Affinage

RAF1

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

Round 2 corrected
Length
648 aa
Mass
73.1 kDa
Annotated
2026-04-28
130 papers in source corpus 44 papers cited in narrative 43 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

RAF1 (c-Raf-1) is a serine/threonine-protein kinase that serves as a central signal transducer in the Ras-MAPK cascade, linking receptor-activated Ras-GTP to the phosphorylation and activation of MEK1/2 and thereby ERK signaling, while also exerting kinase-independent anti-apoptotic and cytoskeletal regulatory functions. Activation requires Ras-GTP-dependent membrane recruitment followed by a multi-step process involving PP2A-mediated dephosphorylation of the inhibitory Ser259 site, dimerization-facilitated autophosphorylation at Ser338, Src-family kinase phosphorylation at Tyr340/341, and stabilization by 14-3-3 proteins and the Hsp90–Cdc37 chaperone complex (PMID:1322500, PMID:8332195, PMID:11756411, PMID:8774884, PMID:10022854, PMID:8601312). Beyond MEK activation, Bcl-2 targets RAF1 to mitochondria where it phosphorylates BAD to suppress apoptosis, and RAF1 inhibits MST2 dimerization and Rok-alpha kinase activity through kinase-independent mechanisms that control apoptosis and cell migration (PMID:8929532, PMID:15618521, PMID:19948477). Gain-of-function mutations in RAF1—particularly those disrupting the Ser259 autoinhibitory 14-3-3 binding motif—cause Noonan syndrome and LEOPARD syndrome with hypertrophic cardiomyopathy (PMID:17603483).

Mechanistic history

Synthesis pass · year-by-year structured walk · 18 steps
  1. 1992 High

    Identifying the first physiological substrate of Raf-1 established it as the kinase directly upstream of MEK in the MAPK cascade, converting Raf-1 from an oncogene product of unknown function to a defined signaling kinase.

    Evidence In vitro kinase reconstitution with purified c-Raf-1 reactivating phosphatase-inactivated MAPK-K (Nature)

    PMID:1322500

    Open questions at the time
    • Substrate specificity beyond MEK not defined
    • In vivo confirmation in intact cells not yet provided
  2. 1993 High

    Demonstrating direct GTP-dependent binding of Ras to the Raf-1 N-terminal cysteine-rich domain identified Raf-1 as a bona fide Ras effector, explaining how receptor tyrosine kinase signals reach the MAPK cascade.

    Evidence GST pulldown with purified Ras-GTP/GDP; effector-loop mutant competition (Nature, two independent labs)

    PMID:8332187 PMID:8332195

    Open questions at the time
    • Structural basis of Ras–Raf interaction not resolved
    • Contribution of CRD versus RBD to membrane engagement unclear
  3. 1993 High

    Mapping the major in vivo phosphorylation sites (Ser43, Ser259, Ser621) and showing PKC-alpha can activate Raf-1 via Ser499 revealed that Raf-1 integrates multiple kinase inputs and that phosphorylation has both activating and inhibitory roles.

    Evidence In vivo phospholabeling, phosphopeptide mapping, site-directed mutagenesis, and in vitro kinase assays (JBC, Nature)

    PMID:8321321 PMID:8349614

    Open questions at the time
    • Kinases responsible for constitutive Ser259 phosphorylation not identified
    • Hierarchical ordering of phosphorylation events unresolved
  4. 1994 High

    Discovery that 14-3-3 proteins bind Raf-1 and modulate its activity, and that Src-family kinases phosphorylate Raf-1 on tyrosine to stimulate kinase activity, revealed two additional layers of Raf-1 regulation beyond Ras binding.

    Evidence Yeast two-hybrid and Xenopus oocyte assays for 14-3-3; SH2 domain pulldown and co-IP with Src/Fyn for tyrosine phosphorylation (Nature, JBC)

    PMID:7517401 PMID:7935795

    Open questions at the time
    • Specific 14-3-3 binding phosphosites on Raf-1 not yet mapped
    • Whether Src phosphorylation is required or merely potentiating in vivo unclear
  5. 1995 High

    Establishing that Ras-dependent membrane recruitment is prerequisite for Src-mediated Tyr340/341 phosphorylation resolved the ordering problem: Ras first recruits Raf-1 to the membrane where co-resident kinases complete activation.

