Affinage

RAF1

RAF proto-oncogene serine/threonine-protein kinase · UniProt P04049

Length
648 aa
Mass
73.1 kDa
Annotated
2026-06-10
100 papers in source corpus 47 papers cited in narrative 48 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 7/7 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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).

Mechanistic history

Synthesis pass · year-by-year structured walk · 16 steps
  1. 1992 High

    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

    PMID:1322500

    Open questions at the time
    • Did not define how RAF1 itself is activated
    • Substrate specificity beyond MAPKK not yet quantified
  2. 1993 High

    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

    PMID:8321321 PMID:8503013

    Open questions at the time
    • Mechanism by which Ras binding activates RAF1 catalysis not resolved
    • Membrane recruitment step not yet defined
  3. 1994 High

    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

    PMID:7517401 PMID:7935389 PMID:7935795 PMID:7969158 PMID:8108400

    Open questions at the time
    • Specific activating phosphosites not yet mapped
    • How 14-3-3 binding affects catalysis mechanistically unresolved
  4. 1995 High

    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

    PMID:7604263 PMID:7623807

    Open questions at the time
    • Order of phosphorylation events not established
    • Identity of membrane phosphatases not defined
  5. 1996 High

    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

    PMID:8637718 PMID:8692945 PMID:8774884 PMID:8774885 PMID:8929532

    Open questions at the time
    • How dimerization mechanistically activates the kinase domain not resolved
    • Relationship between membrane and mitochondrial pools unclear
  6. 1997 High

    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

    PMID:9153224

    Open questions at the time
    • RAF1-side structural determinants not crystallized here
    • Functional consequence on RAF1 conformation addressed only later
  7. 1998 High

    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

    PMID:9689060 PMID:9823899

    Open questions at the time
    • How autoinhibition is relieved by Ras not fully mechanistically resolved
    • Which PAK isoform acts physiologically not yet settled
  8. 1999 High

    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

    PMID:10205168 PMID:10445849 PMID:10490027

    Open questions at the time
    • Spatial coordination of S338 and Y341 phosphorylation not fully resolved
    • RKIP regulation by upstream signals not yet defined
  9. 2001 High

    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

    PMID:11494123 PMID:11733498 PMID:11756411

    Open questions at the time
    • Phosphatase recruitment mechanism to S259 not fully defined
    • Some co-IP findings from single labs
  10. 2001 High

    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

    PMID:11296227

    Open questions at the time
    • Molecular identity of the kinase-independent effector not defined in this study
    • Mechanism of vascular/apoptosis defect unresolved
  11. 2002 High

    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

    PMID:11782426 PMID:11971957

    Open questions at the time
    • Cross-talk between PKA and the dephosphorylation machinery not fully mapped
  12. 2004 High

    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

    PMID:15314064 PMID:15467832 PMID:15753127

    Open questions at the time
    • Structural basis of RAF1-Rok-alpha and RAF1-ASK1 binding not resolved
    • Keratin findings from single lab
  13. 2005 High

    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

    PMID:15664191 PMID:15701972 PMID:15710605 PMID:15845549 PMID:15886202 PMID:16364920

    Open questions at the time
    • Several interaction findings rest on single-lab co-IP
    • Stoichiometry of hetero-oligomers in cells not resolved
  14. 2009 High

    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

    PMID:19595761 PMID:19647225 PMID:19917255

    Open questions at the time
    • Structural basis of paradoxical B-Raf inhibition not fully defined
    • S621 ATP-binding mechanism from single lab
  15. 2014 High

    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

    PMID:24929361

    Open questions at the time
    • Quantitative thresholds for switch behavior context-dependent
    • In vivo relevance of competing-interaction model not tested here
  16. 2022 Medium

    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

    PMID:35948545

    Open questions at the time
    • Single-lab study
    • Physiological/in vivo role of USP7-RAF1 axis not established

Open questions

Synthesis pass · forward-looking unresolved questions
  • 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.
  • 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

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140096 catalytic activity, acting on a protein 5 GO:0098772 molecular function regulator activity 4 GO:0016740 transferase activity 3 GO:0140657 ATP-dependent activity 1
Localization
GO:0005886 plasma membrane 4 GO:0005739 mitochondrion 2 GO:0005829 cytosol 1
Pathway
R-HSA-162582 Signal Transduction 4 R-HSA-5357801 Programmed Cell Death 4 R-HSA-1640170 Cell Cycle 1
Partners
BRAFMAP2K1MST2PDE8ARASRKIPROCK1YWHAZ
Complex memberships
B-Raf/C-Raf heterodimerRAF1:MST2 complexRAF1:Rok-alpha complexRas-RAF1-MEK1 complex

