-oncogene c-RAF","cRaf","Raf-1"],"length_aa":648,"mass_kda":73.1,"function":"Serine/threonine-protein kinase that acts as a regulatory link between the membrane-associated Ras GTPases and the MAPK/ERK cascade, and this critical regulatory link functions as a switch determining cell fate decisions including proliferation, differentiation, apoptosis, survival and oncogenic transformation. RAF1 activation initiates a mitogen-activated protein kinase (MAPK) cascade that comprises a sequential phosphorylation of the dual-specific MAPK kinases (MAP2K1/MEK1 and MAP2K2/MEK2) and the extracellular signal-regulated kinases (MAPK3/ERK1 and MAPK1/ERK2). The phosphorylated form of RAF1 (on residues Ser-338 and Ser-339, by PAK1) phosphorylates BAD/Bcl2-antagonist of cell death at 'Ser-75'. Phosphorylates adenylyl cyclases: ADCY2, ADCY5 and ADCY6, resulting in their activation. Phosphorylates PPP1R12A resulting in inhibition of the phosphatase activity. Phosphorylates TNNT2/cardiac muscle troponin T. Can promote NF-kB activation and inhibit signal transducers involved in motility (ROCK2), apoptosis (MAP3K5/ASK1 and STK3/MST2), proliferation and angiogenesis (RB1). Can protect cells from apoptosis also by translocating to the mitochondria where it binds BCL2 and displaces BAD/Bcl2-antagonist of cell death. Regulates Rho signaling and migration, and is required for normal wound healing. Plays a role in the oncogenic transformation of epithelial cells via repression of the TJ protein, occludin (OCLN) by inducing the up-regulation of a transcriptional repressor SNAI2/SLUG, which induces down-regulation of OCLN. Restricts caspase activation in response to selected stimuli, notably Fas stimulation, pathogen-mediated macrophage apoptosis, and erythroid differentiation","subcellular_location":"Cytoplasm; Cell membrane; Mitochondrion; Nucleus","url":"https://www.uniprot.org/uniprotkb/P04049/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RAF1","classification":"Not Classified","n_dependent_lines":131,"n_total_lines":1208,"dependency_fraction":0.10844370860927152},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000132155","cell_line_id":"CID001255","localizations":[{"compartment":"cytoplasmic","grade":3}],"interactors":[{"gene":"ARAF","stoichiometry":10.0},{"gene":"ARL8B","stoichiometry":0.2},{"gene":"FKBP5","stoichiometry":0.2},{"gene":"MAP2K1","stoichiometry":0.2},{"gene":"MAP2K2","stoichiometry":0.2},{"gene":"PHAX","stoichiometry":0.2},{"gene":"YWHAZ","stoichiometry":0.2},{"gene":"YWHAE","stoichiometry":0.2},{"gene":"YWHAB","stoichiometry":0.2},{"gene":"RBM23","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001255","total_profiled":1310},"omim":[{"mim_id":"621521","title":"SCAFFOLDING CK1-ANCHORING PROTEIN E; SACK1E","url":"https://www.omim.org/entry/621521"},{"mim_id":"621520","title":"SCAFFOLDING CK1-ANCHORING PROTEIN C; SACK1C","url":"https://www.omim.org/entry/621520"},{"mim_id":"621519","title":"SCAFFOLDING CK1-ANCHORING PROTEIN B; SACK1B","url":"https://www.omim.org/entry/621519"},{"mim_id":"619894","title":"ABHYDROLASE DOMAIN-CONTAINING PROTEIN 15; ABHD15","url":"https://www.omim.org/entry/619894"},{"mim_id":"618726","title":"NIMA-RELATED KINASE 10; NEK10","url":"https://www.omim.org/entry/618726"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"skeletal muscle","ntpm":163.1}],"url":"https://www.proteinatlas.org/search/RAF1"},"hgnc":{"alias_symbol":["Raf-1","c-Raf","CRAF"],"prev_symbol":[]},"alphafold":{"accession":"P04049","domains":[{"cath_id":"3.10.20.90","chopping":"56-132","consensus_level":"high","plddt":81.766,"start":56,"end":132},{"cath_id":"3.30.60.20","chopping":"136-192","consensus_level":"high","plddt":78.5349,"start":136,"end":192},{"cath_id":"3.30.200.20","chopping":"350-421","consensus_level":"medium","plddt":87.9076,"start":350,"end":421},{"cath_id":"1.10.510.10","chopping":"428-611","consensus_level":"high","plddt":87.942,"start":428,"end":611}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P04049","model_url":"https://alphafold.ebi.ac.uk/files/AF-P04049-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P04049-F1-predicted_aligned_error_v6.png","plddt_mean":67.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RAF1","jax_strain_url":"https://www.jax.org/strain/search?query=RAF1"},"sequence":{"accession":"P04049","fasta_url":"https://rest.uniprot.org/uniprotkb/P04049.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P04049/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P04049"}},"corpus_meta":[{"pmid":"8321321","id":"PMC_8321321","title":"Protein kinase C alpha activates RAF-1 by direct phosphorylation.","date":"1993","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/8321321","citation_count":1243,"is_preprint":false},{"pmid":"1322500","id":"PMC_1322500","title":"Raf-1 activates MAP kinase-kinase.","date":"1992","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/1322500","citation_count":1215,"is_preprint":false},{"pmid":"8503013","id":"PMC_8503013","title":"Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase.","date":"1993","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/8503013","citation_count":944,"is_preprint":false},{"pmid":"10490027","id":"PMC_10490027","title":"Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP.","date":"1999","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/10490027","citation_count":723,"is_preprint":false},{"pmid":"8929532","id":"PMC_8929532","title":"Bcl-2 targets the protein kinase Raf-1 to mitochondria.","date":"1996","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/8929532","citation_count":720,"is_preprint":false},{"pmid":"9069260","id":"PMC_9069260","title":"The complexity of Raf-1 regulation.","date":"1997","source":"Current opinion in cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/9069260","citation_count":541,"is_preprint":false},{"pmid":"15664191","id":"PMC_15664191","title":"Regulation of Raf-1 by direct feedback phosphorylation.","date":"2005","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/15664191","citation_count":501,"is_preprint":false},{"pmid":"1992343","id":"PMC_1992343","title":"Raf-1 protein kinase is required for growth of induced NIH/3T3 cells.","date":"1991","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/1992343","citation_count":490,"is_preprint":false},{"pmid":"9823899","id":"PMC_9823899","title":"The protein kinase Pak3 positively regulates Raf-1 activity through phosphorylation of serine 338.","date":"1998","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/9823899","citation_count":375,"is_preprint":false},{"pmid":"10205168","id":"PMC_10205168","title":"Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation.","date":"1999","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/10205168","citation_count":371,"is_preprint":false},{"pmid":"16364920","id":"PMC_16364920","title":"Wild-type and mutant B-RAF activate C-RAF through distinct mechanisms involving heterodimerization.","date":"2005","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/16364920","citation_count":346,"is_preprint":false},{"pmid":"11929951","id":"PMC_11929951","title":"Modulation of p53, ErbB1, ErbB2, and Raf-1 expression in lung cancer cells by depsipeptide FR901228.","date":"2002","source":"Journal of the National Cancer Institute","url":"https://pubmed.ncbi.nlm.nih.gov/11929951","citation_count":336,"is_preprint":false},{"pmid":"7935795","id":"PMC_7935795","title":"Activation of Raf-1 by 14-3-3 proteins.","date":"1994","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/7935795","citation_count":334,"is_preprint":false},{"pmid":"8692945","id":"PMC_8692945","title":"Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1.","date":"1996","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/8692945","citation_count":332,"is_preprint":false},{"pmid":"14654844","id":"PMC_14654844","title":"Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2.","date":"2003","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/14654844","citation_count":319,"is_preprint":false},{"pmid":"7935389","id":"PMC_7935389","title":"Mechanism of inhibition of Raf-1 by protein kinase A.","date":"1994","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/7935389","citation_count":293,"is_preprint":false},{"pmid":"8774884","id":"PMC_8774884","title":"Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization.","date":"1996","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/8774884","citation_count":271,"is_preprint":false},{"pmid":"11296227","id":"PMC_11296227","title":"MEK kinase activity is not necessary for Raf-1 function.","date":"2001","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/11296227","citation_count":266,"is_preprint":false},{"pmid":"2188091","id":"PMC_2188091","title":"Mutational activation of c-raf-1 and definition of the minimal transforming sequence.","date":"1990","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/2188091","citation_count":241,"is_preprint":false},{"pmid":"11782426","id":"PMC_11782426","title":"Regulation of Raf-1 activation and signalling by dephosphorylation.","date":"2002","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/11782426","citation_count":239,"is_preprint":false},{"pmid":"14688025","id":"PMC_14688025","title":"Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas.","date":"2003","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/14688025","citation_count":226,"is_preprint":false},{"pmid":"2466340","id":"PMC_2466340","title":"Effect of antisense c-raf-1 on tumorigenicity and radiation sensitivity of a human squamous