    Evidence Mutagenesis of Tyr340/341; subcellular fractionation; mammalian cell co-expression (EMBO J)

    PMID:7542586

    Open questions at the time
    • Identity of the additional Ras-dependent activating event beyond Tyr phosphorylation unresolved
    • Quantitative contribution of each tyrosine site not dissected
  6. 1996 High

    Chemogenetic dimerization experiments proved that Raf-1 oligomerization is sufficient to activate the MAPK cascade, establishing dimerization as a core activation mechanism distinct from simple membrane proximity.

    Evidence Coumermycin/FKBP-Raf and FK1012A dimerization systems with kinase and ERK cascade readouts (Nature, two papers)

    PMID:8774884 PMID:8774885

    Open questions at the time
    • Whether dimerization is with another Raf-1 or with BRAF in physiological settings not addressed
    • Crystal structure of Raf kinase domain dimer not available
  7. 1996 High

    The discovery that Bcl-2 targets Raf-1 to mitochondria where it phosphorylates BAD and prevents apoptosis demonstrated a MEK-independent pro-survival function compartmentalized by subcellular targeting.

    Evidence GFP-Raf-1 localization; mitochondria- vs. plasma-membrane-targeted constructs; BAD phosphorylation and apoptosis assays (Cell)

    PMID:8929532

    Open questions at the time
    • Full spectrum of mitochondrial Raf-1 substrates unknown
    • How Bcl-2–Raf-1 interaction is regulated by upstream signals not resolved
  8. 1996 High

    Defining the RSXpSXP consensus for 14-3-3 binding using Raf-1 phosphopeptides and showing PKA phosphorylates Ser621 to inhibit Raf-1 revealed phosphoserine-dependent scaffolding and cross-talk between cAMP and MAPK pathways.

    Evidence Peptide binding and phosphopeptide competition (Cell); mass spectrometry site identification with PKA co-expression (MCB)

    PMID:8601312 PMID:8816453

    Open questions at the time
    • How dual 14-3-3 occupancy (Ser259 + Ser621) coordinates autoinhibition vs. activation not fully understood
  9. 1997 High

    Identification of the Cdc37–Hsp90 chaperone complex as essential for Raf-1 folding/stability showed that Raf-1 is a client kinase whose steady-state levels and activity depend on chaperone engagement, and that geldanamycin triggers Raf-1 ubiquitination and proteasomal degradation.

    Evidence Dominant-negative Cdc37; geldanamycin disruption; ubiquitin laddering; proteasome inhibitor rescue (MCB, BBRC)

    PMID:10022854 PMID:9367823

    Open questions at the time
    • E3 ligase responsible for Raf-1 ubiquitination not identified
    • Structural basis of Cdc37–Raf-1 kinase domain interaction unknown at this stage
  10. 1999 High

    Demonstrating that Akt directly phosphorylates Raf-1 at Ser259 to inhibit MEK-ERK signaling established a major cross-inhibitory node between PI3K-Akt and Ras-MAPK pathways.

    Evidence Co-immunoprecipitation; in vitro kinase assay; Ser259 mutagenesis; ERK pathway readout (Science)

    PMID:10576742

    Open questions at the time
    • Whether Akt-mediated Ser259 phosphorylation is the dominant mechanism in all cell types not established
  11. 2001 High

    PP2A-mediated dephosphorylation of Ser259 was shown to be the critical switch that licenses Raf-1 membrane accumulation and subsequent Ser338 phosphorylation, ordering the activation cascade: Ser259 dephosphorylation → membrane recruitment → Ser338 phosphorylation.

    Evidence Phosphosite-specific analysis; Ser259 mutagenesis; membrane-tethered Raf-CAAX constructs; kinase assays (JBC, EMBO J)

    PMID:11756411 PMID:11782426

    Open questions at the time
    • How PP2A is recruited to Raf-1 in a stimulus-dependent manner not defined
  12. 2001 High

    Raf-1 knockout mice die embryonically with increased apoptosis, yet kinase-dead knock-in mice (Y340F/Y341F) are viable, proving that Raf-1 has essential kinase-independent functions in vivo beyond MEK-ERK activation.