Evidence

Reading pass · 48 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1992 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. In vitro kinase assay with purified c-Raf-1 and partially purified MAPKK; phosphatase 2A inactivation/reactivation assay; v-raf-transformed cell analysis Nature High 1322500
1993 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. 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 Science High 8503013
1993 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. 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 Nature High 8321321
1994 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. Yeast two-hybrid screen; Xenopus oocyte functional assay; dominant-negative Raf-1 epistasis Nature High 7935795
1994 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. In vitro phosphorylation assays with purified PKA and Raf-1 proteins; kinase domain fragment analysis; sequential activation/inhibition assays Molecular and cellular biology High 7935389
1994 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. Immobilized Ras pulldown from NIH 3T3 cell lysates; MEK-1 and MEK-2 immunodetection; exogenous RAF-1 addition to lysates; MEK-1 mutant analysis Molecular and cellular biology High 7969158
1994 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. Co-immunoprecipitation; SH2 domain binding assay; baculovirus/Sf9 co-expression; site-directed mutagenesis of Src SH2 Arg175; kinase activity assay The Journal of biological chemistry Medium 7517401
1994 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. In vitro kinase assay with purified baculovirus-expressed His-tagged c-Raf-1; Km determination; substrate panel screening; gel-filtration chromatography Proceedings of the National Academy of Sciences of the United States of America High 8108400
1995 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. In vitro activation assay using purified plasma membranes from transformed cells; panel of Raf-1 point and deletion mutants expressed in baculovirus; kinase assays Molecular and cellular biology High 7623807
1995 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. 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 Science Medium 7604263
1996 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. GFP-Raf-1 fusion protein localization; mitochondrial and plasma membrane targeting constructs; BAD phosphorylation assay; cell death assays; kinase-dead Raf-1 mutant Cell High 8929532
1996 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. Co-immunoprecipitation from mammalian cells and baculovirus-infected insect cells; in vitro kinase activation assay with bacterially produced BAG-1; yeast two-hybrid Proceedings of the National Academy of Sciences of the United States of America Medium 8692945
1996 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. Chemical dimerization (coumermycin/gyrase B fusion); MAP kinase cascade activation assay in cells; absence-of-membrane-component controls Nature High 8774884
1996 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. Chemical dimerization (FKBP12-FK1012A system); Raf kinase activity assay; dominant-negative Ras epistasis to show Ras dependence Nature High 8774885
1996 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. In vivo and in vitro binding assays; mutant Raf-1 fragments; co-expression of activated Ras Oncogene Medium 8637718
1997 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. Crystal structure of 14-3-3 zeta; charge-reversal mutagenesis (K49E, R56E, R60E); in vitro binding assays; circular dichroism; partial proteolysis The Journal of biological chemistry High 9153224
1998 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. Site-directed mutagenesis of cysteine-rich domain and Y340D; kinase activity assays; regulatory domain co-expression inhibition experiments Proceedings of the National Academy of Sciences of the United States of America Medium 9689060
1998 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. In vitro kinase assay (PAK3 phosphorylating Raf-1); in vivo phosphorylation assays; phospho-specific antibodies Nature High 9823899
1999 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. Yeast two-hybrid screen; in vitro binding assays; co-immunoprecipitation; confocal microscopy colocalization; antisense RNA and antibody microinjection; reporter gene assays Nature High 10490027
1999 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. Phospho-specific antisera; co-expression of oncogenic Ras and activated Src; mutagenesis of S338, S339, Y340, Y341; kinase activity assays The EMBO journal High 10205168
1999 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. In vitro incubation of Raf-1 with phosphatidylserine; phosphopeptide competition assays; kinase activity measurements Oncogene Medium 10445849
2000 MEKK1 binds endogenous ERK2, MEK1, and Raf-1, suggesting it can assemble all three proteins of the ERK2 MAP kinase module into a complex. Co-immunoprecipitation of endogenous proteins The Journal of biological chemistry Low 10969079
2000 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. GST-Raf-1 pulldown; co-immunoprecipitation; in vitro kinase assays; MEK inhibitor controls; conditional estrogen-regulated Raf-1 mutant system FASEB journal Medium 11023985
2001 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. In vitro Raf-1 activation assay with serine phosphatase inhibitors; S259A Raf-1 mutant; sucrose gradient fractionation of plasma membrane microdomains Oncogene Medium 11494123