carcinoma.","date":"1989","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/2466340","citation_count":211,"is_preprint":false},{"pmid":"8774885","id":"PMC_8774885","title":"Oligomerization activates c-Raf-1 through a Ras-dependent mechanism.","date":"1996","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/8774885","citation_count":206,"is_preprint":false},{"pmid":"11971957","id":"PMC_11971957","title":"Cyclic AMP-dependent kinase regulates Raf-1 kinase mainly by phosphorylation of serine 259.","date":"2002","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/11971957","citation_count":194,"is_preprint":false},{"pmid":"7604263","id":"PMC_7604263","title":"Reversal of Raf-1 activation by purified and membrane-associated protein phosphatases.","date":"1995","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/7604263","citation_count":188,"is_preprint":false},{"pmid":"15753127","id":"PMC_15753127","title":"Raf-1 regulates Rho signaling and cell migration.","date":"2005","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/15753127","citation_count":171,"is_preprint":false},{"pmid":"1869534","id":"PMC_1869534","title":"Erythropoietin induces Raf-1 activation and Raf-1 is required for erythropoietin-mediated proliferation.","date":"1991","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/1869534","citation_count":169,"is_preprint":false},{"pmid":"9689060","id":"PMC_9689060","title":"Autoregulation of the Raf-1 serine/threonine kinase.","date":"1998","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/9689060","citation_count":163,"is_preprint":false},{"pmid":"11494123","id":"PMC_11494123","title":"Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions.","date":"2001","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/11494123","citation_count":163,"is_preprint":false},{"pmid":"15467832","id":"PMC_15467832","title":"Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis.","date":"2004","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/15467832","citation_count":161,"is_preprint":false},{"pmid":"15289381","id":"PMC_15289381","title":"Raf-1 kinase is required for cardiac hypertrophy and cardiomyocyte survival in response to pressure overload.","date":"2004","source":"Circulation","url":"https://pubmed.ncbi.nlm.nih.gov/15289381","citation_count":154,"is_preprint":false},{"pmid":"9111327","id":"PMC_9111327","title":"Induction of cell proliferation in quiescent NIH 3T3 cells by oncogenic c-Raf-1.","date":"1997","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/9111327","citation_count":152,"is_preprint":false},{"pmid":"12127063","id":"PMC_12127063","title":"Untying the regulation of the Raf-1 kinase.","date":"2002","source":"Archives of biochemistry and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/12127063","citation_count":151,"is_preprint":false},{"pmid":"8637718","id":"PMC_8637718","title":"Activated Ras displaces 14-3-3 protein from the amino terminus of c-Raf-1.","date":"1996","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/8637718","citation_count":135,"is_preprint":false},{"pmid":"7623807","id":"PMC_7623807","title":"Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro.","date":"1995","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/7623807","citation_count":133,"is_preprint":false},{"pmid":"9153224","id":"PMC_9153224","title":"Raf-1 kinase and exoenzyme S interact with 14-3-3zeta through a common site involving lysine 49.","date":"1997","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9153224","citation_count":133,"is_preprint":false},{"pmid":"24929361","id":"PMC_24929361","title":"Protein interaction switches coordinate Raf-1 and MST2/Hippo signalling.","date":"2014","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/24929361","citation_count":127,"is_preprint":false},{"pmid":"12369855","id":"PMC_12369855","title":"Design and discovery of small molecules targeting raf-1 kinase.","date":"2002","source":"Current pharmaceutical design","url":"https://pubmed.ncbi.nlm.nih.gov/12369855","citation_count":127,"is_preprint":false},{"pmid":"22043453","id":"PMC_22043453","title":"C-Raf is required for the initiation of lung cancer by K-Ras(G12D).","date":"2011","source":"Cancer discovery","url":"https://pubmed.ncbi.nlm.nih.gov/22043453","citation_count":125,"is_preprint":false},{"pmid":"15710605","id":"PMC_15710605","title":"B-Raf and Raf-1 are regulated by distinct autoregulatory mechanisms.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15710605","citation_count":120,"is_preprint":false},{"pmid":"15943972","id":"PMC_15943972","title":"Second nature: biological functions of the Raf-1 \"kinase\".","date":"2005","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/15943972","citation_count":117,"is_preprint":false},{"pmid":"15886202","id":"PMC_15886202","title":"Raf kinase inhibitory protein regulates Raf-1 but not B-Raf kinase activation.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15886202","citation_count":116,"is_preprint":false},{"pmid":"8390681","id":"PMC_8390681","title":"Raf-1 and p21v-ras cooperate in the activation of mitogen-activated protein kinase.","date":"1993","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/8390681","citation_count":113,"is_preprint":false},{"pmid":"7969158","id":"PMC_7969158","title":"RAS and RAF-1 form a signalling complex with MEK-1 but not MEK-2.","date":"1994","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/7969158","citation_count":110,"is_preprint":false},{"pmid":"11733498","id":"PMC_11733498","title":"Interaction between active Pak1 and Raf-1 is necessary for phosphorylation and activation of Raf-1.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11733498","citation_count":107,"is_preprint":false},{"pmid":"2197271","id":"PMC_2197271","title":"Insulin activates the Raf-1 protein kinase.","date":"1990","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/2197271","citation_count":105,"is_preprint":false},{"pmid":"11756411","id":"PMC_11756411","title":"Dephosphorylation of Ser-259 regulates Raf-1 membrane association.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11756411","citation_count":102,"is_preprint":false},{"pmid":"10969079","id":"PMC_10969079","title":"MEKK1 binds raf-1 and the ERK2 cascade components.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10969079","citation_count":101,"is_preprint":false},{"pmid":"18202127","id":"PMC_18202127","title":"Insulin stimulates primary beta-cell proliferation via Raf-1 kinase.","date":"2008","source":"Endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/18202127","citation_count":91,"is_preprint":false},{"pmid":"19647225","id":"PMC_19647225","title":"Raf-1 addiction in Ras-induced skin carcinogenesis.","date":"2009","source":"Cancer cell","url":"https://pubmed.ncbi.nlm.nih.gov/19647225","citation_count":90,"is_preprint":false},{"pmid":"3550433","id":"PMC_3550433","title":"Rat c-raf oncogene activation by a rearrangement that produces a fused protein.","date":"1987","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/3550433","citation_count":88,"is_preprint":false},{"pmid":"30975481","id":"PMC_30975481","title":"Complete Regression of Advanced Pancreatic Ductal Adenocarcinomas upon Combined Inhibition of EGFR and C-RAF.","date":"2019","source":"Cancer cell","url":"https://pubmed.ncbi.nlm.nih.gov/30975481","citation_count":87,"is_preprint":false},{"pmid":"7517401","id":"PMC_7517401","title":"Raf-1 interacts with Fyn and Src in a non-phosphotyrosine-dependent manner.","date":"1994","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7517401","citation_count":83,"is_preprint":false},{"pmid":"8879680","id":"PMC_8879680","title":"Activation of raf-1, MEK, and MAP kinase in prolactin responsive mammary cells.","date":"1996","source":"Breast cancer research and treatment","url":"https://pubmed.ncbi.nlm.nih.gov/8879680","citation_count":82,"is_preprint":false},{"pmid":"8108400","id":"PMC_8108400","title":"Enzymatic characteristics of the c-Raf-1 protein kinase.","date":"1994","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/8108400","citation_count":77,"is_preprint":false},{"pmid":"10998357","id":"PMC_10998357","title":"Regulation of the Raf-1 kinase domain by phosphorylation and 14-3-3 association.","date":"2000","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/10998357","citation_count":75,"is_preprint":false},{"pmid":"10933809","id":"PMC_10933809","title":"Recruitment and activation of Raf-1 kinase by nitric oxide-activated Ras.","date":"2000","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10933809","citation_count":74,"is_preprint":false},{"pmid":"16861903","id":"PMC_16861903","title":"ERK and beyond: insights from B-Raf and Raf-1 conditional knockouts.","date":"2006","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/16861903","citation_count":71,"is_preprint":false},{"pmid":"2150916","id":"PMC_2150916","title":"The Raf-1 kinase as a transducer of mitogenic signals.","date":"1990","source":"Cancer cells (Cold Spring Harbor, N.Y. : 1989)","url":"https://pubmed.ncbi.nlm.nih.gov/2150916","citation_count":71,"is_preprint":false},{"pmid":"16405965","id":"PMC_16405965","title":"Raf-1 kinase associates with Hepatitis C virus NS5A and regulates viral replication.","date":"2005","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/16405965","citation_count":67,"is_preprint":false},{"pmid":"12438425","id":"PMC_12438425","title":"Raf-1 antagonizes erythroid differentiation by restraining caspase activation.","date":"2002","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/12438425","citation_count":67,"is_preprint":false},{"pmid":"16803888","id":"PMC_16803888","title":"Rheb inhibits C-raf activity and B-raf/C-raf heterodimerization.