    Evidence Gene targeting: Raf-1 knockout and Y340F/Y341F knock-in mice; embryological phenotype analysis (EMBO J)

    PMID:11296227

    Open questions at the time
    • Identity of kinase-independent effectors not yet known
    • Tissue-specific requirements not dissected
  13. 2004 High

    Identifying MST2 as a kinase-independent binding partner of Raf-1 whose dimerization and pro-apoptotic activity are suppressed by Raf-1 explained the apoptosis phenotype of Raf-1-null cells and defined a kinase-independent tumor-suppressive checkpoint controlled by Raf-1.

    Evidence Proteomic complex analysis; co-IP; siRNA rescue of apoptosis in Raf-1−/− cells (Science)

    PMID:15618521

    Open questions at the time
    • Whether Raf-1–MST2 interaction is regulated by Ras or growth factors not fully resolved
    • Structural basis of Raf-1–MST2 complex unknown
  14. 2005 High

    ERK-mediated feedback phosphorylation of Raf-1 at six sites that disrupt Ras binding was shown to desensitize Raf-1, with PP2A and Pin1 required to reset the kinase, establishing the negative feedback loop that limits MAPK signal duration.

    Evidence Mass spectrometry phosphosite mapping; mutagenesis; Ras co-IP; PP2A/Pin1 functional studies (Mol Cell)

    PMID:15664191

    Open questions at the time
    • Kinetics of desensitization and resensitization in single cells not measured
    • Relative contribution of each feedback site not individually quantified
  15. 2005 High

    Raf-1 was found to inhibit Rok-alpha kinase activity in trans through its regulatory domain, promoting Ras-dependent cell migration independently of MEK-ERK, defining a second major kinase-independent effector function.

    Evidence In vitro kinase inhibition assay; domain mapping; rescue of Raf-1−/− cells with regulatory domain alone; migration assay (J Cell Biol)

    PMID:19948477

    Open questions at the time
    • Structural mechanism of Rok-alpha inhibition not resolved at atomic level
    • In vivo relevance in specific tissue contexts not tested
  16. 2007 High

    Identification of gain-of-function RAF1 mutations in Noonan and LEOPARD syndrome patients, with HCM-associated mutants showing enhanced kinase activity and disrupted Ser259-mediated autoinhibition, linked RAF1 hyperactivation directly to human developmental disease.

    Evidence Genotype-phenotype correlation; in vitro and in vivo kinase assays of patient-derived mutants; ERK phosphorylation (Nat Genet)

    PMID:17603483

    Open questions at the time
    • How different mutants produce HCM vs. non-HCM phenotypes mechanistically not resolved
    • Tissue-specific signaling consequences of each mutation not characterized
  17. 2010 High

    The paradoxical activation of wild-type RAF by ATP-competitive RAF inhibitors through drug-induced dimerization and Ras-dependent transactivation of CRAF explained clinical resistance mechanisms and confirmed dimerization as the central switch in RAF activation.

    Evidence Biochemical kinase/dimerization assays; co-IP; membrane fractionation; xenograft models; kinase-dead BRAF–CRAF complex analysis (Nature, Cell)

    PMID:20130576 PMID:20141835

    Open questions at the time
    • Structural basis of drug-induced dimer interface not fully resolved at this time
    • Whether all RAF inhibitor scaffolds induce equivalent paradoxical activation unknown
  18. 2012 High

    Crystallographic determination of the 14-3-3ζ dimer bound simultaneously to pSer233 and pSer259 of C-RAF revealed the structural basis of autoinhibitory cytoplasmic retention, showing how dual phosphosite engagement locks Raf-1 in an inactive state.

    Evidence Crystal structure; ITC; fluorescence polarization; cellular localization (J Mol Biol)

    PMID:22922483

    Open questions at the time
    • Full-length Raf-1 structure in the autoinhibited 14-3-3-bound state not yet available
    • How release from dual 14-3-3 engagement is triggered upon Ras activation remains structurally unresolved

Open questions

Synthesis pass · forward-looking unresolved questions
  • A full-length structure of RAF1 in its autoinhibited and active conformations, the precise mechanism by which Ras-GTP binding triggers release of 14-3-3-mediated autoinhibition, and in vivo quantitative dissection of kinase-dependent versus kinase-independent functions across tissues remain unresolved.
  • No full-length RAF1 structure available
  • Mechanism of stimulus-dependent PP2A recruitment to Raf-1 unknown
  • Quantitative tissue-specific contribution of kinase-independent functions (MST2, Rok-alpha) not established in vivo