2001 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. Phospho-S259 antibody; pharmacological phosphatase inhibition; S259A and Raf-1-CAAX constructs; cell fractionation The Journal of biological chemistry High 11756411
2001 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. Co-immunoprecipitation under physiological conditions; PAK mutant analysis; in vitro phosphorylation of Raf-1 S338; MAPK activation assays The Journal of biological chemistry Medium 11733498
2001 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. Gene targeting (knockout and Y340F/Y341F knock-in mice); in vitro MEK kinase assay; embryonic phenotype analysis; ERK activation assay The EMBO journal High 11296227
2002 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. In vitro PKA phosphorylation mapping; in vivo cAMP stimulation; S259A, S43A mutants; ERK activation assays; cAMP kinetics correlated with ERK deactivation Molecular and cellular biology High 11971957
2002 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. Phospho-S259 antibody; S259A Raf-1 mutant; Ras binding assays; membrane recruitment assays; MEK activation assays The EMBO journal High 11782426
2003 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. Co-immunoprecipitation; RKIP S153 phosphorylation analysis; GRK-2 binding assays; cardiomyocyte functional assays; GPCR signaling readouts Nature High 14654844
2004 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. Conditional Raf-1 gene ablation; cell migration assays; actin/vimentin cytoskeleton imaging; Rok-alpha localization and activity assays; rescue with kinase-dead Raf-1 The Journal of cell biology High 15753127
2004 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. Cre-loxP cardiac-specific knockout; echocardiography; kinase activity assays (MEK, ERK, ASK1, JNK, p38); ASK1 double-knockout rescue The Journal of clinical investigation High 15467832
2004 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. Co-immunoprecipitation; kinase-dead and Ras-binding-defective Raf-1 mutants; in vivo and in vitro phosphorylation assays; 14-3-3 binding-site mutants The Journal of cell biology Medium 15314064
2005 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. 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 Molecular cell High 15664191
2005 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. Co-immunoprecipitation; Ras-dependence assays; kinase activity assays; 14-3-3 binding analysis; activation segment phosphorylation assays Molecular cell High 16364920
2005 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. RKIP overexpression and depletion; Raf-1 phosphomimetic mutants; PAK and Src kinase co-IP; MEK/ERK and DNA synthesis assays The Journal of biological chemistry Medium 15886202
2005 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. Conditional gene ablation; wound healing assay; in vitro cell migration; actin/vimentin cytoskeleton analysis; Rok-alpha localization; kinase-dead rescue The Journal of cell biology High 15753127
2005 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. Raf-1 knockout cells; MST2 siRNA rescue; kinase-dead Raf-1 reconstitution; MST2 dimerization and phosphorylation assays; apoptosis assays Cell cycle Medium 15701972
2005 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. Co-immunoprecipitation; CNK1 siRNA knockdown; ERK activation assays; scaffold dose-response analysis The Journal of biological chemistry Medium 15845549
2005 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. Co-immunoprecipitation of regulatory and catalytic domains; kinase activity assays; mutagenesis of S338/S445; active H-Ras co-expression The Journal of biological chemistry Medium 15710605
2005 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. Co-immunoprecipitation; confocal colocalization; phospho-S338 assay; small molecule and siRNA inhibition of Raf-1; viral replication assay FEBS letters Medium 16405965
2008 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. Co-immunoprecipitation; in vitro S338 phosphorylation assay; subcellular fractionation to mitochondria; kinase activity assay Journal of cellular biochemistry Medium 18465753
2009 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. Co-immunoprecipitation; B-Raf/C-Raf interaction mutants; ERK phosphorylation assays; proliferation assays; C-Raf ectopic expression and depletion Molecular cell High 19917255
2009 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. 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 Cancer cell High 19647225
2013 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. Co-immunoprecipitation; affinity measurement; peptide array mapping; cell-permeable disrupting peptide; catalytically inactive PDE8A dominant negative; PDE8A−/− mice; Drosophila PDE8 deletion Proceedings of the National Academy of Sciences of the United States of America High 23509299
2014 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. Mathematical modelling combined with experimental validation; Raf-1 S259 mutant; MST2 knockdown; Akt phosphorylation assays; LATS1 kinase assays; apoptosis and proliferation readouts Nature cell biology High 24929361
2022 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. Co-immunoprecipitation; ubiquitination assays; USP7 DUB activity assays; phosphorylation assays; cell cycle analysis; proliferation assays Cell death & disease Medium 35948545
2009 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. S621 mutagenesis; ATP-binding assays; 14-3-3 phosphopeptide competition; MEK binding assays; in vitro kinase activity Cellular signalling Medium 19595761