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16803888","citation_count":66,"is_preprint":false},{"pmid":"9583688","id":"PMC_9583688","title":"Abrogation of c-Raf expression induces apoptosis in tumor cells.","date":"1998","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/9583688","citation_count":66,"is_preprint":false},{"pmid":"18472967","id":"PMC_18472967","title":"Constitutive activation of Raf-1 induces glioma formation in mice.","date":"2008","source":"Neoplasia (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/18472967","citation_count":64,"is_preprint":false},{"pmid":"7803760","id":"PMC_7803760","title":"The Raf-1 serine/threonine protein kinase.","date":"1994","source":"Seminars in cancer biology","url":"https://pubmed.ncbi.nlm.nih.gov/7803760","citation_count":62,"is_preprint":false},{"pmid":"8607983","id":"PMC_8607983","title":"Mechanisms regulating Raf-1 activity in signal transduction pathways.","date":"1995","source":"Molecular reproduction and development","url":"https://pubmed.ncbi.nlm.nih.gov/8607983","citation_count":62,"is_preprint":false},{"pmid":"18064632","id":"PMC_18064632","title":"GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis.","date":"2008","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/18064632","citation_count":61,"is_preprint":false},{"pmid":"11742498","id":"PMC_11742498","title":"Association of c-Raf expression with survival and its targeting with antisense oligonucleotides in ovarian cancer.","date":"2001","source":"British journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/11742498","citation_count":61,"is_preprint":false},{"pmid":"17218791","id":"PMC_17218791","title":"Phosphatase and feedback regulation of Raf-1 signaling.","date":"2007","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/17218791","citation_count":58,"is_preprint":false},{"pmid":"10445849","id":"PMC_10445849","title":"Interactions of c-Raf-1 with phosphatidylserine and 14-3-3.","date":"1999","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/10445849","citation_count":58,"is_preprint":false},{"pmid":"11157055","id":"PMC_11157055","title":"Protective role of Raf-1 in Salmonella-induced macrophage apoptosis.","date":"2001","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/11157055","citation_count":58,"is_preprint":false},{"pmid":"19917255","id":"PMC_19917255","title":"C-Raf inhibits MAPK activation and transformation by B-Raf(V600E).","date":"2009","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/19917255","citation_count":57,"is_preprint":false},{"pmid":"19710016","id":"PMC_19710016","title":"Diacylglycerol kinase eta augments C-Raf activity and B-Raf/C-Raf heterodimerization.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19710016","citation_count":56,"is_preprint":false},{"pmid":"11023985","id":"PMC_11023985","title":"The Raf-1 kinase associates with vimentin kinases and regulates the structure of vimentin filaments.","date":"2000","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/11023985","citation_count":55,"is_preprint":false},{"pmid":"15096503","id":"PMC_15096503","title":"Cation diffusion facilitator proteins modulate Raf-1 activity.","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15096503","citation_count":54,"is_preprint":false},{"pmid":"12471618","id":"PMC_12471618","title":"Geldanamycin decreases Raf-1 and Akt levels and induces apoptosis in neuroblastomas.","date":"2003","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/12471618","citation_count":53,"is_preprint":false},{"pmid":"15701972","id":"PMC_15701972","title":"Taming the Hippo: Raf-1 controls apoptosis by suppressing MST2/Hippo.","date":"2005","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/15701972","citation_count":51,"is_preprint":false},{"pmid":"32257057","id":"PMC_32257057","title":"The quaternary assembly of KRas4B with Raf-1 at the membrane.","date":"2020","source":"Computational and structural biotechnology journal","url":"https://pubmed.ncbi.nlm.nih.gov/32257057","citation_count":51,"is_preprint":false},{"pmid":"7799948","id":"PMC_7799948","title":"Raf-1 N-terminal sequences necessary for Ras-Raf interaction and signal transduction.","date":"1995","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/7799948","citation_count":51,"is_preprint":false},{"pmid":"15314064","id":"PMC_15314064","title":"Raf-1 activation disrupts its binding to keratins during cell stress.","date":"2004","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/15314064","citation_count":51,"is_preprint":false},{"pmid":"11971897","id":"PMC_11971897","title":"Activation of c-Raf kinase by ultraviolet light. Regulation by retinoids.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11971897","citation_count":51,"is_preprint":false},{"pmid":"23509299","id":"PMC_23509299","title":"Phosphodiesterase-8A binds to and regulates Raf-1 kinase.","date":"2013","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/23509299","citation_count":50,"is_preprint":false},{"pmid":"18465753","id":"PMC_18465753","title":"p21 activated kinase 5 activates Raf-1 and targets it to mitochondria.","date":"2008","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18465753","citation_count":48,"is_preprint":false},{"pmid":"16527894","id":"PMC_16527894","title":"A balance between Raf-1 and Fas expression sets the pace of erythroid differentiation.","date":"2006","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/16527894","citation_count":48,"is_preprint":false},{"pmid":"29970115","id":"PMC_29970115","title":"Novel oncogene COPS3 interacts with Beclin1 and Raf-1 to regulate metastasis of osteosarcoma through autophagy.","date":"2018","source":"Journal of experimental & clinical cancer research : CR","url":"https://pubmed.ncbi.nlm.nih.gov/29970115","citation_count":47,"is_preprint":false},{"pmid":"2496060","id":"PMC_2496060","title":"Amplification of both c-myc and c-raf-1 oncogenes in a human osteosarcoma.","date":"1989","source":"Japanese journal of cancer research : Gann","url":"https://pubmed.ncbi.nlm.nih.gov/2496060","citation_count":46,"is_preprint":false},{"pmid":"15845549","id":"PMC_15845549","title":"CNK1 is a scaffold protein that regulates Src-mediated Raf-1 activation.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15845549","citation_count":46,"is_preprint":false},{"pmid":"10777790","id":"PMC_10777790","title":"c-Raf regulates cell survival and retinal ganglion cell morphogenesis during neurogenesis.","date":"2000","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/10777790","citation_count":45,"is_preprint":false},{"pmid":"9699002","id":"PMC_9699002","title":"Bc1-2, Raf-1 and mitochondrial regulation of apoptosis.","date":"1998","source":"BioFactors (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/9699002","citation_count":44,"is_preprint":false},{"pmid":"16428931","id":"PMC_16428931","title":"The Raf-1 pathway: a molecular target for treatment of select neuroendocrine tumors?","date":"2006","source":"Anti-cancer drugs","url":"https://pubmed.ncbi.nlm.nih.gov/16428931","citation_count":43,"is_preprint":false},{"pmid":"19595761","id":"PMC_19595761","title":"The C-terminus of Raf-1 acts as a 14-3-3-dependent activation switch.","date":"2009","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/19595761","citation_count":42,"is_preprint":false},{"pmid":"8361757","id":"PMC_8361757","title":"H-ras and raf-1 cooperate in transformation of NIH3T3 fibroblasts.","date":"1993","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/8361757","citation_count":41,"is_preprint":false},{"pmid":"11865188","id":"PMC_11865188","title":"Tangled webs: evidence of cross-talk between c-Raf-1 and Akt.","date":"1999","source":"Science's STKE : signal transduction knowledge environment","url":"https://pubmed.ncbi.nlm.nih.gov/11865188","citation_count":40,"is_preprint":false},{"pmid":"17097642","id":"PMC_17097642","title":"Regulation of RKIP binding to the N-region of the Raf-1 kinase.","date":"2006","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/17097642","citation_count":40,"is_preprint":false},{"pmid":"18212057","id":"PMC_18212057","title":"Survival signaling by C-RAF: mitochondrial reactive oxygen species and Ca2+ are critical targets.","date":"2008","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/18212057","citation_count":40,"is_preprint":false},{"pmid":"23087049","id":"PMC_23087049","title":"Raf-1, actin dynamics, and abelson tyrosine kinase in human airway smooth muscle cells.","date":"2012","source":"American journal of respiratory cell and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/23087049","citation_count":39,"is_preprint":false},{"pmid":"11856330","id":"PMC_11856330","title":"Anti-(Raf-1) RNA aptamers that inhibit Ras-induced Raf-1 activation.","date":"2002","source":"European journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11856330","citation_count":39,"is_preprint":false},{"pmid":"15666389","id":"PMC_15666389","title":"Raf-1 expression may influence progression to androgen insensitive prostate cancer.","date":"2005","source":"The Prostate","url":"https://pubmed.ncbi.nlm.nih.gov/15666389","citation_count":37,"is_preprint":false},{"pmid":"35948545","id":"PMC_35948545","title":"USP7 regulates the ERK1/2 signaling pathway through deubiquitinating Raf-1 in lung adenocarcinoma.","date":"2022","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/35948545","citation_count":36,"is_preprint":false},{"pmid":"19737955","id":"PMC_19737955","title":"C-Raf is associated with disease progression and cell proliferation in a subset of melanomas.","date":"2009","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/19737955","citation_count":36,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":44438,"output_tokens":12289,"usd":0.158825,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":23209,"output_tokens":5530,"usd":0.127147,"stage2_stop_reason":"end_turn"},"total_usd":0.285972,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1992,\n      \"finding\": \"Raf-1 directly phosphorylates and activates MAP kinase-kinase (MAPKK/MEK) at serine/threonine residues in vitro, establishing MAPKK as the first identified physiological substrate of c-Raf-1 and placing Raf-1 as the immediate upstream activator of MAPKK in vivo.