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140096 catalytic activity, acting on a protein 5 GO:0016740 transferase activity 4 GO:0098772 molecular function regulator activity 2
Localization
GO:0005886 plasma membrane 5 GO:0005739 mitochondrion 4 GO:0005829 cytosol 1
Pathway
R-HSA-162582 Signal Transduction 10 R-HSA-5357801 Programmed Cell Death 4 R-HSA-1643685 Disease 1
Partners
BRAFCDC37HSP90AA1KRASMAP2K1ROCK2STK3YWHAZ
Complex memberships
14-3-3–Raf-1 complexBRAF–CRAF heterodimerRaf-1–Hsp90–Cdc37 chaperone complexRaf-1–MEK1 complex

Evidence

Reading pass · 43 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1992 RAF1 (c-Raf-1) directly phosphorylates and activates MAP kinase-kinase (MAPK-K/MEK) at serine/threonine residues in vitro, identifying MAPK-K as the first physiological substrate of the c-raf-1 protooncogene product. In vitro kinase reconstitution assay; purified c-Raf-1 reactivated phosphatase 2A-inactivated MAPK-K with coincident phosphorylation of the 50 kDa MAPK-K polypeptide Nature High 1322500
1993 The amino-terminal cysteine-rich regulatory region of Raf-1 binds directly to GTP-bound (active) Ras with ~50 nM affinity; binding requires the Ras effector region and is blocked by effector-loop mutants of Ras, identifying Raf-1 as a direct effector of Ras. GST pulldown with purified proteins; GTP-/GDP-Ras binding specificity; competition with GAP proteins Nature High 8332187 8332195
1993 PKC-alpha directly phosphorylates and activates Raf-1 both in vitro and in vivo, with a key phosphorylation site at Ser499; mutation of Ser499 or Ser259 abrogates PKC-alpha-mediated but not Ras/Src-mediated Raf-1 activation. In vitro kinase assay with purified PKC-alpha and Raf-1; site-directed mutagenesis; cotransfection in NIH3T3 cells Nature High 8321321
1993 Raf-1 and Mek1 form a stable complex both in vivo and in vitro independently of kinase activity; Raf-1 activates Mek1 >150-fold exclusively through serine phosphorylation at a single site on Mek1. Baculovirus co-infection in Sf9 cells; co-immunoprecipitation; in vitro kinase assay; phosphopeptide mapping PNAS High 8248196
1993 Ser43, Ser259, and Ser621 are the major in vivo phosphorylation sites of Raf-1; Ser259 mutants show ~2-fold increased kinase activity while Ser621 mutants are catalytically inactive, indicating Ser621 is essential for kinase function. Baculovirus expression; in vivo phospholabeling; phosphopeptide mapping; site-directed mutagenesis; in vitro kinase assay Journal of Biological Chemistry High 8349614
1994 14-3-3 proteins (zeta and beta isoforms) interact with the amino-terminal region of Raf-1 and activate Raf-1 kinase activity; expression of 14-3-3 in Xenopus oocytes enhanced Raf-1 activity and promoted Raf-1-dependent oocyte maturation. Yeast two-hybrid screen; Xenopus oocyte functional assay; dominant-negative Raf-1 epistasis Nature High 7935795
1994 Raf-1 interacts with the SH2 domains of Fyn and Src through a serine phosphorylation-dependent (not tyrosine-dependent) mechanism; co-expression with full-length Fyn or Src leads to tyrosine phosphorylation of Raf-1 and stimulation of its kinase activity. SH2 domain pulldown assays; co-immunoprecipitation from Sf9 cells; in vitro kinase assay Journal of Biological Chemistry Medium 7517401
1995 Ras-dependent activation of Raf-1 by oncogenic Src requires Ras-GTP to recruit Raf-1 to the plasma membrane, where it is phosphorylated at Tyr340/341 (likely by Src); an additional activation step beyond tyrosine phosphorylation results from Ras-GTP membrane recruitment itself. Mammalian cell co-expression; mutagenesis of Tyr340/341; subcellular fractionation; kinase assays EMBO Journal High 7542586
1995 14-3-3 beta acts as a Ras-independent activator of Raf-1 in cell-free extracts and in vivo; in quiescent cells 14-3-3 is associated with unactivated Raf-1 but dissociates upon mitogen stimulation. Yeast two-hybrid; purified protein binding; co-immunoprecipitation from NIH 3T3 cells; cell-free kinase assay; PC12 differentiation assay EMBO Journal Medium 7882972
1996 Raf-1 dimerization is sufficient to activate the MAP kinase cascade independently of membrane components; activation via dimerization requires Ras-GTP. Coumermycin-induced chemical dimerization of FKBP-Raf fusion proteins; kinase assays Nature High 8774884