Source papers

Stage 0 corpus · 100 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
1993 Protein kinase C alpha activates RAF-1 by direct phosphorylation. Nature 1243 8321321
1992 Raf-1 activates MAP kinase-kinase. Nature 1215 1322500
1993 Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase. Science (New York, N.Y.) 944 8503013
1999 Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP. Nature 723 10490027
1996 Bcl-2 targets the protein kinase Raf-1 to mitochondria. Cell 720 8929532
1997 The complexity of Raf-1 regulation. Current opinion in cell biology 541 9069260
2005 Regulation of Raf-1 by direct feedback phosphorylation. Molecular cell 501 15664191
1991 Raf-1 protein kinase is required for growth of induced NIH/3T3 cells. Nature 490 1992343
1998 The protein kinase Pak3 positively regulates Raf-1 activity through phosphorylation of serine 338. Nature 375 9823899
1999 Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation. The EMBO journal 371 10205168
2005 Wild-type and mutant B-RAF activate C-RAF through distinct mechanisms involving heterodimerization. Molecular cell 346 16364920
2002 Modulation of p53, ErbB1, ErbB2, and Raf-1 expression in lung cancer cells by depsipeptide FR901228. Journal of the National Cancer Institute 336 11929951
1994 Activation of Raf-1 by 14-3-3 proteins. Nature 334 7935795
1996 Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1. Proceedings of the National Academy of Sciences of the United States of America 332 8692945
2003 Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2. Nature 319 14654844
1994 Mechanism of inhibition of Raf-1 by protein kinase A. Molecular and cellular biology 293 7935389
1996 Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization. Nature 271 8774884
2001 MEK kinase activity is not necessary for Raf-1 function. The EMBO journal 266 11296227
1990 Mutational activation of c-raf-1 and definition of the minimal transforming sequence. Molecular and cellular biology 241 2188091
2002 Regulation of Raf-1 activation and signalling by dephosphorylation. The EMBO journal 239 11782426
2003 Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas. Carcinogenesis 226 14688025
1989 Effect of antisense c-raf-1 on tumorigenicity and radiation sensitivity of a human squamous carcinoma. Science (New York, N.Y.) 211 2466340
1996 Oligomerization activates c-Raf-1 through a Ras-dependent mechanism. Nature 206 8774885
2002 Cyclic AMP-dependent kinase regulates Raf-1 kinase mainly by phosphorylation of serine 259. Molecular and cellular biology 194 11971957
1995 Reversal of Raf-1 activation by purified and membrane-associated protein phosphatases. Science (New York, N.Y.) 188 7604263
2005 Raf-1 regulates Rho signaling and cell migration. The Journal of cell biology 171 15753127
1991 Erythropoietin induces Raf-1 activation and Raf-1 is required for erythropoietin-mediated proliferation. The Journal of biological chemistry 169 1869534
2001 Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions. Oncogene 163 11494123
1998 Autoregulation of the Raf-1 serine/threonine kinase. Proceedings of the National Academy of Sciences of the United States of America 163 9689060
2004 Cardiac-specific disruption of the c-raf-1 gene induces cardiac dysfunction and apoptosis. The Journal of clinical investigation 161 15467832
2004 Raf-1 kinase is required for cardiac hypertrophy and cardiomyocyte survival in response to pressure overload. Circulation 154 15289381
1997 Induction of cell proliferation in quiescent NIH 3T3 cells by oncogenic c-Raf-1. Molecular and cellular biology 152 9111327
2002 Untying the regulation of the Raf-1 kinase. Archives of biochemistry and biophysics 151 12127063
1996 Activated Ras displaces 14-3-3 protein from the amino terminus of c-Raf-1. Oncogene 135 8637718
1997 Raf-1 kinase and exoenzyme S interact with 14-3-3zeta through a common site involving lysine 49. The Journal of biological chemistry 133 9153224
1995 Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro. Molecular and cellular biology 133 7623807