\",\n      \"method\": \"In vitro kinase assay with purified c-Raf-1 and partially purified MAPKK; phosphatase 2A inactivation/reactivation assay; v-raf-transformed cell analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified proteins, replicated across transformed cell and in vitro systems, foundational result confirmed by multiple subsequent studies\",\n      \"pmids\": [\"1322500\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Activated Ras-GTP (but not effector domain mutant Ras-Ile36Ala) specifically binds Raf-1 and is required for formation of complexes containing MAPKK activity, demonstrating that Ras-GTP recruits Raf-1 and MAPKK into a signaling complex in a GTP- and effector-domain-dependent manner.\",\n      \"method\": \"Affinity pulldown using immobilized Ras variants (wild-type, G12V, GMP-PNP-loaded, I36A effector mutant) with cell lysates; direct MAPKK activity assays on Ras-bound complexes\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — biochemical reconstitution with multiple Ras mutants, replicated and confirmed by numerous subsequent studies\",\n      \"pmids\": [\"8503013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"PKC-alpha directly phosphorylates and activates Raf-1 both in vitro and in vivo, including at Ser499; mutations at Ser499 or Ser259 block PKC-alpha-mediated (but not Ras+Lck-mediated) Raf-1 activation, demonstrating a direct PKC-alpha→Raf-1 activation mechanism distinct from Ras-dependent activation.\",\n      \"method\": \"In vitro phosphorylation assay with purified PKC-alpha and Raf-1; site-directed mutagenesis of Ser499 and Ser259; in vivo activation assays in NIH3T3 cells; transformation cooperation assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with purified proteins plus mutagenesis plus in vivo validation, multiple orthogonal methods\",\n      \"pmids\": [\"8321321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"14-3-3 zeta and 14-3-3 beta proteins bind the amino-terminal regulatory region of Raf-1 (identified by yeast two-hybrid), and expression of 14-3-3 proteins in Xenopus oocytes enhances Raf-1 activity and promotes Raf-1-dependent oocyte maturation; dominant-negative Raf-1 blocks these effects.\",\n      \"method\": \"Yeast two-hybrid screen; Xenopus oocyte functional assay; dominant-negative Raf-1 epistasis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid identification plus functional validation in Xenopus oocytes, replicated by multiple subsequent studies\",\n      \"pmids\": [\"7935795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"PKA inhibits Raf-1 by direct phosphorylation of the Raf-1 kinase domain, independently of weakening Raf-1/Ras interaction; PKA phosphorylation can downregulate Raf-1 kinase activity even after prior activation by PKC-alpha or amino-terminal truncation, and the isolated kinase domain lacking the Ras-binding domain is still susceptible.\",\n      \"method\": \"In vitro phosphorylation assays with purified PKA and Raf-1 proteins; kinase domain fragment analysis; sequential activation/inhibition assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified proteins, multiple mechanistic controls (Ras-binding domain deletion, sequential activation experiments), single lab\",\n      \"pmids\": [\"7935389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Raf-1 forms a specific signaling complex with Ras and MEK-1 but not MEK-2; MEK-1 binding to Ras requires RAF-1 as a bridge, and a proline-rich region of MEK-1 containing a phosphorylation site is essential for complex formation.\",\n      \"method\": \"Immobilized Ras pulldown from NIH 3T3 cell lysates; MEK-1 and MEK-2 immunodetection; exogenous RAF-1 addition to lysates; MEK-1 mutant analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical pulldown with multiple controls, specificity for MEK-1 over MEK-2 demonstrated, single lab with orthogonal methods\",\n      \"pmids\": [\"7969158\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Raf-1 associates with Fyn and Src SH2 domains in a serine-phosphorylation-dependent (not tyrosine-phosphorylation-dependent) manner; co-expression of Raf-1 with full-length Fyn/Src results in co-immunoprecipitation, tyrosine phosphorylation of Raf-1, and stimulation of Raf-1 kinase activity.\",\n      \"method\": \"Co-immunoprecipitation; SH2 domain binding assay; baculovirus/Sf9 co-expression; site-directed mutagenesis of Src SH2 Arg175; kinase activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and baculovirus co-expression with multiple domain mutants, single lab\",\n      \"pmids\": [\"7517401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Enzymatic characterization of c-Raf-1 shows Km for ATP of 11.6 µM and for MAPKK of 0.8 µM; c-Raf-1 has highly restricted substrate specificity, with MAPKK as the preferred substrate; active c-Raf-1 elutes as a multimeric complex (>150 kDa) on gel filtration.\",\n      \"method\": \"In vitro kinase assay with purified baculovirus-expressed His-tagged c-Raf-1; Km determination; substrate panel screening; gel-filtration chromatography\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — rigorous in vitro enzyme characterization with purified proteins, multiple substrate specificity controls, single lab\",\n      \"pmids\": [\"8108400\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Raf-1 activation requires its Ras-binding domain (residues 53-132), active kinase function, tyrosine phosphorylation at Y340/Y341, constitutive serine phosphorylation at S621, and an intact zinc finger (C165/C168); S259A mutation reduces but does not abolish activation efficiency; the zinc finger is not required for Ras binding itself.\",\n      \"method\": \"In vitro activation assay using purified plasma membranes from transformed cells; panel of Raf-1 point and deletion mutants expressed in baculovirus; kinase assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution in vitro with purified membranes and extensive mutational analysis, single lab\",\n      \"pmids\": [\"7623807\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Protein phosphatases (both serine/threonine and tyrosine phosphatases) inactivate purified Raf-1; 14-3-3 zeta or HSP90 block phosphatase-mediated inactivation; GTP-loading of plasma membranes from transformed cells inactivates Raf-1 via phosphatases present in the membrane, suggesting membrane-localized phosphatases regulate Raf-1.\",\n      \"method\": \"In vitro phosphatase treatment of purified Raf-1 (from Sf9 cells co-expressing Ras and Src-Y527F); GTP-loading of plasma membranes; phosphatase inhibitor controls\",\n      \"journal\": \"Science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified protein and membranes, single lab\",\n      \"pmids\": [\"7604263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Bcl-2 targets Raf-1 kinase to mitochondria; mitochondria-targeted active Raf-1 protects cells from apoptosis and phosphorylates BAD, whereas plasma membrane-targeted Raf-1 phosphorylates ERK-1/2 but does not protect from apoptosis; kinase-inactive Raf-1 abrogates Bcl-2-mediated apoptosis suppression.\",\n      \"method\": \"GFP-Raf-1 fusion protein localization; mitochondrial and plasma membrane targeting constructs; BAD phosphorylation assay; cell death assays; kinase-dead Raf-1 mutant\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct subcellular targeting experiments with functional readouts, multiple targeting constructs and kinase-dead controls, foundational result replicated in subsequent studies\",\n      \"pmids\": [\"8929532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"BAG-1 specifically binds to and activates Raf-1 kinase; bacterially produced BAG-1 increases Raf-1 kinase activity in vitro; BAG-1 and Raf-1 co-immunoprecipitate from mammalian and insect cells.\",\n      \"method\": \"Co-immunoprecipitation from mammalian cells and baculovirus-infected insect cells; in vitro kinase activation assay with bacterially produced BAG-1; yeast two-hybrid\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, in vitro activation with bacterially produced protein, and yeast two-hybrid, single lab\",\n      \"pmids\": [\"8692945\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Coumermycin-induced dimerization of a modified Raf-1 (fused to gyrase B) is sufficient to activate Raf-1 and stimulate the MAP kinase cascade in the absence of membrane components, indicating that Raf oligomerization per se promotes activation.\",\n      \"method\": \"Chemical dimerization (coumermycin/gyrase B fusion); MAP kinase cascade activation assay in cells; absence-of-membrane-component controls\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — chemical-genetic dimerization system with clean controls, replicated by companion paper (PMID 8774885)\",\n      \"pmids\": [\"8774884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"FK506-induced oligomerization of FKBP12-Raf-1 activates Raf kinase activity in a Ras-GTP-dependent manner, demonstrating that oligomerization promotes Raf activation through a Ras-dependent mechanism.