1996 Oligomerization of c-Raf-1 promoted by the dimeric 14-3-3 scaffold activates Raf kinase activity through a Ras-GTP-dependent mechanism. FKBP-Raf oligomerization with FK1012A dimerizer in COS cells; kinase assays; dominant-negative Ras epistasis Nature High 8774885
1996 PKA phosphorylates Raf-1 at Ser621 within the kinase domain, inhibiting catalytic activity and reducing Ras-binding affinity; Ser621 has a dual role as essential for catalytic activity yet also serving as an inhibitory PKA phosphorylation site. Co-expression with constitutively active PKA catalytic subunit; mass spectrometry; site-directed mutagenesis; in vitro kinase assay Molecular and Cellular Biology High 8816453
1996 Ras-induced activation of Raf-1 requires tyrosine phosphorylation; the active, tyrosine-phosphorylated pool of Raf-1 is membrane-localized and can be inactivated by PTP-1B; 14-3-3 and Hsp90 protect Raf-1 from tyrosine dephosphorylation and inactivation. Baculovirus Sf9 co-expression; PTP-1B inactivation assay; membrane fractionation; Western blotting for phosphotyrosine Molecular and Cellular Biology Medium 8622647
1996 The 14-3-3 protein binding motif defined using Raf-1 phosphopeptides requires phosphoserine; 14-3-3 binds RSXpSXP motifs on Raf-1 (Ser259-region), and phosphopeptides can disrupt 14-3-3 complexes and inhibit Xenopus oocyte maturation. Peptide binding assays; phosphopeptide competition; Xenopus oocyte functional assay Cell High 8601312
1996 Bcl-2 targets Raf-1 to mitochondria via its BH4 domain; mitochondria-targeted Raf-1 phosphorylates BAD and protects cells from apoptosis, whereas plasma membrane-targeted Raf-1 phosphorylates ERK1/2 but does not protect against apoptosis. GFP-Raf-1 localization; outer mitochondrial membrane targeting constructs; apoptosis assays; BAD phosphorylation assay Cell High 8929532
1997 p50(Cdc37) is the primary determinant of Hsp90 recruitment to Raf-1; formation of a ternary Raf-1–Cdc37–Hsp90 complex is required for Raf-1 kinase activity and MAPK pathway signaling; geldanamycin disrupts this complex and blocks Raf-1 activation. Baculovirus Sf9 co-expression; dominant-negative Cdc37 mutant; geldanamycin treatment; kinase assays Molecular and Cellular Biology High 10022854
1997 Geldanamycin disrupts the Raf-1–Hsp90 complex and leads to Raf-1 polyubiquitination and proteasomal degradation; proteasome inhibitors prevent GA-induced Raf-1 loss and partially restore signaling. Western blot; proteasome inhibitors; lysosomal protease inhibitors; ubiquitin laddering assay Biochemical and Biophysical Research Communications Medium 9367823
1997 KSR (kinase suppressor of Ras) translocates to the plasma membrane with activated Ras and enhances Raf-1 activity in a kinase-independent manner via its cysteine-rich CA3 domain. Subcellular localization studies; kinase assays; CA3 domain mutagenesis; mammalian cell transfection PNAS Medium 9371754
1998 Autoinhibition by the N-terminal regulatory region of Raf-1 (mediated through the cysteine-rich domain) suppresses kinase activity; mutations in the CRD or Y340D phosphomimetic mutation relieve this autoinhibition and increase Raf-1 activity. Site-directed mutagenesis; in vitro kinase assays; domain deletion analysis PNAS Medium 9689060
1999 Akt (protein kinase B) directly interacts with Raf-1 and phosphorylates it at a conserved serine residue (Ser259) in its regulatory domain, inhibiting the Raf-MEK-ERK pathway and shifting cellular response from growth arrest to proliferation. Co-immunoprecipitation; in vitro kinase assay; site-directed mutagenesis; ERK pathway readout Science High 10576742
1999 Mitochondria-targeted Raf-1 (activated by Akt in a Ras-independent, PKC-dependent manner) phosphorylates BAD and mediates Akt-dependent anti-apoptotic effects; dominant-negative mitochondria-targeted Raf-1 renders Akt-expressing cells susceptible to apoptosis. Subcellular fractionation; dominant-negative mitochondria-targeted Raf-1; apoptosis assay; BAD phosphorylation assay Cancer Research Medium 10383138
2000 Phosphatidic acid (PA) directly mediates agonist-dependent Raf-1 recruitment to endosomal membranes independently of Ras; mutations disrupting Raf-PA interactions prevent Raf-1 membrane recruitment, while disruption of Ras-Raf interaction does not affect translocation. Subcellular fractionation; GFP-Raf localization; mutagenesis of PA-binding region; dominant-negative Ras Journal of Biological Chemistry Medium 10801816