2014 Protein interaction switches coordinate Raf-1 and MST2/Hippo signalling. Nature cell biology 127 24929361
2002 Design and discovery of small molecules targeting raf-1 kinase. Current pharmaceutical design 127 12369855
2011 C-Raf is required for the initiation of lung cancer by K-Ras(G12D). Cancer discovery 125 22043453
2005 B-Raf and Raf-1 are regulated by distinct autoregulatory mechanisms. The Journal of biological chemistry 120 15710605
2005 Second nature: biological functions of the Raf-1 "kinase". FEBS letters 117 15943972
2005 Raf kinase inhibitory protein regulates Raf-1 but not B-Raf kinase activation. The Journal of biological chemistry 116 15886202
1993 Raf-1 and p21v-ras cooperate in the activation of mitogen-activated protein kinase. Proceedings of the National Academy of Sciences of the United States of America 113 8390681
1994 RAS and RAF-1 form a signalling complex with MEK-1 but not MEK-2. Molecular and cellular biology 110 7969158
2001 Interaction between active Pak1 and Raf-1 is necessary for phosphorylation and activation of Raf-1. The Journal of biological chemistry 107 11733498
1990 Insulin activates the Raf-1 protein kinase. The Journal of biological chemistry 105 2197271
2001 Dephosphorylation of Ser-259 regulates Raf-1 membrane association. The Journal of biological chemistry 102 11756411
2000 MEKK1 binds raf-1 and the ERK2 cascade components. The Journal of biological chemistry 101 10969079
2008 Insulin stimulates primary beta-cell proliferation via Raf-1 kinase. Endocrinology 91 18202127
2009 Raf-1 addiction in Ras-induced skin carcinogenesis. Cancer cell 90 19647225
1987 Rat c-raf oncogene activation by a rearrangement that produces a fused protein. Molecular and cellular biology 88 3550433
2019 Complete Regression of Advanced Pancreatic Ductal Adenocarcinomas upon Combined Inhibition of EGFR and C-RAF. Cancer cell 87 30975481
1994 Raf-1 interacts with Fyn and Src in a non-phosphotyrosine-dependent manner. The Journal of biological chemistry 83 7517401
1996 Activation of raf-1, MEK, and MAP kinase in prolactin responsive mammary cells. Breast cancer research and treatment 82 8879680
1994 Enzymatic characteristics of the c-Raf-1 protein kinase. Proceedings of the National Academy of Sciences of the United States of America 77 8108400
2000 Regulation of the Raf-1 kinase domain by phosphorylation and 14-3-3 association. The Biochemical journal 75 10998357
2000 Recruitment and activation of Raf-1 kinase by nitric oxide-activated Ras. Biochemistry 74 10933809
2006 ERK and beyond: insights from B-Raf and Raf-1 conditional knockouts. Cell cycle (Georgetown, Tex.) 71 16861903
1990 The Raf-1 kinase as a transducer of mitogenic signals. Cancer cells (Cold Spring Harbor, N.Y. : 1989) 71 2150916
2005 Raf-1 kinase associates with Hepatitis C virus NS5A and regulates viral replication. FEBS letters 67 16405965
2002 Raf-1 antagonizes erythroid differentiation by restraining caspase activation. The Journal of experimental medicine 67 12438425
2006 Rheb inhibits C-raf activity and B-raf/C-raf heterodimerization. The Journal of biological chemistry 66 16803888
1998 Abrogation of c-Raf expression induces apoptosis in tumor cells. Oncogene 66 9583688
2008 Constitutive activation of Raf-1 induces glioma formation in mice. Neoplasia (New York, N.Y.) 64 18472967
1995 Mechanisms regulating Raf-1 activity in signal transduction pathways. Molecular reproduction and development 62 8607983
1994 The Raf-1 serine/threonine protein kinase. Seminars in cancer biology 62 7803760
2008 GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis. Journal of cellular physiology 61 18064632
2001 Association of c-Raf expression with survival and its targeting with antisense oligonucleotides in ovarian cancer. British journal of cancer 61 11742498
2007 Phosphatase and feedback regulation of Raf-1 signaling. Cell cycle (Georgetown, Tex.) 58 17218791
2001 Protective role of Raf-1 in Salmonella-induced macrophage apoptosis. The Journal of experimental medicine 58 11157055