\",\n      \"method\": \"Chemical dimerization (FKBP12-FK1012A system); Raf kinase activity assay; dominant-negative Ras epistasis to show Ras dependence\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — chemical-genetic oligomerization system with Ras epistasis controls, companion to PMID 8774884\",\n      \"pmids\": [\"8774885\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"14-3-3 zeta binds bivalently to both the amino- and carboxy-termini of c-Raf-1; activated Ras displaces 14-3-3 zeta specifically from the N-terminal site; S259A mutation in the N-terminal domain prevents 14-3-3 binding at that site; only unphosphorylated 14-3-3 zeta binds the N-terminus of Raf-1.\",\n      \"method\": \"In vivo and in vitro binding assays; mutant Raf-1 fragments; co-expression of activated Ras\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo binding with multiple mutants, single lab\",\n      \"pmids\": [\"8637718\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"The 14-3-3 zeta amphipathic groove (Lys49 being critical) mediates binding to Raf-1; the K49E mutation dramatically disrupts 14-3-3 zeta/Raf-1 interaction; this same site is used to bind exoenzyme S, indicating a common structural binding determinant.\",\n      \"method\": \"Crystal structure of 14-3-3 zeta; charge-reversal mutagenesis (K49E, R56E, R60E); in vitro binding assays; circular dichroism; partial proteolysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure-guided mutagenesis with binding assays and structural validation, single lab\",\n      \"pmids\": [\"9153224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Autoinhibition mediated by the N-terminal regulatory region of Raf-1 (involving the cysteine-rich domain) suppresses kinase activity; disruption of this autoinhibition by cysteine-rich domain mutation or by Y340D phosphomimetic mutation increases Raf-1 activity, demonstrating an intramolecular repression mechanism.\",\n      \"method\": \"Site-directed mutagenesis of cysteine-rich domain and Y340D; kinase activity assays; regulatory domain co-expression inhibition experiments\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with kinase activity readout, single lab, moderate follow-up\",\n      \"pmids\": [\"9689060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"PAK3 phosphorylates Raf-1 on Ser338 both in vitro and in vivo, and this phosphorylation positively regulates Raf-1 activity; PAK3 is regulated by Rho-family GTPases Rac and Cdc42, linking these pathways to Raf-1 activation.\",\n      \"method\": \"In vitro kinase assay (PAK3 phosphorylating Raf-1); in vivo phosphorylation assays; phospho-specific antibodies\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay plus in vivo phosphorylation, replicated by subsequent PAK1 and other PAK studies\",\n      \"pmids\": [\"9823899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"RKIP (Raf kinase inhibitor protein) binds Raf-1, MEK, and ERK in vitro, co-immunoprecipitates with Raf-1 and MEK from cell lysates, and competitively disrupts the Raf-1/MEK interaction without being a substrate; RKIP overexpression inhibits MEK/ERK activation and AP-1-dependent transcription; RKIP downregulation activates MEK/ERK signaling.\",\n      \"method\": \"Yeast two-hybrid screen; in vitro binding assays; co-immunoprecipitation; confocal microscopy colocalization; antisense RNA and antibody microinjection; reporter gene assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (co-IP, in vitro binding, loss-of-function, gain-of-function), replicated by subsequent studies\",\n      \"pmids\": [\"10490027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Activated Raf-1 is phosphorylated on both S338 (by PAK pathway, Ras-dependent) and Y341 (by Src); phosphorylation at both sites is required for full Raf-1 activation; Ras-GTP binding is required for both phosphorylation events to occur, likely at the plasma membrane; B-Raf differs in having constitutive S445 phosphorylation not regulated by Ras.\",\n      \"method\": \"Phospho-specific antisera; co-expression of oncogenic Ras and activated Src; mutagenesis of S338, S339, Y340, Y341; kinase activity assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — phospho-specific antibodies combined with systematic mutagenesis and co-expression experiments, multiple orthogonal methods\",\n      \"pmids\": [\"10205168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Phosphatidylserine (inner plasma membrane phospholipid) displaces 14-3-3 from Raf-1 and increases Raf-1 kinase activity; 14-3-3 removal from activated Raf-1 by phosphopeptides eradicates kinase activity of soluble Raf-1, indicating 14-3-3 maintains Raf-1 activity once activated.\",\n      \"method\": \"In vitro incubation of Raf-1 with phosphatidylserine; phosphopeptide competition assays; kinase activity measurements\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution experiments with multiple conditions, single lab\",\n      \"pmids\": [\"10445849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"MEKK1 binds endogenous ERK2, MEK1, and Raf-1, suggesting it can assemble all three proteins of the ERK2 MAP kinase module into a complex.\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP experiment, no functional validation of the complex, single lab\",\n      \"pmids\": [\"10969079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Raf-1 associates with vimentin via GST-Raf-1 pulldown; vimentin is not a direct Raf-1 substrate but is phosphorylated by Raf-1-associated kinases including casein kinase 2; Raf-1 activation status correlates with vimentin phosphorylation; selective Raf-1 activation induces vimentin network rearrangement independently of MEK/ERK.\",\n      \"method\": \"GST-Raf-1 pulldown; co-immunoprecipitation; in vitro kinase assays; MEK inhibitor controls; conditional estrogen-regulated Raf-1 mutant system\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pulldown, co-IP, kinase assays, and MEK-independent functional readout, single lab\",\n      \"pmids\": [\"11023985\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Ser259 dephosphorylation by PP1 and PP2A is a critical early step in Ras-dependent Raf-1 activation; serine phosphatase inhibition blocks S259 dephosphorylation and prevents Raf-1 activation; S259A Raf-1 mutant is relatively resistant to phosphatase inhibitors and is constitutively membrane-associated.\",\n      \"method\": \"In vitro Raf-1 activation assay with serine phosphatase inhibitors; S259A Raf-1 mutant; sucrose gradient fractionation of plasma membrane microdomains\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro activation system with pharmacological and genetic tools, single lab\",\n      \"pmids\": [\"11494123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Mitogens stimulate Raf-1 S259 dephosphorylation concomitant with Raf-1 membrane accumulation and activation; blocking S259 dephosphorylation inhibits membrane recruitment and activation; S259A mutant is constitutively membrane-localized; membrane-tethered Raf-1-CAAX is activated independently of S259 dephosphorylation, placing S259 dephosphorylation upstream of membrane recruitment.\",\n      \"method\": \"Phospho-S259 antibody; pharmacological phosphatase inhibition; S259A and Raf-1-CAAX constructs; cell fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic and pharmacological tools with clear epistasis, independently confirmed by PMID 11494123\",\n      \"pmids\": [\"11756411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Active PAK1 directly associates with Raf-1 under physiological conditions; active PAK (T423E or N-terminal truncation) binds Raf-1 more strongly than wild-type; kinase-dead PAK barely binds Raf-1; extent of PAK-Raf-1 binding correlates with Raf-1 S338 phosphorylation and MAPK activation; the Raf-1 binding site maps to the C-terminus of the PAK catalytic domain.\",\n      \"method\": \"Co-immunoprecipitation under physiological conditions; PAK mutant analysis; in vitro phosphorylation of Raf-1 S338; MAPK activation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with multiple mutants and correlated kinase activity, single lab\",\n      \"pmids\": [\"11733498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Raf-1 MEK kinase activity (assessed via Y340F/Y341F knock-in mutation abolishing Raf-1 kinase activity toward MEK) is not essential for normal mouse development or ERK activation; however, Raf-1 knockout causes embryonic lethality with vascular defects and increased apoptosis, and ERK activation is normal in both knockout and kinase-dead knock-in cells, revealing a kinase-independent essential function.\",\n      \"method\": \"Gene targeting (knockout and Y340F/Y341F knock-in mice); in vitro MEK kinase assay; embryonic phenotype analysis; ERK activation assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — rigorous gene targeting with two independent alleles (null and kinase-dead knock-in), clean genetic separation of kinase-dependent vs. kinase-independent functions\",\n      \"pmids\": [\"11296227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"PKA phosphorylates Raf-1 on S43, S259, and S621 in vitro and in vivo; S259 phosphorylation is the main mechanism of PKA-mediated Raf-1 inhibition (S259A mutant largely resistant to PKA inhibition); PKA also reduces S338 phosphorylation of Raf-1 in a S259-dependent manner.