2000 Ras dimer formation in lipid membranes is essential for Raf-1 activation; enforced dimerization of unmodified Ras activates Raf-1, and a Ras-dependent Raf-1 activator is still required. Liposome reconstitution; bifunctional crosslinker; protein-fragmentation complementation assay; enforced Ras dimerization constructs Journal of Biological Chemistry Medium 10660519
2001 Dephosphorylation of Raf-1 Ser259 by PP2A is a critical activation step that promotes Raf-1 membrane accumulation and facilitates phosphorylation of the activating site Ser338; Ser259 phosphorylation prevents membrane recruitment rather than directly reducing specific kinase activity. Phosphorylation-state specific analysis; Ser259 mutagenesis; membrane-tethered Raf-1 (Raf-CAAX); kinase assays Journal of Biological Chemistry High 11756411
2001 MEK kinase activity of Raf-1 is not essential for normal mouse development or ERK activation; Raf-1(Y340F/Y341F) knock-in mice lacking detectable MEK kinase activity are viable and fertile, but Raf-1-null mice die embryonically with vascular defects and increased apoptosis. Gene targeting (knockout and knock-in mice); in vitro MEK kinase assay; embryological and cellular phenotype analysis EMBO Journal High 11296227
2002 Ser259 dephosphorylation enhances Raf-1 coupling to Ras and to MEK; Ser259 mutation elevates Raf-1 activity by increasing Ras binding, promoting constitutive membrane recruitment, and facilitating Ser338 phosphorylation. Mutagenesis; co-immunoprecipitation; kinase assays; membrane recruitment assays EMBO Journal High 11782426
2002 C-Raf forms a complex with mitochondrial VDAC (voltage-dependent anion channel) in vivo and blocks VDAC channel reconstitution in planar bilayer membranes in vitro, providing a Bcl-2-independent mechanism for Raf-mediated inhibition of cytochrome c release. Co-immunoprecipitation; planar bilayer electrophysiology; mitochondria-targeted Raf constructs; apoptosis assay BMC Cell Biology Medium 12079506
2004 Raf-1 counteracts apoptosis by suppressing MST2 (mammalian sterile 20-like kinase) through a kinase-independent mechanism: Raf-1 prevents MST2 dimerization and dephosphorylates its activation loop; MST2 depletion rescues apoptosis hypersensitivity of Raf-1-null cells. Proteomic analysis of Raf-1 signaling complexes; co-immunoprecipitation; siRNA knockdown of MST2; apoptosis assays; Raf-1-/- mouse cells Science High 15618521
2005 ERK-mediated direct feedback phosphorylation of Raf-1 at six sites (five proline-directed, ERK targets) after mitogen stimulation inhibits Ras/Raf-1 interaction and desensitizes Raf-1; PP2A and Pin1 subsequently dephosphorylate and restore Raf-1 to a signaling-competent state. Mass spectrometry phosphosite identification; mutagenesis; co-immunoprecipitation; kinase desensitization assays; PP2A and Pin1 functional studies Molecular Cell High 15664191
2005 The Raf-1 cysteine-rich domain (CRD) binds retinoids with high affinity; UV-induced oxidation of CRD cysteine thiols releases zinc and restructures the CRD, priming Raf-1 for Ras-GTP interaction; retinoids modulate these oxidation events to regulate Raf-1 activation. In vitro retinoid binding assay; zinc release assay; kinase activity measurement; mutagenesis Journal of Biological Chemistry Medium 11971897
2005 The Raf-1 regulatory domain (Raf-1reg) inhibits Rok-alpha kinase activity in trans by binding the Rok-alpha kinase domain in a manner analogous to Rok-alpha's own autoinhibitory region; this kinase-independent mechanism promotes cell migration and tumorigenesis downstream of Ras. Co-immunoprecipitation; in vitro kinase assay; domain mapping; Raf-1-/- cell rescue with Raf-1reg; cell migration assay Journal of Cell Biology High 19948477
2005 CNK1 is a scaffold protein that binds preactivated Raf-1 and activated Src simultaneously, mediating Src-dependent tyrosine phosphorylation and activation of Raf-1; CNK1 siRNA knockdown impairs Src-dependent ERK activation. Co-immunoprecipitation; siRNA knockdown; tyrosine phosphorylation assay; ERK activation assay Journal of Biological Chemistry Medium 15845549
2001 Raf-1 phosphorylates the myosin-binding subunit (MBS) of myosin phosphatase and inhibits phosphatase activity; ionizing radiation increases Raf-1–MBS association and suppresses myosin phosphatase activity, implicating Raf-1 in cytoskeletal regulation. Co-immunoprecipitation; in vitro kinase assay with GST-MBS; sequential kinase-phosphatase assay Journal of Biological Chemistry Medium 11719507