1999 Interactions of c-Raf-1 with phosphatidylserine and 14-3-3. Oncogene 58 10445849
2009 C-Raf inhibits MAPK activation and transformation by B-Raf(V600E). Molecular cell 57 19917255
2009 Diacylglycerol kinase eta augments C-Raf activity and B-Raf/C-Raf heterodimerization. The Journal of biological chemistry 56 19710016
2000 The Raf-1 kinase associates with vimentin kinases and regulates the structure of vimentin filaments. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 55 11023985
2004 Cation diffusion facilitator proteins modulate Raf-1 activity. The Journal of biological chemistry 54 15096503
2003 Geldanamycin decreases Raf-1 and Akt levels and induces apoptosis in neuroblastomas. International journal of cancer 53 12471618
2020 The quaternary assembly of KRas4B with Raf-1 at the membrane. Computational and structural biotechnology journal 51 32257057
2005 Taming the Hippo: Raf-1 controls apoptosis by suppressing MST2/Hippo. Cell cycle (Georgetown, Tex.) 51 15701972
2004 Raf-1 activation disrupts its binding to keratins during cell stress. The Journal of cell biology 51 15314064
2002 Activation of c-Raf kinase by ultraviolet light. Regulation by retinoids. The Journal of biological chemistry 51 11971897
1995 Raf-1 N-terminal sequences necessary for Ras-Raf interaction and signal transduction. Molecular and cellular biology 51 7799948
2013 Phosphodiesterase-8A binds to and regulates Raf-1 kinase. Proceedings of the National Academy of Sciences of the United States of America 50 23509299
2008 p21 activated kinase 5 activates Raf-1 and targets it to mitochondria. Journal of cellular biochemistry 48 18465753
2006 A balance between Raf-1 and Fas expression sets the pace of erythroid differentiation. Blood 48 16527894
2018 Novel oncogene COPS3 interacts with Beclin1 and Raf-1 to regulate metastasis of osteosarcoma through autophagy. Journal of experimental & clinical cancer research : CR 47 29970115
2005 CNK1 is a scaffold protein that regulates Src-mediated Raf-1 activation. The Journal of biological chemistry 46 15845549
1989 Amplification of both c-myc and c-raf-1 oncogenes in a human osteosarcoma. Japanese journal of cancer research : Gann 46 2496060
2000 c-Raf regulates cell survival and retinal ganglion cell morphogenesis during neurogenesis. The Journal of neuroscience : the official journal of the Society for Neuroscience 45 10777790
1998 Bc1-2, Raf-1 and mitochondrial regulation of apoptosis. BioFactors (Oxford, England) 44 9699002
2006 The Raf-1 pathway: a molecular target for treatment of select neuroendocrine tumors? Anti-cancer drugs 43 16428931
2009 The C-terminus of Raf-1 acts as a 14-3-3-dependent activation switch. Cellular signalling 42 19595761
1993 H-ras and raf-1 cooperate in transformation of NIH3T3 fibroblasts. Oncogene 41 8361757
2008 Survival signaling by C-RAF: mitochondrial reactive oxygen species and Ca2+ are critical targets. Molecular and cellular biology 40 18212057
2006 Regulation of RKIP binding to the N-region of the Raf-1 kinase. FEBS letters 40 17097642
1999 Tangled webs: evidence of cross-talk between c-Raf-1 and Akt. Science's STKE : signal transduction knowledge environment 40 11865188
2012 Raf-1, actin dynamics, and abelson tyrosine kinase in human airway smooth muscle cells. American journal of respiratory cell and molecular biology 39 23087049
2002 Anti-(Raf-1) RNA aptamers that inhibit Ras-induced Raf-1 activation. European journal of biochemistry 39 11856330
2005 Raf-1 expression may influence progression to androgen insensitive prostate cancer. The Prostate 37 15666389
2022 USP7 regulates the ERK1/2 signaling pathway through deubiquitinating Raf-1 in lung adenocarcinoma. Cell death & disease 36 35948545
2009 C-Raf is associated with disease progression and cell proliferation in a subset of melanomas. Clinical cancer research : an official journal of the American Association for Cancer Research 36 19737955

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