\",\n      \"method\": \"In vitro PKA phosphorylation mapping; in vivo cAMP stimulation; S259A, S43A mutants; ERK activation assays; cAMP kinetics correlated with ERK deactivation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — phosphorylation site mapping in vitro and in vivo, multiple site mutants with clear functional phenotypes, single lab\",\n      \"pmids\": [\"11971957\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Ser259 dephosphorylation is an essential step in Raf-1 activation; phospho-Ser259 Raf-1 is refractory to mitogenic stimulation; S259A mutation elevates kinase activity by enhancing Ras binding and constitutive membrane recruitment, which facilitates S338 phosphorylation; S259A also improves functional coupling to MEK.\",\n      \"method\": \"Phospho-S259 antibody; S259A Raf-1 mutant; Ras binding assays; membrane recruitment assays; MEK activation assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal assays with mechanistic mutants, independently confirmed by companion studies\",\n      \"pmids\": [\"11782426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PKC-dependent phosphorylation of RKIP on Ser153 causes RKIP to dissociate from Raf-1 and instead associate with GRK-2, thereby simultaneously relieving Raf-1 inhibition and blocking GPCR internalization; this switch mechanism was demonstrated in cardiomyocytes.\",\n      \"method\": \"Co-immunoprecipitation; RKIP S153 phosphorylation analysis; GRK-2 binding assays; cardiomyocyte functional assays; GPCR signaling readouts\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mechanistic switch demonstrated with co-IP, mutagenesis, and physiological readout in cardiomyocytes, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"14654844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Raf-1 associates directly with Rok-alpha (a Rho-effector kinase); Raf-1-deficient keratinocytes and fibroblasts show cortical actin bundles, disordered vimentin cytoskeleton, and impaired migration due to hyperactivity and incorrect plasma membrane localization of Rok-alpha; reintroduction of either wild-type or kinase-dead Raf-1 rescues cell shape and migration defects, demonstrating a kinase-independent spatial regulatory role.\",\n      \"method\": \"Conditional Raf-1 gene ablation; cell migration assays; actin/vimentin cytoskeleton imaging; Rok-alpha localization and activity assays; rescue with kinase-dead Raf-1\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout with kinase-dead rescue, multiple cellular phenotype readouts, orthogonal imaging and biochemical analyses\",\n      \"pmids\": [\"15753127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Cardiac-specific Raf-1 knockout causes left ventricular systolic dysfunction and cardiomyocyte apoptosis without affecting MEK/ERK activation; instead, ASK1, JNK, and p38 kinase activities are elevated; ablation of ASK1 rescues the cardiac phenotype, placing Raf-1 upstream of ASK1 suppression in a MEK/ERK-independent survival pathway.\",\n      \"method\": \"Cre-loxP cardiac-specific knockout; echocardiography; kinase activity assays (MEK, ERK, ASK1, JNK, p38); ASK1 double-knockout rescue\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — tissue-specific knockout with genetic rescue and clear epistasis establishing Raf-1→ASK1 suppression independent of MEK/ERK\",\n      \"pmids\": [\"15467832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Raf-1 associates directly with K8 (keratin 8) independently of Raf-1 kinase activity or Ras-Raf interaction; K18 is a physiological Raf-1 substrate; Raf-1 activation during oxidative/toxin stress disrupts keratin-Raf association in a phosphorylation-dependent manner; 14-3-3 residues essential for Raf-1 binding also regulate keratin association.\",\n      \"method\": \"Co-immunoprecipitation; kinase-dead and Ras-binding-defective Raf-1 mutants; in vivo and in vitro phosphorylation assays; 14-3-3 binding-site mutants\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with multiple Raf-1 mutants and substrate phosphorylation assay, single lab\",\n      \"pmids\": [\"15314064\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"ERK-mediated feedback phosphorylation at six proline-directed sites (five are ERK targets) in Raf-1 following mitogen stimulation inhibits the Ras/Raf-1 interaction and desensitizes Raf-1 to further stimuli; dephosphorylation by PP2A and prolyl isomerization by Pin1 return Raf-1 to a signaling-competent state.\",\n      \"method\": \"Mass spectrometry-based phosphorylation site identification; MEK inhibitor treatments; in vitro phosphorylation by ERK; Ras-Raf binding assays; PP2A and Pin1 co-immunoprecipitation and functional assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — mass spectrometry site identification combined with in vitro kinase assays, binding assays, and phosphatase/isomerase functional validation, multiple orthogonal methods\",\n      \"pmids\": [\"15664191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Wild-type B-Raf forms a complex with C-Raf in a Ras-dependent manner, whereas kinase-impaired B-Raf mutants bind C-Raf independently of Ras; B-Raf activates C-Raf through a mechanism involving 14-3-3-mediated hetero-oligomerization and C-Raf transphosphorylation; C-Raf activation segment phosphorylation and 14-3-3 binding to C-Raf are required.\",\n      \"method\": \"Co-immunoprecipitation; Ras-dependence assays; kinase activity assays; 14-3-3 binding analysis; activation segment phosphorylation assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple B-Raf mutants, co-IP, and mechanistic dissection with multiple orthogonal experiments, single lab but comprehensive\",\n      \"pmids\": [\"16364920\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"RKIP inhibits Raf-1 by preventing PAK and Src family kinase phosphorylation of Raf-1 kinase domain (acting after membrane recruitment); phosphomimetic mutations at PAK and Src phosphorylation sites on Raf-1 prevent RKIP association; RKIP has no effect on B-Raf activation despite binding B-Raf.\",\n      \"method\": \"RKIP overexpression and depletion; Raf-1 phosphomimetic mutants; PAK and Src kinase co-IP; MEK/ERK and DNA synthesis assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple phosphomimetic mutants and RKIP depletion, single lab\",\n      \"pmids\": [\"15886202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Raf-1 is required for wound healing in vivo and migration of keratinocytes/fibroblasts in vitro; Raf-1 physically associates with Rok-alpha; Raf-1 loss causes Rok-alpha hyperactivity and mislocalization; these phenotypes are rescued by kinase-dead Raf-1, establishing a kinase-independent function as a spatial regulator of Rho-Rok-alpha signaling.\",\n      \"method\": \"Conditional gene ablation; wound healing assay; in vitro cell migration; actin/vimentin cytoskeleton analysis; Rok-alpha localization; kinase-dead rescue\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout with kinase-dead rescue and multiple cellular readouts (same paper as PMID 15753127)\",\n      \"pmids\": [\"15753127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Raf-1 controls the proapoptotic kinase MST2 by preventing its dimerization and recruiting a phosphatase that removes activating phosphorylations; both functions require Raf-1 binding to MST2 and are independent of Raf-1 kinase activity and the ERK pathway; MST2 siRNA reverts apoptosis hypersensitivity of Raf-1−/− fibroblasts.\",\n      \"method\": \"Raf-1 knockout cells; MST2 siRNA rescue; kinase-dead Raf-1 reconstitution; MST2 dimerization and phosphorylation assays; apoptosis assays\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic rescue with kinase-dead Raf-1 and MST2 siRNA, single lab; companion to the full paper establishing the Raf-1/MST2 interaction\",\n      \"pmids\": [\"15701972\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CNK1 mediates Src-dependent tyrosine phosphorylation and activation of Raf-1 by forming a trimeric complex with preactivated Raf-1 and activated Src; CNK1 regulates Raf-1 activation in a concentration-dependent manner typical of a scaffold protein; CNK1 knockdown by siRNA interferes with Src-dependent ERK activation.\",\n      \"method\": \"Co-immunoprecipitation; CNK1 siRNA knockdown; ERK activation assays; scaffold dose-response analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and siRNA with functional readout, single lab\",\n      \"pmids\": [\"15845549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Raf-1 contains an N-terminal autoinhibitory domain; interaction of this domain with the catalytic domain is blocked by active H-Ras binding; Raf-1 and B-Raf use distinct autoregulatory mechanisms—Raf-1 requires regulated S338 phosphorylation while B-Raf has constitutive S445 phosphorylation.\",\n      \"method\": \"Co-immunoprecipitation of regulatory and catalytic domains; kinase activity assays; mutagenesis of S338/S445; active H-Ras co-expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain co-IP and mutagenesis with kinase activity readouts, single lab\",\n      \"pmids\": [\"15710605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"HCV NS5A binds to the C-terminal domain of NS5A and associates with Raf-1, colocalizing with Raf-1 in the HCV replication complex; NS5A-Raf-1 interaction increases Raf-1 phosphorylation at S338; Raf-1 inhibition by BAY43-9006 or siRNA knockdown attenuates HCV replication.