2004 Raf-1 directly associates with keratin 8 (K8) independently of Raf kinase activity or Ras interaction; K18 is a physiological Raf-1 substrate; Raf-1 activation during oxidative stress disrupts keratin-Raf association in a phosphorylation-dependent manner, and 14-3-3 residues essential for Raf binding also regulate 14-3-3–keratin interaction. Co-immunoprecipitation; in vitro kinase assay; mutagenesis; cell stress experiments; subcellular fractionation Journal of Cell Biology Medium 15314064
2007 Hepatitis B virus X protein (HBx) stimulates mitochondrial translocation of Raf-1 via oxidative stress; this translocation requires Raf-1 phosphorylation at Ser338/339 by PAK1 and at Y340/341 by Src kinase. Subcellular fractionation; co-immunoprecipitation; kinase inhibitor studies; site-specific phosphorylation analysis Journal of Virology Medium 17428866
2007 Gain-of-function missense mutations in RAF1 (affecting the Ser259 autoinhibitory 14-3-3 binding motif and a second hotspot) cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy; HCM-associated mutants show increased kinase activity and enhanced ERK activation, whereas non-HCM mutants are kinase-impaired. In vitro and in vivo kinase assays; mutant RAF1 expression in cells; ERK phosphorylation assays Nature Genetics High 17603483
2008 GRP78 associates and colocalizes with Raf-1 on the outer mitochondrial membrane; GRP78 stabilizes Raf-1 to maintain mitochondrial permeability and prevent ER stress-induced apoptosis, with Raf-1 phosphorylating BAD at Ser75. Co-immunoprecipitation; subcellular fractionation; GRP78/Raf-1 overexpression; BAD phosphorylation assay; apoptosis assay Journal of Cellular Physiology Medium 18064632
2008 Ser338 on Raf-1 is autophosphorylated in response to mitogens: a kinase-dead Raf-1 mutant loses Ser338 phosphorylation under EGF/TPA, active Raf-1 trans-phosphorylates Ser338 on kinase-dead Raf-1 in dimerized complexes, and artificial Raf-1 dimerization induces Ser338 phosphorylation with ERK activation. Site-directed mutagenesis; dimerization constructs; phospho-specific antibodies; ERK activation assay Journal of Biological Chemistry Medium 18775988
2009 The C-terminal 14-3-3 binding site of Raf-1 (Ser621) controls MEK-ERK activation by maintaining Raf-1 in an ATP-binding-competent conformation; 14-3-3 binding to pSer621 completely reverses the inhibitory effect of Ser621 phosphorylation and is required for MEK phosphorylation. Phosphorylation-state analysis; mutagenesis; ATP-binding assay; MEK-binding assay; kinase activity assay Cellular Signalling Medium 19595761
2010 ATP-competitive RAF inhibitors activate wild-type RAF isoforms (including C-RAF) by inducing kinase domain dimerization, membrane localization, and interaction with RAS-GTP in a RAS-dependent manner, paradoxically enhancing MAPK signaling in cells with oncogenic RAS. Biochemical kinase assays; dimerization assays; membrane fractionation; RAS co-immunoprecipitation; xenograft tumor models Nature High 20130576
2010 Kinase-dead BRAF drives tumor progression by forming a RAS-dependent complex with CRAF (RAF1), transactivating CRAF and thereby activating MEK-ERK signaling; RAF inhibitors recapitulate kinase-dead BRAF by driving RAS-dependent BRAF-CRAF binding. Co-immunoprecipitation; MEK-ERK signaling assays; mouse melanoma model with kinase-dead Braf plus oncogenic Ras; RAF inhibitor treatment Cell High 20141835
2012 14-3-3ζ binds the two N-terminal phosphorylation sites of C-RAF (pSer233 and pSer259) simultaneously as a dimer; Ser259 is the high-affinity site and Ser233 is the low-affinity site; simultaneous engagement enhances binding affinity and promotes cytoplasmic retention of C-RAF. Fluorescence polarization; isothermal titration calorimetry; crystal structure of 14-3-3ζ/C-RAFpS233pS259 complex; cellular localization studies Journal of Molecular Biology High 22922483
2020 MD simulations of the quaternary KRas4B–Raf-1 complex show that Raf-1 RBD-CRD stabilizes KRas4B dimer at the membrane and cooperatively enhances affinity; Ras activates Raf by shifting its ensemble toward kinase domain-accessible (dimerization-competent) states rather than by allosteric conformational change. Explicit membrane MD simulations of quaternary KRas4B–Raf-1 RBD-CRD complex Computational and Structural Biotechnology Journal Low 32257057

Source papers

Stage 0 corpus · 130 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2009 Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science (New York, N.Y.) 3312 19608861
2010 Biological, clinical and population relevance of 95 loci for blood lipids. Nature 2873 20686565
2006 Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 2861 17081983
2013 Discovery and refinement of loci associated with lipid levels. Nature genetics 2409 24097068
2004 Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2296 15035987
1996 Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L). Cell 2236 8929531
2012 Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 1718 22658674
2002 Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proceedings of the National Academy of Sciences of the United States of America 1479 12477932
2010 RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 1336 20130576
1993 Protein kinase C alpha activates RAF-1 by direct phosphorylation. Nature 1243 8321321
2010 Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 1221 20141835
1996 Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 1220 8601312
1992 Raf-1 activates MAP kinase-kinase. Nature 1214 1322500
2015 High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities. Cell 1200 26627737
2017 Architecture of the human interactome defines protein communities and disease networks. Nature 1085 28514442
2001 Nerve growth factor signaling, neuroprotection, and neural repair. Annual review of neuroscience 1029 11520933
2015 A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell 1015 26496610
1997 Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell 955 9150145
1999 Phosphorylation and regulation of Raf by Akt (protein kinase B). Science (New York, N.Y.) 910 10576742
2020 A reference map of the human binary protein interactome. Nature 849 32296183
1993 Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1. Nature 830 8332187
2005 Rheb binds and regulates the mTOR kinase. Current biology : CB 795 15854902
1998 Premature senescence involving p53 and p16 is activated in response to constitutive MEK/MAPK mitogenic signaling. Genes & development 779 9765203
2007 Large-scale mapping of human protein-protein interactions by mass spectrometry. Molecular systems biology 733 17353931
1996 Bcl-2 targets the protein kinase Raf-1 to mitochondria. Cell 720 8929532
2012 Quantitative analysis of HSP90-client interactions reveals principles of substrate recognition. Cell 708 22939624
2021 Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. Cell 705 33961781
1993 Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature 695 8332195
2005 An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science (New York, N.Y.) 687 15705808
2001 Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds. Proceedings of the National Academy of Sciences of the United States of America 685 11226259
2011 Phylogenetic-based propagation of functional annotations within the Gene Ontology consortium. Briefings in bioinformatics 656 21873635
1995 Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation. The EMBO journal 548 7542586
1997 The complexity of Raf-1 regulation. Current opinion in cell biology 542 9069260
2007 Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy. Nature genetics 527 17603483
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