\",\n      \"method\": \"Co-immunoprecipitation; confocal colocalization; phospho-S338 assay; small molecule and siRNA inhibition of Raf-1; viral replication assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, colocalization, and functional knockdown, single lab\",\n      \"pmids\": [\"16405965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PAK5 directly associates with Raf-1 (but not A-Raf or B-Raf), phosphorylates Raf-1 at S338, activates Raf-1 kinase activity, and targets a subpopulation of Raf-1 to mitochondria.\",\n      \"method\": \"Co-immunoprecipitation; in vitro S338 phosphorylation assay; subcellular fractionation to mitochondria; kinase activity assay\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, in vitro kinase assay, and subcellular fractionation, single lab\",\n      \"pmids\": [\"18465753\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"C-Raf paradoxically inhibits B-Raf(V600E) kinase activity by forming B-Raf(V600E)-C-Raf complexes; this inhibitory effect is specific to C-Raf among Raf family members; impaired C-Raf binding to B-Raf(V600E) elevates oncogenic potential; oncogenic Ras and sorafenib stabilize B-Raf(V600E)-C-Raf complexes, impairing MAPK activation.\",\n      \"method\": \"Co-immunoprecipitation; B-Raf/C-Raf interaction mutants; ERK phosphorylation assays; proliferation assays; C-Raf ectopic expression and depletion\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple co-IP experiments with binding mutants, functional proliferation and signaling readouts, single lab but comprehensive\",\n      \"pmids\": [\"19917255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Raf-1 functions as an endogenous inhibitor of the Rho-dependent kinase Rok-alpha in the context of a Ras-induced Raf-1:Rok-alpha complex; Raf-1-induced Rok-alpha inhibition allows STAT3 phosphorylation and Myc expression, promoting dedifferentiation in Ras-induced skin tumors; this is kinase-independent.\",\n      \"method\": \"Conditional Raf-1 knockout in Ras-induced skin tumors; Rok-alpha activity and co-IP assays; STAT3 phosphorylation and Myc expression analysis; kinase-dead Raf-1 rescue\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional tumor knockout with molecular mechanistic readouts and kinase-dead validation, single lab but comprehensive in vivo/in vitro approach\",\n      \"pmids\": [\"19647225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PDE8A associates with Raf-1 with picomolar affinity; the PDE8A binding site on Raf-1 maps to amino acids 454-465 of PDE8A; PDE8A protects Raf-1 from PKA-mediated inhibitory phosphorylation at S259, thereby enhancing Raf-1-stimulated ERK signaling; disruption of this interaction reduces ERK activation and the cellular response to EGF.\",\n      \"method\": \"Co-immunoprecipitation; affinity measurement; peptide array mapping; cell-permeable disrupting peptide; catalytically inactive PDE8A dominant negative; PDE8A−/− mice; Drosophila PDE8 deletion\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (co-IP, peptide array, disrupting peptide, genetic knockout in mice and flies), single lab but comprehensive\",\n      \"pmids\": [\"23509299\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Raf-1 regulates both the MST2-LATS and MEK-ERK pathways through competing protein interactions; Akt phosphorylation of MST2 and LATS1 feedback phosphorylation of Raf-1 Ser259 create signaling switches; Raf-1 Ser259 mutation simultaneously drives both apoptosis (MST2) and proliferation (MEK), but concomitant MST2 downregulation switches the outcome to proliferation and transformation.\",\n      \"method\": \"Mathematical modelling combined with experimental validation; Raf-1 S259 mutant; MST2 knockdown; Akt phosphorylation assays; LATS1 kinase assays; apoptosis and proliferation readouts\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — integrated mathematical and experimental approach with multiple genetic tools and functional readouts, single lab\",\n      \"pmids\": [\"24929361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP7 deubiquitinates Raf-1 by binding to the PVDS motif in the CR2 region of Raf-1; USP7 decreases K6, K11, K27, K33, and K48-linked polyubiquitination of Raf-1 and reduces threonine phosphorylation of Raf-1, thereby inhibiting ERK1/2 pathway activation, G2/M transition, and cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assays; USP7 DUB activity assays; phosphorylation assays; cell cycle analysis; proliferation assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, ubiquitination mapping, and functional knockdown with multiple readouts, single lab\",\n      \"pmids\": [\"35948545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The C-terminal 14-3-3 binding site of Raf-1 (S621) acts as an activation switch; mutations preventing 14-3-3 binding at S621 render Raf-1 inactive by specifically disrupting its capacity to bind ATP; 14-3-3 proteins function as critical cofactors that maintain Raf-1 in an ATP-binding-competent conformation.\",\n      \"method\": \"S621 mutagenesis; ATP-binding assays; 14-3-3 phosphopeptide competition; MEK binding assays; in vitro kinase activity\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic mutagenesis with ATP-binding and MEK-binding dissection, single lab\",\n      \"pmids\": [\"19595761\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RAF1 (C-Raf) is a serine/threonine kinase that functions as the canonical link between RAS-GTP (which recruits and promotes RAF1 oligomerization at the plasma membrane) and the MEK-ERK cascade, but also exerts essential kinase-independent functions by physically suppressing the pro-apoptotic kinases MST2 and Rok-alpha; its activity is controlled by a multi-layered phosphorylation code (activating: S338 by PAK1/3, Y341 by Src; inhibitory: S259 by PKA and LATS1, feedback sites by ERK), regulated by 14-3-3 proteins that maintain its ATP-binding-competent conformation, modulated by interacting proteins including RKIP (competitive inhibitor of MEK binding), BAG-1 and PDE8A (activators), and targeted to mitochondria by Bcl-2 where it phosphorylates BAD to suppress apoptosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RAF1 (c-Raf-1) is a serine/threonine kinase that serves as the proximal activator of the MEK-ERK cascade, directly phosphorylating and activating MEK (MAPKK) as its principal physiological substrate with highly restricted specificity [#0, #7]. Its activation is initiated when GTP-loaded Ras binds the RAF1 Ras-binding domain in an effector-domain-dependent manner, recruiting RAF1 and bridging it to MEK-1 to assemble a Ras-RAF1-MEK signaling complex at the plasma membrane [#1, #5, #8]. Activation is gated by a multi-layered phosphorylation code: it requires dephosphorylation of the inhibitory site S259 by PP1/PP2A as an early licensing step that permits membrane recruitment, followed by activating phosphorylation at S338 (by PAK kinases, downstream of Rac/Cdc42) and at Y341 (by Src), with oligomerization itself sufficient to drive activation [#23, #24, #17, #19, #12, #13]. RAF1 is held in an autoinhibited conformation by its N-terminal regulatory/cysteine-rich domain, relieved upon Ras binding, and 14-3-3 proteins bind bivalently to its N- and C-termini to maintain the kinase in an ATP-binding-competent state [#16, #39, #14, #47]. The pathway is negatively regulated by PKA, which inhibits RAF1 chiefly through S259 phosphorylation, by RKIP, which competitively blocks the RAF1-MEK interaction and prevents PAK/Src phosphorylation, and by ERK-mediated feedback phosphorylation that uncouples RAF1 from Ras [#27, #18, #35, #33]. Beyond catalysis, gene-targeting in mice established an essential kinase-independent function: RAF1 promotes cell survival by physically binding and suppressing the pro-apoptotic kinases MST2 and ASK1, and by spatially restraining the Rho-effector kinase Rok-alpha to control cytoskeletal organization and migration [#26, #37, #31, #30, #43]. RAF1 also localizes to mitochondria via Bcl-2, where it phosphorylates BAD to block apoptosis, and integrates pro-survival and pro-proliferative outputs through competing MST2-LATS and MEK-ERK interactions [#10, #45].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Established RAF1's catalytic output by identifying its first physiological substrate, placing it as the direct upstream activator of the MAP kinase cascade.\",\n      \"evidence\": \"In vitro kinase assay with purified c-Raf-1 and MAPKK; v-raf-transformed cell analysis\",\n      \"pmids\": [\"1322500\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how RAF1 itself is activated\", \"Substrate specificity beyond MAPKK not yet quantified\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Connected RAF1 to upstream Ras, showing GTP-loaded Ras directly binds RAF1 to nucleate a signaling complex, and that PKC-alpha provides a Ras-independent activation input.\",\n      \"evidence\": \"Ras-variant affinity pulldowns with MAPKK activity assays; in vitro PKC-alpha phosphorylation and S499/S259 mutagenesis\",\n      \"pmids\": [\"8503013\", \"8321321\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which Ras binding activates RAF1 catalysis not resolved\", \"Membrane recruitment step not yet defined\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Defined the regulatory protein and structural requirements for RAF1 activity — 14-3-3 binding, complex assembly with MEK-1, Src/Fyn association, and the architecture required for activation.\",\n      \"evidence\": \"Yeast two-hybrid and Xenopus oocyte assays; Ras-MEK1 bridging pulldowns; co-IP with Src/Fyn SH2 domains; enzymatic Km characterization\",\n      \"pmids\": [\"7935795\", \"7969158\", \"7517401\", \"8108400\", \"7935389\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific activating phosphosites not yet mapped\", \"How 14-3-3 binding affects catalysis mechanistically unresolved\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Dissected the mutational requirements for RAF1 activation and showed phosphatases and chaperone/14-3-3 dynamically regulate the active state.\",\n      \"evidence\": \"Purified-membrane reconstitution with RAF1 mutant panel; in vitro phosphatase treatment with 14-3-3/HSP90 protection\",\n      \"pmids\": [\"7623807\", \"7604263\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Order of phosphorylation events not established\", \"Identity of membrane phosphatases not defined\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Demonstrated that oligomerization per se drives RAF1 activation and revealed a mitochondrial, MEK-independent pro-survival branch via BAD phosphorylation.\",\n      \"evidence\": \"Coumermycin- and FKBP12/FK506-induced dimerization systems; Bcl-2-mediated mitochondrial targeting with BAD phosphorylation and apoptosis assays; BAG-1 co-IP and in vitro activation\",\n      \"pmids\": [\"8774884\", \"8774885\", \"8929532\", \"8692945\", \"8637718\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How dimerization mechanistically activates the kinase domain not resolved\", \"Relationship between membrane and mitochondrial pools unclear\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Provided the structural basis for 14-3-3/RAF1 recognition by mapping the binding determinant to the 14-3-3 amphipathic groove.\",\n      \"evidence\": \"Crystal structure of 14-3-3 zeta with charge-reversal mutagenesis (K49E) and binding assays\",\n      \"pmids\": [\"9153224\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RAF1-side structural determinants not crystallized here\", \"Functional consequence on RAF1 conformation addressed only later\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Established intramolecular autoinhibition and identified PAK3-mediated S338 phosphorylation as a positive activating input linking Rho-family GTPases to RAF1.\",\n      \"evidence\": \"Cysteine-rich domain and Y340D mutagenesis with kinase assays; in vitro and in vivo PAK3 phosphorylation of S338\",\n      \"pmids\": [\"9689060\", \"9823899\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How autoinhibition is relieved by Ras not fully mechanistically resolved\", \"Which PAK isoform acts physiologically not yet settled\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Integrated the activating phosphorylation code (S338/Y341, Ras-dependent), identified RKIP as a competitive RAF1-MEK inhibitor, and showed 14-3-3 maintains the active conformation.\",\n      \"evidence\": \"Phospho-specific antisera with Ras/Src co-expression and mutagenesis; RKIP yeast two-hybrid, co-IP, and gain/loss of function; phosphatidylserine and phosphopeptide competition assays\",\n      \"pmids\": [\"10205168\", \"10490027\", \"10445849\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Spatial coordination of S338 and Y341 phosphorylation not fully resolved\", \"RKIP regulation by upstream signals not yet defined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Placed S259 dephosphorylation upstream of membrane recruitment in the activation sequence and confirmed direct PAK1-RAF1 association driving S338 phosphorylation.\",\n      \"evidence\": \"Phospho-S259 antibody with phosphatase inhibitors, S259A and RAF1-CAAX constructs, cell fractionation; PAK1 co-IP with mutant panel\",\n      \"pmids\": [\"11756411\", \"11494123\", \"11733498\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphatase recruitment mechanism to S259 not fully defined\", \"Some co-IP findings from single labs\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Genetically separated RAF1's catalytic and non-catalytic roles, revealing an essential kinase-independent survival function despite dispensability of MEK kinase activity for ERK activation and development.\",\n      \"evidence\": \"Knockout and Y340F/Y341F kinase-dead knock-in mice; in vitro MEK kinase and ERK activation assays; embryonic phenotyping\",\n      \"pmids\": [\"11296227\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular identity of the kinase-independent effector not defined in this study\", \"Mechanism of vascular/apoptosis defect unresolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined PKA-mediated inhibition acting primarily through S259 and refined S259 dephosphorylation as the rate-limiting activation step that enhances Ras binding and membrane recruitment.\",\n      \"evidence\": \"In vitro/in vivo PKA phosphorylation site mapping with S259A/S43A mutants; phospho-S259 antibody with Ras binding and MEK activation assays\",\n      \"pmids\": [\"11971957\", \"11782426\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cross-talk between PKA and the dephosphorylation machinery not fully mapped\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified the kinase-independent effectors of RAF1's survival/cytoskeletal roles — spatial suppression of Rok-alpha and suppression of cardiac ASK1 — and a keratin association.\",\n      \"evidence\": \"Conditional RAF1 ablation with kinase-dead rescue, Rok-alpha localization/activity, cytoskeleton imaging; cardiac-specific knockout with ASK1 double-knockout rescue; K8/K18 co-IP and phosphorylation\",\n      \"pmids\": [\"15753127\", \"15467832\", \"15314064\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of RAF1-Rok-alpha and RAF1-ASK1 binding not resolved\", \"Keratin findings from single lab\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Established ERK feedback phosphorylation as a desensitization mechanism, characterized RAF1-MST2 suppression, and defined RAF1-B-Raf hetero-oligomerization and additional scaffolds/regulators.\",\n      \"evidence\": \"Mass-spec phosphosite mapping with Pin1/PP2A functional assays; RAF1 knockout/MST2 siRNA rescue; B-Raf/C-Raf co-IP and 14-3-3 analysis; CNK1 scaffold and RKIP phosphomimetic studies\",\n      \"pmids\": [\"15664191\", \"15701972\", \"16364920\", \"15845549\", \"15886202\", \"15710605\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Several interaction findings rest on single-lab co-IP\", \"Stoichiometry of hetero-oligomers in cells not resolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Revealed RAF1 as a paradoxical inhibitor of oncogenic B-Raf(V600E), a kinase-independent suppressor of Rok-alpha promoting tumor dedifferentiation, and clarified the 14-3-3/S621 ATP-binding switch.\",\n      \"evidence\": \"B-Raf/C-Raf co-IP with binding mutants and proliferation assays; conditional RAF1 knockout in Ras-induced skin tumors with STAT3/Myc readouts; S621 mutagenesis with ATP-binding assays\",\n      \"pmids\": [\"19917255\", \"19647225\", \"19595761\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of paradoxical B-Raf inhibition not fully defined\", \"S621 ATP-binding mechanism from single lab\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Synthesized RAF1 as a node balancing competing MST2-LATS apoptotic and MEK-ERK proliferative outputs through S259-centered switches.\",\n      \"evidence\": \"Mathematical modelling with RAF1 S259 mutant, MST2 knockdown, Akt/LATS1 phosphorylation, and apoptosis/proliferation readouts\",\n      \"pmids\": [\"24929361\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative thresholds for switch behavior context-dependent\", \"In vivo relevance of competing-interaction model not tested here\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified post-translational control of RAF1 stability and ERK output via USP7-mediated deubiquitination.\",\n      \"evidence\": \"Co-IP, ubiquitin-linkage mapping, DUB assays, and cell cycle/proliferation analysis\",\n      \"pmids\": [\"35948545\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study\", \"Physiological/in vivo role of USP7-RAF1 axis not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple regulatory inputs (phosphorylation code, 14-3-3, oligomerization, autoinhibition) are integrated into a single activation trajectory at atomic resolution, and how the catalytic versus scaffolding pools are partitioned in vivo, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No full-length active RAF1 structure in the corpus\", \"Spatial/temporal partitioning of kinase-dependent vs kinase-independent functions not quantified\", \"Mechanism coupling S259 dephosphorylation to conformational activation not defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 7, 10, 17, 19]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 10, 17]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [47]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [37, 30, 31, 43]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 5, 24, 28]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [10, 41]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [20]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 19, 33]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [10, 31, 37, 45]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [46]}\n    ],\n    \"complexes\": [\"Ras-RAF1-MEK1 complex\", \"B-Raf/C-Raf heterodimer\", \"RAF1:Rok-alpha complex\", \"RAF1:MST2 complex\"],\n    \"partners\": [\"RAS\", \"MAP2K1\", \"BRAF\", \"YWHAZ\", \"RKIP\", \"MST2\", \"ROCK1\", \"PDE8A\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}